2-butoxyethyl benzoate

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

July 23, 2015 to February 24, 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: USEPA OPPTS 870.3650 (2000)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

2-butoxyethyl benzoate

EC Number:

226-685-8

EC Name:

2-butoxyethyl benzoate

Cas Number:

5451-76-3

Molecular formula:

C13H18O3

IUPAC Name:

2-butoxyethyl benzoate

Test material form:

other: liquid

Details on test material:

2-Butoxyethyl benzoate, Lot # 201303443-19; Purity 99.2 %

Specific details on test material used for the study:

Test Material Name: 2-Butoxyethyl benzoate
Chemical Name: 2-Butoxyethanol benzoate
Supplier, City, State (Lot, Reference Number): The Dow Chemical Company, Midland, Michigan (Lot # 201303443-19).
Purity/Characterization (Method of Analysis and Reference): The purity of the test material was determined to be 99.2% area (corrected for water) by gas chromatography with identification by nuclear magnetic resonance and gas chromatography mass spectrometry (Gobbi, 2014).
Test Material Stability Under Storage Conditions: The test material was determined to have two years of stability under ambient storage conditions (Wachowicz et al., 2015).

Test animals

Species:

rat

Strain:

other: Crl:CD(SD)

Details on species / strain selection:

Strain and Justification:
Crl:CD(SD) rats were selected because of their general acceptance and suitability for toxicity testing, 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.

Administration / exposure

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on 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.

Details on mating procedure:

Breeding Procedure:
Breeding of the adults commenced after two weeks of treatment. Each female was placed with a single male from the same dose level (1:1 mating) until mating occurred or two weeks had elapsed. During the breeding period, daily vaginal lavage samples were evaluated for the presence of sperm as an indication of mating. The day on which sperm was detected or a vaginal copulatory plug was observed in situ was considered GD 0. The sperm- or plug-positive (presumed pregnant) females were then separated from the males and returned to their home cages. If mating had not occurred after two weeks, the animals were separated without further opportunity for mating.

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.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

500 ppm

Doses / concentrations 2

Dose / conc.:

1 500 ppm

Doses / concentrations 3

Dose / conc.:

5 000 ppm

No. of animals per sex per dose:

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).

Examinations

Parental animals: Observations and examinations:

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

Litter observations:

Litter Data:
Females were observed for signs of parturition beginning on or about GD 20. In so far as possible, parturition was observed for signs of difficulty or unusual duration. The day of parturition was recorded as the first day the presence of the litter was noted and was designated as LD 0. All litters were examined as soon as possible after delivery. The following information was recorded on each litter: the date of parturition, litter size on the day of parturition (LD 0), clinical observations and the number of live and dead pups on days 0, 1, and 4 postpartum, and the sex and body weight of each pup on LD 1 and 4. Any visible physical abnormalities or demeanor changes in the neonates were recorded as they were observed during the lactation period (see animal observations). Any pups found dead or sacrificed in moribund condition were sexed and examined grossly, if possible, for external and visceral defects and then were discarded.

Postmortem examinations (parental animals):

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.

Postmortem examinations (offspring):

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

More detailed explanation of the statistics employed can be found in the "Any other information" section.

Reproductive indices:

Reproductive indices were calculated for all dose level groups as follows:
• Female mating index = (No. females with evidence of mating/No. paired) x 100
• Male mating index = (No. males mated/No. paired) x 100
• Female conception index = (No. females with evidence of pregnancy/No. mated) x 100
• Male conception index = (No. males siring a litter/No. mated) x 100
• Female fertility index = (No. females with evidence of pregnancy/No. paired) x 100
• Male fertility index = (No. males siring a litter/No. paired) x 100
• Gestation index = (No. females delivering a litter/No. females with evidence of pregnancy) x 100
• Gestation survival index = percentage of delivered pups alive at birth
• Post-implantation loss = (No. implants – No. offspring)/(No. implants) x 100 Day 1 or 4 pup survival index = (No. viable pups on day 1 or 4/No. born live) x 100

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

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.

Dermal irritation (if dermal study):

not examined

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):

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).

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

Description (incidence and severity):

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.

Effect levels (P0)

1

Key result

Dose descriptor:

NOEL

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

haematology

clinical biochemistry

organ weights and organ / body weight ratios

histopathology: non-neoplastic

Remarks on result:

other: General Toxicity Results

2

Key result

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

3

Key result

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

Target system / organ toxicity (P0)

Key result

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:

no

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Description (incidence and severity):

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.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

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

Effect levels (F1)

Remarks on result:

other: NOEL was not determined for offspring.

Target system / organ toxicity (F1)

Critical effects observed:

no

Overall reproductive toxicity

Key result

Reproductive effects observed:

no

Any other information on results incl. tables

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%.

Applicant's summary and conclusion

Conclusions:

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). No treatment-related 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). 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). 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 nonadverse, 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). 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 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.

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.