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: The study was conducted according to test guidelines and in accordance with GLP.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report Date:
2012

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
EPA OPPTS 870.1300 (Acute inhalation toxicity)
Deviations:
not specified
Qualifier:
according to
Guideline:
OECD Guideline 403 (Acute Inhalation Toxicity)
Deviations:
not specified
Qualifier:
according to
Guideline:
EU Method B.2 (Acute Toxicity (Inhalation))
Deviations:
not specified
Qualifier:
according to
Guideline:
other: JMAFF, Acute Inhalation Toxicity Study (2000)
Deviations:
not specified
GLP compliance:
yes
Test type:
standard acute method
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Remarks:
migrated information: particulates
Details on test material:
Test material was obtained from SACHEM, Zaltbommel, The Netherlands (Lot# ZMG-184366).

The purity of the test material was determined to be 99.9% (anhydrous basis) by high performance liquid chromatography with identification by nuclear magnetic resonance and liquid chromatography mass spectrometry.

Test animals

Species:
rat
Strain:
Fischer 344/DuCrj
Sex:
male/female
Details on test animals and environmental conditions:
Species and Sex
Rats (male and female)

Strain and Justification
F344/DuCrl rats. Selection of this strain and species was based on a variety of considerations including hardiness, low incidence of respiratory disease, general acceptability for inhalation toxicity studies, and availability of historical control data.

Supplier and Location
Charles River (Kingston, New York)

Age at Study Start
Animals were seven weeks at arrival and eight weeks at the time of exposure.

Physical and Acclimation
During the acclimation period each animal was evaluated by a laboratory veterinarian, or a trained animal/toxicology technician under the direct supervision of a laboratory veterinarian, to determine the general health status and acceptability for study purposes (fully accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International - AAALAC International). The animals were housed twothree
per cage in stainless steel solid bottom cages with corncob bedding and shredding aspen for enrichment, in rooms designed to maintain adequate environmental conditions (temperature, humidity, and photocycle), prior to randomization. Animals were acclimated to the laboratory for at least one week prior to the start of the study. Animals were acclimated to the nose cones for at least two hours on the day preceding exposure to the test material.

Housing
After assignment, animals were housed two to three per cage in stainless steel cages. Cages had solid floors with corncob bedding and shredded aspen for enrichment. Cages contained a hanging feeder and a pressure activated lixit valve-type watering system.

The following environmental conditions were maintained in the animal room.
Temperature: 22 ± 1°C (and a maximum permissible excursion of ± 3°C)
Humidity: 40-70%
Air Changes: 10-15 times/hour (average)
Photoperiod: 12-hour light/dark (on at 6:00 a.m. and off at 6:00 p.m.)

Randomization and Identification
Before administration of test material began, animals were stratified by body weight and then randomly assigned to treatment groups using a computer program designed to increase the probability of uniform group mean weights and standard deviations at the start of the study. Animals placed on study were uniquely identified via subcutaneously implanted transponders (BioMedic Data Systems, Seaford, Delaware) that were correlated to unique alphanumeric identification numbers.

Feed and Water
Animals were provided LabDiet Certified Rodent Diet #5002 (PMI Nutrition International, St. Louis, Missouri) in pelleted form. Feed and municipal water was provided ad libitum except during the 2-hour acclimation period the day prior to exposure and during the 4-hour exposure period. Analyses of the feed were performed by PMI Nutrition International to confirm the diet provides adequate nutrition and to quantify the levels of selected contaminants. Drinking water obtained from the municipal water source was periodically analyzed for chemical parameters and biological contaminants by
the municipal water department. In addition, specific analyses for chemical contaminants were conducted at periodic intervals by an independent testing facility.

Administration / exposure

Route of administration:
inhalation: dust
Type of inhalation exposure:
nose only
Vehicle:
air
Details on inhalation exposure:
Route, Method of Administration, Frequency, Duration and Justification
Inhalation was selected as the route of administration since it is a potential route of human exposure. The test material was administered via a single 4-hour nose-only exposure for each dose.

Exposure Conditions
Exposure room temperature, chamber temperature, humidity, and airflow were monitored continuously and recorded approximately every 30 minutes during the exposure period.

Chambers
A 42-liter, Dow-modified ADG nose-only chamber [30 centimeters (cm) in diameter by 60 cm high] was used for the study (Figure 1). Compressed filtered air supplied to the chamber was at ambient temperature. Airflow through the chamber was determined with a manometer which measured the pressure drop across a calibrated orifice plate and was maintained at approximately 40 liter per minute (LPM) for each exposure, which was sufficient to provide the normal concentration of oxygen to the animals and approximately 57 air changes per hour. The manometer was calibrated with a gas meter (Model DTM-115, Singer Aluminum Diaphragm Meter, American Meter Division, Philadelphia, Pennsylvania) prior to the start of the study. The chamber was operated at a slightly positive pressure relative to the surrounding area and was contained within a secondary vented area. Chamber and exposure room temperature were recorded from two thermocouples attached to an electronic digital thermometer (Control Company, Friendswood, Texas), one thermocouple extended into the exposure chamber and the second was stationed next to the chamber. Chamber relative humidity was monitored by a hygrometer (VWR, West Chester, Pennsylvania) stationed in the interior of the chamber.

Based on the 40 liter per minute flow rate, the theoretical equilibrium time to 99% (T99) of the target concentration was 4.8 minutes. The animals were placed on the chamber after the T99 had elapsed and were removed after 240 minutes of exposure to 0.64 mg/L and all deceased animals were removed by 97 minutes of exposure to 2.03 mg/L.

Generation System
A dust aerosol of benzyltrimethylammonium chloride was generated using a Jet Mill (Model 00 Jet-O-Mizer, Fluid Energy Aljet, Plumsteadville, Pennsylvania). The test material was first ground in a coffee-mill prior to use in order to reduce the particle size. A magnetic feeder (Model F-TO-C, Homer City Automation Homer City, Pennsylvania) was used to feed the test material to the jet mill. The test material did not, however, feed uniformly through the metal funnel and into the jet mill. This required a person, during some of the preliminary work and during the actual exposures, to continuously assist the flow of the test material to the base of the funnel and into the jet mill.

Exposure Concentration
The mass concentration of aerosol present in the chamber was determined gravimetrically 3 times during each exposure period. Samples were taken by drawing air, at 1 L/minute, through a sample probe located in the breathing zone of the animals. Aerosol particles were collected on preweighed glass fiber 47 mm filters (PALL Corporation, Ann Arbor, Michigan). The time-weighted average (TWA) exposure concentration was calculated from the gravimetric measurements, after subtraction of the background measurements.

The nominal concentration was calculated based on the mass of test material fed into the generation system divided by the total chamber airflow.

Particle Size
The aerodynamic particle size was determined twice during the four-hour exposure period during the exposure to 0.64 mg/L by drawing samples from within the animal breathing zone, at a set rate using a constant flow air sampling pump through a multistage mercer-style cascade impactor (Figure 2). The aerodynamic particle size was determined once during the exposure to 2.03 mg/L due to complete mortality within 97 minutes of the exposure period. The mass median aerodynamic diameter (MMAD) and geometric standard deviation (σg) were determined for each sample as well as the average of the two samples taken during the 0.64 exposure.
Analytical verification of test atmosphere concentrations:
yes
Remarks:
mass concentration and particle size
Duration of exposure:
4 h
Concentrations:
0.64 and 2.03 mg/L
No. of animals per sex per dose:
5 males and 5 females/concentration
Control animals:
no
Details on study design:
Animal Observations and Criteria of Response
A cage-side examination was conducted at least once a day, preferably at the same time each day (usually in the morning). This examination was typically performed with the animals in their cages and was designed to detect significant clinical abnormalities that were clearly visible upon a limited examination, and to monitor the general health of the animals. The animals were not hand-held for these observations unless deemed necessary. Significant abnormalities that would be observed include, but were not limited to: decreased/increased activity, repetitive behavior, vocalization, incoordination/limping, injury, neuromuscular function (convulsion, fasciculation, tremor, twitches), altered respiration, blue/pale skin and mucous membranes, severe eye injury (rupture), alterations in fecal consistency, and fecal/urinary quantity. In addition, all animals were observed for morbidity, mortality, and the availability of feed and water at least twice daily.

Animals were weighed and examined prior to exposure to the test material and observed at least every 30 minutes during the exposure period. All surviving rats were weighed on test days 2, 4, 8, 11, and 15 during the two-week post-exposure period.

All rats were submitted for a complete necropsy examination on test day 15. Non-fasted rats submitted alive for necropsy were anesthetized by the inhalation of CO2, their tracheas were exposed and clamped, and the animals were euthanized by decapitation.

A complete necropsy was conducted on all animals by a veterinary pathologist or by a trained technologist qualified to recognize common lesions. The necropsy included an examination of the external tissues and all orifices. The head was removed, the cranial cavity opened and the brain, pituitary and adjacent cervical tissues was examined. The head was split longitudinally to facilitate examination of the nasal passage. The eyes were examined in situ by application of a moistened microscope slide to each cornea. The skin was reflected from the carcass, the thoracic and abdominal cavities were opened and the viscera examined. All visceral tissues were dissected from the carcass, reexamined
and selected tissues were incised. Tissues were not saved and histopathologic examination was not performed unless deemed meaningful on selected tissues based on results of gross pathological examination.

Detailed Clinical Observations
Detailed clinical observations (DCO) were conducted pre-exposure, twice following the exposure and daily thereafter. The DCO was conducted on all animals, at approximately the same time each day according to an established format. The examination included cage-side, hand-held and open-field observations that were recorded categorically or using explicitly defined scales (ranked).

Categorical observations (descriptive) are summarized under the clinical observations table(s).
Statistics:
Means and standard deviations were calculated for descriptive purposes for chamber concentration (mean only), animal body weights, exposure room temperature and chamber temperature, humidity, and airflow. The LC50 was determined using linear interpolation of the arc sine transformed data (Stephan, 1977).

Reference:
Stephan, C. E. (1977). Methods for Calculating an LC50. Aquatic Toxicology and Hazard Evaluation. ASTM 634, Mayer, F. L. and Hamelink, J. L., Eds. American
Society for Testing and Materials, pp. 65-84.

Results and discussion

Effect levels
Sex:
male/female
Dose descriptor:
LC50
Effect level:
ca. 1.14 mg/L air
Based on:
test mat.
Mortality:
2.03 mg/L
Complete mortality was observed 97 minutes into the exposure to the test material.

0.64 mg/L
All animals survived the four-hour exposure to the test material as well as the two week post-exposure period.
Clinical signs:
other: 2.03 mg/L Clinical effects noted during the 2.03 mg/L exposure period were labored breathing in two male and two female rats and soiling of the haircoat in 2 female rats. 0.64 mg/L There were no clinical effects noted during the four-hour exposure. In-l
Body weight:
0.64 mg/L
Mean body weight losses of 1.4 and 2.6% were noted for male and female rats, respectively, on test day 2. Pre-exposure mean body weight values were exceeded by test day 4 and test day 8 for male and female rats, respectively.
Gross pathology:
2.03 mg/L
Liver congestion was noted in all of the rats exposed to 2.03 mg/L benzyltrimethylammonium chloride after the spontaneous deaths during the exposure. Also noted was body and facial soiling.

0.64 mg/L
There were no treatment-related visible lesions noted in any of the rats exposed to 0.64 mg/L benzyltrimethylammonium chloride at the test day 15-scheduled necropsy
Other findings:
2.03 mg/L
The chamber conditions are summarized in Table 2. The resulting time-weighted average concentration was 2.03 mg/L; the nominal concentration was 5.15 mg/L. The difference between the gravimetric and the nominal concentration was due to loss of test material coating the walls of the generation apparatus and exposure chamber, and the inefficiency of the generation system employed.

The average chamber temperature and relative humidity were 21.0 ± 0.1°C and 0 %, respectively. No humidity was added to the exposure system due to the hydroscopic properties of the material. The average exposure room temperature was 20.6 ± 0.2°C. The chamber O2 level was determined to be 20.9% and the CO2 level was determined to be 429 ppm. Airflow was maintained at 40 liters per minute.

All animals died 97 minutes into the exposure; therefore the particle size is based on one determination. The MMAD of the particles was 2.28 microns with a GSD of 2.85 microns. Approximately 27% of the particle mass was contained in a size fraction with an aerodynamic diameter less than 1 micron. Approximately 86% of the particulate mass was present in size fractions with an aerodynamic diameter less than 6 microns.

0.64 mg/L
The resulting time-weighted average concentration was 0.64 mg/L; the nominal concentration was 4.02 mg/L. The difference between the gravimetric and the nominal concentration was due to loss of test material coating the walls of the generation apparatus and exposure chamber, and the inefficiency of the generation system employed.

The average chamber temperature and relative humidity were 20.9 ± 0.8°C and 0%, respectively. No humidity was added to the exposure system due to the hydroscopic properties of the material. The average exposure room temperature was 20.4 ± 0.8°C. The chamber O2 level was determined to be 20.9% and the CO2 level was determined to be 436 ppm. Airflow was maintained at 40 liters per minute

Based on two determinations, the mean MMAD of the particles was 2.67 microns with an average GSD of 1.89. Approximately 26% of the particle mass was contained in a size fraction with an aerodynamic diameter less than 1 micron. Approximately 90% of the particulate mass was present in size fractions with an aerodynamic diameter less than 6 microns.

Any other information on results incl. tables

No additional information available.

Applicant's summary and conclusion

Interpretation of results:
Category 3 based on GHS criteria
Remarks:
Migrated information
Conclusions:
Based on these data, the calculated four-hour LC50 of inhaled benzyltrimethylammonium chloride is 1.14 mg/L for male and female F344/DuCrl rats.
Executive summary:

This study was conducted to determine the acute inhalation toxicological properties of benzyltrimethylammonium chloride. Two groups of five rats/sex were exposed using a nose-only inhalation exposure system, to time-weighted average chamber concentration of 0.64 or 2.03 mg benzyltrimethylammonium chloride per liter of air. The mass median aerodynamic diameter (MMAD) of particulate benzyltrimethylammonium chloride present in the exposure chamber test atmospheres averaged 2.67 microns with an average geometric standard deviation (GSD) of 1.89 for the 0.64 mg/L exposure and 2.28 microns with a GSD of 2.85 for the 2.03 mg/L exposure.

All animals died during exposure to 2.03 mg/L (100% mortality by 97 minutes of exposure. Clinical effects noted prior to death were labored breathing in 2 male and 2 female rats and soiling of the haircoat in 2 female rats. At necropsy all animals were noted to have liver congestion and soiling.

All animals survived the four-hour exposure to 0.64 mg/L as well as the two-week postexposure observation period. There was no in-life observations noted during the four-hour exposure to 0.64 mg/L. Observations noted post-exposure were limited to perineal and/or extensive body soiling in 4 males and 5 females. All rats appeared normal by test day 2. Mean body weight losses of 1.4 and 2.6% were noted for male and female rats, respectively, on test day 2. Pre-exposure mean body weight values were exceeded by test

day 4 and test day 8 for male and female rats, respectively. There were no visible treatment-related lesions noted in any of the rats exposed to 0.64 mg/L benzyltrimethylammonium chloride at the test day 15-scheduled necropsy.

Based on these data, the calculated four-hour LC50 of inhaled particulate benzyltrimethylammonium chloride is 1.14 mg/L for male and female F344/DuCrl rats.