Registration Dossier

Administrative data

Endpoint:
acute toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
June 26, 2015 to November 16, 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 guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 403 (Acute Inhalation Toxicity)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.2 (Acute Toxicity (Inhalation))
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.1300 (Acute inhalation toxicity)
Deviations:
no
GLP compliance:
yes
Test type:
traditional method
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
2-butoxyethyl benzoate
EC Number:
226-685-8
EC Name:
2-butoxyethyl benzoate
Cas Number:
5451-76-3
Molecular formula:
C13H18O3
IUPAC Name:
2-butoxyethyl benzoate
Test material form:
other: liquid
Details on test material:
2-Butoxyethyl benzoate, Lot # 201303443-19; Purity 99.2 %
Specific details on test material used for the study:
Test Material Name: 2-Butoxyethyl benzoate
Chemical Name: 2-Butoxyethanol benzoate
Supplier, City, State (Lot, Reference Number): The Dow Chemical Company, Midland, Michigan (Lot # 201303443-19).
Purity/Characterization (Method of Analysis and Reference): The purity of the test material was determined to be 99.2% area (corrected for water) by gas chromatography with identification by nuclear magnetic resonance and gas chromatography mass spectrometry (Gobbi, 2014).
Test Material Stability Under Storage Conditions: The test material was determined to have two years of stability under ambient storage conditions (Wachowicz et al., 2015).

Test animals

Species:
rat
Strain:
other: F344/DuCrl
Sex:
male/female
Details on test animals or test system 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 9 weeks at the time of both the 3.71 and the 5.39 mg/L exposures.

Physical and Acclimation:
During the acclimation periods each animal was evaluated by a veterinarian trained in the field of Laboratory Animal Medicine, or a trained animal/toxicology technician, to determine the general health status and acceptability for study purposes. The Toxicology and Environmental Research and Consulting Laboratory is fully accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC International). The animals were housed two or three per cage in stainless steel solid bottom cages with corncob bedding, 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 each study. Animals were acclimated to the nose cones for two hours on the day preceding exposure to the test material.

Housing:
After assignment, animals were housed two or three per cage in stainless steel cages. Cages had solid floors with corncob bedding and nylon bone 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 rooms.
Temperature: 22°C with a range of 20°C-26°C
Humidity: 50% with a range of 30-70%
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.)
NOTE: Photoperiod times may change due to study-related activities.

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:
Feed and municipal water was provided ad libitum except during the 2-hour nose-cone acclimation periods and during the 4-hour exposure periods. Animals were provided LabDiet Certified Rodent Diet #5002 (PMI Nutrition International, St. Louis, Missouri) in pelleted form. Analyses of the feed were performed by PMI Nutrition International to confirm the diet provided 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. Copies of these analyses are maintained at the Toxicology and Environmental Research and Consulting laboratory, The Dow Chemical Company, Midland, Michigan.

Animal Welfare:
In accordance with the U.S. Department of Agriculture animal welfare regulations, 9 CFR, Subchapter A, Parts 1-4, the animal care and use activities required for conduct of this study were reviewed and approved by the Institutional Animal Care and Use Committee (IACUC). The IACUC has determined that the proposed Activities were in full accordance with these Final Rules. The IACUC-approved Animal Care and Use Activities used for this study were Acute Tox 01, DCO 01, Humane Endpoints 01, and Animal ID/Animal Care 01.




Administration / exposure

Route of administration:
inhalation: aerosol
Type of inhalation exposure:
nose only
Vehicle:
air
Remark on MMAD/GSD:
The mass median aerodynamic diameter (MMAD) of aerosolized 2-Butoxyethyl benzoate present in the exposure chamber test atmosphere averaged 4.15 microns with an average geometric standard deviation (GSD) of 2.17 microns for the 3.71 mg/L exposure and 2.90 microns with a GSD of 2.04 microns for the 5.39 mg/L exposure.
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 aerosol exposure.

Stability:
The test atmosphere was generated and immediately delivered to the test system; therefore, analysis of the stability of the test material under the conditions of administration was not necessary for this study.

Solubility:
The test material was delivered neat, therefore, solubility testing is not applicable to this study.

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 23 or 25 liters per minute, which was sufficient to provide the normal concentration of oxygen to the animals and approximately 33 or 36 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 negative 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 (Fisher Scientific Co., Pittsburgh, Pennsylvania or Omega Engineering, Inc., Bridgeport, New Jersey), one thermocouple extended into the exposure chamber and the second was stationed next to the chamber. Chamber relative humidity was monitored by a hygrometer (SperScientific., Ltd., Scottsdale, Arizona or VWR, West Chester, Pennsylvania) stationed in the interior of the chamber. Low relative humidity values were expected for exposures of
this type, therefore, air supplied to the chamber was passed through a Model 5-60 DRISTEEM humidifier filled with distilled water to humidify the air to a level more suitable for the animals. Based on the 23 or 25 liter per minute flow rate, the theoretical equilibrium time to 99% (T99) of the target concentration was 8.4 or 7.6 minutes, respectively. The animals were placed on the chamber after the T99 had elapsed and were removed after 240 minutes of exposure.

Generation System:
A liquid aerosol of 2-Butoxyethyl benzoate was generated by metering the test material with a FMI pump (Fluid Metering, Inc., Oyster Bay, New York) into a stainless steel Model 970 Schlick spray nozzle (Orthos, Inc., Schaumburg, Illinois) or a stainless steel ¼-J spray nozzle (Spraying Systems Co., Wheaton, Illinois). The test material was mixed with compressed air in the spray nozzle and aerosol was sprayed into the chamber. The test material was not recycled.

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

Exposure Concentration:
The mass concentration of aerosol present in the chamber was determined gravimetrically six times during the 3.71 mg/L exposure and five times during the 5.39 mg/L exposure. Samples were taken by drawing air, at one liter per minute, through a sample probe located in the breathing zone of the animals (Figure 1). Aerosol particles were collected on preweighed glass fiber 47 mm filters (PALL Corporation, Ann Arbor, Michigan). A XAD-2 sorbent tube (SKC Inc., Eighty Four, Pennsylvania) was added after the filter to capture any test material vapor present in the chamber. Background measurements of vapor in the chamber were taken prior to starting the exposure. After each atmosphere sampling, the filter and tube were reweighed and the average background measurement was then subtracted to obtain the net weight of the particles and vapors collected. The time-weighted average (TWA) exposure concentration was calculated from the gravimetric measurements, after subtraction of the background measurements. The efficiency and performance characteristics of the gravimetric sampling train were assessed before preliminary chamber aerosol samples were measured. Initially, a known mass of undiluted test material was deposited (spiked) onto the glass fiber filter and a known volume of air was pulled through the sample train (filter, and XAD-2 sorbent tubes) to simulate sampling from the exposure chamber. The nominal concentration was calculated based on the amount of test material fed into the generation system divided by the total chamber airflow.

Particle Size Determination:
The aerodynamic particle size distribution was determined three times during the 3.71 mg/L exposure period and two times during the 5.39 mg/L exposure period by drawing samples from within the animal breathing zone, at three liters per minute using a constant flow air sampling pump through a multi-stage mercer-style cascade impactor. The MMAD and geometric standard deviation (GSD) were determined for each sample as well as the average of the samples.

Analytical verification of test atmosphere concentrations:
yes
Duration of exposure:
4 h
Concentrations:
Two groups of five F344/DuCrl rats/sex were exposed for four hours, using a nose-only inhalation exposure system, to time-weighted average (TWA) chamber concentrations of 3.71 or 5.39 mg 2-Butoxyethyl benzoate per liter of air.
No. of animals per sex per dose:
5/sex/dose group
Control animals:
no
Details on study design:
Animal Observations and Criteria of Response:
A cage-side examination was conducted at least once a day, generally 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. Cage-side observations, in which only positive findings were documented, are summarized with clinical observations.
Animals were weighed and examined prior to exposure to the test material and observed at least every 30 minutes during the exposure periods. All rats were weighed on test days 2, 4, 8, 11, and 15 during the two-week post-exposure periods.
All rats were submitted for a complete necropsy examination on test day 15. Non-fasted rats submitted alive for necropsy were anesthetized with a mixture of isoflurane vapors and medical oxygen, and were then placed in a CO2 chamber to continue anesthesia. While under anesthesia, their tracheas were exposed and clamped, and the animals were euthanized by decapitation.
A complete necropsy was conducted on all animals 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 were 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, re-examined 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 prior to exposure, twice following exposure (on test day 1), and daily thereafter. The DCO was conducted on all animals, at approximately the same time each day according to an established format (Table 1). The examination included cage-side, hand-held and open-field observations that were recorded categorically or using explicitly defined scales (ranked).
Statistics:
Means and standard deviations were calculated for descriptive purposes for chamber concentration (mean only), animal body weights, exposure room temperatures and chamber temperature, humidity, and airflow. No statistical analysis to determine the LC50 was performed as no mortality was observed.

Results and discussion

Effect levels
Key result
Sex:
male/female
Dose descriptor:
LC50
Effect level:
> 5.39 mg/L air
Based on:
test mat.
Exp. duration:
4 h
Mortality:
All animals survived the four-hour exposures to 2-Butoxyethyl benzoate as well as the two-week post-exposure periods.
Clinical signs:
other: 3.71 mg/L: Clinical effects noted during the four-hour exposure were limited to soiling of the haircoat in 2/5 female rats and labored breathing in 1/5 female rats. In-life observations noted post-exposure included perineal soiling in 1/5 male and 3/5 fem
Body weight:
3.71 mg/L:
Mean body weight losses of 5.8% and 6.0% were noted for male and female rats, respectively, on test day 2. Pre-exposure mean body weight values were exceeded on test day 8 and all rats continued to gain weight until the scheduled test day 15 necropsy.

5.39 mg/L:
Mean body weight losses of 5.4 and 9.8% were noted for male and female rats, respectively, on test day 2. Pre-exposure mean body weight values were exceeded on test day 8 and all rats continued to gain weight until the scheduled test day 15 necropsy.
Gross pathology:
There were no treatment-related visible lesions noted in any of the rats exposed to 2- Butoxyethyl benzoate at the test day 15-scheduled necropsy.

Any other information on results incl. tables

Chamber Summary Data:

3.71 mg/L:

The resulting time-weighted average concentration was 3.71 mg/L; the nominal concentration was 27.2 mg/L. The difference between the gravimetric and the nominal concentrations was due to the loss of test material coating the walls of the generation apparatus and exposure chamber, and the inefficiency of the generation system employed.

The chamber O2 level was determined to be 20.9% and the CO2 level was determined to be 448 ppm prior to exposure. The average chamber temperature and relative humidity were 22.7 ± 0.5°C and 35.6 ± 2.0%, respectively. The average exposure room temperature was 21.4 ± 0.7°C. Airflow was maintained at 23 liters per minute.

Based on three determinations, the mean MMAD of the particles was 4.15 microns with an average geometric standard deviation of 2.17 microns. Approximately 4.27% of the particle mass was contained in a size fraction with an aerodynamic diameter less than 1 micron. Approximately 70.4% of the particulate mass was present in size fractions with an aerodynamic diameter less than 6 microns.

5.39 mg/L:

The resulting time-weighted average concentration was 5.39 mg/L; the nominal concentration was 6.3 mg/L. The difference between the gravimetric and the nominal concentrations was due to the loss of test material coating the walls of the generation apparatus and exposure chamber, and the inefficiency of the generation system employed.

The chamber O2 level was determined to be 20.8% and the CO2 level was determined to be 488 ppm prior to exposure. The average chamber temperature and relative humidity were 21.1 ± 0.0°C and 24.7 ± 0.5%, respectively. The average exposure room temperature was 21.9 ± 0.1°C. Airflow was maintained at 25 liters per minute.

Based on two determinations, the mean MMAD of the particles was 2.90 microns with an average geometric standard deviation of 2.04 microns. Approximately 9.70% of the particle mass was contained in a size fraction with an aerodynamic diameter less than 1 micron. Approximately 85.9% of the particulate mass was present in size fractions with an aerodynamic diameter less than 6 microns.

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
Based on these data, the four-hour LC50 of inhaled 2-Butoxyethyl benzoate is greater than 5.39 mg/L for male and female F344/DuCrl rats.
Executive summary:

This study was conducted to determine the acute inhalation toxicological properties of 2-Butoxyethyl benzoate. Two groups of five F344/DuCrl rats/sex were exposed for four hours, using a nose-only inhalation exposure system, to time-weighted average (TWA) chamber concentrations of 3.71 or 5.39 mg 2-Butoxyethyl benzoate per liter of air. The mass median aerodynamic diameter (MMAD) of aerosolized 2-Butoxyethyl benzoate present in the exposure chamber test atmosphere averaged 4.15 microns with an average geometric standard deviation (GSD) of 2.17 microns for the 3.71 mg/L exposure and 2.90 microns with a GSD of 2.04 microns for the 5.39 mg/L exposure.

All animals survived the four-hour exposure to 3.71 mg/L 2-Butoxyethyl benzoate as well as the two-week post-exposure period. Clinical effects noted during the four-hour exposure period were limited to soiling of the haircoat in 2/5 female rats and labored breathing in 1/5 female rats. In-life observations noted post-exposure included perineal soiling in 1/5 male and 3/5 female rats and abdominal soiling in 2/5 female rats. Mean body weight losses of 5.8 and 6.0% were noted for male and female rats, respectively, on test day 2. Pre-exposure mean body weight values were exceeded on test day 8. There were no visible treatment-related lesions noted in any of the rats exposed to 2-Butoxyethyl benzoate at the test day 15-scheduled necropsy.

All animals survived the four-hour exposure to 5.39 mg/L 2-Butoxyethyl benzoate as well as the two-week post-exposure period. Clinical effects noted during the four-hour exposure period were limited to soiling of the haircoat in 1/5 female rats. In-life observations noted post-exposure included perineal soiling in 5/5 female rats, abdominal soiling in 1/5 female rats, and extensive body soiling in 1/5 female rats. Additionally, pale skin was noted in 1/5 female rats. All rats appeared normal by test day 7. Mean body weight losses of 5.4 and 9.8% were noted for male and female rats, respectively, on test day 2. Pre-exposure mean body weight values were exceeded on test day 8. There were no visible treatment-related lesions noted in any of the rats exposed to 2-Butoxyethyl benzoate at the test day 15-scheduled necropsy.

Based on these data, the four-hour LC50 of inhaled 2-Butoxyethyl benzoate aerosol is greater than 5.39 mg/L for male and female F344/DuCrl rats.