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

Carcinogenicity

Description of key information

RAT

The no-observed-effect level (NOEL) for both male and female Fischer 344 rats at 24 months was determined to be 1.0 mg/kg bw/day. EPA OPPTS 87.4300, EPA OPPTS 870.3100, OECD 453, OECD 408, EU Method Part B, EU Method B.26, and JMAFF guidelines, Yano et al. 2005.

MOUSE

3 mg/kg/day for males and 30 mg/kg/day for females were considered to be the NOAELs for chronic toxicity. The low dose level of 0.5 mg/kg/day had no effects attributed to treatment and was the NOEL. OECD 451, EU Method B.32 US EPA OPPTS 870.4200 and MAFF, Johnson 2005.

Key value for chemical safety assessment

Carcinogenicity: via oral route

Link to relevant study records

Reference

Reference 1

Endpoint:

carcinogenicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

3 June 2002 - 15 December 2003

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 451 (Carcinogenicity Studies)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.4200 (Carcinogenicity)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.32 (Carcinogenicity Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: MAFF, Ministry of Agriculture, Forestry and Fisheries, 2000

Deviations:

no

GLP compliance:

yes

Species:

mouse

Strain:

CD-1

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: Approximately 6 weeks
- Weight at study initiation: Mean body weight on Day 1 of the test: males 27.4 g; females 22.9 g
- Fasting period before study: No
- Housing: Animals were housed one per cage in stainless steel cages with wire-mesh floors suspended above catch pans. Cages contained feed containers and pressure activated, nipple-type watering systems.
- Diet: ad libitum
- Water: municipal water provided ad libitum
- Acclimation period: Approximately 1 week

ENVIRONMENTAL CONDITIONS
- Temperature: 21.5 - 22.9 °C
- Humidity: 48.1 - 68.0 % (relative)
- Air changes: Approximately 12 - 15 times/hour
- Photoperiod: A 12-hour light/dark photocycle was maintained with lights on at 06:00 and off at 18:00

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on exposure:

DIET PREPARATION
- Rate of preparation of diet (frequency): Premixes were mixed approximately weekly for the first 13 weeks and at approximate monthly intervals thereafter.
- 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 historical body weights and feed consumption data. Subsequently, the concentrations of the test material in the feed were adjusted weekly for the first 13 weeks and at approximately monthly intervals thereafter, based upon the most recent body weight 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 the high-dose male test diets were determined pre-exposure, and at 7.5, 12, and 17 months.
The test material was uniformly distributed throughout the diets by the method of mixing employed, with relative standard deviations (RSDs) ranging from 1.59 to 12.3 % (RSD values were < 5.2 % for all mixing batches examined but the initial batch). These results indicate that the diets were homogeneously mixed over the course of the study.

STABILITY
Data from a thirteen-week toxicity study demonstrated that the test material was stable for at least 42 days in rodent feed at concentrations ranging from 0.005 to 5 %. Furthermore, the stability was also estabilished in a concurrent chronic toxicity/neurotoxicity/oncogenicity study in rats; the test material was shown to be stable for up to 27 days at a concentration of ~ 0.0001 %.
Although the concentrations of the test material in the diets used in this study were anticipated to be within the previously established range, analysis of the lowest concentration of test material in the diet was conducted concurrent with this study to demonstrate at least 35 days stability.
The test material was considered stable in rodent feed over the concentrations used in this study.

CONCENTRATION VERIFICATION
Analyses of all dose levels, plus control and premix were determined pre-exposure, and at 3, 7.5, 12, and 17 months. The method used for analysing the diets was a solvent extraction method followed by analysis using liquid chromatography-mass spectrometry (LC-MS) and solvent standards incorporating an internal standard.
The overall mean concentrations of the test material the individual diets ranged from 93.3 (3 mg/kg/day male diet) to 101 % (30 mg/kg/day female) of target concentrations. The concentrations in the individual diet analyses were within 10 % of targeted concentration on all except four occasions (112, 89.1, 88.7, and 81.0 %).

Duration of treatment / exposure:

18 months

Frequency of treatment:

Continuous in the diet

Dose / conc.:

0 mg/kg bw/day

Remarks:

Basis:
nominal in diet/male and female

Dose / conc.:

0.5 mg/kg bw/day

Remarks:

Basis:
nominal in diet/male and female

Dose / conc.:

3 mg/kg bw/day

Remarks:

Basis:
nominal in diet/male

Dose / conc.:

30 mg/kg bw/day

Remarks:

Basis:
nominal in diet/male and female

Dose / conc.:

100 mg/kg bw/day

Remarks:

Basis:
nominal in diet/female

No. of animals per sex per dose:

50 animals per sex per dose

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: The dose levels were selected following review of the protocol and discussions with the US EPA. The high doses of 30 (males) and 100 (females) mg/kg/day were expected to induce increases in liver weight and microscopic hepatocellular hypertrophy based upon the 90-day study. As there was mortality attributed to treatment at both 30 (males) and 300 mg/kg/day (both sexes) in the 90-day study, it was noted that the high dose levels in the present study could potentially result in excess mortality and exceed a Maximum Tolerated Dose, particularly in the high-dose females. The mid- and low-doses were expected to provide dose response data for the effects induced at the highest dose level, without an increase in mortality.

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: A cage-side (general) clinical examination was conducted at least once a day. This examination was performed with the animals in their cages and was designed to: 1) detect significant clinical abnormalities that were clearly visible upon a limited examination and 2) to monitor the general health of the animals. Significant clinical abnormalities that could be observed on this examination included, but were not limited to: activity, repetitive behaviour, vocalisation, incoordination/lameness, injury, neuromuscular function (convulsion, fasciculation, tremor, twitches), altered respiration, blue/pale skin and mucous membranes, severe eye injury (rupture), faecal consistency, and faecal/urinary quantity. At least twice daily, all animals were observed for morbidity and mortality, and the availability of feed/water.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Detailed clinical observations (DCO) were conducted on all animals pre-exposure (baseline) and at approximately monthly intervals thereafter for the first 10 surviving animals/sex/dose group. These examinations included cage-side, hand-held, and open-field observations that were recorded categorically or using explicitly defined scales (scored).
Clinical examinations, consisting of the categorical portion of the DCO, were conducted on all animals approximately twice each month throughout the study. This examination included a careful, hand-held evaluation of the skin, fur, mucous membranes, respiration, nervous system function (including tremors and convulsions), and animal behaviour.
Animals were observed for general behaviour and appearance, respiration, nervous system function (including tremors and convulsions) and any other signs of clinical toxicity. In addition, all animals were examined for unusual swelling or palpable masses concurrent with the hand-held clinical or detailed clinical observations. The time of onset, location, dimensions, appearance, and progression of each palpable mass was recorded.
The categorical portion of the DCO conducted on the first 10 animals of each dose group/sex fulfilled one occurrence of the clinical observation and palpable mass observation per month.

BODY WEIGHT: Yes
- Time schedule for examinations: All mice were weighed during the pre-exposure period, weekly during the first 13 weeks of the study and then at approximately monthly intervals thereafter. Body weight gains were calculated throughout the study.

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
Feed consumption data were collected weekly during the first 13 weeks of the study and then at approximately monthly intervals for all animals by weighing feed containers at the start and end of a measurement cycle and consumption was calculated using the following equation:
Feed consumption (g/day) = [(initial weight of feed containers - final weight of feed containers)] / [(# of days in measurement cycle) (# of animals per cage)]

The actual test material intake (TMI) was calculated using test material concentrations in the feed, body weights, and feed consumption data using the following equation (expressed as mg/kg/day and ppm):
TMI = [(feed consumption (g/day)*(1000 mg/g)*(% of test material in feed / 100)] / [((current body weight (g) +previous body weight (g)) / 2) / 1000 g/kg]

FOOD EFFICIENCY: No

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: The eyes of all animals were examined by a veterinarian pre-exposure and prior to the scheduled necropsy using indirect ophthalmoscopy. 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 the necropsy using a moistened glass slide pressed to the cornea.

CLINICAL PATHOLOGY
Blood smears were made from all surviving animals via sample collection from the pedal vein (12 months) or orbital sinus (18 months). A white blood cell (WBC) count and differential WBC count were determined from all animals at the terminal sacrifice (18 months).
Blood from moribund animals, anaesthetised with CO₂, was obtained from the orbital sinus or tail vein. Blood smears were not obtained from animals that died spontaneously. A differential white blood cell count, as could be derived from the blood smears, was not determined from animals that were moribund due to the absence of effects in the mice surviving to 18 months.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:

SACRIFICE
Non-fasted mice submitted alive for necropsy were anaesthetised by the inhalation of CO₂, weighed, and blood samples were obtained from the orbital sinus. Tracheas were exposed and clamped, and the animals were euthanised by decapitation.

GROSS PATHOLOGY
A complete necropsy was conducted on all animals. 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.
The brain, liver, kidneys, heart, adrenals, testes, epididymides, ovaries, uterus, and spleen were trimmed and weighed immediately. The ratios of organ weight to terminal body weight were calculated.
Representative samples of the following tissues were collected and preserved in neutral, phosphate-buffered 10 % formalin: adrenals, aorta, auditory sebaceous glands, bone (including joint), bone marrow, brain (cerebrum, brainstem, cerebellum), cecum, cervix, coagulating glands, colon, cranial nerve – optic, duodenum, epididymides, eyes, gross lesions, heart, ileum, jejunum, kidneys, lacrimal/Harderian glands, larynx, liver, lungs, mammary gland - females only, mediastinal lymph node, mediastinal tissues, mesenteric lymph node, mesenteric tissues nasal tissues, oesophagus, oral tissues, ovaries, oviducts, pancreas, parathyroid glands, peripheral nerve -tibial, pituitary, prostate, rectum, salivary glands, seminal vesicles, skeletal muscle, skin and subcutis, spinal cord (cervical, thoracic, lumbar), spleen, stomach, testes, thymus, thyroid gland, tongue, trachea, urinary bladder, uterus and vagina.

Similar necropsy procedures were followed for animals found dead or moribund, except that terminal body weights and organ weights were not obtained.

HISTOPATHOLOGY
The sections from all preserved tissues listed above were processed by standard histologic procedures from all control and high-dose group animals and all animals that died or were sacrificed in a moribund condition. Paraffin embedded tissues were sectioned approximately 6 μm thick, stained with haematoxylin and eosin and examined using a light microscope. The following tissues from the remaining groups were processed and histopathologically examined: liver, kidneys, lungs, and relevant gross lesions.
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 would neither be expected to significantly affect the function of the specific organ/tissue nor would it 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 have been 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 was 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 have been life threatening.
As there was increased mortality in high-dose females, necessitating Peto analysis of tumour data, the neoplasms considered to be the cause of death were identified at histopathologic examination. The “fatal” context for Peto analysis was assigned for mice for which the tumour was the major underlying cause of death or moribund condition. The context “probably fatal” was used for a few animals having one or more advanced neoplasms or a neoplasm and other advanced non- neoplastic disease for which both were considered likely to account for the spontaneous death or moribund condition of the animal. The Peto score was recorded for both males and females, although male mortality was not affected by treatment, to maintain some consistency. All other neoplasms were considered “incidental” or “probably incidental” but this was not recorded in the diagnosis.

Statistics:

Statistical analyses conducted are described below.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

see below

Mortality:

mortality observed, treatment-related

Description (incidence):

see below

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

no effects observed

Haematological findings:

no effects observed

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

see below

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

see below

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

see below

Histopathological findings: neoplastic:

effects observed, treatment-related

Description (incidence and severity):

see below

Other effects:

not examined

Details on results:

MORTALITY
The mortality rates at the end of the study were 22, 20, 20, and 22 % for males in the control, 0.5, 3, and 30 mg/kg/day groups, respectively, and 32, 18, 16, and 56 % for females in the control, 0.5, 30, and 100 mg/kg/day groups, respectively. The increased mortality in females given 100 mg/kg/day was statistically identified and was considered treatment related. The female control group exceeded historical control mortality in the testing laboratory for CD-1 female mice on 18- month dietary toxicity studies; thus, the increased mortality of the high-dose female group is perhaps even more notable than when compared to the concurrent controls.
In the high-dose female group, mortality began on day 67 and progressed steadily through the remainder of the study. Mortality in females given 0.5 or 30 mg/kg/day and for all dose levels of males was similar to controls and occurred much later in the study.
The cause of death or moribund condition for all mice is discussed following the histopathology results. Many of the females given 100 mg/kg/day that were removed early did not have significantly severe histopathologic lesions to ascribe an underlying cause of death or moribundity; rather the increase was ascribed to likely failure to recover from tonoclonic convulsions (although the convulsions were generally not noted for the specific mice at the time of death). While determining an appropriate high-dose level for an oncogenicity study depends upon available data at the time the study is initiated, excess mortality due to causes other than carcinogenicity is considered an indication that the dose level has exceeded a Maximum Tolerated Dose (MTD) under several high-dose selection guidelines (Interagency Regulatory Liaison Group, 1979; OECD, 1981; US EPA, 2003).

CAGE-SIDE AND DETAILED CLINICAL OBSERVATIONS
There were no statistically identified differences in the ranked parameters among the treated animals at any time when compared to their respective controls. There were only isolated incidences (1 mouse/sex/dose group) of changes in extensor thrust response, gait, lacrimation, pupil size (unable to evaluate), muscle tone, responsiveness to touch, and reactivity to stimuli over the course of the study. These observations were generally seen in the last few months of study; the incidence did not increase as the study progressed and was not attributed to the test material.
Most observations from the DCO-categorical, clinical, palpable mass or cage side examinations also were found infrequently. The only observations that occurred in 10 % or more mice/sex in any dose group at any time were tonoclonic convulsions (both sexes), cloudy eyes (females), dermatitis (both sexes), and hair loss (females). An increased incidence of tonoclonic convulsions was noted in the female high-dose group and possibly high-dose group males and was considered treatment related (Table 1). Tonoclonic convulsions, however, are not uncommon in CD-1 mice, usually in relationship to handling during the hands-on clinical examination. From 0 to 3 mice from the control, low- or middle-dose groups were noted with tonoclonic convulsions at the time of the biweekly clinical examinations over the course of the study. Convulsions were first noted on day 43 when a convulsion was noted in a high-dose female. Females given 100 mg/kg/day had a much higher incidence than all other groups at most of the clinical examination times, with the peak being 18 mice with tonoclonic convulsions on day 323. The only time more than three high-dose males had convulsions was on day 337 when seven males given 30 mg/kg/day had convulsions.
Four female mice were noted to have convulsions on four occasions in the 0.5 mg/kg/day dose group and once in the 30 mg/kg/day dose group, but these few instances were not considered treatment related. While the convulsions were similar to those seen in controls, it was noted that convulsions in high-dose females tended to last longer, were more severe in magnitude of the uncontrolled motions, and had a longer post-convulsion recovery period. Most mice recovered from a convulsion although two of the high-dose level females (02A4276, 02A4277) died without recovering from a convulsion. Many of the high-dose female mice found dead had a history of convulsions and convulsions were considered to be the likely cause of increased mortality in this group.
Dermatitis is infrequently noted in CD-1 mice and was present in several mice from all dose groups, including controls, in this study. Dermatitis was commonly initially noted to involve the margin of the ears. In some mice the inflammation remained localised to the pinna, while in others it progressed to involve the neck, head, shoulders, and forelimbs. Progressive dermatitis initially involving the pinna with progression to adjacent sites has been reported in the literature (Slattum et al., 1998). Dermatitis was first noted in males on day 22, and in females on day 127. Dermatitis was not attributed to test material ingestion as the incidence was generally similar among all treated groups. Mice given the test material tended to have a greater incidence than controls at most examination intervals and males also had a generally greater incidence than females. However, the low-dose group had the highest incidence of dermatitis over the course of the study. Overall, the highest incidences of dermatitis in each dose group were 11, 14, 13, and 12 for the males given 0, 0.5, 3, or 30 mg/kg/day, respectively. Females had somewhat lower overall incidences of dermatitis with the highest incidences at any examination interval being 4, 9, 7, and 4 for females given 0, 0.5, 30, or 100 mg/kg/day, respectively. The clinical course of dermatitis was variable. In some mice dermatitis resolved (healed) while in others it persisted for variable intervals. In some males from all groups, including controls, the dermatitis progressed to involve adjacent organs or evidence of septicaemia was present and it was implicated as the underlying cause of death or moribundity.
Two other observations were seen in > 10 % of female mice of any group at some point during the study but were not considered due to the test material. Up to seven control females had a cloudy eye, generally in the last two months of study, but highest incidence seen in treated mice was two. Excessive hair-loss was noted for five high-dose females on days 393 and 407; however, it was also found in four controls at this time and was considered not related to treatment.
Numerous other observations occurred in low incidences (≤4 mice/sex/dose group) over the course of the study. As with the ranked parameters, most of these were seen in the last few months of the study; there was no overall pattern of dose responsiveness; the incidence of any specific observation did not definitively increase with time; and they were not attributed to the test material. These observations were consistent with the type of spontaneous alterations commonly associated with aging mice.

BODY WEIGHTS/BODY WEIGHT GAINS
There were no treatment-related effects in body weights or body weight gains. Body weights and body weight gains of males and females in the treated groups were comparable to the controls throughout the study, with the only statistically identified difference being slightly decreased body weight of mid-dose level males on day 9 of the study.

FEED CONSUMPTION
The feed consumption of males and females in the treated groups was comparable to the controls throughout the study. There were a few instances where the feed consumption was statistically identified. However, these were not present in a consistent manner, either in relationship to dose, direction of change or temporally; and they were considered to represent random variability. Mid-dose level males (3 mg/kg/day) consumed slightly less feed during the first two weeks, which correlated with their slightly decreased body weight on study day 9. After this time point, the mean body weight of this dose group was similar to controls as was their feed consumption except for two intervals.

TEST MATERIAL INTAKE
The mean actual test material intake values were within 2.3 % of the targeted values for all dose groups. The mean test material intake was 0, 0.504, 3.013, or 30.2 mg/kg/day for the male 0. 0.5, 3, and 30 mg/kg/day dose groups, respectively. Mean test material consumption for females was 0, 0.508, 30.5, or 102.3 mg/kg/day for the 0, 0.5, 30, and 100 mg/kg/day dose groups. These values correspond to time-weighted average concentrations of 0, 3.91, 23.9, or 235 ppm for males and 0, 2.99, 184.3, or 613 ppm for females.

OPHTHALMOLOGY
The eyes of all animals were considered normal when examined pre-exposure (day -3). At the ophthalmic examination shortly before study termination (days 546 and 547), there were low incidences of cloudy cornea (3 - 9 mice/sex/dose group), opaque cornea (0 - 2 mice/sex/dose group), cloudy lens (0 - 4 females/dose group), pale fundus (0 - 11 mice/sex/dose group), and isolated instances of missing eye - congenital and phthisis bulbi. All changes were considered to be typical of spontaneous changes in aging mice. There were no ophthalmic effects attributed to the test material. The incidences of most observations were comparable between the various dose groups and controls; however, the mid- and high-dose males had a lower incidence of pale fundus and high-dose females had lower incidence of cloudy cornea than controls.

HAEMATOLOGY
There were no treatment-related changes in the total WBC or the differential WBC counts. As can be expected with aged animals there was marked inter-animal variability which was associated with various disease processes, primarily lymphosarcoma and chronic active dermatitis.
However, individuals having markedly different total WBC or differential WBC counts were found in all groups, including controls.

ORGAN WEIGHTS
As noted for the in-life body weights, there was no effect upon final body weight. The liver was the primary target organ with absolute liver weight increased for the high-dose group mice (both sexes) and relative liver weight (g per 100 g body weight) increased for both the mid- and high-dose groups of each sex (Table 2).
The increase in liver weights due to test material ingestion was similar to that found in the 90-day study; however, there was a difference in the temporal response between the sexes. The increase in relative liver weight for treated male mice at 18 months was approximately double that found at comparable dose levels after 90 days. At 90 days, the relative liver weights were increased 7.4 and 10.3 % for males given 3 or 30 mg/kg/day, respectively, while these increases were 13.5 and 19.1 % at these dose levels after 18 months. In females, the increased liver weight was slightly less at 18 months than that found after 90 days. Females given 30 mg/kg/day had a 13.8 % increased relative liver weight at 90 days and 9.8 % at 18 months. However, the 32.6 % relative liver weight increase for females given 100 mg/kg/day was only slightly less than the 41.6 % found for females given 300 mg/kg/day for 90 days and indicates that this high dose provided a strong induction of the liver response.
The absolute brain weight of females given 100 mg/kg/day was also slightly decreased and was statistically significant while the relative brain weight was similar to controls. The decreased absolute brain weight was considered to be spurious as there was no effect upon relative brain weight, brain weights were not affected in the 90-day study, and there were no concomitant histopathologic findings.
The only other organ for which weights were statistically identified was the uterus, which had decreased absolute and relative uterine weights for females given the low-dose level. There was marked variability in uterine weights in all groups, including controls, due to involvement by cystic endometrial hyperplasia. The decreased weight for the 0.5 mg/kg/day dose group was probably due to somewhat decreased incidence of cystic endometrial hyperplasia and was considered to be incidental and unrelated to treatment with the test material.

GROSS PATHOLOGY
There were no marked differences in incidences of observations between groups that were considered indicative of a treatment-related effect. Most observations had a relative low overall incidence (<10 % in any dose group). Observations found at higher incidence (i.e. cloudy cornea, decreased amount of fat, pale kidneys, ovarian cysts, inflammation of various sites in the skin and subcutis, increased spleen size, and cystic endometrial hyperplasia) were considered typical of spontaneous lesions expected in CD-1 mice of this age and husbandry conditions. The incidence of most of these higher incidence observations did not have a dose-response relationship. Consistent with the clinical observations, mice given the test material, particularly males, had a somewhat greater incidence of dermatitis (particularly of the pinna) but this was not found in a dose-responsive pattern. Mass-nodules, indicative of possible neoplasms, were generally infrequent with the exception of masses of the liver of males and lungs of both sexes. These masses were separated by their locator (anatomic lobe involved). Male mice given 30 mg/kg/day had a greater incidence of liver mass-nodules than controls (14 vs. 3 controls); however, the number was close to that found in the low-dose group (which had 10) and historical control data (3, 9, and 10).
Mass-nodules of the lungs were similar in all dose groups with mass-nodules identified in 6, 9, 7, and 5 males and 7, 4, 10, and 4 females from the control, low-, middle- and high-dose groups, respectively. A few non-neoplastic observations had differences across the dose groups indicative of a potential effect of treatment, but these differences were not definitive; involved common lesions; and required histopathologic diagnosis to assess their relationship to treatment. These observations included decreased bilateral cloudy cornea of the eye of high-dose group females, decreased dark mesenteric lymph nodes in all treated female dose groups, increased bilateral dilatation of the ovarian bursa in the high-dose group females, decreased generalised subcutaneous oedema in all treated mice and decreased cystic endometrial hyperplasia of the uterus in low- and high-dose group females.

HISTOPATHOLOGY
The liver was the primary target of ingested test material. There were increased incidences of very slight or slight hypertrophy of the hepatocytes in centrilobular and midzonal regions of the hepatic lobule in males and females given the high doses of 30 or 100 mg/kg/day, respectively (Table 3). However, there was some background variability in the size of hepatocytes through the hepatic lobule, particularly in male mice and the diagnosis of hepatocyte hypertrophy was not unique to treated mice. Thus, hepatocyte hypertrophy was not found in all treated mice and was also diagnosed for 28 % of control male mice and 8 % of control female mice.
Although females given 100 mg/kg/day had a greater relative liver weight increase than the males given 30 mg/kg/day, hepatocyte hypertrophy was more evident in the males (possibly due to the greater hepatocyte size variability found in control males).
Hepatocyte hypertrophy was also found in the 90-day study and there appeared to be little progression in this histopathologic effect. In the 90-day study, hypertrophy was found in 2/10 male mice given 3 mg/kg/day and 4/10 male mice given 30 mg/kg/day and almost all male mice given 300 or 1000 mg/kg/day, with none present in the controls. In females, hypertrophy was found for most females given 300 or 1000 mg/kg/day but not in those given 3 or 30 mg/kg/day. Additionally, periportal hepatocyte vacuolation and hepatocyte necrosis were observed in some mice given 300 or 1000 mg/kg/day in the 90-day study but were not found at the dose levels tested in the 18-month study.
Since liver tumours are relatively commonly found in mice, it is appropriate to examine the incidence in mice dosed with the test material given the effects upon liver weight and hepatocyte hypertrophy. For males, the number of mice with of liver tumours (adenomas or adenomas plus carcinomas) was slightly increased in males given 30 mg/kg/day (Table 4) but the increase was not statistically significant and the numbers were near the historical control range. The increase was limited to the high dose level as males given the mid-dose of 3 mg/kg/day had slightly fewer hepatic adenomas and total liver tumours than controls. One carcinoma, along with renal disease, was considered to be the cause of death for a high-dose male (02A4082). One hepatic adenoma and the other hepatic carcinoma were considered to be primary factors along with atrial thrombosis in removal due to moribund condition of two other male mice (02A4095, 02A4096) from the high-dose group. All these animals were removed within the last month of the study (days 521, 523, and 533).
Due to increased mortality of the high-dose females, Peto analysis was used to analyse tumour incidence. In high-dose females, adenomas and adenomas plus carcinomas were statistically identified when they were considered in the incidental context. All female hepatic neoplasms were incidental (i.e., were not the cause of death) and were only present in mice from the terminal necropsy. The incidence of adenomas was also within the historical control range. As with the males, there were no effects in the females given mid-dose level (30 mg/kg/day), with no hepatic tumours found in either the controls or mid-dose level females.
Thus, mice given high-dose levels of the test material had marginally increased incidence of primary hepatocellular tumours, but the tumours were only present at dose levels causing a 19 - 33 % increased relative liver weight and were accompanied by mild hepatocyte hypertrophy. Although mice given the middle-dose levels, 3 mg/kg/day for males and 30 mg/kg/day for females, had a 10 - 14 % relative liver weight increase and marginally increased incidence of hepatocyte hypertrophy (males only), increased incidence of hepatocellular neoplasms was not found. The slight increase in hepatocellular neoplasms only in the high-dose groups is not indicative of a robust tumour response, which is supported by the incidence being close to historical control incidence and the lack of effect in the mid-dose groups. As there was significantly increased mortality in the high-dose group females, for which the only statistically identified hepatocellular tumour response was found, it is important to consider the validity of the liver tumour response for risk assessment.
Mice given the high-dose levels (30 or 100 mg/kg/day for males or females, respectively) had a slightly increased incidence of minor changes of the lungs consistent with slight inflammatory response. In females this was seen as small foci of alveoli containing macrophages, with increased multifocal aggregates of alveolar macrophages statistically identified in high-dose females (Table 5). Males given 30 mg/kg/day had slightly more sub-acute to chronic inflammation than controls, but not at a statistically significant level. These foci in males were characterised by accumulations of alveolar macrophages along with low numbers of neutrophils and other mononuclear cells and thickening of the interalveolar septae. The relationship of these lung lesions to test material ingestion is equivocal; however, similar inflammatory changes were found in the lungs of Fischer 344 rats given > 5 mg/kg/day for 12 or 24 months.
Primary lung tumours were the most common neoplasms found in the CD-1 mice in this study (Table 6). There was no apparent dose-response relationship in the occurrence of lung tumours and there were no statistical differences by Yates Chi-square test (males). However, for females given the 100 mg/kg/day high-dose level, bronchiolo-alveolar carcinomas were identified by Peto analysis. The two bronchiolo-alveolar carcinomas present at this dose level were both present in females from the terminal necropsy (i.e., they were incidental, not the cause of death). As this incidence is well within the historical control range for the testing laboratory, this finding was considered spurious.
Very few other histopathologic diagnoses were statistically identified and none were considered related to treatment. Reasons these were not considered adverse treatment-related effects included:
1.) Decreased incidence in the treated mice. These included:
- Brain: Mineralisation, Thalamus, unilateral, focal, very slight. Decreased in females given 100 mg/kg/day.
- Brain: Mineralisation, Thalamus, bilateral, multifocal, very slight. Decreased in males given 30 mg/kg/day.
- Heart: Degeneration, with fibrosis, myocardium, multifocal, very slight or all severities combined. Decreased for females given 100 mg/kg/day.
- Kidney: Nephropathy, bilateral, all severities combined. Decreased for females given 100 mg/kg/day.
- Lacrimal/Harderian Gland: Aggregates of mononuclear cells, bilateral, multifocal, all severities combined or slight severity. Decreased for females given 100 mg/kg/day.
- Ovary: Cyst, bilateral. Decreased for females given 100 mg/kg/day.
- Thyroid gland: Cyst, bilateral, multifocal. Decreased for females given 100 mg/kg/day.
2.) Lack of dose response relationship:
- Kidney: Mineralisation, cortex, multifocal, very slight. Increased in males given 0.5 mg/kg/day but not males given 3 or 30 mg/kg/day.
3.) Increased incidence of a lower level of severity balanced by a decrease in greater severity (potentially indicating a lessening of the disease process):
- Adrenals: Hyperplasia, spindle cell, cortex, bilateral, very slight. Increased for females given 100 mg/kg/day but there was an almost equal decrease for the slight and moderate grades.

Cause of Death
In addition to the lack of difference in overall mortality rate for males as noted above, there were no dose–related differences in the various causes of early removal for males. The most common cause of death was renal disease, either nephropathy or glomerular amyloidosis or a combination of the two, which was considered the underlying factor for early removal of 4 control males and 3 males from each treated dose level. Other common causes were chronic active dermatitis, usually ulcerative with bacterial colonies and likely secondary infection/septicaemia, and urinary tract obstruction and sequellae. As expected for aging mice, several male mice from all groups died of neoplasia. Other than the hepatocyte adenoma and carcinomas as primary cause or co-factors in the early removal of the three high-dose males as noted above, there were no other neoplasms potentially related to treatment. The most frequently diagnosed neoplasms were noted in the skin and subcutis (fibrosarcoma and undifferentiated sarcoma) and lymphosarcoma.
While the incidence of early removal of control females was slightly higher than those given 0.5 or 30 mg/kg/day, the cause of death/moribund condition was similar for female controls and these dose groups. As with males, renal disease was the most frequent non-neoplastic cause of early removal with it being implicated as the underlying cause for 4 controls and 1 to 3 treated females/dose group. Unlike males, progressive dermatitis and urinary tract obstruction were not considered to be the underlying cause of early removal for females. A variety of neoplasms were considered the cause of early removal but there were no treatment-related differences in neoplasms as cause for early removal. As with males, the most frequent neoplasms were lymphosarcoma and neoplasms of the skin and subcutis (including mammary gland). For females, haematopoietic/lymphoid system neoplasms (lymphosarcoma, composite lymphoma or histocytic sarcoma) were the underlying cause of early removal for 6 controls and from 2 to 4 treated females per dose group. As noted in the histopathology section above, all hepatocellular neoplasms and lung bronchioloalveolar carcinomas that were statistically identified by Peto analysis in high-dose females occurred only in mice from the terminal necropsy.
As previously noted, high-dose females had increased mortality. Unlike all other dose groups, male or female, many of the high-dose group females did not have an apparent cause of death/moribund condition following histopathologic examination. For the eight female mice from the high-dose group for which a cause of death/moribund condition was identified, the causes were similar to those found in other groups with lymphosarcoma and renal disease being the most frequent. The other 20 females given 100 mg/kg/day did not have a good cause of death established following histopathologic examination. Almost all of these were spontaneous deaths rather than mice euthanised in moribund condition. These tended to be early, and many had a history of tonoclonic convulsions. As noted previously, two of these mice died without recovering from a convulsion. It is considered likely that many of the other early deaths occurred during a convulsion, although they were not observed by animal caretakers.
Histoautoradiography of ¹⁴C test material failed to demonstrate specific localisation of test material in the brain neuropil of rats, although it was found throughout the choroid plexus of the brain ventricular system. The brains from these mice were evaluated in light of this knowledge but no lesions were found that were considered to relate to the convulsions. Actually, both males and females given the high dose levels in this study had statistically significantly decreased incidence of a spontaneous lesion - focal or multifocal mineralisation within the thalamus.

Palpable Mass Correlates
There were a low number of masses noted in-life; most of which were found to be tumours of the skin or subcutis or associated organs (mammary gland) following histopathologic examination.

Relevance of carcinogenic effects / potential:

Mice at the high-dose level had slightly greater numbers of hepatocyte adenomas and combined adenomas plus carcinomas, with the incidence just above the historical control range. This is considered equivocal evidence of carcinogenic potential as the male liver tumour incidence was not statistically significant and, in females, increased incidence of hepatocellular adenomas or total liver tumours was present only at a dose level that exceeded the MTD and was only statistically identified by Peto analysis, a sensitive technique adjusting for increased mortality.

Key result

Dose descriptor:

NOAEL

Effect level:

3 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

other: see 'Remark'

Remarks on result:

other:

Remarks:

Effect type: other: Toxicity

Dose descriptor:

NOAEL

Effect level:

30 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

other: see 'Remark'

Remarks on result:

other:

Remarks:

Effect type: other: Toxicity

Dose descriptor:

NOAEL

Effect level:

30 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Table 1: Incidence of Tonoclonic Convulsions

(Incidence at selected quarterly intervals and at each interval where any group ≥10 %)

Day	Dose Group (mg/kg/day)
	Males				Females
	0	0.5	3	30	0	0.5	30	100
1	0	0	0	0	0	0	0	0
85	0	0	0	0	0	0	0	0
183	0	0	0	0	0	0	0	1
253	0	0	0	0	2	0	1	5
281	0	1	0	1	1	0	2	7
295	0	1	0	0	0	0	0	8
309	3	0	0	3	3	2	2	11
323	0	1	0	1	1	1	1	18
337	2	1	0	7	2	4	4	12
351	1	2	0	0	3	1	0	5
365	1	0	0	1	0	2	1	2
379	0	0	0	0	2	3	1	17
393	0	0	0	0	2	3	3	12
421	0	0	0	0	3	2	2	5
449	3	0	0	1	2	3	1	7
463	0	0	0	0	1	0	0	2
477	0	1	0	1	0	4	1	7
505	2	1	2	1	3	4	3	8
549	0	0	0	0	1	4	0	3

Bold indicates 10 % or more having tonoclonic convulsions on the examination day.

 

Table 2: Selected Organ Weights

	Dose Group (mg/kg/day)
	Males				Females
	0	0.5	3	30	0	0.5	30	100
Final body weight (g)	45.7	43.9	43.6	44.3	36.8	35.7	36.5	35.5
Liver (g)	2.444	2.612	2.655	2.833†	2.007	2.039	2.181	2.543*
% increase liver weight	-	6.9	8.6	15.9	-	1.6	8.7	26.7
Liver (g/100 g)	5.372	6.013	6.099†	6.399†	5.435	5.721	5.968†	7.208†
% increase rel. liver weight	-	11.9	13.5	19.1	-	5.3	9.8	32.6

Bold values interpreted to be treatment related. Statistically different from control mean by Wilcoxon's (†) or Dunnnett's (*) test, alpha = 0.05.

 

Table 3: Histopathologic Liver Effects

Effect	Dose Group (mg/kg/day)
	Males				Females
	0	0.5	3	30	0	0.5	30	100
Hypertrophy, Hepatocyte, Centrilobular/Midzonal, Diffuse
All severities	14	16	21	30*	4	0	4	11
Very Slight	14	13	18	20	3	0	4	8
Slight	0	3	3	10*	1	0	0	3
Hypertrophy, Hepatocyte, Panlobular, Diffuse
Very Slight	1	0	0	2	0	0	1	3

50 mice were examined per dose level

Bold values interpreted to be treatment related. *Statistically different from control mean by Yates Chi-squared test, alpha = 0.05, two-sided.

 

Table 4: Liver Neoplasms

	Dose Group (mg/kg/day)
	Males				Females
	0	0.5	3	30	0	0.5	30	100
Adenoma, hepatocyte, benign , primary (any death context) (One) (Two) (Three)	5 0 0	3 1 1	2 0 0	8 2 0	0 0 0	0 1 0	0 0 0	3** 0 0
Number of mice with adenoma, hepatocyte, benign, primary (one or more)	5	5	2	10	0	1	0	3**
Carcinoma, hepatocyte, malignant with metastasis, primary	0	0	1	0	0	0	0	0
Carcinoma, hepatocyte, malignant without metastasis, primary (any death context)	0	2	1	2	0	0	0	1
Number of mice with adenoma (one or more) or carcinoma (with or without metastasis)	5	7	4	12	0	1	0	4**

50 mice were examined per dose level

No symbol indicates no statistical difference by Yates Chi-square test, alpha=0.05, two-sided.

** Significantly different from controls by Peto mortality adjusted statistics, alpha =0.05, one-sided.

Death context refers to Fatal or Probably Fatal for Peto analysis.

 

Table 5: Histopathologic Lung Effects

	Dose Group (mg/kg/day)
	Males				Females
	0	0.5	3	30	0	0.5	30	100
Aggregates of alveolar macrophages, focal All severities combined Very slight Slight	3 3 0	0 0 0	0 0 0	5 2 0	0 0 0	0 0 0	4 4 0	4 3 1
Aggregates of alveolar macrophages, multifocal All severities combined Very slight Slight Moderate	0 0 0 0	0 0 0 0	1 0 1 0	1 0 1 0	0 0 0 0	1 1 0 0	4 3 1 0	6* 4 1 1
Inflammation, subacute to chronic, focal All severities combined Very slight Slight	2 1 1	3 2 1	5 2 3	5 2 3	1 1 0	1 0 1	0 0 0	2 1 1
Inflammation, subacute to chronic, multifocal All severities combined Very slight Slight Moderate	0 0 0 0	3 0 1 2	1 0 1 0	6 4 0 2	4 1 3 0	3 1 2 0	0 0 0 0	2 1 1 0

50 mice were examined per dose level

Bold values interpreted to be treatment related.

* Statistically different from control mean by Yates Chi-squared test, alpha = 0.05, two-sided.

 

Table 6: Bronchiolo-alveolar Neoplasms

	Dose Group (mg/kg/day)
	Males†				Females
	0	0.5	3	30	0	0.5	30	100
Adenoma, bronchiolo-alveolar benign, primary (one)	8	11	12	12	9	9	13	9
Adenoma, bronchiolo- alveolar benign, primary (two)	1	0	1	0	2	1	2	1
Adenoma, bronchiolo-alveolar benign, primary (three)	0	1	0	0	0	0	0	0
Carcinomas, malignant without metastasis, primary	1	1	2	0	0	0	1	2**
Number of mice with adenoma or carcinoma	10	13	15	12	11	10	16	12

50 mice were examined per dose level

† No statistical difference by Yates Chi-square test, alpha=0.05, two-sided.

** Significantly different from controls by Peto mortality adjusted statistics, alpha =0.05, one-sided.

Conclusions:

Under the conditions of this study, 3 mg/kg/day for males and 30 mg/kg/day for females were considered to be the NOAELs. The low dose level of 0.5 mg/kg/day had no effects attributed to treatment and was the NOEL.

Executive summary:

A study was conducted to assess the potential oncogenicity and chronic toxicity of the test material in accordance with the standardised guidelines OECD 451, EU Mehotd B.32 US EPA OPPTS 870.4200 and MAFF, Ministry of Agriculture, Forestry and Fisheries, 2000 under GLP conditions.

Groups of CD-1 mice (50/sex) were given diets formulated to provide 0 (controls), 0.5, 3 (males only), 30, or 100 (females only) mg /kg body weight/day for up to 18 months. Animals were evaluated over the course of the study by daily cage side and periodic hand-held clinical examinations, body weight, and feed consumption. Ophthalmic examinations were conducted pre-exposure and prior to necropsy. All mice had a complete necropsy examination with white blood cell (WBC) and differential WBC counts and weights of selected organs at the scheduled necropsy. An extensive set of organs was examined histopathologically from all controls and high-dose group mice as well as all mice removed from study prior to the scheduled termination. Histopathologic examination was also conducted for target organs, selected major organs, and all gross lesions of mice from the low-and intermediate-dose groups from the terminal necropsy.

There was statistically significant increased mortality for females given the 100 mg/kg/day high dose (56 % cumulative mortality vs. 32 % in controls at 18 months). There was an increased incidence of tonoclonic convulsions in this dose group as compared to controls (though this strain of mouse is prone to convulsions), and convulsions were noted at some time point for many of the high-dose females that died spontaneously. Convulsions were considered the probable cause of increased early mortality, although convulsions were not noted immediately prior to death for most mice. Increased mortality not due to neoplasia is an indicator that this dose level exceeded a maximum tolerated dose (MTD).

There were no treatment-related effects noted during the in-life phase of the study for male mice from any dose level or females given 0.5 or 30 mg/kg/day. At necropsy, the absolute weights of the livers of high-dose group mice (both sexes) were increased and the liver weights relative to body weight were increased in both high- and mid-dose group mice (both sexes). Minor microscopic liver changes consisting of very slight or slight hepatocyte hypertrophy were noted for high-dose level mice (both sexes) and mid-dose level males, although only the male high-dose incidence was statistically significant.

Mice at the high-dose level had slightly greater numbers of hepatocyte adenomas and combined adenomas plus carcinomas, with the incidence just above the historical control range. .

Although relative liver weight was slightly increased at the middle dose levels and minimal hepatocellular hypertrophy was present in males, these were considered likely adaptive effects to metabolism of this test material and these levels (3 mg/kg/day for males and 30 mg/kg/day for females) were considered to be the no-observed-adverse-effect-level (NOAEL). The incidence of hepatocellular tumours for mid-dose level mice (both sexes) was equal to or less than the concurrent controls. The low dose level of 0.5 mg/kg/day had no effects attributed to treatment and was the no-observed-effect level (NOEL).

Reference 2

Endpoint:

carcinogenicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

09 April 2002 - 22 April 2005

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:

other: EPA OPPTS 870.4300 (Combined Chronic Toxicity / Carcinogenicity) and EPA OPPTS 870.3100 (90-Day Oral Toxicity in Rodents)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: OECD Guideline 453 (Combined Chronic Toxicity / Carcinogenicity Studies) and OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EU Method Part B. (Combined Chronic Toxicity / Carcinogenicity Test) and EU Method B.26 (Sub-Chronic Oral Toxicity Test: Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: JMAFF, Combined Chronic Toxicity/Oncogenicity Study and Subchronic Oral Toxicity Study) 1985

Deviations:

no

GLP compliance:

yes

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: The animals were housed 2-3 per cage during acclimatisation. During the study animals were housed 2 per cage in in stainless steel cages.
- Diet: Certified rodent diet in meal form, provided ad libitum.
- Water: Municipal water, provided ad libitum.
- Acclimation period: Animals were acclimated to the laboratory for 7 days prior to the start of the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.8 to 22.8 °C, with the exception of one day when the temperature was 27.7 °C.
- Humidity (%): 40 to 69 %, with the exception of three occasions when the humidity was 38 %.
- Air changes (per hr): A 12-hour light/dark photocycle was maintained for all animal rooms with lights on at 6:00 a.m. and off at 6:00 p.m.
- Photoperiod (hrs dark / hrs light): approximately 12-15 times/hour.

IN-LIFE DATES: From: April 18, 2002 (males) and April 19, 2002 (females). To: All surviving male and female oncogenicity rats were necropsied on April 19-27, 2004 (April 19, 21 and 23 or days 733, 735 and 737 for males and April 20, 22, 26 and 27 or days 733, 735, 739, and 740 for females).

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on exposure:

DIET PREPARATION
Diets were prepared by serially diluting a concentrated test material- feed mixture (pre- mix) with ground feed. Premixes were mixed periodically throughout the study based on stability data. Initial concentrations of test material in the diet were calculated from historical body weights and feed consumption data. Subsequently, the concentrations of the test material in the feed were adjusted weekly for the first 13-weeks of the study and at 4-week intervals thereafter based upon the most recent body weight 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 prior to the start of dosing and at approximately 1.5, 3, 8, 12, 18, and 24 months. Additional analytical analyses were conducted at approximately 8, 9, 14, and 20 months to verify homogeneity of the diet mixes.
- Stability: Previous 90-day toxicity study in rats (Yano and Dryzga, 2002) demonstrated that the test material was stable for at least 42 days in rodent chow at concentrations ranging from 0.005 % to 5 %. In this study, an additional stability evaluation was conducted at 0.0001 % to cover the concentration ranges of the administered diets.
- Concentration Verification: Analyses of the premix, all dose levels and the 0 (control) mg/kg bw/day diets were determined prior to the start of exposure and at approximately 1.5, 3, 8, 12, 18, and 24 months. Additional concentration analyses were also conducted at approximately 14, 18, and 20 months. The method for analysing the test material in feed was a solvent extraction method followed by analyses using liquid chromatography- mass spectrometry (LC-MS) and solvent standards incorporating an internal standard.
- Retainer Samples: Reference samples (one/sex/dose/mix) were retained and stored in sealed vials in a manner consistent with the sample retention policy of the laboratory.

Duration of treatment / exposure:

Two years (oncogenicity group).

Frequency of treatment:

Daily.

Post exposure period:

There was no post exposure period. The in-life phase of the study was terminated at the end of exposure.

Dose / conc.:

0 mg/kg bw/day

Remarks:

Basis:
nominal in diet

Dose / conc.:

0.1 mg/kg bw/day

Remarks:

Basis:
nominal in diet

Dose / conc.:

1 mg/kg bw/day

Remarks:

Basis:
nominal in diet

Dose / conc.:

75 mg/kg bw/day

Remarks:

Basis:
nominal in diet

Dose / conc.:

300 mg/kg bw/day

Remarks:

Basis:
nominal in diet

No. of animals per sex per dose:

Fifty per sex per dose.

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: Dose levels for this study were selected based on the toxicity data from a subchronic toxicity study (Yano and Dryzga, 2001), a non-guideline pharmacokinetic study (Saghir and Mendrala, 2002) and written and verbal consultations with the U.S. E.P.A.
For the chronic toxicity/oncogenicity portion of the study the targeted dose levels were 0, 0.1, 1.0, 75, and 300 mg/kg bw/day.
Based on the data from the 90-day dietary toxicity study, as well as the pharmacokinetic study, proposed dose levels were 0, 0.1, 1.0, 10, and 75 mg/kg bw/day. Data from these studies indicated that gastrointestinal absorption was nearly saturated at an actual dose of 78-86 mg/kg (targeted concentration of 100 mg/kg). Numerous toxicology data points, especially liver weights, supported this conclusion. In the 90-day dietary toxicity study, absolute and relative liver weights were increased 17 % and 21 % for males and 30 % and 34 % for females at the 100 mg/kg bw/day dose level. In subsequent discussions with the EPA, the Agency noted that the original proposed dose levels may be sufficient to produce adequate toxicity in the chronic study, but that a more conservative approach would be to use a higher dose which would provide greater assurance of safety (EPA, 2002). Therefore, the highest-dose level was selected at 300 mg/kg bw/day because additional test material was absorbed at a dose higher than 100 mg/kg bw/day, although the dose was not proportional to toxicity or the area under the curves (AUCs). As previously noted in discussions with the EPA, this reflected a conservative approach to dose level selection. Treatment-related effects induced in rats given 300 mg/kg bw/day were expected to be as follows: 1) slight reduction in body weight, 2) increased liver weights, and 3) possible alterations in clinical chemistry parameters.
Similar effects were expected to occur in rats given 75 mg/kg bw/day as would occur in rats given 300 mg/kg bw/day due to a saturation of absorption at a dose of approximately 75 mg/kg bw/day.
- Dosing route selection rationale: Potential routes of human exposure to the test material would be via 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.

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: A cage-side (general) clinical examination was conducted at least once a day, typically at the same time each day (usually in the morning). Moribund animals not expected to survive until the next observation periods were humanely euthanised that day. In addition, all animals were observed for morbidity, mortality, and the availability of feed and water at least twice daily.
- Cage side observations included, but not limited to: Decreased/increased activity, repetitive behaviour, vocalisation, incoordination/limping, injury, neuromuscular function (convulsion, fasciculation, tremor, twitches), altered respiration, blue/pale skin and mucous membranes, severe eye injury (rupture), alterations in faecal consistency, and faecal/urinary quantity.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: A complete detailed clinical observation (DCO) was conducted at pre-exposure and monthly for 12 months then at 15, 18, 21, and 24 months on the first 10 surviving animals from the oncogenicity group. Baseline clinical observations were conducted on all animals (day 1) not receiving DCOs. The DCOs were conducted on animals at approximately the same time each examination day according to an established format.
- Parameters checked include:
> Inside home cage: abnormal movements or behaviour, gait evaluation, and palpebral closure.
> Outside home cage: postural thrust response, responsiveness to touch, bilateral eye movement, pupil reactivity, pupil reactivity, lacrimation (clear periocular wetness), salivation (clear perioral wetness), muscle tone, visual response, and reactivity to observer.
> Categorical observations: abnormal behaviour, abnormalities of the eye, abnormal urine and faeces, abnormalities of the gastrointestinal (GI) tract, injury, missing extremity, abnormal muscle movements, palpable mass/swellings, abnormal posture, abnormalities of the reproductive tract, abnormal respiration, abnormal skin or hair-coat/mucous membranes, excessive soiling, and general abnormalities.

PALPABLE TUMOR OBSERVATIONS: Yes
- Time schedule: Palpable tumor observations (categorical observations) were conducted monthly from months 12-24 on all animals. Palpable mass observations were conducted on animals at approximately the same time each examination day according to an established format.

BODY WEIGHT: Yes
- Time schedule for examinations: The rats were weighed during the pre-exposure period, weekly during the first 13 weeks of the study and then at approximately monthly intervals during the remainder of the study. Body weight gains were calculated throughout the study.

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
- Food consumption: Feed consumption data were collected weekly during the first 13 weeks of the study and then at approximately monthly intervals thereafter for all animals. Feed containers were weighed at the start and end of a measurement cycle and consumption was calculated using the following equation:
Feed consumption (g/day) = (initial weight of feed containers - final weight of feed containers) / [(No. of days in measurement cycle)(No. of animals per cage)]
- Test material intake (TMI): TMI was calculated for 0-90 days, 0-12 months and 12-24 months using test material concentrations in the feed, actual body weights and measured feed consumption in the following equation:
TMI = [(feed consumption) * (1000) * (% of test material in feed/100)] / {[(current BW + previous BW) / 2] / 1000}

FOOD EFFICIENCY:
Feed efficiency was calculated using body weight gain and feed consumption data from the first 13 weeks of the study using the following equation:
Feed efficiency = (g feed consumed/day) / (g body weight gain/day)

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: The eyes of all animals were examined by a veterinarian pre-exposure and prior to termination using indirect ophthalmoscopy.
- Dose groups that were examined: All groups 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. A prosector also examined eyes 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 of anesthetised animals at 18 and 24 months. The blood samples were taken after the FOB testing was completed on the subset of this group.
- Anaesthetic used for blood collection: Yes (CO2).
- Animals fasted: Yes
- How many animals: These samples were taken from the first 10 surviving rats/sex/dose group. Additional blood samples were obtained from 4/rats/sex/dose from the one-year chronic toxicity groups and the plasma were frozen and stored. These samples were not subsequently analysed.
- Haematology sample preparation: Blood samples for a complete blood count from the main group 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.
Blood was collected from all remaining surviving rats from the oncogenicity group at the scheduled necropsy at 24 months. A total WBC and differential WBC were conducted (Technicon H.1E Haematology Analyser). Blood smears prepared and archived from the first 10 rats/sex/dose at 18 months, all moribund rats and all remaining rats in the oncogencity group.
- Coagulation sample preparation: Blood samples for coagulation were collected in sodium citrate tubes, centrifuged and plasma collected and assayed using an ACL9000.
- Haematology parameters checked: Haematocrit (Hct), haemoglobin (Hgb) concentration, red blood cell (RBC) count, total white blood cell (WBC) count, platelet (PLAT) count, differential WBC count, and RBC indices - mean corpuscular hemoglobin concentration (MCH), mean corpuscular volume (MCV), and mean corpuscular haemoglobin concentration (MCHC).
- Coagulation parameters checked: Prothrombin time (PT).

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Blood samples were collected from the orbital sinus of anesthetised animals at 18 and 24 months.
- Animals fasted: Yes
- How many animals: Samples were taken from the first 10 surviving rats/sex/dose group. Additional blood samples were obtained from 4/rats/sex/dose from the one-year chronic toxicity groups and the plasma were frozen and stored. These samples were not subsequently analysed.
- 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).

URINALYSIS: Yes
- Time schedule for collection of urine: Urine was collected from the first 10 surviving rats/sex/dose group from the oncogenicity group at 18 and 24 months. (Note - One control female (animal # 02A2147) from the neurotoxicity group died and was replaced with the first surviving rat from the shared chronic/neurotoxicity group (animal # 02A2141) for neuropathologic effects. Urine from female 02A2147 was collected, analysed and reported with the chronic toxicity/oncogenicity group). Urine was also collected by manual compression of the bladder prior to the necropsy for characterisation of the microsediment using a pooled sample from each dose group/sex.
- Metabolism cages used for collection of urine: Yes, animals were housed in metabolism cages and urine collected overnight (approximately 16 hours).
- Animals fasted: No
- Parameters checked:
> Assays: Colour, appearance, specific gravity (refractometer) and urine volume.
> Semiquantitative analysis of: pH, bilirubin, glucose, proteins, ketones, blood, and urobilinogen.

Sacrifice and pathology:

NECROPSY
Fasted rodents, submitted alive, were anesthetised by the inhalation of CO2, weighed, and blood samples were obtained from the orbital sinus. Their tracheas were exposed and clamped, and the animals were euthanised 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.
Similar necropsy procedures were followed for animals found dead or moribund, except that body weights, organ weights, standard blood samples (only blood smears on moribund animals), and urine samples were not obtained.
Moribund animals that were not expected to survive until the next observation period, and any animals found dead were necropsied on that day. However, animals euthanised in moribund condition or found dead after routine working hours or on weekends or holidays were refrigerated until the next scheduled work day at which time they were necropsied.

GROSS PATHOLOGY: Yes
- Organ weights: The brain, liver, kidneys, heart, adrenals, thymus (90 days, only), testes, epididymides, ovaries, uterus, and spleen.

HISTOPATHOLOGY: Yes
The tissues listed below were processed by standard histologic procedures from control and high-dose group animals and all animals that died or were sacrificed in a moribund condition. (Note - One control female (animal # 02A2147) from the neurotoxicity group died and was replaced with the first surviving rat from the shared chronic/neurotoxicity group (animal # 02A2141) for neuropathologic effects. Histology data from control female 02A2147 was examined and reported with the chronic toxicity/oncogenicity data sets). The following tissues from the remaining groups were processed and examined: liver, lungs, kidneys, relevant gross lesions, target organs and other selected organs.
> 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, lacrimal/harderian gland, 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, seminal vesicles, skeletal muscle, skin and subcutis, spinal cord (cervical, thoracic, lumbar), spleen, stomach, testes, thymus, thyroid gland, tongue, trachea, urinary bladder, uterus and vagina.

Statistics:

For details on the statistics used in this study see below in the field "Any other information on materials and methods incl. tables".

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

No treatment related effects in the first year. Treatment related clinical observations recorded during the second year of dosing; higher incidence of missing parts of the tail (males given 300 mg/kg bw/day).

Mortality:

mortality observed, treatment-related

Description (incidence):

No treatment related effects in the first year. Treatment related clinical observations recorded during the second year of dosing; higher incidence of missing parts of the tail (males given 300 mg/kg bw/day).

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Lower body weights and body weight gains (males and females given 75 and 300 mg/kg bw/day).

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

No treatment related effects.

Food efficiency:

no effects observed

Description (incidence and severity):

No treatment related effects.

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

not examined

Ophthalmological findings:

no effects observed

Description (incidence and severity):

No treatment related effects.

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Increased prothrombin time (males given 75 and 300 mg/kg bw/day).

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Increased cholesterol levels (females given 75 and 300 mg/kg bw/day); higher alkaline phosphatase activities (males and females given 75 and 300 mg/kg bw/day).

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

Higher urine volume (males and females given 75 and 300 mg/kg bw/day); lower urine specific gravity (males and females given 75 and 300 mg/kg bw/day).

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Treatment related effects observed in males and females given 75 and 300 mg/kg bw/day, see below “Results” for more details.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Treatment related effects observed in males and females exposed to the test material, see below “Results” for more details.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Increased incidences of non-neoplastic effects were observed in males and females given 75 and 300 mg/kg bw/day.

Histopathological findings: neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Increased incidence of neoplasms were observed in males given 300 mg/kg bw/day and females given 75 and 300 mg/kg bw/day.

Other effects:

not examined

Details on results:

CLINICAL SIGNS AND MORTALITY
- Mortality: Over the course of the study, 19, 12, 16, 16, and 14 males died spontaneously or were humanely euthanised due to their moribund condition from the 0, 0.1, 1, 75, or 300 mg/kg bw/day dose groups, respectively. For females, 9, 14, 16, 11, and 4 rats died or were humanely euthanised from the 0, 0.1, 1, 75, or 300 mg/kg bw/day groups, respectively. There were no statistically identified differences in cumulative mortality for either males or females.
- Detailed Clinical Observations: Isolated observations in males and females involving unilateral or bilateral increases in pupil size and decreased amounts of faeces were interpreted not to be treatment related due to the low incidence and lack of a dose-response relationship.
- Clinical Observations: During the first year, all observations were interpreted as not being treatment related. Four treated males (1.0 mg/kg bw/day) and six females (0.1 mg/kg bw/day) had periocular soiling, and up to four females had urine soiling (0.1 mg/kg bw/day). These findings were sporadic, transient, lacked a dose response and were interpreted not to be treatment related. Observations other than those previously discussed occurred in only one or two rats/sex/dose group and/or there was no pattern suggesting an increased occurrence in relation to exposure to the test material.
During the second year of the study, there were greater numbers and variety of observations than noted through the first year. This was an expected finding due to geriatric diseases as these rats neared the end of their life span. Increasing numbers of rats had cloudy eyes, preputial or clitoral gland swelling, papules/pustules of the skin, soiling - urine and periocular, and missing part of the tail. Papules/pustules of the skin (generalised distribution) affected the tail and were higher in incidence than the controls for males given 0.1, 1.0, 75, and 300 mg/kg bw/day and females given 300 mg/kg bw/day. The increased incidence of papules/pustules of the skin of males given 0.1 and 1.0 mg/kg bw/day were not dose related and were interpreted not to be treatment related. Males given 300 mg/kg bw/day also had a higher incidence of missing part of the tail than the control or other dosed rats. The interpretation of these differences is discussed in the histopathology section of the report.
Cloudy eyes (males and females), preputial/clitoral gland swellings and soiling (urine and periocular) were not dose related and were interpreted not related to the test material. Urine soiling was observed more often in females given 75 and 300 mg/kg bw/day around day 568; however, the incidence decreased markedly by the end of the study, suggesting that these nonspecific observations were not toxicologically significant. All other in-life observations not previously discussed were also interpreted not to be treatment related.
Masses were infrequently found on the categorical portion of DCO examinations through the first 12 months of study, with only one male given 300 mg/kg bw/day having a small mass on the nose that later resolved. As the study progressed, the incidence of palpable masses increased; however, there was no evidence of a dose-response relationship in the incidence of palpable masses. The resolution of palpable masses is addressed following the gross and histopathologic examination of these lesions.

BODY WEIGHT AND WEIGHT GAIN
Body weights for the male rats given 75 or 300 mg/kg bw/day were lower than controls and were interpreted to be treatment related. The differences from controls were first statistically identified at day 85, when the males given 75 or 300 mg/kg/day weighed 2.5 % and 2.7 % less than controls, respectively. Body weights of these males remained lower than the controls throughout the study and were frequently statistically identified as different from the controls. The body weights of males given 300 mg/kg bw/day were 3.7 % lower than controls on day 363 and males given 75 mg/kg bw/day weighed 3.5 % less on day 363. Males given 75 or 300 mg/kg bw/day weighed 7.0 and 9.7 % less than controls, respectively, on day 729, when the in-life phase of the study was complete. Body weights of male rats given 1.0 mg/kg bw/day were slightly lower than controls throughout the study; however, the differences were not attributed to treatment, as they were generally within 2 % of the control body weights. Body weights of males given 0.1 mg/kg bw/day were comparable to the controls throughout the study.
Body weights for the females given 75 or 300 mg/kg bw/day were lower than controls and were treatment related. Decreases from controls were first statistically identified on day 176, when the females given 300 mg/kg bw/day weighed 2.5 % less than controls. Body weights for females given 75 mg/kg bw/day were statistically identified and 3.2 % lower than controls on day 316. Body weights for the 75 and 300 mg/kg bw/day groups continued to decrease and were statistically identified for the remainder of the study. Body weights of female rats given 75 or 300 mg/kg bw/day were 4.0 % and 6.0 % lower than controls on day 363, and were 14.0 % and 14.7 % less than controls, respectively on day 729. Body weights for female rats given 0.1 or 1.0 mg/kg bw/day were comparable to controls throughout the study.
Differences in body weights were reflected in lower body weight gains for males and females given 75 or 300 mg/kg bw/day.
Body weight gains of males given 75 or 300 mg/kg bw/day were 4.6 % and 5.1 % less than controls, respectively on day 363. These decreases remained until study termination, at which time they were 9.5 % and 13.9 % lower than controls for the 75 and 300 mg/kg bw/day groups, respectively. Body weight gains for females given 75 or 300 mg/kg bw/day were 6.5 % and 9.8 % lower than controls on day 363 and 21.8 % and 22.4 % lower than controls at study termination. The lower body weight gains of males given 300 mg/kg bw/day and females given 75 or 300 mg/kg bw/day clearly exceed the 10 % body weight gain decrement criteria, as defined by several regulatory authorities (EPA, 2003; HED, 2003; Commission Directive, 2001), and males given 75 mg/kg bw/day almost meet the 10 % criteria and may confound the interpretation of tumorous effects at these dose levels.

FOOD CONSUMPTION AND COMPOUND INTAKE
- Feed Consumption: Feed consumption values for males given 1 or 300 mg/kg bw/day were higher than controls (< 6 %) and frequently statistically identified through day 407; however, from day 407 to study termination, feed consumption for males given 300 mg/kg bw/day were comparable to controls at most time points. In contrast, feed consumption for males given 1.0 mg/kg bw/day continued to be higher than the controls and statistically identified and was 8.4 % higher at study termination. Feed consumption for males given 0.1 or 75 mg/kg bw/day was considered comparable to controls throughout the study. The cause of the slightly higher feed consumption observed through day 407 for males given 300 mg/kg bw/day was not determined.
Feed consumption values for females given 1.0, 75, or 300 mg/kg bw/day were higher (< 7 %) than controls from day 1-379 and frequently statistically identified. After day 379, feed consumption values of females given 75 or 300 mg/kg bw/day were lower than controls and statistically identified. In contrast, feed consumption values of females given 1.0 mg/kg bw/day, continued to be higher than the controls and statistically identified throughout the remainder of the study. The cause of the feed consumption variability observed for females given 75 or 300 mg/kg bw/day was not determined. Feed consumption of females given 0.1 mg/kg bw/day was comparable to the controls.
These apparent differences in feed consumption may reflect normal biologic variability between groups and were interpreted not to be treatment related. The apparent increase in feed consumption observed at 1.0 mg/kg bw/day was considered unrelated to treatment because of a lack of a dose-response relationship.
- Test Material Intake: The test material intakes were very close to the targeted dose for all levels over the course of the study. The time weighted average amounts of ingested test material (mg/kg bw/day) were within 5 % of the targeted dosages for all dose groups.
Months 0-12: The actual doses received were 0.100 (± 0.006), 1.00 (± 0.06), 75.3 (± 4.2) and 303 (± 17) mg/kg bw/day for males and 0.099 (± 0.005), 0.99 (± 0.07), 74.5 (± 3.3) and 298 (± 14) for females dosed at 0.1, 1.0, 75 and 300 mg/kg bw/day, respectively.
Months 0-24: The actual doses received were 0.101 (± 0.006), 1.01 (± 0.06), 78.7 (± 13.4) and 303 (± 15) mg/kg bw/day for males and 0.101 (± 0.005), 1.01 (± 0.06), 75.5 (± 3.0) and 303 (± 13) for females dosed at 0.1, 1.0, 75 and 300 mg/kg bw/day, respectively.

FOOD EFFICIENCY
Feed efficiency between the control and treated males and females were generally comparable during most time points. On occasion there were instances of apparent increased or decreased feed efficiency. These differences were not consistently dose related and were interpreted not to be treatment related.

OPHTHALMOSCOPIC EXAMINATION
Pre-exposure examination of rats placed on study indicated all rats were within normal limits. Variable numbers of male and female rats had a pale fundus, cloudy cornea, periocular soiling, opaque cornea, opaque lens, phthisis bulbi, incomplete pupil dilatation, or missing, enlarged eye or microphthalmia at the 12- and 24- month intervals. Periocular soiling was considered to be a non-specific observation, while eyes with pale fundus, opaque cornea, opaque lens, or cloudy cornea were interpreted to be spontaneous, age-related changes.

HAEMATOLOGY
- Treatment Related Effects
Prothrombin time was consistently elevated in males given 75 or 300 mg/kg bw/day and was interpreted to be treatment related. Males given 300 mg/kg bw/day had statistically identified elevations in prothrombin time beginning at 3 months, while in males given 75 mg/kg bw/day, this occurred at 6 months. The elevations in prothrombin time were evident until 18 months in both of these treatment groups. At no time during the study were there clinical observations of haemorrhage or bruising that could reflect an adverse effect of this slightly longer prothrombin time.
Prothrombin times at 24 months in males given 75 or 300 mg/kg bw/day were similar to the controls. The lack of a difference in prothrombin time between males given 0 mg/kg bw/day and those given 75 or 300 mg/kg bw/day at 24 months may be due to increased variability in prothrombin times associated with confounding disease processes in rats of this age.
- Effects Not Related To Treatment
Prothrombin times in females were slightly lower than the controls and statistically identified at 18 months in the 75 and 300 mg/kg bw/day dose groups. These differences were not observed at 3, 6, 12, or 24 months, and were interpreted not to be treatment related. Moreover, slight decreases in prothrombin time would not normally be considered toxicologically significant.
Males given 75 or 300 mg/kg bw/day had haematocrits that were slightly higher than the controls at 18 and 24 months and these differences were statistically identified. However, these values were within the historical control range. Males given 75 or 300 mg/kg bw/day also had statistically identified higher RBC counts observed at these time points. The MCV values (ratio of the haematocrit to RBC count), were slightly lower in males given 75 or 300 mg/kg bw/day at 18 and 24 months, relative to the concurrent controls; however, they were close to the historical control values and therefore interpreted not to be treatment related. Moreover, the mean concurrent control MCV (61.2) of males at 24 months was unusually high compared to historical controls, further suggesting that the alterations in MCV were not treatment related.
Additionally, there were no treatment-related changes in the bone marrow suggestive of increased output of red blood cells. Haemoglobin values were also similarly elevated and statistically identified in males given 75 or 300 mg/kg bw/day at 18 and 24 months. Haemoglobin values were within or in close proximity to historical control values. In addition, the concurrent controls were outside the range (lower) of historical controls and the increases in haemoglobin values were not physiologically or toxicologically significant. Therefore, the increased haematocrit, increased RBC counts and increased haemoglobin values observed at the 18 and 24 months in males given 75 or 300 mg/kg bw/day were interpreted not to be treatment related.
Statistically identified changes that were considered spurious included slightly lower values in the 12- month haematocrit and haemoglobin of females given 300 mg/kg bw/day, a slightly lower value in the 18- month haemoglobin concentration of females given 75 mg/kg bw/day and a slight increase in the 6- month haemoglobin concentration of males given 75 mg/kg bw/day. Similarly, higher platelet counts in males given 300 mg/kg bw/day at 3 and 6 months, and females given 75 or 300 mg/kg bw/day at 6 months were also considered spurious. These statistically identified changes were considered not toxicologically relevant due to lack of consistency across time points and/or lack of dose responsiveness.

CLINICAL CHEMISTRY
- Treatment Related Effects
Females given 75 or 300 mg/kg bw/day had slightly higher cholesterol (CHOL) levels than the controls throughout the study. These differences in CHOL levels were higher than the historical control ranges and were interpreted to be treatment related, but were not associated with a clear adverse effect.
Males and females given 75 or 300 mg/kg bw/day had higher alkaline phosphatase activities (ALP) than the controls throughout the study, were frequently statistically identified and included: males and females given 75 mg/kg bw/day at 3, 6, 12, and 18 months, and males and females given 300 mg/kg bw/day at 3, 6, 12 (males only), and 18 (females only) months. These differences were higher than the concurrent controls, generally were outside of the historical control ranges, associated with higher liver weights and hepatocellular hypertrophy, and were interpreted to be treatment related.
- Effects Not Related To Treatment
Males given 75 or 300 mg/kg bw/day had higher CHOL levels than the controls at selected times during the study, which were frequently statistically identified, and included: 75 mg/kg bw/day at 3 months, and 300 mg/kg bw/day at 3 and 12 months. The CHOL differences in males were not observed at 18 and 24 months and were not attributed to the test material.
Males given 75 or 300 mg/kg bw/day had higher total protein (TP) than the controls throughout the study, which were frequently statistically identified and included: 75 mg/kg bw/day at 3, 6, 12, 18, and 24 months and males given 300 mg/kg bw/day at 3, 6, and 12 months. These differences were higher than the concurrent controls but in close proximity to the high end of the historical control ranges and were interpreted not to be treatment related.
Males given the test material had higher urea nitrogen (UN) concentrations throughout the study, were frequently statistically identified and included: 1.0 mg/kg bw/day at 3 months, 75 mg/kg bw/day at 3, 6, 12, and 18 months, and 300 mg/kg bw/day at 3, 6, 12, 18, and 24 months. In general, these differences were only slightly higher than the concurrent controls and frequently within the historical control ranges. Creatinine (CREAT) values of males given the test material were similar to the controls throughout the study, although the values for two dose levels (0.1 and 300 mg/kg bw/day) at 3 months were statistically different from the controls. These differences were interpreted not to be treatment related, because of a lack of dose response, the failure for the effect to occur with prolonged exposure, and the absence of a significant degree of renal disease that would correlate with these slightly higher values.
Females given 75 or 300 mg/kg bw/day also had higher UN values than the controls at selected times during the study, were frequently statistically identified and included: 75 and 300 mg/kg bw/day at 6 and 18 months. These differences were only slightly higher than the concurrent controls and/or frequently within the historical control ranges and were not attributed to the test material. Creatinine (CREAT) values of females given the test material were similar to the controls throughout the study, not statistically identified as different and were not indicative of kidney disease.
Males given 75 or 300 mg/kg bw/day had lower potassium concentration than the controls at 3 months and were statistically identified. These differences were lower than the historical control range; however, similar differences were not observed at the longer exposure periods (6-24 months) and were interpreted not to be treatment related.
Males given 75 or 300 mg/kg bw/day had higher albumin levels than the controls at 3 months and were statistically identified. These differences were higher than the concurrent controls but were within the historical control range, similar differences were not observed at the longer exposure periods (6-24 months), and were therefore interpreted not to be treatment related.

URINALYSIS
- Treatment-Related Effects
Males given 75 or 300 mg/kg bw/day had higher urine volume than the controls throughout the study, were occasionally statistically identified and included: 75 mg/kg bw/day at 3, 6 (statistically identified), 12, and 18 months, and 300 mg/kg bw/day at 3, 6 (statistically identified), 12, 18 (statistically identified), and 24 months. There were a few instances when males given 1.0 mg/kg bw/day had increased urine volumes as compared to the controls. The differences in males given 300 mg/kg bw/day were almost always higher than the historical control ranges and were interpreted to be treatment related. Differences in males given 1.0 or 75 mg/kg bw/day were not consistently elevated above concurrent or historical control values and were not attributed to treatment with the test material.
Females given 75 or 300 mg/kg bw/day had higher urine volume than the controls throughout the study, were frequently statistically identified and included: 75 mg/kg bw/day at 3, 6, 12, 18, and 24 months, and 300 mg/kg bw/day at 3, 6, 12, 18, and 24 months. There were also a few instances when females given 1.0 mg/kg bw/day had increased urine volumes.
The urine volume differences in females given 75 or 300 mg/kg bw/day were attributed to treatment with the test material because the values were outside of the concurrent and historical control values. Urine volumes of females given 1.0 mg/kg bw/day were interpreted to be within normal limits.
Males and females given 75 or 300 mg/kg bw/day had lower urine specific gravity than the controls throughout the study, were frequently statistically identified and included: 75 mg/kg bw/day at 3 (males and females), 6 (males and females), 12 (males and females), 18 (males and females) and 24 (females) months, and 300 mg/kg bw/day at 3 (males and females), 6 (males and females), 12 (males and females), 18 (males and females) and 24 (males and females) months. In addition, there were a few instances when males and females given 0.1 or 1.0 mg/kg bw/day had lower urine specific gravity.
The differences in males and females given 75 or 300 mg/kg bw/day were almost always lower than the historical control ranges and were interpreted to be treatment related. The pathogenesis of the increased urine volume and decreased specific gravity was not clear as these rats did not have a toxicologically significant degree of renal disease and the UN and CREAT concentrations were unaffected by the test material.
- Effects Not Related To Treatment
Males given 75 or 300 mg/kg bw/day had a higher incidence of a more alkaline urine pH at all time points throughout the study, as compared to the concurrent controls. Differences were most apparent during the first 18 months of the study and were only marginally different at the end of the study.
Male urine pH data from three recently completed studies indicated that the urine pH of males given 75 or 300 mg/kg bw/day were outside of the historical control data at 3 months, but were within or in close proximity to the historical control values at 6, 12, 18, and 24 months. Based on these data, the differences in urine pH observed in this study were interpreted not to be treatment related.
Urine pH of females given the test material was similar to the concurrent controls throughout the study, with the exception of the 300 mg/kg bw/day group at 18 months. However, urine pH values at this time were similar to the historical control data. Therefore, the isolated occurrence of alkaline (pH 7) urine was not attributed to treatment to the test material.

ORGAN WEIGHTS
- Primary Treatment Related Effects
Final body weights of males given 75 or 300 mg/kg bw/day for 12 and 24 months were lower than the controls, statistically identified (24 months), and consistent with lower body weights of these males during the in- life phase of the study. Males given 75 or 300 mg/kg bw/day for 12 and 24 months had higher absolute and relative weights of the liver and kidneys. These differences were dose related, statistically identified, outside of the historical control values and were interpreted to be treatment related. Males given 75 or 300 mg/kg bw/day also had higher absolute and relative testes weights and lower absolute and relative epididymal weights at 24 months. The testes had not been previously identified to be a target organ of the test material; however, the testes weights were clearly outside of the historical control range and the differences in weight were attributed to the occurrence of larger interstitial cell tumors in males given 75 or 300 mg/kg bw/day, with no difference in the incidence of these tumors at these dose levels (see Histopathology section below). Lower epididymal weights were interpreted to be a secondary effect of the larger testicular neoplasms, resulting in a greater degree of seminiferous tubule atrophy and ultimately less spermatic elements in the epididymides.
Females given 75 or 300 mg/kg bw/day for 12 and 24 months also had higher absolute and relative weights of the liver and adrenals. These differences in liver weight were dose related, whereas the adrenal weights were similar. These organ weights were statistically identified, outside of the historical control values and were interpreted to be treatment related.
- Secondary Treatment Related Effects
Final body weights of males given 75 or 300 mg/kg bw/day for 12 and 24 months were lower than the controls, statistically identified (24 months), and consistent with lower body weights of these males during the in-life phase of the study. These lower body weights were associated with higher relative weights of the heart (12 and 24 months) and brain (24 months). Males given 75 or 300 mg/kg bw/day at 24 months also had lower absolute and relative spleen weights than the concurrent or historical controls. The lower spleen weight of these males was attributed to a lower incidence of Fischer 344 rat leukemia.
Final body weights of females given 75 or 300 mg/kg bw/day for 12 and 24 months were a) lower than the controls, b) statistically identified (24 months), c) were consistent with lower body weights of these females during the in-life phase of the study, and d) were interpreted to be treatment related. These lower body weights were associated with higher relative weights of the heart (12 and 24 months), kidneys (12 and 24 months), and brain (24 months). Females given 300 mg/kg bw/day had a higher relative spleen weight (12 months), and females given 75 or 300 mg/kg bw/day had lower absolute and relative spleen weights at 24 months. The lower spleen weights of these females at 24 months were attributed to a lower incidence of Fischer 344 rat leukemia.
- Effects Not Related to Treatment
The weights of the adrenal glands (absolute – males given 300 mg/kg bw/day; relative – males given 75 or 300 mg/kg bw/day) at 24 months were higher than the concurrent control values and were interpreted not to be treatment related because the values were within the historical control range.
Females given 75 or 300 mg/kg bw/day for 24 months had lower absolute and relative uterine weights than the concurrent controls. Uterine weights were similarly interpreted not to be treatment related because the values were within or in close proximity to the historical control values and the concurrent values appeared to be an outlier, since those values were outside of the historical controls.

GROSS PATHOLOGY
> 12 Months:
- Treatment-Related Effects
The presence of foci on the surface of the lungs in the majority of males and almost all females given 300 or 75 mg/kg bw/day was interpreted to be treatment related. These foci were pale, focal or multifocal, either confined to one lobe or noted in multiple lobes. Such foci were seen on the lungs of 6 of 10 males given 75 or 300 mg/kg bw/day and in 8 of 10 and 10 of 10 in females given 75 or 300 mg/kg bw/day, respectively. These foci on the lungs represented small areas of chronic inflammation (see Histopathology section below). One male given 300 mg/kg bw/day for 12 months had a mass nodule on the liver that was diagnosed as an adenoma (liver adenomas were interpreted to be treatment related, see Histopathology section). This mass nodule on the liver was interpreted to be treatment related because liver tumors were not observed at 12 months in three previous chronic toxicity/oncogenicity studies and because of the significant increase in liver tumors seen at 24 months at this dose level.
- Effects Not Related To Treatment
The right optic nerves in some animals across all dose groups and in both sexes were either decreased in size or missing. This finding was not associated with the exposure to the test article and was attributed to the technical artifact of repeated retro-orbital bleeding at 3-, 6-, and 12-months. Grossly observed mass nodules unrelated to treatment consisted of 3, 1, 0, and 1 uterine polyp(s) in females given 0.1, 1.0, 75, or 300 mg/kg bw/day, respectively. The low incidence and lack of dose response indicated that the uterine polyps were not treatment related at 12 months. Additionally, one control female (animal #02A2147) designated for the 12- month necropsy that was euthanised on day 232 due its moribund condition, had mass nodules in the mesenteric and mediastinal tissues. Data from this rat were incorporated in the gross and histopathology data from the 12-month necropsy. Moribundity in this animal was attributed to the presence of the mass nodule in the mesenteric tissue, which was diagnosed histologically to be an invasive and malignant rhabdomyosarcoma of the skeletal muscle of the diaphragm. All other gross observations were considered spontaneous changes unrelated to treatment, commonly found in Fischer 344 rats of this age and husbandry conditions.
> 24 Months:
- Treatment-Related Effects
There was an increased incidence of one or more foci on the surface of the lungs in majority of males and females given 75 or 300 mg/kg bw/day for up to 24 months. This was similar to what was observed at 12 months. Foci were pale, focal, or multifocal; either confined to one lobe or noted in multiple lobes. They represented small areas of chronic inflammation (see Histopathology section below). In the liver, there was a higher incidence of small foci (pale/dark/focal/multifocal) on one or more lobes of the liver in males given 300 mg/kg bw/day. In general, many of these grossly observed foci represented eosinophilic and/or basophilic foci of cellular (hepatocyte) alteration and occasional hepatocellular adenomas (see Histopathology section for resolution of these lesions).
Females given 75 or 300 mg/kg bw/day had increased incidence of mass nodules of the uterus, particularly those that were diagnosed as stromal polyps (see Histopathology section below). The tails of males given 0.1, 75, and 300 mg/kg bw/day and females given 300 mg/kg bw/day had an increased incidence of lesions diagnosed as papules (focal and multifocal). The incidences in males given 0, 0.1, 1.0, 75, and 300 mg/kg/day were 5,14, 7, 21, and 25, respectively and in females were 1, 1, 1, 0, and 8, respectively. These lesions generally represented focal hyperkeratosis with or without inflammation (see Histopathology section below). Additionally, males given 300 mg/kg bw/day (9/50) also had necrosis of the tail tip. Females given 300 mg/kg bw/day appeared to have a higher incidence of uterine mass/nodules – polypoid, 0.2 – 0.5 cm than the controls. These mass/nodules were subsequently interpreted to be treatment related following histopathology (see Histopathology section).
- Effects Not Related to Treatment
The right optic nerve of females given 300 mg/kg bw/day showed a slightly higher incidence (6 vs. 1 control) of decreased size as compared to the controls. This lesion in the females was not considered treatment related because of its unilateral distribution (see Histopathology section below).
Numerous other gross observations, including several mass-nodules were noted at 24 months; however, none of these gross observations were considered indicative of treatment-related gross effects and were generally consistent with expected geriatric conditions of Fischer 344 rats of this age.

Relevance of carcinogenic effects / potential:

Increased incidences of neoplasms were observed in males treated with the test material at 300 mg/kg bw/day and females treated at 75 and 300 mg/kg bw/day. However the excessive high doses used in the study and the subsequent saturation of absorption are considered to confound the interpretation of tumorous effects at these dose levels. Based on the findings in this study the oncogenic effect of the test material is unclear.

Key result

Dose descriptor:

NOAEL

Effect level:

1 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Decrease in body weight, liver and kidney effects, and lung effects.

Remarks on result:

other: Effect type: toxicity (migrated information)

Dose descriptor:

NOAEL

Effect level:

75 mg/kg bw/day

Based on:

test mat.

Sex:

male

Basis for effect level:

other: Increase in liver adenomas

Remarks on result:

other: Effect type: carcinogenicity (migrated information)

Dose descriptor:

NOAEL

Effect level:

1 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

other: Increase in liver adenomas, and uterine stromal polyps

Remarks on result:

other: Effect type: carcinogenicity (migrated information)

Analytical Chemistry

The homogeneity of the test material in rodent feed was determined on seven separate mixings (mixed prior to study start and at approximately 1.5, 3, 8, 12, 18, and 24 months) for the 0.1 mg/kg bw/day female and 300 mg/kg bw/day male diets, the lowest and highest concentrations used in the study. Homogeneity analyses were also conducted on the female 0.1 mg/kg bw/day diets (mixed on 1/14/03 and 5/31/03) and on the female 0.1 mg/kg bw/day and male 300 mg/kg bw/day diets (mixed on 12/01/03), to address homogeneity values from the previous analysis that were outside the laboratory’s acceptable range of ± 15 %. Follow-up analyses on diet mixes using the same procedures resulted in relative standard deviations (RSD) of < 10 %, indicating the diets were homogeneous, with the exception of the female 0.1 mg/kg bw/day diet that had a reported RSD of 18.2 % (analysed on 10/6/03). No specific reason for the variability in homogeneity of the 0.1 mg/kg bw/day female diets was determined. The overall RSDs for the female 0.1 mg/kg bw/day and male 300 mg/kg bw/day diets were 15.1 % and 4.08 %, respectively; therefore, the diets containing the test material were considered to be homogeneous.

The concentrations of the test material were determined for the control, premix, and diets from all treatment levels on seven separate mixings (mixed prior to study start and at approximately 1.5, 3, 8, 12, 18, and 24 months) and were acceptable. Analyses were also conducted (1/14/03, 5/31/03, and 12/01/03) to address homogeneity issues (previously described above) and were conducted (5/31/03 and 12/01/03) to address high-dose female and low-dose female concentration values outside the laboratory’s acceptable range of ± 15 %. Follow-up analyses on diets prepared from similar procedures resulted in acceptable target concentrations for these diets. Analytical results varied from 86.8 % to 123 % of the target concentration of the test material for individual samples, but only three analysed concentrations had a difference greater than 15 % from target. Mean concentrations for each dose level for all analytical time points ranged from 96.3 % to 105 % of target.

Stability of the test material was determined in the 0.1 mg/kg bw/day diet (0.0001 %, lowest concentration). The test material was stable in the 0.1 mg/kg bw/day diet for at least 27 days, at which time it was 111 % of the initial concentration.

Histopathology (non-neoplastic and neoplastic)

> 12 Months:

- Treatment Related Effects

Treatment related histological findings were confined to the lungs and liver. The livers of males given 300 or 75 mg/kg bw/day had slight centrilobular to midzonal hepatocyte hypertrophy and this change corresponded with the statistically identified increases in the liver weights.

In the lungs of males and females given 300 or 75 mg/kg bw/day, there were small areas of very slight or slight chronic inflammation either focal or multifocal, distributed in one or more lobes, which corresponded to the pale foci, observed grossly. Generally, these foci consisted of aggregates of large foamy alveolar macrophages variably admixed with small numbers of neutrophils and occasionally some lymphocytes.

Portions of the alveolar walls within these foci were variably thickened with increased amounts of collagen, infiltrating inflammatory cells with or without hyperplastic type-2 pneumocytes and macrophages. Additionally, in some animals, cholesterol clefts surrounded by macrophages were also seen in these foci of chronic inflammation. The grades very slight and slight were used to indicate the severity and/or extent of distribution (approximately 1 % or less, and 1-3 % of pulmonary parenchyma for grades ‘very slight’ and ‘slight’, respectively) but were clearly not adverse or detrimental to the overall health of the animals.

Two males given 300 mg/kg bw/day had benign hepatocellular adenomas in the liver. This was interpreted to be likely related to treatment due to 1) the absence of its occurrence in 12- months historical control data from three recent 2-year studies done in this laboratory and 2) elevated incidence of hepatic adenomas at 24 months (see Histopathology 24 Months section).

- Effects Not Related To Treatment

Eosinophilic foci (1-5 range) were seen in the livers of one male given 75 and in one male given 300 mg/kg bw/day. This low incidence and lack of dose response, and its absence in the females indicated that it was not treatment related. Similarly, there was a higher incidence of basophilic foci (6-10 range) in males given 75 mg/kg bw/day (4 of 10), but only one male given 300 mg/kg bw/day showed the same change. Again, this lack of dose response indicated that the higher incidence of basophilic foci (6-10 range) in males given 75 mg/kg bw/day was not related to treatment.

Very slight or moderate focal alveolar epithelial hyperplasia was seen in the lungs of one male given 75 mg/kg bw/day, one male given 300 mg/kg bw/day, and in one female given 300 mg/kg bw/day. Although this change was not seen in the controls, the low incidence in the 75 and 300 mg/kg bw/day dose groups was indicative that this alteration was not treatment related.

All other histopathologic observations, with the exception of the optic nerve lesions observed in rats from the 12- month interim necropsy were interpreted to be spontaneous changes typically seen in Fischer 344 rats of this age and husbandry conditions. The degeneration of the optic nerve was generally unilateral, and of varying severity including some in which little or no nerve tissue was even found among the fragments of connective tissue and retrobulbar vasculature (termed decreased size, severe). There was no relationship between the optic nerve lesions and exposure to the test material. The degeneration of the optic nerve was secondary to the repeated retro-orbital blood collection. In some of the rats, these iatrogenic optic nerve lesions were also accompanied by unilateral atrophy of the optic tract in the brain and unilateral retinal atrophy in the eye.

Neoplastic changes, both malignant and benign were observed at the 12- month interim necropsy, but were considered unrelated to exposure to the test material due to low incidence and/or lack of dose response. There were two instances of malignancy in the males of the 300 mg/kg bw/day dose group. They were 1) a poorly differentiated carcinoma in the mediastinal tissue, with metastasis to the lung and invasion into the adjacent skeletal musculature surrounding the thoracic vertebrae in one male and, 2) a thyroid follicular cell adenocarcinoma (without metastasis) in another male. Another instance of malignancy, which was an invasive rhabdomyosarcoma (without metastasis) of the diaphragm extending into the mesenteric and mediastinal tissue was observed in one control female (animal # 02A2147) that was humanely euthanized on day 232 due to its moribund status. Benign tumors diagnosed and interpreted to be unrelated to treatment, included a bronchiolo-alveolar adenoma in the lung of one female given 300 mg/kg bw/day, a cortical adenoma in a control female, ganglioneuroma of the adrenal gland in a female given 75 mg/kg bw/day, adenomas of the pituitary gland in 2, 1, and 2 males given 0, 1, or 75 mg/kg bw/day, respectively, and in two females given 0.1 mg/kg bw/day, and endometrial stromal polyps in the uteri of 3, 1, and 1 females given 0.1, 1, or 300 mg/kg/day, respectively.

> 24 Months:

- Treatment Related Effects (Neoplasms)

A higher incidence of hepatocellular adenomas was observed in the livers of males given 300 mg/kg bw/day (16 animals) relative to the controls (6 animals) and was statistically identified. The increase in the incidence was largely driven by higher incidence of males with one or two hepatocellular adenomas/animal. In addition, one male given 300 mg/kg bw/day had three hepatocellular adenomas. The liver tumor data indicated that the higher number of hepatocellular adenomas was not associated with an increase in the incidences of hepatocellular carcinomas, as the incidence of hepatocellular carcinomas between males given 0 or 300 mg/kg bw/day were comparable.

Historical control data from three recently completed 2-year studies in this laboratory show the range of the total number of males with hepatocellular adenomas was 1 or 2 out of 50 or 55 animals.

Since the incidence of hepatocellular adenomas in the 300 mg/kg bw/day dose group was higher than the concurrent control and historical control values, this change was interpreted to be treatment related. It should be noted that the incidence of hepatocellular tumors in the control group of this study was three times higher than that observed in the historical controls. The reason for this difference in background liver tumor incidence in control males and the significance of this apparent drift in the background liver tumor incidence was not determined.

Historical control liver tumor incidence from the National Toxicology Program (NTP) and Charles River Laboratories, indicates that the Toxicology & Environmental Research & Consulting historical control liver tumor data are similar to the mean data observed at other institutions.

Although hepatocellular carcinomas were seen in some animals, the incidence was low across all dose groups, namely 1, 1, 1, 0, and 2 males given 0, 0.1, 1.0, 75, or 300 mg/kg bw/day, respectively, and hence were interpreted to be unrelated to treatment.

Females given 300 or 75 mg/kg bw/day had a higher incidence of uterine polyps than the control females that were statistically identified. The increase in incidence was primarily due to a higher incidence of females having one and two uterine polyps/animal. The incidences of animals having three uterine polyps were somewhat similar with the controls having 1/50 and the 75 and 300 mg/kg bw/day groups having an incidence of 1/50 and 3/50, respectively.

In the historical controls from three recently reported two-year carcinogenicity studies, the range of the total number of females with endometrial stromal polyps was between 11 and 19, with some variability in the incidence of multiple polyps.

Historical control endometrial stromal polyp incidence data from the National Toxicology Program and Charles River Laboratories indicates that the Toxicology & Environmental Research and Consulting historical control data for endometrial stromal polyps are similar to the mean data observed at other institutions.

The incidences of the uterine polyps in females given 300 or 75 mg/kg bw/day were outside the concurrent and historical control data, suggesting that these differences were treatment related. Uterine stromal polyps were not observed with increased frequency in females given the test material at any dose level for 12 months, indicating that these uterine tumors did not have an early onset. At no time during the toxicologic evaluation of the test material was the uterus identified as a target organ in Fischer 344 rats (28 days – Lick et al., 1997; 90 days – Yano and Dryzga, 2002; and 12 months – see RESULTS previously discussed), Sprague-Dawley (two-generation reproduction study – Carney et al., 2004), CD-1 mice (28 days – Yano and Day, 2001, 90 days, Yano and Day, 2002 or 18 months - Johnson, 2005) or Beagle dogs (28 days – Stebbins et al., 2002, 90 days – Stebbins et al., 2003, one year – Stebbins et al., 2004).

Males and females given 75 or 300 mg/kg bw/day had lower incidences of leukemia than were observed in rats given 0, 0.1 or 1.0 mg/kg bw/day. The majority of these differences were statistically identified and were lower than historical control incidences. The mechanism for these differences was not determined.

- Treatment-Related Effects (Non-Neoplastic)

Treatment related histologic changes were observed in livers, lungs, and tail in both males and females, and in kidneys, testes and epididymides of males given the test material.

Treatment related hypertrophy of the hepatocytes was observed in males and females given 300 or 75 mg/kg bw/day and statistically identified. In the males, the hypertrophy was panlobular and in females it was mostly centrilobular/midzonal (with altered tinctorial properties – increased eosinophilia) in distribution. A few females given 300 or 75 mg/kg bw/day also had hepatocellular hypertrophy involving the entire hepatic lobule (panlobular); however, the difference in incidence between the controls did not clearly indicate that these differences were treatment related.

In the liver of males given 75 or 300 mg/kg bw/day, there was a treatment related increase in the incidence of basophilic foci (21 or more) relative to respective controls that was treatment related. The incidence of eosinophilic foci (6-10) was elevated in males given 75 mg/kg bw/day relative to that of controls, but due to the lack of dose response in the 300 mg/kg bw/day dose group, this was considered not treatment-related. In females, on the other hand, there was a decrease in the incidence of basophilic foci (21 or more) in the 75 and 300 mg/kg bw/day dose groups relative to the controls that were statistically identified. The relevance of these increases and decreases in foci was unclear.

As was noted at 12 months, the lungs of the majority of males and females given 75 or 300 mg/kg bw/day had small areas of very slight to slight chronic inflammation of alveoli, either focal or multifocal, distributed in one or more lobes, and generally corresponded to the pale foci observed grossly. These differences were statistically identified (multifocal slight, multifocal very slight and slight and all severities and distributions combined). In general, these foci consisted of aggregates of large foamy alveolar macrophages variably admixed with small numbers of neutrophils, occasionally some lymphocytes and multinucleated giant cells. Portions of the alveolar walls within these foci were variably thickened with increased amounts of collagen, infiltrating inflammatory cells with or without hyperplastic type-2 pneumocytes and macrophages. As a part of this chronic inflammation, small numbers of ciliated cells lined portions of alveolar walls in occasional animals. Additionally, cholesterol clefts surrounded by variable numbers of macrophages with or without occasional multinucleated giant cells were also seen, in some animals, within these foci of chronic inflammation. The grades very slight and slight were used to indicate the severity and/or extent of distribution (approximately 1 % or less, and 1-3 % of pulmonary parenchyma for grades ‘very slight’ and ‘slight’, respectively) but were clearly not adverse or detrimental to the overall health of the animals.

In the kidneys of males given 300 or 75 mg/kg bw/day, there were statistically identified higher incidences of unilateral hyperplasia of the pelvic epithelium and mineralisation of the pelvic epithelium. In general, these changes were mild, frequently focal and were interpreted to be treatment related.

Historical controls from three recent studies conducted in this laboratory indicated wide variations in the incidence of hyperplasia of the pelvic epithelium (1-11 out of 50-55) and mineralisation of the pelvic epithelium (1-19 out of 50-55). The combined incidence of very slight and slight hyperplasia of the pelvic epithelium in males given 300 mg/kg bw/day was elevated relative to the concurrent and historical controls and statistically identified, and this effect was considered treatment-related. However, although males given 75 mg/kg bw/day had statistically identified higher incidence of ‘very slight’ hyperplasia of the pelvic epithelium, it was within historical control range and the combined incidence across all grades was not statistically identified and hence, was considered not treatment-related.

The combined incidence of all grades of mineralisation of the pelvic epithelium of the kidneys were clearly elevated compared to controls and statistically identified in males given 300 or 75 mg/kg bw/day. In addition, its incidence was outside the historical controls and hence, these differences were considered treatment related.

In the testis, there was a statistically identified, increased incidence in severe bilateral atrophy of the seminiferous tubules in males given 300 mg/kg bw/day. This condition, in the majority of the affected animals was a secondary consequence to the presence of larger/heavier interstitial cell tumors (Leydig cell tumor) of the testis that corresponded to higher weights of testes in males given 300 or 75 mg/kg bw/day (absolute and/or relative testicular weights). Interstitial cell tumors are very common in Fischer 344 rats and nearly 100 % of Fischer 344 rats develop multiple hyperplasias or adenomas at the end of two-year studies (Boorman et al., 1990). Indeed, in the current study, the incidence of interstitial cell tumors (unilateral and bilateral combined), including controls, was 90 – 96 %.

Increased testicular weights (attributed to the interstitial cell tumors) and its secondary effect of increased incidence of severe bilateral atrophy of the seminiferous tubules were present in males given 300 mg/kg bw/day, and hence associated with treatment.

These effects likely reflect an exacerbation of a pre-existing high incidence of interstitial cell tumors and severe seminiferous tubule atrophy. Since effects on interstitial cells were not observed after 12 months of exposure to the test material, it appears that these alterations occur late in life. In addition, studies of variable duration evaluating the toxicity of the test material in Fischer 344 and Sprague-Dawley rats, CD-1 mice and Beagle dogs have not demonstrated any treatment-related effects involving the testes.

The consequence of the increased incidence of severe bilateral atrophy of the seminiferous tubules was evident in the epididymides in the form of an increased incidence of bilateral aspermia, findings which both occur commonly in aging Fischer 344 rats as evident in the concurrent control group. Statistically identified higher incidence of bilateral aspermia in the epididymides was seen in 37/49 and 38/49 males given 300 or 75 mg/kg bw/day. Again, this was interpreted generally to be a consequence of larger interstitial tumors (inferred from increased testicular weights), leading to an increased incidence of severe atrophy of seminiferous tubules with resultant lack of sperm in the epididymides of males given 300 or 75 mg/kg bw/day.

As mentioned earlier under the section ‘Gross Pathology – 24 Months’, there was an increased incidence of focal or multifocal lesions on the tails grossly identified as papules in males given 0.1, 75, and 300 mg/kg bw/day and females given 300 mg/kg bw/day (see Gross Pathology – 24 Month section). Histologically, the grossly recognised tail lesions were identified as focal or multifocal areas of hyperkeratosis with or without inflammation. In general, in the affected areas of the tail, there were variable degrees of focal/multifocal superficial dilated cystic structures lined by keratinised stratified squamous epithelium, and interpreted to be superficial hair follicles due to the presence of hair shafts and opening to the exterior. These hair follicles were generally distended with abundant keratin, some free hair shafts, variable amounts of inflammatory cells and debris, and frequently, these dilated follicles communicated with the surface of the skin. A chronic inflammatory response, when seen, was variably accompanied by focal epidermal hyperplasia, and focal dermal fibrosis. In some instances, focal epidermal ulceration and necrosis were also observed as part of the inflammatory process. The combined incidences of focal and multifocal hyperkeratosis in the tail of males and females given 300 mg/kg bw/day were interpreted to be treatment related, although the pathogenesis remains unclear.

Necrosis of the tail (tip) was another gross observation that was increased in incidence at necropsy mainly in males given 300 mg/kg bw/day. In general, in these affected tails, there was focally extensive chronic active inflammation, with or without ulceration. Usually the inflammation extended deep into the subcutis around a coccygeal vertebra, with reactive periosteal bone formation. In some cases there was inflammation of some arterioles causing thrombosis. In the affected area (tail tip) there was also full thickness necrosis of the tail tip involving the bone, bone marrow, connective tissue, and skin, consistent with infarction of the tail tip. Focally extensive inflammation in the tail of males given 300 mg/kg bw/day was statistically identified and considered related to treatment. In two males given 300 mg/kg bw/day, the histological diagnosis was ‘necrosis’ due to full thickness necrosis of the tail tip without any inflammation that was also considered a part of the spectrum of tail effects. The combined incidence of focally extensive inflammation and necrosis of tails in males given 300 mg/kg bw/day was increased and interpreted to be treatment related.

The thrombosis leading to infarction and necrosis of the tail tip was localized in nature, as there was no evidence of systemic thrombosis in other organs. The local thrombosis was consistent with a primary injury to the tail tip, leading to infection, moderate/severe focally extensive inflammation and necrosis.

As the tail was not a protocol required organ for routine histological analysis, only those tails that were grossly identified as having focal or multifocal papules or with other relevant gross observations at the time of 24- month necropsy were examined for histological correlation.

- Effects Not Related To Treatment (Non-Neoplastic)

In the kidneys, male rats given 1, 75, or 300 mg/kg bw/day had increased incidence of very slight and slight chronic progressive glomerulonephropathy (CPGN) that were statistically identified. CPGN is a common spontaneous geriatric kidney lesion in aging rats with males being much more affected than females (Montgomery and Seely, 1990; Short and Goldstein, 1992). Rat strains vary in the degree to which they spontaneously develop CPGN with Fischer 344 rats being one of the more affected strains.

The majority of the controls and those given 0.1 mg/kg bw/day had very slight CPGN (25 % or less involvement of the kidneys). 25/50, 25/50 and 28/50 of the male rats given 1, 75, or 300 mg/kg bw/day showed slight CPGN (26-50 % involvement of the kidneys), while the number diagnosed as moderate CPGN (51 -75 % involvement of the kidneys) was low and comparable across all dose levels including controls. Severe CPGN (> 75 % involvement) was seen in one male each in 75 and 300 mg/kg bw/day dose groups. The majority of females, on the other hand, showed only very slight CPGN across all dose groups including controls.

The apparent increase in the incidence of slight CPGN in males given 1, 75, or 300 mg/kg bw/day, although statistically identified, was considered not related to treatment for the following reasons:

1) Lack of convincing dose response. The fact that the incidence of slight CPGN remained virtually unchanged, with no progression to increased severity such as ‘moderate or severe’ with a 75 fold and 300 fold increase in dose levels.

2) The number of control males with ‘very slight’ CPGN in the present study (34) was higher as compared to many of the historical controls (0-33) and therefore, resulted in a proportional decrease in higher grades of severity as seen in the historical controls. Earlier studies done in this laboratory (except for one) within the past 10 years have shown variability in the incidence and severity of CPGN in control Fischer 344 rats at 24 months. Johnson et al., 2002, in his report on a 2-year carcinogenicity study, summarised historical controls on incidence and severity of CPGN in Fischer 344 rats observed in this laboratory (except for one).

Moreover, historical control data from three two-year studies conducted very recently in this laboratory under identical conditions indicated the wide variations in the incidence of severity. The incidence of slight CPGN in males given 1, 75, or 300 mg/kg bw/day in the current study (25–28) was in close proximity to the historical controls (11-25).

Females given 300 mg/kg bw/day had a higher incidence of severe unilateral atrophy of the optic nerve, unilateral cataract and severe unilateral atrophy of the retina, relative to control females and were statistically identified. Low incidences of severe unilateral atrophy of the optic nerve, unilateral cataracts, and severe unilateral retinal atrophy occurred in a non-dose related manner in females given 0.1, 1.0, and 75 mg/kg bw/day. The incidences of bilateral cataracts or bilateral severe retinal atrophy were low and did not indicate any treatment-related pattern. These data, in addition to the implausibility of a unilateral treatment-related effect on the optic nerve, lens, and retina strongly suggest that these differences were not treatment related and were likely the result of spontaneous disease processes commonly observed in Fischer 344 rats (Boorman et al.,1990).

Females given 75 or 300 mg/kg bw/day had higher incidences of very slight, multifocal atrophy of the pancreatic acini than the control females. The difference in incidence for females given 300 mg/kg bw/day were statistically identified. There was no increase in the incidence of acinar atrophy of slight, moderate, or severe degree indicating that the differences in the incidences of very slight, multifocal acinar atrophy were not treatment related and were likely the result of variability of spontaneous disease processes.

There were a number of other histopathologic lesions that were increased or decreased compared to the controls, and were statistically identified but were interpreted not to be treatment related for the following reason(s):

> Decreased Incidence of a Unilateral Lesion

Eye inflammation chronic active cornea unilateral focal – males given 300 mg/kg bw/day).

> Lack of a Dose-Response Relationship

Liver aggregates of macrophages/histiocytes, multifocal, very slight – males given 75 mg/kg bw/day

Eye mineralisation, cornea, unilateral multifocal, very slight – females given 75 mg/kg bw/day

Liver hypertrophy hepatocyte centrilobular/midzonal slight – females given 1.0 mg/kg bw/day

Liver necrosis hepatocyte multifocal – females given 0.1, 75, or 300 mg/kg bw/day

Liver vacuolization consistent with fatty change individual cells – females given ≥ 0.1 mg/kg bw/day

Spleen extramedullary hematopoiesis – females given 300 mg/kg bw/day Uterus cyst endometrium multifocal – females given 75 mg/kg bw/day.

Conclusions:

Under the conditions of the test, the no-observed-effect level (NOEL) for both male and female Fischer 344 rats at 24 months was determined to be 1.0 mg/kg bw/day.

Executive summary:

The carcinogenicity of the test material was assessed in a 2 year dietary toxicity study using Fischer 344 rats. The study was performed under GLP conditions and in accordance with the standardised guidelines EPA OPPTS 87.4300, EPA OPPTS 870.3100, OECD 453, OECD 408, EU Method Part B, EU Method B.26, and JMAFF guidelines.

Groups of 65 males and 65 female Fischer 344 rats were fed diets formulated to provide 0, 0.1, 1.0, 75, or 300 mg/kg bw/day for up to two years. An additional 10 rats/sex were given 0 or 1.0 mg/kg bw/day for 90 days to evaluate subchronic toxicity. Ten rats/sex/dose were necropsied after one year (chronic toxicity group), five rats/sex/dose were necropsied after one year (chronic neurotoxicity group) and the remaining 50 rats/sex/dose were fed the respective diets for up to two years and necropsied (oncogenicity group).

Daily cage-side examinations, weekly detailed clinical observations, and periodic body weight and feed consumption were monitored throughout the study on all rats.

Haematology, clinical chemistry and urinalysis parameters were evaluated at regular intervals throughout the study. Ophthalmic examinations were conducted on all rats pre-exposure and all surviving rats prior to the scheduled necropsies. All rats had a complete necropsy and organ weights were obtained from all rats at the scheduled necropsies. Histopathologic examinations of an extensive set of tissues were performed on all control and high-dose level rats and all rats that died spontaneously or were euthanised due to their moribund condition. Histopathologic examination of the surviving animals from the low- and intermediate-dose levels was limited to target organs, suspected target organs, and gross lesions.

Chronic Toxicity/Oncogenicity: Males given 300 mg/kg bw/day and females given 75 or 300 mg/kg bw/day had increased incidences of liver adenomas and uterine stromal polyps, respectively, that were attributed to treatment with the test material. Lower incidences of leukemia occurred in males and females given 75 or 300 mg/kg bw/day, compared to the controls. Males given 75 or 300 mg/kg bw/day had apparently larger testicular interstitial cell (Leydig cell) tumors than the controls, based on higher testicular weights; however, the incidences of interstitial cell tumors were similar at all dose levels, including the controls. Increased incidences of neoplasms were not observed in males given ≤ 75 mg/kg bw/day or females given ≤ 1.0 mg/kg bw/day.

Males and females given 75 or 300 mg/kg bw/day had treatment-related decreases in body weight and body weight gains, and increased liver and kidney (males only) weights. Other treatment related effects in males and females given 75 or 300 mg/kg bw/day consisted of increases in the incidence of 1) lung inflammation, and 2) hyperkeratosis of the tail (300 mg/kg bw/day only). Males given 75 or 300 mg/kg bw/day had increased incidence of mineralisation of renal pelvic epithelium and increased incidence of basophilic liver foci. Males given 300 mg/kg bw/day also had increased incidence of hyperplasia of renal pelvic epithelium, and necrosis of the tail tip and secondary increases in the incidences of testicular seminiferous tubule atrophy and epididymal aspermia. Females given 75 or 300 mg/kg bw/day had a decreased incidence of basophilic liver foci. There were also minor treatment related alterations in clinical pathology parameters in males and/or females given 75 or 300 mg/kg bw/day in prothrombin time, cholesterol, alkaline phosphatase activity, urine volume, and urine specific gravity. Treatment related non-neoplastic effects were not observed in males or females given ≤ 1.0 mg/kg bw/day.

Collectively, the findings from this study should be considered in light of the possibility that excessive high doses (e.g., as evidenced by significant decrements in body weight gain) and subsequent saturation of absorption (e.g., as shown by earlier pharmacokinetic studies as well as by increased relative liver weights that were not proportional to administered dose) may confound the interpretation of tumorous effects at these dose levels.

The no-observed-effect level (NOEL) for both males and females at 24 months was 1.0 mg/kg bw/day.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

1 mg/kg bw/day

Study duration:

chronic

Species:

rat

Quality of whole database:

Two studies are available; both are conducted in accordance with standardised guidelines under GLP conditions. The quality of the database is therefore considered to be high.

Carcinogenicity: via inhalation route

Endpoint conclusion

Endpoint conclusion:

no study available

Carcinogenicity: via dermal route

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

In accordance with the criteria for classification as defined in Annex I (Classification and Labelling Requirements for Hazardous Substances and Mixtures), Regulation (EC) No. 1272/2008 (CLP), the substance does not require classification with respect to carcinogenicity because in both rats and mice no carcinogenic response was obtained at appropriate dose levels not exceeding the MTD, and with linear toxicokinetic.

Additional information

Two studies are available; both are conducted in accordance with standardised guidelines under GLP conditions and both were awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

In the first study, the carcinogenicity of the test material was assessed in a 2 year dietary toxicity study using Fischer 344 rats. The study was performed under GLP conditions and in accordance with the standardised guidelines EPA OPPTS 87.4300, EPA OPPTS 870.3100, OECD 453, OECD 408, EU Method Part B, EU Method B.26, and JMAFF guidelines.

Groups of 65 males and 65 female Fischer 344 rats were fed diets formulated to provide 0, 0.1, 1.0, 75, or 300 mg/kg bw/day for up to two years. An additional 10 rats/sex were given 0 or 1.0 mg/kg bw/day for 90 days to evaluate subchronic toxicity. Ten rats/sex/dose were necropsied after one year (chronic toxicity group), five rats/sex/dose were necropsied after one year (chronic neurotoxicity group) and the remaining 50 rats/sex/dose were fed the respective diets for up to two years and necropsied (oncogenicity group).

Daily cage-side examinations, weekly detailed clinical observations, and periodic body weight and feed consumption were monitored throughout the study on all rats.

Haematology, clinical chemistry and urinalysis parameters were evaluated at regular intervals throughout the study. Ophthalmic examinations were conducted on all rats pre-exposure and all surviving rats prior to the scheduled necropsies. All rats had a complete necropsy and organ weights were obtained from all rats at the scheduled necropsies. Histopathologic examinations of an extensive set of tissues were performed on all control and high-dose level rats and all rats that died spontaneously or were euthanised due to their moribund condition. Histopathologic examination of the surviving animals from the low- and intermediate-dose levels was limited to target organs, suspected target organs, and gross lesions.

Chronic Toxicity/Oncogenicity:

Males given 300 mg/kg bw/day and females given 75 or 300 mg/kg bw/day had increased incidences of liver adenomas and uterine stromal polyps, respectively, that were attributed to treatment with the test material. Lower incidences of leukemia occurred in males and females given 75 or 300 mg/kg bw/day, compared to the controls. Males given 75 or 300 mg/kg bw/day had apparently larger testicular interstitial cell (Leydig cell) tumors than the controls, which was related to higher testicular weights. However, interstitial cell tumors are very common in Fischer 344 rats and nearly 100% of Fischer 344 rats develop multiple hyperplasias or adenomas at the end of two-year studies (Boorman et al., 1990). The incidences of interstitial cell tumors (unilateral and bilateral combined) were similar at all dose levels, including the controls, below 100% (90 – 96%) and without any dose-relationship. No increase in the incidence of any neoplasms was observed in males given ≤ 75 mg/kg bw/day or females given ≤ 1.0 mg/kg bw/day.

Collectively, the findings from this study should be considered in light of the possibility that excessive high doses (as evidenced by significant decrements in body weight gain) and subsequent saturation of absorption (as shown by earlier pharmacokinetic studies as well as by increased relative liver weights that were not proportional to administered dose) may confound the interpretation of carcinogenic effects at these dose levels.

Males and females given 75 or 300 mg/kg bw/day had treatment-related decreases in body weight and body weight gains. Body weight gains of males given 75 or 300 mg/kg/day were 4.6% and 5.1% less than controls, respectively on day 363. These decreases remained until study termination, at which time they were 9.5% and 13.9% lower than controls for the 75 and 300 mg/kg/day groups, respectively. Body weight gains for females given 75 or 300 mg/kg/day were 6.5% and 9.8% lower than controls on day 363 and 21.8% and 22.4% lower than controls at study termination. The lower body weight gains of males given 300 mg/kg/day and females given 75 or 300 mg/kg/day clearly exceed the 10% body weight gain decrement criterion for themaximal tolerated dose (MTD dose),which should not be exceededto enable hazard identification and hazard characterisation. Males given 75 mg/kg/day almost meet the 10% criterion. In addition, in a previously conducted toxicokinetic study (Saghir 2002) with single oral doses of 10, 100 or 250 mg/kg, only a fraction (0.7-5.0%) of the administered dose was recovered in the urine. The total amount of noviflumuron-related radioactivity (mg-equivalents) excreted in urine through 72 hr after dosing was identical at 100 and 250 mg/kg (0.33 vs. 0.34 mg-eq. for males and 0.43 vs. 0.42 mg-eq. for females at 100 and 250 mg/kg, respectively). The study also demonstrated no dose proportionalities in the area under the concentration-time course profiles (AUC) or the maximum plasma concentrations (Cmax) among doses. The approximate 10-fold increase in dose from 10 to 100 mg/kg produced AUCs that were only 6- to 7-fold larger and the approximate 25-fold increase in dose from 10 to 250 mg/kg produced AUCs that were only 7- to 9-fold larger. Similar patterns were observed when Cmax among the doses were compared. A 150% increase in dose (from 100 to 250 mg/kg) increased AUC only by 20% to 36% and Cmax by 25%. An identical amount of urinary excretion of noviflumuron-derived radioactivity at two doses that were approximately 2.5-fold apart and a drop in urinary clearance are indications of saturation of absorption and/or metabolism in animals. Similarly, non-proportionalities in AUC and Cmax and higher faecal elimination suggest saturation of absorption. The interpretation of any carcinogenic effects at 75 or 300 mg/kg bw/day was therefore confounded by excessive toxicity and subsequent non-linearity of toxicokinetic.

Males and females given 75 or 300 mg/kg bw/day had treatment-related increased liver and kidney (males only) weights. Although dose-related, the increased relative liver weights were not proportional to administered dose. Other treatment related effects in males and females given 75 or 300 mg/kg bw/day consisted of increases in the incidence of 1) lung inflammation, and 2) hyperkeratosis of the tail (300 mg/kg bw/day only). Males given 75 or 300 mg/kg bw/day had increased incidence of mineralisation of renal pelvic epithelium and increased incidence of basophilic liver foci. Males given 300 mg/kg bw/day also had increased incidence of hyperplasia of renal pelvic epithelium, and necrosis of the tail tip and secondary increases in the incidences of testicular seminiferous tubule atrophy and epididymal aspermia. Females given 75 or 300 mg/kg bw/day had a decreased incidence of basophilic liver foci. There were also minor treatment related alterations in clinical pathology parameters in males and/or females given 75 or 300 mg/kg bw/day in prothrombin time, cholesterol, alkaline phosphatase activity, urine volume, and urine specific gravity. Treatment related non-neoplastic effects were not observed in males or females given ≤ 1.0 mg/kg bw/day.

 

The no-observed-effect level (NOEL) for both males and females at 24 months was 1.0 mg/kg bw/day.

In the second key study, a study was conducted to assess the potential oncogenicity of the test material in accordance with the standardised guidelines OECD 451, EU Method B.32 US EPA OPPTS 870.4200 and MAFF, Ministry of Agriculture, Forestry and Fisheries, 2000 under GLP conditions.

Groups of CD-1 mice (50/sex) were given diets formulated to provide 0 (controls), 0.5, 3 (males only), 30, or 100 (females only) mg/kg body weight/day for up to 18 months. Animals were evaluated over the course of the study by daily cage side and periodic hand-held clinical examinations, body weight, and feed consumption. Ophthalmic examinations were conducted pre-exposure and prior to necropsy. All mice had a complete necropsy examination with white blood cell (WBC) and differential WBC counts and weights of selected organs at the scheduled necropsy. An extensive set of organs was examined histopathologically from all controls and high-dose group mice as well as all mice removed from study prior to the scheduled termination. Histopathologic examination was also conducted for target organs, selected major organs, and all gross lesions of mice from the low-and intermediate-dose groups from the terminal necropsy.

There was statistically significant increased mortality for females given the high dose of 100 mg/kg/day (56% cumulative mortality vs. 32% in controls at 18 months), which also exceeded the testing facility HC values. There was an increased incidence of tonoclonic convulsions in this dose group as compared to controls (though this strain of mouse is prone to convulsions), and convulsions were noted at some time points for many of the high-dose females that died spontaneously. Convulsions were considered the probable cause of increased early mortality, although convulsions were not noted immediately prior to death for most mice. Increased mortality not due to neoplasia is an indicator that this dose level exceeded a maximum tolerated dose (MTD).

There were no treatment-related effects noted during the in-life phase of the study for male mice from any dose level or females given 0.5 or 30 mg/kg/day. At necropsy, the absolute weights of the livers of high-dose group mice (both sexes) were increased and the liver weights relative to body weight were increased in both high- and mid-dose group mice (both sexes). Minor microscopic liver changes consisting of very slight or slight hepatocyte hypertrophy were noted for high-dose level mice (both sexes) and mid-dose level males, although only the male high-dose incidence was statistically significant.

Mice at the high-dose level had slightly greater numbers of hepatocyte adenomas and combined adenomas plus carcinomas, with the incidences just above the historical control range. This is considered equivocal evidence of carcinogenic potential as the male liver tumour incidence was not statistically significant and, in females, increased incidence of hepatocellular adenomas or total liver tumours was present only at a dose level that exceeded the MTD and was only statistically identified by Peto analysis, a sensitive technique adjusting for increased mortality.

Although relative liver weight was slightly increased at the middle dose levels and minimal hepatocellular hypertrophy was present in males, these were considered likely adaptive effects to metabolism of this test material and these levels (3 mg/kg/day for males and 30 mg/kg/day for females) were considered to be the no-observed-adverse-effect-level (NOAEL) for chronic toxicity. The incidence of hepatocellular tumours for mid-dose level mice (both sexes) was equal to or less than the concurrent controls and 30 mg/kg/day was therefore the NOAEL for carcinogenicity. The low dose level of 0.5 mg/kg/day had no effects attributed to treatment and was the no-observed-effect level (NOEL).

Justification for selection of carcinogenicity via oral route endpoint:

Liver adenomas and uterine stromal polyps in female rats only at dose levels exceeding the MTD and with non-linear toxicokinetics

Carcinogenicity: via oral route (target organ): digestive: liver