2-butoxyethyl benzoate

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

There is one key GLP-study available for 2-butoxyethyl benzoate. An OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproductive/Developmental Toxicity Screening Test).

Link to relevant study records

Reference

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

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: USEPA OPPTS 870.3650 (2000)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Specific details on test material used for the study:

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

Species:

rat

Strain:

other: Crl:CD(SD)

Details on species / strain selection:

Strain and Justification:
Crl:CD(SD) rats were selected because of their general acceptance and suitability for toxicity testing, availability of historical control data and the reliability of the commercial supplier.

Sex:

male/female

Details on test animals or test system and environmental conditions:

Species and Sex: Rats (male and female)
Strain and Justification: Crl:CD(SD) rats were selected because of their general acceptance and suitability for toxicity testing, availability of historical control data and the reliability of the commercial supplier.
Supplier and Location: Charles River (Raleigh, North Carolina)
Age at Study Start: Approximately eight weeks of age at initiation of treatment.

Physical and Acclimation:
During the acclimation period each animal was evaluated by a veterinarian trained in the field of Laboratory Animal Medicine, or a trained animal/toxicology technician, to determine the general health status and acceptability for study purposes. The Toxicology and Environmental Research and Consulting Laboratory was fully accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC International). Prior to randomization, the animals were housed two per cage in stainless steel solid bottom cages with corncob bedding, in rooms designed to maintain adequate conditions (temperature, humidity, and photocycle). Animals were acclimated to the laboratory for at least one week prior to the start of the study.

Housing:
After assignment to the study, animals were single housed in solid bottom stainless steel cages containing ground corn cob bedding, except during breeding and during the gestation and littering phases of the study. During breeding, one male and one female were placed in stainless steel cages with wire mesh floors that were suspended above catch pans in order to better visualize copulation and plugs. During gestation and littering, dams (and their litters) were housed in plastic cages provided with ground corn cob bedding from approximately GD 0 until LD 4. Cages contained a feed crock and a pressure activated lixit valve-type watering system. The following environmental conditions were maintained in the animal room.
Temperature: 22°C with a range of 20°C-26°C
Humidity: 50% with a range of 30-70%
Air Changes: 10-15 times/hour (average)
Photoperiod: 12-hour light/dark (on at 6:00 a.m. and off at 6:00 p.m.)

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

Randomization and Identification:
Prior to test material administration, animals were stratified by body weight and then randomly assigned to treatment groups using a computer program designed to increase the probability of uniform group mean weights and standard deviations at the start of the study. Animals that were placed on study were uniquely identified via subcutaneously implanted transponders (BioMedic Data Systems, Seaford, Delaware) that were correlated to unique alphanumeric identification numbers (Text Table 2). If a transponder stopped functioning or was lost, it was replaced with a new transponder that was correlated with the unique animal number.

Feed and Water:
Feed and municipal water were provided ad libitum. Animals were provided LabDiet Certified Rodent Diet #5002 (PMI Nutrition International, St. Louis, Missouri) in meal form. Analyses of the feed were performed by PMI Nutrition International to confirm the diet provided adequate nutrition and to quantify the levels of selected contaminants. Drinking water obtained from the municipal water source was periodically analyzed for chemical parameters and biological contaminants by the municipal water department. In addition, specific analyses for chemical contaminants were conducted at periodic intervals by an independent testing facility. Copies of these analyses are maintained in the study file.

Animal Welfare:
In accordance with the U.S. Department of Agriculture animal welfare regulations, 9 CFR, Subchapter A, Parts 1-4, the animal care and use activities required for conduct of this study were reviewed and approved by the Institutional Animal Care and Use Committee (IACUC). The IACUC determined that the proposed Activities were in full accordance with these Final Rules. The IACUC-approved Animal Care and Use Activities used for this study were DART 01, DCO 01, Neuro Tox 01, Humane Endpoints 01, Blood Collection 01, Tissue Collection 01 and Animal ID 01.

Route of administration:

oral: feed

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.

Dose / conc.:

500 ppm

Dose / conc.:

1 500 ppm

Dose / conc.:

5 000 ppm

No. of animals per sex per dose:

12/sex/dose group

Control animals:

yes, plain diet

Details on study design:

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

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

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

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: 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.

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

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

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

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

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.

Remarks on result:

other: NOEL was not determined for offspring.

Critical effects observed:

no

Key result

Reproductive effects observed:

no

Analytical Chemistry:

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

Conclusions:

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.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

350 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

GLP/Guideline study.

Additional information

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

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. 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.

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 (see Specific Investigations: Other Studies - Haematoxicity for human relevance). Females given 5000 ppm also had a treatment-related higher platelet count, which may have been caused by a generalized increase in platelet production within the bone marrow in association with the reticulocytosis.

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

Treatment-related effects on organ weight were observed only in the liver of females at the high dose level (5000 ppm). 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 5000 ppm. The hepatocellular hypertrophy was interpreted to be a non-adverse and adaptive effect.

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.

Effects on developmental toxicity

Description of key information

There is one key GLP-study available for 2-butoxyethyl benzoate. An OECD Guideline 414 (Prenatal Develpmental Toxicity Study).

Link to relevant study records

Reference

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

October 19, 2015 to July 27, 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.31 (Prenatal Developmental Toxicity Study)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Specific details on test material used for the study:

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

Species:

rat

Strain:

other: Crl:CD(SD)

Details on test animals or test system and environmental conditions:

Species and Sex: Rats, time-mated females
Strain and Justification: Crl:CD(SD) rats were selected because of their general acceptance and suitability for toxicity testing, availability of historical background data, and the reliability of the commercial supplier.
Supplier and Location: Charles River Laboratories (Raleigh, North Carolina)
Age and Weight at Study Start: Sexually mature adult weighing approximately 200-250 g

Physical and Acclimation:
During the acclimation period each animal was evaluated by a veterinarian trained in the field of Laboratory Animal Medicine, or a trained animal/toxicology technician, to determine the general health status and acceptability for study purposes. The Toxicology and Environmental Research and Consulting Laboratory is fully accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC International). The animals were housed one per cage in stainless steel cages, in rooms designed to maintain adequate conditions (temperature, humidity, and photocycle), and acclimated to the laboratory for at least four days prior to the start of dosing.

Housing:
After assignment, animals were housed one per cage in stainless steel cages. Cages had solid floors with corncob bedding. Cages contained a feed crock and a pressure activated lixit valve-type watering system. The following environmental conditions were targeted in the animal room, however temporary excursions from these environmental conditions may have occurred on an infrequent basis; all observed ranges were documented in the study file.
Temperature: 22°C with a range of 20°C-26°C
Humidity: 50% with a range of 30-70%
Air Changes: 10-15 times/hour (average)
Photoperiod: 12-hour light/dark (on at 6:00 a.m. and off at 6:00 p.m.)
Note: Photoperiod times may have changed due to study-related activities.

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 cage contained paper nesting material.

Randomization and Identification:
Animals were stratified by GD 0 body weight and then randomly assigned to treatment groups using a computer program designed to increase the probability of uniform mean group 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. 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:
Animals were provided LabDiet Certified Rodent Diet #5002 (PMI Nutrition International, St. Louis, Missouri) in meal form. Feed and municipal water were provided ad libitum. Analyses of the feed were performed by PMI Nutrition International to confirm the diet provided adequate nutrition and to quantify the levels of selected contaminants. Drinking water obtained from the municipal water source was periodically analysed for chemical parameters and biological contaminants by the municipal water department. In addition, specific analyses for chemical contaminants were conducted at periodic intervals by an independent testing facility. There were no contaminants in either the feed or water at levels that would have adversely impacted the results or interpretation of this study. Copies of these analyses are maintained in the study file.

Animal Welfare:
In accordance with the U.S. Department of Agriculture animal welfare regulations, 9 CFR, Subchapter A, Parts 1-4, the animal care and use activities required for conduct of this study were reviewed and approved by the Institutional Animal Care and Use Committee (IACUC). The IACUC determined that the proposed Activities were in full accordance with these Final Rules. The IACUC-approved Animal Care and Use Activities used for this study were DART 02, Humane Endpoints 01, and Animal ID 01.

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on exposure:

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

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analysis:
Concentration Verification and Homogeneity:
Dose confirmation analyses of all dose levels, plus control and premix, were determined pre-exposure. The homogeneity of the low-dose and the high-dose test diets was 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) has shown 2-butoxyethyl benzoate to be stable in diet for at least 65 days at concentration
levels ranging from 0.0005 to 10%. The established concentration range and duration spanned those used in this study; therefore, additional stability analyses
were not conducted.

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

Details on mating procedure:

Breeding Procedures:
Sexually mature virgin females were naturally mated with males of the same strain (one male:one female) at the supplier’s facility. Females were checked for in situ copulation plugs the following morning and those found with such a plug were removed from the males' cages. The day on which a vaginal plug was detected was considered GD 0. GD 0 body weights were provided by the supplier, and maintained in the study record. Rats
arrived in our laboratory on GD 1 or 2.

Duration of treatment / exposure:

Groups of 24 time-mated female Crl:CD(SD) rats were administered 2-butoxyethyl benzoate in the diet at dose levels of 0, 500, 1500, or 5000 ppm on GD 6-21.

Frequency of treatment:

Test material was administered in the diet seven days/week from GD 6-21 as recommended by the applicable guideline.

Duration of test:

Rats were administered 2-butoxyethyl benzoate in the diet on GD 6-21 and were necropsied on GD 21.

Dose / conc.:

500 ppm

Dose / conc.:

1 500 ppm

Dose / conc.:

5 000 ppm

No. of animals per sex per dose:

24 females/dose group.

Control animals:

yes, plain diet

Details on study design:

Route, Method of Administration, Frequency, Duration and Justification:
Test material was administered in the diet from GD 6-21 as recommended by the applicable guideline. Oral exposure is a possible route of human 2-butoxyethyl benzoate exposure; therefore, oral administration of 2-butoxyethyl benzoate to rats via diet represented an appropriate means of exposure. Compared to gavage administration, additional advantages of the dietary route of oral exposure include the provision of a stable and consistent systemic exposure throughout all stages of embryo/fetal development (Rasoulpour et al., 2010), reductions in stress, avoidance of potential vehicle-induced confounding effects, and the elimination of potential dosing-related injuries to the maternal animals (Cooper et al., 2006).

Dose Levels and Justification:
Dose levels for this study were selected on the basis of the developmental toxicity probe study discussed previously. The high dose is expected to induce signs of maternal toxicity, including changes in hematologic parameters indicative of anemia and/or increased spleen weight. The lower dose levels were selected to provide dose response data for any toxicity that may be observed among the high-dose group rats.

Maternal examinations:

Daily Observations:
A cage-side examination was conducted twice daily, approximately at the same time each day. This examination was typically performed with the animals in their cages and was designed to detect significant clinical abnormalities that are 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 have been 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.

Clinical Observations:
Clinical observations were conducted on all animals at least once daily. These examinations were performed at approximately the same time each examination day. 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.

Body Weights/Body Weight Gains:
Body weights were recorded on GD 0 by the supplier and then daily from GD 6-21, at the conducting lab. Statistical analysis of body weights was performed using data collected on GD 0, 6, 9, 12, 15, 18, and 21. Statistical analysis of body weight gains was conducted for the following intervals: GD 0-6, 6-9, 9-12, 12-15, 15-18, 18-21, 6-21, and 0-21.

Feed Consumption:
Feed consumption was recorded and statistically analyzed for all animals every three days from GD 3-21 by weighing feed containers at the start and end of a measurement
cycle. Feed consumption was calculated using the following equation:
Feed consumption (g/day) = (initial weight of feed container - final weight of feed container)/(# of days in measurement cycle)

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

Toxicokinetic Data:
A terminal blood sample was taken from the first four pregnant dams/group that did not have scratched feed at the time of collection (16 total) and their respective litters at necropsy, on GD 21. Dam blood was collected via the orbital sinus following anesthesia with a mixture of isoflurane vapors and medical oxygen at the scheduled necropsy and fetal blood was collected (maximum of 60 μL/fetus) via the umbilical cord (pooled by litter). Blood samples were collected into tubes with solvent (acetonitrile) and stored in the -80°C freezer until analysis. Analyses of the blood samples were conducted to determine parent compound, 2-butoxyethyl benzoate, and expected major metabolites, 2-butoxyethanol and 2-butoxyacetic acid, using liquid chromatography with tandem mass spectrometry detection (LC/MS-MS) (Perala, 2016).
Toxicokinetic evaluation of the chemical analysis results was conducted to evaluate dose proportionality and systemic levels of 2-butoxyethyl benzoate and the expected major metabolites in dams and fetuses by the dietary route of administration.

Clinical Pathology:
Animals were not 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.
Hematology:
Sample Preparation:
Blood samples for a complete blood count were mixed with ethylenediaminetetraacetic acid (EDTA). Blood smears were prepared, stained with Wright- Giemsa stain, cover-slipped and archived for potential future evaluation if warranted.
Hematologic parameters were assayed using the Advia 120 Hematology Analyzer (Siemens Healthcare Diagnostics, Tarrytown, New York).

Necropsy:
On GD 21, all surviving animals (not fasted) were anesthetized with a mixture of isoflurane vapors and medical oxygen, and a blood sample was obtained from the orbital sinus. The animals were then euthanized by carbon dioxide inhalation, and a limited gross pathologic examination (necropsy) was performed. The sequence of the maternal necropsies were counterbalanced across groups (e.g., control, high, middle, low) to control for potential confounding influences of timing on fetal growth and skeletal ossification.
The maternal necropsy included an examination of the external tissues and all orifices. The skin was reflected from the carcass, the thoracic and abdominal cavities were opened and the viscera were examined. The liver, kidneys, and spleen were dissected from the carcass and were incised. Any obvious gross pathologic alterations were recorded, and the weight of the liver, kidneys, spleen, and gravid uterus were recorded. The ratios of liver, kidney, and spleen weights to terminal body weight were calculated. Representative portions of liver, kidneys, spleen, and gross lesions were preserved in neutral, phosphate-buffered 10% formalin. Microscopic examination of the liver, kidneys, spleen and gross lesions was not conducted. Transponders were removed and placed in with the preserved tissues.

Ovaries and uterine content:

A detailed examination of the reproductive tract was performed, and the number and position of implantations, viable fetuses, dead fetuses, and resorptions was recorded. Resorptions were classified as either “early” or “late” based on the presence (late resorption) or absence (early resorption) of grossly recognizable embryonic/fetal form, while a “dead fetus” indicates a very recent death as evidenced by a lack of external degenerative changes. For females with one or more viable fetuses, the number of ovarian corpora lutea was counted. The uteri of females lacking visible implantations were stained with a 10% aqueous solution of
sodium sulfide based on (Kopf et al., 1964) and examined for evidence of early resorptions in order to verify pregnancy status.

Fetal examinations:

The sex and body weight of all viable fetuses were recorded. All fetuses were given an external examination that included observations on body proportions, the head and face (including closure of the palate), abdomen, spine, extremities, genitalia, rectum and tail. All viable fetuses were euthanized by sublingual oral administration of sodium pentobarbital solution. Approximately one half of all the fetuses in each litter were chosen randomly via computer for visceral examination conducted by dissection under a low power stereomicroscope for evidence of visceral alterations (Staples, 1974; Stuckhardt and Poppe, 1984). The visceral examination included observation of the thymus, trachea, esophagus, lungs, great vessels, heart (external and internal), liver, gastrointestinal tract, pancreas, spleen, kidney (sectioned), adrenal glands, ureters, bladder and reproductive organs. The heads of these fetuses were removed, placed in Bouin’s fixative and serially sectioned to allow for inspection of the eyes, brain, nasal passages and tongue (Wilson, 1965). The remaining fetuses not selected for visceral examination were then skinned, eviscerated, preserved in alcohol and double stained with Alcian Blue and Alizarin Red S for cartilage and bone according to methods based on Trueman
et al. (1999) and Zablotny (2002). A thorough evaluation of the fetal skeleton was conducted on the remaining fetuses not selected for visceral examination.
However, a fetus may have been intentionally changed from one selected for visceral examination to one processed for skeletal examination (and vice versa) if it was deemed that such examination provided more meaningful data about a suspected abnormality.
All fetal alterations were classified as a variation or malformation. A variation is defined as a divergence beyond the normal range of structural constitution that may not adversely affect survival or health. A malformation is defined as a permanent structural change that may adversely affect survival, development or function and/or which occurs at a relatively low incidence in the specific species/strain. The maternal necropsy and fetal examinations were conducted such that investigators were blind to treatment group assignment.

Statistics:

For detailed description of the statistics performed, see "Any other infomation on materials and methods inlc. tables" section.

The following is the list of statistical measurements used:
Bartlett’s test (alpha = 0.01; Winer, 1971)
Parametric (Steel and Torrie, 1960) or nonparametric (Hollander and Wolfe, 1973) analysis of variance (ANOVA)
Dunnett's test (alpha = 0.05; Winer, 1971)
Wilcoxon Rank-Sum test (alpha = 0.05; Hollander and Wolfe, 1973)
Bonferroni's correction (Miller, 1966)
censored Wilcoxon test (Haseman and Hoel,1974)
Fisher exact probability test (alpha = 0.05; Siegel, 1956)
Binomial distribution test
Statistical outliers (alpha = 0.02) were identified by the sequential method of Grubbs (1969)
Descriptive statistics (i.e., mean ± standard deviation)
Linear regression

Indices:

Rregnancy rates, litter size, numbers of corpora lutea or implantations, percent preimplantation loss, percent postimplantation loss, fetal sex ratios, fetal body weights and gravid uterine weights.

Historical control data:

Historical control data was used in the evaluation of selected organ weights and mean fetal weights.

Clinical signs:

no effects observed

Description (incidence and severity):

Examinations performed on all animals revealed no treatment-related findings. 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 cageside observations.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Description (incidence):

All animals survived until scheduled termination.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

When compared to controls, dams provided with 5000 ppm 2-butoxyethyl benzoate had statistically-identified and treatment-related decreases in maternal body weight gain during the GD 18-21 interval, along with a 10.5% reduction in body weight gain over the GD 6-21 period. Additionally, the largest decrease (22.6%) in body weight gain occurred during the GD 18-21 interval. The body weight gain decrements at 5000 ppm correlated with decreases in feed consumption during the GD 18-21 interval. There were no treatment-related decreases in gestation body weights at any dose level tested or body weight gains at 500 or 1500 ppm.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Dams in the 5000 ppm group had treatment-related decreases in feed consumption during the GD 18-21 interval that was statistically identified and 13% lower than controls. There were no treatment-related effects on feed consumption in animals in the 500 and 1500 ppm groups. Rats were given 0, 500, 1500, or 5000 ppm 2-butoxyethyl benzoate in rodent feed, which corresponded to time-weighted average doses of 0, 37.4, 109, or 352 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):

Treatment-related hematologic effects in dams given 5000 ppm consisted of statistically identified lower mean red blood cell count and hemoglobin concentration, hematocrit, higher mean MCV, lower mean MCHC, and a statistically identified higher mean reticulocyte count. These hematologic effects were representative of regenerative anemia in dams given 5000 ppm, and were interpreted to be adverse. There were no treatment-related hematologic effects in dams given 500 or 1500 ppm. Absolute and differential white blood cell counts did not reveal any treatment-related differences at any dose level.

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

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):

There were no statistically-significant or treatment-related differences in final body weight for females. Dams administered 5000 ppm had statistically significant and treatment-related increases in absolute and relative spleen weights of 31.9% and 35.3%, respectively. Relative liver and kidney weights in dams given 5000 ppm were statistically-identified, but were interpreted to be unrelated to treatment as weights were near or within historical control values from this laboratory. There were no treatment-related organ weight effects in dams given 500 or 1500 ppm.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Dams administered 5000 ppm had treatment-related increases in the incidence of dark spleen and increased size of the spleen in four or two dams, respectively. All other gross pathologic observations were considered to be spontaneous alterations, unassociated with exposure.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Details on results:

Maternal toxicity was limited to dams given 5000 ppm and consisted of the following treatment-related effects: decreases in body weight gain, feed consumption, increases in spleen weights, and hematological effects. See attachment for relevant tables.

Number of abortions:

no effects observed

Pre- and post-implantation loss:

no effects observed

Total litter losses by resorption:

no effects observed

Early or late resorptions:

no effects observed

Dead fetuses:

no effects observed

Changes in pregnancy duration:

not examined

Description (incidence and severity):

Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): not examined

Changes in number of pregnant:

no effects observed

Details on maternal toxic effects:

There were no significant treatment related effects on pregnancy rates, litter size, numbers of corpora lutea or implantations, percent preimplantation loss, percent postimplantation loss, fetal sex ratios, fetal body weights, or gravid uterine weights at any dose level.

Key result

Dose descriptor:

NOEL

Effect level:

1 500 ppm

Based on:

test mat.

Basis for effect level:

body weight and weight gain

food consumption and compound intake

haematology

organ weights and organ / body weight ratios

Key result

Abnormalities:

no effects observed

Fetal body weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

The mean male, female, and combined fetal body weights in the 5000 ppm group were statistically identified as decreased relative to controls. These findings were considered to be unrelated to treatment as the male, female, and combined fetal weights were within the laboratory recent historical control range.
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): not examined

Reduction in number of live offspring:

no effects observed

Changes in sex ratio:

no effects observed

Changes in litter size and weights:

not examined

Changes in postnatal survival:

not examined

External malformations:

no effects observed

Description (incidence and severity):

There were no treatment-related external alterations in any dose group. Incidental findings bearing no relationship to treatment included the malformation anasarca in one fetus in the control group. There were no craniofacial alterations recorded in any dose group.

Skeletal malformations:

no effects observed

Description (incidence and severity):

There were no treatment-related skeletal alterations in any dose group. No skeletal malformations were noted in any dose group. Incidental findings bearing no
relationship to treatment included the variations of delayed ossification (DO) zygomatic, DO thoracic centra, DO ribs, DO sternebrae, calloused ribs, class I and
II wavy ribs, an irregular pattern of ossification sternebrae, and an extra site of ossification sternebra. Given that these observations occurred in the control group, at low frequencies, and/or lacked a dose response, these observations were considered spurious and unrelated to treatment.

Visceral malformations:

no effects observed

Description (incidence and severity):

There were no treatment-related visceral alterations in any dose group. Incidental findings bearing no relationship to treatment included the malformation situs
inversus observed in one fetus in the 1500 ppm dose group and the malformations ectopic ovary and ectopic uterus observed in one fetus in the 500 ppm dose group. Additional incidental findings bearing no relationship to treatment included the variations of supernumerary hepatic lobule, hemorrhage of the liver or adrenal, and a bifurcated renal vein. Given that these observations occurred at low frequencies, and/or lacked a dose response, these observations were considered spurious and unrelated to treatment.

Key result

Dose descriptor:

NOEL

Effect level:

5 000 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Administration of 2-butoxyethyl benzoate in the diet at dose levels up to and including 5000 ppm produced no indications of embryo/fetal toxicity or teratogenicity.

Key result

Abnormalities:

no effects observed

Key result

Developmental effects observed:

no

Analytical:

Analyses of all test diets from the initial mix of the study revealed acceptable mean concentrations ranging from 97.5 to 97.8% of targeted concentrations. Analyses of the

low- and high-dose test diets indicated that the test material was homogeneously distributed based on relative standard deviations of ≤ 1.1%.

Fetal Examination Summary:

There were no statistically identified or treatment-related differences in the incidence of any fetal alteration in any of the treated groups compared to controls. The small number

of alterations observed in fetuses from dams administered 2-butoxyethyl benzoate either occurred at low frequencies and/or were not dose related.

Toxicokinetic Data:

Collected blood samples of all non-fasted dams and fetuses on GD 21 were quantitatively analyzed by LC/MS-MS to determine the terminal blood concentrations of parent

compound, 2-butoxyethyl benzoate, and the expected major metabolites, 2-butoxyethanol and 2-butoxyacetic acid (McFadden, 2016). The blood concentrations of

parent 2-butoxyethyl benzoate and metabolites (2-butoxyethanol and 2-butoxyacetic acid) were analyzed against the dietary dose levels via the toxicokinetic analysis method

described above.

Overall, the toxicokinetic results for the metabolite, 2-butoxyacetic acid in both dam and fetal plasma samples are suggestive of possible induction of both alcohol dehydrogenase

and aldehyde dehydrogenase as the metabolite 2-butoxyacetic acid was formed from 2-butoxyethanol via these enzymes (Ghanayem et al., 1987). The possible induction of

both alcohol dehydrogenase and aldehyde dehydrogenase could impact the metabolic pathways of 2-butoxyethyl benzoate. As the metabolite 2-butoxyacetic acid showed

nonlinear kinetics (supralinear) at the high dose (5000 ppm) in both dam and fetal blood samples, subsequent studies in rats should be conducted at a kinetically-derived maximum dose level of less than 5000 ppm 2-butoxyethyl benzoate.

Conclusions:

Maternal toxicity was limited to dams given 5000 ppm and consisted of the following treatment-related effects: decreases in body weight gain, feed consumption, increases in spleen weights, and hematological effects. Dams provided 5000 ppm had a statisticallyidentified treatment-related 10.5% decrease in maternal body weight gain throughout the GD 6-21 treatment period and a 22.6% decrease during the GD 18-21 interval. These body weight gain effects correlated with decreases in feed consumption during the GD 18-21 interval. Treatment-related hematological effects consisted of statisticallyidentified lower mean red blood cell count and hemoglobin concentration, hematocrit, higher mean corpuscular volume (MCV), lower mean corpuscular hemoglobin concentration (MCHC), and a statistically identified higher mean reticulocyte count. These hematological effects were representative of regenerative anemia. At necropsy, there were treatment-related increases in absolute and relative spleen weights of 31.9% and 35.3%, respectively. Treatment-related gross pathological changes included dark spleens in four dams and an increased size of the spleen in two of the four dams. There was no treatment-related maternal toxicity in the 500 or 1500 ppm dose groups.
Administration of 2-butoxyethyl benzoate in the diet at dose levels up to and including 5000 ppm produced no indications of embryo/fetal toxicity or teratogenicity.
Chemical analysis results showed that quantifiable 2-butoxyethyl benzoate was not present in the control dam and fetal blood samples but was quantifiable at very low levels in blood from a single 1500 ppm-treated dam and four of the twelve treated pooled fetal samples (two litters at 500 ppm, one litter at 1500 ppm, and one litter at 5000 ppm). The blood levels of 2-butoxyethyl benzoate these samples ranged from 0.0181 μg/g (5000 ppm litter) to 0.0303 μg/g (1500 ppm dam) at the LOQ (Limit of Quantitation) ranging from 0.0138 to 0.0243 μg/g. Possible contamination or matrix effect for contributing these low levels of 2-butoxyethyl benzoate could not be definitively discounted. Quantifiable 2-butoxyethanol was not present in the control dam and fetal blood samples but was present in all treated dam samples (0.0283-2.46 μg/g), and most of treated fetal samples (0.0381-3.98 μg/g where present). Quantifiable 2-butoxyacetic acid was present in all treated dam samples and pooled fetal samples (1.05-41.7 μg/g), and a single control dam and its corresponding pooled fetal sample at low levels (0.0355 μg/g and 0.0303 μg/g, respectively). Possible analytical matrix effects and/or sample contamination could not be definitively discounted for the quantifiable levels of 2-butoxyacetic acid in these control samples. Similar levels of both 2-butoxyethanol and 2-butoxyacetic acid were observed between dams and the corresponding fetuses. 2-Butoxyethanol in blood exhibited linear kinetics across all dose levels in both fetuses and dams. However, 2-butoxyacetic acid in blood exhibited supralinear kinetics at 5000 ppm in both fetuses and dams.
Therefore, under the conditions of this study, the no-observed-effect level (NOEL) for maternal toxicity was 1500 ppm, and the embryo/fetal NOEL was 5000 ppm.

Executive summary:

The purpose of this study was to evaluate the maternal and developmental toxicity of 2-butoxyethyl benzoate in Crl:CD(SD) rats following dietary administration. Groups of 24

time-mated female Crl:CD(SD) rats were administered 2-butoxyethyl benzoate in the diet at concentrations of 0, 500, 1500, or 5000 ppm on gestation day (GD) 6-21, which

corresponded to time-weighted average doses of 0, 37.4, 109, or 352 mg/kg/day. In-life maternal study parameters included clinical observations, body weight, body weight gain

and feed consumption. On GD 21, all surviving rats were bled for a hematological evaluation, euthanized and examined for gross pathologic alterations. Liver, kidneys, spleen, and gravid uterine weights were recorded, along with the number of corpora lutea, uterine implantations, resorptions, and live/dead fetuses. All fetuses were weighed, sexed and examined for external alterations. Approximately one half of the fetuses were examined for visceral alterations while skeletal examinations were conducted on the remaining fetuses.

In addition, chemical analyses of terminal blood samples were conducted to determine parent compound, 2-butoxyethyl benzoate, and suspected major metabolites, 2-butoxyethanol and 2-butoxyacetic acid.

Maternal toxicity was limited to dams given 5000 ppm and consisted of the following treatment-related effects: decreases in body weight gain, feed consumption, increases in spleen weights, and hematological effects. Dams provided 5000 ppm had a statisticallyidentified treatment-related 10.5% decrease in maternal body weight gain throughout the GD 6-21 treatment period and a 22.6% decrease during the GD 18-21 interval. These body weight gain effects correlated with decreases in feed consumption during the GD 18-21 interval. Treatment-related hematological effects consisted of statistically-identified lower mean red blood cell count and hemoglobin concentration, hematocrit, higher mean corpuscular volume (MCV), lower mean corpuscular hemoglobin concentration (MCHC), and a statistically identified higher mean reticulocyte count. These hematological effects were representative of regenerative anemia. At necropsy, there were treatment-related increases in absolute and relative spleen weights of 31.9% and 35.3%, respectively.

Treatment-related gross pathological changes included dark spleens in four dams and an increased size of the spleen in two of the four dams. There was no treatment-related

maternal toxicity in the 500 or 1500 ppm dose groups.

Administration of 2-butoxyethyl benzoate in the diet at dose levels up to and including 5000 ppm produced no indications of embryo/fetal toxicity or teratogenicity.

Chemical analysis results showed that quantifiable 2-butoxyethyl benzoate was not present in the control dam and fetal blood samples but was quantifiable at very low levels in blood

from a single 1500 ppm-treated dam and four of the twelve treated pooled fetal samples (two litters at 500 ppm, one litter at 1500 ppm, and one litter at 5000 ppm). The blood levels of 2-butoxyethyl benzoate these samples ranged from 0.0181 μg/g (5000 ppm litter) to 0.0303 μg/g (1500 ppm dam) at the LOQ (Limit of Quantitation) ranging from 0.0138 to 0.0243 μg/g. Possible contamination or matrix effect for contributing these low levels of 2-butoxyethyl benzoate could not be definitively discounted. Quantifiable 2-butoxyethanol was not present in the control dam and fetal blood samples but was present in all treated dam samples (0.0283-2.46 μg/g), and most of treated fetal samples (0.0381-3.98 μg/g where present). Quantifiable 2-butoxyacetic acid was present in all treated dam samples and pooled fetal samples (1.05-41.7 μg/g), and a single control dam and its corresponding pooled fetal sample at low levels (0.0355 μg/g and 0.0303 μg/g, respectively). Possible analytical matrix effects and/or sample contamination could not be definitively discounted for the quantifiable levels of 2-butoxyacetic acid in these control samples. Similar levels of both 2-butoxyethanol and 2-butoxyacetic acid were observed between dams and the corresponding fetuses. 2-Butoxyethanol in blood exhibited linear kinetics across all dose levels in both fetuses and dams. However, 2-butoxyacetic acid in blood exhibited supralinear kinetics at 5000 ppm in both fetuses and dams.

Therefore, under the conditions of this study, the no-observed-effect level (NOEL) for maternal toxicity was 1500 ppm, and the embryo/fetal NOEL was 5000 ppm.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

350 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

GLP/Guideline study.

Additional information

OECD Guideline 414 (Prenatal Develpmental Toxicity Study):

Maternal toxicity was limited to dams given 5000 ppm and consisted of the following treatment-related effects: decreases in body weight gain, feed consumption, increases in spleen weights, and hematological effects. Dams provided 5000 ppm had a statistically identified treatment-related 10.5% decrease in maternal body weight gain throughout the GD 6-21 treatment period and a 22.6% decrease during the GD 18-21 interval. These body weight gain effects correlated with decreases in feed consumption during the GD 18-21 interval. Treatment-related hematological effects consisted of statistically identified lower mean red blood cell count and hemoglobin concentration, hematocrit, higher mean corpuscular volume (MCV), lower mean corpuscular hemoglobin concentration (MCHC), and a statistically identified higher mean reticulocyte count. These hematological effects were representative of regenerative anemia (see Specific Investigations: Other Studies - Haematoxicity for human relevance).

At necropsy, there were treatment-related increases in absolute and relative spleen weights of 31.9% and 35.3%, respectively. Treatment-related gross pathological changes included dark spleens in four dams and an increased size of the spleen in two of the four dams. There was no treatment-related maternal toxicity in the 500 or 1500 ppm dose groups.

Administration of 2-butoxyethyl benzoate in the diet at dose levels up to and including 5000 ppm produced no indications of embryo/fetal toxicity or teratogenicity.

Therefore, under the conditions of this study, the no-observed-effect level (NOEL) for maternal toxicity was 1500 ppm, and the embryo/fetal NOEL was 5000 ppm.

Justification for classification or non-classification

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

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 NOEL for reproductive and neurological effects in both males and females was 5000 ppm, the highest dose level tested.

OECD Guideline 414 (Prenatal Develpmental Toxicity Study):

Administration of 2-butoxyethyl benzoate in the diet at dose levels up to and including 5000 ppm produced no indications of embryo/fetal toxicity or teratogenicity.

Therefore, under the conditions of this study, the embryo/fetal NOEL was 5000 ppm.