2-butoxyethyl benzoate

Endpoint summary

Link to relevant study record(s)

Reference

Endpoint:

basic toxicokinetics in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

July 16, 2015 to August 2, 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Objective of study:

absorption

distribution

excretion

metabolism

Qualifier:

according to guideline

Guideline:

OECD Guideline 417 (Toxicokinetics)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.36 (Toxicokinetics)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.7485 (Metabolism and Pharmacokinetics)

Deviations:

no

GLP compliance:

yes

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):
Non-Radiolabeled: The Dow Chemical Company, Midland, Michigan (Lot #
201303443-19).
Radiolabeled: Lot # HL00623-011-50

Purity/Characterization (Method of Analysis and Reference):
Non-radiolabeled purity: 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).
Radiochemical purity: The GLP radiochemical purity was conducted concurrently with the study. The purity was determined to be 100% by liquid chromatography with radiochemical detection with identification by liquid chromatography with mass spectrometry detection.

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).

Specific Activity of Radiolabeled Test Material: 50.68 mCi/mmol (QC Supervisor, 2015)

Radiolabelling:

yes

Species:

rat

Strain:

other: F344/DuCrl

Details on species / strain selection:

Strain and Justification:
F344/DuCrl rats were selected because of their general acceptance and suitability for toxicity testing, availability of historical background data, the reliability of the commercial supplier, and the rats is the preferred species for pharmacokinetic/metabolism studies.

Sex:

male/female

Details on test animals or test system and environmental conditions:

Species and Sex: Rat (male and female)
Strain and Justification: F344/DuCrl rats were selected because of their general acceptance and suitability for toxicity testing, availability of historical background data, the reliability of the commercial supplier, and the rats is the preferred species for pharmacokinetic/metabolism studies.
Supplier and Location: Charles River (Kingston, New York)
Age at Study Start: 9 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). Non-cannulated animals, used for repeated dosing, were housed 2-3 per cage in stainless steel solid bottom cages with corncob bedding prior to randomization. Animals were acclimated to the laboratory environment for at least one week prior to use, including at least two days in metabolism cages. Cages contained a hanging feeder and a pressure activated lixit valve-type watering system. Jugular vein cannulated rats, used for time-course groups, (surgery performed by the supplier) were acclimated in metabolism cages for at least one week prior to use.

Housing:
Following administration of 14C-test material, the animals were housed singly in glass Roth-type metabolism cages, which were designed for the separation and collection of urine, feces, CO2, and organic volatiles. Air was drawn through the metabolism cages at ~ 850 mL/minute.
Temperature: 22°C with a range of 19°C-23°C
Humidity: 50% with a range of 30-75%
Photoperiod: 12-hour light/dark (on at 6:00 a.m. and off at 6:00 p.m.)
Note: The collection of the 12 hour specimens for all groups interrupted the normal light cycle period. Daylight savings time occurred during an acclimation period.

Randomization and Identification:
Animals were stratified by body weight and/or patency of their cannulae, and then randomly assigned to treatment groups using a computer program. Animals placed on study were uniquely identified with alphanumeric identification.

Feed and Water:
Animals were given LabDiet Certified Rodent Diet #5002 (PMI Nutrition International, St. Louis, Missouri) in pelleted form. Feed and municipal water were provided ad libitum except that access to feed was restricted approximately 16 hours prior to the administration of test material and was returned about 4 hours post-dosing with the exception of animals in Group 3 (repeat), which was restricted only prior to the last dose (radiolabeled test material). Analyses of the feed were performed by PMI Nutrition International to confirm the diet provides adequate nutrition and to quantify the levels of selected contaminants. Drinking water obtained from the municipal water source is periodically analyzed for chemical parameters and biological contaminants by the municipal water department. In addition, specific analyses for chemical contaminants are conducted at periodic intervals by an independent testing facility. Copies of these analyses are maintained in the study file.

Enrichment:
The animals from Group 3 (repeated dose) had nylon bones in their cages during acclimation and during repeat cold-dose period only. The remaining animals were jugular vein cannulated and also had nylon bones in their cages during the acclimation period which were removed prior to fasting. After dosing, all cannulated animals were housed in glass Roth type cages so they were able to have visual contact only.

Animal Welfare:
In accordance with the U.S. Department of Agriculture animal welfare regulations, 9 CFR, Subchapter A, Parts 1-4, the animal care and use activities required for conduct of this study were reviewed and approved by the Institutional Animal Care and Use Committee (IACUC). The IACUC has determined that the proposed activities are in full accordance with these Final Rules. The IACUC-approved animal care and use activities to be used for this study are Metabolism 01, Blood Collection 01, Humane Endpoints 01, Subchronic 01 and Animal ID 01. Animals were observed daily (with the exception of one day, whereas only the AM cageside was recorded) while on the study and signs of toxicity and other relevant clinical features recorded.

Cannulation:
Jugular Vein:
Rats in which the plasma/RBC 14C time-course was determined were obtained already cannulated in the jugular vein by the supplier.

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

Dose Preparation:
Based on the solubility study results, 2-butoxyethyl benzoate was soluble in corn oil at 500 mg/mL (Ji et al., 2015). Therefore, corn oil was used for the dose carrier. The amount of dose solution was administered at a target volume of ~5 mL/kg body weight; the target radioactivity was ~500 μCi/kg. Appropriate amounts of 14C-labeled and/or non-radiolabeled 2-butoxyethyl benzoate were added to obtain the target doses of 25 or 250 mg 2-butoxyethyl benzoate/kg body weight. Confirmation of the test material concentration in the dose solutions was conducted. Radioactivity in the dose solutions were quantified by LSS as described below.

Dose Formulation Analysis:
Dose Confirmation and Homogeneity:
Chemical analysis was conducted concurrently with the study to determine the concentration of 2-butoxyethyl benzoate and chemical homogeneity of the test material in all dose solutions by high performance liquid chromatography with
ultraviolet detection (HPLC/UV). The respective dose solutions for Groups 1-3, had their radiochemical homogeneity determined prior to administration (via LSS). The analytical method validation was performed concurrently with the study.

Stability:
Stability of 2-butoxyethyl benzoate was conducted for at least 21 days in corn oil at concentrations of 0.25, 2.5 and 250 mg/mL (Perala, 2016).

Solubility:
The solubility of the test substance 2-butoxyethyl benzoate was determined in corn oil at 500 mg/mL (Ji et al., 2015).

Duration and frequency of treatment / exposure:

Oral Dose Administration:
The oral dose was administered via a ball-tipped gavage needle attached to a glass syringe. Animals from Groups 1, 2 and 4 received a single oral dose of radiolabeled test material. Group 3 animals received 14-daily doses of non-radiolabeled test material. On day 15, each Group 3 animal received a single dose of radiolabeled dose formulation. Group 5 animals received a single oral dose of corn oil (dose carrier only).

Dose / conc.:

25 other: mg/kg bw

Remarks:

Single and repeat dose

Dose / conc.:

250 other: mg/kg bw

Remarks:

Single dose

No. of animals per sex per dose / concentration:

Test Groups 1-3: 4/sex/group
Test Groups 4 and 5: 1/sex/group

Control animals:

yes, concurrent vehicle

Details on study design:

Dose Route and Justification, Method of Administration, Frequency and Duration:
A possible route of human exposure to the test material would be via occupational and consumer use of products. Thus, administration to rats via oral gavage represented an appropriate means of exposure. Groups 1-2 (see Study Design above) received a single oral dose of 14C-2-butoxyethyl benzoate. Group 3 received 14 daily oral doses of unlabeled 2-butoxyethyl benzoate followed by a single oral dose of 14C-2-butoxyethyl benzoate.

Dose Levels and Justification:
The dose levels selected for this pharmacokinetic/metabolism study (Text Table 1) were based on previous oral toxicity information described above. The high dose selected, 250 mg/kg, was a level where minimal effects have been observed in previous toxicity studies (Johnson et al., 2015; Zablotny and Stebbins, 2016) after repeated dose. The low dose selected of 25 mg/kg was below current toxic effect levels (Kumar, 2013).

Details on dosing and sampling:

Oral Dose Administration:
The oral dose was administered via a ball-tipped gavage needle attached to a glass syringe. Animals from Groups 1, 2 and 4 received a single oral dose of radiolabeled test material. Group 3 animals received 14-daily doses of non-radiolabeled test material. On day 15, each Group 3 animal received a single dose of radiolabeled dose formulation. Group 5 animals received a single oral dose of corn oil (dose carrier only).
The volume of radiolabeled dose formulation administered to each animal was calculated based on the body weight taken on the day of dose administration, with the exception of Group 3 (repeat). The volume of dose administered to each Group 3 animal was based on the body weight taken on day 1 (for dosing days 1-7) and day 8 (for dosing days 8-14) of dose administration. The volume of radiolabeled dose administered on day 15 was based on individual body weights on day 15. The actual amount of radiolabeled test material administered to each animal was determined by weighing the dose syringe before and after dose administration. The targeted volume of the dose formulation was 5 mL/kg bw. The amount of non-radiolabeled test material administered to Group 3 (days 1-14) animals was determined by target volume, not syringe weight.

Time-Course Blood and Plasma Collection:
Group 1 and 2 rats were fitted with indwelling jugular vein cannulae, which were used to collect blood for the determination of time course 14C concentration of the test materials in plasma and RBC.
Blood (approximately 0.1-0.2 mL from Group 1 and 2 animals) was collected from the jugular vein cannula and transferred into tubes containing sodium heparin anticoagulant at 0.08, 0.17, 0.25, 0.5, 1, 2, 3, 6, 12, 24, and every 24 hours post-dosing thereafter from each animal until 168 hours. Radioactivity was determined in appropriate aliquots of the plasma and RBC of each sample collected from each group. Weighed aliquots of RBC were oxidized and the plasma and RBC analyzed for radioactivity by LSS.

Specimen Collection:
Urine:
All urine voided during the study was collected in dry-ice cooled traps. The urine traps were changed at 12-, 24- and 48-hour post-dosing followed by 24-hour intervals for the remainder of the study (Historically, rats do not reproducibly void a sufficient volume of urine by 6 hours post-dosing to allow for radioactivity determination via LSS and for chemical analysis. Therefore, the first collection interval was 12 hours post-dosing). The cages were rinsed with water at the time the traps were changed and the rinse collected. Each urine specimen and urine/cage rinse was weighed, and a weighed aliquot of each sample was analyzed for radioactivity by LSS as described below. Equal volume aliquots of urine samples (per time, dose and sex) from the 0-12-hour, 12-24-hour and 24-48-hour collection intervals were pooled and stored at -80ºC and 0-12-hour and 12-24-hour urine samples underwent chemical analysis, as the majority of the radioactivity was eliminated in those intervals.

Feces:
Feces were collected in dry-ice chilled containers at 24-hour intervals. An aqueous homogenate (~ 25% w/w) was prepared (shaken for > 4 hours) and weighed aliquots of these homogenates were oxidized, (OxiMate 80 Sample Oxidizer, PerkinElmer Life Sciences, Inc., Boston, Massachusetts) and quantitated for radioactivity by LSS. In addition, equal volume aliquots of fecal homogenates from each animal were taken from the 0-24-hour and 24-48-hour collection intervals and pooled (per dose and sex). These pooled samples were stored at -80ºC and 0-24-hour fecal homogenate samples underwent chemical analysis, as the majority of the radioactivity was eliminated in that interval.

Expired Volatiles:
Air was drawn through the cage at approximately 850 mL/minute. Upon exiting the cage, the air passed through charcoal to trap expired volatiles. The charcoal traps were changed at 24-hour intervals up to 48-hours. The radioactivity was <1% of the administered dose thereafter. The charcoal was ground with a blender. Weighed aliquots of the charcoal were oxidized and analyzed for radioactivity by LSS.

Expired CO2:
Following the charcoal trap (described above) the expired air was passed through a solution of monoethanolamine:1-methoxy-2-propanol (3:7 v/v) to trap expired CO2 and analyzed for radioactivity by LSS. The CO2 trap was changed at the 6-, 12- and 24-hour intervals and at 24-hour intervals thereafter.

Terminal Sacrifice:
At the specified time post-dosing the animals were anaesthetized with CO2/O2 and sacrificed by exsanguination. Following sacrifice the Roth cages were washed and the cage wash analyzed for radioactivity by LSS.

Tissues:
The following tissues from animals of Groups 1-3 were collected at sacrifice:
adrenal, bladder, blood (terminal), bone (femur), bone marrow (femur), brain carcass (residual), fat (perirenal), GI tract (with contents), heart, kidney, liver, lung, lymph node, muscle (skeletal), ovary, pancreas, pituitary, plasma (terminal), RBC (red blood cells), skin, spleen, testis, thymus, thyroid, uterus

The brain, carcass, GI tract with contents, kidney, liver and testis were collected, homogenized (~ 33% homogenate with water), a weighed aliquot solubilized or oxidized, and analyzed for radioactivity by LSS. Bone was solubilized with sodium hydroxide and analyzed for radioactivity by LSS. Blood was centrifuged to obtain plasma and the plasma analyzed for radioactivity by LSS. RBC was oxidized and analyzed for radioactivity by LSS. The skin was removed from the carcass and a representative skin sample oxidized and analyzed for radioactivity by LSS. The remaining tissues were directly oxidized without homogenization and analyzed for radioactivity by LSS.

Final Cage Wash:
Following the terminal sacrifice of the animals, a final cage wash (FCW) was performed with tap water and detergent. The final cage wash and contents were collected and the weight of the sample determined. The FCW and contents were shaken for > 4 hours, and a weighed aliquot was analyzed for radioactivity by LSS.

Terminal Plasma and RBC:
Terminal blood (Groups 1-3) was obtained at sacrifice via cardiac puncture. The blood was centrifuged to obtain plasma and RBC and analyzed for radioactivity via LSS. Group 4 animal blood was placed in vials containing acetonitrile with 1% acetic acid (Ratio of blood versus acetonitrile was 1:4), vortexed and chemically analyzed following SOP MTH-ACL-8.

Control Samples:
A male and female rat was dosed with the corn oil carrier. Carrier control urine (0-12 and 12-24 hour intervals) and feces (0-24 hour interval) were collected in dry-ice cooled traps. The control animals were sacrificed and blood collected by the same procedure as the dosed animals.

Sample Analysis:
14C Analysis:
Radioactivity was quantified in a liquid scintillation spectrometer (Packard Tri- Carb 2900TR). Counts per minute (cpm) were corrected for quench and converted to disintegrations per minute (dpm). Descriptive statistics were conducted, (i.e., mean ± standard deviation [± SD]). Samples with dpm less than twice the concurrently run background (blanks) were considered to contain insufficient radioactivity to reliably quantify (less than the limit of quantitation; those samples were denoted with an “NQ” in the Results Tables). Non-quantifiable excreta samples (urine/rinse/feces) were identified in the tables as NQ and a value of '0' used in calculations. For tissues, when a sample is non-quantifiable, that sample was assigned the quantitation limit (QL) for calculations and displayed as NQ with the quantitation limit in parenthesis. The mean was calculated from actual values and calculated QL values and were presented as ± SD, unless greater than ½ of the values are presented as NQ, in which case the mean was expressed as NQ (QL) ± SD. If all tissue values were NQ the mean was presented as NQ (QL) with no SD displayed. Values reported as 0.00 were actually <0.005% of the administered dose.

Metabolite Profiles:
Pooled samples (Groups 1-3 and 5; urine and feces) from selected intervals (0-12-, 12-24-hour urine and 0-24-hour feces) and Cmax blood (Group 4) were analyzed via high performance liquid chromatography (HPLC) with in-line radiochemical detection or via fraction collection with LSS analysis following SOP MTH-ACL-8.

Metabolite Identification:
An attempt was made to identify, by appropriate analytical methodologies, both parent test material and major metabolite(s) that exceeded 5% of the administered dose in selected urine, fecal and Cmax blood specimens following SOP MTH-ACL-8. Analysis of excreta and blood samples from these animals aided in metabolite identification.

Statistics:

Descriptive statistics were used, i.e., mean ± standard deviation. All calculations in the database were conducted using Microsoft Excel (Microsoft Corporation, Redmond, Washington) spreadsheets and databases in full precision mode (15 digits of accuracy). Pharmacokinetic parameters were calculated for plasma and RBC data, including AUC (area-under-the-curve), Cmax, ½Cmax, and elimination rate constants, using PK Solutions (v.2.0.6., Summit Research Services, Montrose, Colorado), a pharmacokinetic computer modeling program.

Type:

absorption

Results:

Orally administered 14C-2-butoxyethyl benzoate was absorbed rapidly without any apparent lag time. The total recovery of radioactivity from all animals was an average of 98 ±4%.

Type:

distribution

Results:

Between 8-15% of the 14C-2-butoxyethyl benzoate remained in the tissues after 168 hours post-dosing in all of the groups. Results indicate a low bioaccumulation potential of 14C-2-butoxyethyl benzoate in rats.

Type:

metabolism

Results:

The major metabolites were identified as ethylene glycol, 2-butoxyacetic acid, glucuronide of 2-butoxyethanol and 2-butoxyethanol.

Type:

excretion

Results:

The majority of the administered 14C-2-butoxyethyl benzoate derived radioactivity was rapidly excreted in urine without any significant difference between the dose levels. Most of the remaining oral dose was eliminated in expired CO2.

Details on absorption:

Absorption of Orally Administered Dose:
A conservative estimate of absorption of the oral doses was made from the percent of administered radioactivity recovered in urine (including the cage rinses and FCW) and tissues at sacrifice (except GI tract). This estimate may be low, since it does not account for net biliary elimination, which was not measured in the current study. The nominal 25 or 250 mg/kg dose group animals (Groups 1-3) showed high absorption of 14C-2-butoxyethyl benzoate (at least 88-95% of the administered dose), based on recovery in urine, rinse, FCW, expired CO2 and non-GI tissues.

Time-Course Concentration of Radioactivity and Pharmacokinetics in Plasma:
The kinetic parameters derived from the plasma concentration time-course of 14C-2-butoxyethyl benzoate were determined using a two-compartmental model to accommodate the biphasic manner of decline in plasma radioactivity after Tmax.
After an oral gavage dose of 25 or 250 mg/kg bw, 14C-2-butoxyethyl benzoate radioactivity was rapidly absorbed without any apparent lag time and achieved the highest measured concentration (Cmax), on average, at 3 hours (Tmax). As mentioned above, after Tmax, plasma concentrations declined in a biphasic manner. The mean half-life for the rapidly declining plasma alpha phase (t½α) averaged between 3 and 6 hours for both dose groups. The more slowly declining beta phase plasma half-life (t½β) averaged ~47-52 hours for both low and high dose groups. The Cmax parameters were greater than dose-proportional in high dose male rats relative to the low dose group; however, the AUC was less than dose-proportional in the males; for females, the Cmax and AUC values were approximately dose proportional across the two dose levels.
One female rat (15A3706) from Group 1 became non-patent at the 12 hour time-point, so no blood was collected for remaining collection time-points. The animal looked moribund at the 96-hour collection time-point and the veterinarian assessment was conducted. The assessment concluded that the most likely cause of morbidity was renal disease (sepsis), potentially associated with venous catheterization; therefore, no pharmacokinetic parameters were derived from this animal. No blood samples were collected from male rat 15A3696 (Group 2) at the 144 hr timepoint and female rat 15A3708 (Group 2) at the 96-144 hr time-points due to patency issues.

Time-Course Concentration of Radioactivity and Pharmacokinetics in RBC:
The RBC 14C-2-butoxyethyl benzoate concentrations of Groups 1 and 2 were approximately 2-3x lower than their plasma concentrations. Similarly, the RBC Cmax data were 2-3-fold lower than that of plasma.
Double peaks were observed in RBC concentration time-course data in all dosed rats. The double peaks were observed before and after 12-hr timepoint with the first peak reaching a maximum concentration at 2-3 hours and the second broader peak achieving at maximum concentration at 48-72 hours post-dosing. However, since this phenomenon was not present in the plasma samples it is unlikely to be enterohepatic recirculation but potentially due to metabolite-related binding to the RBC.
Due to the elimination portion of the time-course flattening out at 12-168 hours postdosing, the RBC kinetic parameters (Tmax, Cmax Absorption t½, and Elimination t½) were calculated based on the RBC samples collected during the first 12 hours post-dosing.
The Cmax parameters were greater than dose-proportional in high dose rats relative to low dose values; however, the AUC was approximately dose-proportional in both male and female rats.

Details on distribution in tissues:

Disposition of the Administered 14C-2-Butoxyethyl Benzoate in Tissues:
Group 1. Single Oral Low Dose (25 mg/kg bw) 14C-2-butoxyethyl benzoate:
Following a single oral dose of 14C-2-butoxyethyl benzoate, a mean total of 14 and 11% of the administered dose was recovered in tissues of male and female rats, respectively. The majority (73-74%) of the administered dose in tissues was recovered in the skin and carcass. The next highest tissue was the liver with <2% of the administered dose.

Group 2. Single Oral High Dose (250 mg/kg bw) of 14C-2-butoxyethyl benzoate:
Following a single oral dose of 14C-2-butoxyethyl benzoate, a mean total of 10 and 8% of the administered dose was recovered in tissues of male and female rats, respectively. The majority (77-79%) of the administered dose in tissues was recovered in the skin and carcass. The next highest tissue was the liver with ≤0.95% of the administered dose.

Group 3. Repeat Oral Low Dose (25 mg/kg bw) 14C-2-butoxyethyl benzoate:
Following a repeat (14 days) oral dose of 25 mg/kg 2-butoxyethyl benzoate and single oral dose of 25 mg/kg 14C-2-butoxyethyl benzoate, a mean total of 15 and 11% of the administered dose was recovered in tissues of male and female rats, respectively. The majority (78-82%) of the administered dose in tissues was recovered in the skin and carcass. The next highest tissue was the liver with <1.4% of the administered dose.

Details on excretion:

Urinary Elimination of the Administered 14C-2-Butoxyethyl Benzoate:
Group 1. Single Oral Low Dose (25 mg/kg bw) 14C-2-butoxyethyl benzoate:
Following a single oral dose of 14C-2-butoxyethyl benzoate, a mean total of 52 and 56% of the administered dose was recovered in urine (including the rinse) of male and female rats, respectively. Radioactivity was rapidly excreted with 95% of total urinary elimination occurring within 24 hours post-dosing.

Group 2. Single Oral High Dose (250 mg/kg bw) of 14C-2-butoxyethyl benzoate:
Following a single oral dose of 14C-2-butoxyethyl benzoate, a mean total of 68% of the administered dose was recovered in urine (including the rinse) of both male and female rats. Radioactivity was rapidly excreted with 91-94% of total urinary elimination occurring within 24 hours post-dosing.

Group 3. Repeat Oral Low Dose (25 mg/kg bw) 14C-2-butoxyethyl benzoate:
Following a repeat (14 days) oral dose of 25 mg/kg 2-butoxyethyl benzoate and single oral dose of 25 mg/kg 14C-2-butoxyethyl benzoate on test day 15, a mean total of 60 and 59% of the administered dose was recovered in urine (including the rinse) of male and female rats, respectively. Radioactivity was rapidly excreted with 95% of total urinary elimination occurring within 24 hours post-dosing.

Fecal Elimination of the Administered 14C-2-Butoxyethyl Benzoate:
Group 1. Single Oral Low Dose (25 mg/kg bw) 14C-2-butoxyethyl benzoate:
Following a single oral dose of 25 mg/kg 14C-2-butoxyethyl benzoate, a mean total of 5 and 4% of the administered dose was recovered in the feces of male and female rats, respectively. Radioactivity was rapidly excreted with 76-79% of total fecal elimination occurring within 48 hours post-dosing.

Group 2. Single Oral High Dose (250 mg/kg bw) of 14C-2-butoxyethyl benzoate:
Following a single oral dose of 250 mg/kg 14C-2-butoxyethyl benzoate, a mean total of 3% of the administered dose was recovered in the feces of both male and female rats. Individual animal data are presented in Appendix Table 1. Radioactivity was rapidly excreted with 74-81% of total fecal elimination occurring within 48 hours post-dosing.

Group 3. Repeat Oral Low Dose (25 mg/kg bw) 14C-2-butoxyethyl benzoate:
Following a repeat oral dose of 25 mg/kg 2-butoxyethyl benzoate and single oral dose of 25 mg/kg 14C-2-butoxyethyl benzoate on test day 15, a mean total of 6 and 5% of the administered dose was recovered in the feces of male and female rats, respectively. Radioactivity was rapidly excreted with 81-83% of total fecal elimination occurring within 24 hours post-dosing.

Expired Air Elimination of the Administered 14C-2-Butoxyethyl Benzoate:
Group 1. Single Oral Low Dose (25 mg/kg bw) 14C-2-butoxyethyl benzoate:
Following a single oral dose of 14C-2-butoxyethyl benzoate, a mean total of 23 and 26% of the administered dose was recovered in expired CO2 of male and female rats, respectively. Radioactivity was rapidly excreted with 84-87% of total expired CO2 elimination occurring within 48 hours post-dosing.
In addition to the expired CO2, a mean total of 0.42 and 0.21% of the administered dose was recovered in other expired volatiles of male and female rats, respectively.

Group 2. Single Oral High Dose (250 mg/kg bw, 1.125 mmol/kg bw) of 14C-2-butoxyethyl benzoate:
Following a single oral dose of 14C-2-butoxyethyl benzoate, a mean total of 16 and 18% of the administered dose was recovered in expired CO2 of male and female rats, respectively. Radioactivity was rapidly excreted with 83-85% of total expired CO2 elimination occurring within 48 hours post-dosing. In addition to the expired CO2, a mean total of 0.16 and 0.18% of the administered dose was recovered in expired volatiles of male and female rats, respectively. Overall, approximately 13% of administered dose was recovered as CO2 within 48 hours post-dosing in male rats. In a similar ADME study for 2-butoxyethanol, 125 mg/kg bw (1.059 mmol/kg bw) was orally administered to male F344 rats via the same dose vehicle as used in the current study, approximately 18% of administered dose was recovered as CO2 within 48 hours post-dosing in male rats (Ghanayem et al., 1987). The similar radioactivity recovery as CO2 within 48 hours post-dosing in male rats with similar mole equivalent dosage in male rats, indicated that both 2-butoxyethyl benzoate and 2-butoxyethanol had similar absorption and elimination.

Group 3. Repeat Oral Low Dose (25 mg/kg bw) 14C-2-butoxyethyl benzoate:
Following a repeat (14 days) oral dose of 25 mg/kg 2-butoxyethyl benzoate and single oral dose of 25 mg/kg 14C-2-butoxyethyl benzoate on test day 15, a mean total of 20 and 23% of the administered dose was recovered in in expired CO2 of male and female rats, respectively. Radioactivity was rapidly excreted with 83-84% of total expired CO2 elimination occurring within 48 hours post-dosing.
In addition to the expired CO2, a mean total of 0.15 and 0.07% of the administered dose was recovered in expired volatiles of male and female rats, respectively.

Toxicokinetic parameters:

other: See attachment Kinetic Parameters Results.

Metabolites identified:

yes

Details on metabolites:

Metabolite(s) Profile and Quantitation:
Metabolite profiling and quantitation were conducted on select samples for Groups 1-5.
Selected samples of excreta and Cmax blood from F344/DuCrl rats orally-dosed with 14C- 2-butoxyethyl benzoate (as a single 25 mg/kg low dose, single 550 mg/kg high dose or 25 mg/kg repeat dose) were submitted for radiochemical profiling and identification of major metabolites. Radiochemical profile analysis was achieved via high performance liquid chromatography (HPLC) separation with in-line radiochemical detection (RAM) or fraction collection and liquid scintillation spectrometry (LSS). Representative urine, fecal homogenate extract and blood extract samples were also analyzed by HPLC with electrospray ionization and accurate mass/time-of-flight tandem mass spectrometry detection (HPLC/ESI/TripleTOF-MS/MS) to identify metabolites.
A total of up to 14 radiochemical peaks were detected in the urine samples, 3 of which were only detected in urine samples. No parent 2-butoxyethyl benzoate was detected in the urine samples. The most abundant urinary metabolite in all groups (ranging from 13.19 to 31.44% of the administered dose) was positively identified as 2-butoxyacetic acid. Three additional metabolites were present in at least one group/gender total urine radiochemical profile at a concentration greater than 5% of the administered dose. Those metabolites were tentatively identified as ethylene glycol (3.02 – 5.58% of the administered dose in the urine samples), the glucuronide conjugate of 2-butoxyethanol (3.39 – 7.59% of the administered dose in the urine samples) and the glycine conjugate of 2-butoxyacetic acid (1.28 – 5.25% of the administered dose in the urine samples).
A total of up to 13 radiochemical peaks were detected in the fecal homogenate extracts, of which 4 metabolites were only observed in the fecal samples. Parent 2-butoxyethyl benzoate was detected in four of the six pooled fecal homogenate extract samples and comprised less than 0.5% of the administered dose. The most abundant fecal metabolite across all fecal homogenate extract samples (comprising up to 2.37% of the administered dose) was positively identified as butoxyacetic acid.
A total of up to 8 radiochemical peaks (4 unique to blood samples) were detected in the Cmax blood extracts. No parent 2-butoxyethyl benzoate was detected in the Cmax blood extract samples. Across both sexes, the most abundant blood metabolite was also positively identified as butoxyacetic acid with females having an approximately equal amount of ethylene glycol present.
Parent 2-butoxyethyl benzoate was well metabolized in the rats orally dosed with 14Clabeled test material. In addition to the metabolites discussed above, metabolism resulting from hydroxylation and oxidation of the glycol side-chain were observed as well as the corresponding glucuronide and glycine conjugates. Of all 22 metabolites observed across the urine, fecal and blood samples, there were 11 metabolites common between urine and fecal samples and 4 blood metabolites that were also observed in urine/fecal samples.
The major metabolites (ethylene glycol, 2-butoxyacetic acid, glucuronide of 2- butoxyethanol and 2-butoxyethanol) were also identified as the same major metabolites from previous ADME study (Ghanayem et al., 1987; Medinsky et al., 1990) where rats were orally administered with molar equivalent dosage of 2-butoxyethanol, indicating that 2-butoxyethyl benzoate and 2-butoxyethanol were metabolized in similar metabolic pathways in rats.

Dose Solution (Concentration, Stability, Administration and Recovery of the Dose):

The concentration of radioactivity in each of the dose solutions was 112-122% of the target concentration. The deviation of the target radioactivity concentrations was deemed to have no impact on the overall interpretation of the study. The measured concentration of total test material in each dose solution was 99-102% of the target concentration. The homogeneity analysis revealed acceptable homogeneity with 0.2- 0.9% relative standard deviation for all groups. Previous work showed that 2-butoxyethyl benzoate was stable in corn oil for at least 21 days at concentrations of 0.25, 2.5 and 250 mg/mL (Perala, 2016); therefore, no stability study was conducted in this study.

At the time of 14C-2-butoxyethyl benzoate administration, the body weights ranged 0.150 to 0.183 kg and 0.112-0.126 kg for male and female rats, respectively. The mean dose of 14C-2-butoxyethyl benzoate administered to the groups was 21-31 and 259-268 mg/kg bw for the targeted 25 and 250 mg/kg dose groups, respectively. Though the doses administered were slightly above or below the target, this was deemed to have no impact on the overall interpretation of the study. A range of 473-619 μCi/kg was administered to male and female rats for all dose groups. The actual radioactivity administered was 95-124% of the targeted dose of ~500 μCi/kg.

Total recovery of radioactivity from all the animals (Groups 1-3) averaged 98±4%.

There were no signs of toxicity observed in any animals following oral administration of 25 or 250 mg/kg bw 2-butoxyethyl benzoate.

Conclusions:

A pharmacokinetic study was conducted in F344/DuCrl rats for 168 hours (7 days) postdosing to determine absorption, distribution and excretion of 14C-2-butoxyethyl benzoate following oral exposure (single dose of 25 or 250 mg/kg bw or repeat dose of 25 mg/kg bw).
Orally administered 14C-2-butoxyethyl benzoate was absorbed rapidly without any apparent lag time. The percent absorption of the orally administered dose in the low and high dose groups was at least 88-95%, based on summarized total radioactivity recoveries from urine, rinse, final cage wash, expired CO2 and non-GI tissues. The total recovery of radioactivity from all animals was an average of 98 ±4%.
Similar pharmacokinetic parameters were observed in rats for either dose level. The time-course of 14C-2-butoxyethyl benzoate-derived radioactivity in plasma exhibited a biphasic decline after reaching Cmax and was therefore fit to a two-compartment pharmacokinetic model. Plasma radioactivity declined rapidly during the α phase (t½α = 3-6 hours), followed by a slower decline during the terminal β phase (t½β = 47-52 hours).
Between 8 and 15% of the orally administered 14C-2-butoxyethyl benzoate remained in the tissues after 168 hours (7 days) post-dosing in all of the groups. The majority (73-82%) however, was recovered in the non-systemic organ tissues (skin and carcass). The highest levels in any systemic organs (i.e. liver) were only 1-2% indicating low bioaccumulation potential of 14C-2-butoxyethyl benzoate in rats.
The majority (52-68%) of the administered 14C-2-butoxyethyl benzoate derived radioactivity was rapidly excreted in urine without any significant difference between the dose levels. Also, the majority of the urinary elimination (91-95%) occurred within the first 24 hours post-dosing. Most of the remaining oral dose (16-26%) was eliminated in expired CO2, with the majority of the expired CO2 elimination (83-87%) occurring within the first 48 hours post-dosing.
A total of nineteen radiochemical peaks were detected in the urine and/or fecal samples across the profiles of all 3 groups (Groups 1-3). A total of eight radiochemical peaks were detected in Cmax blood samples. Only three major peaks of nineteen radiochemical peaks in the urine and/or fecal samples accounted for more than 5% of the administered dose. No parent 2-butoxyethyl benzoate was detected in the urine or Cmax blood samples.
2-butoxyethyl benzoate was detected in the fecal samples at the level of less than 0.5% of administered dose.
Among these three major peaks detected in urine and /or fecal samples, the most abundant metabolite (Peak 17) accounted for ~14 % to ~32 % of the administered dose in all 3 groups and was identified as 2-butoxyacetic acid, the second most abundant metabolite (Peak 13) accounted for ~3.4 % to ~7.6 % of the administered dose in all 3 groups and was identified as glucuronide conjugate of 2-butoxyethanol, and the third most abundant metabolite (Peak 2) accounted for ~3.3 % to ~6.2 % of the administered dose in all 3 groups and was identified as ethylene glycol. Both ethylene glycol and 2-butoxyacetic acid were also present in Cmax blood samples. Across both sexes, 2-butoxyacetic acid was the most abundant metabolite in Cmax blood samples, although females had approximately equal amounts of ethylene glycol present as well.
Other identified urine and/or fecal minor metabolites, which accounted for less than 5% of administered dose, included (3-oxobutoxy)acetic acid, glucuronide conjugate of 4-(2- hydroxyethoxy)butan-2-ol and glycine conjugate of 2-butoxyacetic acid. None of these minor metabolites were present in Cmax blood samples.
The major metabolites (ethylene glycol, 2-butoxyacetic acid, glucuronide of 2-butoxyethanol and 2-butoxyethanol) were identified as the same major metabolites from previous ADME study where rats were orally administered with molar equivalent dosage of 2-butoxyethanol, indicating that 2-butoxyethyl benzoate and 2-butoxyethanol were metabolized in similar metabolic pathways in rats.
In summary, administered 14C-2-butoxyethyl benzoate-derived radioactivity was quickly absorbed, highly metabolized and eliminated in urine and CO2 from the rat, with tissue residues in the skin and carcass. Absorption, distribution, metabolism and elimination appeared to be similar with single and repeat dosing. Similar rates of absorption distribution, and elimination were observed between male and female rats for either dose group. 2-butoxyethyl benzoate and 2-butoxyethanol were metabolized in similar metabolic pathways in rats.

Executive summary:

A pharmacokinetic study was conducted in F344/DuCrl rats for 168 hours (7 days) postdosing to determine absorption, distribution and excretion of 14C-2-butoxyethyl benzoate following oral exposure (single dose of 25 or 250 mg/kg bw or repeat dose of 25 mg/kg bw).

Orally administered 14C-2-butoxyethyl benzoate was absorbed rapidly without any apparent lag time. The percent absorption of the orally administered dose in the low and high dose groups was at least 88-95%, based on summarized total radioactivity recoveries from urine, rinse, final cage wash, expired CO2 and non-GI tissues. The total recovery of radioactivity from all animals was an average of 98 ±4%.

Similar pharmacokinetic parameters were observed in rats for either dose level. The timecourse of 14C-2-butoxyethyl benzoate-derived radioactivity in plasma exhibited a biphasic decline after reaching Cmax and was therefore fit to a two-compartment pharmacokinetic model. Plasma radioactivity declined rapidly during the α phase (t½α = 3-6 hours), followed

by a slower decline during the terminal β phase (t½β = 47-52 hours).

Between 8 and 15% of the orally administered 14C-2-butoxyethyl benzoate remained in the tissues after 168 hours (7 days) post-dosing in all of the groups. The majority (73-82%) however, was recovered in the non-systemic organ tissues (skin and carcass). The highest levels in any systemic organs (i.e. liver) were only 1-2% indicating low bioaccumulation potential of 14C-2-butoxyethyl benzoate in rats.

The majority (52-68%) of the administered 14C-2-butoxyethyl benzoate derived radioactivity was rapidly excreted in urine without any significant difference between the dose levels. Also, the majority of the urinary elimination (91-95%) occurred within the first 24 hours post-dosing. Most of the remaining oral dose (16-26%) was eliminated in expired CO2, with the majority of the expired CO2 elimination (83-87%) occurring within the first 48 hours post-dosing.

A total of nineteen radiochemical peaks were detected in the urine and/or fecal samples across the profiles of all 3 groups (Groups 1-3). A total of eight radiochemical peaks were detected in Cmax blood samples. Only three major peaks of nineteen radiochemical peaks in the urine and/or fecal samples accounted for more than 5% of the administered dose. No parent 2-butoxyethyl benzoate was detected in the urine or Cmax blood samples. 2-butoxyethyl benzoate was detected in the fecal samples at the level of less than 0.5% of administered dose.

Among these three major peaks detected in urine and /or fecal samples, the most abundant metabolite (Peak 17) accounted for ~14 % to ~32 % of the administered dose in all 3 groups and was identified as 2-butoxyacetic acid, the second most abundant metabolite (Peak 13) accounted for ~3.4 % to ~7.6 % of the administered dose in all 3 groups and was identified as glucuronide conjugate of 2-butoxyethanol, and the third most abundant metabolite (Peak 2) accounted for ~3.3 % to ~6.2 % of the administered dose in all 3 groups and was identified as ethylene glycol. Both ethylene glycol and 2-butoxyacetic acid were also present in Cmax blood samples. Across both sexes, 2-butoxyacetic acid was the most abundant metabolite in Cmax blood samples, although females had approximately equal amounts of ethylene glycol present as well.

Other identified urine and/or fecal minor metabolites, which accounted for less than 5% of administered dose, included (3-oxobutoxy)acetic acid, glucuronide conjugate of 4-(2-hydroxyethoxy)butan-2-ol and glycine conjugate of 2-butoxyacetic acid. None of these minor metabolites were present in Cmax blood samples.

The major metabolites (ethylene glycol, 2-butoxyacetic acid, glucuronide of 2-butoxyethanol and 2-butoxyethanol) were identified as the same major metabolites from previous ADME study where rats were orally administered with molar equivalent dosage of 2-butoxyethanol, indicating that 2-butoxyethyl benzoate and 2-butoxyethanol were metabolized in similar metabolic pathways in rats.

In summary, administered 14C-2-butoxyethyl benzoate-derived radioactivity was quickly absorbed, highly metabolized and eliminated in urine and CO2 from the rat, with tissue residues in the skin and carcass. Absorption, distribution, metabolism and elimination appeared to be similar with single and repeat dosing. Similar rates of absorption distribution, and elimination were observed between male and female rats for either dose group. 2-butoxyethyl benzoate and 2-butoxyethanol were metabolized in similar metabolic pathways in rats.

Description of key information

A pharmacokinetic study was conducted in F344/DuCrl rats for 168 hours (7 days) postdosing to determine absorption, distribution and excretion of 14C-2-butoxyethyl benzoate following oral exposure (single dose of 25 or 250 mg/kg bw or repeat dose of 25 mg/kg bw).

Orally administered 14C-2-butoxyethyl benzoate was absorbed rapidly without any apparent lag time. The percent absorption of the orally administered dose in the low and high dose groups was at least 88-95%, based on summarized total radioactivity recoveries from urine, rinse, final cage wash, expired CO2 and non-GI tissues. The total recovery of radioactivity from all animals was an average of 98 ±4%.

Between 8 and 15% of the orally administered 14C-2-butoxyethyl benzoate remained in the tissues after 168 hours (7 days) post-dosing in all of the groups. The majority (73-82%) however, was recovered in the non-systemic organ tissues (skin and carcass). The highest levels in any systemic organs (i.e. liver) were only 1-2% indicating low bioaccumulation potential of 14C-2-butoxyethyl benzoate in rats.

The majority (52-68%) of the administered 14C-2-butoxyethyl benzoate derived radioactivity was rapidly excreted in urine without any significant difference between the dose levels. Also, the majority of the urinary elimination (91-95%) occurred within the first 24 hours post-dosing. Most of the remaining oral dose (16-26%) was eliminated in expired CO2, with the majority of the expired CO2 elimination (83-87%) occurring within the first 48 hours post-dosing.

Only three major peaks of the nineteen radiochemical peaks in the urine and/or fecal samples accounted for more than 5% of the administered dose. No parent 2-butoxyethyl benzoate was detected in the urine or Cmax blood samples. 2-butoxyethyl benzoate was detected in the fecal samples at the level of less than 0.5% of administered dose.

Among these three major peaks detected in urine and /or fecal samples, the most abundant metabolite accounted for ~14 % to ~32 % of the administered dose and was identified as 2-butoxyacetic acid, the second most abundant metabolite accounted for ~3.4 % to ~7.6 % of the administered dose and was identified as glucuronide conjugate of 2-butoxyethanol, and the third most abundant metabolite accounted for ~3.3 % to ~6.2 % of the administered dose and was identified as ethylene glycol. Both ethylene glycol and 2-butoxyacetic acid were also present in Cmax blood samples.

The major metabolites (ethylene glycol, 2-butoxyacetic acid, glucuronide of 2-butoxyethanol and 2-butoxyethanol) were identified as the same major metabolites from previous ADME studies where rats were orally administered with molar equivalent dosage of 2-butoxyethanol, indicating that 2-butoxyethyl benzoate and 2-butoxyethanol were metabolized in similar metabolic pathways in rats.

Key value for chemical safety assessment

Bioaccumulation potential:

low bioaccumulation potential