N-butyl-N-[(triethoxysilyl)methyl]butan-1-amine

Skin irritation / corrosion

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:

other: The study was conducted according to the appropriate OECD test guideline, and in compliance with GLP.

Data source

Reference

Title:

Unnamed

Year:

2015

Report date:

2015

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)

Deviations:

yes

Remarks:

The deviations did not influence the quality or integrity of the present study.

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

N-butyl-N-[(triethoxysilyl)methyl]butan-1-amine

EC Number:

810-490-5

Cas Number:

35501-23-6

Molecular formula:

C15H35NO3Si

IUPAC Name:

N-butyl-N-[(triethoxysilyl)methyl]butan-1-amine

Constituent 2

Reference substance name:

N,N-Dibutylaminomethyl-triethoxysilan

IUPAC Name:

N,N-Dibutylaminomethyl-triethoxysilan

Test material form:

other: liquid

Details on test material:

Name: N,N-Dibutylaminomethyl-triethoxysilan
CAS No: 35501-23-6
Lot No: 310113
Physical State: liquid
Colour: colourless to yellowish
Storage Conditions: at room temperature, protected from light,
protected from water/ humidity
Purity: 97.7% (GC)
Stability: rapid hydrolysis in water
Date of analysis: 31 January 2013
Expiry Date: 21 January 2015

Test system

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): The liquid test item was applied undiluted. 30 µL of the test item were dispensed directly atop the EpiDerm(TM) tissue. Afterwards, a nylon mesh was used as spreading tool and carefully placed on the tissue surface. The compatibility of the test item with the nylon mesh was checked in a pre-test.

Details on study design:

Test System:
The test was carried out with the reconstituted three-dimensional human skin model EpiDerm(TM) (MatTek). This skin model consists of normal (non-cancerous), human-derived epidermal keratinocytes (NHEK) which have been cultured to form a multilayered, highly differentiated model of the human epidermis. The NHEK are cultured on chemically modified, collagen-coated cell culture inserts (Millicell(R)). The EpiDerm(TM) 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.

Provided Materials:
The EpiDerm(TM) tissues were provided as kits (EPI-200-SIT, MatTek), consisting of the following components relevant for this study:
1x sealed 24-well plate containing 24 inserts with tissues on Agarose (Lot: 19684 Kit C)
2x 24-well plates
8x 6-well plates
1x bottle of assay medium (DMEM-based medium, Lot: 100914 TMD)
1x bottle of DPBS Rinse Solution
1x 1 vial 5% SDS Solution (TC-SDS-5%)
25 pieces Nylon Mesh circles (8 mm diameter, 200 µm pore)
For MTT reduction assay the MTT assay kit (MTT-100; MatTek) was provided, consisting of:
1 vial MTT concentrate (Lot 092314MHA)
1 vial MTT diluent (supplemented DMEM; Lot 1553587)
1 bottle extractant solution (Isopropanol; Lot 072914ZSA)
 
Pre-Experiments:
To check the 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 in an incubator at 37 ± 1 °C. If the mixture turns blue/purple, the test item is presumed to have reduced MTT.
To check the colouring potential of the test item 30 µL of the test item were mixed per 300 µL aqua dest. in a transparent recipient and incubated at 37 ± 1°C for 60 min.
Liquid test item was tested for mesh compatibility by applying 30 µL of the test item to a nylon mesh, placed on a slide. After 60 min. the mesh was examined microscopically. If the test item interacts with the mesh, it was applied without mesh as spreading aid.

Experimental Procedure:
Upon receipt of the EpiDerm(TM), the tissues were transferred into 6-well plates containing 0.9 mL pre-warmed assay medium per well. The 6-well plates were incubated overnight in a humidified incubator at 37 ± 1 °C, 5.0% CO2.
After this pre-incubation the tissues were treated with each dose group in triplicate, starting with the negative control. Start time was recorded with dosing of the first tissue staggered in one-minute intervals. After dosing of all tissues, all plates were transferred to the incubator for 35 ± 1 min. After 35 min, all plates were removed from the incubator and placed under the 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 inserts were placed in a prepared 6-well plate containing 0.9 mL pre-warmed 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.
After this incubation period the inserts were transferred in 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 ± 5 min MTT incubation period the tissues were rinsed three times with DPBS and afterwards placed on blotting paper to dry the tissues. The tissues were immersed in 2 mL isopropanol, sealed to inhibit evaporation and incubated at room temperature for at least two hours. At the end of the formazan extraction period the plate was mixed by shaking until the solution colour became homogeneous. Per each tissue 2 x 100 µL aliquots of the extract were mixed with 100 µL isopropanol and transferred into a 96-well plate and OD was measured at 550 nm without reference wavelength in a plate spectrophotometer.
 
Data Analysis:
Irritant potential of the test item was predicted from the relative mean tissue viabilities compared to the negative control tissues concurrently treated with DPBS. The test item is considered to be irritant to skin in accordance with regulation EC 1272/2008 (UN GHS Category 2), if the tissue viability after 15 min of exposure and 42 h of post-incubation is less or equal to 50%. The test substance may be considered as non-irritant to skin in accordance with UN GHS No Category if the tissue viability after exposure and post-treatment incubation is higher than 50%.

Mean tissue viability Prediction I / NI
(% negative control)
≤ 50 % Irritant (I): UN GHS Category 2
> 50 % Non-Irritant (NI): UN GHS No Category

Test Acceptance Criteria:
The test meets acceptance criteria if:
- mean OD550 nm of the three negative control tissues is ≥ 0.8 and ≤ 2.8
- mean relative tissue viability of the three positive control tissues is ≤ 20%
- standard deviation (SD) of relative tissue viability obtained from each three concurrently tested tissues is < 18%.

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

other: other: relative tissue viability

Value:

62.8

Remarks on result:

other:

Remarks:

Basis: other: mean optical density. Time point: After 60 min exposure and 42 h post incubation period . Max. score: 100.0. (migrated information)

In vivo

Irritant / corrosive response data:

The test item showed no irritant effects. The mean relative tissue viability (% negative control) was > 50% (62.8%) after 60 min treatment and 42 h post incubation.

The controls confirmed the validity of the study. The mean absolute OD550 of the three negative control tissues was ≥ 1 and ≤ 2.5. The mean relative tissue viability (% negative control) of the positive control was ≤ 20% (2.7%). The maximum standard deviation of viability of replicate tissues of all dose groups was < 18% (0.2% - 5.4%).

Other effects:

The mixture of 30 µL test item per 1 mL MTT medium showed no reduction of MTT compared to the solvent. The mixture did not turn blue/purple.
The mixture of 30 µL / 25 mg of the test item per 300 µl aqua dest. showed no colouring detectable by unaided eye-assessment.
Microscopical examination of a nylon mesh treated for 60 min. with 30 µL of the test item showed no interactions. Therefore, the nylon mesh was used as spreading aid.

Applicant's summary and conclusion

Interpretation of results:

not irritating

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

In this study under the given conditions the test item showed no irritant effects. The test item is therefore classified as “non-irritant” in accordance with UN GHS “No Category”.

Executive summary:

SUMMARY

In the present study the skin irritant potential of N,N-Dibutylaminomethyl-triethoxysilan was analysed.The EpiDerm™-Standard Model (EPI-200™), a reconstituted three-dimensional human epidermis model, was used as a replacement for the Draize Skin Irritation Test (OECD TG 404) to distinguish between UN GHS “Category 2” skin irritating test substances and not categorized test substances (“No Category”) which may be considered as non-irritant. Hereby, the test item was applied topically. Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT after a60 min exposure and 42 h post incubation periodand compared to those of the concurrent negative controls.

In this study under the given conditions the test item showed no irritant effects. The relative mean tissue viability after 60 min of exposure and 42 h post incubation was> 50%. The test item is therefore classified as “non-irritant” in accordance with UN GHS “No Category”.