Registration Dossier

Toxicological information

Skin irritation / corrosion

Currently viewing:

Administrative data

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
July 11, 2012 - July 13, 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Test method according to OECD Guideline 439. GLP study.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report date:
2012

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 (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
5-ethyl-2,8-dimethyl-5-[(propan-2-ylideneamino)oxy]-4,6-dioxa-3,7-diaza-5-silanona-2,7-diene
EC Number:
611-631-1
Cas Number:
58190-57-1
Molecular formula:
C11H23N3O3Si
IUPAC Name:
5-ethyl-2,8-dimethyl-5-[(propan-2-ylideneamino)oxy]-4,6-dioxa-3,7-diaza-5-silanona-2,7-diene
Details on test material:
- Name of test material (as cited in study report): EAC3
- Physical state: Liquid
- Purity: 92.13%
- Lot/batch No.: 1000061820
- Expiration date of the lot/batch: 11 January 2013
- Storage condition of test material: Controlled Room Temperature (15-25°C, below 70 RH%), Protected from humidity

Test animals

Species:
other: In vitro: Human skin
Details on test animals or test system and environmental conditions:
The test system used was EPISKIN-SM, a three-dimensional human epidermis model.

Test system

Type of coverage:
other: In-vitro test
Preparation of test site:
other: In-vitro test
Vehicle:
unchanged (no vehicle)
Controls:
other: In-vitro test. Negative and positive controls were conducted.
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 50 µL
Duration of treatment / exposure:
15 minutes.
Observation period:
After approx. 42 hours.
Number of animals:
Not applicable: In-vitro test.
3 replicate wells for the test item and 3 negative controls + 3 positive controls were used
Details on study design:
PRE-INCUBATION (Day -1):
The assay platewells were filled with a pre-warmed medium (2 mL per well, 37 ºC). The epidermis units were placed, with the media below in contact with the epidermis, into each prepared well and then incubated overnight at 37°C in an incubator with 5% CO2.

APPLICATION AND RINSING (Day 0):
- Test skin units: 50 μL of test item (3 replicates)
- Negative control skin units: 50 μL Phosphate Buffered Saline (PBS) (3 replicates)
- Positive control skin units: 50 μL Sodium Dodecyl Sulphate (SDS) 5% aq. solution (3 replicates)
- For additional control for staining effects of the test item, 50 μL of the test item was applied evenly to the epidermal surface.
The treated plates were incubated for 15 min. exposure time at room temperature (21.7-22.3 ºC). Then, the EPISKIN-SM units were removed and rinsed with PBS 1x solution (0.9%). The units were place into the plate well with fresh pre-warmed maintenance medium (2mL/well) and incubated for 42 h at 37 ºC with 5% CO2.

MTT TEST (Day 2):
The EPISKIN-SM units were transferred into the MTT solution filled wells (2 mL of 0.3 mg/mL MTT per well). The staining control well instead, was transferred to well filled with fresh assay medium. The units were incubated for 3 h at 37 ºC with 5% CO2, protected from light.

FORMAZAN EXTRACTION (Day 2):
The epidermises from each disk were separated with the aid of forceps and both parts (epidermis and collagen matrix) and were placed into a tube of 500 µL acidified isopropanol. The capped tubes were thoroughly mixed by using a vortex mixer and then incubated for 2 hours at room temperature protected from light with gentle agitation (~150 rpm) for formazan extraction.

CELL VIABILITY MEASUREMENT (Day 2):
2×200 μL samples from each tube were placed into the wells of a 96-well plate. The OD (Absorbance / Optical Density) of the samples in a 96-well plate spectrophotometer was read at a wavelength 540 nm, using acidified isopropanol solution blank (6×200 μL). The viability was expressed as a % relative to negative control.

Results and discussion

In vivo

Irritant / corrosive response data:
The mean relative viability of test item EAC3 after 15 minutes exposure and 42 hours post incubation period (based on the optical activity measured at 540 nm) was 115% (SD± 8.89). According to EU classification, the substance was determined to be non-irritant since the mean tissue viability % was >50.

Any other information on results incl. tables

CELL VIABILITY:

The results of the optical density (OD) measured at 540 nm of each extract and the calculated % viability of the cells:

Substance

Optical density (OD)

Viability (%)

Negative control (PBS)

1

0.797

104

2

0.744

97

3

0.753

98

Mean

0.765

100

Stand. dev. (SD)

3.79

Positive control

(SDS 5%)

1

0.246

32

2

0.207

27

3

0.083

11

Mean

0.179

23

Stand. dev. (SD)

10.97

Test ítem

(EAC3)

1

0.856

112

2

0.959

125

3

0.823

108

Mean

0.879

115

Stand. dev. (SD)

8.89

VALIDITY OF THE TEST:

The mean OD value of the three negative control tissues was 0.765.

The positive control result showed a mean of 23% viability.

Each standard deviation value (SD) of the % viability was below 18.

Control OD values were below historically established boundaries.

The SD calculated from individual % tissues viabilities of the three identically test item treated replicates was 8.89.

All validity criteria were within acceptable limits and therefore the study can be considered as valid.

INDICATOR FOR POTENTIAL FALSE VIABILITY:

The test material did not interact with the MTT (based on no colour change observed).

The non specific colour % was calculated as 3.5% (below the threshold of 5.0) and therefore additional data calculation was not necessary.

Applicant's summary and conclusion

Interpretation of results:
not irritating
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
The mean relative viability of test item EAC3 after 15 minutes exposure and 42 hours post incubation period was 115% (SD± 8.89). According to EU classification, the substance is considered to be non-irritant since the mean tissue viability % was >50.
Executive summary:

An in-vitro skin irritation test in the EPISKIN model was performed on the substance EAC3 in accordance with OECD Guideline 439 and GLP. Disks of EPISKIN (three units / chemical) were treated with EAC3 and incubated for 15 minutes at room temperature. Exposure of test material was terminated by rinsing with PBS. Epidermis units were then 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 5% CO2 protected from light. The formazan extract in acidified isopropanol was then spectrophotometrically evaluated for optical density (OD) and quantified. SDS 5% and PBS treated epidermis were used as positive and negative controls respectively. For each treated tissue, optical density (OD) was calculated and the tissue viability was expressed as a % relative to negative control. Following exposure with EAC3, the mean treated skin value was 115% and therefore non-irritant to the skin (>50%). All validity criteria were within acceptable limits and therefore the study can be considered as valid.