Ammonium 2,3,3,3-tetrafluoro-2-(heptafluoropropoxy)propanoate

Repeated dose toxicity: oral

Administrative data

Endpoint:

chronic toxicity: oral

Remarks:

combined repeated dose and carcinogenicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

This study was selected as the key study because the information provided for the hazard endpoint is sufficient for the purpose of classification and labeling and/or risk assessment. Although data provided have a report year after 2008, the study was performed to fulfill needs required by government regulators and/or for product stewardship purposes. DuPont’s stewardship principle states that “We will adhere to the highest standards for the safe operation of facilities and the protection of our environment, our employees, our customers and the people of the communities in which we do business”. The study was carried out in accordance with our internal Product Stewardship standard which is part of the American Chemical Council’s “Responsible Care Program”. This study was not performed to fulfill an information requirement under REACH, but since the test data were already available they were provided as part of the REACH submission.

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 453 (Combined Chronic Toxicity / Carcinogenicity Studies)

Deviations:

no

Remarks:

Conducted according to the guideline in effect at the time of study conduct.

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: EPA OPPTS 870.4300 (Combined Chronic Toxicity / Carcinogenicity)

Deviations:

no

Remarks:

Conducted according to the guideline in effect at the time of study conduct.

Test guideline 3

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Remarks:

Conducted according to the guideline in effect at the time of study conduct.

Test guideline 4

Qualifier:

according to guideline

Guideline:

other: JMAFF Japan Agricultural Chemicals Regulation Law 12 Nousan No. 8147

Deviations:

no

Remarks:

Conducted according to the guideline in effect at the time of study conduct.

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Reference substance 002

Cas Number:

62037-80-3

Details on test material:

- Purity: 84%

Test animals

Species:

rat

Strain:

other: Crl:CD(SD)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source:
- Age at study initiation: approximatley 7 weeks
- Weight at study initiation: 131-154 g for males; 97-128 g for females
- Fasting period before study: No
- Housing: Pair-housed (same-sex) in polyboxes with non-aromatic bedding in an environmentally controlled room
- Diet (e.g. ad libitum): ad libitum, except during designated periods
- Water (e.g. ad libitum): tap water, ad libitum
- Acclimation period: 14 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18-26°C (reported as 64-79°F)
- Humidity (%): 30-70%
- Air changes (per hr): Not reported
- Photoperiod (hrs dark / hrs light): 12 hrs/12 hrs (the dark cycle was interrupted intermittently due to study-related activities

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

other: deionized water

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS: The test article was used as received from the Sponsor. The test article formulations were adjusted for purity of 84%. The test article was mixed with the appropriate amount of deionized water to achieve the desired concentrations. Formulations of the test article were prepared weekly at nominal concentrations of 0.01, 0.1, 5, and 50 mg/mL, and were stored at room temperature.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The dosing formulations prepared for Week 1 at each concentration were evaluated for homogeneity. Samples from the top, middle, and bottom were collected from each dosing group. The results showed all concentrations met the homogeneity acceptance criteria (100 ±10% average recovery; ≤5% RSD). The average concentrations of the homogeneity samples ranged from 93.2 to 95.3 of nominal with a RSD of 0.710 to 1.98%.

Weeks 1, 2, 3, 4, 17, 30, 44, 47, 48, 56, 69, 82, and 95 dose formulations were analysed for concentration verification. Duplicate samples from the middle stratum at the 0.0 mg/mL (vehicle control), 0.01 mg/mL, 0.1 mg/mL, 5.0 mg/mL, and 50 mg/mL concentration were injected and analysed. Duplicate samples from the top, middle, bottom stratum at the 0.01 mg/mL concentration from the Week 44 dosing formulations were also injected and analysed. Samples for each level met the sample analysis acceptance criteria for accuracy and precision (100 ±10% average recovery; ≤5% RSD through Week 48, ≤10% RSD from Week 48 through the end of the study), with a few exceptions. Most of the deviations from acceptability criteria were small and were not considered to have impacted the study. The Week 44 samples (original and backup samples) of the 0.01 mg/mL formulation were approximately double the nominal concentration. These values support that animals in this (male only) group received approximately double the nominal dose of test substance during that week. This deviation did not impact the overall integrity of the study as this dose was below the NOAEL for males, and no adverse effects were observed that were attributed exposure to this higher than nominal dose.

Duration of treatment / exposure:

up to 104 weeks in males and up to 101 weeks in females

Frequency of treatment:

Daily

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
0.1, 1, and 50 mg/kg/day for males
Basis:
actual ingested

Doses / concentrations 2

Remarks:

Doses / Concentrations:
1, 50, and 500 mg/kg/day for females
Basis:
actual ingested

No. of animals per sex per dose:

80

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: In a previous study, Crl:CD(SD) rats (10/sex/dose) were dosed with the test substance by oral gavage for at least 90 days at daily doses of 0, 0.1, 10, or 100 mg/kg/day for males and 0, 10, 100, or 1000 mg/kg/day for females. In the 1000 mg/kg/day group, three females died prior to scheduled sacrifice and others displayed clinical signs. No other test substance-related effects were observed in surviving animals in all groups on body weight or nutritional parameters, clinical or ophthalmological observations, or neurobehavioral parameters. Test substance-related findings included regenerative anaemia (males: 100 mg/kg/day; females: 1000 mg/kg/day), clinical chemistry effects consistent with PPARα activation (males: >10 mg/kg/day; females: 100-1000 mg/kg/day), and increased liver weights and associated hepatocellular hypertrophy (males: >10 mg/kg/day; females: 1000 mg/kg/day). Similar liver effects were observed at >3 mg/kg/day in males and 300 mg/kg/day in females in a rat 28-day gavage study. Increased kidney weights were observed in males and females at >10 mg/kg/day. In females, renal papillary necrosis and/or renal tubular necrosis were observed in the two females found dead prior to scheduled sacrifice and in one female that survived to the scheduled sacrifice. Clinical and anatomic pathology parameters were fully or partially (male haematology effects; liver weights) reversible after an approximate 4-week recovery period. Based on the results of the 90-day and 28-day studies, doses selected for this study were 0, 0.1, 1, and 50 mg/kg/day in males and 0, 1, 50, and 500 mg/kg/day in females. The high dose was expected to produce effects on clinical chemistry and liver weight and microscopic pathology in males and females, without producing excessive liver toxicity. The middle dose was expected to possibly produce liver and clinical chemistry effects in either sex or to be a no-observed-adverse effect level (NOAEL). The low dose was expected to be a NOAEL in both males and females.

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily; the afternoon cage side observation was conducted at approximately the same time each day (±2 hours). Beginning at Week 53, a third cage side observation was conducted daily in the evening.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Prior to randomization and weekly during the study. On occasion, clinical observations were recorded at unscheduled intervals. The observations included, but were not limited to, evaluation of the skin, fur, eyes, ears, nose, oral cavity, thorax, abdomen, external genitalia, limbs and feet, respiratory and circulatory effects, autonomic effects such as salivation, and nervous system effects including tremors, convulsions, reactivity to handling, and bizarre behaviour, and the palpation of masses.

BODY WEIGHT: Yes
- Time schedule for examinations: The day after receipt, prior to randomization (Week -2), weekly for the first 14 weeks starting on Day 1 (prior to dosing), and every other week thereafter during the study.

FOOD CONSUMPTION: Yes
- Time schedule: Pretest (Week -1), weekly during the first 13 weeks, and for 2 week intervals starting on Week 14 during the study. The food consumption was measured for the cage and divided by the number of surviving animals. Food consumption was calculated weekly, the first quarter (Weeks 1 to 13), the first year (Weeks 1 to 52), and for the entire study (Weeks 1 to 102 for males and Weeks 1 to 100 for females).

FOOD EFFICIENCY: Yes
- Time schedule: Weekly, the first quarter (Weeks 1 to 13), the first year (Weeks 1 to 52), and for the entire study (Weeks 1 to 102 for males and Weeks 1 to 100 for females).

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Pretest and on all surviving animals prior to the interim and terminal necropsies.
- Dose groups that were examined: All animals

HAEMATOLOGY: Yes
- Time schedule for collection of blood: 3, 6, and 12 months for haematology; 12 and 18 months and prior to terminal necropsy for blood smears
- Anaesthetic used for blood collection: No data
- Animals fasted: Yes
- How many animals: 10/sex/group
- Parameters checked in table No. 1 were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: 3, 6, and 12 months
- Animals fasted: Yes
- How many animals: 10/sex/group
- Parameters checked in table No. 2 were examined.

URINALYSIS: Yes
- Time schedule for collection of urine: 6 and 12 months
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters checked in table No.3 were examined.

NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes (see table 4)
HISTOPATHOLOGY: Yes (see table 4)

Necropsy examinations were performed on all animals dying spontaneously, euthanized in extremis, or euthanized at scheduled necropsies. Animals were examined carefully for external abnormalities including palpable masses. The skin was reflected from a ventral midline incision, and any subcutaneous masses were identified and correlated with antemortem findings. The abdominal, thoracic, and cranial cavities were examined for abnormalities and the organs removed, examined, and, where required, placed in fixative. The pituitary was fixed in situ. The eyes and testes were fixed using a modified Davidson’s fixative. All other tissues were fixed in neutral buffered formalin. Formalin was infused into the lung via the trachea and into the urinary bladder. Body weight and the organ weights identified in Table 4 were recorded for all animals at scheduled necropsies and appropriate organ weight ratios were calculated (relative to body and brain weights). Paired organs were weighed together. A combined weight of the thyroid gland with the bilateral parathyroid post fixation was obtained. Organs were not weighed for animals dying spontaneously or euthanized in extremis. Microscopic examination of fixed haematoxylin and eosin-stained paraffin sections were performed on sections of tissues and from the groups identified in Table 4 and all animals dying spontaneously or euthanized in extremis.

Statistics:

See Table 5

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

effects observed, treatment-related

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

not examined

Ophthalmological findings:

no effects observed

Haematological findings:

effects observed, treatment-related

Clinical biochemistry findings:

effects observed, treatment-related

Urinalysis findings:

effects observed, treatment-related

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Gross pathological findings:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

effects observed, treatment-related

Details on results:

CLINICAL SIGNS AND MORTALITY: There were no test article-related effects on survival over the course of this study. A single test article-related cause of death/moribundity was inflammation/necrosis of the kidneys which occurred in seven of the 500 mg/kg/day females and was characterized by papillary necrosis. In males the most common causes of death/moribundity were pituitary tumours and undetermined. In females the most common causes of death/moribundity were mammary tumour and pituitary tumour. Females were terminated during Week 101, prior to scheduled termination, due to low survival in all female dose groups, especially control and 50 mg/kg/day groups. However, this did not impact the study as this was approximately 2 years of test article exposure. Even though survival among all female groups was low there were no statistically significant differences and survival was comparable among all groups. There were no test article-related clinical observations. All observations were transient or common in these species and were not considered test article-related. There was no test-article related increase in masses or mass findings.

BODY WEIGHT AND WEIGHT GAIN: Exposure to the test substance produced no adverse reductions in body weight and body weight gain in males. Mean body weight in 50 mg/kg/day males was statistically significantly below control over most of the first year, although mean body weight was only 4% below control in males at Week 52 (not statistically significant), and exceeded the control value at termination. Mean body weight gain in this group was 6% below control in males over Weeks 1 to 52 and exceeded the control value over the two year period. Based on the small magnitude of the changes, the effect among males at 50 mg/kg/day was not considered adverse. Exposure to 500 mg/kg/day of the test substance produced adverse reductions in body weight and body weight gain in females. In this group, statistically significantly lower mean body weight was observed from weeks 30 through 86. Mean body weight was 13% below control at Week 52, and mean body weight gain was 20% below controls over Weeks 1 to 52 (both statistically significant). Mean final body weight (week 100) and overall body weight gain (Weeks 1-100) were comparable to the control value. However, these body weight changes were considered adverse at this dose based on the difference during the first year on study. No adverse effects on body weight were observed in any other dose group. Mean body weight and body weight gain was lower in females at 1 mg/kg/day over most of the first year of the study. This finding was not considered test article-related as no dose response was noted, the small magnitude of the difference, and the fact that it did not persist over the remainder of the study. Statistically significant differences in body weight or body weight gain were observed in all dose groups during weekly/biweekly measurements, but were not attributed to test article exposure as they were intermittent, included both higher and lower values (relative to control), and did not demonstrate a consistent dose-response.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): There were no adverse test article-related effects on food consumption in either sex or in food efficiency in males at any dose. A few statistically significant differences in food consumption were observed in all dose groups during weekly/biweekly measurements, but were not attributed to test article exposure as they were intermittent and did not demonstrate a consistent temporal- or dose-response.

FOOD EFFICIENCY: Adverse effects on food efficiency were observed in 500 mg/kg/day females. In this group, food efficiency was 23% below control (statistically significant) over the first year and 11% below control (statistically significant) overall (Weeks 1-100). Lower mean food efficiency was noted over the first year in males at 50 mg/kg/day. However, overall (Weeks 1-104) food efficiency was comparable to controls. No effects were noted in any other dose group. A few statistically significant differences in food efficiency were observed in all dose groups during weekly/biweekly measurements, but were not attributed to test article exposure as they were intermittent and did not demonstrate a consistent temporal- or dose-response.

OPHTHALMOSCOPIC EXAMINATION: No test article-related findings were noted in the interim or terminal ophthalmoscopic examination.

HAEMATOLOGY: At the 3, 6, and 12 month intervals there were mild decreases in red cell mass (erythrocytes, haemoglobin, and hematocrit in females receiving 500 mg/kg/day. Effects were mild in females (up to 28% less than control) and were not associated with any test article-related effects on erythrocyte morphology. Appropriate increases in reticulocytes (106% above respective) occurred in response to the decreases in red cell mass. The increases in reticulocytes were associated with expected decreases in MCHC and increases MCV. This collection of findings is suggestive of red cell loss or haemolysis although the exact mechanisms involved are unknown. All of these findings were considered test article-related and adverse. Statistically significant decreases in red cell mass were also present in males receiving 50 mg/kg/day at the 3- and 6-month interval. However, the decreases were small (decreases were less than 10% from control except for haemoglobin at 3 months) and did not induce statistically significant changes in reticulocytes. In addition, red mass changes were transient— at the 12 month interval there were no statistically significant changes in any red cell mass parameter, and values in individual animals in the 50 mg/kg/day group were similar to controls. Therefore, the red cell mass changes in 50 mg/kg/day males were considered to be test article-related but non-adverse. All other mean and individual haematology values were considered to be within an acceptable range for biologic and procedure-related variation. These include the following:

- Group mean erythrocytes were statistically lower in the 50 mg/kg/day female group at 12 months. This finding was considered unrelated to treatment, as the difference relative to control was small (6%), there were no statistically significant changes in haemoglobin or hematocrit in this group at this time point, and there were no statistically significant changes in any red cell mass parameter in this group at the 3- and 6-month time point.

- Statistically lower haemoglobin in the 1 mg/kg/day female groups at 3 months did not occur in a dose-related manner, was not associated with statistically significant changes in other red cell mass parameters at any time point, and was thus also considered a spurious finding.

No test article-related effects among leukocytes were observed in either sex at any interval (12, 18 and 24 months) in which these evaluations were performed. There were no test article-related effects among coagulation times in either sex at any dose level. All mean and individual values were considered within an acceptable range for biologic and/or procedure-related variation. Statistically decreased APTT was present in the 500 mg/kg/day female group at the 12-month time point. These changes were not considered toxicologically significant based on the direction of change (decreased rather than prolonged) and absence of correlative findings in other coagulation parameters.

CLINICAL CHEMISTRY:

Liver Enzymes: At the 12-month interval in males receiving 50 mg/kg/day, there were mild increases relative to controls in enzymes indicative of liver injury including alkaline phosphatase, ALT, AST and sorbitol dehydrogenase (sorbitol dehydrogenase and AST not statistically significant). These enzyme changes correlated with microscopic findings of minimal cystic degeneration and minimal to mild focal necrosis in the liver of males at 50 mg/kg/day. Therefore, these enzymes changes were considered test article-related and adverse. Minimal but statistically significant increases in alkaline phosphatase were also present at the 3- and 6-month intervals in the 50 mg/kg/day male group. At these intervals, increases in alkaline phosphatase were less than those present at 12 months and were not associated with statistically significant changes in other enzymes indicative of hepatic or hepatobiliary injury. Therefore, the changes in alkaline phosphatase in the 50 mg/kg/day male group at the 3 and 6 month intervals may be due in part or in whole to test article-related enzyme induction, as the test article was previously shown to produce an increase in total P450 enzyme activity in male rats at 30 mg/kg/day. There were no test article-related changes in liver enzymes in males receiving 1 or 0.1 mg/kg/day or in females at any of the dose levels tested (up to 500 mg/kg/day).

Serum Proteins: Minimal, statistically significant increases in albumin were present in males receiving 50 mg/kg/day at all intervals (up to 16% above controls) and in females receiving 500 mg/kg/day at the 3-month interval (10% above controls). In addition, statistically significant decreases (of up to 17% below control) in globulin were present in females at 500 mg/kg/day at all intervals (an associated decrease in total protein was also present in this group at the 6-month interval). No statistically significant decreases in globulin were present in males at any dose or interval, small decreases in individual values for these parameters in individual animals in the 50 mg/kg/day male group may have been test article-related. The changes in albumin and globulin in the high-dose male and female groups also resulted in statistically significant increases in albumin/globulin ratio in these groups at all intervals. The test article is a peroxisome proliferator, and the pattern of change in serum proteins observed in high dose males and females-lower globulin and higher albumin-is a well-established response to PPARα activation. Peroxisome proliferators are anti-inflammatory, producing decreases in acute phase proteins (which contribute to the globulin fraction), and increases in negative acute phase protein (albumin). However, no adverse biological outcomes have been associated with such changes in these serum proteins. Therefore, these changes in serum proteins in high dose males and females were considered test article-related although they were not considered biologically relevant based on their small magnitude and lack of association with known adverse outcomes. In addition to the serum protein changes noted above, minimal, statistically significant increases in albumin/globulin ratio were present in the 1 mg/kg/day males and 50 mg/kg/day females at all intervals. Also, in some individual animals in these groups, albumin tended to be higher and globulin lower than controls. However, group mean albumin and globulin in these groups were not statistically different from controls (with the exception of elevated albumin in the 1 mg/kg/day male group at 12 months and decreased globulin in the 50 mg/kg/day female group at 6 months), and differences from control group means for both albumin and globulin were ≤8% at all intervals. Therefore, the statistically significant changes in albumin/globulin ratio in these groups were also considered to be test article-related but non-adverse based on the minimal nature of the changes.

Other: All other mean and individual clinical chemistry values were considered within an acceptable range for biologic and procedure-related variation. There were other statistically significant changes among clinical chemistry analytes that were not considered of any additional relevance to the test article based on their small magnitude, sporadic nature, direction of change, relation to changes already discussed, and/or lack of a dose response. These are discussed in more detail below.
• Phosphorus was statistically higher than control in the 500 mg/kg/day female group at the 12-month interval. The relationship to treatment for this difference is uncertain; however values in individual rats in this group were similar to controls except for one animal, and there were no statistically significant changes in phosphorus in any treated group at any other time point. Therefore, based on the minimal nature of these changes, they were not considered to be adverse.
• Phosphorus was also statistically higher in the 0.1 and 50 mg/kg male groups at the 3-month interval. These differences were considered to be unrelated to test article administration since they did not occur in a dose-related manner and there were no statistically significant differences in phosphorus in any treated group relative to control at the 6- and 12- month intervals.
• Calcium was statistically higher in the 50 mg/kg/day males at the 12-month interval. One fraction of serum calcium exists as “bound” to albumin, and increases in albumin are necessarily associated with physiologically appropriate increases in calcium. Changes in bound calcium have no effect on unbound (“ionized”) calcium, which is the physiologically active form of calcium. Therefore, the increase in calcium in the 50 mg/kg/day male group at 12 months was considered to be secondary to albumin changes, physiologically irrelevant, and thus non-adverse.
• Urea nitrogen was statistically higher than the respective control in the 1 mg/kg/day male group at the 12-month interval and in the 50 mg/kg/day male group at the 6-month interval. These differences were not dose-related and/or were not consistent across time, and there were no correlative changes in related clinical chemistry parameters or with microscopic changes in the kidneys. Therefore, these differences were considered spurious and unrelated to administration of the test article.
• Chloride was statistically higher than control in females at 1 and 500 mg/kg/day (but not at 50 mg/kg/day) at the 6-month interval. These differences were not considered to be test article-related as they very slight (only 2% above control), did not occur in a dose-related manner, and were not associated with changes in chloride at any other interval.

URINALYSIS: In females receiving 500 mg/kg/day, minimal, statistically significant increases in urine volume and decreases in urine specific gravity-suggestive of a minimal diuresis-were present at both the 6- and 12-month intervals. Although minimal and not associated with changes in kidney-related chemistry parameters (e.g., urea nitrogen, creatinine), these changes may be correlative to increased incidences and severity of chronic progressive nephropathy observed microscopically in this dose group at the 1-year interim sacrifice. Urine pH was increased in males at all dose levels and in females receiving 1 or 500 mg/kg/day. These changes in urine pH are of uncertain relationship to administration of the test article based on the lack of a clear dose response across the affected groups. However, based on the lack of any correlative findings suggestive of an effect on the urogenital system (except in the kidneys of the 500 mg/kg/day females, as noted above), the changes in urine pH were considered non-adverse. All other individual urinalysis values were considered within an acceptable range for biologic and procedure-related variation.

ORGAN WEIGHTS:

Interim: Test article-related organ weight changes were limited to the high dose groups. Increased liver weights occurred in males at 50 mg/kg/day and in female rats at 500 mg/kg/day. In males, the increase was small and only the mean liver relative to body weight was statistically significantly increased (14.53% above control). In females, the liver weight increase was larger (mean liver relative to body weight was 66.75% above control) and all parameters (absolute and relative to both brain and body weight) were statistically significantly increased. The liver weight changes in the affected male and female groups were associated with microscopic changes in the liver (discussed below). Mean final body weight at the interim necropsy was 19.51% less than control in the 500 mg/kg/day females. As a result of this decrement in mean final body weight, the brain, kidney, and thyroid/parathyroid relative to body weight were statistically significantly increased. Aside from a slight increase in severity of chronic progressive nephropathy in the kidneys, there were no microscopic changes in these organs associated with the increased weights, and mean absolute weights were not increased. Thus, these changes were considered secondary to the body weight decrement at 500 mg/kg/day. Additionally, mean absolute and relative to brain weights of the spleen in the 500 mg/kg/day females were statistically significantly lower than controls. These differences were not considered test article-related, as there were no microscopic changes in the spleen in either sex.

Terminal: No test article-related or statistically significant organ weight changes occurred in males. In females, the only test article-related effect on organ weights was an increase in liver weights at 500 mg/kg/day. Mean absolute and relative to both body and brain weights were increased compared to control, with mean liver relative to body weight 41.61% greater than control. There were several test article-related microscopic changes to account for the increased weights, as described below.

GROSS PATHOLOGY

Interim: A test article-related macroscopic observation, “irregular surface” of the kidneys, was noted in the kidneys of one 500 mg/kg/day (high dose) female. This observation correlated with mild chronic progressive nephropathy in this animal and was indicative of a slight increase in severity of chronic progressive nephropathy in the 500 mg/kg/day female group at one year. Other macroscopic observations were considered incidental and typical of lesions seen in rats of this strain and age.

Terminal: No test article-related macroscopic observations were noted in males. In females, test article-related macroscopic observations were noted in the kidneys and liver. In the kidneys, “irregular surface” was noted in 16 of 70 animals at 500 mg/kg/day (not present in controls or any of the lower dose groups), while in the liver, “tan focus/foci” was noted in 1, 1, 1, and 8 of 70 animals each at 0, 1, 50, and 500 mg/kg/day, respectively, and “mass/nodule” was noted in 14 of 70 animals at 500 mg/kg/day (not present in controls or any of the lower dose groups). These macroscopic observations were correlative to test article-related microscopic findings described below.

HISTOPATHOLOGY: NON-NEOPLASTIC:

Interim: Test article-related microscopic findings were noted in the liver of both male and female rats, and in the kidneys of females, in the high-dose groups (50 and 500 mg/kg/day for males and females, respectively). In males, there was a slight increase in minimal focal cystic degeneration of the liver (0, 0, 0, and 3 at 0, 0.1, 1, and 50 mg/kg/day, respectively). This finding was more pronounced in the terminal portion of the study. Also in males, there was a slight increase in minimal to mild focal necrosis of the liver (1, 1, 0, and 5 at 0, 0.1, 1, and 50 mg/kg/day, respectively). In females, the only microscopic finding in the liver was centrilobular hypertrophy, which occurred in all 10 of the 500 mg/kg/day females. This change was of minimal severity and was characterized primarily by a slight increase in size of centrilobular hepatocytes with increased red granularity to the cytoplasm and is consistent with peroxisome proliferation. Also in females, there was a very slight increase in incidence and severity of chronic progressive nephropathy in the kidneys at 500 mg/kg/day. This change was characterized by foci of basophilic tubules, some with thickening of basement membranes. In the 500 mg/kg/day group, most incidences were of mild severity, while in the other groups, including controls, the incidences were primarily of minimal severity, although in a single control female the incidence was of moderate severity (see table in any other information on results below). In males, there was a single interstitial cell adenoma of the testes at 50 mg/kg/day; incidences of interstitial cell hyperplasia were 1, 0, 0, and 3 at 0, 0.1, 1, and 50 mg/kg/day. The incidences of these changes in treated groups were not statistically different from controls (historical data for rats of this age were not available). Proliferative interstitial cell lesions are discussed in more detail under microscopic findings for the terminal sacrifice. All other microscopic findings were considered incidental, and typical of those seen in rats of this strain and age.

Terminal: Test article-related non-neoplastic microscopic changes were observed in the liver of males and in the liver, kidneys, nonglandular stomach (limiting ridge), and tongue of females at the highest doses tested, 50 mg/kg/day in males and 500 mg/kg/day in females.

Liver: In the liver of males at 50 mg/kg/day there were statistically significantly increased incidences of focal cystic degeneration, centrilobular hepatocellular hypertrophy, and centrilobular hepatocellular necrosis. Cystic degeneration was characterized by the presence of multilocular cystic spaces containing finely granular or flocculent material without endothelial or epithelial cells lining the spaces. Centrilobular hypertrophy, morphologically consistent with peroxisome proliferation, was characterized by hepatocytes with red granular cytoplasm sometimes containing small amounts of pigment morphologically compatible with lipofuscin. Centrilobular hepatocellular necrosis was typically of the coagulative type with strongly eosinophilic-staining cytoplasm and pyknotic nuclei. Test article-related findings in the liver of females at 500 mg/kg/day were similar to those noted in males at 50 mg/kg/day, and also included low incidences of panlobular hepatocellular hypertrophy and individual cell hepatocellular necrosis. Panlobular hepatocellular hypertrophy was characterized by enlargement of hepatocytes (as described above for centrilobular hypertrophy) throughout the entire liver. Individual cell necrosis was characterized by the presence of scattered single hepatocytes with features characteristic of apoptosis. See table in any other information on results below.

Kidneys: Statistically significantly increased microscopic findings in the kidneys of females at 500 mg/kg/day included tubular dilatation, oedema of the renal papilla, transitional cell hyperplasia in the renal pelvis, tubular mineralization, renal papillary necrosis, and chronic progressive nephropathy. Tubular dilatation frequently occurred in an ascending pattern extending from the papilla to the outer cortex, while at other times it was more prominent in the papilla. Oedema of the papilla was characterized by increased rarefaction or myxomatous change in the papillary interstitium, sometimes with polypoid protrusions from the lateral surface of the papilla. The oedema and tubular dilatation were often associated with hyperplasia of the transitional cell epithelium lining the papilla and pelvis. In some animals, necrosis of the tip of the papilla was present. In some 500 mg/kg/day females with the renal papillary changes, lesions diagnosed as chronic progressive nephropathy (CPN) were comprised of dilated tubules (often in an ascending pattern as described above), mononuclear cell infiltrates, and basophilic tubules, but with less thickening of tubular basement membranes than typically seen in CPN. In these animals, the constellation of lesions diagnosed as CPN may be more representative of retrograde nephropathy, rather than typical CPN. See table in any other information on results below.

Stomach: The nonglandular stomach (limiting ridge only) and the tongue had statistically significantly increased incidences of hyperplasia of squamous epithelium at 500 mg/kg/day. In the tongue, subacute/chronic inflammation occurred in association with squamous epithelial cell hyperplasia. There is no data describing incidence of epithelial hyperplasia of the limiting ridge of the nonglandular stomach in the historical control database for 2 year studies. The incidence of squamous cell hyperplasia of the tongue at 500 mg/kg/day (18.6%) exceeds the historical control range of 0-3.3%. There was also a single incidence of squamous cell carcinoma (1.4%) in the tongue of females at 500 mg/kg/day. This is well within the historical control range of 0-1.7% and the finding of a single such tumour was not considered a direct result of test article administration. See table in any other information on results below.

Other: A statistically significant increase in the incidence of alveolar histiocytosis was present in females at 500 mg/kg/day. The incidences were 22, 20, 21, 42 (61%) at 0, 1, 50, and 500 mg/kg/day, respectively. The incidence at 500 mg/kg/day was statistically significant by both the Fisher Exact test and the Cochran-Armitage trend test and is at the upper end of the historical control range of 9.2-61.7%. The increased incidence of this common background finding may be secondary to aspiration of dosing formulation at this high concentration; however, a definitive mechanism for this increase could not be determined. A slight but statistically higher (by the Cochran-Armitage Trend test) incidence of pancreatic acinar cell hyperplasia occurred in females at 50 and 500 mg/kg/day; incidences were 0, 2, 5, 5 (7.1%) at 0, 1, 50, and 500 mg/kg/day, respectively. The incidences of acinar cell hyperplasia at the two highest doses slightly exceeded the historical control range of 0-4.6%, but were not significant by the Fisher Exact test and were not associated with pancreatic acinar cell tumours. In addition, acinar cell hyperplasia did not occur in a clear dose response manner, as incidences in the 50 and 500 mg/kg/day groups were the same despite the order of magnitude difference in dose. In contrast, all other test article-related changes observed at 500 mg/kg/day occurred with a clear dose response. Therefore, the slight increase in acinar cell hyperplasia in the 50 and 500 mg/kg/day females was considered most likely spurious and not test article-related. A statistically significant increase (by both the Fisher Exact test and the Cochran-Armitage trend test) in the incidence of alopecia/hypotrichosis was present in females at 500 mg/kg/day. The incidences were 1/70, 2/48, 5/55, and 9/70 (12.9%). However, the relevance of alopecia/hypotrichosis is more appropriately made by interpretation of the incidence of this finding in the clinical observations of the study rather than the microscopic observations. Therefore, for microscopic purposes, this was not considered at potential target organ.

Finally, incidences of cataract of the lens of the eye, pelvic mineralization of the kidney, and angiectasis of the liver were statistically significantly increased. Cataract of the eye and angiectasis of the liver were statistically significantly increased by the Cochran-Armitage trend test at 500 mg/kg/day while pelvic mineralization of the kidney was statistically significantly increased by the Cochran-Armitage trend test and Fisher’s exact test at 500 mg/kg/day, and Fisher’s exact test at 1 mg/kg/day. Incidences of cataract of the eye were 0/69, 0/48, 0/55, and 3/70 (4.29%) at 0, 1, 50, and 500 mg/kg/day, respectively. The historical control range for cataract is 0 to 10.8%.15 Incidences of pelvic mineralization of the kidney were 52/70, 63/70, 58/70, and 63/70 (90.0%) at 0, 1, 50, and 500 mg/kg/day, respectively. The historical control range is 45.0 to 87.7% (note: two studies in the historical control database with an incidence of 0/60 reflect that this change was simply not tracked as pelvic mineralization in the studies). Incidences of angiectasis of the liver were 1/70, 0/70, 3/70, and 5/70 (7.14%) at 0, 1, 50, and 500 mg/kg/day, respectively. The historical control range is 0 to 10.0%. For each of the changes, the incidence was well within the historical control range, except pelvic mineralization, which is a very common background finding, only slightly exceeded the historical control range. Thus, these changes were not considered test article-related. All other non-neoplastic microscopic observations were of the type typically seen in rats of this strain and age, and were considered incidental and not related to test article administration.

HISTOPATHOLOGY: NEOPLASTIC : See any other information on results below for details.

Effect levels

1

Dose descriptor:

NOAEL

Effect level:

1 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male

Basis for effect level:

other: see 'Remark'

2

Dose descriptor:

NOAEL

Effect level:

50 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

female

Basis for effect level:

other: see 'Remark'

Target system / organ toxicity

Critical effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

Male NOAEL: 1 mg/kg
Female NOAEL: 50 mg/kg
This study and the conclusions which are drawn from it fulfill the quality criteria (validity, reliability, repeatability).

Executive summary:

This study was conducted to evaluate the potential chronic toxicity and oncogenicity of the test article when administered via oral gavage over the major portion of the life span of the test animals. Four treatment groups of 80 male and 80 female rats were administered the test article at respective dose levels of 0.1 (males only), 1, 50, and 500 (female only) mg/kg/day. One additional group of 80 animals/sex served as the control and received the vehicle, deionized water. The vehicle or test article was administered to all groups via oral gavage, once a day for up to 104 weeks, at a dose volume of 10 mL/kg/dose. Due to poor overall survival in females, all female dose groups were sacrificed at Week 101. In this report, this terminal interval will be referred to as 24 month.

Observations for morbidity, mortality, injury, and the availability of food and water were conducted twice daily (three times daily beginning in Week 53) for all animals. Observations for clinical signs and masses were conducted weekly. Body weight and body weight change were measured and recorded weekly for the first 14 weeks and then every other week thereafter. Food consumption was measured and recorded pretest (Week -1), weekly during the first 13 weeks, and for 2 week intervals starting on Week 14, and food efficiency was calculated. Ophthalmoscopic examinations were conducted all animals pretest and on all surviving animals prior to the interim and terminal necropsies. Blood and urine samples for clinical pathology evaluations were collected from designated animals at 3 (no coagulation or urine), 6, and 12 months. Peripheral blood smears were collected from designated animals at 12 and 18 months and prior to termination. At study termination (12 and 24 months), necropsy examinations were performed, organ weights were recorded, and tissues were microscopically examined.

There were no test article-related effects on survival. There were no adverse clinical or ophthalmological observations attributed to test article exposure. No adverse, test article-related effects on body weight or nutritional parameters were observed in males at any dose. Mean food consumption and food efficiency values were generally comparable to control throughout the study. Exposure to 500 mg/kg/day (females) produced adverse reductions in body weight, body weight gain, and food efficiency. Mean body weight in this group was 13% below control at Week 52 (statistically significant), but was generally comparable to the control value at termination. Mean body weight gain in this group was 20% below controls over Weeks 1 to 52 but only slightly lower than the control value (not statistically significant) over the two year period. There was no test article-related effect on food consumption. The reduced body weight gain was associated with lower mean food efficiency over the first year although overall (2-year) food efficiency was comparable to controls. The body weight, body weight gain, and food efficiency differences were considered adverse at this dose based on the magnitude of difference during the first year of exposure. No adverse effects on body weight or nutritional parameters were observed in any other dose group.

There were no test article-related effects on survival over the course of this study. The only test article-related cause of death/morbidity was inflammation/necrosis of the kidneys in 500 mg/kg/day females. This cause of death/morbidity was assigned to animals whose early death was considered to be the result of renal papillary necrosis observed microscopically. There was no test-article related increase in masses or mass findings.

At the 3, 6, and 12 month intervals, there were mild decreases in red cell mass (erythrocytes, haemoglobin, and hematocrit) in females receiving 500 mg/kg/day (up to 28% below control). These changes were associated with an appropriate increase in reticulocytes (up to 106% above control). There were no effects on erythrocyte morphology. This collection of findings is suggestive of red cell loss or increased red cell turnover (haemolysis) although the exact mechanisms involved are unknown. All of these findings were considered test article related and adverse. Statistically significant decreases in red cell mass were also present in males receiving 50 mg/kg/day at the 3- and 6-month interval. However, the decreases were small, did not induce statistically significant changes in reticulocytes, and were transient (no statistically significant differences at 12 months), and values in individual animals in the 50 mg/kg/day group were similar to controls. Therefore, the red cell mass changes in 50 mg/kg/day males were considered to be test article-related but nonadverse. No other effects on haematological parameters were attributed to test article exposure in either sex at any dose or interval. There were no test article-related effects among coagulation times in either sex at any dose level. No test article-related effects among leukocytes were observed in either sex.

Increases in enzymes indicative of liver injury were observed at 12 months in males at 50 mg/kg/day, including mild increases in alanine aminotransferase (ALT) and sorbitol dehydrogenase, and were correlated with microscopic findings of minimal cystic degeneration and minimal to mild focal necrosis in the liver of males at 50 mg/kg/day. Therefore, these enzymes changes were considered test article-related and adverse. There were also mild increases in alkaline phosphatase at the 3 and 6 month intervals in males at this dose level; these increases were less than those present at 12 months and were not associated with statistically significant changes in other enzymes indicative of hepatic or hepatobiliary injury at these time points. Therefore, the changes in alkaline phosphatase at the 3 and 6 month intervals may be due in part or in whole to test article-related enzyme induction, as the test article was previously shown to produce an increase in total P450 enzyme activity in male rats at 30 mg/kg/day. There were no test article-related changes in liver enzymes in males receiving 1 or 0.1 mg/kg/day or in females at any of the dose levels tested (up to 500 mg/kg/day). Increases in albumin were present in 50 mg/kg/day males at all intervals and in 500 mg/kg/day females at the 3-month interval. Decreases in globulin were present in 500 mg/kg/day females at all intervals (with an associated decrease in total protein at the 6-month interval). No statistically significant decreases in group means for globulin were present in males at any dose or interval; however, small decreases in individual values for these parameters in individual animals in the 50 mg/kg/day male group may have been test article-related. The changes in albumin and globulin in the high-dose male and female groups also resulted in statistically significant increases in albumin/globulin ratio in these groups at all intervals. These changes in serum proteins in high dose males and females were considered test article-related but were not considered biologically relevant based on their small magnitude and lack of association with known adverse outcomes. Statistically significant increases in urine volume and decreases in urine specific gravity were observed at 6- and 12-months in 500 mg/kg/day females, suggestive of a minimal diuresis. Although minimal and not associated with changes in kidney-related chemistry parameters, these differences may be correlative to increased incidences and severity of chronic progressive nephropathy observed in this dose group at the 1-year interim sacrifice.

At the interim necropsy, test article-related effects included irregular surface of the kidney (500 mg/kg/day females) and increased liver weights (50 mg/kg/day males and 500 mg/kg/day females). Microscopic pathology findings in the interim sacrifice groups included minimal focal cystic degeneration and minimal to mild focal necrosis of the liver (50 mg/kg/day males), centrilobular hypertrophy of the liver (500 mg/kg/day females), and increased incidence and severity of chronic progressive nephropathy in the kidney (500 mg/kg/day females).

At termination (2-year), test article-related increases in liver weight and macroscopic observations in the kidneys (irregular surface) and liver (tan focus/foci and mass/nodule) were observed in 500 mg/kg/day females. These macroscopic observations were correlative to test article-related microscopic findings described below.

At termination, test article-related non-neoplastic microscopic changes were observed at the highest doses tested in each sex: in the liver of 50 mg/kg/day males and in the liver, kidneys, nonglandular stomach (limiting ridge), and tongue of 500 mg/kg/day females. In the liver of males at 50 mg/kg/day, there were statistically significantly increased incidences of focal cystic degeneration, centrilobular hepatocellular hypertrophy, and centrilobular hepatocellular necrosis. Test article-related findings in the liver of 500 mg/kg/day females were similar to those noted in 50 mg/kg/day males, and also included low incidences of panlobular hepatocellular hypertrophy and individual cell hepatocellular necrosis. Microscopic findings in the kidneys of 500 mg/kg/day females included tubular dilatation, oedema of the renal papilla, transitional cell hyperplasia in the renal pelvis, tubular mineralization, renal papillary necrosis, and chronic progressive nephropathy (CPN). In some 500 mg/kg/day females, the constellation of lesions diagnosed as CPN may be more representative of retrograde nephropathy, rather than typical CPN. Statistically significantly increased incidences of hyperplasia of squamous epithelium were observed in the nonglandular stomach (limiting ridge only) and the tongue in females at 500 mg/kg/day. In the tongue, subacute/chronic inflammation occurred in association with squamous epithelial cell hyperplasia. Test article-related neoplastic changes occurred in the liver of females administered 500 mg/kg/day, and consisted of an increased incidence of hepatocellular adenoma and hepatocellular carcinoma. The increased incidences of hepatocellular tumours occurred in association with degenerative/necrotic changes in the liver at this dose level (see above under discussion of non-neoplastic lesions). No hepatocellular tumours and no test article-related degenerative or necrotic changes were observed in lower dose females and the incidence of hepatocellular tumours in males was comparable between the controls and the 50 mg/kg/day group. Equivocal increases in pancreatic acinar cell tumours and testicular interstitial (Leydig) cell tumours occurred in males administered 50 mg/kg/day. In males at 50 mg/kg/day, a statistically significant increase was observed in the incidence of pancreatic acinar cell adenoma/carcinoma combined, but not adenoma or carcinoma alone. In addition, the incidences of acinar cell hyperplasia were not significantly different from controls in any of the treated male groups. However, based on the known PPARα agonist activity of the test article, the marginal increase in pancreatic acinar cell tumours in the 50 mg/kg/day male group provides equivocal evidence of a test article-related effect. The incidence of interstitial cell adenoma of the testes was increased in males at 50 mg/kg/day, and one interstitial cell adenoma was also present in one male in the 50 mg/kg/day group at the interim necropsy. The incidence of interstitial cell hyperplasia at 50 mg/kg/day was also higher than in control males. Since PPARα agonists are known to produce proliferative interstitial cell lesions (hyperplasia and adenoma) in the testes of rats, a relationship to treatment for these findings in the 50 mg/kg/day male group cannot be ruled out. However, based on the marginal nature of the increased incidences of these lesions, their lack of statistical significance, and the relatively high incidence of these lesions in concurrent controls, the relationship to treatment for these findings is equivocal. The incidences of proliferative interstitial cell lesions of the testes in the 0.1 and 1 mg/kg/day groups, were similar to or less than controls. No adverse pathology findings occurred in male rats administered 0.1 or 1 mg/kg/day or in females administered 1 or 50 mg/kg/day.

Under the conditions of this study, the no-observed-adverse-effect level (NOAEL) for chronic toxicity of the test substance was 1 mg/kg/day in male rats and 50 mg/kg/day in females. The NOAEL in males is based on increases in focal cystic degeneration, focal necrosis, and centrilobular necrosis of the liver, with associated increases in cytotoxic liver enzymes, and equivocal increases in pancreatic acinar cell tumours and testicular interstitial (Leydig) cell tumours, all observed at 50 mg/kg/day. In females the NOAEL is based on reductions in body weight, body weight gain, and food efficiency; mild decreases in red cell mass; increases in individual cell necrosis in the liver, hyperplasia and/or inflammation in the nonglandular stomach and tongue; an increase in incidence and severity of microscopic pathology in the kidneys; and an increase in hepatocellular adenomas and carcinomas, all observed at 500 mg/kg/day.

Test article-related increases in hepatocellular adenoma and hepatocellular carcinoma were observed in females at 500 mg/kg/day. Equivocal increases in pancreatic acinar cell tumours and testicular interstitial (Leydig) cell tumours occurred in males administered 50 mg/kg/day. Clear thresholds were established for all of these tumour types, as test article-related tumour responses occurred only at the highest doses tested in males and females. Most research indicates that induction of these specific tumours in rats by non-genotoxic peroxisome proliferators likely has little or no relevance in humans, especially in plausible human exposure scenarios.