N-butyl phenyl ether

Endpoint summary

Description of key information

OECD 422 combined repeated dose/reproductive screening study in rats (oral gavage)

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

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.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

LOAEL

30 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

good

System:

urinary

Organ:

bladder

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

There is a single repeated dose toxicity study available for Butyl Phenyl Ether.

Oral OECD 422:

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.

Summary:

With the exception of effects on bodyweight in top dose males and what appears to be adaptive liver weight increases in high dose males and females, the critical effect observed in thsi study was irritation/inflammation in the urinary bladder. This effect occurred at all dose levels, but with greater severity in males versus females. In the females there was some evidence of a dose response, particularly with the hyperplasia and inflammation, however in the males all dose groups appeared to be affected equally in terms of both severity and frequency.

This effect in the bladder did not appear to compromise the functioning of this organ nor was it associated with any other effects on the urinary tract or on the composition of the urine.

It is possible that this effect is related to the cytotoxicity/irritation of the parent compound or its metabolites (cytotoxicity was a consistent finding in all in vitro assays). However no clear conclusions on mode of action can be drawn from this study.

Due to the presence of this bladder finding at all dose levels it was not possible to derive a NOAEL for this study. Consequently the LOAEL is the lowest dose of 30 mg/kg bw/day) and this will be taken forward to the risk characterisation.

Justification for classification or non-classification

Although the effects on the bladder occurred at a dose level less than 100 mg/kg bw/day and without a clear no effect level, the nature of the effect (slight inflammation/irritation) was not severe, nor did it compromise the functioning of the urinary bladder or result in significant morbidity. Consequently it is concluded that the classification criteria for repeated dose toxicity are not met and no classification for this endpoint is warranted.