2-butoxyethyl benzoate

Developmental toxicity / teratogenicity

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.31 (Prenatal Developmental Toxicity Study)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

2-butoxyethyl benzoate

EC Number:

226-685-8

EC Name:

2-butoxyethyl benzoate

Cas Number:

5451-76-3

Molecular formula:

C13H18O3

IUPAC Name:

2-butoxyethyl benzoate

Test material form:

other: liquid

Details on test material:

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

Specific details on test material used for the study:

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

Test animals

Species:

rat

Strain:

other: Crl:CD(SD)

Details on 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.

Administration / exposure

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.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

500 ppm

Doses / concentrations 2

Dose / conc.:

1 500 ppm

Doses / concentrations 3

Dose / conc.:

5 000 ppm

No. of animals per sex per dose:

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.

Examinations

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.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

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.

Maternal developmental toxicity

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.

Effect levels (maternal animals)

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

Maternal abnormalities

Key result

Abnormalities:

no effects observed

Results (fetuses)

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.

Effect levels (fetuses)

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.

Fetal abnormalities

Key result

Abnormalities:

no effects observed

Overall developmental toxicity

Key result

Developmental effects observed:

no

Any other information on results incl. tables

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.

Applicant's summary and conclusion

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.