2-butoxyethyl benzoate

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

November 12, 2015 to August 18, 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.26 (Sub-Chronic Oral Toxicity Test: Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3100 (90-Day Oral Toxicity in Rodents)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

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: Crl:CD(SD)

Details on species / strain selection:

Strain and Justification:
Crl:CD(SD) rats were selected because of their general acceptance and suitability for toxicity testing, previous toxicity studies on 2-butoxyethyl benzoate have used this strain, availability of historical background data and the reliability of the commercial supplier.

Sex:

male/female

Details on test animals or test system and environmental conditions:

Species and Sex: Rats (male and female)
Strain and Justification: Crl:CD(SD) rats were selected because of their general acceptance and suitability for toxicity testing, previous toxicity studies on 2-butoxyethyl benzoate have used this strain, availability of historical background data and the reliability of the commercial supplier.
Supplier and Location: Charles River (Raleigh, North Carolina)
Age at Study Start: 6-8 weeks

Physical and Acclimation:
During the acclimation period 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-three per cage in stainless steel solid bottom cages with corncob bedding, in rooms designed to maintain adequate 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.

Housing:
After assignment, animals were housed two per cage in stainless steel cages. Cages had solid floors with corncob bedding, and nylon bones for enrichment. Cages contained a feed crock and a pressure activated lixit valve-type watering system. The following environmental conditions were maintained in the animal room.
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.
Temporary excursions from these ranges for temperature and humidity may have occurred on an infrequent basis, and photoperiod times changed due to study-related activities; all observed ranges were documented in the study file.

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 meal form. Feed and municipal water were provided ad libitum. 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. There were no contaminants in either the feed or water at levels that would have adversely impacted the results or interpretation of this study. Copies of these analyses are maintained in the study file.

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 determined that the proposed Activities were in full accordance with these Final Rules. The IACUC-approved Animal Care and Use Activity used for this study was Subchronic/Chronic Tox 01.

Administration / exposure

Route of administration:

oral: feed

Details on route of administration:

Route and Justification, Method of Administration, Frequency and Duration:
Oral exposure is a possible route of human 2-butoxyethyl benzoate exposure; therefore, oral administration of 2-butoxyethyl benzoate to rats via diet represented an appropriate means of exposure. Animals were fed treated diets seven days/week for at least 90 days.

Vehicle:

unchanged (no vehicle)

Details on oral exposure:

Dose Preparation:
Test material was administered as a constant fixed percent in the diet. Premixes were mixed periodically throughout the study based on stability data. The high-dose diet was prepared by mixing premix with control lab diet to obtain the desired percent of test material in the diet. The low-dose diet was prepared by serially diluting the high-dose level diet with control lab diet. The concentrations were not adjusted for purity.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analysis:
Dose Confirmation and Homogeneity:
Dose confirmation analyses of all dose levels, plus control and premix, was determined pre-exposure, near the middle, and end of the study. The homogeneity of the low-dose female and the high-dose male or 500 and 5000 ppm test materialfeed mixtures were determined concurrent with dose confirmation. The method
used for analyzing the test material in the diet was high performance liquid chromatography mass spectrometry in positive ionization mode (HPLC/ESIMS/ MS) (Ito and McFadden, 2014).

Stability:
A previously conducted stability study (Ito and McFadden, 2014) showed 2-butoxyethyl benzoate to be stable in diet for at least 65 days at concentration levels ranging from 0.0005 to 10%. The established concentration range and duration spanned those used in this study; therefore, additional stability analyses were not conducted.

Retainer Samples:
Retained samples (one/sex/dose/mix plus control and premix) were stored in sealed vials in a manner consistent with the sample retention policy of the laboratory.

Duration of treatment / exposure:

Animals were fed treated diets seven days/week for at least 90 days.

Frequency of treatment:

Animals were fed treated diets seven days/week for at least 90 days.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

500 ppm

Doses / concentrations 2

Dose / conc.:

1 500 ppm

Doses / concentrations 3

Dose / conc.:

5 000 ppm

No. of animals per sex per dose:

10/sex/dose group

Control animals:

yes, plain diet

Details on study design:

Dose Levels and Justification:
The high dose of 5000 ppm was expected to produce decreased feed consumption, some body weight depression and possible organ weight and hematology effects. The middose (1500 ppm) and low-dose level (500 ppm) were expected to provide dose response data for any treatment-related effects observed in the high-dose group. The low-dose was expected to be a no-observed-effect level (NOEL).

Examinations

Observations and examinations performed and frequency:

Daily Observations:
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. Cage-side observations, in which only positive findings were documented, are summarized with clinical observations.

Detailed Clinical Observations:
Detailed clinical observations (DCO) were conducted on all animals pre-exposure and once per week throughout the study. 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).

Ophthalmology:
The eyes of all animals were examined by a veterinarian pre-exposure and prior to the scheduled necropsy using indirect ophthalmoscopy. One drop of 0.5% tropicamide ophthalmic solution was instilled topically in each eye to produce mydriasis prior to the indirect ophthalmic examinations. Eyes were also examined by a prosecutor during the necropsy using a moistened glass slide pressed to the cornea.

Body Weights/Body Weight Gains:
All rats were weighed during the pre-exposure period and at least weekly during the dosing period. Body weight gains were calculated.

Feed Consumption:
Feed consumed was determined pre-exposure and at least twice weekly for all animals by weighing feed containers at the start and end of a measurement cycle. Feed consumption was calculated using the following equation:
Feed consumption (g/day) = (initial weight of feed container - final weight of feed container)/(# of days in measurement cycle) (# of animals per cage)

Test Material Intake:
The test material intake (TMI, expressed as mg/kg bw/day) was calculated upon completion of the study using test material concentrations in the feed, body weights and feed consumption data.

Toxicokinetics:
Blood sample collection and analysis
Urine sample collection and analysis

Clinical Pathology:
Animals were fasted overnight prior to blood collection. Blood samples were obtained from the orbital sinus following anesthesia via inhalation of O2/isoflurane at the scheduled necropsy.

Hematology:
Sample Preparation:
Blood samples for a complete blood count were mixed with ethylenediaminetetraacetic acid (EDTA). Blood smears were prepared, stained with Wright-Giemsa stain, cover-slipped and archived for potential future evaluation if warranted.
Hematologic parameters were assayed using the Advia 120 Hematology Analyzer (Siemens Healthcare Diagnostics, Tarrytown, New York).

Coagulation:
Sample Preparation:
Blood samples were collected in sodium citrate tubes, centrifuged, plasma collected, and assayed using the ACL9000 Analyzer (Instrumentation Laboratory, Bedford, Massachusetts).

Clinical Chemistry:
Sample Preparation:
Blood samples were collected and serum was separated from cells as soon as possible. Serum parameters were measured using a cobas c311 Clinical Chemistry Analyzer (Roche Diagnostics, Indianapolis, Indiana).

Urinalysis:
Urine samples were obtained from all animals the week prior to the scheduled necropsy. Animals were housed in metabolism cages and the urine collected overnight (approximately 16 hours). Feed and water was available during this procedure.

Sacrifice and pathology:

Anatomic Pathology:
Necropsy:
Fasted rats submitted alive for necropsy were anesthetized with a mixture of isoflurane vapors and medical grade oxygen. While under anesthesia, blood was collected from the orbital sinus. The animals were then placed in a CO2 chamber to continue anesthesia. Under a deep plane of anesthesia, 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 assisted by a team of trained individuals. 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 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. The nasal cavity was
flushed via the nasopharyngeal duct and the lungs were distended to an approximately normal inspiratory volume with neutral, phosphate-buffered 10% formalin using a hand-held syringe and blunt needle.
The brain, liver, kidneys, heart, adrenals, testes, epididymides, uterus, ovaries, thymus and spleen were trimmed and weighed immediately. The thyroid with the parathyroid gland(s) were trimmed and weighed following fixation and dissection from the trachea. The ratios of organ weight to terminal body weight were calculated.
Representative samples of tissues were collected and preserved in neutral, phosphate-buffered 10% formalin. Thymuses from animals 5383 (control) and 5392 (low dose) were inadvertently placed in the same jar at necropsy. Both thymuses were examined histopathologically and found to be within normal limits. Therefore, “within normal limits” was recorded separately for the thymus of each animal. Transponders were removed and placed in jars with the tissues.

Histopathology:
Sections from all preserved tissues were processed by standard histologic procedures from control and high-dose group animals and all animals that died or were sacrificed in a moribund condition. Paraffin embedded tissues were sectioned approximately six μm thick, stained with hematoxylin and eosin and examined by a veterinary pathologist using a light microscope. Relevant gross lesions were microscopically examined from all animals in the low- and intermediate dose groups.
Selected histopathologic findings were graded to reflect the severity of specific lesions to evaluate: 1) the contribution of a specific lesion to the health status of an animal, 2) exacerbation of common naturally occurring lesions as a result of the test material, and 3) dose-response relationships for treatment-related effects. Very slight and slight grades were used for conditions that were altered from the normal textbook appearance of an organ/tissue, but were of minimal severity and usually with less than 25% involvement of the parenchyma. This type of change was neither expected to significantly affect the function of the specific organ/tissue nor have a significant effect on the overall health of the animal. A moderate grade was used for conditions that were of sufficient severity and/or extent (up to 50% of the parenchyma) that the function of the organ/tissue was adversely affected, but not to the point of organ failure. The health status of the animal may or may not have been affected, depending on the organ/tissue involved, but generally lesions graded as moderate were not life threatening.

Statistics:

Means and standard deviations
Bartlett's test
Parametric (Steel and Torrie, 1960) or nonparametric (Hollander and Wolfe, 1973) analysis of variance (ANOVA)
Dunnett's test
Wilcoxon Rank-Sum test with a Bonferroni correction
Z-test of proportions
Statistical outliers were identified by a sequential test
Descriptive statistical (i.e., mean ± standard deviation) analyses were performed on the toxicokinetic data using Microsoft Excel
Squares regression analysis

See "Any other information on materials and methods incl. tables" for more detail on statistics.

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Description (incidence and severity):

There were no treatment-related clinical findings or scored DCO observations in males or females given 2-butoxyethyl benzoate at any dose level.

Mortality:

no mortality observed

Description (incidence):

All rodents survived the 90-day test period.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Females given 5000 ppm had a slight decrease in body weight gains (up to 12.2% less than controls) throughout the duration of the study, which corresponded to decreased feed consumption and was considered treatmen-trelated. There were no effects on body weights or body weight gains in males at any dose level or in females given 500 or 1500 ppm.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Females given 5000 ppm had treatment-related decreases in feed consumption which were statistically identified on days 8-18, 25-29, 32-39, 53-57, 60-71, and 89-93 (See attachment). There were no treatment-related effects on feed consumption in males at any dose level or in females given 500 or 1500 ppm. There were a few spurious statisitical findings in male feed consumption across groups that were considered unrelated to treamtent.
Male and female rats were given 0, 500, 1500, or 5000 ppm. These values correspond to time-weighted average doses of 0, 28.9, 88.1, or 285 mg/kg/day for males and 0, 32.6, 94.9, or 310 mg/kg/day for females, respectively.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

no effects observed

Description (incidence and severity):

Ophthalmologic examinations indicated all rats were within normal limits at pre-exposure and prior to study termination.

Haematological findings:

no effects observed

Description (incidence and severity):

There were no significant changes in any of the hematologic parameters for male and female rats. There were no treatment-related alterations in the prothrombin times of male and female rats.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Male rats given 5000 ppm had a very slight, statistically-identified decrement in sodium that was interpreted to be associated with treatment. The minimal decrease in sodium is not biologically significant and is therefore interpreted to be non-adverse.
Male rats given 5000 ppm had a slightly decreased total protein that was statistically identified. Decrements in total protein were interpreted to be unrelated to treatment as they were within recent historical control values for the laboratory, were not corroborated by histological findings, and occurred in isolation from associated clinical chemistry alterations.

Urinalysis findings:

no effects observed

Description (incidence and severity):

There were no treatment-related alterations in the urinalysis parameters for male and female rats.

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

There were no treatment-related alterations in organ weights of male and female rats.

Gross pathological findings:

no effects observed

Description (incidence and severity):

There were no treatment-related gross pathologic observations. All gross pathologic observations were considered to be spontaneous alterations, unassociated with exposure.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

There were no treatment-related histopathologic observations. All observations were considered to be spontaneous alterations unassociated with exposure.

Histopathological findings: neoplastic:

not examined

Other effects:

effects observed, treatment-related

Description (incidence and severity):

Toxicokinetics:
Chemical analysis results showed that no quantifiable 2-butoxyethyl benzoate and its expected metabolites (2- butoxyethanol and 2-butoxy acetic acid) were present in any of the control blood or urine samples.

Kinetics of 2-butoxyethyl benzoate in blood and urine samples:
2-Butoxyethyl benzoate was not quantifiable in blood samples from treated rats; therefore, no blood toxicokinetic analysis was performed for 2- butoxyethyl benzoate.
Conversely, 2-butoxyethyl benzoate was present and quantifiable in most treated urine samples of male and female rats except the low dose treated male rat group where 2- butoxyethyl benzoate was quantifiable in only one of six rats.

Kinetics of 2-butoxyethanol in blood and urine samples:
2-Butoxyethanol was present and quantifiable in most treated blood samples except the 500 ppm and 1500 ppm treated female blood samples where 2-butoxyethanol was not quantifiable in some of the blood samples.
Conversely, 2-butoxyethanol was present and quantifiable in all treated urine samples of male and female rats.

Kinetics of 2-butoxyacetic acid in blood and urine samples:
2-Butoxyacetic acid was present and quantifiable in all treated blood samples of male and female rats.
Similarly, 2-butoxyacetic acid was present and quantifiable in all treated urine samples of male and female rats.

Overall, the toxicokinetic results for 2-butoxyethyl benzoate and its expected metabolites (2-butoxyethanol and 2-butoxy acetic acid) in both male and female rats are dose proportional (linear) at all dose levels.

Effect levels

1

Key result

Dose descriptor:

NOEL

Effect level:

1 500 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

clinical biochemistry

food consumption and compound intake

2

Key result

Dose descriptor:

NOAEL

Effect level:

1 500 ppm

Based on:

test mat.

Sex:

female

Basis for effect level:

body weight and weight gain

food consumption and compound intake

3

Key result

Dose descriptor:

NOAEL

Effect level:

5 000 ppm

Based on:

test mat.

Sex:

male

Basis for effect level:

other: Male rats given 5000 ppm had a very slight, statistically-identified decrement in sodium that was interpreted to be associated with treatment. The minimal decrease in sodium is not biologically significant and is therefore interpreted to be non-adverse.

Target system / organ toxicity

Critical effects observed:

no

Any other information on results incl. tables

Dose Analysis:

The concentrations and homogeneity of 2-butoxyethyl benzoate in the diets were determined for the control and all treatment levels pre-exposure, near the middle, and end of the study. Analyses of all 2-butoxyethyl benzoate test diets and premixes indicated the mean concentration for each dose level ranged from 95.7 to 104.9% of targeted concentrations, indicating acceptable concentrations of 2-butoxyethyl benzoate.

The homogeneity of 2-butoxyethyl benzoate in diets was determined pre-exposure, near the middle, and at the end of the study for the 500 and 5000 ppm diets, the lowest and highest concentrations used in the study. The relative standard deviations for all diets sampled were between 0.9 and 3.9% indicating that all diets were homogeneously mixed.

Toxicokinetics:

Chemical analysis results showed that no quantifiable 2-butoxyethyl benzoate and its expected metabolites (2- butoxyethanol and 2-butoxy acetic acid) were present in any of the control blood or urine samples.

Kinetics of 2-butoxyethyl benzoate in blood and urine samples:

2-Butoxyethyl benzoate was not quantifiable in blood samples from treated rats; therefore, no blood toxicokinetic analysis was performed for 2-butoxyethyl benzoate.

Conversely, 2-butoxyethyl benzoate was present and quantifiable in most treated urine samples of male and female rats except the low dose treated male rat group where 2-butoxyethyl benzoate was quantifiable in only one of six rats (animal # 15A5350). The levels of 2-butoxyethyl benzoate in 24-hr urine samples from male and female rats accounted for equal to or less than 0.294% and 0.599%, respectively, of the administered dose (the daily intake of 2-butoxyethyl benzoate) from all treatment groups. Toxicokinetic analysis showed that 2-butoxyethyl benzoate levels in urine were dose-proportional (linear) in females (at all dose levels) and in males (only at middle and high dose levels due to the lack of enough quantifiable urine levels in male rats treated with low dose of 2-butoxyethyl benzoate). There was large variability in urine levels of 2-butoxyethyl benzoate within the dose groups (i.e., in 4 of 5 cases, standard deviations were greater than or approximately equal to group means).

It is unusual to have detectable levels of 2-butoxyethyl benzoate in urine without having quantifiable levels in blood when animals have reached steady state. The most likely explanations are the following:

1) Loss of 2-butoxyethyl benzoate in blood due to degradation or poor recovery. This explanation is unlikely because the stability and recovery of 2-butoxyethyl benzoate in blood was verified in a separate stability study, which included internal standard/spikes to verify 2-butoxyethyl benzoate stability in blood under the collection and storage conditions used in this study (Perala, 2016).

2) Enhanced detection of 2-butoxyethyl benzoate in urine. This explanation is unlikely because the lower limit of quantitation was less for 2-butoxyethyl benzoate in blood (~0.03 μg/g) than in urine (~0.25 μg/g).

3) Contamination of urine samples with test diet containing 2-butoxyethyl benzoate. This explanation seems most likely based on exclusion of the above hypotheses as well as the high variability of the urine samples.

Kinetics of 2-butoxyethanol in blood and urine samples:

2-Butoxyethanol was present and quantifiable in most treated blood samples except the 500 ppm and 1500 ppm treated female blood samples where 2-butoxyethanol was not quantifiable in some of the blood samples. Toxicokinetic analysis showed that blood AUC24 values of 2-butoxyethanol were dose-proportional (linear) in male rats (at all dose levels) but linearity could not be determined in the female rats due to the lack of enough quantifiable blood levels of 2-butoxyethanol for calculating blood AUC24h in female rats treated with low- and mid-dose of 2-butoxyethyl benzoate). Conversely, 2-butoxyethanol was present and quantifiable in all treated urine samples of male and female rats. The 24-hr urinary elimination of 2-butoxyethanol in male and female rats accounted for equal to or less than 0.333% and 0.412%, respectively, of the administered dose (the daily intake of 2-butoxyethyl benzoate) from all treatment groups. Toxicokinetic analysis showed that 2-butoxyethanol levels in urine were dose-proportional (linear) in both male and female rats at all dose levels.

Kinetics of 2-butoxyacetic acid in blood and urine samples:

2-Butoxyacetic acid was present and quantifiable in all treated blood samples of male and female rats. Overall, the averaged AUC24hr value in female rats was higher than that from male rats at the same dose level. Toxicokinetic analysis showed that blood AUC24hr values of 2-butoxyacetic acid were dose-proportional (linear) in both male and female rats at all dose levels. Similarly, 2-butoxyacetic acid was present and quantifiable in all treated urine samples of male and female rats. The 24-hr urinary elimination of 2-butoxyacetic acid in male and female rats accounted for 57.9% and 77.5%, respectively, of the administered dose (the daily intake of 2-butoxyethyl benzoate) from all treatment groups, which may be related to timing of sample collection relative to dietary intake. Overall, the percent of administered dose for 2-butoxyacetic acid in 24-hr urine samples from female rats was higher than that from male rats. Toxicokinetic analysis showed that 2-butoxyacetic acid levels in urine were also dose-proportional (linear) in both male and female rats at all dose levels.

Overall, the toxicokinetic results for 2-butoxyethyl benzoate and its expected metabolites (2-butoxyethanol and 2-butoxy acetic acid) in both male and female rats are dose proportional (linear) at all dose levels.

Applicant's summary and conclusion

Conclusions:

Dietary administration of 2-butoxyethyl benzoate to Crl:CD(SD) rats resulted in treatmentrelated decreases in female body weight gains and feed consumption only at the high dose level (5000 ppm). No treatment-related differences in body weights/body weight gains or feed consumption were observed for females at 500 or 1500 ppm or for males at any dose level throughout the duration of the study.
There were no treatment-related effects in clinical signs, ophthalmic, hematology, prothrombin time, or urinalysis parameters. There were no treatment related organ weight effects, gross or histopathologic observations. Male rats given 5000 ppm had a very slight and statistically-identified decrement in sodium that was interpreted to be associated with treatment but was considered non-adverse.
Chemical analysis results showed that no quantifiable 2-butoxyethyl benzoate or its expected metabolites (2-butoxyethanol and 2-butoxyacetic acid) were present in any of the control blood or urine samples.

Blood kinetics:
2-Butoxyethyl benzoate was not quantifiable in any of the treated blood samples. 2-butoxyethanol was quantifiable in most treated blood samples except in the female samples where twelve of eighteen low-dose and four of eighteen mid-dose blood samples had non-quantifiable 2-butoxethanol; 2-butoxyacetic acid was present and quantifiable in all treated blood samples. Due to the fact that 2-butoxyethyl benzoate was not quantifiable in any of the treated blood samples, toxicokinetic evaluations for the blood samples were only conducted for the expected metabolites (2-butoxyethanol and 2-butoxyacetic acid) and results showed that 2-butoxyacetic acid was dose-proportional (linear) across all dose levels in both male and female rats and 2-butoxyethanol was doseproportional
(linear) across all dose levels only in male rats. The number of nonquantifiable 2-butoxyethanol samples in the female precluded toxicokinetic analysis.

Urine kinetics:
2-Butoxyethyl benzoate was quantifiable in most of the treated urine samples except in the male low dose-treated urine samples where 2-butoxyethyl benzoate was quantifiable in one of the six samples. The levels of 2-butoxyethyl benzoate in 24-hr urine samples from male and female rats accounted for equal to or less than 0.294% and 0.599%, respectively, of the administered dose (the daily intake of 2-butoxyethyl benzoate) from all treatment groups. As 2-butoxyethyl benzoate was not quantifiable in any of the treated blood samples, the presence of 2-butoxyethyl benzoate in urine samples was possibly attributed to the contamination of urine samples with 2-butoxyethyl benzoate test diet. 2-butoxyethanol and 2-butoxyacetic acid were quantifiable in all treated urine samples.
Toxicokinetic evaluations showed that 2-butoxyethyl benzoate, 2-butoxyethanol, and 2-butoxyacetic acid were all dose-proportional (linear) across all dose levels in both male and female rats.

The no-observed-effect level (NOEL) for Crl:CD(SD) rats of either sex was 1500 ppm 2-butoxyethyl benzoate based on decreases in body weight gain and feed consumption in 5000 ppm females and decrements in serum sodium levels in 5000 ppm males. The noobserved-adverse effect level (NOAEL) was 1500 ppm in females and 5000 ppm in males.

Executive summary:

Ten male and ten female Crl:CD(SD) rats per group were given test diets formulated to supply 0, 500, 1500, or 5000 ppm 2-butoxyethyl benzoate for at least 90 days. These values correspond to time-weighted average doses of 0, 28.9, 88.1, or 285 mg/kg/day for males and 0, 32.6, 94.9, or 310 mg/kg/day for females, respectively. Parameters evaluated were daily cage-side observations, weekly detailed clinical observations, ophthalmic examinations, body weights/body weight gains, feed consumption, hematology, prothrombin time, clinical chemistry, urinalysis, selected organ weights, and gross and histopathologic examinations.

Dietary administration of 2-butoxyethyl benzoate to Crl:CD(SD) rats resulted in treatmentrelated decreases in female body weight gains and feed consumption only at the high dose level (5000 ppm). No treatment-related differences in body weights/body weight gains or feed consumption were observed for females at 500 or 1500 ppm or for males at any dose level throughout the duration of the study.

There were no treatment-related effects in clinical signs, ophthalmic, hematology, prothrombin time, or urinalysis parameters. There were no treatment related organ weight effects, gross or histopathologic observations. Male rats given 5000 ppm had a very slight and statistically-identified decrement in sodium that was interpreted to be associated with treatment but was considered non-adverse.

Chemical analysis results showed that no quantifiable 2-butoxyethyl benzoate or its expected metabolites (2-butoxyethanol and 2-butoxyacetic acid) were present in any of the control blood or urine samples.

Blood kinetics:

2-Butoxyethyl benzoate was not quantifiable in any of the treated blood samples. 2-butoxyethanol was quantifiable in most treated blood samples except in the female samples where twelve of eighteen low-dose and four of eighteen mid-dose blood samples had non-quantifiable 2-butoxethanol; 2-butoxyacetic acid was present and quantifiable in all treated blood samples. Due to the fact that 2-butoxyethyl benzoate was not quantifiable in any of the treated blood samples, toxicokinetic evaluations for the blood samples were only conducted for the expected metabolites (2-butoxyethanol and 2-butoxyacetic acid) and results showed that 2-butoxyacetic acid was dose-proportional (linear) across all dose levels in both male and female rats and 2-butoxyethanol was doseproportional (linear) across all dose levels only in male rats. The number of nonquantifiable 2-butoxyethanol samples in the female precluded toxicokinetic analysis.

Urine kinetics:

2-Butoxyethyl benzoate was quantifiable in most of the treated urine samples except in the male low dose-treated urine samples where 2-butoxyethyl benzoate was quantifiable in one of the six samples. The levels of 2-butoxyethyl benzoate in 24-hr urine samples from male and female rats accounted for equal to or less than 0.294% and 0.599%, respectively, of the administered dose (the daily intake of 2-butoxyethyl benzoate) from all treatment groups. As 2-butoxyethyl benzoate was not quantifiable in any of the treated blood samples, the presence of 2-butoxyethyl benzoate in urine samples was possibly attributed to the contamination of urine samples with 2-butoxyethyl benzoate test diet. 2-butoxyethanol and 2-butoxyacetic acid were quantifiable in all treated urine samples.

Toxicokinetic evaluations showed that 2-butoxyethyl benzoate, 2-butoxyethanol, and 2- butoxyacetic acid were all dose-proportional (linear) across all dose levels in both male and female rats.

The no-observed-effect level (NOEL) for Crl:CD(SD) rats of either sex was 1500 ppm 2-butoxyethyl benzoate based on decreases in body weight gain and feed consumption in 5000 ppm females and decrements in serum sodium levels in 5000 ppm males. The no observed-adverse effect level (NOAEL) was 1500 ppm in females and 5000 ppm in males.