1,2-Benzenediol, 3,5-dialkyl-, 1,2-dicarboxylate

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

12 June - 12 July 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study was conducted according to test guidelines and in accordance with GLP.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

other: USEPA, OPPTS 870.3050

Deviations:

not specified

Test guideline 2

Qualifier:

according to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Deviations:

not specified

Test guideline 3

Qualifier:

according to guideline

Guideline:

EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))

Deviations:

not specified

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Test animals

Species:

rat

Strain:

Fischer 344/DuCrj

Sex:

male/female

Details on test animals or test system and environmental conditions:

Species and Sex
Rats (male and female) (nulliparous and non-pregnant)

Strain and Justification
F344/DuCrl rats were selected because of their general acceptance and suitability for toxicity testing, availability of historical background data and the reliability of the commercial supplier.

Supplier and Location
Charles River (Kingston, New York)

Age at Study Start
Approximately 9 weeks

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

Housing
After assignment to the study, animals were housed one per cage in stainless steel, solid bottom cages. Ground corn cob and shredded Aspen bedding were used in the cages (Harlan Laboratories, Inc., Indianapolis, Indiana). Cages contained a feed crock and a pressure activated lixit valve-type watering system. The following environmental conditions were maintained in the animal room.
Temperature: 22°C with a tolerance of ± 1°C (and a maximum permissible excursion of ± 3°C)
Humidity: 40-70%
Air Changes: 10-15 times/hour (average)
Photoperiod: 12-hour light/dark (on at 6:00 a.m. and off at 6:00 p.m.)

Enrichment
Enrichment for rats included the use of ground corn cob and shredded Aspen bedding and open areas on the cage sides for visualization of other rats.

Randomization and Identification
Before administration of test material began, 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 placed on study were uniquely identified via subcutaneously implanted transponders (BioMedic Data Systems, Seaford, Delaware) that were correlated to unique alphanumeric identification numbers.

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

Administration / exposure

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on oral exposure:

Route and Justification, Method of Administration, Frequency and Duration
The oral route was selected because it represented a possible route of human exposure. Thus, oral administration of the test material to rats via the diet represented an appropriate means of exposure. Animals were fed diets containing the test substance seven days/week for at least 28 days.

Dose Preparation
Diets were prepared by serially diluting a concentrated test material-feed mixture (premix) with ground feed. Premixes and diets were mixed weekly and dietary concentrations were adjusted based upon the most recent body weight and feed consumption data. Initial concentrations of test material in the diet were calculated from historical body weight and feed consumption data.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Dose Confirmation and Homogeneity
Dose confirmation analyses of all dose levels, plus control and premix, were determined pre-exposure from the first mix. The homogeneity of the low-dose female and the high-dose male test material-feed mixtures were determined concurrent with dose confirmation. The method used for analyzing the test material in the diet was a solvent extraction method followed by analysis using liquid chromatography-mass spectrometry (LCMS).

Stability
Stability analyses were conducted prior to the start of the study and concurrent with the study. The test substance was stable for 50 days in the feed at concentrations ranging from 0.005 to 10%. The established concentration range and duration of stability spanned those used in this study.

Results
The concentrations and homogeneity of the test substance in the diets were determined for the control, premix, and all treatment levels pre-exposure. Analyses of all test diets indicated the mean concentration ranged from 92.3 to 109.7% of targeted concentrations indicating acceptable concentrations of the test substance. The relative standard deviations for all diets sampled were between 2.5 and 8.4% indicating that diets were homogeneously mixed.

Duration of treatment / exposure:

Animals were fed diets containing the test substance seven days/week for at least 28 days.

Frequency of treatment:

Daily

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 100, 300 and 1000 mg/kg
Basis:
actual ingested

No. of animals per sex per dose:

10/sex/dose

Control animals:

yes, concurrent no treatment

Details on study design:

Experimental Design and Critical Dates
Although the test guidelines require only 5 animals/sex/dose group, the inclusion of functional tests in this study necessitated the use of a larger sample size due to the typically higher inter-animal variability in the neurobehavioral endpoints as compared to other response-variables. All in-life data and gross pathological observations at necropsy were obtained from 10/rats/sex/dose, while all other data collected post-mortem were confined to five rats/sex/dose as detailed below.

Groups of ten male and ten female F344/DuCrl rats were fed diets supplying 0, 100, 300, or 1000 mg/kg body weight/day (mg/kg/day) for at least 28 days to evaluate the potential for systemic toxicity. Parameters evaluated on all rats were daily cage-side clinical observations, weekly detailed clinical observations, functional tests, ophthalmic examinations, body weights, body weight gains, feed consumption and gross pathologic observations at the scheduled necropsy. Hematology, prothrombin time, clinical chemistry, urinalysis, and selected organ weights were conducted on the first five rats/sex/dose group. An extensive histopathological evaluation of organs/tissues was conducted on the first five rats/sex of the control and high-dose groups. In addition, histopathology was also conducted on liver tissue from the first five males of the lowand intermediate-dose groups.

Test material administration for all animals began on June 12, 2012. Rats were necropsied on July 11 and 12, 2012 (test days 30 and 31).

Positive control:

No data.

Examinations

Observations and examinations performed and frequency:

Daily Observations
A cage-side examination was conducted at least once a day, at approximately the same time each day (usually in the morning). This examination was typically performed with the animals in their cages and was designed to detect significant clinical abnormalities that were clearly visible upon a limited examination, and to monitor the general health of the animals. The animals were not hand-held for these observations unless deemed
necessary. Significant abnormalities that could have been observed include, but were not limited to: decreased/increased activity, repetitive behavior, vocalization, incoordination/limping, injury, neuromuscular function (convulsion, fasciculation, tremor, and 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.

Detailed Clinical Observations
Detailed clinical observations (DCO) were conducted on all animals pre-exposure and once per week throughout the study. The DCO was conducted on all animals, at approximately the same time each day according to an established format. The examination included cage-side, hand-held and open-field observations that were recorded categorically or using explicitly defined scales (ranked). Categorical observations (descriptive) are summarized under the clinical observations

Functional Tests
The functional tests (sensory evaluation, rectal temperature, grip performance and motor activity) were conducted on all animals pre-exposure and during the last week of the treatment period.

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, during and after the study. The instrument was recalibrated if the temperature recordings differed from the reference thermometer by more than ± 0.5°C.

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 were 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, is acceptable).

Motor Activity
An automated system was used for motor activity (MA) data collection. No entry into the MA test room was allowed during the testing period. Each
test session consisted of six 8-minute epochs, totaling 48 minutes of testing per animal per test session. This duration was chosen based on the results of a validation study indicating that performance of control animals approached asymptote in 30-40 minutes in F344/DuCrl rats (The Dow Chemical Company, 2007). Activity counts for each epoch were recorded.

Motor Activity Cage Calibration: Cages used for testing were calibrated prior to testing each day. Calibration was performed with a rod attached to a rotary motor that breaks the infrared beam at a constant speed. The duration of each beam break was calculated to ensure equivalence across chambers.

Motor Activity Cage Allocation: Rats were allocated to the motor activity cages in such a way that the counterbalancing of treatment groups and sexes across cages and test times was maximized.

Ophthalmology
The eyes of all animals were examined by a veterinarian pre-exposure and prior to the scheduled necropsy (day 29) using indirect ophthalmoscopy. One drop of 0.5% tropicamide ophthalmic solution was instilled topically in each eye to produce mydriasis prior to the indirect ophthalmic examinations. Eyes were also examined by a prosector during the necropsy using a moistened glass slide pressed to the cornea.

Body Weights/Body Weight Gains
All rats were weighed pre-exposure, twice during the first week and at least weekly throughout the study. Body weight gains were calculated relative to day 1.

Feed Consumption
The amount of feed consumed was determined pre-exposure, twice during the first week and at least weekly for all animals by weighing feed containers at the start and end of a measurement cycle. Feed consumption was calculated using the following equation:
Feed consumption (g/day) = (initial weight of feed container - final weight of feed container)/(# of days in measurement cycle) (# of animals per cage)

Test Material Intake
The test material intake (TMI, expressed as mg/kg body weight/day) was calculated using feed concentrations, body weights and feed consumption data in the following equation:

TMI = [(feed consumption (g/day))(1000 mg/g)(% of test material in feed)/100]/[((Current BW (g) + Previous BW (g))/2)/1000g/kg]

References
Mattsson, J. L., Johnson, K. A., and Albee, R. R. (1986). Lack of neuropathologic consequences of repeated dermal exposure to 2,4-dichlorophenoxyacetic acid in rats. Fund. Appl. Toxicol. 6, 175-181.

The Dow Chemical Company (2007). Motor Activity Validation Study Using Positive Controls: Effects of Amphetamine and Chlorpromazine. Report of Toxicology & Environmental Research and Consulting, The Dow Chemical Company, Midland, Michigan.

Sacrifice and pathology:

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 from the first five rats/sex/dose group.

Hematology
Sample Preparation
Blood samples for a complete blood count were mixed with ethylenediaminetetraacetic acid (EDTA). Blood smears were prepared, stained with Wright-Giemsa stain, cover-slipped and archived for potential future evaluation, if warranted.

Hematologic parameters were assayed using the Advia 120 Hematology Analyzer (Siemens Healthcare Diagnostics, Tarrytown, New York).
Assays
Hematocrit (HCT)
Hemoglobin (HGB) concentration
Red blood cell (RBC) count
Total white blood cell (WBC) count
Differential WBC count
Platelet (PLT) count
Reticulocyte (RET) count
RBC indices:
Mean Corpuscular Hemoglobin (MCH)
Mean Corpuscular Volume (MCV)
Mean Corpuscular Hemoglobin Concentration (MCHC)
Platelet counts were obtained from undiluted and diluted blood samples of all rats. Platelet counts from all blood samples analyzed including those of the controls, were found to be uniformly slightly lower than expected values despite all routine calibration/validation procedures confirming the hematology analyzer was in good working condition. However, this unexpected phenomenon disappeared when the blood samples were diluted
1:10 with isotonic saline resulting in platelet counts that were within the normal reference intervals noted in 90-day studies in F344 rats recently conducted in this laboratory (see Results section). All other hematology parameters evaluated were within expected ranges. The hematology analyzer was subsequently inspected by the manufacturer’s authorized personnel and a new laser was installed. Rodent blood samples (unrelated to those of this study) subsequently analyzed yielded normal expected results including platelet counts indicating that the original defect in rodent platelet counts was associated with the laser. Because all the blood samples were handled, diluted and analyzed in the same manner consistently with control and treatmentgroups, the platelet counts generated did not impact the interpretation of the hematology results.

Coagulation
Sample Preparation
Blood samples were collected in sodium citrate tubes, centrifuged, plasma collected, and assayed using the Instrumentation Laboratory ACL9000
Analyzer (Beckman Coulter, Inc., Miami, Florida).
Assay
Prothrombin time (PT)
Clinical Chemistry
Sample Preparation
Blood samples were collected and serum was separated from cells as soon as possible. The following 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)
Gamma-glutamyl transpeptidase (GGT)
Concentrations of:
Albumin (ALB)
Albumin/Globulin Ratio (A/G) - calculated
Cholesterol (CHOL)
Creatinine (CREA)
Electrolytes (NA, K, PHOS, CL and CA)
Globulin (GLOB) – calculated (TP-ALB)
Glucose (GLUC)
Total bile acids (TBA)
Total bilirubin (TBIL)
Total protein (TP)
Triglycerides (TRIG)
Urea nitrogen (UN)

Urinalysis
Urine samples were obtained from the first five rats/sex/dose group the week prior to the scheduled necropsy. Rats 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 the first five rats/sex/dose group by gentle manual compression of the urinary bladder. The urine samples were pooled from each group, and the microsediments were characterized microscopically.

Anatomic Pathology
Necropsy
Fasted rats submitted alive for necropsy were weighed (prior to necropsy in the animal room), anesthetized by the inhalation of CO2/O2, and blood samples obtained from the orbital sinus (from the first five rats/sex/dose group). Their tracheas were exposed and clamped, and the rats were euthanized by decapitation. A complete necropsy was conducted on all rats by a veterinary pathologist 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.

From the first five rats/sex/dose group, the brain, liver, kidneys, heart, adrenals, testes, epididymides, prostate + seminal vesicles with coagulating glands (and fluids), prostate, thymus and spleen were trimmed and weighed immediately. The thyroid glands (with the parathyroid) were weighed post fixation. The ratios of organ weight to terminal body weight were calculated.

Representative samples of tissues were collected and preserved in neutral, phosphate-buffered 10% formalin from all rats (as per guidelines). Testes and epididymides were preserved in Bouin’s fixative. Transponders were removed and placed in with the tissues.

Histopathology
The number of sections from all preserved tissues were processed by standard histologic procedures from the first five control and high-dose group
rats. 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 following tissues from the first five rats of the remaining groups were processed, sectioned, and stained: liver, kidneys, and relevant gross lesions. Relevant gross lesions were microscopically examined from all rats. All target tissues (liver, males only) were microscopically examined from the low- and intermediate-dose group rats to define a NOEL. Liver (females) and kidneys from the low- and intermediate-dose group rats were, however, not microscopically examined because there were no treatment-related histopathological changes in the liver of females and kidneys of males and females of the high-dose group. 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 may be 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 would not be 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 may be life threatening.

Other examinations:

No additional information available.

Statistics:

Means and standard deviations were calculated for all continuous data. All parameters examined statistically (feed consumption was addressed below) were first tested for equality of variance using Bartlett's test (Winer, 1971). If the results from Bartlett's test were significant at alpha = 0.01, then the data for the parameter may have been subjected to a transformation to obtain equality of the variances. The transformations that were examined were the common log, the inverse, and the square root, in that order. The data were reviewed and an appropriate form of the data was selected. The selected form of the data was then subjected to the appropriate parametric analysis as described below.

In-life body weights were evaluated using a repeated measures (RM) analysis of variance (ANOVA), the multivariate approach, for time (the repeated factor), sex, and dose (Winer, 1971). In the repeated measures ANOVA with a pre-exposure data point, the time-dose interaction assessed the true effect of treatment (Bonate, 2000).

The first examination in the RM-ANOVA was of the time-sex-dose interaction. If significant at alpha = 0.02, the analysis was repeated separately for each sex without examining the results of other factors. The time-dose interaction was examined next. If the time-dose interaction was statistically significant at alpha = 0.05, linear contrasts tested the time-dose interaction for the comparisons of each dose group to the control group. A Bonferroni correction (Miller, 1966) was applied to the alpha level to compensate for the multiple comparisons with the control group. This correction controlled the experiment-wise error rate. The corrected comparison-wise alpha level of 0.02 was reported so direct comparison could be made to the Pillai’s Trace p-values generated.

(continued below)

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not specified

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

not examined

Ophthalmological findings:

no effects observed

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

no effects observed

Behaviour (functional findings):

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

male rats at 300 and 1000 mg/kg/day

Details on results:

Mortality
There was no mortality. All rats survived to the scheduled necropsy.

Detailed Clinical and Cage-side Observations
There were no treatment-related observations in any group during the study period. The only observation noted was red periocular soiling in one rat (# 4440) in the 300 mg/kg/day group from test day 15-22. This was considered spurious and unrelated to treatment due to the isolated occurrence and lack of a dose-response relationship.

Functional Tests
Sensory Evaluation
There were no treatment-related effects in the sensory evaluation. There were two observations that were statistically identified in the z-test of proportions at alpha = 0.05, and both of these observations were in females at the high-dose level under baseline conditions (responsiveness to tail pinch, minimal; and responsiveness to tail pinch, moderate). These observations were considered to be spurious and unrelated to treatment as they occurred under baseline conditions (pre-exposure).

Rectal Temperature
There were no treatment-related effects in rectal temperature. The treatment by sex interaction was not significant (p = 0.4930), indicating that there were no treatment-related sex differences in rectal temperature. The main effect of treatment was also not significant (p = 0.4824), indicating that the test substance did not affect rectal temperature.

Grip Performance
There were no treatment-related effects in grip performance. The treatment by sex interaction was not significant for hindlimb (p = 0.2397) or forelimb (p = 0.6849) grip performance, which indicated that there were no treatment-related sex differences in grip performance. The main effect of treatment was also not significant for hindlimb (p = 0.9172) or forelimb (p = 0.8501) grip performance, which indicated that the test substance did not affect grip performance in rats.

Motor Activity
There were no treatment-related effects seen in motor activity. The treatment by time by sex interaction was not significant (p = 0.8151), which indicated that there was not a treatment-related sex difference in motor activity at either time point. The treatment by time interaction was also not significant (p = 0.7240), which indicated that the test substance did not affect motor activity in rats. The treatment by time by epoch interaction was not significant (p = 0.5658), which indicated that the test substance did not affect the within-session distribution of motor activity counts at either time interval.

Ophthalmology
Pre-exposure examination of all rats placed on study indicated that they were suitable for study assignment. Observations such as, pale fundus, and cloudy cornea were noted in some rats in the control and 100 mkd groups prior to study termination (test day 29); however, these were interpreted to be spontaneous and unrelated to treatment due to the low incidence and lack of a dose-response relationship.

Body Weights/Body Weight Gain
There were no statistically identified or treatment-related differences in the body weights of male or female rats at any dose level as compared to their respective controls. Body weight gains (relative to test day 1) of males and females at all dose levels were similar to those of the controls.

Feed Consumption
There were no statistically identified or treatment-related differences in feed consumption of male or female rats at any dose level as compared to their respective controls.

Test Material Intake
Males and females were given diets formulated to supply 0, 100, 300, or 1000 mg/kg/day, which corresponded to time-weighted average doses of 0, 94.5, 286, or 963 mg/kg/day for males and 0, 92.4, 284, or 938 mg/kg/day for females, respectively.

Clinical Pathology
Hematology
There were no statistically identified or treatment-related changes in any of the hematologic parameters of males or females at any dose level
as compared to their respective controls.

Platelet counts from all blood samples analyzed including those of the controls, were found to be uniformly slightly lower than the expected values despite all routine calibration/validation procedures confirming the hematology analyzer was in good working condition. However, this unexpected phenomenon (subsequently found to be associated with the laser; see Methods section) that was restricted to only platelet counts, was rectified when the blood samples were diluted 1:10 with isotonic saline that resulted in platelet counts which were comparable to the normal reference intervals noted in 90-day studies with F344 rats conducted recently in this laboratory. Because all the blood samples were handled, diluted and analyzed in the same manner consistently with control and treatment-groups, the platelet counts generated did not impact the interpretation of the hematology results.

There were no statistically identified or treatment-related changes in platelet counts of males or females either in the undiluted or diluted blood samples at any dose-level.

Prothrombin Time
There were no statistically identified or treatment-related changes in prothrombin times of males or females at any dose level as compared to their respective controls.

Clinical Chemistry
There were no treatment-related alterations in any of the clinical chemistry parameters of males or females at any dose level.

The only statistically identified difference noted was a higher mean cholesterol level of mid-dose males. Males given 300 mg/kg/day had a statistically identified, 25% higher serum cholesterol concentration when compared to that of the controls. This was however, interpreted to be unrelated to treatment due to lack of a doseresponse relationship.

Urinalysis
There were no treatment-related alterations in any of the urinalysis parameters for male and female rats at any of the dose levels.

Anatomic Pathology
Organ Weight
Treatment-related, statisticallyidentified increases in the mean relative liver weights were noted in male rats given 300 (17.7 %) and 1000 mg/kg/day (14.3 %), relative to controls (Table 1). The higher relative liver weights corresponded with hypertrophy of centrilobular and midzonal hepatocytes at these dose levels (see Histopathology section). In addition, the absolute liver weights of male and female rats given 300 or 1000 mg/kg/day were statistically-identified (sexes combined for statistical analysis) as higher than that of the controls. The higher mean absolute liver weights of female rats given 300 or 1000 mg/kg/day were interpreted to be unrelated to treatment because 1) lack of histopathologic correlate, 2) lack of dose-response relationship and 3) the relative liver weights were not statistically-identified.

In both males and females (sexes combined for statistical analysis), the absolute kidney weight at 300 and the relative kidney weights at 300 or 1000 mg/kg/day were statistically identified as slightly higher than those of the controls. These alterations in kidney weights were interpreted to be unrelated to treatment because 1) there was no histopathologic evidence of kidney disease and/or any indication of renal dysfunction (based on normal blood urea nitrogen and creatinine values) that could correlate to the increases in kidney weights, 2) there was no clear dose response relationship, and 3) the kidney weights were within or near historical controls ranges.

Gross Pathology
There were no treatment-related gross pathologic observations. All gross pathologic observations were interpreted to be spontaneous alterations, unassociated with exposure to the test substance.

Histopathology
Male rats given 300 or 1000 mg/kg/day had treatment-related very slight hypertrophy of the centrilobular and midzonal hepatocytes. The hypertrophied hepatocytes had increased cytoplasmic eosinophilia when compared to those of the controls. The severity of this alteration was not dose-dependent. The hepatocellular hypertrophy with increased cytoplasmic eosinophilia corresponded to the higher relative liver weights noted in male rats given 300 or 1000 mg/kg/day. The hepatocellular hypertrophy along with the modest increases in relative liver weights of males given 300 (17.7 %) or 1000 mg/kg/day (14.3 %), relative to controls were interpreted to be an adaptive hepatocellular response to accommodate increased functional load following the ingestion of the test material. This interpretation was based on the absence of treatment-related changes in liver enzyme activities (serum ALT, AST and/or GGT) or any other clinical chemistry parameters or histopathologic evidence of necrosis, apoptosis or cell proliferation, vacuolization or any other degenerative changes indicative of liver toxicity (Williams and Iatropoulous, 2002, Hall et al., 2012). Therefore, the increased liver weights and the hepatocellular hypertrophy with increased cytoplasmic eosinophilia in males given 300 or 1000 mg/kg/day in the absence of histopathologic or clinical chemistry alterations indicative of liver toxicity was interpreted to be an adaptive and non-adverse change.

There were no treatment-related histopathologic changes in males given 100 mg/kg/day or in females at any dose level.

All other histopathologic observations in the liver and other organs examined were interpreted to be spontaneous alterations unassociated with exposure to the test substance.

References
Hall, A. P., Elcombe, C. R., Foster, J. R., Harada, T., Kaufmann, W., Knippel, A., Küttler, K., Malarkey, D. E., Maronpot, R. R., Nishikawa, A., Nolte, T., Schulte,
A., Strauss, V., and York, M. J. (2012). Liver Hypertrophy: A Review of Adaptive (Adverse and Non-adverse) Changes-Conclusions from the 3rd
International ESTP Expert Workshop. Toxicologic Pathology published online 21 June 2012, (http://tpx.sagepub.com/content/early/2012/06/20/0192623312448935).

William, G. M. and Iatropoulos, M. J. (2002). Alteration of Liver Cell Function and Proliferation: Differentiation Between Adaptation and Toxicity. Toxicologic Pathology 30: 41-53.

Effect levels

1

Dose descriptor:

NOEL

Effect level:

100 mg/kg bw (total dose)

Based on:

test mat.

Sex:

male

Basis for effect level:

other: Based on the liver effects noted in males given 300 or 1000 mg/kg/day.

2

Dose descriptor:

NOEL

Effect level:

1 000 mg/kg bw (total dose)

Based on:

test mat.

Sex:

female

Basis for effect level:

other: No effects noted in female rats receiving 1000 mg/kg/day, the highest dose examined.

3

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw (total dose)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: As the liver effects observed in males given 300 or 1000 mg/kg/day were non-adverse, the No-Observed-Adverse-Effect Level (NOAEL) was determined to be 1000 mg/kg/day in male and female rats.

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

Table 1 Final Body and Organ Weights

Sex	Males
Dose (mg/kg/day)	Historical Control^@	0	100	300	1000
Final Body wt (g)	188.6 - 221.8	246.0	242.0	247.7	248.5
Liver (g)	6.320 - 6.888	6.893	7.231	8.189*	7.963*
Liver (g/100)	2.980 - 3.492	2.803	2.987	3.300^a	3.204^a
Kidneys (g)	1.490 - 1.635	1.681	1.726	1.890*	1.823
Kidneys (g/100)	0.717 - 0.790	0.685	0.713	0.762*	0.734*
Sex	Females
Dose (mg/kg/day)	Historical Control^@	0	100	300	1000
Final Body wt (g)	129.7 - 136.9	157.4	152.1	157.6	156.6
Liver (g)	3.793 - 4.199	4.138	4.027	4.302*	4.267*
Liver (g/100)	2.847 - 3.120	2.630	2.647	2.730	2.723
Kidneys (g)	1.006 - 1.139	1.089	1.038	1.116*	1.131
Kidneys (g/100)	0.759 - 0.843	0.692	0.682	0.708*	0.722*

@Historical control data obtained from seven 28-day dietary studies conducted in this laboratory in 2009 – 2011.

* Statistically different from control mean, males and females analyzed together, by Dunnett’s test, alpha = 0.05.

^a Statistically different from control mean, males analyzed separately, by Dunnett’s test, alpha = 0.05. Bold type indicates the effects judged to be treatment related.

Applicant's summary and conclusion

Conclusions:

Under the conditions of this study, based on the liver effects noted in males given 300 or 1000 mg/kg/day, the No-Observed-Effect Level (NOEL) for the test substance in F344/DuCrl rats was the targeted dietary dose of 100 mg/kg/day in males and 1000 mg/kg/day in females. As the liver effects observed in males given 300 or 1000 mg/kg/day were non-adverse, the No-Observed-Adverse-Effect Level (NOAEL) was determined to be 1000 mg/kg/day in male and female rats.

Executive summary:

The purpose of this study was to evaluate the potential toxicity of the test substance in rats following dietary administration for at least 28 days. Ten male and ten female F344/DuCrl rats per group were given test diets formulated to supply 0, 100, 300, or 1000 milligrams/kilogram body weight/day (mg/kg/day, mkd) for at least 28 days. Parameters evaluated were daily cage-side clinical observations, weekly detailed clinical observations, functional tests, ophthalmic examinations, body weights, body weight gains, feed consumption and gross pathologic observations at the scheduled necropsy. Hematology, prothrombin time, urinalysis, clinical chemistry and selected organ weights were evaluated on the first five males and females per dose-group. An extensive histopathologic evaluation was conducted on the first five males and females of the control and high-dose groups. In addition, histopathologic evaluation of liver tissue from the first five male rats in the low- and intermediate dose-groups were conducted.

There were no treatment-related effects in clinical signs, functional tests, body weights/body weight gains, feed consumption, ophthalmic examinations, hematology, prothrombin time, clinical chemistry, urine parameters or gross pathological observations of male or female rats at any dose level as compared to their respective controls.

Treatment-related, statistically-identified higher mean relative liver weights were noted only in male rats given 300 (17.7 %) or 1000 mg/kg/day (14.3 %), as compared to that of the controls and corresponded to treatment-related very slight hypertrophy of the centrilobular and midzonal hepatocytes. The hypertrophied hepatocytes had increased cytoplasmic eosinophilia when compared to those of the controls. The microscopic liver effects and the modest liver weight increases noted in males given 300 or 1000 mkd were interpreted to be non-adverse adaptive changes since there were no associated treatment-related changes in clinical chemistry parameters or histopathological evidence of treatment-related hepatocellular necrosis, proliferation, apoptosis or any other degenerative changes indicative of liver toxicity.

Under the conditions of this study, based on the liver effects noted in males given 300 or 1000 mg/kg/day, the No-Observed-Effect Level (NOEL) in F344/DuCrl rats was the targeted dietary dose of 100 mg/kg/day in males and 1000 mg/kg/day in females. As the liver effects observed in males given 300 or 1000 mg/kg/day were non-adverse, the No-Observed-Adverse-Effect Level (NOAEL) was determined to be 1000 mg/kg/day in male and female rats.