N-[[3,5-dichloro-2-fluoro-4-(1,1,2,3,3,3-hexafluoropropoxy)phenyl]carbamoyl]-2,6-difluorobenzamide

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

25 January 2001 - 03 January 2002

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2002

Report date:

2002

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.26 (Sub-Chronic Oral Toxicity Test: Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3100 (90-Day Oral Toxicity in Rodents)

Deviations:

no

Test guideline 4

Qualifier:

according to guideline

Guideline:

other: JMAFF Testing Guidelines Subchronic Oral Toxicity Study

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

1-[3,5-dichloro-2-fluoro-4-(1,1,2,3,3,3-hexafluoropropoxy)phenyl]-3-(2,6-difluorobenzoyl)urea

EC Number:

601-779-5

Cas Number:

121451-02-3

Molecular formula:

C17H7Cl2F9N2O3

IUPAC Name:

1-[3,5-dichloro-2-fluoro-4-(1,1,2,3,3,3-hexafluoropropoxy)phenyl]-3-(2,6-difluorobenzoyl)urea

Test material form:

solid: particulate/powder

Details on test material:

- Physical state: White, solid/powder

Test animals

Species:

rat

Strain:

Fischer 344

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: 6 weeks.
- Housing: Individually, in stainless steel cages.
- Diet: Rodent diet in meal form, provided ad libitum.
- Water: Municipal water, provided ad libitum.
- Acclimation period: Acclimated to the laboratory for one week prior to the start of the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21.7 - 22.7 °C
- Humidity (%): 45 - 52 %
- Air changes (per hr): Approximately 12-15 times/hour.
- Photoperiod (hrs dark / hrs light): 12-hour light/dark photocycle was maintained for all animal room(s) with lights on at 6:00 a.m. and off at 6:00 p.m.

IN-LIFE DATES: From: 25 January 2001 To: 26 April 2001 (males) and 27 April 2001 (females), respectively (test days 92 and 93). Recovery group animals were necropsied on 25 May 2001 (test day 121).

Administration / exposure

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on oral exposure:

DIET PREPARATION
- Rate of preparation of diet (frequency): Premixes were mixed periodically throughout the study based on stability data. Diets were prepared weekly based upon the most recent body weight and feed consumption data.
- Mixing appropriate amounts with (Type of food): Diets were prepared by serially diluting a concentrated test material-feed mixture (premix) with ground feed. Initial concentrations of test material in the diet were calculated from pre-exposure body weights and feed consumption data.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

- Homogeneity: The homogeneity of the low-dose female and high-dose male test material-feed mixtures were determined pre-exposure, near the middle, and end of the study. Aliquots were taken from multiple areas within the containers. The method for analysis of the test material in feed was a solvent extraction method followed by analysis using liquid chromatography-mass spectrometry (LC-MS) and solvent standards incorporating an internal standard.
- Stability: The stability of the test material in rodent feed was determined pre-exposure over the concentration range used in this study (low-dose female diet and the high-dose male diet).
- Concentration verification: Analyses of all dose levels for the test material were conducted pre-exposure, near the middle, and end of the study. The method used for analysing the test material in feed was as described above.
- Retainer samples: Reference samples (one/sex/dose/mix and premix) were retained and stored at ambient temperature in sealed vials in a manner consistent with the sample retention policy of the laboratory.

Duration of treatment / exposure:

At least 90 consecutive days.

Frequency of treatment:

Daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Basis:
nominal in diet

Doses / concentrations 2

Dose / conc.:

10 mg/kg bw/day (nominal)

Remarks:

Basis:
nominal in diet

Doses / concentrations 3

Dose / conc.:

100 mg/kg bw/day (nominal)

Remarks:

Basis:
nominal in diet

Doses / concentrations 4

Dose / conc.:

500 mg/kg bw/day (nominal)

Remarks:

Basis:
nominal in diet

Doses / concentrations 5

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Remarks:

Basis:
nominal in diet

No. of animals per sex per dose:

Ten per sex per dose

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: The high-dose of 1000 mg/kg bw/day was selected based on results of the 28-day study. The high-dose was expected to produce effects in the liver and kidney. The mid- and low-dose levels of 500, 100, and 10 mg/kg bw/day were expected to provide dose-response data for any treatment-related effects observed in the high-dose group. The low-dose was expected to be a no-observed-effect level (NOEL).
- Dosing route rational: Probable routes of human exposure to the test material would be via accidental ingestion during application or manufacture. Thus, administration of the test material to rats via the diet represented an appropriate means of exposure.
- Rationale for animal assignment: Animals were stratified by pre-exposure body weight and then randomly assigned to treatment groups using a computer program.
- Post-exposure recovery period in satellite groups: Additional groups (ten/sex/dose) were given exposed to the test material at either 0 or 1000 mg/kg bw/day for 90 days and were then given control feed for an additional 28 days to assess the reversibility of treatment-related effects induced following exposure to the test material.

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice each day.
- Cage side observations (where possible): Skin, fur, mucous membranes, respiration, nervous system function (including tremors and convulsions), animal behaviour, moribundity, mortality, and the availability of feed and water.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Detailed clinical observations (DCO) were conducted pre-exposure and weekly throughout the study. DCO were conducted on all animals, at approximately the same time each examination day. The data for all in-life parameters were separated between the rats designated for the main and the recovery groups to facilitate computerized data management.
- DCO examinations: Cage-side, hand-held and open-field observations that were recorded categorically or using explicitly defined scales (scored).

BODY WEIGHT: Yes
- Time schedule for examinations: All rats were weighed during the pre-exposure period and at least weekly during the remainder of the study. Body weight gains were also calculated.

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
- Food consumption: Feed consumption data were collected pre-exposure and at least weekly thereafter for all animals. Feed containers were weighed at the start and end of a measurement cycle and feed consumption was calculated using the following equation:
Feed consumption (g/day) = (initial weight of feeder - final weight of feeder) /[(No. of days in measurement cycle)(No. of animals per cage)]
- Test material intake: TMI was calculated using actual feed concentrations, body weights and feed consumption data in the following equation:
TMI = [(feed consumption) * (1000) * (% of test material in feed/100)] / {[(current BW + previous BW) / 2] / 1000}

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: A veterinarian examined the eyes pre-exposure and prior to the scheduled necropsy (main group only) using indirect ophthalmoscopy.
- Dose groups that were examined: The eyes of all animals were examined.
- Method: One drop of 0.5 % tropicamide ophthalmic solution was instilled in each eye to produce mydriasis prior to the indirect ophthalmic examinations. Eyes were also examined by a prosector during necropsy through a moistened glass slide pressed to the cornea.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Blood samples were collected from the orbital sinus following exposure to CO2, at the scheduled necropsy.
- Anaesthetic used for blood collection: No data
- Animals fasted: Yes
- How many animals: All animals.
- Sample preparation:
> Complete blood count: Blood samples for a complete blood count (main group only) were mixed with ethylenediamine-tetraacetic acid (EDTA). Blood smears were prepared, stained with Wright’s stain, and archived. Haematologic parameters were assayed using a Technicon H•1E Haematology Analyser.
> Coagulation: Blood samples for coagulation were collected in sodium citrate tubes, centrifuged and plasma collected and assayed using an MLA Electra 700, Automatic Coagulation Timer. Prothrombin time was also evaluated in male rats from the recovery group.
- Parameters checked:
> Complete blood count: Haematocrit (Hct), haemoglobin (Hgb) concentration, red blood cell (RBC) count, total white blood cell (WBC) count, platelet (PLAT) count, differential WBC count, RBC indices (MCH, MCV and MCHC), and methaemoglobin concentration.
> Coagulation: Prothrombin time (PT).

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Blood samples were collected from the orbital sinus following exposure to CO2, at the scheduled necropsy.
- Animals fasted: Yes
- How many animals: All animals.
- Sample preparation: Blood samples were collected in glass tubes and sera were separated from cells as soon as possible following blood collection. Serum parameters were measured using a Hitachi 914 Clinical Chemistry Analyser.
- Parameters checked:
> Enzyme Activities of: Alkaline phosphatase (AP), alanine aminotransferase (ALT), and aspartate aminotransferase (AST).
> Concentrations of: Albumin (ALB), cholesterol (CHOL), creatinine (CREAT), electrolytes (Na, K, PO4, Cl and Ca), glucose (GLU), total bilirubin (TBILI), total protein (TP), and urea nitrogen (UN).
> Recovery group: Alkaline phosphatase, aspartate aminotransferase, glucose, total protein and cholesterol were evaluated in males from the recovery groups and alkaline phosphatase, glucose, albumin, cholesterol, and calcium were evaluated in females from the recovery groups.

URINALYSIS: Yes
- Time schedule for collection of urine: Urine was collected during the week prior to necropsy (~16-hour period). Urine was also collected by manual compression of the bladder prior to the necropsy for characterization of the microsediment.
A timed urine volume collection with complete urinalysis was conducted as previously described, but without microscopic observations of the microsediment, from the recovery groups.
- Metabolism cages used for collection of urine: Yes
- Animals fasted: No
- Parameters checked: Colour, appearance and specific gravity (refractometer), and urine volume. Semiquantitative analysis of: pH, bilirubin, glucose, proteins, ketones, blood, and urobilinogen.

Sacrifice and pathology:

Fasted rodents were exposed to CO2, weighed, and blood samples were obtained from the orbital sinus. 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 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 glass 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.

GROSS PATHOLOGY: Yes
- Dose groups examined: All animals.
- Organ weights examined: The brain, liver, kidneys, heart, adrenals, testes, epididymides, uterus, ovaries, thymus, and spleen were trimmed and weighed immediately. The ratios of organ weight to terminal body weight were calculated. The brain, liver, and kidneys from recovery group males, and the liver, adrenals, and spleen from recovery groups females were also weighed.

HISTOPATHOLOGY: Yes
- Dose groups examined: All control and high-dose group animals. The livers from the recovery groups were also histologically examined.
- Tissues examined: Adrenals, aorta, auditory sebaceous glands, bone (including joint), bone marrow, brain (cerebrum, brainstem, cerebellum), cecum, cervix, coagulating glands, colon, cranial nerve – optic, duodenum, epididymides, esophagus, eyes, gross lesions, heart, ileum, jejunum, kidneys, larynx, liver, lungs, mammary gland (females only), mediastinal lymph node, mediastinal tissues, mesenteric lymph node, mesenteric tissues, nasal tissues, oral tissues, ovaries, oviducts, pancreas, parathyroid glands, peripheral nerve - tibial, pituitary, prostate, rectum, salivary glands, skeletal muscle, skin and subcutis, spinal cord (cervical, thoracic, lumbar), spleen, stomach, testes, thymus, thyroid gland, tongue, trachea, urinary bladder, uterus and vagina.

Other examinations:

ENZYMOLOGY
- Sample collection: Representative samples of liver were collected during the necropsy after approximately 90 days of exposure to the test material.
- Sample collection: Tissues were flash frozen in liquid nitrogen, and stored at -80 °C. Frozen tissues were subsequently processed by appropriate methods to obtain microsomal fractions from each animal. The relative activities of several mixed function oxidase enzymes (MFO - CYP1A1, CYP1A2, CYP2B1/2, and CYP2E1) were measured.
> Microsome Isolation
- Dose groups examined: Five animals per sex from the control and high dose groups were examined.
- Method: Total microsomal proteins for individual animal and pooled samples were determined using the Pierce BCA™ method.
> In Vitro Enzyme Assays
- Dose groups examined: Animals in the high dose group only.
- Method: The activities of selected MFO enzymes were measured in vitro using generally accepted methodologies. CYP1A1, CYP1A2, and CYP2B1/2 activities were measured as ethoxyresorufin (EROD), methoxyresorufin (MROD) and pentoxyresorufin (PROD) O-dealkylase activities, respectively, using the microplate fluorometric method outlined by Kennedy and Jones (1994). These methods involved the measurement of the in vitro O-dealkylation of 7-alkoxyresorufins to a highly fluorescent product, resorufin, by relatively closely related microsomal MFOs (reviewed by Burke and Mayer, 1983). CYP2E1 activity was measured as the hydroxylation of ρ-nitrophenol (p-NPH) using the spectrophotometric method outlined by Reinke and Moyer (1985).

ELECTRON MICROSCOPY
- Dose groups examined: Three rats/sex from the control and high-dose groups.
- Sample collection/preparation: Liver samples (left lateral lobe) were diced into approximately 1 mm³ cubes to aid rapid fixation and placed in phosphate buffered solution of 2 % glutaraldehyde/2 % formaldehyde for 1-2 days on a rotator in a cold room. Tissue specimens were post fixed in 1 % osmium tetroxide, dehydrated in graded alcohols and propylene oxide, and embedded in epon/araldite epoxy resin. Thick sections (1-2 µ) were cut with a diamond microtome, and stained with toluidine blue. The centrilobular areas of sections demonstrating well defined hepatic lobules were selected using light microscopy, and thin sections (60-90 mµ) were cut from the corresponding blocks using a Sorvall®MT-2B ultramicrotome.
These sections were picked up on 200 mesh copper grids, air dried, and stained with uranyl acetate and lead citrate. Tissue specimens from 2-3 rats/sex/dose were evaluated using a Hitachi H-600 transmission electron microscope and representative hepatocytes were digitally photographed.
- Evaluation: Electron micrographs were printed and evaluated by a veterinary pathologist.

URINE METABOLITES
- Dose groups examined: Pooled sample from each dose group/sex.
- Sample collection: Urine was collected, by manual compression of the bladder prior to the necropsy, for preliminary metabolite identification.
- Analysis: Urine samples were analysed using high performance liquid chromatography/negative electrospray ionization/mass spectrometry.

Statistics:

Means and standard deviations were calculated for all continuous data. Body weights, feed consumption, organ weights, urine volume, urine specific gravity, clinical chemistry data, coagulation and appropriate haematologic data were evaluated by Bartlett's test (alpha = 0.01; Winer, 1971) for equality of variances. Based on the outcome of Bartlett's test, exploratory data analysis was performed by a parametric (Steel and Torrie, 1960) or nonparametric (Hollander and Wolfe, 1973) analysis of variance (ANOVA). If significant at alpha = 0.05, the ANOVA were followed respectively by Dunnett's test (alpha = 0.05; Winer, 1971) or the Wilcoxon Rank-Sum test (alpha = 0.05; Hollander and Wolfe, 1973) with a Bonferroni correction (Miller, 1966) for multiple comparisons to the control. The experiment-wise α level was reported for these two tests. DCO incidence scores were statistically analysed by a z-test of proportions comparing each treated group to the control group (alpha = 0.05; Bruning and Kintz, 1987). Data collected at different time points were analysed separately. Descriptive statistics only (means and standard deviations) were reported for body weight gains, RBC indices, and differential WBC counts. Statistical outliers were identified by a sequential test (alpha = 0.02; Grubbs, 1969), but routinely excluded only from feed consumption calculations. Outliers may have been excluded from other analyses only for documented, scientifically sound reasons.
Because numerous measurements were statistically compared in the same group of animals, the overall false positive rate (Type I errors) were greater than the nominal alpha levels. Therefore, the final interpretation of the data will consider statistical analyses along with other factors, such as dose-response relationships and whether the results were consistent with other biological and pathological findings and historical control values.

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Description (incidence and severity):

No toxicologically significant effects.

Mortality:

no mortality observed

Description (incidence):

No toxicologically significant effects.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Reduced body weight and body weight gains (males and females given ≥ 100 mg/kg bw/day).

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

No toxicologically significant effects.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

no effects observed

Description (incidence and severity):

No toxicologically significant effects.

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Increased prohrombin time (males given ≥ 100 mg/kg bw/day).

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Increased ALP and cholesterol (males and females given ≥ 100 mg/kg bw/day); deceased AST (males and females ≥ 100 mg/kg bw/day); decreased glucose (males ≥ 100 mg/kg bw/day and females at 1000 mg/kg bw/day).

Urinalysis findings:

no effects observed

Description (incidence and severity):

No toxicologically significant effects.

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Increased liver weights (males and females given ≥ 100 mg/kg bw/day); increased relative liver weights (males and females given ≥ 10 mg/kg bw/day); increased adrenal weights and relative adrenal weights (females given ≥ 100 mg/kg bw/day).

Gross pathological findings:

no effects observed

Description (incidence and severity):

No toxicologically significant effects.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Microscopic liver hypertrophy with increased eosinophilia, centrilobular to midzonal (males given ≥ 10 mg/kg bw/day and females given ≥ 100 mg/kg bw/day).

Histopathological findings: neoplastic:

not examined

Details on results:

> MAIN GROUP

CLINICAL SIGNS AND MORTALITY
- Mortality: There was no mortality.
- Cage-Side Observations: On day 82, one female given 10 mg/kg bw/day had maloccluded incisors and blood in the cage. These findings were interpreted not to be treatment related because of the low incidence and lack of a dose-response relationship.
- Detailed Clinical Observations: Examinations performed on all animals revealed a low and sporadic incidence of palpebral closure, cloudy eye, maloccluded teeth, and perineal and periocular soiling. Increased lacrimation was also observed but occurred more in controls than in rats exposed to the test material. Two females given 500 mg/kg bw/day had ulcerated areas (palpable masses) on the muzzles that were transient. All of these observations were low in incidence, not statistically identified, or dose related and were interpreted not to be related treatment.

BODY WEIGHT AND WEIGHT GAIN
Male and female rats given 100, 500, or 1000 mg/kg bw/day weighed less than the controls from approximately days 43 (males) and 43 (females) until the end of the study. The differences in body weights were only 3 - 4 % lower than the controls at the end of the study, were occasionally statistically identified, and were not dose related. Differences in body weights were also reflected in lower body weight gains for rats given 100, 500, or 1000 mg/kg bw/day. The differences in body weight gains were 4.7 - 8.8 % (males) and 6.9 - 13.4 % (females) lower than the controls at the end of the study. These differences in body weights and body weight gains, although not dose related were attributed to the test material.

FOOD CONSUMPTION AND COMPOUND INTAKE
- Feed Consumption: Male rats given 100, 500, or 1000 mg/kg bw/day had a slightly lower feed consumption compared to the controls during the later half of the dosing phase of the study, and these differences were occasionally statistically identified. Differences in feed consumption were consistent with the slightly lower body weights of these male rats. Slightly lower feed consumption values were also noted for females given 10, 100, 500, or 1000 mg/kg bw/day, starting the first month of the study and occasionally occurring during the remainder of the dosing phase of the study. These differences were occasionally statistically identified, however, they were not dose related and were interpreted not to be a clear effect of treatment.
- Test Material Intake: Male and female rats given 10, 100, 500, or 1000 mg/kg bw/day received levels of test material that were acceptably close to the targeted concentrations for each dose group.

OPHTHALMOSCOPIC EXAMINATION
The eyes of all rats were within normal limits at the pre-exposure examination. Variable numbers of males and two females had a cloudy cornea at the end of the study, with the controls and the lower-dose levels having the highest incidences. A pale fundus and periocular soiling were infrequently observed in isolated rats from the main group at the end of the study. These observations were interpreted not to be treatment related due to the low incidence and lack of a dose-response relationship.

HAEMATOLOGY
- Haematology: Females given 10, 100, 500 or 1000 mg/kg bw/day had hemoglobin concentrations that were slightly lower than the control value and statistically identified. These differences were interpreted not to be treatment related because the values for rats given the test material were well within the historical control range and the concurrent controls were slightly higher than the historical control range. There were no significant changes in any of the haematologic parameters for male rats or any other changes for female rats.
- Prothrombin Time: Male rats given 100, 500, or 1000 mg/kg bw/day had prothrombin times that were greater than the controls, and the values for the 500 and 1000 mg/kg bw/day groups were statistically identified.
Prothrombin time differences at 100, 500, and 1000 mg/kg bw/day were interpreted to be treatment related. However these effects were not toxicologically significant, because there was no indication that these prolonged prothrombin times were associated with any clinical bleeding abnormalities. There were no significant differences in the prothrombin times of females at any dose level.

CLINICAL CHEMISTRY
Male and female rats given 100, 500, or 1000 mg/kg bw/day had higher alkaline phosphatase (ALP) activities and cholesterol values, and lower aspartate aminotransferase (AST) activities than the controls. Glucose levels of males given 100, 500, or 1000 mg/kg bw/day and females given 1000 mg/kg bw/day were also slightly lower than the controls. These differences were statistically identified, except for the female and the 500 mg/kg bw/day male AST activities, interpreted to be treatment related, but not adverse based on the absence of clinical disease, and were likely secondary to alterations in liver metabolism.
A number of other clinical chemistry parameters were statistically different from the controls but were interpreted not to be treatment related because the values were:
1) Within or consistent with the historical control range (higher total protein (TP) for males given 500 or 1000 mg/kg bw/day; lower total bilirubin for females given ≥10 mg/kg bw/day),
2) Lacked a dose-response relationship (lower potassium for females given 500 mg/kg bw/day; lower albumin and calcium for females given ≥100 mg/kg bw/day),
3) Concurrent control value was not consistent with historical controls (total bilirubin for females),
4) The differences between the control and treated groups were minor (lower albumin and calcium for females given ≥ 100 mg/kg bw/day).

URINALYSIS
Urine volumes of male rats given 10, 100, 500, or 1000 mg/kg bw/day and female rats given 100, 500, or 1000 mg/kg bw/day were higher than the controls and some of these differences were statistically identified. Values for females were somewhat dose related, but the values were not dose related for males. A historical control urine volume for males indicated that the concurrent control value was low and the differences were not meaningful. Urine volume for females given 100, 500, or 1000 mg/kg bw/day were higher than the historical control value and could possibly be treatment related. Lower urine specific gravities were noted for males given 10, 100, 500, or 1000 mg/kg bw/day and females given 100, 500 or 1000 mg/kg bw/day and were statistically identified. Specific gravity values for the test material males and females were within or in close proximity to the historical control values and indicated that the differences in specific gravity were not biologically significant. In addition, microscopic treatment-related changes were not observed in the kidneys of either male or females rats given the test material. Urine pH appeared more alkaline in males and females given the test material. These differences may be associated with the excretion of alkaline metabolites of the test material, presence of compounds that interfere with the pH test strip, or reflect the normal variability in this parameter.

ORGAN WEIGHTS
The final body weights of male and female rats given 100, 500, or 1000 mg/kg bw/day were slightly lower than the controls (2 – 5 %) and were attributed to treatment, however; only the body weights of males given 500 mg/kg bw/day were statistically identified. Liver weights (absolute and relative) of males and females given 100, 500, or 1000 mg/kg bw/day were increased compared to the controls and statistically identified. The relative liver weights of males and females given 10 mg/kg bw/day were also increased and statistically identified. The adrenal gland weights (absolute and relative) of females given 100, 500, or 1000 mg/kg bw/day were increased compared to the controls and statistically identified. These organ weight changes were interpreted to be treatment related.
The relative liver weights of males given 10, 100, 500, or 1000 mg/kg bw/day were 7.4, 20.7, 21.2, or 20.5 % larger than the controls; whereas, the relative liver weights of females given 10, 100, 500, or 1000 mg/kg bw/day were 7.8, 34.4, 43.3, or 40.3 % larger than the controls. The percentage increases in liver weights were not proportional to the dose of test material administered and clearly indicated a lack of a dose relationship in the increased liver weights of males and females given 100, 500, or 1000 mg/kg bw/day. A similar lack in the proportionality was also evident in the increases in adrenal gland weights (absolute and relative) in female rats given 100, 500, or 1000 mg/kg bw/day.
The following organ weights were higher or lower than the controls in rats given the test material and were frequently statistically identified:
1) Lower absolute epididymal weights in males given 500 or 1000 mg/kg bw/day,
2) Higher relative heart and spleen weights of females given 100, 500, or 1000 mg/kg bw/day,
3) Higher relative thymus weight of females given 1000 mg/kg bw/day,
4) Higher relative kidneys weight of males given 100, 500, or 1000 mg/kg bw/day and females given 500 mg/kg bw/day,
5) Higher relative brain weights of males and females given 100, 500, or 1000 mg/kg bw/day.
These differences were interpreted to be secondary to the lower body weights of these rats and were interpreted not to be a direct effect of treatment with the test material.

GROSS PATHOLOGY
There were no treatment-related gross pathologic observations. All gross pathologic observations were interpreted to be spontaneous alterations (cloudy corneas, strangulated fat, liver hernia, gingival abscess, malocclusion, and facial soiling).

HISTOPATHOLOGY: NON-NEOPLASTIC
The liver was identified as a primary target organ in males given 10, 100, 500 or 1000 mg/kg bw/day and female rats given 100, 500, or 1000 mg/kg bw/day. Microscopic changes were characterized by an increase in hepatocellular size (hypertrophy) in the centrilobular and midzonal region of the hepatic lobule. In addition, these hepatocytes had an increase in eosinophilic staining of the cytoplasm (altered tinctorial properties).
All other observations were interpreted to be the result of spontaneous alterations, unassociated with the test material.

ELECTRON MICROSCOPY
Male and female rats given 1000 mg/kg bw/day had slightly increased amounts of smooth endoplasmic reticulum. All other organelles appeared qualitatively and quantitatively normal.

LIVER ENZYMES
The activities of the hepatic CYP1A subfamily of mixed function oxygenase enzymes were statistically identified as elevated in high-dose group rats relative to controls. A moderate 4.4 - 4.5 fold increase in MROD, indicative of CYP1A2 activity, and a 2.0 - 2.2 fold increase in EROD, indicative of CYP1A1 activity, was observed in high-dose males and females. In addition, minimal, yet occasionally statistically identified increases (1.2 - 1.7 fold) in PROD and p-NPH activities, indicative of CYP2B1/2 and CYP2E1 activities, respectively, were also noted in high-dose group rats. Taken together, these changes in MFO activities correlated with increased liver weights, light and electron microscopic findings of treated rats and were consistent with an adaptive response to the increased metabolic load placed upon this organ.

CHEMICAL ANALYSIS OF URINE
Urine samples from the 500 and 1000 mg/kg bw/day males had test material levels above the low standard (0.091 µg/mL), which was set at the detection limit for this analysis. The amount of test material in these samples was less than 0.03 % of the administered dose. Samples were also analysed for the free aniline metabolite of the test material, however; no useful data were obtained from these analyses.
Urine samples from male and female rats given 1000 mg/kg bw/day were screened for potential metabolites based on the known metabolic pathway for a similar molecule. No definitive metabolite identification data were obtained from these samples due to the occurrence of numerous peaks in the chromatogram, which could not be discriminated.

Effect levels

Key result

Dose descriptor:

NOAEL

Effect level:

10 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Based on an increase in relative liver weights (males and females) and microscopic hepatocellular hypertrophy in males.

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

Analytical Chemistry

The homogeneity of the test material in rodent feed was determined for three separate mixing batches (mixed prior to study start, middle and near the conclusion of the study) for the 10 mg/kg bw/day female and 1000 mg/kg bw/day male test diets, the lowest and highest concentrations used in the study. The diets were homogeneous, with relative standard deviations for all diets sampled between 1.64 % and 6.37 %.

The concentrations of the test material were determined for the control, premix and test diets from all treatment levels at the three time points and were found to be acceptable. LC/MS analysis indicated 87 - 118 % of the target concentration of the test material for each individual sample. Target concentrations greater than 110 % were a single occurrence over the study for some diet mixes but mean values for the three analytical time points were within 98 – 108 %. The mean concentrations for each dose level ranged from 98.0 to 107.7 % of targeted concentration. No test material was found in the control diet.

The test material was stable for at least 42 days in the feed at concentrations ranging from 0.005 to 5 %.

Recovery Group Results

- Detailed clinical and Cage-Side Observations:

DCOs for recovery animals during the dosing phase of the study were addressed with the discussion for the main group animals (above). There were no DCOs conducted during the recovery phase of the study because there were no treatment-related effects in the main group animals. There were no treatment-related effects.

- Ophthalmology:

Ophthalmic findings for recovery animals observed during the dosing phase of the study were addressed with the discussion for the main group animals. There were no ophthalmic examinations conducted during the recovery phase of the study due to the absence of treatment-related effects in the main group animals.

- Body Weights and Body Weight Gains:

The lower body weights apparent at the end of the dosing period in the main group males (-3.9 %) and females (-3.3 %) given 1000 mg/kg bw/day were still present at the end of the 28-day recovery period in males (-4.6 %) and females (-4.4 %) given 1000 mg/kg bw/day during the dosing phase of the study.

- Feed Consumption:

Male rats given 1000 mg/kg bw/day consumed slightly less feed than the controls during the recovery phase of the study. These differences were not statistically identified and were interpreted not to be toxicologically significant. There were no significant differences in feed consumption for females.

- Prothrombin Time:

There were no significant differences in prothrombin time for male rats.

- Clinical Chemistry:

Female rats given 1000 mg/kg bw/day had a marginally higher ALP activity than the controls. This difference was statistically identified but was interpreted not to be treatment related because the value for the high-dose group was within the historical control data for recovery group female rats. The other chemistry values (ALP - males, AST, glucose, and total protein -males; cholesterol, calcium - females and albumin - females) were similar between control and high-dose rats indicating a reversal of effects seen following 90 days of exposure to the test material.

- Urinalysis:

Urine volume and specific gravity of males and females previously given 1000 mg/kg bw/day were similar to the controls. Male and female rats given 1000 mg/kg bw/day had a slightly higher pH than the controls. This may reflect the continued excretion of alkaline metabolites of the test material or interference with the test strip. There were no other differences in any of the urinary parameters for either males or females.

- Organ Weights:

The final body weights of males and females given 1000 mg/kg bw/day were lower than the controls and statistically identified. In addition, males given 1000 mg/kg bw/day had a higher relative brain weight, lower absolute epididymal weight, higher relative kidney weight, and higher relative liver weight; while females given 1000 mg/kg bw/day had higher absolute and relative adrenal weights, higher relative heart weight, higher absolute and relative liver weights, and lower absolute spleen weight.

The lower final body weights and increased liver and adrenal gland weights were interpreted to be treatment related and these effects did not completely return to control levels following 28 days of being given control feed. Final body weights for males and females given 1000 mg/kg bw/day were approximately 5 % lower and relative liver weights were 5 % and 13 % higher than the control values, while the relative adrenal weights for females given 1000 mg/kg bw/day were approximately 25 % higher than the controls. Lower body weight values were similar at 90 days and following 28 days of control feed. Increased relative liver weights decreased from ~20 % for males given 1000 mg/kg bw/day and 40 % for females given 1000 mg/kg bw/day at day 90 to values only 5 % and 13 % above controls at recovery day 28, indicating partial reversal of the liver weight increase. Adrenal weights (relative) decreased from ~33 % for females given 1000 mg/kg bw/day in the main group to 25 % above controls at recovery day 28, indicating a partial reversal of the adrenal weight increase.

The alterations in brain, kidney and heart weights were interpreted to be secondary to the lower body weight and were not target organs of the test material. A lower, absolute spleen weight in females was interpreted not to be treatment related since female rats given 1000 mg/kg bw/day, main group, from the dosing phase of the study had higher absolute spleen weights.

- Gross Pathology:

There were no treatment-related gross pathologic observations. All gross pathologic observations were considered spontaneous alterations, unassociated with exposure to the test material.

- Histopahology:

The livers of male and female rats given 0 or 1000 mg/kg bw/day from the recovery phase of the study were microscopically examined because treatment-related hepatocellular hypertrophy was noted in rats given 1000 mg/kg bw/day during the dosing phase of the study. Hepatocellular hypertrophy was noted in the livers of male rats but not female rats from the recovery phase of the study that were given 1000 mg/kg bw/day.

Treatment-related hypertrophy induced in females following 90 days of ingesting the test material was reversible after 28 days of control feed. A similar complete reversal did not occur in males given 1000 mg/kgbw/day, as hypertrophic changes were present in the majority of the males. Although not completely reversed, the effect was somewhat reduced in degree in that the initial hypertrophic changes involved the centrilobular to midzonal regions of the liver, whereas the effects following the exposure to control feed only involved the centrilobular region of the liver. The involvement of only the centrilobular region of the liver lobule versus the centrilobular and midzonal regions indicates some degree of reversibility and correlated with the lesser weight increase.

Conclusions

Table 1: Summary of Treatment Related Effects – Main Group

Dose (mg/kg bw/day)	10	100	500	1000
↓ Body weight	-	MF	MF	MF
↑ Liver weight (g)	-	MF	MF	MF
↑ Liver, weight (g/100)	MF	MF	MF	MF
↑ Adrenal weight (g)	-	F	F	F
↑ Adrenal weight (g/100)	-	F	F	F
↑ ALP	-	MF	MF	MF
↓ AST	-	MF	MF	MF
↓ Glucose	-	M	M	MF
↑ Cholesterol	-	MF	MF	MF
↑ Prothrombin time	-	M	M	M
Microscopic liver hypertrophy with increased eosinophilia, centrilobular to midzonal	M	MF	MF	MF
Electron microscopic increase in smooth endoplasmic reticulum	ND	ND	ND	MF
↑ Liver mixed function oxidase enzymes	ND	ND	ND	MF

- absence of an effect, M - male, F - female, ND = no data

 

Table 2: Summary of Salient Findings – Recovery Group

Recovery Status	Complete Recovery	Partial Recovery	No recovery
↓ Body weight (g)			MF
↑ Liver weight (g)	M	F
↑ Liver weight (g/100)	-	MF
↑ Adrenal weight (g)		F
↑ Adrenal weight (g/100)		F
ALP	MF
AST	M
Glucose	MF
Cholesterol	MF
Prothrombin time	M
Urine pH - alkaline		F	M
Microscopic hepatocellula hypertrophy	F	M

M - male, F - female

Applicant's summary and conclusion

Conclusions:

Under the conditions of the test, the lowest-observed-effect level (LOEL) and no-observed-adverse effect level (NOAEL) were 10 mg/kg/day based on an increase in relative liver weights (males and females) and microscopic hepatocellular hypertrophy in males. A no- observed-effect level was not determined.

Executive summary:

The subchronic toxicity of the test material was assessed in a 13 week dietary toxicity study using Fischer 344 rats. The study was performed under GLP conditions and in accordance with the standardised guidelines OECD 408, EU Method B.26, EPA OPPTS 870.3100 and JMAFF testing guidelines subchronic oral toxicity study.

Ten male and ten female Fischer 344 rats per group were given test diets formulated to supply 0, 10, 100, 500, or 1000 mg/kg bw/day for at least 90 days. Additional groups (ten/sex) were given either 0 or 1000 mg/kg bw/day for 90 days and were then given control feed for an additional 28 days to assess the reversibility of treatment-related effects induced following exposure to the test material. Parameters evaluated were daily observations, detailed clinical observations, ophthalmologic examinations, body weight, feed consumption, haematology, clinical chemistry, urinalysis, selected organ weights, gross and histopathologic examinations, and hepatic mixed function oxidase levels.

Male and female rats given 100, 500, or 1000 mg/kg bw/day had decreases in body weights (3-6 %) and body weight gains (4.7-13.4 %) compared to the controls which lacked a dose-response relationship. Liver weights of male and female rats given 10, 100, 500, or 1000 mg/kg bw/day were increased and statistically identified. Liver weight increases in rats given 100, 500, or 1000 mg/kg bw/day were not proportional to the dose administered. Microscopic effects consisting of an increase in hepatocytes size with an increase in eosinophilic staining of the hepatocyte cytoplasm occurred in males given ≥ 10 mg/kgbw/day and females given ≥ 100 mg/kg bw/day. Enzyme determinations indicated that the increases in liver weights were associated with a modest induction of a number of liver enzymes. In addition, the electron microscopic examination of the livers indicated that the smooth endoplasmic reticulum of centrilobular hepatocytes was increased in amount. Other effects of no or minor toxicologic significance occurred in male and/or female rats given ≥ 100 mg/kg bw/day during the dosing phase of the study and consisted of: 1) increased adrenal gland weights (females only), 2) minor increases in alkaline phosphatase activity, 3) minor decreases in aspartate aminotransferase activity and glucose levels (primarily males), and minor increases in cholesterol and prothrombin time (males only).

Complete recovery of treatment-related effects during the dosing phase of the study occurred with the following parameters: microscopic hepatocellular hypertrophy (females), and numerous alterations in clinical pathology parameters. A few of the parameters affected during the dosing phase of the study partially reversed during the recovery phase of the study and included: liver weights and adrenal weight increases, urine pH (females), and microscopic hepatocellular hypertrophy (males). The lower body weights identified during the dosing phase of the study persisted throughout the recovery phase of the study indicating that this effect was not reversible in 28 days.

Under the conditions of the test, the lowest-observed-effect level (LOEL) and no-observed-adverse effect level (NOAEL) were 10 mg/kg/day based on an increase in relative liver weights (males and females) and microscopic hepatocellular hypertrophy in males. A no- observed-effect level was not determined.