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:
other: GLP guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2014
Report Date:
2015

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 403 (Acute Inhalation Toxicity)
Version / remarks:
(2009)
Qualifier:
according to
Guideline:
other: OECD Guidance Document No. 39 (2009)
GLP compliance:
yes (incl. certificate)
Test type:
standard acute method

Test material

Reference
Name:
Unnamed
Type:
Constituent

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-Nederland, AD Horst, The 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. Goennert, 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; during this period, rats were also acclimatized to the restraining tubes.


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- 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
Vehicle:
air
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Mode of exposure: Animals were nose-only exposed to the aerosolized test article 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. 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.
- Exposure apparatus: Plexiglas exposure restrainers (TSE, Bad Homburg, Germany). Each inhalation chamber segment was suitable to accommodate 20 rats at the perimeter location. For validation see Pauluhn, Journal of Applied Toxicology 14, 1994, 55-62 and Pauluhn & Thiel, Journal of Applied Toxicology 27, 2007, 160-167.
- 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: Under dynamic conditions the various concentrations of the test article were atomized into the baffle (pre-separator) of the inhalation chamber. For atomization a binary nozzle and conditioned compressed air (15 L/min) was used. The representative dispersion pressure was approximately 600 kPa (constant liquid feed through the nozzle maintained at room temperature). The test article was fed into the nozzle system using a digitally controlled pump (Harvard PHD 2000 infusion pump).
- Optimization of respirability: In order to increase the efficiency of the generation of fine particles through evaporation of the vehicle and to prevent larger particles from entering the chamber a pre-separator (baffle) system was used.
- 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) = 237, 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 RAS-2 real-time aerosol photometer (MIE, Bedford, Massachusetts, USA).
- Brief description of analytical method used: gravimetric analysis of filter samples (filter: Glass-Fibre-Filter, Sartorius, Göttingen, Germany; digital balance).
- 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: 89.3 % (109 mg/m³), 86.6 % (166 mg/m³), and 86.1 % (428 mg/m³)
- MMAD (Mass median aerodynamic diameter) / GSD (Geometric st. dev.): The aerosol was generated so that it was respirable to rats, i.e. the average mass median aerodynamic diameter (MMAD) throughout the groups was 1.59 – 1.68 µm, the geometric standard deviation (GSD) was 1.67 – 1.73.
Analytical verification of test atmosphere concentrations:
yes
Remarks:
gravimetric analysis
Duration of exposure:
4 h
Concentrations:
109, 166, and 428 mg/m³
No. of animals per sex per dose:
5
Control animals:
other: yes, 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 behaviour 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:
For necropsy findings: pair-wise Fisher test after the R x C chi-squared test.
Analysis of variance (ANOVA) was used for statistical evaluation.
Calculation of LC50 was performed according to Rosiello et al. (1977; Rosiello, Essigmann and Wogan, Tox and Environ. Health, 3, 797) as modified by Pauluhn (1983). It is based on the maximum likelihood method of Bliss (1983; Q.J.Pharm.Pharmacol., 11, 192).

Results and discussion

Effect levelsopen allclose all
Sex:
female
Dose descriptor:
LC50
Effect level:
ca. 135 mg/m³ air
Exp. duration:
4 h
Sex:
male/female
Dose descriptor:
LC50
Effect level:
ca. 161 mg/m³ air
Exp. duration:
4 h
Mortality:
Mortality occurred at 109, 166 and 428 mg/m³ (males: 0/5 at 0 mg/m³, 1/5 at 109 mg/m³, 1/5 at 166 mg/m³, 5/5 at 428 mg/m³; females: 0/5 at 0 mg/m³, 1/5 at 109 mg/m³, 4/5 at 166 mg/m³, 5/5 at 428 mg/m³).
Female rats seem to be more susceptible than male rats due to the fact that at 166 mg/m³ only one male rat out of five died whereas four of five female rats died at this concentration. All rats exposed to 428 mg/m³ died on day 0.
Clinical signs:
All rats exposed to 109 and 166 mg/m³ showed clinical signs (e.g. irregular breathing, bradypnea, laboured breathing, dyspnea, breathing sounds, stridor, nose red encrusted, nostrils with red encrustations, motility reduced, tremor, atony, high-legged gait, piloerection, haircoat ungroomed, ears: pallor, cyanosis, emaciation, nasal discharge (serous), reduced reflexes).
Body weight:
Significant decreased body weights were found at 109 mg/m³ and above.
Gross pathology:
Necropsy findings were predominantly unremarkable in surviving rats whereas in rats that succumbed in the course of study the following findings of toxicological importance at 109 mg/m³ and above were observed (e.g. nostrils: brownish encrustations; nose: red encrusted, whitish viscous content; trachea: white foamy content; lung: less collapsed, light coloured, dark-red, dark-red marbled; pleural cavity: clear liquid content).
Other findings:
Significant decreased body temperatures were found at 109 mg/m³ and above.

Any other information on results incl. tables

The mortality patterns were typical of an irritation-related acute lung edema (e.g.: white foamy discharge in the nose, white foamy content in the trachea, less collapsed and dark-red marbled lungs, bradypnea, labored breathing, breathing sounds, stridor, cyanosis, pallor).

Applicant's summary and conclusion

Executive summary:

An acute inhalation toxicity study in rats was recently conducted for reasons of product safety (the substance is used in spray applications). In this study, conducted according to OECD TG 403 and OECD GD 39, male and female rats were nose-only exposed to the liquid aerosol of the substance at 0 (air control), 109, 166, and 428 mg/m³. The respirability of the aerosol was in compliance with the respective test guidelines (the MMAD throughout the groups was 1.59 – 1.68 µm, the GSD was 1.67 – 1.73).

All rats exposed to 109 and 166 mg/m³ showed clinical signs (e.g. irregular breathing, bradypnea, stridor, nose and nostrils with red encrustations, motility reduced, tremor, atony, piloerection, haircoat ungroomed, emaciation, nasal discharge (serous), reduced reflexes). Significant decreased body temperatures and body weights were found at 109 mg/m³ and above. Mortality occurred at 109, 166 and 428 mg/m³. Female rats seem to be more susceptible than male rats due to the fact that at 166 mg/m³ only one male rat out of five died whereas four of five females died at this concentration. All rats exposed to 428 mg/m³ died on day 0. The mortality patterns were typical of an irritation-related acute lung oedema (e.g.: white foamy discharge in the nose, white foamy content in the trachea, less collapsed and dark-red marbled lungs, bradypnea, labored breathing, breathing sounds, stridor, cyanosis, pallor). Necropsy revealed findings of toxicological importance at 109 mg/m³ and above (e.g. nose: red encrusted, whitish viscous content; trachea: white foamy content; lung: less collapsed, light coloured, dark-red, dark-red marbled).

Due to the fact that a gender-specific mortality pattern was observed at 166 mg/m³, the LC50 for the more susceptible females is taken forward: LC50 (4 h, females) 135 mg/m³.