Registration Dossier

Administrative data

Endpoint:
acute toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
January/February 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

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

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:
traditional method
Limit test:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid - solid: mixture of
Specific details on test material used for the study:
- Stability under test conditions: Analytical investigations verified the stability of the substance in the vehicle used.
- The test substance is a solution in approx. 30 % ethyl acetate. For the generation of a test atmosphere further ethyl acetate was used to decrease viscosity. Since the active ingredient of the test item itself is a solid, the gravimetric concentrations measured during the study reflect the active ingredient of the test item.

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, 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 with gnawing sticks. The legal requirements for housing experimental animals (Directive 2010/63/EU) are followed.
- 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: aerosol
Type of inhalation exposure:
nose only
Vehicle:
other: ethyl acetate
Mass median aerodynamic diameter (MMAD):
1.61 µm
Geometric standard deviation (GSD):
2.22
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).
- 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). 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 85-90 % 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: Conditioned (dry, oil free) compressed 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 BEKO RA 55 compressed air dryer.
- System of generating particulates/aerosols: Atmosphere for inhalation exposure was generated under dynamic conditions using a digitally controlled Harvard PHD 2000 infusion pump and a modified Schlick nozzle (Schlick GmbH, Coburg, Germany). All liquid containing parts of the nozzle were maintained at the required temperatures (selected during the pre-test phase) using a water jacket connected to a digitally controlled thermostat. Temperature of nozzle was adjusted at 5°C to maintain temporally stable conditions (evaporation of solvent in the nozzle).
- 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: The particle-size distribution was analyzed using a 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: The test-substance concentration was determined by gravimetric analysis (filter: glass-fiber filter, Sartorius, Gottingen, Germany; digital balance, post-sampling drying period of 30 min at 70°C). This method was used to define the actual concentration.
- Determination of volatile components: The relative proportion of constituents prone to evaporate is determined as follows: aliquots of the test item were added onto glass fiber filters and the filters were allowed to dry under specified conditions (at 70°C drying temperature) over a time period of maximal 3 hours. During this time course that time of drying was defined at which stable conditions are attained. The factor (f) to multiply filter analyses to calculate the actual concentration of the test item including the solvent is determined (factor 1.23).
- 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: 78.6 % at 3498 mg/m³ (gravimetrical conc.).
- 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 1.61 µm at at 3498 mg/m³ (gravimetrical conc.); GSD was 2.22.
Analytical verification of test atmosphere concentrations:
yes
Duration of exposure:
4 h
Concentrations:
Target concentration: 5000 mg/m³
The maximum technically attainable concentration was:
- for active ingredient (gravimetric determination): 3498 mg/m³
- for actual test item (substance in approx. 30 % solvent, conc. re-calculated): 4303 mg/m³
No. of animals per sex per dose:
3, additionally 5 animals per sex for vehicle control
Control animals:
yes
Remarks:
Vehicle control (ethyl acetate)
Details on study design:
- Duration of observation period following administration: 2 weeks
- Frequency of observations and weighing: Body weights were measured before exposure (day 0), on days 1, 3, 7, and 14. The appearance and behavior of each rat were examined carefully at least two 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.
- 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:
Body weights: Means and single standard deviations of body weights are calculated. Mean body weights are also depicted graphically as a function of time
(see result section). 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.
Particle size analysis: described in detail in report.
Physiological data: Data of rectal temperature measurements are statistically evaluated using the ANOVA procedure (vide infra).
Randomization: A computerized list of random numbers served the purpose to assign animals at random to the treatment groups.
Analysis of variance (ANOVA): 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) = 95 % (p = 0.05). 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. This program was originally obtained from BCTIC.

Results and discussion

Effect levels
Sex:
male/female
Dose descriptor:
LC50
Effect level:
> 3 498 mg/m³ air
Based on:
act. ingr.
Exp. duration:
4 h
Remarks on result:
other: maximal technical attainable concentration
Mortality:
Mortality did not occur up the technical maximal producible concentration (3498 mg/m³ based on active ingredient).
Clinical signs:
Animals of the control group revealed slightly bradypnea and slightly irregular breathing on day 0 after the exposure session. For animals exposed to the test substance the following clinical signs were observed (day 0 - day 1): bradypnea, labored breathing, irregular breathing, motility reduced, atony, high-legged gait, piloerection, haircoat ungroomed, nose/muzzle red encrusted, nostrils with red encrustations.
Body weight:
Comparisons between the control and the exposure groups revealed significant decrease in the incremental body weight gain on day 1. There is evidence of recovery during the postexposure period.
Gross pathology:
No macroscopic findings were observed for animals of the control group. For animals exposed to the test substance the following was detected at necropsy: Two male rats showed few red foci in the lungs and one female rat revealed few light colored areas in the lung.
Other findings:
There were no findings during reflex measurements in rats exposed to the test item.
Statistical comparisons between the control and the exposure groups revealed significant changes in body temperature at 3498 mg/m³ (active ingredient of the test item).

Applicant's summary and conclusion

Executive summary:

A study on the acute inhalation toxicity of the test item (solution of the substance at approx. 70 % in 30 % ethyl acetate) has been conducted in accordance with OECD TG 403 and also OECD OECD GD 39. In that study a group of rats was nose-only exposed to the liquid aerosol of the test item at the maximal technically attainable concentration of 4303 mg/m³ (test item including ethylacetate), which corresponds to 3498 mg/m³ pure substance (= "active ingredient"). Since the pure substance itself, without ethylacetate, is solid, gravimetric concentrations were used to reflect the toxicological study results. Further ethyl acetate has been used as vehicle to decrease viscosity. Rats of the control group were exposed to the vehicle (ethyl acetate) under otherwise identical circumstances.

The respirability of the aerosol was adequate and in compliance with test guidelines (MMAD 1.61 µm, GSD 2.22). All rats exposed to 3498 mg/m³ active ingredient showed clinical signs (bradypnea, labored breathing, irregular breathing, motility reduced, atony, high-legged gait, piloerection, haircoat ungroomed, nose/muzzle red encrusted, nostrils with red encrustations). Significantly decreased body temperatures and incremental body weight gain were found at 3498 mg/m³ active ingredient. Mortality did not occur up to the technically maximal producible concentration. After the recovery period, red foci were seen in the lungs of two male rats and few light colored areas were found in the lung of a female rat exposed to the test item.

The LC50 (4 h, both sexes) was therefore concluded to be > 3498 mg/m³ active ingredient (maximal technical attainable concentration) of the test substance.