Registration Dossier

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 guidelineopen allclose all
according to
OECD Guideline 403 (Acute Inhalation Toxicity)
other: test procedures were adapted so as to comply also with the EU Directive 92/69/EEC and OECD Guidance Document No. 39 (2009)
GLP compliance:
yes (incl. certificate)
Test type:
standard acute method

Test material

Test material form:
solid: particulate/powder

Test animals

Details on test animals and environmental conditions:
- Strain: HsdRCCHan: Wist (SPF bred)
- Source: Harlan-Nederland (NL), AD Horst
- 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 ± 3 °C
- Humidity (%): 40 - 60 %
- Air changes (per hr): approximately 10
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
inhalation: dust
Type of inhalation exposure:
nose only
other: unchanged (no vehicle)
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 minimizes rebreathing of the exhaled test atmosphere.
- Generation of atmosphere and exposure techniques: Test atmosphere was generated using a WRIGHT DUST FEEDER system (BG IInc.,Waltham, MA 02154, USA). For dry powder dispersion, conditioned compressed dry air (28 liters/min; generic dispersion pressure: 120 kPa) was used. The principle
performance of the WRIGHT DUST FEEDER dust generating system can be described as follows: the test substance was metered in a reservoir and then was compressed to a pellet using approximately 1 metric ton by a carva laboratory press (F. S. Carver Inc., Wabash, IN 46992, USA). From this pellet defined amounts of test substance were scraped off and entrained into the main air flow. The airborne powder was then conveyed into the inner cylinder of the inhalation chamber.
- Inhalation chamber equilibrium concentration: The test atmosphere generation conditions provide an adequate number of air exchanges per hour [28 L/min x 60 min/(3.8 L) = 422, continuous generation of test atmosphere]. Based on OECD GD39 the equilibrium concentration (t95) can be calculated after McFarland, 1976. Thus, under the test conditions used chamber equilibrium is attained in less than one minute of exposure. At each exposure port a minimal air flow rate of 0.75 L/min was provided. The test atmosphere can by no means be diluted by bias-air-flows.
- 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.
- Exhaust air treatment: The exhaust air was purified via filter systems.
- Temperature and humidity measurements were performed by a computerized Data Acquisition and Control System using HC-S3 sensors (Rotronic). The position of the probe was at the exposure location of rats. Temperature and humidity data are integrated for 30-seconds and displayed accordingly. The humidity sensors are calibrated using saturated salt solutions according to Greenspan (1977) and Pauluhn (1994) in a two-point calibration at 33 % (MgCI2) and at 75 % (NaCI) relative humidity. The calibration of the temperature sensors is also checked at two temperatures
using reference thermometers.

- Nominal concentration: The nominal concentration was not calculated from the ratio of the total quantity of test article consumed and the total throughput of air through the inhalation chamber as this would have required a dismantling of the dust generator.
- Samples taken from breathing zone: yes
- Gravimetric concentation: gravimetric analysis of filter samples (filter: Glass-Fibre-Filter, Sartorius, Göttingen, Germany; digital balance).
- Particle size distribution: The particle-size distribution was analyzed using an ANDERSON critical orifice cascade impactor. An adhesive stage coating (silicone spray) was omitted due to the adhesive properties of the test article. Gravimetric analyses of filters used a digital balance.
- MMAD (Mass median aerodynamic diameter): 3.5 µm; GSD (Geometric st. dev.): 2.4
Analytical verification of test atmosphere concentrations:
Duration of exposure:
4 h
5142 mg/m³ (actual concentration as dry powder aerosol)
No. of animals per sex per dose:
Control animals:
other: Comparisons with an appropriate historical air control group were performed. This control was exposed to an atmosphere using essentially similar exposure conditions as were used for the test substance (15 L air/min; conditioned air; 4 h exposure)
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 1, 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.
- Body weights: Means and single standard deviations of body weights are calculated. Mean body weights are also depicted graphically as a function of time. Since in acute studies individual group means may differ prior to commencement of the first exposure, the body weight gain was statistically evaluated for each group. For these evaluations a one-way ANOVA (vide infra) is used.
- Physiological data: Data of rectal temperature measurements are statistically evaluated using the ANOVA procedure (vide infra).
- Calculation of the LCso: If calculation of a median lethal concentration (LCSO) is possible, it is performed by computer (PC) according to the method of Rosiello et al. (1977) as modified by Pauluhn (1983). This method is based on the maximumlikeli hood method of Bliss (1938). If only 2 pairs of values with greater than 0 % lethality and less than 100 % are available then the first linear approximation is based on these values and a l-homogeneity test is not performed. In this case the interpolated concentration at SO% lethality is designated the approximate LCSO. Additionally, the moving average interpolation according to Schaper et al. (1994) is used for calculation, if applicable.
- Analysis of variance (A NO VA): This parametric method checks for normal distribution of data by comparing the median and mean. The groups are compared at a confidence level of (1-a) = 9S % (p = O.OS). The test for the between-group homogeneity of the variance employed Box's test if more than 2 study groups were compared with each other. If the above F-test shows that the intra-group variability is greater than the inter-group variability, this is shown in the Appendix as "no statistical difference between the groups". If a difference is found then a pairwise post-hoc comparison is conducted (1- and 2-sided) using the Games and Howell modification of the Tukey-Kramer significance test.

Results and discussion

Effect levelsopen allclose all
Dose descriptor:
Effect level:
5 142 mg/m³ air
Based on:
test mat.
Exp. duration:
4 h
Remarks on result:
other: clinical signs: irregular and laboured breathing
Dose descriptor:
Effect level:
> 6 597 mg/m³ air
Based on:
act. ingr.
Exp. duration:
4 h
Dose descriptor:
other: NOAEL
Effect level:
< 1 554 mg/m³ air
Based on:
act. ingr.
Exp. duration:
4 h
Mortality did not occur up to the maximum technically attainable level.
Clinical signs:
other: Irregular and labored breathing pattern, bradypnea, dyspnea, breathing sounds, motility reduced, atony, high-legged gait, piloerection, and cyanosis.
Body weight:
Comparisons between the control and the exposure groups revealed transient changes (decreases) in body weights.
Gross pathology:
Animals sacrificed at the end of the observation period: The macroscopic findings of extrapulmonary organs were essentially indistinguishable between the test article exposure and control group. In the test group local discolorations of the lung and enlarged lung-associated lymph nodes were observed.
Other findings:
A battery of reflex measurements was made on the first post-exposure day. Differences between the rats of the control and the trated group were not observed.
Statistical comparisons between the control and the exposure groups revealed significant changes in body temperatures indicative of hypothermia.

Applicant's summary and conclusion

Executive summary:
A study on the acute inhalation toxicity on rats has been conducted in accordance with OECD TG 403. One group of Wistar rats (5 males and 5 female) were nose only exposed (4 h) to the solid aerosol of the test article in the maximum technically achievable concentration of 5142 mg/m³. The aerosol was generated so that it was respirable to rats (average MMAD of 3.5 µm / GSD 2.4). Mortality did not occur. Clinical observation showed irregular and labored breathing patterns as the lead pathodiagnostic effect. Based on the results the LC50 was determined with 5142 mg/m³ for males and females. In summary, the test substance (solid aerosol) proved to have a low acute inhalation toxicity in rats.