Registration Dossier

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:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2014
Report Date:
2014

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 403 (Acute Inhalation Toxicity)
Version / remarks:
(2009)
GLP compliance:
yes (incl. certificate)
Test type:
standard acute method

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Strain: Hsd Cpb:WU (SPF)
- Source: Harlan, AD Horst, Netherlands
- 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 (based on A. Spiegel and R. Gönnert, Zschr. Versuchstierkunde, 1, 38, 1961 and G. Meister, Zschr. Versuchstierkunde, 7, 144-153, 1965).
- Diet and water: ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- 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: mixture of vapour and aerosol / mist
Type of inhalation exposure:
nose only
Vehicle:
air
Mass median aerodynamic diameter (MMAD):
>= 1.25 - <= 2.5 µm
Geometric standard deviation (GSD):
>= 1.79 - <= 2.51
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Mode of exposure: Animals were exposed to the aerosolized test substance in restrainers made of Plexiglas. Restrainers were chosen that accommodated the animals' size. The type of exposure principle is comparable with a directed-flow exposure design (Moss and Asgharian, Respiratory Drug Delivery IV, 1994, 197) and minimizes re-breathing of exhaled test-atmosphere.
- Exposure apparatus: The chambers used are commercially available (TSE, Bad Homburg, Germany) and the performance as well as their validation has been published (Pauluhn, Journal of Applied Toxicology 14, 55-62, 1994, and Pauluhn & Thiel, Journal of Applied Toxicology 27, 160-167, 2007). 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). Each inhalation chamber segment was suitable to accommodate 20 rats at the perimeter location. The ratio between supply and exhaust air was selected so that 90 % of the supplied air was extracted via the exhaust air location and, if applicable, via sampling ports. The slight positive balance between the air volume supplied and extracted ensured that no passive influx of air into the exposure chamber occurred.
- Source and rate of air: Dry conditioned air, 15 L/min
- Method of conditioning air: Compressed air was supplied by Boge compressors and was conditioned (freed from water, dust and oil) automatically by a VIA compressed air dryer.
- System of generating particulates/aerosols: A modified BGI 3-nozzle Collison nebulizer (Type CN-25 MRE, BGI Inc., Waltham MA, USA) was used in order to attain a high and temporally stable concentration of exposure atmospheres. The temperature was maintained at 5 °C using a digitally controlled cryostat. Nebulization used conditioned, pressurized air (15 L/min; dispersion pressure approximately 200 kPa). The test atmosphere was directly entrained into the inhalation chamber with additional dilution of air (pull/push dilatation cascade which maintains the specified total flow rate into the inhalation chamber).
- Optimization of respirability: In order to increase the efficiency of the generation of respirable particles and prevent larger particles from entering the chamber a pre-separator (baffle) system was used (Tillery, Environmental Health Perspectives, 16, 25-40, 1976).
- Inhalation chamber equilibrium concentration: The test atmosphere generation conditions provide an adequate number of air exchanges per hour (15 L/min x 60 min/(3.8 L) > 200, continuous generation of test atmosphere). Under such test conditions 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.
- Method of particle size determination: Cascade impactor (Berner critical orifice cascade impactor)
- Treatment of exhaust air: The exhaust air was purified via filter systems.
- Temperature, humidity: Temperature and humidity measurements were performed by the computerized Data Acquisition and Control System using HC-S3 sensors (Rotronic Messgeräte GmbH, Ettlingen, Germany). The position of the probe was at the exposure location of rats.

TEST ATMOSPHERE
- The integrity end stability of the aerosol generation and exposure system was measured by using a Microdust Pro real-time aerosol photometer (Casella, USA), if technically feasible.
- Brief description of analytical method used: gravimetric analysis of filter samples (filter: Glass-Fibre-Filter, Sartorius, Göttingen, Germany; digital balance). Additionally, a derivatization nitro-reagent method was used (Adsorption tubes containing glass powder coated N-4-Nitrobenzyi-N-npropylamine (nitro reagent). The eluated urea-derivatives were quantified by HPLC.
- Samples taken from breathing zone: yes
- Particle size distribution: The particle size distribution was analysed using a BERNER critical orifice cascade impactor. Aerosol mass < 3 µm: 83.4 % at 35 mg/m³, 83.1 at 69 mg/m³, 64.9 at 114 mg/m³, and 62.4 at 211 mg/m³.
- MMAD (Mass median aerodynamic diameter) / GSD (Geometric st. dev.): The respirability of the aerosol was adequate and in compliance with test guidelines, i.e. the average mass median aerodynamic diameter (MMAD) was 1.25 µm at 35 mg/m³, 1.36 µm at 69 mg/m³, 2.33 µm at 114 mg/m³, and 2.5 µm at 211 mg/m³; GSD was in the range of 2.51-1.79.
Analytical verification of test atmosphere concentrations:
yes
Duration of exposure:
4 h
Concentrations:
Target concentrations: 10, 20, 40, 75, and 150 mg/m³
Analytical concentrations (HPLC): 19, 35, 69, 114, and 211 mg/m³
No. of animals per sex per dose:
5
Control animals:
yes
Remarks:
air control
Details on study design:
- Duration of observation period following administration: 2 weeks
- Frequency of observations and weighing: Bodyweights were recorded prior to exposure and 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.
- Necropsy of survivors performed: yes
- Other examinations performed: Reflexes were tested, based on recommendations made by Irwin (Psychopharmacologica 13, 1968, 222-257). Rectal temperatures were measured shortly after cessation of exposure (approximately within ½hour after the end of exposure) using a digital thermometer with a rectal probe for rats.
Statistics:
Pair-wise Fisher test after the R x C chi-squared test used for necropsy findings (Procedure in accordance with Gad and Weil, Statistics for Toxicologists. Principles and Methods of Toxicology, ed. A.W. Hayes, Raven Press, New York, 280, 1982).
Analysis of variance (ANOVA) used for statistical evaluation (e.g. body weights, rectal temperatures).
If calculation of a LC50 is possible, it is performed by computer (PC) according to the method of Rosiello et al. (J. Toxicol. Environ. Health 3, 797-809, 1977) as modified by Pauluhn (1983). This method is based on the maximumlikelihood method of Bliss (Q.J. Pharm. Pharmacol. 11, 192-216, 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 X²-homogeneity test is not performed. In this case the interpolated concentration at 50 % lethality is designated the approximate LC50. Additionally, the moving average interpolation according to Schaper et al. (Arch. Toxicol. 68, 332-337, 1994) is used for calculation, if applicable.

Results and discussion

Effect levelsopen allclose all
Sex:
male
Dose descriptor:
LC50
Effect level:
84 mg/m³ air
95% CL:
52 - 135
Exp. duration:
4 h
Key result
Sex:
female
Dose descriptor:
LC50
Effect level:
53 mg/m³ air
95% CL:
35 - 81
Exp. duration:
4 h
Mortality:
Mortality occurred at and above exposure concentrations of 114 and 35 mg/m³ in male and female rats, respectively, from the day of exposure up to the postexposure day 4 (one rat succumbed on day 18). Thus, female rats displayed a higher susceptibility.
mortalities - males (time of onset): 4/5 at 114 mg/m³ (<24 h, 2 d, 18 d), 5/5 at 211 mg/m³ (< 24 h).
mortalities - females (time of onset): 2/5 at 35 mg/m³ (1 d, 2 d), 3/5 at 69 mg/m³ (< 24 h, 1 d, 4 d), 4/5 at 114 mg/m³ (< 24 h, 1 d, 4 d), 5/5 at 211 mg/m³ (< 24 h, 1 d, 2 d).
Clinical signs:
Clinical observations revealed the following signs: bradypnea, irregular breathing pattern, labored breathing pattern, dyspnea, breathing sounds, stridor, motility reduced, atony, tremor, high-legged gait, squatting posture, piloerection, haircoat ungroomed, cyanosis, nose reddened, nasal discharge (serous), nostrils with red encrustations, nose/muzzle area red encrusted, emaciation, prone position. Signs persisted up to the end of the postexposure period.
Body weight:
Significantly decreased body weights after exposure with evidence of recovery during the postexposure period.
Gross pathology:
Gross necropsy findings considered to be causal for death by an acute lung edema. The macroscopic findings of extrapulmonary organs were essentially indistinguishable amongst exposure and control groups.
Findings for animals sacrificed at the end of the observation period: Lung discoloration and enlarged lymph nodes draining the lung.
Findings for animals succumbed during observation period: The findings show evidence of lung injury and airway irritation.
Other findings:
Reduced reflexes and hypothermia were observed.

Any other information on results incl. tables

The respirability of the aerosol was adequate to achieve the objective of study. However, in the range of the LC50 the predominating exposure phase was vapour rather than aerosol.

Applicant's summary and conclusion

Executive summary:

A rat study on the acute inhalation toxicity of the substance 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). In this study groups of rats were nose-only exposed to the liquid aerosol of the test article at actual concentrations of 19, 35, 69, 114, and 211 mg/m³. The respirability of the aerosol was adequate to achieve the objective of study. However, in the range of the LC50 the predominating exposure phase was vapour rather than aerosol.

The LC50 in this study was 84 mg/m³ for male, and 53 mg/m³ for female rats. Mortality occurred at and above exposure concentrations of 114 and 35 mg/m³ in male and female rats, respectively. Thus, female rats displayed a higher susceptibility. Clinical observations revealed signs reflective of respiratory tract irritation. Mortality occurred from the exposure day up to the postexposure day 4 (one rat succumbed on day 18). Signs persisted up to the end of the postexposure period. Gross necropsy findings (lung injury and airway irritation) considered to be causal for death by an acute lung edema.