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

Endpoint summary

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Endpoint:

skin sensitisation: in vivo (LLNA)

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.

Qualifier:

according to guideline

Guideline:

OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Species:

mouse

Strain:

CBA

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Species/strain: healthy CBA/CaOlaHsD mice
- Source: Harlan Laboratories GmbH, 5800 AN Venray, The Netherlands
- Sex: female (nulliparous and non-pregnant)
- Age at the beginning
of the study: 8-9 weeks
- Number of animals: 5 mice / group
5 mice / prescreen test

The animals were derived from a controlled full-barrier maintained breeding system (SPF). According to Art. 9.2, No. 7 of the German Act on Animal Welfare the animals are bred for experimental purposes.

ENVIRONMENTAL CONDITIONS
- Full barrier in an air-conditioned room
- Temperature: 22 ± 3 °C
- Relative humidity: 55 ± 10%
- Artificial light, sequence being 12 hours light, 12 hours dark
- Air change: at least 10 x / hour
- Free access to Altromin 1324 maintenance diet for rats and mice (prescreen test and main study: lot no. 1526)
- Free access to tap water, sulphur acidified to a pH value of approx. 2.8 (drinking water, municipal residue control, microbiological controls at regular intervals)
- The animals were kept in groups of 5 animals in IVC cages, type II L, polysulphone cages on Altromin saw fibre bedding (prescreen test and main study: lot no. 02102140609)
- Certificates of food, water and bedding are filed at BSL BIOSERVICE
- Adequate acclimatisation period (at least five days) under laboratory conditions

Vehicle:

acetone/olive oil (4:1 v/v)

Concentration:

Based on the results observed in the prescreen test the following test item concentrations were selected for the main study:
25%, 50% (each diluted with AOO 4:1, v/v) and 100% (undiluted test item)
The preparations were made immediately prior to each dosing.

No. of animals per dose:

5 mice / group

Details on study design:

RANGE FINDING TESTS:
- Compound solubility: Before the initiation of the prescreen test, a solubility test was performed to define the vehicle and the maximum concentration which is technically applicable to the animals. The maximum technically applicable concentration of the test was found to be 100%. Two animals were treated by topical application with the test item on three consecutive days at a concentration of 100% (undiluted) to the entire dorsal surface of each ear. Two animals were treated by topical application with the test item on three consecutive days at a concentration of 50% (diluted with AOO) to the entire dorsal surface of each ear. One further animal was treated with 100% AOO and served as negative control.

- Irritation: Neither signs of systemic toxicity nor erythema formation (E0) at any application site could be detected in any animals. The animals showed furthermore a normal ear swelling, which corresponded to an increase of the ear thickness ≤ 20% at the end (day 6) of the prescreen test.


MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: LLNA
- Criteria used to consider a positive response: Stimulation Index

TREATMENT PREPARATION AND ADMINISTRATION:

Topical Application:
Immediately before the first application the thickness of both ears of all animals was measured. Each mouse was treated by topical application of 25 µL of the selected solution to the entire dorsal surface of each ear. A second measurement of the ear thickness of all animals was carried out approximately 48 hours after the first application. Topical applications were performed once daily over three consecutive days.

Administration of 3H-Methyl Thymidine:
Five days after the first topical application all mice were dosed with 20 µCi 3H-methyl thymidine by intravenous injection (tail vein) of 250µL of 3H-methyl thymidine, diluted to a working concentration of 80µCi/mL.

Preparation of Cell Suspension:
Approximately 5 hours after the injection of 3H-methyl thymidine all mice were sacrificed by cervical dislocation. Shortly before sacrificing the thickness of the ears of all animals was measured for a third time. The draining auricular lymph nodes were excised, individually pooled for each animal (2 lymph nodes per animal) and collected in phosphate buffered saline (PBS). A single cell suspension of pooled lymph node cells was prepared by gentle mechanical disaggregation through polyamide gauze (200 mesh size). After washing the gauze with PBS the cell suspension was pelleted in a centrifuge. The supernatant was discarded and the pellets were resuspended with PBS. This washing procedure was repeated. After the final wash each pellet was resuspended in approx. 1 mL 5% TCA at approx. 4° C for approximately 18 hours for precipitation of macromolecules. Each precipitate was once washed again, resuspended in 1 mL 5% TCA and 7 mL scintillation fluid was added. Then this solution was transferred into scintillation vials and stored at room temperature overnight.

Determination of Incorporated 3H-Methyl Thymidine
The 3H-methyl thymidine – incorporation was measured in a ß-counter and expressed as the number of disintegrations per minute (DPM). Similarly, background 3H-methyl thymidine levels were also measured (5% TCA). Determination of radioactivity was performed individually for each animal.
 
Reliability Check
The recent reliability check was performed in September 2014. The raw data of this study are kept in the BSL archives (BSL Project ID 134702 G).
Positive-control substance: P-Phenylenediamine (CAS 106-50-3, Sigma, purity > 98%; Lot No.: SLBC7171V) 1%
Vehicle: AOO (4:1 (v/v) acetone/olive oil)
Species/strain: healthy CBA/CaOlaHsd mice
Source: Harlan Laboratories GmbH, 5800 AN Venray, The Netherlands
Concentrations: 1% on three consecutive days

Positive control substance(s):

other: P-Phenylenediamine (CAS 106-50-3)

Statistics:

The proliferative response of lymph node cells was expressed as the number of radioactive disintegrations per minute per lymph node (DPM/NODE) and as the ratio of 3H-methyl thymidine - incorporation into lymph node cells of test group animals relative to that recorded for control group animals (STIMULATION INDEX). Before DPM/NODE values were determined, background values were subtracted.
EC3 values, calculated concentrations which induce stimulation indices of three, are determined by linear interpolation {EC3=c+[(3-d)/(b-d)]x(a c)}, between two points of the stimulation indices axis, one above (a,b) and one below (c,d) the stimulation index of three. If all measured points are above or below the stimulation index of three, no EC3 value can be stated.
A substance is regarded as a 'sensitiser' in the LLNA if at least one concentration of the test item results in a 3-fold or greater increase in 3H-methyl thymidine - incorporation into lymph node cells of the test group animals, relative to that recorded for the lymph nodes of control group animals (Stimulation Index equal to or greater than 3.0).

Parameter:

SI

Remarks on result:

other: The stimulation index at a concentration of 25% was 1.7 The stimulation index at a concentration of 50% was 1.9 The stimulation index at a concentration of 100% was 5.0

Interpretation of results:

sensitising

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

The EC3 value (derived by linear interpolation) was calculated to be at a test item concentration of 67.74%. The measurement of the ear thickness did not reveal any relevant increase of the ear thickness in any dosage group. According to Commission Regulation (EU) No 286/2011 as well as GHS (Globally Harmonized Classification System) the test item N,N-Dibutylaminomethyl-triethoxysilan has obligatory labelling requirement for skin sensitisation and is classified into Category 1B.

Executive summary:

SUMMARY

Based on the results of the prescreen test the test item was assessed for sensitising properties at concentrations of 25%, 50% (v/v) , each diluted with AOO 4:1, (v/v) and 100% (undiluted test item).

At the daily clinical observation animals showedsticky fur, hair loss and bald skin. No case of mortality was observed.

Species/strain:                     Mice, CBA/CaOlaHsd

Number of animals:              20/main test

Vehicle:                               AOO (4:1 (v/v) acetone/olive oil)

 

Summary Results:

One of the three tested concentrations exceeded the stimulation index of 3.

The stimulation index at a concentration of        25%    was     1.7

The stimulation index at a concentration of        50%    was     1.9

The stimulation index at a concentration of        100%  was     5.0

 

The mean ear thickness on	day 1	day 3	day 6
for the 25% test group was	0.19 mm	0.19 mm	0.18 mm
for the 50% test group was	0.18 mm	0.18 mm	0.19 mm
for the 100% test group was	0.18 mm	0.18 mm	0.19 mm
for the negative control group was	0.18 mm	0.18 mm	0.18 mm

 

Conclusion

The EC3 value (derived by linear interpolation) was calculated to be at a test item concentration of 67.74%.

The measurement of the ear thickness did not revealany relevant increase of the ear thickness in any dosage group. According to Commission Regulation (EU) No 286/2011as well as GHS (Globally Harmonized Classification System) the test item N,N-Dibutylaminomethyl-triethoxysilan has obligatory labelling requirement for skin sensitisation and is classified into Category 1B.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

A skin sensitization study according to OECD 429 was performed with N,N-Dibutylaminomethyl-triethoxysilane. Based on the results of the prescreen test the test item was assessed for sensitising properties at concentrations of 25%, 50% (v/v) , each diluted with acetone/olive oil 4:1, (v/v) and 100% (undiluted test item) on CBA/CaOlaHsd mice. At the daily clinical observation animals showed sticky fur, hair loss and bald skin. No case of mortality was observed. The EC3 value (derived by linear interpolation) was calculated to be at a test item concentration of 67.74%. According to Commission Regulation (EU) No 286/2011as well as GHS (Globally Harmonized Classification System) the test item N,N-Dibutylaminomethyl-triethoxysilan was a moderate skin sensitiser and has obligatory labelling requirement for skin sensitisation and is classified into Category 1B.

Justification for selection of skin sensitisation endpoint:
The reliable GLP compliant OECD Guideline study was chosen.

Justification for classification or non-classification

Based on the reliable data provided for this endpoint N,N-Dibutylaminomethyl-triethoxysilan was a moderate skin sensitiser and has obligatory labelling requirement for skin sensitisation and is classified into Category 1B.