N-butyl phenyl ether

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / bone marrow chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

1996 version

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Details on species / strain selection:

Crl:CD(SD)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories (Raleigh, North Carolina)
- Females (if applicable) nulliparous and non-pregnant: Yes
- Age at study initiation: P - 8 weeks
- Weight at study initiation: (P) Males: 277-281 g; Females: 197-200 g
- Fasting period before study: none
- Housing: Upon arrival animals were housed two or three per cage in solid bottom stainless steel cages. The solid bottom cages contained ground corn cob bedding. Cages contained a feed crock and a pressure activated lixit valve-type watering system.
After assignment to study, animals were housed singly in solid bottom stainless steel cages, except during breeding and during the gestation and littering phases of the study. The solid bottom cages contained ground corn cob bedding. During breeding, one male and one female were placed in stainless steel cages with wire mesh floors that were suspended above absorbent paper in order to better visualize copulation plugs. During gestation and littering, dams (and their litters) were housed in plastic cages provided with irradiated ground corn cob bedding from approximately GD 0 until completion of lactation (LD 4). Cages contained a feed crock and a pressure activated lixit valve-type watering system.
- Enrichment: Enrichment for rats was provided from the day of arrival until necropsy. The cage contained nylon bones during the pre-breeding phase (males and females) and following mating (all males) or paper nesting material during the gestation and lactation phases of the study.
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: at least one week
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22°C with a range of 20°C-26°C
- Humidity (%): 50% with a range of 30-70%
- Air changes (per hr): 10-15 times/hour (average)
- Photoperiod (hrs dark / hrs light):12-hour light/dark (on at 6:00 a.m. and off at 6:00 p.m.)
IN-LIFE DATES: From: To: Dosing started: June 01, 2016; Male necropsy July 6-7, 2016; Female necropsy July 13 -19, 2016. Pups euthenized on Post natal day 4.

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSE: Butyl phenyl ether was administered in corn oil and CP in distilled water, such that a dose volume of 4 ml/kg body weight yielded the targeted dose. Dose volumes were adjusted using the most current body weight. Dose solutions were not corrected for purity. Butyl phenyl dose solutions were prepared within the 12 day stability limit.


VEHICLE
- Supplier: Corn Oil supplied by Sigma-Aldrich, St. Louis, Missouri
- Justification for use and choice of vehicle (if other than water): Vehicle choice determined by solubility.

Details on mating procedure:

Breeding of the adults commenced after approximately two weeks of butyl phenyl ether treatment. Each female was placed with a single male from the same dose level (1:1 mating) until pregnancy 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 to determine concentration of butyl phenyl ether of all dosing solutions from the first mix of the study was initiated prior to the start of dosing. The low- and high-dose solutions from the first mix of the study were analyzed to confirm homogeneous distribution of the test material concurrent with dose confirmation. Analysis was performed using gas chromatography (GC) with mass spectrometry (MS) detection (Marty, in progress). Details of the analyses are maintained in the study files.
Stability
Stability of the test material in the vehicle was determined prior to the start of the study at concentrations ranging from 0.25-500 mg/ml.
Retainer Samples
A sample of the solid test material was retained, but samples of the dose solutions were not retained.

Duration of treatment / exposure:

Male rats were dosed daily for 14 days prior to mating and continuing throughout breeding for up to 36 days. Female rats were dosed once daily for 14 days prior to breeding, and continuing through breeding (two weeks), gestation (three weeks), and lactation (four days).

Frequency of treatment:

Daily

Dose / conc.:

0 mg/kg bw/day (actual dose received)

Dose / conc.:

30 mg/kg bw/day (actual dose received)

Dose / conc.:

100 mg/kg bw/day (actual dose received)

Dose / conc.:

350 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

12

Control animals:

yes, concurrent vehicle

Details on study design:

Selection of the high-dose level (350 mg/kg/day) was based upon data obtained from the range-finding study and was expected to induce some toxic effects (decreased body weights and feed consumption, increased absolute and relative liver weights, and/or increased relative kidney weights) but not death or obvious suffering. The lower dose levels were selected to provide dose-response data for any toxicity that may be observed among the high-dose group.

Assignment of animals to treatment groups: 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.

Positive control:

yes - for the genotoxicity part of the study - cyclophosphamide. See In vivo Genetox section of IUCLID.

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 were 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 conducted on dams and their litters, at least twice daily. These examinations were performed as described above.
Clinical Observations
Clinical observations were conducted on all animals pre-exposure (including positive control animals) and at least daily throughout the study (excluding positive control animals). During the exposure period, these examinations were conducted approximately one hour after dosing. Females were observed for signs of parturition beginning on or about gestation day (GD) 20 (see litter data). 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 males pre-exposure (excluding positive control animals) and weekly throughout the exposure period (excluding positive control animals). DCO were conducted on all females pre-exposure and weekly throughout the pre-breeding and breeding periods. Mated (sperm-positive or plug-positive) females received DCO examinations on GD 0, 7, 14, and 20. Females that deliver litters were subsequently evaluated on LD 3. DCO were not conducted on females that failed to mate or deliver a litter during the gestation and lactation phases of the study. The DCO 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 (excluding positive controls) and during the last week of the treatment period (excluding positive control animals). 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.

Sensory Evaluation
The sensory evaluation included a test for nociception (responsiveness to tail pinch) and for startle response (responsiveness to sharp noise). The evaluation was conducted in a clear plastic box.
Rectal Temperature
Rectal temperature was measured by carefully placing a rectal thermistor (Physitemp, Clifton, New Jersey) approximately 4 cm into the rectum for approximately 10 seconds. Temperature was then recorded. The thermistor was validated at 37oC before and after the study. The instrument was re-calibrated if the temperature recordings differed from the reference thermometer by more than +/- 0.5oC.
Grip Performance
Hindlimb grip performance was tested according to the procedure described by Mattsson et al. (1986). Briefly, the observer placed the rat’s forelegs on a plastic bench and the hindfeet are set on a horizontal screen attached to an electronic strain gauge (Chatillon, Greensboro, North Carolina). The observer then smoothly but firmly pulled backward on the tail until the rat’s grip on the screen was broken. An electronic strain gauge was used to record the rat’s resistance to the pull in grams. The average of three trials was used for statistical analysis. Forelimb grip performance was similarly tested. In this application, a bench was not used, and the rats were placed so that the forefeet were on the screen and the hindfeet were suspended approximately 10 cm above the plastic platform.
Instrument Validation: A standard 500-gram weight attached to a fine-gauge wire was suspended from the load cell and was checked just before and just after testing
(a 1% tolerance, i.e., 500 +/- 5 grams, was acceptable).
Motor Activity
A commercially-available automated test system (MotorMonitor, Kinder Scientific, Inc., Poway, CA) was used for motor activity (MA) data collection. Each animal was tested individually in one of 24 available square acrylic enclosures (approximately 22” X 22”) which contained a grid of infra-red beams
(32 total beams, 16x and 16y) near the floor of the enclosure. The MA test room was maintained under white noise conditions (55-65 dB) and with the lighting off during testing. No entry into the MA test room was allowed during the testing period. Each test session consisted of six 10-minute intervals, totaling 60 minutes of testing per animal per test session (Andrus et al., 2015). All beam breaks “activity counts” for each interval were recorded.
Motor Activity Enclosure Allocation: Rats were allocated to the motor activity enclosures in such a way that the counterbalancing of treatment groups and sexes across enclosures and test times was maximized.
Body Weights/Body Weight Gains
All rats were weighed once during the pre-exposure period (including positive control animals), twice during the first week of the study and once during the second week (excluding positive control animals). Male body weights continued to be recorded weekly throughout the study (excluding positive control animals). Females were weighed weekly during the pre-breeding and breeding periods (excluding positive control animals). 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 weighed at least weekly until termination. Body weight gains were determined for the following intervals: GD 0-7, 7-14, 14-20, 0-20 and LD 1-4.
Feed Consumption
Feed consumption for all animals (excluding positive control animals) was determined twice during the first week by weighing feed containers at the start and end of a measurement cycle. Thereafter, feed consumption was measured weekly during the pre-breeding phase (excluding positive control animals). 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. For females delivering litters, feed consumption was measured on LD 1 and 4. Feed consumption was not measured for females that failed to mate or deliver a litter.

Litter observations:

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: date of parturition, litter size on the day of parturition (LD 0), the number of live and dead pups on days 0, 1, and 4 postpartum, and the sex and the 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). In addition, pup clinical observations were recorded on days 0, 1, and 4 postpartum. Any pups found dead were sexed and examined grossly, if possible, for external and visual defects and then discarded.

Postmortem examinations (parental animals):

Clinical Pathology
Animals were fasted overnight prior to blood collection. Blood samples were obtained from the orbital sinus following anesthesia with CO2/O2 at the scheduled necropsy. Blood was not obtained from animals that died or were euthanized in a moribund condition prior to their scheduled necropsy. Blood samples were not obtained from females that failed to deliver a litter.
Hematology
Sample Preparation
Blood samples for a complete blood count were mixed with ethylenediamine-tetraacetic 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).
Assays
Hematocrit (HCT)
Hemoglobin (HGB) concentration
Red blood cell (RBC) count
Total white blood cell (WBC) count
Differential WBC count
Neutrophils (NEUT)
Lymphocytes (LYMP)
Monocytes (MONO)
Eosinophils (EOS)
Basophils (BASO)
Large Unstained Cells (LUC) which include, atypical lymphocytes,
large lymphocytes, plasma cells, and blasts
Platelet (PLT) count
Reticulocyte (RET) count
RBC indices:
Mean Corpuscular Hemoglobin (MCH)
Mean Corpuscular Volume (MCV)
Mean Corpuscular Hemoglobin Concentration (MCHC)

Coagulation
Sample Preparation
Blood samples were collected in sodium citrate tubes, centrifuged, plasma collected, and assayed using the ACL9000 Analyzer (Instrumentation Laboratory, Bedford, Massachusetts).
Assay
Prothrombin time (PT)

Clinical Chemistry
Sample Preparation
Blood samples were collected and serum was separated from cells as soon as possible. Serum parameters were measured using a cobas c311 Clinical Chemistry Analyzer (Roche Diagnostics, Indianapolis, Indiana).
Enzyme Activities of:
Alkaline phosphatase (ALP)
Alanine aminotransferase (ALT)
Aspartate aminotransferase (AST)
Concentrations of:
Albumin (ALB)
Albumin/Globulin Ratio (A/G) - calculated
Cholesterol (CHOL)
Creatinine (CREA)
Electrolytes
Calcium (CA)
Phosphorus (PHOS)
Sodium (NA)
Potassium (K)
Chloride (CL)
Globulin (GLOB) - calculated
Glucose (GLUC)
Total bile acids (TBA)
Total bilirubin (TBIL)
Total protein (TP)
Triglycerides (TRIG)
Urea nitrogen (UN)

Urinalysis
Urine samples were obtained from all males (excluding positive control animals) the week prior to the scheduled necropsy. Animals were housed in metabolism cages and the urine collected overnight (approximately 16 hours). Feed and water were available during this procedure.
Assays
Color, appearance, specific gravity (refractometer) and urine volume
Semiquantitative analysis of the following was conducted using Siemens Multistix Reagent Strips on the Clinitek Advantus Analyzer (Siemens Healthcare Diagnostics, Tarrytown, New York):
pH
Bilirubin
Glucose
Protein
Ketones
Blood
Urobilinogen

Microscopic Exam:
Urine samples were collected from each male by manual compression of the urinary bladder. The urine samples were pooled from each group, and the microsediment were characterized microscopically.

Anatomic Pathology
Adult Necropsy
Adult males (fasted) were submitted for necropsy after at least four weeks of exposure. Adult females (fasted) were terminated on LD 5, or at least 24 days after the end of the mating 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 by the inhalation of CO2/O2. Blood was collected from the orbital sinus (all males, all females that littered), 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 examined for the presence and number of implantation sites. 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, thyroid with parathyroids (weighed after fixation) were recorded, and organ:body weight ratios calculated.
Representative samples of tissues listed in Table 4 were collected and preserved in neutral, phosphate-buffered 10% formalin, with the exception of the testes and epididymides that were fixed in Bouin’s. Transponders were removed and placed in jars with the tissues.


Histopathology
Histopathologic examination of the tissues indicated in Table 4 (see attached) was conducted on all control and high-dose adult rats. Examination of tissues from the remaining groups was limited to those tissues that demonstrated treatment-related histologic effects at the high dose (liver and urinary bladder in males and females) and relevant gross lesions. Paraffin embedded tissues were sectioned approximately 6 µm thick, stained with hematoxylin and eosin and examined by a veterinary pathologist using a light microscope.
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 would have been 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):

All pups surviving to LD 4 were euthanized by an intraperitoneal injection of sodium pentobarbital solution, examined for gross external alterations, and then discarded. Any pups found dead or which were euthanized in moribund condition were examined to the extent possible and discarded.

Statistics:

see below

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

Offspring viability indices:

• 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

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, males in the 350 mg/kg/day group had treatment-related decreases in body weights throughout the treatment period (4.7-9.3%). These decreases were statistically identified on TD 29 and 35. No treatment-related differences in body weights were observed for males at 30 or 100 mg/kg/day, or in females at any dose level throughout the duration of the study. No treatment-related differences in body weight gains were observed for females at any exposure level tested throughout gestation or lactation.
Males given 350 mg/kg/day had a treatment-related and statistically-identified lower mean final body weight (10%), relative to controls.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

When compared to controls, males in the 350 mg/kg/day group had treatment-related and statistically identified decreases in feed consumption during the TD 1-4 interval (18.6%). No significant differences in feed consumption were observed for males at 30 or 100 mg/kg/day, or in females at any exposure level throughout the duration of the study.

Food efficiency:

no effects observed

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

no effects observed

Ophthalmological findings:

no effects observed

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Males given 350 mg/kg/day had a statistically-identified higher mean reticulocyte count. The higher reticulocyte value in this dose group was interpreted to be treatment related because the mean value was higher than the historical control range, and 11/12 individual rats given 350 mg/kg/day had reticulocyte counts that were higher than the mean reticulocyte count of the male control group. However, the higher reticulocyte count was interpreted to be a non-adverse effect because of the lack of any other treatment-related alterations in hematologic parameters at any dose level, and the lack of histopathologic effects in the bone marrow and spleen of males given 350 mg/kg/day. Females given 30 mg/kg/day had a statistically- identified higher mean platelet count that was interpreted to be unrelated to treatment because of the lack of a dose response

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

no effects observed

Behaviour (functional findings):

no effects observed

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Treatment-related increases in the incidence of very slight hypertrophy of centribolublar/midzonal hepatocytes, with increased cytoplasmic eosinophilia, were present in the liver of males and females given 350 mg/kg/day. 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 ALP), and the absence of treatment-related histopathologic alterations such as necrosis, increased apoptosis, inflammation, proliferative or degenerative changes in the liver of males and females at 350 mg/kg/day.
Treatment-related adverse effects of the urinary bladder were present in males and females at all dose levels. The majority of males and some of the females given 30, 100 or 350 mg/kg/day had slight diffuse hyperplasia of the transitional epithelium and very slight multifocal chronic inflammation of the lamina propria of the urinary bladder. One male given 30 mg/kg/day had slight chronic inflammation of the lamina propria of the urinary bladder. Additional treatment-related observations of the urinary bladder consisted of slight diffuse edema of the lamina propria and muscularis and slight multifocal chronic active inflammation of the lamina propria in one female given 30 mg/kg/day and in two females given 100 mg/kg/day, focal or multifocal acute (recent) hemorrhage of the lamina propria in one female given 30 mg/kg/day and in one female given 100 mg/kg/day, and slight multifocal erosions of the transitional epithelium in one female given 100 mg/kg/day.

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

Description (incidence and severity):

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. The percent postimplantation loss (percentage of live born pups relative to total uterine implantation sites) was higher in the 350 mg/kg/day group (13.88%) relative to the control group (5.35%). Although the higher percent postimplantation loss value was slightly outside of the recent laboratory historical control range for OECD 421 and OECD 422 studies, it was considered spurious and not attributable to treatment as the higher value was not statistically identified and was primarily driven by a single litter (1558) having its only pup born

Key result

Dose descriptor:

LOAEL

Effect level:

30 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

Key result

Dose descriptor:

NOAEL

Effect level:

350 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

reproductive performance

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

30 mg/kg bw/day (actual dose received)

System:

urinary

Organ:

bladder

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Histopathological findings:

not examined

Other effects:

no effects observed

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

350 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

viability

other: general toxicity

Key result

Reproductive effects observed:

no

Conclusions:

Gavage administration of butyl phenyl ether to Crl:CD(SD) rats resulted in treatment-related decreases in male body weight throughout the treatment period (4.7-9.3%) only at the high dose level (350 mg/kg/day). These decreases were statistically identified on TD 29 and 35. No significant differences in body weights were observed for males at 30 or 100 mg/kg/day, or in females at any dose 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.
Similar to body weight effects, treatment-related and statistically identified decreases in feed consumption were observed in males at the high dose level (350 mg/kg/day) during the TD 1-4 interval (18.6%). No significant differences in feed consumption were observed for males at 30 or 100 mg/kg/day or in females at any exposure level throughout the duration of the study.
Males given 350 mg/kg/day had a treatment-related and statistically-identified higher mean reticulocyte count. However, the higher reticulocyte count was interpreted to be a non-adverse effect. There were no treatment-related hematologic effects in males given 300 or 100 mg/kg/day or in females at any dose level.
There were no treatment-related changes in prothrombin times, clinical chemistry parameters, or urinalysis parametersfor males and females at any dose level.
Males given 350 mg/kg/day had a treatment-related statistically-identified lower mean final body weight (10%) relative to controls. Males and females given 350 mg/kg/day had treatment-related and statistically -identifiedt higher mean relative liver weights (7.3% and 7.9%, respectively) as compared to controls.
The higher relative liver weights corresponded to treatment-related increased incidence of very slight hypertrophy of centrilobular/midzonal hepatocytes with increased cytoplasmic eosinophilia in males and females given 350 mg/kg/day. The hepatocellular hypertrophy was interpreted to be a non-adverse and adaptive effect.
In addition to the liver effects, there were treatment-related adverse effects of the urinary bladder present in males and females at all dose levels. The majority of males and some of the females given 30, 100 or 350 mg/kg/day had slight diffuse hyperplasia of the transitional epithelium and very slight multifocal chronic inflammation of the lamina propria of the urinary bladder. One male given 30 mg/kg/day had slight chronic inflammation of the lamina propria of the urinary bladder. Additional treatment-related observations of the urinary bladder consisted of slight diffuse edema of the lamina propria and muscularis and slight multifocal chronic active inflammation of the lamina propria in one female given 30 mg/kg/day and in two females given 100 mg/kg/day, focal or multifocal acute (recent) hemorrhage of the lamina propria in one female given 30 mg/kg/day and in one female given 100 mg/kg/day, and slight multifocal erosions of the transitional epithelium in one female given 100 mg/kg/day.
There were no treatment-related effects of butyl phenyl ether on neurological or reproductive function or prenatal/early neonatal growth and survival of the offspring.
Based on these results, the no-observed adverse-effect level (NOAEL) for general toxicity was not established due to the occurrence of treatment-related adverse effects of the urinary bladder present in males and females at all dose levels. The no-observed-effect level (NOEL) for reproductive and neurological effects was 350 mg/kg/day, the highest dose level tested.

Executive summary:

Groups of 12 male and 12 female Crl:CD(SD) rats were administered butyl phenyl ether daily by gavage in corn oil at dose levels of 0 (control), 30, 100, or 350 mg/kg/day. Females were dosed once daily for two weeks prior to breeding, through breeding (two weeks), gestation (three weeks), and through postpartum day 4. Females were necropsied on postpartum day 5. Males were dosed two weeks prior to breeding and continuing through breeding (two weeks) until necropsy (test days 36 or 37). Effects on reproductive and neurological function as well as general toxicity were evaluated. In addition, postmortem examinations included a gross necropsy of the adults with collection of organ weights and histopathologic examination of tissues. Litter size, pup survival, sex, body weight, and the presence of gross external abnormalities were also assessed. In addition, bone marrow was collected at necropsy and cytogenic effects were evaluated using the erythrocyte micronucleus test (MNT) in males. 

Gavage administration of butyl phenyl ether to Crl:CD(SD) rats resulted in treatment-related decreases in male body weight throughout the treatment period (4.7-9.3%) only at the high dose level (350 mg/kg/day). These decreases were statistically identified on TD 29 and 35. No significant differences in body weights were observed for males at 30 or 100 mg/kg/day, or in females at any dose 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.

Similar to body weight effects, treatment-related and statistically identified decreases in feed consumption were observed in males at the high dose level (350 mg/kg/day) during the TD 1-4 interval (18.6%). No significant differences in feed consumption were observed for males at 30 or 100 mg/kg/day or in females at any exposure level throughout the duration of the study.

Males given 350 mg/kg/day had a treatment-related and statistically-identified higher mean reticulocyte count. However, the higher reticulocyte count was interpreted to be a non-adverse effect. There were no treatment-related hematologic effects in males given 30 or 100 mg/kg/day or in females at any dose level.

There were no treatment-related changes in prothrombin times, clinical chemistry parameters, or urinalysis parameters for males and females at any dose level. 

Males given 350 mg/kg/day had a treatment-related statistically-identified lower mean final body weight (10%) relative to controls. Males and females given 350 mg/kg/day had treatment-related and statistically -identified higher mean relative liver weights (7.3% and 7.9%, respectively) as compared to controls. 

The higher relative liver weights corresponded to treatment-related increased incidence of very slight hypertrophy of centrilobular/midzonal hepatocytes with increased cytoplasmic eosinophilia in males and females given 350 mg/kg/day. The hepatocellular hypertrophy was interpreted to be a non-adverse and adaptive effect. 

In addition to the liver effects, there were treatment-related adverse effects of the urinary bladder present in males and females at all dose levels. The majority of males and some of the females given 30, 100 or 350 mg/kg/day had slight diffuse hyperplasia of the transitional epithelium and very slight multifocal chronic inflammation of the lamina propria of the urinary bladder. One male given 30 mg/kg/day had slight chronic inflammation of the lamina propria of the urinary bladder. Additional treatment-related observations of the urinary bladder consisted of slight diffuse edema of the lamina propria and muscularis and slight multifocal chronic active inflammation of the lamina propria in one female given 30 mg/kg/day and in two females given 100 mg/kg/day, focal or multifocal acute (recent) hemorrhage of the lamina propria in one female given 30 mg/kg/day and in one female given 100 mg/kg/day, and slight multifocal erosions of the transitional epithelium in one female given 100 mg/kg/day.

There were no treatment-related effects of butyl phenyl ether on neurological or reproductive function or prenatal/early neonatal growth and survival of the offspring.

There were no treatment-related effects in micronucleated polychromatic erythrocytes (MN-PCE) frequency for males at any dose. Therefore, butyl phenyl ether was negative in this bone marrow erythrocyte MNT under the experimental conditions used. 

Based on these results, the no-observed adverse-effect level (NOAEL) for general toxicity was not established due to the occurrence of treatment-related adverse effects of the urinary bladder present in males and females at all dose levels. The no-observed-effect level (NOEL) for reproductive and neurological effects was 350 mg/kg/day, the highest dose level tested.

Cross-reference 2

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

1996 version of guideline

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Details on species / strain selection:

Crl:CD(SD)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories (Raleigh, North Carolina)
- Females (if applicable) nulliparous and non-pregnant: Yes
- Age at study initiation: P - 8 weeks
- Weight at study initiation: (P) Males: 277-281 g; Females: 197-200 g
- Fasting period before study: none
- Housing: Upon arrival animals were housed two or three per cage in solid bottom stainless steel cages.
The solid bottom cages contained ground corn cob bedding. Cages contained a feed crock and a press
ure activated lixit valve-type watering system.
After assignment to study, animals were housed singly in solid bottom stainless steel cages, except durin g breeding and during the gestation and littering phases of the study. The solid bottom cages contained ground corn cob bedding. During breeding, one male and one female were placed in stainless steel
cages with wire mesh floors that were suspended above absorbent paper in order to better visualize copulation plugs. During gestation and littering, dams (and their litters) were housed in plastic cages provided with irradiated ground corn cob bedding from approximately GD 0 until completion of lactation
(LD 4). Cages contained a feed crock and a pressure activated lixit valve-type watering system. - Enrichment: Enrichment for rats was provided from the day of arrival until necropsy. The cage contai ned nylon bones during the pre-breeding phase (males and females) and following mating (all males) or
paper nesting material during the gestation and lactation phases of the study.
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: at least one week
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22°C with a range of 20°C-26°C
- Humidity (%): 50% with a range of 30-70%
- Air changes (per hr): 10-15 times/hour (average)
- Photoperiod (hrs dark / hrs light):12-hour light/dark (on at 6:00 a.m. and off at 6:00 p.m.)
IN-LIFE DATES: From: To: Dosing started: June 01, 2016; Male necropsy July 6-7, 2016; Female
necropsy July 13 -19, 2016. Pups euthenized on Post natal day 4.

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on oral exposure:

PREPARATION OF DOSE: Butyl phenyl ether was administered in corn oil and CP in distilled water, such that a dose volume of 4 ml/kg body weight yielded the targeted dose. Dose volumes were adjusted using the most current body weight. Dose solutions were not corrected for purity. Butyl phenyl dose solutions were prepared within the 12 day stability limit.
VEHICLE
- Supplier: Corn Oil supplied by Sigma-Aldrich, St. Louis, Missouri
- Justification for use and choice of vehicle (if other than water): Vehicle choice determined by solubility.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analysis to determine concentration of butyl phenyl ether of all dosing solutions from the first mix of the
study was initiated prior to the start of dosing. The low- and high-dose solutions from the first mix of
the study were analyzed to confirm homogeneous distribution of the test material concurrent with dose
confirmation. Analysis was performed using gas chromatography (GC) with mass spectrometry (MS) det
ection (Marty, in progress). Details of the analyses are maintained in the study files.
Stability
Stability of the test material in the vehicle was determined prior to the start of the study at concentrations
ranging from 0.25-500 mg/ml.
Retainer Samples
A sample of the solid test material was retained, but samples of the dose solutions were not retained.

Duration of treatment / exposure:

Male rats were dosed daily for 14 days prior to mating and continuing throughout breeding for up to 36
days. Female rats were dosed once daily for 14 days prior to breeding, and continuing through breeding
(two weeks), gestation (three weeks), and lactation (four days).

Frequency of treatment:

Saily

Dose / conc.:

0 mg/kg bw/day (actual dose received)

Dose / conc.:

30 mg/kg bw/day (actual dose received)

Dose / conc.:

100 mg/kg bw/day (actual dose received)

Dose / conc.:

350 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

12

Control animals:

yes, concurrent vehicle

Details on study design:

Selection of the high-dose level (350 mg/kg/day) was based upon data obtained from the range-finding study and was expected to induce some toxic effects (decreased body weights and feed consumption, increased absolute and relative liver weights, and/or increased relative kidney weights) but not deathobvious suffering. The lower dose levels were selected to provide dose-response data for any toxicity that may be observed among the high-dose group.
Assignment of animals to treatment groups: 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.

Positive control:

yes - for the genotoxicity part of the study - cyclophosphamide. See In vivo Genetox section of IUCLID.

Observations and examinations performed and frequency:

Daily In-Life Observations
A cage-side examination was conducted at least twice daily. This examination was typically performed with the animals in their cages and was designed to detect significant clinical abnormalities that were cl early 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 were 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 conducted on dams and their litters, at least twice daily. These examinations were performed as described above.
Clinical Observations
Clinical observations were conducted on all animals pre-exposure (including positive control animals) and at least daily throughout the study (excluding positive control animals). During the exposure period, these examinations were conducted approximately one hour after dosing. Females were observed for signs of parturition beginning on or about gestation day (GD) 20 (see litter data). 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 males pre-exposure (excluding positive control animals) and weekly throughout the exposure period (excluding positive control animals). DCO were conducted on all females pre-exposure and weekly throughout the pre-breeding and breeding periods. Mated (sperm-positive or plug-positive) females received DCO examinations on GD 0, 7, 14, and 20. Females that deliver litters were subsequently evaluated on LD 3. DCO were not conducted on females that failed to mate or deliver a litter during the gestation and lactation phases of the study. The DCO 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 (excluding positive controls) and during the last week of the treatment period (excluding positive control animals). 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.
Sensory Evaluation
The sensory evaluation included a test for nociception (responsiveness to tail pinch) and for startle response (responsiveness to sharp noise). The evaluation was conducted in a clear plastic box.
Rectal Temperature
Rectal temperature was measured by carefully placing a rectal thermistor (Physitemp, Clifton, New Jersey) approximately 4 cm into the rectum for approximately 10 seconds. Temperature was then recorded. The thermistor was validated at 37#C before and after the study. The instrument was re-calibrated if the temperature recordings differed from the reference thermometer by more than 0.5oC.
Grip Performance
Hindlimb grip performance was tested according to the procedure described by Mattsson et al. (1986). Briefly, the observer placed the rat’s forelegs on a plastic bench and the hindfeet are set on a horizontal screen attached to an electronic strain gauge (Chatillon, Greensboro, North Carolina). The observer then smoothly but firmly pulled backward on the tail until the rat’s grip on the screen was broken. An electronic strain gauge was used to record the rat’s resistance to the pull in grams. The average of three trials was used for statistical analysis. Forelimb grip performance was similarly tested. In this application, a bench was not used, and the rats were placed so that the forefeet were on the screen and the hindfeet were suspended approximately 10 cm above the plastic platform. Instrument Validation: A standard 500-gram weight attached to a fine-gauge wire was suspended from
the load cell and was checked just before and just after testing (a 1% tolerance, i.e., 500 # 5 grams, was acceptable).
Motor Activity
A commercially-available automated test system (MotorMonitor, Kinder Scientific, Inc., Poway, CA) was used for motor activity (MA) data collection. Each animal was tested individually in one of 24 available square acrylic enclosures (approximately 22” X 22”) which contained a grid of infra-red beams (32 total beams, 16x and 16y) near the floor of the enclosure. The MA test room was maintained under white noise conditions (55-65 dB) and with the lighting off during testing. No entry into the MA test room was allowed during the testing period. Each test session consisted of six 10-minute intervals, totaling 60 minutes of testing per animal per test session (Andrus et al., 2015). All beam breaks “activity counts” for
each interval were recorded.
Motor Activity Enclosure Allocation: Rats were allocated to the motor activity enclosures in such a way that the counterbalancing of treatment groups and sexes across enclosures and test times was maximized.
Body Weights/Body Weight Gains
All rats were weighed once during the pre-exposure period (including positive control animals), twice during the first week of the study and once during the second week (excluding positive control animals).
Male body weights continued to be recorded weekly throughout the study (excluding positive control animals). Females were weighed weekly during the pre-breeding and breeding periods (excluding positive control animals). 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 weighed at least weekly until termination. Body weight gains were determined forthe following intervals: GD 0-7, 7-14, 14-20, 0-20 and LD 1-4.
Feed Consumption
Feed consumption for all animals (excluding positive control animals) was determined twice during the first week by weighing feed containers at the start and end of a measurement cycle. Thereafter, feed consumption was measured weekly during the pre-breeding phase (excluding positive control animals). 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. For females delivering litters, feed consumption was measured on LD1 and 4. Feed consumption was not measured for females that failed to mate or deliver a litter.

Sacrifice and pathology:

Clinical Pathology
Animals were fasted overnight prior to blood collection. Blood samples were obtained from the orbi
tal sinus following anesthesia with CO2/O2 at the scheduled necropsy. Blood was not obtained from
animals that died or were euthanized in a moribund condition prior to their scheduled necropsy. Blood
samples were not obtained from females that failed to deliver a litter.
Hematology
Sample Preparation
Blood samples for a complete blood count were mixed with ethylenediamine-tetraacetic acid (EDTA). Bl
ood 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).
Assays
Hematocrit (HCT)
Hemoglobin (HGB) concentration
Red blood cell (RBC) count
Total white blood cell (WBC) count
Differential WBC count
Neutrophils (NEUT)
Lymphocytes (LYMP)
Monocytes (MONO)
Eosinophils (EOS)
Basophils (BASO)
Large Unstained Cells (LUC) which include, atypical lymphocytes,
large lymphocytes, plasma cells, and blasts
Platelet (PLT) count
Reticulocyte (RET) count
RBC indices:
Mean Corpuscular Hemoglobin (MCH)
Mean Corpuscular Volume (MCV)
Mean Corpuscular Hemoglobin Concentration (MCHC)
Coagulation
Sample Preparation
Blood samples were collected in sodium citrate tubes, centrifuged, plasma collected, and assayed using
the ACL9000 Analyzer (Instrumentation Laboratory, Bedford, Massachusetts).
Assay
Prothrombin time (PT)
Clinical Chemistry
Sample Preparation
Blood samples were collected and serum was separated from cells as soon as possible. Serum pa
rameters were measured using a cobas c311 Clinical Chemistry Analyzer (Roche Diagnostics, Indianapo
lis, Indiana).
Enzyme Activities of:
Alkaline phosphatase (ALP)
Alanine aminotransferase (ALT)
Aspartate aminotransferase (AST)
Concentrations of:
Albumin (ALB)
Albumin/Globulin Ratio (A/G) - calculated
Cholesterol (CHOL)
Creatinine (CREA)
Electrolytes
Calcium (CA)
Phosphorus (PHOS)
Sodium (NA)
Potassium (K)
Chloride (CL)
Globulin (GLOB) - calculated
Glucose (GLUC)
Total bile acids (TBA)
Total bilirubin (TBIL)
Total protein (TP)
Triglycerides (TRIG)
Urea nitrogen (UN)
Urinalysis
Urine samples were obtained from all males (excluding positive control animals) the week prior to the
scheduled necropsy. Animals were housed in metabolism cages and the urine collected overnight (
approximately 16 hours). Feed and water were available during this procedure.
Assays
Color, appearance, specific gravity (refractometer) and urine volume
Semiquantitative analysis of the following was conducted using Siemens Multistix Reagent Strips on the
Clinitek Advantus Analyzer (Siemens Healthcare Diagnostics, Tarrytown, New York):
pH
Bilirubin
Glucose
Protein
Ketones
Blood
Urobilinogen
Microscopic Exam:
Urine samples were collected from each male by manual compression of the urinary bladder. The urine
samples were pooled from each group, and the microsediment were characterized microscopically.
Anatomic Pathology
Adult Necropsy
Adult males (fasted) were submitted for necropsy after at least four weeks of exposure. Adult females
(fasted) were terminated on LD 5, or at least 24 days after the end of the mating period for females no
t 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 by the inhalation of CO2/O2. Blo
od was collected from the orbital sinus (all males, all females that littered), 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 na
sopharyngeal duct and the lungs were distended to an approximately normal inspiratory volume with n
eutral, 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 examined for the presence and number of implantation
sites. 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, thyroid with
parathyroids (weighed after fixation) were recorded, and organ:body weight ratios calculated.
Representative samples of tissues listed in Table 4 were collected and preserved in neutral, phosphate
-buffered 10% formalin, with the exception of the testes and epididymides that were fixed in Bouin’s.
Transponders were removed and placed in jars with the tissues.
Histopathology
Histopathologic examination of the tissues indicated in Table 4 (see attached) was conducted on all cont
rol and high-dose adult rats. Examination of tissues from the remaining groups was limited to those
tissues that demonstrated treatment-related histologic effects at the high dose (liver and urinary bladder
in males and females) and relevant gross lesions. Paraffin embedded tissues were sectioned approx
imately 6 μm thick, stained with hematoxylin and eosin and examined by a veterinary pathologist using a
light microscope.
The histopathological examination of the testes included a qualitative assessment of stages of sperma
togenesis. 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-hematoxyli
n. 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, infla
mmatory 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 would have been 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.

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

When compared to controls, males in the 350 mg/kg/day group had treatment-related decreases in body
weights throughout the treatment period (4.7-9.3%). These decreases were statistically identified on TD
29 and 35. No treatment-related differences in body weights were observed for males at 30 or 100 mg/k
g/day, or in females at any dose level throughout the duration of the study. No treatment-related differenc
es in body weight gains were observed for females at any exposure level tested throughout gestation or
lactation.
Males given 350 mg/kg/day had a treatment-related and statistically-identified lower mean final body
weight (10%), relative to controls.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

When compared to controls, males in the 350 mg/kg/day group had treatment-related and statistically
identified decreases in feed consumption during the TD 1-4 interval (18.6%). No significant differences in
feed consumption were observed for males at 30 or 100 mg/kg/day, or in females at any exposure level
throughout the duration of the study.

Food efficiency:

no effects observed

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

no effects observed

Ophthalmological findings:

no effects observed

Haematological findings:

no effects observed

Description (incidence and severity):

Males given 350 mg/kg/day had a statistically-identified higher mean reticulocyte count. The higher
reticulocyte value in this dose group was interpreted to be treatment related because the mean value
was higher than the historical control range, and 11/12 individual rats given 350 mg/kg/day had reticuloc
yte counts that were higher than the mean reticulocyte count of the male control group. However, the
higher reticulocyte count was interpreted to be a non-adverse effect because of the lack of any other
treatment-related alterations in hematologic parameters at any dose level, and the lack of histopathol
ogic effects in the bone marrow and spleen of males given 350 mg/kg/day. Females given 30 mg/kg/day
had a statistically- identified higher mean platelet count that was interpreted to be unrelated to treatment
because of the lack of a dose response

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

no effects observed

Behaviour (functional findings):

no effects observed

Immunological findings:

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Males and females given 350 mg/kg/day had treatment-related and statistically-identified higher mean
relative liver weights (7.3% and 7.9%, respectively), as compared to controls. The higher relative liver
weights corresponded to increases in the incidence of very slight hypertrophy of centrilobular/midzo
nal hepatocytes, with increased cytoplasmic eosinophilia, in males and females given 350 mg/kg/day.
Alterations in organ weights that were interpreted to be reflective of the lower mean final body weight
of males given 350 mg/kg/day consisted of a statistically-identified lower mean absolute adrenal gland
weight, and statistically identified higher relative kidney, brain and epididymides weights. Females give
n 350 mg/kg/day had a statistically-identified higher mean relative kidney weight that was interpreted to
be unrelated to treatment because the value was within the historical control range, and there were no
corresponding histopathologic alterations of the kidneys. There were no treatment-related alterations in
final body weights or organ weights in males or females given 30 or 100 mg/kg/day.

Gross pathological findings:

no effects observed

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Treatment-related increases in the incidence of very slight hypertrophy of centribolublar/midzonal h epatocytes, with increased cytoplasmic eosinophilia, were present in the liver of males and females given 350 mg/kg/day. The hepatocellular hypertrophy was interpreted to be a non-adverse and adaptive effec
t, based on the modest corresponding increase in liver weights, along with the absence of any treatmentrelated changes in liver enzyme activities (ALT, AST and ALP), and the absence of treatment-related histopathologic alterations such as necrosis, increased apoptosis, inflammation, proliferative or dege
nerative changes in the liver of males and females at 350 mg/kg/day.
Treatment-related adverse effects of the urinary bladder were present in males and females at all dose levels. The majority of males and some of the females given 30, 100 or 350 mg/kg/day had slight diffuse hyperplasia of the transitional epithelium and very slight multifocal chronic inflammation of the lamina propria of the urinary bladder. One male given 30 mg/kg/day had slight chronic inflammation of the lamina propria of the urinary bladder. Additional treatment-related observations of the urinary bladder consisted of slight diffuse edema of the lamina propria and muscularis and slight multifocal chronic active inflammation of the lamina propria in one female given 30 mg/kg/day and in two females given 100 mg/kg/day, focal or multifocal acute (recent) hemorrhage of the lamina propria in one female given 30 mg/kg/day and in one female given 100 mg/kg/day, and slight multifocal erosions of the transitional epithelium in one female given 100 mg/kg/day.

Results are provided in the table below.

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Key result

Dose descriptor:

LOAEL

Effect level:

30 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

organ weights and organ / body weight ratios

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

30 mg/kg bw/day (actual dose received)

System:

urinary

Organ:

bladder

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

350 mg/kg bw/day (actual dose received)

System:

hepatobiliary

Organ:

liver

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

Histopathological findings in the urinary bladder

Sex	Males				Females
Dose Level (mg/kg/day)	0	30	100	350	0	30	100	350
URINARY BLADDER (number examined)	12	12	12	12	12	12	12	12
Edema, lamina propria, muscularis, diffuse                                                                -slight	0	0	0	0	0	1	2	0
Hemorrhage, acute (recent), lamina propria, focal                                               -very slight	0	0	0	0	0	0	1	0
Hemorrhage, acute (recent), lamina propria, multifocal                                       -very slight	0	0	0	0	0	1	0	0
Hyperplasia, transitional epithelium, diffuse                                                        -very slight	0	1	0	1	2	2	4	4
-slight	0	10	11	11	0	3	1	7
Inflammation, chronic, lamina propria, multifocal                                       -very slight	0	11	12	12	0	3	4	8
-slight	0	1	0	0	0	0	0	0
Inflammation, chronic active, lamina propria, multifocal                                               -slight	0	0	0	0	0	1	2	0
Erosion, transitional epithelium, multifocal                                                                -slight	0	0	0	0	0	0	1	0

Bold typeindicates the effect was interpreted to be treatment related

Conclusions:

Gavage administration of butyl phenyl ether to Crl:CD(SD) rats resulted in treatment-related decreases in male body weight throughout the treatment period (4.7-9.3%) only at the high dose level (350 mg/kg/day). These decreases were statistically identified on TD 29 and 35. No significant differences in body weights were observed for males at 30 or 100 mg/kg/day, or in females at any dose 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.
Similar to body weight effects, treatment-related and statistically identified decreases in feed consumption were observed in males at the high dose level (350 mg/kg/day) during the TD 1-4 interval (18.6%). No significant differences in feed consumption were observed for males at 30 or 100 mg/kg/day or in females at any exposure level throughout the duration of the study.
Males given 350 mg/kg/day had a treatment-related and statistically-identified higher mean reticulocyte count. However, the higher reticulocyte count was interpreted to be a non-adverse effect. There were no treatment-related hematologic effects in males given 300 or 100 mg/kg/day or in females at any dose level.
There were no treatment-related changes in prothrombin times, clinical chemistry parameters, or urinalysis parametersfor males and females at any dose level.
Males given 350 mg/kg/day had a treatment-related statistically-identified lower mean final body weight (10%) relative to controls. Males and females given 350 mg/kg/day had treatment-related and statistically -identifiedt higher mean relative liver weights (7.3% and 7.9%, respectively) as compared to controls.
The higher relative liver weights corresponded to treatment-related increased incidence of very slight hypertrophy of centrilobular/midzonal hepatocytes with increased cytoplasmic eosinophilia in males and females given 350 mg/kg/day. The hepatocellular hypertrophy was interpreted to be a non-adverse and adaptive effect.
In addition to the liver effects, there were treatment-related adverse effects of the urinary bladder present in males and females at all dose levels. The majority of males and some of the females given 30, 100 or 350 mg/kg/day had slight diffuse hyperplasia of the transitional epithelium and very slight multifocal chronic inflammation of the lamina propria of the urinary bladder. One male given 30 mg/kg/day had slight chronic inflammation of the lamina propria of the urinary bladder. Additional treatment-related observations of the urinary bladder consisted of slight diffuse edema of the lamina propria and muscularis and slight multifocal chronic active inflammation of the lamina propria in one female given 30 mg/kg/day and in two females given 100 mg/kg/day, focal or multifocal acute (recent) hemorrhage of the lamina propria in one female given 30 mg/kg/day and in one female given 100 mg/kg/day, and slight multifocal erosions of the transitional epithelium in one female given 100 mg/kg/day.
There were no treatment-related effects of butyl phenyl ether on neurological or reproductive function or prenatal/early neonatal growth and survival of the offspring.

Based on these results, the no-observed adverse-effect level (NOAEL) for general toxicity was not established due to the occurrence of treatment-related adverse effects of the urinary bladder present in males and females at all dose levels. The no-observed-effect level (NOEL) for reproductive and neurological effects was 350 mg/kg/day, the highest dose level tested.

Executive summary:

Groups of 12 male and 12 female Crl:CD(SD) rats were administered butyl phenyl ether daily by gavage in corn oil at dose levels of 0 (control), 30, 100, or 350 mg/kg/day. Females were dosed once daily for two weeks prior to breeding, through breeding (two weeks), gestation (three weeks), and through postpartum day 4. Females were necropsied on postpartum day 5. Males were dosed two weeks prior to breeding and continuing through breeding (two weeks) until necropsy (test days 36 or 37). Effects on reproductive and neurological function as well as general toxicity were evaluated. In addition, postmortem examinations included a gross necropsy of the adults with collection of organ weights and histopathologic examination of tissues. Litter size, pup survival, sex, body weight, and the presence of gross external abnormalities were also assessed. In addition, bone marrow was collected at necropsy and cytogenic effects were evaluated using the erythrocyte micronucleus test (MNT) in males. 

Gavage administration of butyl phenyl ether to Crl:CD(SD) rats resulted in treatment-related decreases in male body weight throughout the treatment period (4.7-9.3%) only at the high dose level (350 mg/kg/day). These decreases were statistically identified on TD 29 and 35. No significant differences in body weights were observed for males at 30 or 100 mg/kg/day, or in females at any dose 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.

Similar to body weight effects, treatment-related and statistically identified decreases in feed consumption were observed in males at the high dose level (350 mg/kg/day) during the TD 1-4 interval (18.6%). No significant differences in feed consumption were observed for males at 30 or 100 mg/kg/day or in females at any exposure level throughout the duration of the study.

Males given 350 mg/kg/day had a treatment-related and statistically-identified higher mean reticulocyte count. However, the higher reticulocyte count was interpreted to be a non-adverse effect. There were no treatment-related hematologic effects in males given 30 or 100 mg/kg/day or in females at any dose level.

There were no treatment-related changes in prothrombin times, clinical chemistry parameters, or urinalysis parameters for males and females at any dose level. 

Males given 350 mg/kg/day had a treatment-related statistically-identified lower mean final body weight (10%) relative to controls. Males and females given 350 mg/kg/day had treatment-related and statistically -identified higher mean relative liver weights (7.3% and 7.9%, respectively) as compared to controls. 

The higher relative liver weights corresponded to treatment-related increased incidence of very slight hypertrophy of centrilobular/midzonal hepatocytes with increased cytoplasmic eosinophilia in males and females given 350 mg/kg/day. The hepatocellular hypertrophy was interpreted to be a non-adverse and adaptive effect. 

In addition to the liver effects, there were treatment-related adverse effects of the urinary bladder present in males and females at all dose levels. The majority of males and some of the females given 30, 100 or 350 mg/kg/day had slight diffuse hyperplasia of the transitional epithelium and very slight multifocal chronic inflammation of the lamina propria of the urinary bladder. One male given 30 mg/kg/day had slight chronic inflammation of the lamina propria of the urinary bladder. Additional treatment-related observations of the urinary bladder consisted of slight diffuse edema of the lamina propria and muscularis and slight multifocal chronic active inflammation of the lamina propria in one female given 30 mg/kg/day and in two females given 100 mg/kg/day, focal or multifocal acute (recent) hemorrhage of the lamina propria in one female given 30 mg/kg/day and in one female given 100 mg/kg/day, and slight multifocal erosions of the transitional epithelium in one female given 100 mg/kg/day.

There were no treatment-related effects of butyl phenyl ether on neurological or reproductive function or prenatal/early neonatal growth and survival of the offspring.

Based on these results, the no-observed adverse-effect level (NOAEL) for general toxicity was not established due to the occurrence of treatment-related adverse effects of the urinary bladder present in males and females at all dose levels. The no-observed-effect level (NOEL) for reproductive and neurological effects was 350 mg/kg/day, the highest dose level tested.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 475 (Mammalian Bone Marrow Chromosome Aberration Test)

Version / remarks:

Assay incorporated into OECD 422 assay

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian bone marrow chromosome aberration test

Test material

Test material information

Constituent 1

Reference substance name:

N-butyl phenyl ether

EC Number:

214-426-1

EC Name:

N-butyl phenyl ether

Cas Number:

1126-79-0

Molecular formula:

C10H14O

IUPAC Name:

butoxybenzene

Details on test material:

Test Material Name Butyl phenyl ether
Lot/Reference/Batch Number 22466
Purity/Characterization (Method of Analysis and Reference): 99.41% ± 0.01% by gas chromatography after correction for water content. Identification was by gas
chromatography mass spectrometry and Fourier transform infrared spectroscopy (Ferruzzi, 2016a).
Test Material Stability Under Storage Conditions: Butyl phenyl ether, lot 22466, was determined to be stable for 2 weeks at 54°C which is equivalent to 24 months under ambient storage conditions as tested under U.S. EPA OPPTS Guideline 830.6317. (Ferruzzi, 2016b).

Test animals

Species:

rat

Strain:

Sprague-Dawley

Details on species / strain selection:

Crl:CD(SD)

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories (Raleigh, North Carolina)
- Females (if applicable) nulliparous and non-pregnant: Yes
- Age at study initiation: P - 8 weeks
- Weight at study initiation: (P) Males: 277-281 g; Females: 197-200 g
- Fasting period before study: none
- Housing: Upon arrival animals were housed two or three per cage in solid bottom stainless steel cages.
The solid bottom cages contained ground corn cob bedding. Cages contained a feed crock and a press
ure activated lixit valve-type watering system.
After assignment to study, animals were housed singly in solid bottom stainless steel cages, except durin
g breeding and during the gestation and littering phases of the study. The solid bottom cages contained
ground corn cob bedding. During breeding, one male and one female were placed in stainless steel
cages with wire mesh floors that were suspended above absorbent paper in order to better visualize
copulation plugs. During gestation and littering, dams (and their litters) were housed in plastic cages
provided with irradiated ground corn cob bedding from approximately GD 0 until completion of lactation
(LD 4). Cages contained a feed crock and a pressure activated lixit valve-type watering system.
- Enrichment: Enrichment for rats was provided from the day of arrival until necropsy. The cage contai
ned nylon bones during the pre-breeding phase (males and females) and following mating (all males) or
paper nesting material during the gestation and lactation phases of the study.
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: at least one week
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22°C with a range of 20°C-26°C
- Humidity (%): 50% with a range of 30-70%
- Air changes (per hr): 10-15 times/hour (average)
- Photoperiod (hrs dark / hrs light):12-hour light/dark (on at 6:00 a.m. and off at 6:00 p.m.)
IN-LIFE DATES: From: To: Dosing started: June 01, 2016; Male necropsy July 6-7, 2016; Female
necropsy July 13 -19, 2016. Pups euthenized on Post natal day 4.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSE: Butyl phenyl ether was administered in corn oil and CP in distilled water, such
that a dose volume of 4 ml/kg body weight yielded the targeted dose. Dose volumes were adjusted using
the most current body weight. Dose solutions were not corrected for purity. Butyl phenyl dose solutions
were prepared within the 12 day stability limit.
VEHICLE
- Supplier: Corn Oil supplied by Sigma-Aldrich, St. Louis, Missouri
- Justification for use and choice of vehicle (if other than water): Vehicle choice determined by solubility.

Duration of treatment / exposure:

For the test material: 36 days
for the positive control a single dose 24 hours prior to scheduled necropsy.

Frequency of treatment:

daily

Post exposure period:

24 hours

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (actual dose received)

Doses / concentrations 2

Dose / conc.:

30 mg/kg bw/day (actual dose received)

Doses / concentrations 3

Dose / conc.:

100 mg/kg bw/day (actual dose received)

Doses / concentrations 4

Dose / conc.:

350 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

5 males

Control animals:

yes, concurrent vehicle

Positive control(s):

Cyclophosphamide, 20 mg/kg bw/day, 5 males

Examinations

Tissues and cell types examined:

Bone marrow

Details of tissue and slide preparation:

The bone marrow from the first five male rats of the vehicle control group , the butyl phenyl ether treated groups, and the five male rats in the CP group were removed at the scheduled necropsy on test day 36 from the left femur by aspiration into fetal calf serum and centrifuged at 6000-10000 rpm for five minutes. The cell pellet was resuspended in a drop of serum and a wedge film was prepared on a microscope slide. The slides were allowed to air dry and then fixed with methanol prior to staining with Acridine Orange. Only slides from the male rats were scored since no apparent difference in toxicity (e.g., relative liver weight and effects on urinary bladder) between the sexes was observed in the study and decreased food consumption and body weight and increased reticulocyte counts were observed in males only. Slides were coded, scored, and decoded upon completion to control for bias.

Evaluation criteria:

Whenever possible, a minimum of 4000 polychromatic erythrocytes (PCE) were examined from each animal and the number of MN-PCE were recorded. The proportion of PCE among total erythrocytes (PCE and normal chromatic erythrocytes (NCE)) in the bone marrow were determined by examining at least 500 erythrocytes. The percentage of PCE was expressed as PCE /PCE+NCE x 100.

Statistics:

MN-PCE and percent PCE were tested for equality of variance using Bartlett's test (alpha = 0.01; Winer, 1971). If the results from Bartlett's test were significant, then the data for the parameter might have been subjected to a transformation to obtain equality of the variances. The transformations that were examined are the common log, the inverse, and the square root in that order. The data were reviewed and an appropriate form of the data selected and subjected to the following analysis.
The MN-PCE data and the data on percent PCE were analyzed by a one-way analysis (Winer, 1971). Pairwise comparisons of treated vs. control groups were done, if the dose effect was significant, by Dunnett’s t-test, one-sided (upper) for MN-PCE and two-sided for the percent PCE (Winer 1971). Linear dose-related trend tests would be performed if any of the pairwise comparisons yielded significant differences. The alpha level at which all tests was conducted was 0.05.
The final interpretation of biological significance of the responses was based on both statistical outcome and scientific judgment. A test was considered valid if all of the following conditions were met:
• There was a significant increase in the incidence of MN-PCE in the positive control treatment as compared to the concurrent negative controls.
• The mean for percent PCE value in one or more of the test material treated groups should be  20% of the control value indicating no undue effect on erythropoiesis (toxicity).
A test material was considered positive in this assay if the following criterion was met:
• Statistically significant increase in MN-PCE frequency at one or more dose levels accompanied by a dose response.
A test material was considered negative in this assay if the following criterion is met:
• No statistically significant dose-related increase in MN-PCE when compared to the negative control.

Results and discussion

Test results

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

yes

Remarks:

general toxicity (systemic - liver wt increase, effects in bladder)

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Any other information on results incl. tables

The %PCE observed in the groups treated with butyl phenyl ether was not significantly different from the vehicle control group. As expected, the %PCE of the positive control group was significantly lower than that of the vehicle control group. The MN-PCE frequency (/1000) in the vehicle control group was 3.30 ± 1.10 (mean ± SD). The MN-PCE frequencies in the 30, 100, and 350 mg/kg bw/day treatment groups were 3.30 ± 1.32, 3.69 ± 0.85, and 3.72 ± 1.01, respectively. There were no significant differences in MN-PCE frequency among the groups treated with butyl phenyl ether and the vehicle control group. The adequacy of the experimental conditions for the detection of induced micronuclei was confirmed with the observation of a significant increase in the frequencies of MN-PCE in the positive control group (35.65 ± 3.74). 

The treatment-related effects on liver and urinary bladder (see below) demonstrated the systematic distribution of the test material and/or its metabolites after oral gavage. The treatment-related hematologic effects (e.g., statistically significant higher mean reticulocyte count in the 350 mg/kg/day males) further demonstrated the adequate bone marrow exposure to the test material and/or its metabolites. Therefore, butyl phenyl ether was negative in this bone marrow erythrocyte MNT under the experimental conditions used. 

Table: Summary of Micronucleated Reticulocytes (MN-RET) Frequencies and Percent Reticulocytes (%RET) - Males

Exposure (mg/kg bw/day)	N^a	MN-PCE/1000^b	% PCE^c
0^d	5	3.30 ± 1.10	63.1 ± 4.0
30	5	3.30 ± 1.32	66.4 ± 6.2
100	5	3.69 ± 0.85	63.9 ± 2.0
350	5	3.72 ± 1.01	68.4 ± 9.3
Positive Control (20 mg/kg CP^e)	5	35.65 ± 3.74 *	43.8 ± 9.7 *
^aThe number of animals per dose group for the micronucleus assay. ^bMN-PCE frequency: MN-PCE per 1000 PCEs. 4000 PCEs were examined per animal except that 712 PCEs were scored for Animal 1494 in the 100 mkd group and 2100 PCEs were scored for Animal 1508 in the 350 mkd group due to smear quality. ^cPCE percentage: 500 erythrocytes were examined per animal and % PCE = PCE/(PCE+NCE) × 100. ^dAnimals were dosed with the vehicle (corn oil). ^eCP = Cyclophosphamide monohydrate * Significantly different from the negative control (alpha=0.05).

Applicant's summary and conclusion

Conclusions:

There were no treatment-related effects in micronucleated polychromatic erythrocytes (MN-PCE) frequency for males at any dose. Therefore, butyl phenyl ether was negative in this bone marrow erythrocyte MNT under the experimental conditions used.