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

Data source

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

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

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

Test animals

Species:
other: EPISKIN-SM tissue.

Test system

Vehicle:
unchanged (no vehicle)
Controls:
other: positive and negative control tissues
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 10 μl (26.3 μl/cm2)
- Concentration (if solution): undiluted

CONTROLS: Controls were set up in parallel to the test item cultures in order to confirm the validity of the test.
Negative control: Phosphate Buffered Saline (PBS; Gibco, Cat. No. 14040-091, Lot No. 1424317). 10 μl (26.3 μl/cm2)
Positive control: 5% sodium dodecyl sulfate (SDS; AppliChem, Art.-No. A1112,0500, CAS No.: 151-21-3, Lot No. 1V010396) in distilled water 10 μl (26.3 μl/cm2)
Duration of treatment / exposure:
15 ± 0.5 minute.
Observation period:
Treated tissue was post-incubated at 37 ± 1 °C, 5.0% CO2 for 42 ± 1 h
Number of animals:
Three tissues for each of test, negative control and positive control groups.
Details on study design:
TEST SYSTEM: reconstituted three-dimensional human skin model EPISKIN-SM™ (SkinEthic). This skin model consists of normal (non-cancerous), adult 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. 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.

PRE-EXPERIMENT
To check the non-specific MTT-reducing capability of the test item 10 μl of the test item were mixed per 2 ml MTT medium and incubated for 3 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]

EXPERIMENTAL PROCEDURE
Tissues were pre-incubated for at least 24 h 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 15 ± 0.5 minutes, the tissues were washed with PBS to remove residual test item, then excess PFS removed with blotting paper. The tissues were then placed in fresh MTT medium and incubated for a further 3 hours.

After incubation the tissues were dried on blotting paper and a biopsy punch was used and the epidermis was separated from the collagen matrix. Epidermis and collagen matrix were extracted with 500 μl of acidic isopropanol. Extraction was carried out protected from light over the weekend at 2 - 8°C, then the tubes were mixed by vortexing. Any visible fragments were removed by centrifugation.

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

Results and discussion

In vitro

Results
Irritation / corrosion parameter:
% tissue viability
Value:
113.9
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Remarks:
% tissue viability is the mean optical density relative to control. The standard deviation was +/- 18.2 % This slightly exceeds the acceptance criterion of 18%. Since each value is far beyond the 50% cut-off value, this standard deviation is accepted.
Other effects / acceptance of results:
PRE-EXPERIMENT-test item showed no reduction of MTT relative to negative control. No colouring was detected by unaided eye when 10 μl of the test item were mixed with 90 μl distilled water.

EXPERIMENT: see Table 2 below.

QUALITY CRITERIA: see Table 3 below.

Any other information on results incl. tables

Table 2: Experimental Results

Name

Negative Control

Positive Control

Test Item

Tissue

1

2

3

1

2

3

1

2

3

Mean OD550 of the duplicates (blank-corrected)

0.711

0.816

0.746

0.050

0.106

0.052

0.930

0.704

0.954

Total mean OD550 of 3 replicate tissues (blank-corrected)

0.758*

0.069

0.863

SD OD550

0.049

0.029

0.125

Mean relative tissue viability[%]

100

 

9.2**

113.9

SD tissue viability[%]***

7.0

4.2

18.2

CV [% viability]

7.0

45.9

16.0

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

** Mean relative tissue viability of the three positive control tissues is ≤ 40%.

*** The standard deviation (SD) obtained from the three concurrently tested tissues is < 18%. This criterion failed for the test item treated tissues.

NC negative control

OD550 Optical density at 550 nm

Table 3: Quality Criteria

 

value

cut off

pass/fail

Mean  

OD550 nm blank

0.044

< 0.1

pass

Mean absolute OD550 nm NC

0.802

0.6 ≤ NC ≤1.5

pass

mean % viability PC

9.2

≤ 40%

pass

SD of % viability

4.2 – 18.2*

< 18%

 fail*

 

* Criterion failed for the test item treated tissues.

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
[2-(perfluorohexyl)ethyl]trimethoxysilane has been tested in a reliable in vitro study for skin irritation conducted according to OECD 439 and in compliance with GLP using EPISKIN-SM tissue. The mean tissue viability of the test-item treated tissues was not reduced relative to the negative controls. The positive control produced the expected reduction in viability. It is concluded that the test item is not irritant under the conditions of the test.