2-Pyridinecarboxylic acid, 4-amino-3,6-dichloro-

Carcinogenicity

Description of key information

ORAL
NOAEL (carcinogenicity) = 1000 mg/kg/day (males and females), NOEL (toxicity) = 50 mg/kg bw/day (males and females); NOAEL (toxicity)  500 mg/kg/day (females), NOAEL (toxicity) = 50 mg/kg/day (males), rat, OECD 453, EU Method B.33, EPA OPPTS 870.4300 and JMAFF, Johnson & Dryzga (2005)
NOEL = 1000 mg/kg bw/day (males); NOEL = 250 mg/kg bw/day (females), mouse, EPA OPPTS 870.4200, OECD 451, EEC Part B (Carcinogenicity Test), JMAFF (2000), Stebbins & Day (2003)

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:

21 July 2001 to 4 March 2004

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 453 (Combined Chronic Toxicity / Carcinogenicity Studies)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.33 (Combined Chronic Toxicity / Carcinogenicity Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.4300 (Combined Chronic Toxicity / Carcinogenicity)

Deviations:

yes

Remarks:

- Detailed clinical observations were evaluated monthly for the first 12 months and quarterly from 12-24 months on ten rats/sex/dose group from the oncogenicity group.

Qualifier:

according to guideline

Guideline:

other: JMAFF, 2000

Deviations:

no

GLP compliance:

yes

Specific details on test material used for the study:

Purity: 94.5%

Species:

rat

Strain:

Fischer 344

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: 7 weeks
- Weight on study day 1: 149.4 g for males and 113.4 g for females
- Housing: During acclimation animals were housed two-three per cage in stainless steel cages. During the main study, animals were housed two per cage in stainless steel cages. Cages had wire mesh floors and were suspended above catch pans. Cages contained a feed container and pressure activated nipple-type watering systems.
- Diet: ad libitum
- Water: ad libitum (municipal water)
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature: 20.4 - 24.7 °C
- Humidity: 40-69 % (relative)
- Air changes: 12-15 changes per hour
- Photoperiod: 12 hour light/dark photocycle

IN-LIFE DATES
- From: Test material administration began on the 14 August 2001 for the males and the 15 August 2001 for the females
- To: Necropsies took place on the 15, 19 and 20 August 2003 for the males and the 18, 20, 21, 22 and 15 August 2003 for the females

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on exposure:

DIET PREPARATION
- Rate of preparation of diet (frequency): Diet mixes were fed to the animals for a 2-week period before a new diet mix was prepared.
- Mixing appropriate amounts with (Type of food): 7 % premix was mixed with the diet to form the test diets.
- Storage temperature of food: Ambient temperature in sealed vessels.

Diets were prepared by serially diluting a concentrated test material- feed mixture (premix) 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 pre-exposure 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 four-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:

Analyses of the premixes and all the mixes, including the control diet, were performed prior to dosing and at approximately 4, 8, 12, 18 and 24 months of the study. The test material was extracted with solvent and analysed using liquid chromatography-mass spectrometry (LC-MS).
The homogeneity of the mixtures was determined in the low dose diet for the females and the high dose male test diet. The analyses were performed prior to dosing and then again at around 4, 8, 12, 18 and 24 months. Additional homogeneity determinations were performed when the pre-mix concentration was changed from 3 to 7 %.
Data on stability was available from a previous four-week study performed in mice which demonstrated that the test material was stable for at least 21 days in rodent chow. An additional stability test for the 7 % premix study was conducted up to 55 days.
One reference sample per sex per dose per mix were retained and stored at ambient temperature in sealed vials.

RESULTS
The concentrations of the test material were determined for the control and test diets from nine time points and were found to be acceptable. The mean concentrations for each dose level over the course of the study ranged from 96.9 to 105 % of targeted concentration.
No test material was found in the control diet. LC-MS analysis of each individual sample indicated 78.4-121 % of the target concentration, with the exception of one value of 135 %. A follow-up analysis using the same diet mixing instructions resulted in 90.7 % of the target concentration.
The homogeneity in rodent feed was determined for nine separate mixing batches for the 5 mg/kg/day female and 1000 mg/kg/day male test diets. In addition, homogeneity was conducted on the 50 and 1000 mg/kg/day female diet mixes and the 7 % premix and 1000 mg/kg/day female diet mix. The homogeneity of the diets was considered acceptable, with relative standard deviations for all diets sampled between 2.33 and 15.9 %, with the exception of one analysis where the relative standard deviation was 43.71 % for a sample from the 5 mg/kg/day female dose group. The relative standard deviation for the majority of samples was < 10 %.
Stability of the test material was determined in the 7 % premix and was determined to be stable in the premix for at least 55 days, at which it was 96.5 % of the concentration found initially.

Duration of treatment / exposure:

Up to 24 months

Frequency of treatment:

Daily; continuous in the diet

Dose / conc.:

0 mg/kg bw/day (nominal)

Dose / conc.:

5 mg/kg bw/day (nominal)

Dose / conc.:

50 mg/kg bw/day (nominal)

Dose / conc.:

500 mg/kg bw/day (nominal)

Dose / conc.:

1 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

50 animals/sex/dose

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: The high dose was selected based on preliminary results of a subchronic dietary toxicity study with a four week recovery period. The cecal weights were around 2.8 times the control values for males and females dosed with 1000 mg/kg/day for 13 weeks. Males had very slight epithelial hyperplasia of the cecum and ileum. The high dose selected was therefore expected to produce increased weight of the cecum and a decreased urine pH. The other doses were expected to provide a dose response relationship for any effect that might be observed at the highest dose tested for this study. The low dose was expected to produce a NOEL.

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Animals were observed for mortality and moribundity at least twice daily. Other observations were carried out at least once a day.
- Cage side observations: Cage side observations included but were 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: Pre-exposure then monthly for 12 months, then at 15, 18, 21 and 24 months on the first 10 surviving animals.

BODY WEIGHT: Yes
- Time schedule for examinations: Pre-exposure, weekly for the first 13 weeks and then monthly thereafter.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes

FOOD EFFICIENCY:
- Calculated as (g/feed consumed/day)/( g bodyweight gain): Yes (first 13 weeks of the study)

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: pre-exposure then at 12 and 24 months using indirect ophthalmoscopy
- Dose groups that were examined: All groups

HAEMATOLOGY: Yes
- Time schedule for collection of blood: 3, 6 and 12 months from the chronic toxicity group included in the combined study and at 18 and 24 months from the oncogenicity animals.
- Anaesthetic used for blood collection: Yes, CO₂
- Animals fasted: Yes
- How many animals: 10 rats/sex/dose
- Parameters checked: Haematocrit, haemoglobin concentration, red blood cell count, total white blood cell count, platelet count, differential white blood cell count, mean corpuscular volume, mean corpuscular haemoglobin, mean corpuscular haemoglobin concentration

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: 3, 6 and 12 months from the chronic toxicity group included in the combined study and at 18 and 24 months from the oncogenicity animals.
- Anaesthetic used for blood collection: Yes, CO₂
- Animals fasted: Yes
- How many animals: 10 rats/sex/dose
- Parameters checked: Prothrombin time, alkaline phosphatase activity, alanine aminotransferase activity, aspartate aminotransferase activity, albumin, cholesterol, creatinine, electrolytes (Ca, Na, K, phosphate and Cl), glucose, total bilirubin, total protein and urea nitrogen.

URINALYSIS: Yes
- Time schedule for collection of urine: 3, 6 and 12 months from the chronic toxicity group included in the combined study and at 18 and 24 months from the oncogenicity animals.
- Metabolism cages used for collection of urine: Yes (overnight, 16 hours). Urine was also collected by manual compression of the urinary bladder.
- Animals fasted: No
- Parameters checked: colour, appearance, specific gravity, urine volume, pH, bilirubin, glucose, proteins, ketones, blood and urobilinogen

NEUROBEHAVIOURAL EXAMINATION: No (addressed in a separate group)

Sacrifice and pathology:

NECROPSY
Fasted rodents submitted alive for necropsy were anaesthetised by the inhalation of CO₂. The tracheas were exposed and clamped and the animals were euthanised by decapitation.
A complete necropsy was conducted on all animals and 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.
The brain, cecum (full and empty), 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.

GROSS PATHOLOGY: Yes. Representative samples of tissues listed in Table 1 were collected and preserved in neutral, phosphate-buffered 10 % formalin.
HISTOPATHOLOGY: Yes. The number of sections from all preserved tissues listed in Table 1 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. 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, cecum, spleen and all relevant gross lesions. The target organ (cecum) was microscopically examined from the low- and intermediate-dose group animals to define a NOEL.

Statistics:

Bodyweights, feed consumption, organ weights, urine volume and specific gravity, clinical chemistry, coagulation and haematologic data were evaluated using Bartlett’s test for equality of variances (α=0.01). Parametric or non-parametric analysis of variance (ANOVA) (α=0.05) were performed based on these results. A Dunnett’s test or the Wilcoxon Rank-Sum test with a Bonferroni correction for multiple comparisons was performed (α=0.05) if significant.
Clinical observations were analysed by a z-test of proportion (α = 0.05). Descriptive statistics were reported for bodyweight gains, feed efficiency, RBC indices and differential WBC counts. Statistical outliers were identified by a sequential test (α = 0.02) but only excluded from feed consumption and feed efficiency statistics.
Statistical analyses were conducted on bodyweight, feed consumption, haematologic parameters, clinical chemistry parameters, urine volume, urine specific gravity and organ weight data throughout the study (geriatric changes were accounted for). Cumulative histopathologic incidences for all animals were used in the statistical analyses.
For tissues where all animals were examined, specific histopathological incidences were evaluated for deviation from linearity (α=0.01) using ordinal spacing of the doses. Linear data were tested with the Cochran-Armitage Trend test. If statistically significant (α=0.02) or significant deviation from linearity was observed, incidences were compared to the control using a pair-wise Chi-square test with Yates’ continuity correction (α = 0.05, two sided). For tissues evaluated from all control and high dose rats, but not all rats from intermediate doses, statistical analysis consisted of pairwise comparisons of the control and high dose group. Rare tumours (background incidence ≤1%) were considered significant in the Chi-square test with Yates’ continuity correction (α=0.10, two sided). Gehan-Wilcoxon procedure was used to evaluate the differences in mortality.

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Lower for males and females at 1000 mg/kg and lower for males at 500 mg/kg

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Increased in males at 1000 mg/kg/day

Food efficiency:

no effects observed

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

not examined

Ophthalmological findings:

no effects observed

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

AST levels were increased in 1000 mg/kg females at 3 and 6 months; the toxicological significance of this could not be determined.

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

Consistent changes for both sexes at 500 and 1000 mg/kg (increased volume, decreased specific gravity, pH, protein and ketones)

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

500 and 1000 mg/kg/day males and females had increased absolute and relative full and empty cecal weights

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Enlarged cecum at 500 and 1000 mg/kg/day

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Slight diffuse hyperplasia of the mucosal epithelium of the cecum in male rats at 500 and 1000 mg/kg/day and females at 1000 mg/kg/day

Histopathological findings: neoplastic:

no effects observed

Description (incidence and severity):

All tumours observed were considered to be incidental.

Details on results:

CLINICAL SIGNS AND MORTALITY
No statistically significant differences in mortality rates for males and females at any dose group. Due to the nature of the study and the selection of the animal model used, very little mortality was observed for the first 16-18 months, after which this increased in all groups.
There were no statistically significant or treatment related changes in clinical or detailed clinical observations in any treated group. An increase in the incidence of clinical observations occurred during the second year of the study; however this was attributed to geriatric diseases.Increasing numbers of rats were observed with periocular soiling, cloudy eyes, preputial or clitoral gland swelling, dermatitis, or papules of the skin or tail. However, these findings were sporadic and/or lacked a dose response and were considered spontaneous changes unrelated to test material administration. All other in- life observations were considered to be unrelated to treatment.

BODY WEIGHT AND WEIGHT GAIN
Bodyweights for males dosed with 500 and 1000 mg/kg/day were lower than controls. The difference between treated animals and the control developed gradually during the study and reached statistical significance at day 120 where the males at 500 and 1000 mg/kg/day weighed 2.4 and 3.8 % less than controls, respectively. These groups were generally statistically identified for the remainder of the study. Males at 50 mg/kg/day had slightly lower bodyweights than controls; however, this was not attributed to treatment as they were within 97 % of the control bodyweights and were only transiently statistically identified. Males at 5 mg/kg/day had slightly lower bodyweights, but this was not statistically identified at any point. The initial bodyweights of male rats dosed with 5 or 50 mg/kg/day were slightly lower than the rats in the other dosing groups.
Bodyweights for females dosed with 1000 mg/kg/day were slightly lower than the controls and remained so throughout the study. These were considered to be treatment related. The decrement developed gradually and was maintained at approximately the same level throughout the study. This was statistically identified at day 36 (2.0 % lower than controls) and remained 2-3 % lower throughout the study, but were identified as statistically significant at around half the time. Over the last several months of the study, the bodyweights of the 1000 mg/kg/day females were 4-5 % less than the controls (3.7 % at termination). Bodyweights for all other treated groups for the females rats were comparable to those observed for the control female group.
These differences in males and females were concurrent with lower bodyweight gains at 1000 mg/kg/day for males and females and males at 500 m/kg/day. At 12 months, gains were 4.5 and 7.7 % lower for 500 and 1000 mg/kg/day males, respectively. These continued until termination at 24 months at which point the gains were 7.7 and 6.3 % lower for 500 and 1000 mg/kg/day males, respectively. Bodyweights for females dosed with 1000 mg/kg/day were 2.8 % and 3.7 % lower than control females at 12 and 24 months, respectively.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
Feed consumption for males was found to be increased in the 1000 mg/kg/day dose group at almost every examination and was often statistically significant. This was considered to be an effect of treatment. At 12 months, this was increased 3.6 % over control values. This continued to increase until study termination at which point it was recorded to be 7.1 % greater than control consumption.
Feed consumption for the 500 mg/kg/day group was comparable to controls for the first year of the study; however, from day 372 onwards, the feed consumption for this group was slightly increased over control values and was sporadically statistically identified. This was also identified as an effect of treatment. At 50 mg/kg/day, feed consumption was slightly elevated above control values from 1 year to the end of the study. This was slight, and not considered to be related to treatment. Feed consumption for the low dose group males was comparable to controls.
Females fed diet containing the test material tended to have increased feed consumption, though this was decreased initially up to day 15. After day 15, feed consumption remained slightly elevated compared to controls up to day 435. At the end of the first year, food consumption at 50, 500 and 1000 mg/kg was 2.6, 5.2 and 2.6 % greater than controls, respectively. From day 456, feed consumption of females in the low dose group was comparable to controls until the end of the study. For all other groups, elevated consumption continued to the end of the study and was statistically identified for most observations. The increases were not dose related, with the highest dose group often showing the smallest increase. At the end of the study, food consumption in the 50, 500 and 1000 mg/kg/day groups were increased by 3.8, 1.5 and 4.5 %, respectively.
Test material intake was consistent with the target concentrations for all dose levels in the study. Over the course of the entire study, the time weighted average amount of ingested test material (mg/kg/day) was within 2.5 % of the targeted dosages for all dose groups.

FOOD EFFICIENCY
Although feed consumption was highly variable, no clear pattern emerged in relation to administration of the test material.
Feed efficiency decreased over the 13-week time period in both sexes and all dose groups due to slowing of the growth rate while maintaining relatively constant feed consumption. Although slightly increased feed consumption was noted for males given 1000 mg/kg/day and a non-dose related increase for all female dose groups, these minor changes did not result in a consistent pattern of treatment-related altered feed efficiency.

OPHTHALMOSCOPIC EXAMINATION
Ocular haemorrhage, engorged blood vessels, pale fundus, cloudy cornea, periocular soiling, cloudy lens, opaque cornea, opaque lens, phthisis bulbi, missing eye and enlarged or protruding eye were observed at the 12 and 24 month observation points. These were not treatment related due to their low incidence and lack of dose-response. Periocular soiling was considered to be a non-specific clinical sign, while eyes with pale fundus, opaque cornea, opaque lens, cloudy cornea or cloudy lens were considered to be age related changes. Phthisis bulbi and missing eye were considered to be secondary to disease or blood collection.

HAEMATOLOGY
No treatment related changes were observed in haematological parameters for treated rats. There was slight inter-animal variability at 18 months and marked variability, particularly in WBC count and differential count, at 24 months. However mean values were not affected for any parameter in a dose-responsive manner. The individual differences were attributed to spontaneous disease (primarily leukaemia and its stage of involvement).
The following observations reached statistical significance. At 5 mg/kg/day, males had an increased WBC count for 12 months. Females had increased platelets at 5, 500 and 1000 mg/kg/day for 12 months. This was not dose responsive. At 18 months females in the 500 mg/kg/day group had decreased haemoglobin, and females at 1000 mg/kg/day had decreased haematocrit.
Prothrombin time was equivocally affected for high dose animals and for males only at 500 mg/kg/day. Due to the difference in response between the sexes, the minor differences observed, no association with other clotting disorders and the time frame this was observed (only in the first 12 months), this was not considered to be toxicologically relevant. Males had slightly longer prothrombin times, whereas the affected females had slightly shorter times.

CLINICAL CHEMISTRY
The only statistically significant difference observed in males was decreased cholesterol in males dosed with 500 mg/kg/day for 18 months.
The AST levels of females in the 1000 mg/kg/day group were slightly increased over the first year of the study and were attributed to dosing. The AST levels reached statistical significance at 3 and 6 months. This was not considered to be toxicologically significant as the AST levels were not increased at 18 and 24 months, this effect had not been demonstrated in previous toxicity studies, and the males in this study did not exhibit the same effect despite being the more sensitive of the sexes to effects. This increase also did not accompany any histopathological effects in any organ that could be correlated. There was minimal inter-animal variability in AST levels through 12 months, but variability increased thereafter. This was found to be moderate at 24 months and was usually secondary to hepatic involvement by leukaemia.
Other changes noted in females that were statistically identified were considered to be incidental. Decreased total protein and cholesterol was noted for females at 1000 mg/kg/day at the three month observation. Increased glucose was noted in females at 6 months in the low dose group. Increased cholesterol was observed in the 50 and 500 mg/kg/day groups at 12 months. At 24 months, all groups dosed with the test material (except the high dose group) had decreased albumin levels at 24 months.
There were no electrolyte treatment related changes in either sex at any dose level or time point. Males and females had increased calcium at 500 mg/kg/day for 6 months.

URINALYSIS
At 500 and 1000 mg/kg/day increased urine volume and decreased urine specific gravity, pH, protein and ketones were observed. These effects were treatment related but variable in dose-response relationship and statistical significance. These developed gradually and were less definitive at the end of the study (attributed to geriatric changes). These were unaccompanied by treatment related renal histopathologic changes and were considered to be non-adverse.
The urine changes were considered to be adaptive. The ceca at the two highest doses were enlarged by semi-solid ingesta. These formed normal pellet faeces. This was expected to be as a result in increased colonic water resorption with compensatory renal excretion of the additional water which led to increased urine volume and decreased specific gravity. Decreased urine pH was attributed to renal excretion of the test material. Decreased protein and ketone levels were not considered to be adverse and attributed to the dilution of the normal levels of these normally present in rat urine. Increased urine volume and decreased specific gravity is typically associated with renal disease, however this is usually accompanied by increased proteinuria. Rats dosed with the test material demonstrated less chronic renal disease that that typically associated with male Fischer 344 rats. The renal effects were considered adaptive to altered water balance.

ORGAN WEIGHTS
At 500 and 1000 mg/kg/day, males and females had statistically significant increases in absolute and relative full (including contents) and empty cecal weights. The full cecal weights in 1000 mg/kg/day males was around 10.3 g (3.0 fold) above the control weights, while for females it was 6.4 g (2.6 fold). The full cecal weight for males dosed with 500 mg/kg/day was 3.8 g (1.7 fold) above controls whereas for females it was 1.7 g (1.4 fold). At 1000 mg/kg/day, empty cecal weights were increased approximately 1.5 fold for males and 1.4 fold for females. Although terminal bodyweights were not statistically different, the bodyweights for males given 500 mg/kg/day and both sexes dosed with 1000 mg/kg/day were considered to be negatively affected by test material administration.
The relative weights of the heart, liver and ovaries were statistically different for females dosed with 500 and 1000 mg/kg/day. This was attributed to decreased terminal bodyweight.
Males had decreased absolute heart weight at 500 mg/kg/day and males dosed with 50 mg/kg/day had decreased absolute and relative weights of the epididymides. These were statistically significant, but lacked a dose response-relationship and therefore were not considered to be treatment related.
A large variability in organ weights was observed in the rats at study termination. This was largely due to neoplasia, particularly in the spleen which coincided with the frequent occurrence of leukaemia. Adrenal glands, ovaries and uterus weights were also affected by the presence of tumours. The incidence of neoplasia in these organs was much lower than the occurrence of leukaemia, but did affect the mean and standard deviation of these organ weights.

GROSS PATHOLOGY
Mass nodules were observed at necropsy, however these were not considered treatment related and were consistent with the geriatric condition of the rats.

HISTOPATHOLOGY: NON-NEOPLASTIC
Very slight hyperplasia of the cecal mucosa was observed, and was only statistically significant in males dosed with 1000 mg/kg/day.
There were a large number of histopathological diagnoses made for the rats at 24 months; this was attributed to the geriatric condition of the rats.
There were a number of histopathological observations that reached statistical significance, however these were not considered to be toxicologically significant. Males in the 1000 mg/kg/day group were found to have severely decreased spermatic elements (epididymis) and very slight multifocal hyperplasia of the liver (bile duct) which occurred with or without inflammation. In addition to these findings, males in the 1000 mg/kg/day dose group were found to have a decreased incidence in myocardium degeneration with fibrosis and at 50 and 1000 mg/kg/day there was an observed decrease in males with slight multifocal hyperplasia of the liver (bile duct) which occurred with or without inflammation. Females in the 1000 mg/kg/day dose group had a decreased incidence of primary benign pituitary gland (pars distalis) adenomas and at 500 and 1000 mg/kg/day, females were found to have a decreased incidence of basophilic cellular alteration in hepatocytes.

HISTOPATHOLOGY: NEOPLASTIC (if applicable)
Early euthanasia of the animals was largely due to the neoplastic condition of the animals in all groups, including the control. Large granular lymphocyte leukaemia (predominantly males) and adenomas of the pituitary gland were the most common neoplastic findings. These are both historically associated with the animal model used in the study and the incidence of which in all groups of this study were found to be unremarkable.
In addition to the large granular lymphocyte leukaemia and adenomas of the pituitary gland, a leading cause for removal from the study was large masses of the skin and subcutis (including mammary and other subcutaneous glands). These were considered typical of common tumours observed in the Fischer 344 rat, despite the diverse histotypes recorded.
The low numbers of other neoplastic conditions that resulted in the animals being euthanised were typical of spontaneous disease in the rat model. Most palpable masses examined were neoplasms. The most common neoplasms in males were derived from the connective tissues of the subcutis (fibromas) but epithelial origin tumours were also present. Most in- life masses in females originated in the mammary gland with the most frequent in all dose groups being fibroadenomas, a benign tumour. Lower numbers of malignant neoplasms were found and also neoplasms of organs associated with the skin.

HISTORICAL CONTROL DATA (if applicable)
The rat model was selected based on the availability of historical background data. There were no reported deviations for the rats used in the study in comparison to the historical background data.

Relevance of carcinogenic effects / potential:

No increase in neoplasms was observed in either male or female rats at any dose level indicating that the test material did not have an oncogenic potential under the conditions of this study.

Key result

Dose descriptor:

NOAEL

Remarks:

carcinogenicity

Effect level:

1 000 mg/kg diet

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No neoplastic changes were observed that were associated with administration of the test material.

Key result

Dose descriptor:

NOEL

Remarks:

toxicity

Effect level:

50 mg/kg diet

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No treatment related effects were noted in either sex at this level.

Key result

Dose descriptor:

NOAEL

Remarks:

toxicity

Effect level:

500 mg/kg diet

Based on:

test mat.

Sex:

female

Basis for effect level:

other: Slightly decreased bodyweights at 1000 mg/kg/day; Slight diffuse hyperplasia of the cecal mucosal epithelium at 1000 mg/kg/day; Grossly enlarged cecal weights at 500 and 1000 mg/kg/day accompanied by adaptive urinalysis changes.

Key result

Dose descriptor:

NOAEL

Remarks:

toxicity

Effect level:

50 mg/kg diet

Based on:

test mat.

Sex:

male

Basis for effect level:

other: see 'Remark'

Table 2: Mean selected organ and body weights

Parameter	Dose Level (mg/kg/day)
	0	5	50	500	1000
Males
Bodyweight (g)	412.3	400.5	399.6	389.3*	387.6*
Full cecum (g)	5.209	4.983	5.519	9.049*	15.501*
relative (g/100 g)	1.266	1.245	1.385	2.334*	3.995*
Empty cecum (g)	2.400	2.375	2.340	3.123*	3.573*
relative (g/100 g)	0.584	0.592	0.586	0.807*	0.925*
Females
Bodyweight (g)	278.9	275.9	286.2	269.9	266.4
Full cecum (g)	3.999	4.174	4.208	5.721*	10.438*
relative (g/100 g)	1.441	1.522	1.474	2.119*	3.942*
Empty cecum (g)	1.862	1.889	1.957	2.141*	2.592*
relative (g/100 g)	0.671	0.687	0.685	0.795*	0.980*
Heart (g)	0.867	0.881	0.897	0.887	0.863
relative (g/100 g)	0.313	0.320	0.314	0.331*	0.326*
Liver (g)	7.613	7.850	7.731	7.707	7.415
relative (g/100 g)	2.754	2.860	2.714	2.884*	2.789*
Ovaries (g)	0.082	0.083	0.081	0.090	0.086
relative (g/100 g)	0.030	0.030	0.028	0.034	0.033*
* Statistically significant (α = 0.05)

Table 3: Number of rats with very slight diffuse hyperplasia of the cecal mucosa

Sex	Dose Level (mg/kg/day)
	0	5	50	500	1000
Males	3	2	4	2	12*
Females	3	2	0	0	8

* Statistically significant (α = 0.05)

Table 4: Mean urine volume (mL)

Time point (months)	Dose Level (mg/kg/day)
	0	5	50	500	1000
Males
3	3.9	3.4	3.6	3.7	5.0
6	3.1	3.3	3.7	5.0*	5.7*
12	4.9	5.1	5.7	9.0*	8.6*
18	3.8	3.9	3.8	5.6	6.2
24	7.3	6.3	5.7	8.5	11.3*
Females
3	2.8	4.2	3.4	4.4*	5.5*
6	3.7	4.3	4.4	5.5	4.8
12	5.7	9.6	6.8	8.1	6.6
18	5.1	5.8	5.8	9.9*	7.3*
24	8.3	8.8	10.0	12.9	12.4
* Statistically significant (α = 0.05)

Table 5: Mean urine specific gravity

Time point (months)	Dose Level (mg/kg/day)
	0	5	50	500	1000
Males
3	1.081	1.084	1.080	1.083	1.075
6	1.082	1.078	1.075	1.063*	1.064*
12	1.065	1.066	1.061	1.043*	1.049*
18	1.076	1.078	1.070	1.058*	1.065
24	1.057	1.060	1.056	1.044	1.041*
Females
3	1.072	1.062	1.062	1.057*	1.052*
6	1.060	1.063	1.057	1.049	1.054
12	1.050	1.041	1.048	1.038	1.044
18	1.060	1.055	1.057	1.041*	1.053
24	1.038	1.041	1.037	1.032	1.033
* Statistically significant (α = 0.05)

Conclusions:

Under the conditions of the test, no increase in neoplasms was observed in the test animals at any dose level. The NOAEL for carcinogenicity was therefore considered to be 1000 mg/kg/day, the highest dose tested. Administration of the test material was associated with reduced bodyweights, increased cecal weights (empty and full) and hyperplasia of the cecal mucosa in the highest doses tested. The NOEL for toxicity for both sexes was determined to be 50 mg/kg/day as no test material related effects were noted at this level for males and females. The NOAEL for females was determined to be 500 mg/kg/day and the NOAEL for males was 50 mg/kg/day.

Executive summary:

The carcinogenicity of the test material was evaluated in male and female Fischer 344 rats in a study conducted in accordance with the standardised guidelines OECD 453, EU Method B.33, EPA OPPTS 870.4300 and JMAFF under GLP conditions.

The rats were administered the test material for 2 years in the diet at 0, 50, 500 and 1000 mg/kg/day (50 rats per sex, per dose), which was supplied ad libitum. The animals were observed throughout the study for clinical signs of toxicity, bodyweight and feed consumption, ophthalmological effects, haematological and clinical chemistry changes and at the end of the two year study underwent gross necropsy with detailed histopathological examinations for neoplastic and non-neoplastic changes.

No effects on survival were observed with test material administration, and the animals did not demonstrate clinical signs associated with toxicity. Bodyweights for males and females dosed with 1000 mg/kg/day were found to be lower than controls, as was the bodyweights of male rats dosed with 500 mg/kg/day. In addition to lower bodyweights, 500 and 1000 mg/kg/day males and females had increased absolute and relative full and empty cecal weights, which were observed to be enlarged at gross necropsy. Slight diffuse hyperplasia of the mucosal epithelium of the cecum in male rats was observed at 500 and 1000 mg/kg/day and also in females at 1000 mg/kg/day. The effects noted in the urinalysis results for animals dosed with 500 and 1000 mg/kg/day were considered to be secondary to the affects noted in the cecum.

Under the conditions of the test, no increase in neoplasms was observed in the test animals at any dose level. The NOAEL for carcinogenicity was therefore considered to be 1000 mg/kg/day, the highest dose tested. Administration of the test material was associated with reduced bodyweights, increased cecal weights (empty and full) and hyperplasia of the cecal mucosa in the highest doses tested. The NOEL for toxicity for both sexes was determined to be 50 mg/kg/day as no test material related effects were noted at this level for males and females. The NOAEL for females was determined to be 500 mg/kg/day and the NOAEL for males was 50 mg/kg/day.

Reference 2

Endpoint:

carcinogenicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

31 October 2001 to 19 December 2003

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.4200 (Carcinogenicity)

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 451 (Carcinogenicity Studies)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EEC, Part B (Carcinogenicity Test), 87/302/EEC, 1988

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Specific details on test material used for the study:

Purity: 94.5%

Species:

mouse

Strain:

CD-1

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: 6 weeks
- Weight at study initiation (group mean values): 29.8 - 30.1 g (males); 23.6 - 23.9 g (females)
- Housing: Animals were individually housed in stainless steel cages with wire-mesh floors suspended above catch pans and contain a feed container and a pressure activated nipple-type watering system.
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature: 21.6 - 22.2 °C
- Humidity: 44.9 - 62.1 % (relative)
- Air changes: 12 - 15 air changes per hour
- Photoperiod: 12 hours of darkness / 12 hours of light (06:00 to 18:00)

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 (premix) with ground feed. Premixes were mixed approximately bi-monthly for the first 90 days of the study, and at approximate monthly intervals thereafter. 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 90 days of the study 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 was determined pre-exposure, and during months 3, 8, 13 and 17.
Homogeneity of the test material was evaluated in the low-dose female and high-dose male diets from 5 time points. The test material was uniformly distributed throughout the diets with the method of mixing used, with relative standard deviations ranging from 2.34 to 12.6 % (majority of relative standard deviation values were < 5 % ). These results indicate that the diets were homogeneously mixed throughout the study.

- Stability
A previous 4-week toxicity study had shown the test material to be stable for at least 21 days in the rodent feed at concentrations ranging from 0.0005 to 3 %, which bracketed the concentrations used in this study. Additional stability data was evaluated for at least 34 days at concentrations of 0.00258 and 5 % to enable monthly mixing procedures.
The test material was stable in the rodent feed at concentrations ranging from 0.0258 to 5 %. Approximately 113 % of the initial concentration of the 0.0258 % (50 mg/kg/day female diet) sample was present in the feed after 35 days, and approximately 81.7 % of the initial concentration of the 5 % premix was present in the feed after 35 days. These concentrations bracketed those fed to the animals during the study.

- Concentration Verification
Analyses of all dose levels, plus control and premix were determined pre-exposure, and during months 3, 8, 13 and 17. 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 concentrations of test material in the individual diets ranged from 85.7 to 125 % of targeted values with overall mean concentrations ranging from 97.4 to 111 % of target concentrations.

Duration of treatment / exposure:

Animals were treated for up to 18 months.

Frequency of treatment:

Continuous (in diet).

Post exposure period:

Necropsies were conducted on surviving males on days 547-549 and on females on study days 548, 552, and 553.

Dose / conc.:

0 mg/kg bw/day (nominal)

Dose / conc.:

50 mg/kg bw/day (nominal)

Dose / conc.:

250 mg/kg bw/day (nominal)

Dose / conc.:

1 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

50 males and 50 females per dose.

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: The limit test of 1000 mg/kg/day was chosen as the high-dose based on the results of a 90-day mouse study in which adverse effects were not observed. The intermediate and low-dose levels were expected to provide dose response data for any treatment-related effects observed in the high-dose group. The low dose was also expected to be a no-observed-effect level (NOEL).
- Rationale for animal assignment: Animals were stratified by pre-exposure body weights and then randomly assigned to treatment groups using a computer program.

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: 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 were observed include, but were not limited to: activity, repetitive behaviour, vocalisation, inco-ordination/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, usually at the beginning and end of each day, all animals were observed for morbidity and mortality, and the availability of feed/water.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: detailed clinical observations were conducted on all animals on days 7 for females and 8 for males (baseline) and at approximately monthly intervals for months 1 - 9. During months 9 - 12, month 17 and at study termination, the first 10 surviving animals/sex/dose group were evaluated for detailed clinical examination parameters. These examinations included cage-side, hand-held and open-field observations that were recorded categorically or using explicitly defined scales.
In addition, all animals were examined for palpable masses once per month, starting at month 6 and continuing through month 18. The time of onset, location, dimensions, appearance and progression of each palpable mass were recorded. These observations were recorded concurrently with the categorical portion of the detailed clinical observation examination for those animals which had both observation types.

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
- Time schedule for examinations: Feed consumption data were collected weekly during the first 13 weeks of the study then at approximately month intervals thereafter for all animals. Feeders 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 feeder - final weight of feeder) / (number of days in measurement cycle x number of animals per cage)
Actual test material intake (TMI) was calculated using actual feed concentrations, body weights and feed consumption in the following formula:
TMI = [feed consumption (g/day) x 1000 mg/g x (% of test material in feed / 100)] / [((current body weight (g) + previous body weight (g)) / 2) / 1000 g/kg]

FOOD EFFICIENCY: Yes
- Time schedule for examinations: Feed efficiency was calculated using mean body weight gain (bwg) data and mean feed consumption data from the first 13 weeks of the study using the following equation:
Feed Efficiency (g/g gain) = (g feed consumed / day) / (g bwg / day)

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: The eyes of all animals were examined 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.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Blood smears were made from all surviving animals via sample collection from the pedal vein (12 months) or orbital sinus (18 months). Blood from moribund animals was obtained from the pedal vein or tail. Blood smears were not obtained from animals that died spontaneously. A white blood cell count and differential white blood count were determined from all animals in the treated and control groups at the terminal sacrifice (18 months). A differential white blood cell count, as derived from the blood smears, was not determined from animals that were moribund due to the absence of effects at 18 months.
- Anaesthetic used for blood collection: Yes (CO₂)

CLINICAL CHEMISTRY: No

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:

SACRIFICE
Non-fasted rodents 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: Yes
A complete necropsy was conducted on all animals. 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.
Similar necropsy procedures were followed for animals found dead or moribund, except that terminal body weights and organ weights 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 found dead after routine working hours or on weekends, and holidays were refrigerated until the next scheduled workday at which time they were necropsied.

HISTOPATHOLOGY: Yes
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, colon, cranial nerve - optic, duodenum, epididymides, eyes, gallbladder, 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/pharynx, oesophagus, oral tissues, ovaries, oviducts, pancreas, parathyroid glands, peripheral nerve - tibial, pituitary, prostate, rectum, salivary glands, skeletal muscle, skin and subcutis, spinal cord (cervical, thoracic, lumbar), spleen, stomach, testes, thymus, thyroid gland, tongue, trachea, urinary bladder, uterus, vagina.
Sections from all preserved tissues listed 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. Paraffin embedded tissues were sectioned approximately 6 μm thick, stained with haematoxylin and eosin and examined by a veterinary pathologist using a light microscope. The following tissues from the remaining groups were processed and histopathologically examined: liver, kidneys, lungs, and relevant gross lesions.

Statistics:

See "Any other information on materials and methods incl. tables" for information.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

See "Details on results" for information

Mortality:

mortality observed, treatment-related

Description (incidence):

See "Details on results" for information

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

no effects observed

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

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Description (incidence and severity):

No increase in neoplasms was observed in either male or female mice at any dose level

Details on results:

MORTALITY
The mortality rates at the end of the study were 38, 32, 34, and 42 % for males in the control, 50, 250, and 1000 mg/kg/day groups, respectively, and 16, 34, 30, and 42 % for females in the control, 50, 250, and 1000 mg/kg/day groups, respectively. The incidence of mortality in females given 50 or 1000 mg/kg/day was statistically identified as increased, relative to controls. There were no statistically identified differences in the incidence of mortality in females given 250 mg/kg/day, nor in males at any dose level. The higher mortality rate of females given 1000 mg/kg/day (high-dose) was interpreted to be treatment related. The most common cause of death in high-dose females was nephropathy. However, the overall incidence and severity of nephropathy was not increased in males or females from any dose group. Therefore, the increased number of high-dose females that died or were euthanised moribund due to nephropathy was interpreted to be unrelated to treatment. The statistically identified increase in mortality of females given 50 mg/kg/day was interpreted to not be treatment related because of the lack of a dose response, and the absence of any treatment-related histopathologic effects at this dose level.

CAGE-SIDE AND 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 sporadic incidences ( ≤ 2 mice/dose group) of extensor thrust response changes, changes in gait, lacrimation, palpebral closure and pupil size (unable to evaluate) throughout the study. These observations were not dose related and were not attributed to treatment with the test material.
Categorical observations included sporadic incidences ( ≤ 5 mice/dose group) of behaviour-decreased activity, repetitive circling behaviour, enlarged or protruding eyes, swollen ocular membranes, cloudy eyes, eyelid closure, altered faeces, altered gait, maloccluded incisors, not eating or drinking, accidental injury, distended abdomen, missing part of ears or tail, thin appearance, cold to touch, blood in cage, generalised oedema, swollen lower lip, altered posture, unretracted penis, vulvar discharge, changes in respiration, excessive hairloss, discoloured skin/mucous membranes, ungroomed appearance, papules/pustules, dehydration, flaking/scaling or ulceration of skin, abnormally soft or limp muscles, convulsions and body soiling and swellings. These observations were consistent with the type of spontaneous alterations commonly associated with aging mice and were not attributed to treatment with the test material. The only observation that occurred at a greater frequency (> 5 mice/dose group) was dermatitis. Dermatitis was first noted in males on test day 35, and in females on test day 63. This alteration did not occur in a dose related incidence and therefore was interpreted to be not treatment related. Males from the control group had the highest incidence of dermatitis over the course of the study.

BODY WEIGHTS
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 no statistically identified differences.

FEED CONSUMPTION AND TEST MATERIAL INTAKE
There were no treatment-related effects in feed consumption. The feed consumption of males and females in the treated groups were comparable to the controls throughout the study. The only statistically identified difference (increase) in feed consumption occurred on days 7 - 14 for males given 1000 mg/kg/day.
The actual test material intake values met the targeted values. Males consumed 0, 50.2, 251, or 1000 mg/kg/day and females consumed 0, 50.9, 252, or 1010 mg/kg/day. These values correspond to time-weighted average concentrations of 0, 367, 1870, or 7350 ppm for males and 0, 319, 1590, or 6130 ppm for females.

FEED EFFICIENCY
Calculated feed efficiencies were highly variable within and between dose levels and were likely a reflection of the normal variability in feed consumption and body weight gain. There were no treatment-related effects on feed efficiency.

OPHTHALMOLOGY
Examinations performed on all animals pre-exposure and at study termination revealed isolated occurrences of pale fundus, cloudy cornea, cloudy lens, opaque cornea, opaque lens, eyelids partially closed, irregular corneal surface, enlarged or protruding eye, anterior synechia or phthisis bulbi. These observations were interpreted not to be treatment related due to the lack of a dose-response pattern and the low frequency of occurrence.

CLINICAL PATHOLOGY
- Haematology: There were no treatment related changes in the total white blood cell or the differential white blood cell counts.

ANATOMIC PATHOLOGY
- Organ Weights: There were no treatment-related alterations in terminal body weights or organ weights of male or female mice from any dose level. The only statistically identified alterations in organ weights were elevated mean absolute and relative ovary weights of mice given 50 or 250 mg/kg/day. The elevated ovary weights from these dose levels were reflective of an increase in the incidence of individual animals with grossly observed fluid-filled ovarian cysts. These cysts were recorded as dilatation of the ovarian bursa or as haematocysts at gross necropsy, and were not drained prior to weighing so that the structural integrity of the ovaries was maintained for histological examination. Microscopic evaluation of the ovaries from all females revealed no treatment-related increases in the number of animals with ovarian cysts or haematocysts. The mean absolute and relative ovary weights of mice given 1000 mg/kg/day were also higher than controls, but not statistically identified. The elevated mean ovary weights of mice given 1000 mg/kg/day were reflective of one female with markedly distended fluid-filled cysts of both ovaries. If the ovary weight (6.798 g) of this individual animal was excluded from statistical analysis, the mean absolute and relative ovary weights of mice given 1000 mg/kg/day would have been comparable (0.119 g) to the controls. The alterations in ovary weights were interpreted to not be treatment related because of the lack of a dose response, and the absence of any histopathologic ovarian effects. The higher mean ovarian weights in the 50 and 250 mg/kg/day groups were interpreted to be reflective of the variability in the size of ovarian cysts or haematocysts. If the ovarian weights of animals from the scheduled terminal necropsy that had grossly observed dilatation of the ovarian bursa or haematocysts were excluded from analysis, the ovarian weights of all dose levels were comparable to controls.

- Gross Pathology: There were no treatment-related gross pathologic observations in males from any dose level. The increased mortality in females given 1000 mg/kg/day was associated with a few gross pathologic alterations. Females given 1000 mg/kg/day had an increased incidence of pale kidneys, relative to controls. Most of the animals with pale kidneys had moderate or severe nephropathy. Other gross observations that were more frequent in females given 1000 mg/kg/day consisted of decreased amount of body fat, the presence of haemolysed blood in the gastrointestinal tract, pulmonary atelectasis and perineal soiling. These gross observations were interpreted to reflect debility and/or stress of the animals that died spontaneously or were euthanised moribund, and not primary treatment-related alterations. Females given 50, 250 or 1000 mg/kg/day had an increased incidence of dilatation of the ovarian bursa. Microscopic evaluation of the ovaries revealed that most of the fluid-filled ovarian structures were not dilated bursas, but rather, were ovarian cysts derived from anovulatory follicles, or from epithelial cords of the interstitium of the ovary. The increased incidence of dilatation of the ovarian bursa in females given 50, 250 or 1000 mg/kg/day was interpreted to not be treatment-related because of the lack of a dose response, and the absence of any histopathologic ovarian effects.

- Histopathology: There were no treatment-related statistically identified histopathologic effects in males or females at any dose level. The overall incidence of nephropathy in treated mice from all modes of death (spontaneous death, moribund, and scheduled terminal sacrifice) was comparable to controls at all dose levels. However, in females given 1000 mg/kg/day (high-dose) that died spontaneously or were euthanised moribund, there was an increased incidence of moderate or severe nephropathy as a contributory factor of their deaths. There were 11 mice of early death or moribund status with nephropathy at the 1000 mg/kg/day dose level, versus 4, 2, and 4 mice of early death or moribund status with nephropathy at the 0, 50, or 250 mg/kg/day dose levels, respectively. The increased incidence of moderate or severe nephropathy in high-dose females that died or were euthanised moribund was interpreted to be unrelated to treatment, because the overall incidence and severity of nephropathy was not increased in males or females from any dose group.

Relevance of carcinogenic effects / potential:

No increase in neoplasms was observed in either male or female mice at any dose level indicating that the test material did not have an oncogenic potential under the conditions of this study.

Key result

Dose descriptor:

NOEL

Remarks:

toxicity

Effect level:

1 000 other: mg/kg/day

Based on:

test mat.

Sex:

male

Key result

Dose descriptor:

NOEL

Remarks:

toxicity

Effect level:

250 other: mg/kg/day

Based on:

test mat.

Sex:

female

Table 1: Terminal Body and Ovary Weights

Dose (mg/kg/day)	0	50	250	1000
Terminal body weight (g)	37.3	38.3	38.4	37.8
Ovary weight (g)	0.161 ± 0.342	0.236 ± 0.279*	0.359 ± 0.566*	0.349 ± 1.259
Ovary weight (g/100)	0.438 ± 0.938	0.628 ± 0.734*	0.976 ± 1.611*	0.886 ± 3.055
Ovary weight (g) excluding mice with dilated bursa or haematocyst	0.043 ± 0.030	0.052 ± 0.054	0.049 ± 0.027	0.047 ± 0.058
Ovary weight (g/100) excluding mice with bursa or haematocyst	0.116 ± 0.077	0.136 ± 0.143	0.128 ± 0.069	0.123 ± 0.148

 *Statistically different from control mean by Wilcoxon's test, alpha = 0.05

Table 2: Gross Pathologic Observations in Females

Dose (mg/kg/day)	0	50	250	1000
Kidneys, pale, bilateral	5	6	7	13
Decreased amount of fat	4	5	8	9
Haemolysed blood, gastrointestinal tract	1	5	1	10
Atelectasis	0	0	2	4
Perineal soiling	2	5	3	8

Table 3: Gross Pathology, Dilation of the Ovarian Bursa

Dose (mg/kg/day)	0	50	250	1000
Dilation, ovarian bursa, left	3	8	9	4
Dilation, ovarian bursa, right	5	6	6	4
Dilation, ovarian bursa, bilateral	3	11	12	8
Dilation, ovarian bursa, any symmetry	11	25	27	16

Conclusions:

Under the conditions of the study the NOEL for males was 1000 mg/kg/day and 250 mg/kg/day for females.

Executive summary:

The oncogenicity of the test material was investigated in a study which was conducted under GLP conditions and in accordance with the standardised guidelines EPA OPPTS 870.4200, OECD 451, EEC Part B (Carcinogenicity Test), and JMAFF (2000).

During the study groups of 50 male and 50 female CD-1 mice were given test material in feed. Males and females were given targeted dosages of 0, 50, 250 or 1000 mg/kg/day for up to 18 months. Data were collected on clinical appearance and behaviour, body weights, and feed consumption at regular intervals throughout the study, and on ophthalmology at study start and termination. Blood samples were collected from all mice at terminal sacrifice for white blood cell and differential white cell counts. Organ weights and gross pathology were evaluated in all animals. Histopathologic examinations were conducted on a complete set of tissues from control and high-dosage animals and all animals that died or were sacrificed moribund prior to study termination. The histopathologic examinations of animals from the low- and mid-dosage groups were limited to liver, kidneys, lungs, and relevant gross lesions.

There were no treatment-related effects in males at any dose level. Females given 1000 mg/kg/day (high-dose) had a statistically identified increase in mortality which was interpreted to be treatment related. The most common cause of death in high-dose females was nephropathy. However, the overall incidence and severity of nephropathy was not increased in males or females from any dose group. Therefore, the increased number of high-dose females that died or were euthanised moribund due to nephropathy was interpreted to be unrelated to treatment. There were no other treatment-related effects in females at any dose level. No increase in neoplasms was observed in either male or female mice at any dose level indicating that the test material did not have an oncogenic potential under the conditions of this study. The NOEL for males was 1000 mg/kg/day and 250 mg/kg/day for females.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

50 mg/kg bw/day

Study duration:

chronic

Species:

rat

Quality of whole database:

Two studies were available, both were performed in accordance with OECD guidelines and under GLP conditions. The tests were performed in the standard species for the guideline. The studies were reported to a high standard with sufficient detail to assess the presented data. The studies contained no deviations or anomalies that were thought to affect the quality of the data. 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, Regulation (EC) No. 1272/2008, the substance does not require classification with respect to carcinogenicity.

In accordance with the criteria for classification as defined in Annex VI, Directive 67/548/EEC (DSD), the substance does not require classification with respect to carcinogenicity.

Additional information

ORAL

In the key study by Johnson & Dryzga (2005), the carcinogenicity of the test material was evaluated in male and female Fischer 344 rats in a study conducted in accordance with the standardised guidelines OECD 453, EU Method B.33, EPA OPPTS 870.4300 and JMAFF under GLP conditions.

The rats were administered the test material for 2 years in the diet at 0, 50, 500 and 1000 mg/kg/day (50 rats per sex, per dose), which was supplied ad libitum. The animals were observed throughout the study for clinical signs of toxicity, bodyweight and feed consumption, ophthalmological effects, haematological and clinical chemistry changes and at the end of the two year study underwent gross necropsy with detailed histopathological examinations for neoplastic and non-neoplastic changes.

No effects on survival were observed with test material administration, and the animals did not demonstrate clinical signs associated with toxicity. Bodyweights for males and females dosed with 1000 mg/kg/day were found to be lower than controls, as was the bodyweights of male rats dosed with 500 mg/kg/day. In addition to lower bodyweights, 500 and 1000 mg/kg/day males and females had increased absolute and relative full and empty cecal weights, which were observed to be enlarged at gross necropsy. Slight diffuse hyperplasia of the mucosal epithelium of the cecum in male rats was observed at 500 and 1000 mg/kg/day and also in females at 1000 mg/kg/day. The effects noted in the urinalysis results for animals dosed with 500 and 1000 mg/kg/day were considered to be secondary to the affects noted in the cecum.

Under the conditions of the test, no increase in neoplasms was observed in the test animals at any dose level. The NOAEL for carcinogenicity was therefore considered to be 1000 mg/kg/day, the highest dose tested. Administration of the test material was associated with reduced bodyweights, increased cecal weights (empty and full) and hyperplasia of the cecal mucosa in the highest doses tested. The NOEL for toxicity for both sexes was determined to be 50 mg/kg/day as no test material related effects were noted at this level for males and females. The NOAEL for females was determined to be 500 mg/kg/day and the NOAEL for males was 50 mg/kg/day.

In the second key study, by Stebbins & Day (2003), the oncogenicity of the test material was investigated in a study which was conducted under GLP conditions and in accordance with the standardised guidelines EPA OPPTS 870.4200, OECD 451, EEC Part B (Carcinogenicity Test), and JMAFF (2000).

During the study groups of 50 male and 50 female CD-1 mice were given test material in feed. Males and females were given targeted dosages of 0, 50, 250 or 1000 mg/kg/day for up to 18 months. Data were collected on clinical appearance and behaviour, body weights, and feed consumption at regular intervals throughout the study, and on ophthalmology at study start and termination. Blood samples were collected from all mice at terminal sacrifice for white blood cell and differential white cell counts. Organ weights and gross pathology were evaluated in all animals. Histopathologic examinations were conducted on a complete set of tissues from control and high-dosage animals and all animals that died or were sacrificed moribund prior to study termination. The histopathologic examinations of animals from the low- and mid-dosage groups were limited to liver, kidneys, lungs, and relevant gross lesions.

There were no treatment-related effects in males at any dose level. Females given 1000 mg/kg/day (high-dose) had a statistically identified increase in mortality which was interpreted to be treatment related. The most common cause of death in high-dose females was nephropathy. However, the overall incidence and severity of nephropathy was not increased in males or females from any dose group. Therefore, the increased number of high-dose females that died or were euthanised moribund due to nephropathy was interpreted to be unrelated to treatment. There were no other treatment-related effects in females at any dose level. No increase in neoplasms was observed in either male or female mice at any dose level indicating that the test material did not have an oncogenic potential under the conditions of this study. The NOEL for males was 1000 mg/kg/day and 250 mg/kg/day for females.

Justification for selection of carcinogenicity via oral route endpoint:
The key study was selected based on the choice of the animal model used. The rat is the preferred species for testing in line with the OECD guidelines.