2-butoxyethyl benzoate

Endpoint summary

Description of key information

There is a key GLP-study according to OECD guideline 408 and a supporting GLP-study according to OECD guideline 422 available for 2-butoxyethyl benzoate.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Reference 1

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

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

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

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

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: 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.

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.

Dose / conc.:

500 ppm

Dose / conc.:

1 500 ppm

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).

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.

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.

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

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

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.

Critical effects observed:

no

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.

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.

Reference 2

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

July 23, 2015 to February 24, 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: USEPA OPPTS 870.3650

Deviations:

no

GLP compliance:

yes

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: 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, availability of historical control 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, availability of historical control data and the reliability of the commercial supplier.
Supplier and Location: Charles River (Raleigh, North Carolina)
Age at Study Start: Approximately eight weeks of age at initiation of treatment.

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 was fully accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC International). Prior to randomization, the animals were housed two per cage in stainless steel solid bottom cages with corncob bedding, in rooms designed to maintain adequate conditions (temperature, humidity, and photocycle). Animals were acclimated to the laboratory for at least one week prior to the start of the study.

Housing:
After assignment to the study, animals were single housed in solid bottom stainless steel cages containing ground corn cob bedding, except during breeding and during the gestation and littering phases of the study. During breeding, one male and one female were placed in stainless steel cages with wire mesh floors that were suspended above catch pans in order to better visualize copulation and plugs. During gestation and littering, dams (and their litters) were housed in plastic cages provided with ground corn cob bedding from approximately GD 0 until LD 4. 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.)

Enrichment:
Enrichment for rats included the use of ground corn cob bedding and open areas on the cage sides for visualization of other rats. In addition, the cages may have contained nylon bones or paper nesting material.

Randomization and Identification:
Prior to test material administration, 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 that were placed on study were uniquely identified via subcutaneously implanted transponders (BioMedic Data Systems, Seaford, Delaware) that were correlated to unique alphanumeric identification numbers (Text Table 2). If a transponder stopped functioning or was lost, it was replaced with a new transponder that was correlated with the unique animal number.

Feed and Water:
Feed and municipal water were provided ad libitum. Animals were provided LabDiet Certified Rodent Diet #5002 (PMI Nutrition International, St. Louis, Missouri) in meal 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 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 Activities used for this study were DART 01, DCO 01, Neuro Tox 01, Humane Endpoints 01, Blood Collection 01, Tissue Collection 01 and Animal ID 01.

Route of administration:

oral: feed

Details on route of administration:

Oral was the preferred route of exposure according to OECD Guideline 422. Exposure by diet was selected in the event that a positive finding warranted a subsequent twogeneration reproductive toxicity study that would be conducted using the dietary route.
Females were dosed by dietary exposure for 14 days prior to breeding, and continuing through breeding (up to two weeks), gestation (three weeks), and lactation (four days). Males were dosed via the diet for 14 days prior to mating and continuing through breeding (up to two weeks) until test day 35.

Vehicle:

unchanged (no vehicle)

Details on oral exposure:

Dose Preparation:
Diets were prepared by serially diluting a concentrated test material-feed mixture (premix) with ground feed. Premixes were prepared periodically throughout the study based on stability data. Diets were prepared as a fixed percent of test material in rodent feed. The concentrations of the diets were not adjusted for purity.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analysis:
Concentration Verification and Homogeneity:
Dose confirmation analyses of all dose levels, plus control and premix, were determined pre-exposure. The homogeneity of the low-dose and the high-dose test diets were determined concurrent with dose confirmation. Analysis was performed by high performance liquid chromatography mass spectrometry in positive ionization mode (HPLC/ESI-MS/MS) (Ito and McFadden, 2014).

Stability:
A previously conducted toxicity study (Ito and McFadden, 2014) showed 2-butoxyethyl benzoate to be stable in rodent 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/dose/mix) were stored in sealed vials in a manner consistent with the sample retention policy of the laboratory.

Duration of treatment / exposure:

Groups of 12 male and 12 female Crl:CD(SD) rats were fed diets supplying concentrations of 0, 500, 1500, or 5000 ppm. The females were dosed daily for approximately two weeks prior to breeding, continuing through breeding (up to two weeks), gestation (three weeks), and through postpartum day 4. The males were dosed beginning approximately two weeks prior to breeding and continuing through breeding (up to two weeks) until test day 35.

Frequency of treatment:

Rats were fed diets supplying the assigned concentrations seven days/week for the duration of the exposure period.

Dose / conc.:

500 ppm

Dose / conc.:

1 500 ppm

Dose / conc.:

5 000 ppm

No. of animals per sex per dose:

12/sex/dose group

Control animals:

yes, plain diet

Details on study design:

Route, Method of Administration, Frequency, Duration and Justification:
Oral was the preferred route of exposure according to OECD Guideline 422. Exposure by diet was selected in the event that a positive finding warranted a subsequent twogeneration reproductive toxicity study that would be conducted using the dietary route. Females were dosed by dietary exposure for 14 days prior to breeding, and continuing through breeding (up to two weeks), gestation (three weeks), and lactation (four days). Males were dosed via the diet for 14 days prior to mating and continuing through breeding (up to two weeks) until test day 35.

Dose Levels and Justification:
The high-dose level of 5000 ppm was expected to induce some toxic effects but not death or obvious suffering. In the developmental toxicity probe study, exposure of time-mated female Crl:CD(SD) rats to 5000 ppm 2-butoxyethyl benzoate produced treatment-related hematological toxicity accompanied by increased absolute and relative spleen weights. The hematological effects in females at 3500 ppm were equivocal in the range-finding study and minimal in the developmental toxicity probe study, and were not accompanied by increased spleen weights. Therefore, the 5000 ppm dose level was expected to induce toxicity manifest as alterations in hematological parameters and/or increased spleen weight. The lower dose levels were selected to provide dose response data for any toxicity that may have been observed among the high-dose group rats and to establish a no-observable-effect-level (NOEL).

Observations and examinations performed and frequency:

Daily In-Life Observations:
A cage-side examination was conducted at least twice daily. 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 could be observed included, 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 examinations were also conducted on dams and their litters, at least twice daily. These examinations were conducted as described above.

Clinical Observations:
Clinical observations were conducted on all animals pre-exposure. Females were observed for signs of parturition beginning on or about gestation day (GD) 20 (see Litter Data). Females that delivered litters were subsequently evaluated on lactation day (LD) 0, 1, and 4. Clinical observations were not conducted on females that failed to deliver a litter, unless deemed appropriate based on cage-side observations. Clinical observations included a careful, hand-held examination of the animal with an evaluation of abnormalities in the eyes, urine, feces, gastrointestinal tract, extremities, movement, posture, reproductive system, respiration, skin/hair-coat, and mucous membranes, as well as an assessment of general behavior, injuries or palpable mass/swellings.

Detailed Clinical Observations:
Detailed clinical observations (DCO) were conducted on all animals pre-exposure and once per week throughout the study. Mated females received DCO examinations on GD 0, 7, 14, and 20, and LD 3. For females that failed to mate or deliver a litter a DCO was conducted at least weekly. In contrast to the daily cageside and clinical observations which were designed to detect acute changes, the DCO was designed to detect more persistent neurological changes and, therefore, was conducted at approximately the same time each examination day, according to an established format. The examination included cage-side, hand-held and open-field observations, which were recorded categorically or using explicitly defined scales (ranks).

Functional Tests:
The functional tests (sensory evaluation, rectal temperature, grip performance and motor activity) were conducted pre-exposure and during the last week of the treatment period. For the females, this took place on LD 4. Females that failed to deliver did not undergo functional testing during the last week of treatment.

Body Weights:
All rats were weighed pre-exposure, twice during the first week of study and once during the second week. Male body weights continued to be recorded weekly throughout the study. Females were weighed weekly during the breeding period. During gestation, females were weighed on gestation days (GD) 0, 7, 14, and 20. Females that delivered litters were weighed on lactation days (LD) 1 and 4. Females that failed to mate or deliver a litter were not weighed during the gestation or lactation phases. Body weights were determined for the following intervals: GD 0, 7, 14, 20 and LD 1 and 4. Body weight gains were determined for the following intervals: GD 0-7, 7-14, 14-20, 0-20, and LD 1-4.

Feed Consumption:
For males and females, feed consumption was determined twice during the first week by weighing feed crocks at the start and end of a measurement cycle. Thereafter, feed crocks were measured weekly during the pre-breeding phase. During breeding, feed consumption was not measured in males or females due to co-housing. Following breeding, feed consumption for males was not measured. For mated females, feed consumption was measured on GD 0, 7, 14, and 20. After parturition, feed consumption was measured on LD 1 and 4. Feed consumption was not recorded for females that failed to mate or deliver a litter. Feed consumption was calculated using the following equation:
Feed consumption (g/day) = (initial weight of crock - final weight of crock)/(# of days in measurement cycle)

Test Material Intake:
Test material intake (TMI) was calculated for the adults using test material concentrations in the feed, actual body weights, and measured feed consumption data.

Toxicokinetics:
Kinetic Analysis of Blood – Pre-mating Phase:
Blood samples were collected from the first four non-fasted rats/sex near the end of the pre-breeding phase (TD 14) via tail nick. Analyses of the blood samples were conducted to quantify levels of parent compound (2-butoxyethyl benzoate) and expected major metabolites (2-butoxyethanol and 2-butoxyacetic acid).
Sample Analysis:
The blood was analyzed using liquid chromatography with tandem mass spectrometry detection (LC/MS-MS) and gas chromatography with tandem mass spectrometry detection (GC/MS-MS) to evaluate dose proportionality and systemic exposure.

Clinical Pathology:
Animals were fasted overnight prior to blood collection. Blood samples were obtained from the orbital sinus following anesthesia with a mixture of isoflurane vapors and medical oxygen at the scheduled necropsy. Blood samples were not obtained from females that failed to deliver a litter.
The following analysis were performed: Hematology, Coagulation, Clinical Chemistry, Urinalysis

Sacrifice and pathology:

Anatomic Pathology:
Adult Necropsy:
Adult males (fasted) were submitted for necropsy after 35 days of exposure. Adult females (fasted) were terminated on LD 5, or at least 24 days after the end of the breeding period for females not producing a litter. On the morning of the scheduled necropsy, fasted rats were weighed in the animal room and submitted alive for necropsy. The animals were anesthetized with a mixture of isoflurane vapors and medical oxygen. While under anesthesia, blood was collected from the orbital sinus (all males and all females that littered). The animals were 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 or a technician qualified to recognize lesions, 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 uteri of all females were stained with an aqueous solution of 10% sodium sulfide stain based on Kopf et al., 1964 for approximately one minute and were examined for the presence and number of implantation sites. After evaluation, uteri were gently rinsed with saline and preserved in neutral phosphate-buffered 10% formalin.
Weights of the adrenals, brain, epididymides, heart, kidneys, liver, spleen, testes, thymus, and thyroid with parathyroids (weighed after fixation) were recorded, and organ:body weight ratios calculated.
Representative samples of tissues listed in in the attachment "Repro-Dev Histopath Table 3" were collected and preserved in neutral, phosphate-buffered 10% formalin, with the exception of the testes and epididymides that were fixed in Bouin’s fixative. Transponders were removed and placed in jars with the tissues.

Histopathology:
The number of sections from all preserved tissues listed in in the attachment "Repro-Dev Histopath Table 3" were processed by standard histologic procedures from control and high-dose group animals. 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, and all potential target tissues (spleen and liver of females) were microscopically examined from all animals in the low- and intermediate dose groups. The parathyroid gland from animal 3542 (5000 ppm group) was not present on the slide(s) submitted and was therefore not examined histologically. Also, one optic nerve of the pair from animal 3596 (5000 ppm group) was missing, therefore only one optic nerve was examined histologically. There were sufficient tissues from the remaining animals in these groups for diagnosis, indicating
there was no negative impact on study.
The histopathological examination of the testes included a qualitative assessment of stages of spermatogenesis. A cross section through the approximate center of both testes of control and high-dose males was embedded in paraffin, sectioned at 5 μm and stained with modified periodic acid-Schiffs-hematoxylin. The presence and integrity of the stages of spermatogenesis was qualitatively evaluated following the criteria and guidance of Russell et al. (1990). Microscopic evaluation included a qualitative assessment of the relationships between spermatogonia, spermatocytes, spermatids, and spermatozoa seen in cross sections of the seminiferous tubules. The progression of these cellular associations defined the cycle of spermatogenesis. In addition, sections of both testes were examined for the presence of degenerative changes (e.g., vacuolation of the germinal epithelium, a preponderance of Sertoli cells, sperm stasis, inflammatory changes, mineralization, and fibrosis).
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. A severe grade was used for conditions that were extensive enough to cause significant organ/tissue dysfunction or failure. This degree of change in a critical organ/tissue could have been life threatening.

Off-Spring Necropsy:
All pups surviving to LD 4 were euthanized by an oral dose of sodium pentobarbital solution, followed by decapitation. They were examined for gross external alterations and discarded. Any pups found dead or which were euthanized in moribund condition were examined to the extent possible and discarded.

Statistics:

The following means of statistical analysis were employed:
Descriptive statistical (i.e., mean ± standard deviation) analyses
AUC24h calculations were performed via the trapezoidal rule
A least squares regression analysis
Bartlett's test (alpha = 0.01; Winer, 1971) for equality of variances
Parametric (Steel and Torrie, 1960) or nonparametric (Hollander and Wolfe, 1973) analysis of variance (ANOVA)
Dunnett's test (alpha = 0.05; Winer, 1971) or the Wilcoxon Rank-Sum (alpha = 0.05; Hollander and Wolfe, 1973) test with Bonferroni's correction (Miller, 1966)
Nonparametric ANOVA
Wilcoxon Rank-Sum test with Bonferroni's correction
Sequential method of Grubbs (1969)
Fisher exact probability test (alpha = 0.05; Siegel, 1956) with Bonferroni's correction
Binomial distribution test (alpha = 0.05; Steel and Torrie, 1960)
Censored Wilcoxon test (alpha = 0.05; Hollander and Wolfe, 1973) as modified by Haseman and Hoel (1974) with Bonferroni’s correction
Z-test of proportions
Analysis of covariance (ANCOVA)
Pillai Trace statistic

Clinical signs:

no effects observed

Description (incidence and severity):

In-Life Observations:
No treatment-related effects on behavior or demeanor were observed at any dose level during the study. Observations recorded in the dose groups occurred at low frequency and bore no relationship to treatment. There were no notable observations made during the cage-side observations.

Detailed Clinical Observations:
Examinations performed on all animals prior to the study revealed that all animals were in good health for study purposes. Examinations performed on all animals weekly throughout the study revealed no treatment-related findings.

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

When compared to controls, females in the 5000 ppm group had treatment-related decreases in body weights on test days 4, 8, and 15 (3.7-6.8%) during prebreeding, as well as GD 0, 7, 14, and 20 (3.6-7.8%), and LD 1 and 4 (7.6-8.4%) ( See Attachment Body Weight and Food Consumption-Text Table 3 and 4). These decreases were statistically identified on TD 8 and 15, GD 0 and 7, and LD 1 and 4. No significant differences in body weights were observed for females at 500 or 1500 ppm, or males at any exposure level, throughout the duration of the study. No significant differences in body weight gains were observed for females at any exposure level tested throughout gestation or lactation.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Feed Consumption:
When compared to controls, females in the 5000 ppm group had treatment-related decreases in feed consumption during the intervals of TD 1-4, 4-8 and 8-15 (9.5-12.4%) during prebreeding, and LD 1-4 (9.2%) (See Attachment Body Weight and Food Consumption-Text Table 3 and 4). These decreases were statistically identified on TD 1-4, 4-8 and 8-15. No significant differences in feed consumption were observed for females at any exposure level tested throughout gestation. No significant differences in feed consumption were observed for females at 500 or 1500 ppm or males at any dose level throughout the duration of the study.

Test Material Intake:
For the 0, 500, 1500, and 5000 ppm groups, respective time-weighted average dose levels of 2-butoxyethyl benzoate during the pre-breeding phase were 0, 39.2, 114, or 379 mg/kg/day for males and 0, 38.7, 117, or 377 mg/kg/day for females. During the gestation and lactation phases, respective time-weighted average dose levels of 2-butoxyethyl benzoate for females were 0, 36.5, 109, or 381 mg/kg/day and 0, 56.0, 170, or 565 mg/kg/day.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Hematology:
Treatment-related hematologic effects in females given 5000 ppm consisted of statistically identified lower mean red blood cell count and hemoglobin concentration, higher mean MCV and MCH, lower mean MCHC, and a statistically identified higher mean reticulocyte count (See Attachment Hematology-Text Table 5). These hematologic effects were representative of regenerative anemia in females given 5000 ppm, and were interpreted to be adverse. Females given 5000 ppm also had a treatment-related higher platelet count, which may have been caused by a generalized increase in platelet production within the bone marrow in association with the reticulocytosis. There were no treatment-related hematologic effects in females given 500 or 1500 ppm, or in males from any exposure level. Males given 500 ppm had a statistically significant lower mean red blood cell count that was interpreted to be unrelated to treatment because of the lack of a dose response.

Coagulation:
There were no treatment-related changes in prothrombin times for males and females at any exposure level.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Clinical Chemistry:
Treatment-related clinical chemistry effects in females given 5000 ppm consisted of statistically-identified higher mean urea nitrogen, triglyceride, creatinine and phosphorus concentrations (See Attachment Clinical Chemistry-Text Table 6). The statistically-identified higher mean urea nitrogen and triglyceride concentrations were above the historical control ranges from studies recently conducted at this laboratory. The higher creatinine and phosphorus concentrations were within the recent historical control ranges. However, when the individual animal creatinine and phosphorus concentrations are compared to the concurrent individual animal control group data, there is clearly a trend of minimally higher values for these parameters in the female 5000 ppm group. Therefore, higher phosphorus and creatinine concentrations were interpreted to be treatment-related effects. All of the treatment-related elevations in clinical chemistry parameters were interpreted to be non-adverse, because there were no corresponding alterations in organ weights, and no histopathologic correlates. Females given 5000 ppm had a statistically-identified higher mean sodium concentration that was interpreted to be unrelated to treatment because the value was of minimal difference from the concurrent control group, was within the historical control range, and did not demonstrate a clear dose response. There were no treatment-related clinical chemistry effects in females given 500 or 1500 ppm, or in males from any exposure level. Males given 5000 ppm had a statistically-identified higher mean urea nitrogen concentration. Males given 500 ppm had a statistically-identified lower mean aspartate aminotransferase activity. Males given 1500 ppm had a statisticallyidentified lower mean calcium concentration. The statistically-identified clinical chemistry alterations in males were interpreted to be unrelated to treatment because of the lack of a clear dose response and/or the values were within historical control ranges.

Urinalysis findings:

no effects observed

Description (incidence and severity):

There were no treatment-related changes in urinalysis parameters for males at any exposure level.

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Females given 5000 ppm had a treatment-related lower mean final body weight (6.1%), relative to controls. Females given 5000 ppm had a treatment-related higher mean relative liver weight (5.1%), relative to controls (See Attachment Final Body Weights and Organ Weight Differences-Text table 7). The higher relative liver weight corresponded to the histopathologic observation of very slight hypertrophy of centrilobular/midzonal hepatocytes, with increased cytoplasmic eosinophilia, in females given 5000 ppm. The lower mean final body weight and higher mean relative liver weight in females given 5000 ppm were interpreted to be non-adverse. There were no treatment-related alterations in final body weights or organ weights in females given 500 or 1500 ppm, or in males from any exposure level.

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 dietary administration of 2-butoxyethyl benzoate.

Neuropathological findings:

no effects observed

Description (incidence and severity):

Functional Tests:
Sensory Evaluation:
Examinations performed on males and females revealed no treatment-related findings.

Rectal Temperature:
There were no treatment-related effects on rectal temperature in males (p = 0.4887) or females (p = 0.3758).

Grip Performance:
There were no treatment-related effects on hindlimb grip performance either in males (p = 0.7096) or females (p = 0.4400). Similarly, there were no treatment-related effects on forelimb grip performance either in males (p = 0.1340) or females (p = 0.8978).

Motor Activity:
There were no treatment-related effects on motor activity. Treatment did not affect motor activity total counts (treatment-by-time interaction) either in males (p = 0.7571) or in females (p = 0.7004). Similarly, the distribution of the motor activity counts within session (treatment x time x interval interaction) was not affected by treatment either in males (p = 0.1290) or in females (p = 0.7891).

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Treatment-related very slight hypertrophy of centribolublar/midzonal hepatocytes, with increased cytoplasmic eosinophilia, was present in the liver of 11/12 females given 5000 ppm. The hepatocellular hypertrophy was interpreted to be a non-adverse and adaptive effect, based on the modest corresponding increase in liver weights, along with the absence of any treatment-related changes in liver enzyme activities (ALT, AST and GGT), and the absence of necrosis, increased apoptosis, inflammation, proliferative or degenerative changes in the liver of females at this dose level.

All females that delivered a litter from the control, 500 ppm, 1000 ppm and 5000 ppm groups had very slight, slight or moderate erythrocytic extramedullary hematopoiesis of the spleen. The incidence of the various severities of this alteration did not follow a dose-responsive progression. The erythrocytic extramedullary hematopoiesis of the spleen was interpreted to be reflective of hemorrhage associated with parturition, and not a treatment-related effect. All other histopathologic observations were considered to be spontaneous alterations, unassociated with dietary administration of 2-butoxyethyl benzoate.

Histopathological findings: neoplastic:

not examined

Other effects:

effects observed, treatment-related

Description (incidence and severity):

Toxicokinetics:
Overall, the results showed that 2-butoxyethyl benzoate was hydrolyzed to 2-butoxyethanol after dietary intake in both male and female rats. The resulting hydrolyzed product 2-butoxyethanol was further metabolized to 2-butoxyacetic acid. The hydrolysis of the parent compound was efficient, with less than 1% of the sum of the two metabolites found as parent compound in the high dose blood samples (based on comparison of AUC values of group mean blood levels). Toxicokinetic analysis showed that the blood levels of 2-butoxyethanol in both male and female rats were linear across all dose levels. However, unlike male rats, the blood levels of 2-butoxyacetic acid in female rats exhibited supralinear kinetics at the high dose (5000 ppm).

Dose descriptor:

NOEL

Effect level:

1 500 ppm

Based on:

test mat.

Sex:

female

Basis for effect level:

body weight and weight gain

clinical biochemistry

food consumption and compound intake

haematology

histopathology: non-neoplastic

organ weights and organ / body weight ratios

Remarks on result:

other: General Toxicity Results

Dose descriptor:

NOEL

Effect level:

5 000 ppm

Based on:

test mat.

Sex:

male

Basis for effect level:

other: There were no treatment-related effects in males at any dose level.

Remarks on result:

other: General Toxicity Results

Dose descriptor:

NOEL

Effect level:

5 000 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No reproductive or neurological effects observed in males or females at any dose tested.

Remarks on result:

other: Reproductive and Neurological effects

Critical effects observed:

yes

Lowest effective dose / conc.:

5 000 ppm

System:

haematopoietic

Organ:

blood

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

Reproductive Indices, Pup Survival, and Sex Ratio:

There were no treatment-related effects at any dose level on mating, conception, fertility, gestation indices, time to mating, gestation length, post-implantation loss, pup survival or pup sex ratio.

Litter Observations:

Observations recorded in the offspring occurred at low frequency and bore no relationship to treatment. There were no visible external morphologic alterations noted in any of the offspring.

Litter Size and Pup Body Weights:

There were no treatment-related effects on litter size or pup body weights at any dose level tested.

Analytical Chemistry:

Data on test material concentration and homogeneity are represented in Appendix D. Analyses of all test diets from the first mix of the main study revealed acceptable mean concentrations ranging from 88.7 to 103.4% of targeted concentrations. Analyses of the low-dose female and high-dose male diet indicated that the test material was homogeneously distributed based on relative standard deviations of ≤ 3.9%.

Conclusions:

Based on the results, the no-observed-effect level (NOEL) for general toxicity was 1500 ppm in females and 5000 ppm in males. The NOEL for reproductive and neurological effects in both males and females was 5000 ppm, the highest dose level tested.

Executive summary:

The purpose of this study was to evaluate the potential effects of 2-butoxyethyl benzoate following rat dietary administration on general toxicity, neurological and reproductive function, and prenatal/early neonatal growth and offspring survival. This study evaluated 2-butoxyethyl benzoate in the OECD 422 design. Groups of 12 male and 12 female Crl:CD(SD) rats were administered 2-butoxyethyl benzoate via the diet at concentrations supplying 0, 500, 1500, and 5000 ppm. Females were dosed daily for two weeks prior to breeding, through breeding (up to two weeks), gestation (three weeks), and through postpartum day 4. Females were necropsied on post-partum day 5. The males were dosed for two weeks prior to breeding, through breeding and until test day 35. Effects on reproductive and neurological function as well as general toxicity were evaluated. In addition, post-mortem examinations included a gross necropsy of the adults with collection of organ weights and extensive histopathologic examination of tissues. Litter size, pup survival, sex, body weight, and the presence of gross external abnormalities were also assessed.

Dietary administration of 2-butoxyethyl benzoate to Crl:CD(SD) rats resulted in treatment-related decreases in female body weight only at the high dose level (5000 ppm). At the 5000 ppm dose level, treatment-related decreases in female body weights were observed on test days 4, 8 and 15 during prebreeding, as well as gestation days 0, 7, 14, and 20, and lactation days 1 and 4. These decreases were statistically identified on TD 8 and 15, GD 0 and 7, and LD 1 and 4. No treatmentrelated differences in body weight gains were observed for females at any dose level tested throughout gestation or lactation. No treatment-related differences in body weights or body weight gains were observed for females at 500 or 1500 ppm or for males at any dose level throughout the duration of the study.

Similar to body weight effects, treatment-related decreases in feed consumption were only observed in females at the high dose level (5000 ppm). Females in the 5000 ppm group had treatment-related decreases in feed consumption during the intervals of TD 1-4, 4-8 and 8-15 during prebreeding, and LD 1-4, which correlated to the observed body weight decreases. These feed consumption decreases were statistically identified on TD 1-4, 4-8 and 8-15. No treatment-related differences in feed consumption were observed for females in the 5000 ppm group throughout gestation. No treatment-related differences in feed consumption were observed for females at 500 or 1500 ppm or for males at any dose level throughout the duration of the study.

Blood toxicokinetic results showed that quantifiable 2-butoxyethyl benzoate was not present in any control blood samples, but was present in some treated blood samples at very low levels. Quantifiable blood levels of two 2-butoxyethyl benzoate metabolites (2-butoxyethanol and 2-butoxyacetic acid) were present in all treated rats at high concentrations. 2-Butoxyethanol exhibited linear kinetics in blood across all dose levels. The blood levels of 2-butoxyethanol in male rats were higher than the blood levels of 2-butoxyethanol in female rats at each dose level. 2-Butoxyacetic acid exhibited linear kinetics in male rats; however, blood levels of this metabolite exhibited supralinear kinetics at 5000 ppm in female rats. The blood levels of 2-butoxyacetic acid in female rats at the high dose level were higher than the blood levels in male rats.

Treatment-related hematologic effects were observed only in females at the high dose level (5000 ppm). Females given 5000 ppm had treatment-related and statistically identified lower mean red blood cell count and hemoglobin concentration, higher mean MCV and MCH, lower mean MCHC, and higher mean reticulocyte count. These hematologic effects were representative of regenerative anemia in females given 5000 ppm, and were interpreted to be adverse. Females given 5000 ppm also had a treatment-related higher platelet count, which may have been caused by a generalized increase in platelet production within the bone marrow in association with the reticulocytosis. There were no treatment-related hematologic effects in females given 500 or 1500 ppm, or in males at any dose level.

There were no treatment-related changes in prothrombin times for males and females at any exposure level.

Treatment-related clinical chemistry effects were observed only in females at the high dose level (5000 ppm). Females given 5000 ppm had statistically identified higher mean urea nitrogen, triglyceride, creatinine and phosphorus concentrations. Higher phosphorus and creatinine concentrations were interpreted to be treatment-related effects. All of the treatment-related elevations in clinical chemistry parameters were interpreted to be non-adverse, because there were no corresponding alterations in organ weights, and no histopathologic correlates. There were no treatment-related clinical chemistry effects in females given 500 or 1500 ppm, or in males at any dose level.

There were no treatment-related changes in urinalysis parameters for males at any dose level.

Treatment-related effects on organ weight were observed only in the liver of females at the high dose level (5000 ppm). Females given 5000 ppm had a treatment-related lower mean final body weight (6.1%), relative to controls. Females given 5000 ppm had a treatment-related higher mean relative liver weight (5.1%), relative to controls. The higher relative liver weight corresponded to

the histopathologic observation of very slight hypertrophy of centrilobular/midzonal hepatocytes, with increased cytoplasmic eosinophilia, in females given 5000 ppm. The lower mean final body weight and higher mean relative liver weight in females given 5000 ppm were interpreted to be nonadverse. There were no treatment-related alterations in final body weights or organ weights in

females given 500 or 1500 ppm, or in males at any dose level.

There were no treatment-related gross pathologic observations.

A treatment-related liver histopathologic change was observed only in females at the high dose level (5000 ppm). Treatment-related very slight hypertrophy of centribolublar/midzonal hepatocytes, with increased cytoplasmic eosinophilia, was present in the liver of 11/12 females given 5000 ppm. The hepatocellular hypertrophy was interpreted to be a non-adverse and adaptive effect, based on the modest corresponding increase in liver weights, along with the absence of any treatment-related changes in liver enzyme activities (ALT, AST and GGT), and the absence of necrosis, increased apoptosis, inflammation, proliferative or degenerative changes in the liver of females at this dose level.

There were no treatment-related effects of 2-butoxyethyl benzoate on neurological or reproductive function, or prenatal/early neonatal growth and survival of the offspring.

Based on these results, the no-observed-effect level (NOEL) for general toxicity was 1500 ppm in females and 5000 ppm in males. The NOEL for reproductive and neurological effects in both males and females was 5000 ppm, the highest dose level tested.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

94.9 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

GLP/Guideline study.

Additional information

Repeated Dose Oral (Key Study):

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.

Dietary administration of 2-butoxyethyl benzoate to Crl:CD(SD) rats resulted in treatment-related decreases in female body weight gains and feed consumption only at the high dose level (5000 ppm). 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.

Blood kinetics:

2-Butoxyethyl benzoate was not quantifiable in any of the treated blood samples. 2-butoxyethanol was quantifiable in most treated blood samples and 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).

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. 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.

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.

Repeated Dose Oral (Supporting Study):

The purpose of this study was to evaluate the potential effects of 2-butoxyethyl benzoate following rat dietary administration on general toxicity, neurological and reproductive function, and prenatal/early neonatal growth and offspring survival. Dietary administration of 2-butoxyethyl benzoate to Crl:CD(SD) rats resulted in treatment-related decreases in female body weight only at the high dose level (5000 ppm). Similar to body weight effects, treatment-related decreases in feed consumption were only observed in females at the high dose level (5000 ppm).

Blood toxicokinetic results showed that quantifiable 2-butoxyethyl benzoate was present in some treated blood samples at very low levels. Quantifiable blood levels of two 2-butoxyethyl benzoate metabolites (2-butoxyethanol and 2-butoxyacetic acid) were present in all treated rats at high concentrations.

Treatment-related hematologic effects were observed only in females at the high dose level (5000 ppm). Females given 5000 ppm had treatment-related and statistically identified lower mean red blood cell count and hemoglobin concentration, higher mean MCV and MCH, lower mean MCHC, and higher mean reticulocyte count. These hematologic effects were representative of regenerative anemia in females given 5000 ppm, and were interpreted to be adverse

(see Specific Investigations: Other Studies - Haematoxicity for human relevance). Females given 5000 ppm also had a treatment-related higher platelet count, which may have been caused by a generalized increase in platelet production within the bone marrow in association with the reticulocytosis. Treatment-related clinical chemistry effects were observed only in females at the high dose level (5000 ppm). Females given 5000 ppm had statistically identified higher mean urea nitrogen, triglyceride, creatinine and phosphorus concentrations. Higher phosphorus and creatinine concentrations were interpreted to be treatment-related effects. All of the treatment-related elevations in clinical chemistry parameters were interpreted to be non-adverse, because there were no corresponding alterations in organ weights, and no histopathologic correlates.

Treatment-related effects on organ weight were observed only in the liver of females at the high dose level (5000 ppm). The higher relative liver weight corresponded to the histopathologic observation of very slight hypertrophy of centrilobular/midzonal hepatocytes, with increased cytoplasmic eosinophilia, in females given 5000 ppm. The higher mean relative liver weight and hepatocellular hypertrophy in females given 5000 ppm were interpreted to be nonadverse.

Based on these results, the no-observed-effect level (NOEL) for general toxicity was 1500 ppm in females and 5000 ppm in males. The NOEL for reproductive and neurological effects in both males and females was 5000 ppm, the highest dose level tested.

Justification for classification or non-classification

Repeated Dose Oral:

In the key study, 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.

In the supporting study, the no-observed-effect level (NOEL) for general toxicity was 1500 ppm in females and 5000 ppm in males. The NOEL for reproductive and neurological effects in both males and females was 5000 ppm, the highest dose level tested.

The effects observed in these studies are not considered significant toxic effects or are not considered to be relevant for humans, therefore, no classification under GHS is warranted.