Registration Dossier

Administrative data

Description of key information

GLP-studies according to OECD guidelines 402, 403 and 425 are available for 2-butoxyethyl benzoate.

Key value for chemical safety assessment

Acute toxicity: via oral route

Link to relevant study records
Reference
Endpoint:
acute toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was conducted according to OECD TG 425, EPA TG OPPTS 870.1100 and in accordance with the Principles of Good Laboratory Practice (GLP)
Reason / purpose for cross-reference:
reference to same study
Reason / purpose for cross-reference:
reference to other study
Qualifier:
according to guideline
Guideline:
OECD Guideline 425 (Acute Oral Toxicity: Up-and-Down Procedure)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.1100 (Acute Oral Toxicity)
Deviations:
no
Principles of method if other than guideline:
not applicable
GLP compliance:
yes
Test type:
up-and-down procedure
Limit test:
no
Species:
rat
Strain:
Wistar
Sex:
female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: inhouse bred (outbred)
- Age at study initiation: 8-2 weeks
- Weight at study initiation: 197.4 - 208.5 g
- Fasting period before study: overnight fasting
- Housing: Rats were housed individually in standard polysulfone cages (Size: approximately L 425 x B 266 x H 175 mm), with stainless steel top grills having facilities for pelletted food and drinking water.
- Diet (ad libitum): Ssniff® rats/mice pellet food-maintenance meal manufactured by Ssniff Spezialdiäten GmbH., Ferdinand-Gabriel-Weg 16, D-59494 SÖest, Germany, was provided to the animals.
- Water (ad libitum): Deep bore-well water passed through activated charcoal filter and exposed to UV rays in Aquaguard on-line water filter-cum-purifier manufactured by Eureka Forbes Ltd., Mumbai 400 001, India, was provided to animals in polycarbonate bottles with stainless steel sipper tubes. The water bottles were replenished once daily and the water bottles were changed once a week.
- Acclimation period: five to twenty six days under standard laboratory conditions

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-22°C
- Humidity (%): 56 – 66%
- Air changes (per hr): 12-15 air changes/hour
- Photoperiod (hrs dark / hrs light): 12 hours light and 12 hours dark cycle
Route of administration:
oral: gavage
Vehicle:
unchanged (no vehicle)
Details on oral exposure:
Undiluted test substance was administered as such.
Doses:
Single animals were dosed in sequence. Each animal was dosed by oral gavage using a stainless steel ball-tipped gavage needle attached to a syringe.
Steps 1-8: The test substance was administered at the dose of 2000 mg/kg (1.97 mL/kg), 175 mg/kg (0.17 mL/kg), 550 mg/kg (0.54 mL/kg), 2000 mg/kg
(1.97 mL/kg), 550 mg/kg (0.54 mL/kg), 2000 mg/kg (1.97 mL/kg), 550 mg/kg (0.54 mL/kg) and 2000 mg/kg (1.97 mL/kg) body weight for Steps 1- 8, respectively, as a single oral gavage to the rats fasted for 16 to 17 hours (access to water was not interrupted). The animals were fed immediately after dosing.
No. of animals per sex per dose:
single
Control animals:
no
Details on study design:
- Duration of observation period following administration: 14 days
- Frequency of observations and weighing: The animals were observed five times on test day 1 (day of administration) i.e., at 30 minutes, 1, 2, 3 and 4 hours after dose administration and once daily during days 2 – 15. Individual body weights of animals were recorded on test day 1 (pre-administration), day 8 (7 days post administration) and day 15 (14 days post administration) or at death.
- Necropsy of survivors performed: yes
Statistics:
Acute Oral Toxicity Software Program; AOT425StatPgm; AOT 425StatPgm Program User’s Manual; and Simulation Results for the AOT425StatPgm Program
Preliminary study:
not applicable
Sex:
female
Dose descriptor:
LD50
Effect level:
939.8 mg/kg bw
Based on:
test mat.
95% CL:
550 - 2 000
Remarks on result:
other: Based on the present study results, the estimated acute oral LD50 of 2-butoxyethyl benzoate is 939.8 mg/kg body weight with a 95% confidence interval of 550 to 2000 mg/kg body weight in female Wistar rats.
Mortality:
Step 1: One female rat was treated with the test substance at a limit dose of 2000 mg/kg (1.97 mL/kg) body weight. Slight piloerection, lethargy and/or red
discoloration of urine were observed in the rat on days 1 to 5 and it died on day 6.
Step 2: The second female rat was treated with the test substance at the dose of 175 mg/kg (0.17 mL/kg) body weight. There were no clinical signs of toxicity or mortality.
Step 3: The third female rat was treated with the test substance at the dose of 550 mg/kg (0.54 mL/kg) body weight. There were no clinical signs of toxicity or
mortality.
Step 4: The fourth female rat was treated with the test substance at the dose of 2000 mg/kg (1.97 mL/kg) body weight. Moderate piloerection, lethargy and/or red discoloration of urine were observed in the rat on days 2 to 4 and it died on day 4.
Step 5: The fifth female rat was treated with the test substance at the dose of 550 mg/kg (0.54 mL/kg) body weight. There were no clinical signs of toxicity or
mortality.
Step 6: The sixth female rat was treated with the test substance at the dose of 2000 mg/kg (1.97 mL/kg) body weight. Slight piloerection, lethargy and/or red
discoloration of urine were observed in the rat on days 1 to 3 and it died on day 3.
Step 7: The seventh female rat was treated with the test substance at the dose of 550 mg/kg (0.54 mL/kg) body weight. There were no clinical signs of toxicity or mortality.
Step 8: The eighth female rat was treated with the test substance at the dose of 2000 mg/kg (1.97 mL/kg) body weight. Slight piloerection, lethargy and/or red
discoloration of urine were observed in the rat on days 1 to 3 and it died on day 4.
Clinical signs:
Step 1: One female rat was treated with the test substance at a limit dose of 2000 mg/kg (1.97 mL/kg) body weight. Slight piloerection, lethargy and/or red
discoloration of urine were observed in the rat on days 1 to 5 and it died on day 6.
Step 2: The second female rat was treated with the test substance at the dose of 175 mg/kg (0.17 mL/kg) body weight. There were no clinical signs of toxicity or mortality.
Step 3: The third female rat was treated with the test substance at the dose of 550 mg/kg (0.54 mL/kg) body weight. There were no clinical signs of toxicity or
mortality.
Step 4: The fourth female rat was treated with the test substance at the dose of 2000 mg/kg (1.97 mL/kg) body weight. Moderate piloerection, lethargy and/or red discoloration of urine were observed in the rat on days 2 to 4 and it died on day 4.
Step 5: The fifth female rat was treated with the test substance at the dose of 550 mg/kg (0.54 mL/kg) body weight. There were no clinical signs of toxicity or
mortality.
Step 6: The sixth female rat was treated with the test substance at the dose of 2000 mg/kg (1.97 mL/kg) body weight. Slight piloerection, lethargy and/or red
discoloration of urine were observed in the rat on days 1 to 3 and it died on day 3.
Step 7: The seventh female rat was treated with the test substance at the dose of 550 mg/kg (0.54 mL/kg) body weight. There were no clinical signs of toxicity or mortality.
Step 8: The eighth female rat was treated with the test substance at the dose of 2000 mg/kg (1.97 mL/kg) body weight. Slight piloerection, lethargy and/or red
discoloration of urine were observed in the rat on days 1 to 3 and it died on day 4.
Body weight:
Step 1 [2000 mg/kg (1.97 mL/kg) body weight] : The rat had lost weight at death when compared to its initial weighing.
Step 2 [175 mg/kg (0.17 mL/kg) body weight] : The rat gained weight throughout the observation period.
Step 3 [550 mg/kg (0.54 mL/kg) body weight] : The rat gained weight throughout the observation period.
Step 4 [2000 mg/kg (1.97 mL/kg) body weight] : The rat had lost weight at death when compared to its initial weighing.
Step 5 [550 mg/kg (0.54 mL/kg) body weight]: The rat gained weight throughout the observation period.
Step 6 [2000 mg/kg (1.97 mL/kg) body weight] : The rat had lost weight at death when compared to its initial weighing.
Step 7 [550 mg/kg (0.54 mL/kg) body weight] : The rat gained weight throughout the observation period.
Step 8 [2000 mg/kg (1.97 mL/kg) body weight] : The rat had lost weight at death when compared to its initial weighing
Gross pathology:
Step 1 [2000 mg/kg (1.97 mL/kg) body weight] : Kidney – discolored red, multifocal; urinary bladder – distended with red colour content; stomach glandular
mucosa erosion, discolored red multifocal were observed at necropsy.
Step 2 [175 mg/kg (0.17 mL/kg) body weight] : There were no abnormalities detected at necropsy.
Step 3 [550 mg/kg (0.54 mL/kg) body weight] : There were no abnormalities detected at necropsy.
Step 4 [2000 mg/kg (1.97 mL/kg) body weight] : Liver pale; kidneys - discoloration, red multifocal; urinary bladder – distended with red colour content;
stomach - gladular mucosa - erosions, discoloration red multifocal were observed at necropsy.
Step 5 [550 mg/kg (0.54 mL/kg) body weight]: There were no abnormalities detected at necropsy.
Step 6 [2000 mg/kg (1.97 mL/kg) body weight] : Liver pale, all lobes; kidneys - discoloration red, multifocal; urinary bladder – distended with red content; stomach glandular mucosa - erosions; stomach non glandular mucosa, discoloration red multifocal were observed at necropsy.
Step 7 [550 mg/kg (0.54 mL/kg) body weight] : There were no abnormalities detected at necropsy
Step 8 [2000 mg/kg (1.97 mL/kg) body weight] : Kidneys discoloration, red multifocal and urinary bladder distended with red colour content; stomach glandular mucosa erosion, discoloration red multifocal were observed at necropsy.
Other findings:
None

None

Interpretation of results:
Toxicity Category IV
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
Based on the present study results, the estimated acute oral LD50 of 2-butoxyethyl benzoate was 939.8 mg/kg body weight with a 95% confidence interval of 550 to 2000 mg/kg body weight in female Wistar rats. According to Guidance to regulation (EC) No. 1272/2008 on classification, labelling and packaging (CLP) of substances and mixtures, 2-butoxyethyl benzoate will be classified under Acute toxicty, oral, Category 4.
Executive summary:

An acute oral toxicity test (Up-and-Down Procedure) was conducted with female Wistar rats to determine the potential of 2-butoxyethyl benzoate to produce toxicity from a single dose via the oral route.

The study was initiated with a limit test at 2000 mg/kg (step 1) with one female rat. The undiluted test substance was administered at 2000 mg/kg body weight as a single oral dose to fasted (16 - 17 hours) rats. The animal exhibited clinical signs of toxicity such as slight piloerection, lethargy and red discoloration of urine. The animal died on day 6. As per the AOT 425 statpgm, the main test was then conducted with the dose of 175 mg/kg (step 2). The rat survived with no clinical signs of toxicity, hence the next dose of 550

mg/kg (step 3) was administered to one female rat. There were no clinical signs of toxicity and the rat survived. The next dose of 2000 mg/kg (limit dose) (step 4) was then administered to one female rat. The animal exhibited clinical signs of toxicity such as moderate piloerection, lethargy and red discoloration of urine. The animal died on day 4. Hence the dosing was continued with 550 mg/kg (steps 5 and 7) and 2000 mg/kg (steps 6 and 8). There were no clinical signs of toxicity at 550 mg/kg (steps 5 and 7).

Slight piloerection, lethargy and red discoloration of urine were observed at 2000 mg/kg (steps 6 and 8) and the rats died on day 3 and day 4, respectively. The surviving rats were observed for 14 days post treatment. All the surviving rats gained body weight during the 14- day observation period. The preterminally dead rats lost weight when compared to their initial body weight.

All surviving animals were sacrificed on the scheduled Day 15 and subjected to a gross necropsy. There were no gross abnormalities detected in any of the rats at necropsy.

Liver pale; kidneys - discoloration, red multifocal; urinary bladder – distended with red colour content; stomach - glandular mucosa - erosions, discoloration red multifocal and/or stomach non glandular mucosa, discoloration red multifocal were observed in all pre-terminally dead rats at necropsy. The LD50 was determined after the 14-day observation period using the AOT 425 statpgm.

Based on the present study results, the estimated acute oral LD50 of 2-butoxyethyl benzoate was 939.8 mg/kg body weight with a 95% confidence interval of 550 to 2000 mg/kg body weight in female Wistar rats.According to Guidance to regulation (EC) No. 1272/2008 on classification, labelling and packaging (CLP) of substances and mixtures, 2-butoxyethyl benzoate will be classified under Acute toxicty, oral, Category 4.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
LD50
Value:
939.8 mg/kg bw
Quality of whole database:
GLP/Guideline study.

Acute toxicity: via inhalation route

Link to relevant study records
Reference
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
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
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).
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.




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.
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.

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.

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.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Quality of whole database:
GLP/Guideline study.

Acute toxicity: via dermal route

Link to relevant study records
Reference
Endpoint:
acute toxicity: dermal
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was conducted according to OECD TG 402, EPA TG OPPTS 870.1200 and in accordance with the Principles of Good Laboratory Practice (GLP)
Reason / purpose for cross-reference:
reference to same study
Reason / purpose for cross-reference:
reference to other study
Qualifier:
according to guideline
Guideline:
OECD Guideline 402 (Acute Dermal Toxicity)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.1200 (Acute Dermal Toxicity)
Deviations:
no
Principles of method if other than guideline:
not applicable
GLP compliance:
yes
Test type:
standard acute method
Limit test:
yes
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: inhouse bred (outbred)
- Age at study initiation: 11-12 weeks
- Weight at study initiation: males - 242.6 - 266.2 g, females - 211.6 - 222.3 g
- Housing: Rats were housed individually in standard polysulfone cages (Size: approximately L 425 x B 266 x H 175 mm), with stainless steel top grills having facilities for pelletted food and drinking water
- Diet (ad libitum): Ssniff® rats / mice pellet food - maintenance meal manufactured by Ssniff Spezialdiäten GmbH., Ferdinand-Gabriel-Weg 16, D-59494 SÖest, Germany, was provided to the animals.
- Water (ad libitum): Deep bore-well water passed through activated charcoal filter and exposed to UV rays in Aquaguard on-line water filter-cum-purifier manufactured by Eureka Forbes Ltd., Mumbai 400 001, India, was provided to animals in polycarbonate bottles with stainless steel sipper tubes. The water bottles were replenished once daily and the water bottles were changed once a week.
- Acclimation period: 5-7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 22°C
- Humidity (%): 56 - 66 %
- Air changes (per hr): 12 - 15 air changes/hour
- Photoperiod (hrs dark / hrs light): 12 hours light and 12 hours dark cycle
Type of coverage:
occlusive
Vehicle:
unchanged (no vehicle)
Details on dermal exposure:
Approximately 24 hours prior to treatment, the hair on the dorsolateral thoracic surface of the skin was clipped (approximately 8 x 10 cm) with an electric clipper (Aesculap® - Germany).
Based on the individual body weight the undiluted test substance at the dose of 2000 mg/kg body weight [1.97 mL/kg based on the density of the test substance of 1.0164 g/mL] was applied directly to the dorsolateral thoracic surface of the skin of the animal to cover about 10% of body surface and covered with cotton gauze (size: Males: 9 x 6 cm; Females: 8 x 5 cm of 6 ply gauze) and secured in position by nonallergenic surgical tape wound around the torso. The test substance was in contact with the skin for 24 hours.
After the 24-hour contact period, the patches were removed and the application sites were wiped with wet clean towels to remove any residual test substance.
Duration of exposure:
24 hours
Doses:
2000 mg/kg body weight
No. of animals per sex per dose:
5 males + 5 females
Control animals:
not required
Details on study design:
- Duration of observation period following administration: 14 days
- Frequency of observations and weighing: The animals were observed for clinical signs of toxicity five times during day 1 (day of administration) i.e., at about 30 minutes, 60 minutes (post administration) and subsequently three times at hourly intervals and once daily during days 2 - 15. Individual body weights of animals were recorded on test days 1 (pre-application), 8 (7 days post application), and 15 (14 days post application).
- Necropsy of survivors performed: yes - At the end of the observation period, all rats were euthanised using isoflurane anaesthesia followed by exsanguination. The external surface of the body, all orifices, tissues and organs of the thoracic and abdominal cavities of all animals were examined for gross pathological changes. All findings were recorded.
- Other examinations performed: The site of application was observed for skin reactions once daily during the 14-day observation period.
Statistics:
None
Preliminary study:
not applicable
Sex:
male/female
Dose descriptor:
LD0
Effect level:
> 2 000 mg/kg bw
Based on:
test mat.
Remarks on result:
other: Under the conditions of this study, the acute dermal LD50 of 2-butoxyethyl benzoate is greater than 2000 mg/kg body weight in male and female Wistar rats.
Mortality:
There were no mortality observed during the study.
Clinical signs:
There were no clinical signs of toxicity or mortality observed during the study.
Body weight:
All rats gained body weight throughout the observation period.
Gross pathology:
There was no abnormality detected at the necropsy.
Other findings:
There were no skin reactions observed at the site of application.

None

Interpretation of results:
not classified
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
Under the conditions of this study, the acute dermal LD50 of 2-butoxyethyl benzoate was greater than 2000 mg/kg body weight in male and female Wistar rats. According to Guidance to regulation (EC) No. 1272/2008 on classification, labelling and packaging (CLP) of substances and mixtures, 2-butoxyethyl benzoate will not be classified.
Executive summary:

An acute dermal toxicity test was conducted with male and female Wistar rats to determine the potential for 2-butoxyethyl benzoate to produce toxicity from a short term exposure via the dermal route.

Based on the individual body weight the undiluted test substance at the dose of 2000 mg/kg body weight was applied directly to the dorsolateral thoracic surface of the skin of the animal to cover about 10% of body surface and covered with cotton gauze (size: Males: 9 x 6 cm; Females: 8 x 5 cm of 6 ply gauze) and secured in position by non-allergenic surgical tape wound around the torso. After a 24 -hour contact period with the skin, the unabsorbed test substance at the site of application was removed by wiping with a wet clean towel.

The test was initiated with five female rats at the dose of 2000 mg/kg body weight. As there were no clinical signs of toxicity, local skin reactions or mortality, the treatment was then conducted in 5 male rats at the same dose. All the rats were observed for clinical signs of toxicity and mortality for 14 days post application. There were no clinical signs of toxicity, local skin reactions or mortality. All animals had gained body weight during the 14- day observation period. At the end of observation period, all animals were euthanized and subjected to necropsy. There were no gross pathological abnormalities detected at the necropsy.

Under the conditions of this study, the acute dermal LD50 of 2-butoxyethyl benzoate was greater than 2000 mg/kg body weight in male and female Wistar rats. According to Guidance to regulation (EC) No. 1272/2008 on classification, labelling and packaging (CLP) of substances and mixtures, 2-butoxyethyl benzoate will not be classified.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Quality of whole database:
GLP/Guideline study.

Additional information

Acute Oral Toxicity:

An acute oral toxicity test (Up-and-Down Procedure) was conducted with female Wistar rats to determine the potential of 2 -butoxyethyl benzoate to produce toxicity from a single dose via the oral route. The LD50 was determined after the 14-day observation period using the AOT 425 statpgm. Based on the present study results, the estimated acute oral LD50 of 2 -butoxyethyl benzoate was 939.8 mg/kg body weight with a 95% confidence interval of 550 to 2000 mg/kg body weight in female Wistar rats.According to Guidance to regulation (EC) No. 1272/2008 on classification, labelling and packaging (CLP) of substances and mixtures, 2 -butoxyethyl benzoate will be classified under Acute toxicty, oral, Category 4.

Acute Inhalation Toxicity:

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 at both concentration along with the two-week post-exposure period. Based on this, the four-hour LC50 of inhaled 2-Butoxyethyl benzoate aerosol is greater than 5.39 mg/L for male and female F344/DuCrl rats.

Acute Dermal Toxicity:

An acute dermal toxicity test was conducted with male and female Wistar rats to determine the potential for 2 -butoxyethyl benzoate to produce toxicity from a short term exposure via the dermal route.

Based on the individual body weight the undiluted test substance at the dose of 2000 mg/kg body weight was applied, for 24 hours, directly to the dorsolateral thoracic surface of the skin of the animal to cover about 10% of body surface. There were no clinical signs of toxicity, local skin reactions or mortality during the 14 -day post-exposure period. Under the conditions of this study, the acute dermal LD50 of 2 -butoxyethyl benzoate was greater than 2000 mg/kg body weight in male and female Wistar rats.

Justification for classification or non-classification

Acute Oral Toxicity:

Based on the study results, the estimated acute oral LD50 of 2 -butoxyetheyl benzoate was 939.8 mg/kg body weight with a 95% confidence interval of 550 to 2000 mg/kg body weight in female Wistar rats. According to Guidance to regulation (EC) No. 1272/2008 on classification, labelling and packaging (CLP) of substances and mixtures, 2 -butoxyethyl benzoate will be classified under Acute toxicty, oral, Category 4.

Acute Inhalation Toxicity:

Based on the 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.

According to Guidance to regulation (EC) No. 1272/2008 on classification, labelling and packaging (CLP) of substances and mixtures, 2 -butoxyethyl benzoate will not be classified.

Acute Dermal Toxicity:

Under the conditions of this study, the acute dermal LD50 of 2 -butoxyethyl benzoate was greater than 2000 mg/kg body weight in male and female Wistar rats. According to Guidance to regulation (EC) No. 1272/2008 on classification, labelling and packaging (CLP) of substances and mixtures, 2 -butoxyethyl benzoate will not be classified.