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Toxicological information

Acute Toxicity: inhalation

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Administrative data

acute toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guideline
according to guideline
OECD Guideline 403 (Acute Inhalation Toxicity)
GLP compliance:
yes (incl. QA statement)
Test type:
standard acute method

Test material

Constituent 1
Chemical structure
Reference substance name:
N-[6-(3-{6-[3-(6-formamidohexyl)-2,4-dioxo-1,3-diazetidin-1-yl]hexyl}-2,4-dioxo-1,3-diazetidin-1-yl)hexyl]formamide; N-{6-[3-(6-formamidohexyl)-2,4-dioxo-1,3-diazetidin-1-yl]hexyl}formamide
EC Number:
Molecular formula:
N-[6-(3-{6-[3-(6-formamidohexyl)-2,4-dioxo-1,3-diazetidin-1-yl]hexyl}-2,4-dioxo-1,3-diazetidin-1-yl)hexyl]formamide; N-{6-[3-(6-formamidohexyl)-2,4-dioxo-1,3-diazetidin-1-yl]hexyl}formamide
Constituent 2
Reference substance name:
Cas Number:

Test animals

Details on test animals or test system and environmental conditions:
- Strain: Hsd Cpb:WU (SPF)
- Source: Harlan-Winkelmann GmbH, Borchen, Germany
- Age at study initiation: approximately 2 months
- Weight at study initiation: At the study start the variation of individual weights did not exceed ± 10 per cent of the mean for each sex
- Housing: singly in conventional Makrolon® Type IIIH cages
- Diet and water: ad libitum
- Acclimation period: at least 5 days

- Temperature (°C): 22 ± 2°C
- Humidity (%): 40 - 60 %
- Air changes (per hr): approximately 10
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
inhalation: aerosol
Type of inhalation exposure:
nose only
Details on inhalation exposure:
- Mode of exposure: Animals were exposed to the aerosolized test article in restrainers made of Plexiglas. The design of the directed-flow inhalation chamber prevents rebreathing of the test atmosphere.
- Generation of atmosphere: Atmospheres were generated under dynamic conditions using a digitally controlled Harvard S/N infusion pump and a modified Schlick-nozzle Type 970, form-S 3 (Schlick GmbH, COburg, Germany).
- Generation of aerosol: The test substance was nebulized neat using conditioned (dry, oil-free) compressed air (dispersion pressure approximately 600 kPa, 10-30 µl/min solution of the test substance, 15 L air/min and inhalation chamber segment (1 segment used). The respective concentration was achieved by applying extraction/dilution cascades.
- Inhalation chamber: One segment of the aluminum inhalation chamber has the following dimensions: inner diameter = 14 cm, outer diameter = 35 cm (two-chamber system), height = 25 cm (internal volume = about 3.8 L). Details of this modular chamber and its validation have been published previously (Pauluhn, Journal of Applied Toxicology, 14, 55-62, 1994).
- Optimization of respirability: In order to increase the efficiency of the generation of respirable particles and to prevent larger particles from entering the chamber a preseparator/ baffle system was used.
- Conditioning the compressed air: Compressed air was supplied by Boge compressors and was conditioned (i.e. freed from water, dust, and oil) automatically by a VIA compressed air dryer. Adequate control devices were employed to control supply pressure.
- Inhalation chamber steady-state concentration: The test atmosphere generation conditions provide an adequate number of air exchanges per hour (ca. 474 x, continuous generation of test atmosphere). Under such test conditions steady state is attained within the first minute of exposure (t99% = 4.6 x chamber volume/flow rate). The ratio between the air supplied and exhausted was chosen so that approximately 90% of the supplied air is removed via the exhaust system. The remainder provides adequate dead-space ventilation for the exposure tubes. At each exposure port a minimal air flow rate of 0.75 I/min was provided. The test atmosphere can by no means be diluted by bias-air-flows.
- Exhaust air treatment: The exhaust air was purified by passage through aseries of aerosol filters.
- Temperature and humidity measurements are also performed by the computerized HP 3852A Data Acquisition and Control System using FTF sensors (ELKA ELEKTRONIK, Lüdenscheid, Germany). The position of the measuring probe was at the exposure location of rats.

- The integrity end stability of the aerosol generation and exposure system was measured by using a RAS-2 real-time aerosol photometer (MIE, Bedford, Massachusetts, USA).
- Samples taken from breathing zone: yes
- Brief description of analytical method used: gravimetric analysis of filter samples (filter: Glass-Fibre-Filter, Sartorius, Göttingen, Germany; digital balance).
- Particle size distribution: The particle-size distribution was analyzed using a BERNER-TYPE AERAS lowpressure criticai orifice cascade impactor (Hauke, Gmunden, Austria). Aerosol mass < 3 µm: 85.9 % for 65 mg/m³, 87.3 % for 117 mg/m³, 85.2 % for 168 mg/m³, 88 % for 357 mg/m³.
- MMAD (Mass median aerodynamic diameter): < 4 µm (mean MMAD = 1.5-1.8 µm) / GSD (Geometric st. dev.): 1.7
Analytical verification of test atmosphere concentrations:
Duration of exposure:
4 h
65, 117, 168, and 357 mg/m³
No. of animals per sex per dose:
Control animals:
other: yes, controls were exposed to conditioned dry air
Details on study design:
- Duration of observation period following administration: 14 days
- Frequency of observations and weighing: Body weights were measured before exposure, on days 3 and 7, and weekly thereafter. The appearance and behavior of each rat were examined carefully several times on the day of exposure and at least once daily thereafter. Weekend assessments were
made once a day (morning). Assessments from restraining tubes were made only if unequivocal signs occurred (e.g. spasms, abnormal movements, and severe respiratory signs).
- Necropsy of survivors performed: yes
- Other examinations performed: The rectaI temperatures were measured shortly after cessation of exposure using a digital thermometer with a rectal probe for rats. A battery of reflex measurements was made on the first postexposure day.
For necropsy findings: pair-wise Fisher test after the R x C chi-squared test in accordance with Gad and Weil (Statistics for Toxicologists. Principles and Methods of Toxicology, ed. AW. Hayes, Raven Press, New York, 280, 1982). For statistical evaluation of the body weight gain for each group a one-way ANOVA (vide infra) is used. Data of rectaI temperature measurements are statistically evaluated using the ANOVA procedure (vide infra). For LC50 calculation: method of Rosiello et al. (J. Tox. and Environ. Health, 3, 797-809, 1977) as modified by Pauluhn (Computer-Aided Estimation of the LD50/LC50 BAYER AG Report No. 11835, dated May 18, 1983).

Results and discussion

Effect levelsopen allclose all
Dose descriptor:
Effect level:
ca. 158 mg/m³ air
Exp. duration:
4 h
Dose descriptor:
other: NO(A)EL
Effect level:
< 65 mg/m³ air
Exp. duration:
4 h
Mortality did not occur at 65 mg/m³ whereas at higher concentration rats succumbed on the exposure day or first postexposure days in a concentration-dependent manner.
Animals that died (onset of mortality): males 0/5 at 65 mg/m³, 0/5 at 117 mg/m³, 3/5 at 168 mg/m³ (0d, 1d), 5/5 at 357 mg/m³ (0d, 1d); females 0/5 at 65 mg/m³, 1/5 at 117 mg/m³ (1d), 3/5 at 168 mg/m³ (1d), 5/5 at 357 mg/m³ (0d, 1d).
Clinical signs:
other: Dose group 65 mg/m³: all rats tolerated the exposure without specific signs. Rats exhibited clinical signs in dose group 117 mg/m³ and above. The following signs occured: irregular breathing patterns, labored breathing patterns, tachypnea, piloerection, h
Body weight:
Comparisons between the control and the exposure groups revealed significant, although transient changes (decreases) in body weights.
Gross pathology:
Necropsy findings were unremarkable in surviving rats whereas in rats that succumbed intercurrently the following findings predominated: nose: serous discharge, lung less collapsed and edema, hydrothorax, consolidation, discoloration of parenchymatous organs.
Other findings:
Reflexes: In comparison to the rats of the control group, some rats of group 3 and 4 displayed a reduced righting response.
Rectal temperature: Statistical comparisons between the control and the exposure groups revealed significant changes in body temperatures indicative of hypothermia.

Any other information on results incl. tables

The rats displayed signs suggestive of respiratory irritation. Necropsy findings support the conclusion that mortality is causally related to acute lower respiratory tract irritation, i.e., lung edema.

Applicant's summary and conclusion

Executive summary:

An Acute Inhalation Toxicity study according to OECD TG 403 was conducted on 5 rats per sex, which were nose-only exposed to concentrations of 65, 117, 168 and 357 mg/m³ test substance aerosol. The aerosol was of adequate respirability for the rats (mean MMAD 1.5 -1.8 µm / GSD 1.7).

Mortality did not occur at 65 mg/m³ whereas at higher concentration rats succumbed on the exposure day or first postexposure days in a concentration-dependent manner. Based on the result of the probit analysis, the approximate LC50 was 158.3 mg/m³. The rats displayed signs suggestive of respiratory irritation. Necropsy findings (lung edema) support the conclusion that mortality is causally related to acute lower respiratory tract irritation.