1-Butanamine, N-[3-(trimethoxysilyl)propyl]-, reaction products with (trimethoxysilyl)-N-[(trimethoxysilyl)propyl]-1-propanamine and 1,6-diisocyanatohexane homopolymer

Acute Toxicity: inhalation

Administrative data

Endpoint:

acute toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP compliant, guideline study, available as unpublished report, fully adequate for assessment

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 403 (Acute Inhalation Toxicity)

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.2 (Acute Toxicity (Inhalation))

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.1300 (Acute inhalation toxicity)

GLP compliance:

yes (incl. QA statement)

Test type:

standard acute method

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

iGloss Crosslinker (ZQ54-2211)

IUPAC Name:

iGloss Crosslinker (ZQ54-2211)

Details on test material:

- Name of test substance: iGloss Crosslinker (ZQ54-2211)
- Test substance No.: 11/0715-1
- Batch No.: 294222
- Purity: Mn 1200 Da; Mw 1800 Da; PDI 1.5; relative RI-area of compounds; with M 250-500 Da: 1.7%; with M 250-1000 Da: 29.1%; 1,6-hexamethylene diisocyanate: 98 mg/kg. The test item showed the expected IR-bands. The 1H-NMR and 13C-NMR spectra showed the expected signals for the given reaction products.
- Stability: under storage conditions
- Date of production: 01 Aug 2011
- Physical state / appearance: Liquid / yellowish, clear
- Storage conditions: Room temperature, no direct sunlight, protect against humidity

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan Laboratories B.V., Kreuzelweg 53, 5961 NM Horst, The Netherlands
- Age at study initiation: males: approx. 7 - 8 weeks; females: approx. 9 – 10 weeks
- Weight at study initiation: 189.8 g - 203. g
- Housing: Makrolon cages, type M III (floor area about 800 cm2) (for single housing) or Polysulfon cages (H-Temp [PSU]), floor area about 2065 cm2 (610x435x215 mm); supplied by TECNIPLAST, Hohenpeißenberg, Germany (caged in groups, if the animals were free from clinical signs and findings)
- Number of animals per cage: Single housing or up to 5 animals
- Enrichment: Wooden gnawing blocks (Typ NGM E-022); Abedd ® Lab. and Vet. Service GmbH Vienna, Austria
- Bedding: Dust-free wooden bedding
- Diet (e.g. ad libitum): Kliba-Labordiät (Maus / Ratte Haltung “GLP”), Provimi Kliba SA, Kaiseraugst, Basel, Switzerland, ad libitum
- Water (e.g. ad libitum): Tap water ad libitum
- Acclimation period: at least 5 days before exposure.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24
- Humidity (%): 30 - 70
- Air changes (per hr): 15
- Day / night rhythm: 12 h / 12 h

The room was completely disinfected using a disinfector ("AUTEX", fully automatic, formalinammonia-based terminal disinfector) before the start of the study. The floor and the walls were cleaned once a week with water containing an appropriate disinfectant.

Administration / exposure

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

nose/head only

Vehicle:

other: anhydrous acetone

Details on inhalation exposure:

SUBSTANCE VEHICLE: anhydrous acetone

GENERATION OF THE INHALATION ATMOSPHERE
- Test-substance preparation: Test group 1: A test-substance dilution of one part anhydrous acetone + seven parts test substance (w/w) was used. Test group 2 and 3: A test-substance dilution of two parts anhydrous acetone + seven parts test substance (w/w) was used.
- Equipment: Two-component atomizer Mod. 970 (stainless steel, Schlick) Magnetic stirrer. Continuous infusion pump Perfusor (Harvard PHD Ultra, Tyco/Healthcare, Kendall, Monojet)
- Generation technique: Liquid aerosols were generated.For each test group the aerosols were produced by continuously pumping amounts of the test substance preparation to a two-component atomizer. Using compressed air, the aerosol was produced with the atomizer inside the exposure system.

EXPOSURE
Head-nose inhalation system INA 20 (glass-steel construction, BASF SE, volume V ≈ 55 L): the animals were restrained in glass tubes and their snouts projected into the inhalation system. The homogenous distribution of test substance atmosphere/s in this inhalation system has been verified with model aerosols. Central air conditioning system provides cold air of about 15°C. This cold air will pass through an activated charcoal filter, be adjusted to room temperature of 20 to 24°C and pass through a second particle filter (H13 (HEPA) Camfil Farr, Germany). The so generated conditioned air is used to generate inhalation atmospheres.
Compressed air is produced by an oil-free compressor (HT 6, Josef Mehrer GmbH & Co KG, Germany). For this purpose, air is filtered by an inlet air strainer and introduced into the compressor. After passed through an second ultra filter (SMF 5/3, 108 mm, Donalson), the compressed air (15 bar) is stored in a storage of 1500 or 5000 L. The compressed air is conducted to the labs via pipes, where the pressure is reduced to 6 bar. In the lab, the compressed air can be taken as required.
The exhaust air is filtered and conducted into the exhaust air of the building. The exposure system was located inside an exhaust cabin in an air-conditioned laboratory. During each exposure, the following parameters were recorded four times at about 1-hour intervals: Supply air flows (compressed air) (from a central air-conditioning system): 1.5 m³/h. The flows were adjusted and continuously measured with a flowmeter (Yokogawa); Exhaust air flows: 1.35 m³/h. The flows were adjusted and continuously measured with a flowmeter (Yokogawa).
The lower amounts of exhaust air, which were adjusted by means of a separate exhaust air system, achieved positive pressures inside the exposure systems. This ensured that the mixtures of test substance and air were not diluted with laboratory air in the breathing zones of the animals. Air changes of about 27 times per hour can be calculated by dividing the supply air flows by the volume of the inhalation systems. The animals were exposed to the inhalation atmosphere for 4 hours plus equilibration time of the inhalation systems (t99 about 10 min). No surveillance of the oxygen content in the inhalation system was performed. The air change was judged to be sufficient to prevent oxygen depletion by the breathing of the animals, and the concentration of the test substance used could not have a substantial influence on oxygen partial pressure.
Temperatures: The temperatures in the inhalation system were measured continuously with a digital thermometer.
Humidities: The humidities in the inhalation system were measured with a dielectric probe (Testo).

ANALYTICAL INVESTIGATIONS
- The nominal concentrations were calculated from the amounts of test substance dosed and the supply air flows.
- Gravimetric measurement of the inhalation atmosphere concentration: In technical trials it was shown, that the test substance was quantitatively recovered, when the preparation was dried on gravimetric filters. Vacuum pump (Millipore); Sampling devices: Filtration equipment with probe, internal diameter: 7 mm, (Millipore); Filter: MN 85/90 BF (d = 47 mm); Equipment: Balance Mettler AT 250; Sampling position: immediately adjacent to the animals' noses at a separate spare port; Sampling flow: 3 L/min; Sampling velocity: 1.25 m/s; Sampling frequency: 4 samples at about hourly intervals; Test group:Sample volume (L) 1:30, 2:12, 3:6
In technical trials it was shown, that the test substance was quantitatively recovered, when the preparation was dried on gravimetric filters. Preweighed filters were placed into the filtration equipment. By means of the vacuum pump metered volumes of the aerosol were drawn through the filter. For each sample the aerosol concentration in mg/L was calculated from the difference between the preweight of the filter and the weight of the filter after sampling, with reference to the sample volume of the inhalation atmospheres. Mean and standard deviation were calculated for each concentration from the results of the individual measurements.
- Particle size analyses: Equipment: Stack Sampler Mark III (Andersen); Vacuum pump (Millipore); Sampling probe (internal diameter 6.9 mm); Limiting orifice 3 L/min; Balance: Sartorius M3P and Sartorius LC 1201S
Before sampling, the impactor stages were assembled with preweighed glass-fiber collecting discs and equipped with a backup particle filter. The impactor was connected to the vacuum pump, and for each test group samples were taken from the breathing zone of the animals according to the following tables. Sampling occurred 30 minutes (or later) after the beginning of the exposure. After sampling the impactor was taken apart. The collecting discs and the backup particle filter were re-weighed. Test group:Number of samples:Sample volume (L), 1:2:30, 2:2:12, 3:2:6
- The feed used in the study was assayed for chemical as well as for microbiological contaminants. In view of the aim and duration of the study, the contaminants occurring in commercial feed should not influence the results. Deviations from these specifications that might have had any adverse effect on the results are reported in the results section.
- Analysis of drinking water: The drinking water is regularly assayed for chemical contaminants both by the municipal authorities of Frankenthal and by the Environmental Analytics Water/Steam Monitoring of BASF SE as well as for bacteria by a contract laboratory. The Drinking Water Regulation will serve as the guideline for maximum tolerable contaminants.
- The bedding and enrichment are regularly assayed for contaminants (chlorinated hydrocarbons and heavy metals). On the basis of the analytical findings the bedding was found to be suitable.

Analytical verification of test atmosphere concentrations:

yes

Duration of exposure:

4 h

Remarks on duration:

+ about 10 min equilibration time of the inhalation systems

Concentrations:

1.257, 2.869 and 5.134 mg/L

No. of animals per sex per dose:

5

Control animals:

no

Details on study design:

EXPERIMENTAL PROCEDURE
Observation period: At least 14 days.
Feed and Water: twice a day on workdays and once daily on weekends and public holidays.
Body weight determination: once during the acclimatization period, shortly before exposure (day 0) and at least on days 1, 3 and 7, and before the sacrifice of the animals at the end of the observation period.
Signs: Detailed clinical observations were recorded for each animal separately several times during exposure and at least once daily on the pre-exposure day and during the observation period.
Mortality: twice each workday and once on Saturdays, Sundays and on public holidays.
Pathology: At the end of the observation period the surviving animals were killed with CO2-inhalation in a chamber with increasing concentration over time, and were subjected to gross-pathological examination as well as the animal which died or were killed in a moribund state before. To clarify the gross-pathological findings, selected organs of individual animals were examined histopathologically. Each moribund animal, which most probably would have died on account of the toxic effects of the test substance was killed during the observation period according to the German Animal Protection Law (BGBL I, 22 Aug 1986).

Statistics:

By the data the LC50 was calculated by Probit analysis (FINNEY, D.J. (1971): "Probit Analysis" Cambridge University Press). For results of the type ”LC50 greater than”, ”LC50 approx.”, or ”LC50 smaller than”, the binomial test was used for statistical evaluation.
The calculation of the particle size distribution was carried out in the inhalation laboratory on the basis of mathematical methods for evaluating particle measurements, according to DIN 66141 and DIN 66161.

Results and discussion

Effect levels

Sex:

male/female

Dose descriptor:

LC50

Effect level:

5.031 mg/L air (analytical)

Based on:

test mat.

Exp. duration:

4 h

Mortality:

Test group 1 (1.257 mg/L): No lethality was observed in male and female animals during the study period of 14 days.
Test group 2 (2.869 mg/L): No lethality was observed in male and female animals during the study period of 14 days.
Test group 3 (5.134 mg/L): Three of five female animals but no males died or were killed in a moribund state during the post exposure observation period.
(one female was found dead: in-life day 1; one female was sacrificed moribund: in-life day 1; one female was sacrificed moribund: in-life day 6)

Clinical signs:

other: Clinical signs of toxicity in animals exposed to 1.257 mg/L comprised accelerated and abdominal respiration, respiration sounds, colorless discharge and red encrusted nose, piloerection and substance contaminated. These various findings were observed from

Body weight:

Test group 1 (1.257 mg/L): the mean body weights of the animals decreased during the first post exposure observation day and increased from study day 3 onward.
Test group 2 (2.869 mg/L): the mean body weights of the animals decreased during the first post exposure observation day and increased from study day 3 onward.
Test group 3 (5.134 mg/L): the mean body weights of the male animals decreased during the first post exposure observation day and increased from study day 3 onward. The mean body weights of the female animals decreased during the first few post exposure observations days and increased from study day 7 onward.

Gross pathology:

No gross pathological abnormalities were noted during the necropsy at the termination of the post exposure observation period in animals exposed to 1.257 mg/L and 2.869 mg/L. In anaimals exposed to 5.134 mg/L of the one female animal that was found dead on study day 1, dark-red discoloration in all lung lobes, edema and red encrusted nose were noted during necropsy. Necropsy findings of the one female animal that were killed in a moribund state on study day 1 comprised dark-red discoloration in all lung lobes, edema of the lung and. Necropsy findings of the one female animal that were killed in a moribund state on study day 6 comprised at the edge of the right cranial lung lobe a focus, red encrusted nose, alopecia on the left nose region and substance contaminated fur. The remaining animals showed no gross pathological abnormalities during the necropsy at the termination of the post exposure observation period.

Any other information on results incl. tables

The following clinical signs and findings were observed during (up to 4 hours) and after exposure (> 4 hours).

Table 1: Duration of signs (test group 1)

Test group 1 (1.257 mg/L)	Male animals	Female animals
Fur, substance contaminated	d0 – d1	d0 – d1
Nose, colorless discharge	d1 – d5	d1 – d5
Nose, red encrusted	d0 – d4	d0
Respiration, abdominal	d0 – d2	d0 – d4
Respiration, accelerated	h4	h4
Respiration, sounds	d0 – d1	-
Piloerection	d0 – d5	d0 – d4

No clinical signs and findings were observed from study day 6 onwards.

 

Table 2: Duration of signs (test group 2)

Test group 2 (2.869 mg/L)	Male animals	Female animals
Alopecia	-	d6 – d14
Fur, substance contaminated	d0 – d6	d0 – d14
Nose, discharge colorless	d1 – d2	d1 – d2
Nose, red discharge	d1 – d2	-
Nose, red encrusted	d1 – d2	-
Respiration, abdominal	d0 – d2	d0 – d2
Respiration, accelerated	h3 – d2	h3 – d2
Respiration, sounds	-	d0 – d2
Piloerection	d0 – d3	d0 – d2

No clinical signs and findings were observed in the male animals from study day 7 onwards.

The clinical signs and finding were observed in the female animals during the whole study period.

 

Table 3: Duration of signs (test group 3)

Test group 3 (5.134 mg/L)	Male animals	Female animals
Lethality (number of animals)	-	3
Alopecia	d2 – d14	d5 – d14
Eye, red encrusted	-	d0 – d4
Eyelid, closed	-	d1 – d4
Dehydrated general condition	-	d2 – d4
Fur, substance contaminated	d0 – d12	d0 – d6
Gasping	-	d0 – d6
Nose, colorless discharge	d2 – d6	-
Nose, red discharge	-	d1
Nose, red encrusted	d1	d1
Nose, swelling	d0 – d1	d0 – d1
Respiration, abdominal	d0 – d2	d0 – d5
Respiration, accelerated	h1 – h2	h1 – h2; d1 – d2
Respiration, labored	h3 – h4	h3 – h4
Respiration, sounds	-	d2 – d6
Piloerection	d0 – d14	d0 – d7
Poor general condition	-	d1 – d6
Salivation	d0 – d4	d0 – d4
Skin, pale	-	d1

 

The clinical signs and finding were observed during the whole study period.

PATHOLOGY

Necropsy findings:

Table 4: Animals that died or were killed in a moribund state during the study period

Findings	test group 3
Number of animals	3 females
Lung: dark-red discoloration in all lobes	2 females
Lung: Edema	2 females
Lung: at the edge of the right cranial lobe a focus (ᴓ4 mm)	1 female
Nose: red encrusted	3 females
Fur: left nose region alopecia	1 female
Fur: substance contaminated	1 female

 

To further evaluate the macroscopic findings, histopathological examination was carried out of the respiration tract (nasal cavity, larynx, trachea, lung) from female animal no. 609 which was killed in a moribund state on study day 6. The lung showed a minimal to slight diffuse acute congestion and a minimal to slight diffuse alveolar histiocytosis. The nasal cavity showed a slight to moderate multifocal necrotizing and purulent rhinitis with degeneration and regeneration. The larynx and trachea were without findings.

 

Table 5: Necropsy of the animals at termination of the post exposure periods

Findings	test group 1	test group 2	test group 3
Number of animals	5 males + 5 females	5 males + 5 females	5 males + 2 females
Organs without particular findings	5 males + 5 females	5 males + 5 females	5 males + 2 females