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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: 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:
Test type:
fixed concentration procedure
Limit test:

Test material

Constituent 1
Chemical structure
Reference substance name:
EC Number:
EC Name:
Cas Number:
Molecular formula:
Details on test material:
- Name of test material (as cited in study report): Methylharnstoff, technisch (methylurea, technical grade)
- Physical state: solid (powder) / white
- Analytical purity: 98.5%
- Lot/batch No.: 07849
- Stability under test conditions: was ensured for at least the study period.
- Storage condition of test material: room temperature excluded from air and protected from light.
- Other: no data on contaminants

Test animals

Details on test animals or test system and environmental conditions:
- Source: Dr. K. Thomae GmbH, Biberach, Germany
- Age at study initiation: approx. 8 - 9 weeks
- Mean weight at study initiation (± standard deviation): male animals: 275 ± 10.8 g, female animals: 183 ± 12.5 g.
- Fasting period before study: no data
- Housing: in groups of five animals in cages type D III
- Diet: KLIBA rat/mouse laboratory diet 24-343-4 10 mm pellets, Klingentalmuehle AG, Kaiseraugst, Switzerland, ad libitum.
- Water: ad libitum
- Acclimation period: no data

- Temperature (°C): 20-24°C
- Humidity (%): 30-70 %
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12 h/ 12h

Administration / exposure

Route of administration:
inhalation: aerosol
Type of inhalation exposure:
nose/head only
Details on inhalation exposure:
- Exposure apparatus: Head-nose inhalation system INA 20 (glass-steel construction, BASF AG)
- Exposure chamber volume: ca. 55 L
- Method of holding animals in test chamber: animals were restrained in tubes and their snouts projected into the inhalation chamber.
- Source and rate of air: The air flow (supply air) was set with compressed air to 1500 l/ h
- System of generating particulates/aerosols: For technical reasons a 20 weight % solution of the test substance in bidest. water was applied. A liquid aerosol was generated by means of a piston metering pump KP 2000 (Desaga) and a two-component atomizer Mod. 970 (Schlick). The test substance was supplied to the two-component atomizer by means of the metering pump. By means of compressed air the aerosol was generated, which was passed into the inhalation system. By means of an exhaust air system the pressure ratios in the inhalation system were adjusted in such a way that the amount of exhaust air was about 10% lower (excess pressure). This ensured that the mixture of test substance and air was not diluted with laboratory air in the breathing zones of the animals.
- Method of particle size determination: Particle size analysis was performed with an Andersen Stack Sampler Mark III. Before the sampling, the impactor was equipped with glass-fiber collecting discs and a backup particle filter. The impactor was connected to the pump and the test apparatus, and 30 minutes after the beginning of the test at the earliest, one sample (12 l) was taken. The impactor was taken apart. The collecting discs and the backup particle filter were dried in a drying kiln with a temperature of 60 °C for 90 minutes. After the drying the collecting discs and the backup particle filter were weighed. The contents of the pre-impactor as well as the amounts of the material adsorbed on the walls of the impactor and in the sampling probe (wall losses) were also determined quantitatively. The mass median aerodynamic diameter (MMAD) was calculated from the results of the particle size analysis.
- Temperature, humidity, pressure in air chamber: The exposure system was placed in an air-conditioned laboratory. Temperatures in the exposure system were 19 - 25°C. Deviations from this specification which would have had any adverse effect on the results of the study did not occur.

- Brief description of analytical method used:
• Nominal concentration: The nominal concentration was calculated from the amount of substance consumed and the air flow.
• The analytical concentration of the inhalation atmosphere was determined gravimetrically (Mettler AE 240): Two preweighed filters were placed into the filtration equipment. By means of a vacuum compressed air pump a volume of the aerosol was drawn through the filter (Filter: MN 85/90 Bf [d = 4 .7 cm]) with a sampling velocity of 1.25 m/s, a sampling amount of 3 l, and a sampling frequency of 1 sample per hour. After the sample was taken, the filters were dried in a drying kiln with a temperature of 60 °C for 90 minutes. The dust concentration in mg/l was calculated from the difference between the preweight of the filter and the weight of the filter after drying, with reference to the sample volume of the inhalation atmosphere. The mass median aerodynamic diameter (MMAD) was calculated from the results of the particle size analysis.
- Samples taken from breathing zone: yes, in a position immediately to the animals noses.

- Composition of vehicle (if applicable): bidest. water
- Concentration of test material in vehicle (if applicable): 20 %
- Justification of choice of vehicle: The test substance was a powder. Therefore a solution of the test compound in water was used.

- Particle size distribution: see table attached.
Respirable dust reaching the alveoli is the amount of dust that passes through a separation system, the effect of which corresponds to the theoretical separation function of a sedimentation separator which separates 50% of the particles with an aerodynamic diameter of 5 µm. Stage 4 of the cascade impactor used separates 50% of the particles with an aerodynamic diameter of 5.5 µm. Particles collected in this stage and in the following stages are respirable aerosol reaching the alveolar region.
- MMAD 50 % (Mass median aerodynamic diameter) / GSD (Geometric st. dev.): 1.6 µm / 1.7
A respirable aerosol fraction that might reach the alveolar region of about 100% as obtained from the results of the particle size analysis.

Analytical verification of test atmosphere concentrations:
Duration of exposure:
4 h
The mean analytically measured concentration of the test substance was 5.73 ± 0.43 mg/l (mean ± SD). The selection of the concentration of the test group was based on the limit test, OECD-Guidelines, method 403.
No. of animals per sex per dose:
Control animals:
Details on study design:
- Duration of observation period following administration: 14 days
- Frequency of observations and weighing: The body weight of the animals was checked before the beginning of the test, after 7 days and at the end of the observation period. Clinical findings were recorded several times during exposure and at least once on each workday in the observation period. A check for dead animals was made daily.
- Necropsy of survivors performed: yes, at the end of the 14-day observation period the animals were sacrificed with CO2 and were subjected to grosspathological examination.
The statistical evaluation of the concentration/effect relationship was carried out on the basis of the binomial test (Wittig, H: Mathematische Statistik 1974, pp. 32 - 35) in accordance with the BASF Computer Center.
The calculation of the particle size distribution was carried out in the Department of Toxicology of BASF AG on the basis of mathematical methods for evaluating particle measurements (DIN 66141: Darstellung von Korngrößenverteilungen, DIN 66161: Partikelgrößenanalyse)

Results and discussion

Effect levels
Dose descriptor:
Effect level:
> 5.7 mg/L air
Exp. duration:
4 h
Remarks on result:
other: No mortality
No deaths occurred throughout the study.
Clinical signs:
other: During exposure, accelerated respiration was observed in 4/10, 4/10, 3/10, and 5/10 animals (after 1, 2, 3, and 4 hours of exposure, respectively). No clinical signs were observed during the first 30 minutes of exposure. Accelerated respiration persist
Body weight:
Body weight gain of males and females was slightly retarded during the second week of observation when compared with historical control data. At 7 days post treatment, mean body weight was 302 g for males and 193 g for females. At 13 days post dose, mean body weight was 325 g for males and 204 g for females.
Control data:
before dosing: 248 g (males), 177 g (females);
after 7 days: 285 g (males), 196 g (females);
after 14 days: 318 g (males), 211 g (females);
Gross pathology:
No pathological findings were noted at necropsy.
Other findings:
No other findings were observed.

Applicant's summary and conclusion

Based on the results of this study, the test substance does not have to be classified according to 67/548/EEC and Regulation (EU) No. 1272/2008.