2H-Azepin-2-one, hexahydro-, polymer with 1,6-diisocyanatohexane

Acute Toxicity: inhalation

Administrative data

Endpoint:

acute toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2012-06-27 to 2012-07-24

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study

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)

Version / remarks:

2009

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes

Test type:

standard acute method

Limit test:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Copolymer of hexahydro-2H-azepin-2-one and 1,6-diisocyanatohexane

EC Number:

607-997-7

Cas Number:

26776-30-7

Molecular formula:

Exact identification is not feasible

IUPAC Name:

Copolymer of hexahydro-2H-azepin-2-one and 1,6-diisocyanatohexane

Test material form:

other: solid, translucent resin

Details on test material:

>99% oligomer (UVCB substance)

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ORGANISMS: 
- Strain: WISTAR HsdCpd:WU
- Source: Harlan-Nederland
- Age: young adult
- Weight at study initiation: males: 170.0 - 200.0 g, females: 166.0 - 183.0 g
- Number of animals: 13 males and 13 females
- Housing: singly in Makrolon Type III cages
- Diet: ad libitum, ssniff R/M-H
- Water: ad libitum, tap water
- Acclimatisation period: at least 5 days, during this period, rats were also acclimatized to the restraining tubes
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22°C +/- 3 °C
- Humidity (%): 40 - 60 %
- Photoperiod (hrs dark / hrs light): 12 hours darkness, 12 hours artifical light
- Ventilation: approx. 10 air changes per hour

Administration / exposure

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

nose only

Vehicle:

other: ethyl acetate and dry compressed air

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Type of exposure: nose-only, using a dynamic inhalation apparatus, comparable with a directed-flow exposure
- Method of holding animals in test chamber: separately in Plexiglas exposure restrainers
- Source of air: dry compressed filtered air
- Method of conditioning air: manometer and air-flow meter (TSI Mass Flow-meter 4040), flow rates were monitored continuously by flow meters
and if necessary readjusted
- Type or preparation of particles: Portions of the solid test article were scrapped-off from the resin and were dissolved in ethyl acetate at a targeted concentration of 20 %(w/v). Stability was verified over 7 days. Nebulization of the resin was attempted up to 80°C (decomposition temperature
100°C). Despite these effects, the test article remained in a physical state of high viscosity with glue-like properties. The last resource was to
aerosolize the test article in ethyl acetate at relative low concentration (20%) and low nozzle accordingly.
Under dynamic conditions the test substance is atomized into the baffle (pre-separator) of the inhalation chamber. For atomization a binary
Schlick-type nozzle and conditioned compressed air (15 Llmin) was used. The representative dispersion pressure was in the range of 600 kPa. The nozzle was maintained at 10°C by a water jacket connected to a digitally controlled water bath. The test article was injected into the nozzle system
using a digitally controlled pump (Harvard PHD 2000 infusion pump). In the control group conditioned dry air only was used. The same
aerosolization principles were used in the vehicle and test article groups. The atomization temperature and concentration of the test article in the
vehicle was optimized to attain stable and highly respirable aerosol concentrations at the expense of a high concentration of vehicle.
- Analysis of the aerosol concentration: The actual aerosol concentration in the inhalation chamber was measured gravimetrically with an air sample
filter (Minisart SM 17598 0.45 µm) and pump (Vacuubrand, MZ 2C ) controlled by a rotameter. Dust samples were taken once every hour during the exposure
- Method of particle size distribution: The particle-size distribution was analyzed using a BERNER critical orifice cascade impactor. Each impactor
stage was covered by an aluminum foil and an additional glass fiber filter to prevent liquid run-off from the filter. Gravimetric analyses of filters
used a digital balance. Prior to gravimetric analysis the filter was dried for 30 min @70°C. Due to the glue-like properties, no additional coating
agent was used to minimize bouncing and re-entrainment of aerosol. The samples for the analysis of the particle-size distribution were taken in the
vicinity of the breathing zone. During each exposure two samples were taken, which was the maximum feasible due to the analytical samples taken.
- Temperature, humidity: T: 21.8 - 22.5 °C, H:
TEST ATMOSPHERE
- Concentrations: 3069 mg/m³ 
Air flow entrance (L/min): 15
Air flow exit (L/min): 12.8
Air change (changes per hour): 237
- Analytical method used: The nominal concentration was calculated from the ratio of the quantity of the atomized liquid and the total throughput of air through the inhalation chamber.The test-substance concentration was determined by gravimetric analysis (filter: glass-fiber filter, Sartorius,
Gottingen, Germany; digital balance). This method was used to define the actual concentration.
- Samples taken from breathing zone: Chamber samples were taken in the vicinity of the breathing zone
- MMAD (Mass median aerodynamic diameter): 2.7 µm
- GSD (Geometric st. dev.): 2.6

VEHICLE
- Composition of vehicle: etyl acetate purum, an atomization without the carrier ethyl acetate at an already mildly toxic concentration
(3069 mg/m3 ) was technically not feasible

CONTROL ANIMALS
- 5m/5f: conditioned air
- 5m/5f: vehicle ethyl acetate

Analytical verification of test atmosphere concentrations:

yes

Remarks:

The nominal concentration was calculated from the ratio of the quantity of the atomized liquid and the total throughput of air through the inhalation chamber.

Duration of exposure:

4 h

Concentrations:

Gravimetric concentration of 3069 mg test item/m3
The respirability of the aerosol was adequate to achieve the objective of study, i.e. the average mass median aerodynamic diameter (MMAD) was
2.7 µm, the average geometric standard deviation (GSD) was 2.6.

No. of animals per sex per dose:

3

Control animals:

yes

Details on study design:

EXAMINATIONS: 
- Post dose observation period: 14 days
- body weights: before exposure, on days 1, 3,  7 and 14 after treatment   
- mortality: time of death is recorded as precisely as possible
- clinical signs: several times on the day of exposure and at least once daily thereafter, no specific assessment was performed during exposure while animals were restrained, only if unequivocal signs occurred e.g. spasms, abnormal movements, and severe respiratory signs.
Following exposure, observations are made and recorded systematically; individual records are maintained for each animal. Cage-side
observations included, but were not limited to, changes in the skin and fur, eyes, mucus membranes, respiratory, circulatory, autonomic and
central nervous system, and somatomotor activity and behavior pattern. Particular attention was directed to observation of tremors, convulsions,
salivation, diarrhea, lethargy, somnolence and prostration.
The following reflexes were tested: visual placing response and grip strength on wire mesh, abdominal muscle tone, corneal and pupillary reflexes, pinnal reflex, righting reflex, tail-pinch response, startle reflex with respect to behavioral changes stimulated by sounds (finger snapping) and
touch (back).
- rectal temperatures: were measured shortly after cessation of exposure (approximately within 1/2 hour after the end of exposure) using a digital thermometer with a rectal probe for rats.
- Necropsy: All rats, irrespective of the day of death, were given a gross-pathological examination, with particular reference to changes related to
the respiratory tract.

Statistics:

Data of body weights and rectal temperature measurements are statistically evaluated using the ANOVA procedure.

Results and discussion

Preliminary study:

The maximum technically attainable concentration is 3069 mg test item/m3.

Effect levels

Sex:

male/female

Dose descriptor:

LC50

Effect level:

> 3 069 mg/m³ air

Based on:

test mat.

Exp. duration:

4 h

Remarks on result:

other: maximum technically achievable concentration

Mortality:

No animal died prematurely.

Clinical signs:

other: Group 1-males & females: No specific signs observed. Group 2-males: Bradypnea, labored breathing patterns, piloerection. Group 2-females: Bradypnea labored breathing patterns, piloerection, motility reduced, atony Group 3-males: Bradypnea, labored breathi

Body weight:

Comparisons between the vehicle control and the test article exposure group did not reveal significant differences of body weight gains.

Gross pathology:

There were no observable findings at the end of the study. The macroscopic findings of extrapulmonary organs were essentially indistinguishable
amongst the groups.

Other findings:

A battery of reflex measurements was made on the first post-exposure day, differences between groups 1 to 3 were not observed.

Applicant's summary and conclusion

Conclusions:

In summary, the aerosolized test substance 2H-Azepin-2-one, hexahydro-, polymer with 1,6 diisocyanatohexane proved to have a low acute inhalation toxicity to rats. Due to the similarity of the vehicle and test article group, it seems that the glue-like resin aerosol may have acted as carrier for the liquid vehicle which aggravated the response observed. An atomization without the carrier ethyl acetate at an already mildly toxic concentration (3069 mg/m3) was
technically not feasible.
LC50inhalation (liquid aerosol, 4 h): LC50 males & females: >3069 mg/m3
NO(A)EL: males & females: <3069 mg/m3 air 

Executive summary:

The aim of the present experiment was to obtain information on the acute toxicity and LC50, following a single 4-hour inhalation exposure in an acute inhalation study in WISTAR rats.

A study on the acute inhalation toxicity of 2H-Azepin-2-one, hexahydro-, polymer with 1,6 diisocyanatohexane on rats has been conducted in accordance with OECD TG 403 (2009). Test procedures were adapted so as to comply also with the EU Directive 92/69/EEC, and especially OECD GD 39 (2009). One group of rats was nose-only exposed to a concentration of 3069 mg/m3 (maximum technically achievable concentration). The aerosol was generated using the vehicle ethyl acetate. Test article exposed rats were compared to a historical air control group and a concurrent vehicle control group. The results can be summarized as follows:

LC50inhalation (liquid aerosol, 4 h): LC50 males & females: >3069 mg/m3 air

NO(A)EL: males & females: < 3069 mg/m3 air 

Mortality did not occur up to the maximum technically attainable concentration of 3069 mg/m3.  The clinical signs observed in the vehicle control (bradypnea, labored breathing patterns, piloerection, motility reduced, atony) and in the test article group (bradypnea, labored breathing patterns, piloerection, motility reduced, atony, gait high-legged, muzzle area: red encrustations, hair-coat: clotted and depilated areas) were somewhat similar but differed markedly in their duration (up to the first postexposure day in the vehicle group and first postexposure week in the test article group. The changes in body weights and rectal temperature (hypothermia) were essentially indistinguishable between these groups. The hair-coat with clotted and depilated areas was clearly related to the glue-like physical properties of the test article.

The respirability of the aerosol was adequate to achieve the objective of study, i.e. the average mass median aerodynamic diameter (MMAD) was 2.7 µm, the average geometric standard deviation (GSD) was 2.6.

 

In summary, the aerosolized test substance proved to have a low acute inhalation toxicity to rats. Due to the similarity of the vehicle and test article group, it seems that the glue-like resin aerosol may have acted as carrier for the liquid vehicle which aggravated the response observed. An atomization without the carrier ethyl acetate at an already mildly toxic concentration (3069 mg/m3) was technically not feasible.

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