Registration Dossier

Diss Factsheets

Toxicological information

Skin irritation / corrosion

Currently viewing:

Administrative data

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
2015-10-12 to 2015-10-19
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
Qualifier:
according to guideline
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Deviations:
no
GLP compliance:
yes

Test material

Constituent 1
Chemical structure
Reference substance name:
-
EC Number:
439-510-7
EC Name:
-
Cas Number:
149048-48-6
Molecular formula:
Constituent 1: C14H26N2O3Si Constituent 2: C13H22N2O2Si
IUPAC Name:
1-(3-{13-[3-(aminomethyl)phenyl]-6,6,8,8-tetramethoxy-7-oxa-2,12-diaza-6,8-disilatridecan-1-yl}phenyl)methanamine; 1-[3-({[3-(trimethoxysilyl)propyl]amino}methyl)phenyl]methanamine
Test material form:
liquid

Test animals

Species:
other: Human skin model
Strain:
other: EPISKIN Small ModelTM
Details on test animals or test system and environmental conditions:
- Test system: EPISKIN Small ModelTM is a three-dimensional human epidermis model, which consists of adult human-derived epidermal keratinocytes. The adult human-derived epidermal keratinocytes have been seeded on a dermal substitute consisting of a collagen type I matrix coated with type IV collagen. The keratinocytes were grown for 13 days in culture, resulting in highly differentiated and stratified epidermis model comprising the main basal, supra basal, spinous and granular layers and a functional stratum corneum.
- Preincubation: Tissues were preincubated with Maintenance Medium for 27 hours at 37°C.
- Killed tissues: Living epidermis was transferred into 12 well plates and incubated with 2 ml Milli-Q for 48 ± 1 hours. After incubation, killed epidermis was stored at ≤ -15°C. Killed tissues were thawed by placing them for 1 hour at room temperature in 12 well plates on 2 ml maintenance medium.
- MTT medium: MTT concentrate was diluted in assay medium to result in final concentration of 0.3 mg/ml.
- Environmental conditions: All incubations, excluding incubation with test item were carried out in a controlled environment with humid atmosphere of 80 - 100% (actual range 70 - 91%), containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C (actual range 36.5 - 37.3°C).

Test system

Vehicle:
unchanged (no vehicle)
Controls:
other: negative control: 25 μl PBS; positive control: 25 μl 5% SDS
Amount / concentration applied:
25 μl of undiluted test item
Duration of treatment / exposure:
15 +/- 5 min at room temperature
Observation period:
incubated for 42 hours at 37°C
Number of animals:
3 tissues per test item together with negative and positive controls
Details on study design:
TREATMENT:
- The test item was applied undiluted on top of the skin model.
- Three killed tissues treated with test item and three killed non treated tissues were used for the cytotoxicity evaluation with MTT.
- The positive control was re-spread after 7 minutes contact time.
- After exposure period the tissues were washed with phosphate buffered saline to remove residual test item.
- After rinsing, the cell culture inserts were dried carefully and moved to a new well on 2 ml pre-warmed maintenance medium until all tissues were dosed and rinsed.

CELL VIABILITY MEASUREMENT:
- After incubation, cell culture inserts were dried carefully to remove excess medium and were transferred into a 12-wells plate prefilled with 2 ml MTT-medium (0.3 mg/ml) and incubated for 3 h at 37°C and dried afterwards.
- Total biopsy was performed
- Epidermis was separated from the collagen matrix and both parts were placed in prelabelled microtubes and extracted with 500 μL isopropanol
- Tubes were stored refrigerated and protected from light for 72 hours
- The amount of extracted formazan was determined spectrophotometrically at 570 nm in duplicate with the TECAN Infinite® M200 Pro Plate Reader.

Results and discussion

In vitro

Results
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
15 ± 0.5 minutes treatment
Value:
124
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Remarks:
Mean % tissue viability was >= 50 %

Any other information on results incl. tables

Irritant / corrosive response data

The relative mean tissue viability obtained after 15 ± 0.5 minutes treatment with the submission substance compared to the negative control tissues was 124%.

Other effects

-               Test for colour interference: a colour change was observed by adding MTT-medium it was concluded that the submission substance did interact with the MTT endpoint

-               Test for reduction of MTT by the test item: The non-specific reduction of MTT by the submission substance was -5% of the negative control tissues

Table 1: Mean absorption in the in vitro skin irritation test

 

Mean

(OD570)

SD

Negative control

0.878

+/- 0.122

Test substance

1.088

+/- 0.110

Positive control

0.107

+/- 0.028

OD = optical density

SD = Standard deviation

Table 2: Mean tissue viability in the in vitro skin irritation test

 

Mean tissue viability (percentage of control)

Negative control

100

Test substance

124

Positive control

12

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
In the in vitro skin irritation study, the submission substance was concluded to be not irritating to human skin model EPISKIN-SMTM.