3-anilino-5-methyl-5-(4-phenoxyphenyl)-1,3-oxazolidine-2,4-dione;famoxadone (ISO)

Carcinogenicity

Description of key information

2-Year Rat Diet: Not Carcinogenic. OECD 453, OPP 83-5, JMAFF 59-NohSan-4200; Reliability = 1

18-Month Mouse Diet: Not Carcinogenic. OECD 451, OPP 83-2, JMAFF 59-NohSan-4200; Reliability = 1

18-Month Mouse Diet: Not Carcinogenic. OPPTS 870.4200; Reliability = 1

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:

supporting study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.4200 (Carcinogenicity)

Deviations:

no

GLP compliance:

yes

Specific details on test material used for the study:

Substance name: Famoxadone (DPX-JE874) Technical
Lot #: DPX-JE874-221
Purity: 97.3% by reanalysis

Species:

mouse

Strain:

other: Crl:CD-1®(ICR)BR

Sex:

male/female

Route of administration:

oral: feed

Vehicle:

other: acetone and diet

Analytical verification of doses or concentrations:

yes

Duration of treatment / exposure:

18 months

Frequency of treatment:

Daily

Dose / conc.:

2 000 ppm

Dose / conc.:

7 000 ppm

No. of animals per sex per dose:

50

Control animals:

yes, plain diet

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

In 7000 ppm male mice, there was a statistically significant increase in the incidence of pale mice and of stained fur/skin, and a statistically significant decrease in the incidence of hair loss. In 7000 ppm female mice, there was a statistically significant decrease in the incidence of superficial wounds of the ear and neck and of misshapen ears.

The increase in pallor in male mice may be compound related, as mild Heinz body anemia was observed in a previous study in male and female mice exposed to 7000 ppm for 90 days. However, the significance of this observation is not clear. Evaluation of red blood cell parameters is not required by carcinogenicity test guidelines and, therefore, was not evaluated in this current study. Furthermore, no increased incidence of pallor was observed in female mice. The increase in stained fur/skin was mainly due to an increase in stained perineum. This observation was not considered adverse, as there was no associated histopathology. It is not known whether the other statistically significant differences in clinical observations were compound related. However, they were not adverse as they all represented reductions in the incidence of these signs.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

No compound-related effects on survival were produced by dietary exposure to famoxadone in male or female mice. The percent survival on the final weigh day (test day 546) was 69%, 76%, and 64% in male mice and 68%, 73%, and 78% in female mice in the 0, 2000 ppm and 7000 ppm groups, respectively.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Mild but adverse, compound-related reductions (compared to control) in body weight and body weight gain were observed in male mice exposed to 7000 ppm. Mean body weight and body weight gain over the first 3 months of dietary exposure were generally comparable to control. Mean body weight on test day 91 and mean body weight gain over test days 0-91 were 97% and 88% of control, respectively (neither statistically significant). Body weight gain in this group was variable throughout the study, with mean body weight gain demonstrating statistically significant increases, statistically significant decreases, or no significant differences over individual weekly/biweekly intervals. However, mean body weight in this group was significantly below control on test days 49, 63, and 77, and on most days from test day 119 to the end of the study. Mean body weight on test days 273 and 546 (final weigh day) was 94% and 93%, respectively, of control (both statistically significant). Mean body weight gain was 80% (statistically significant) over test days 0-273 and 0-546.
No adverse compound-related differences in mean body weight or body weight gain were observed in the 2000 ppm male group. Mean body weight in this group was significantly below control on 2 test days (63 and 175), but mean body weight on test days 91, 273, and 546 was 97%, 97%, and 99% of control, respectively (none statistically significant). Mean body weight gain over test day 0-91, 0-273, and 0-546 was 83%, 89%, and 98% of control, respectively. The differences were not statistically significant over test days 0-273 or 0-546. This difference over test days 0-91 was statistically significant, but was not considered to be compound related, as there was no dose response over this period. Furthermore, individual weekly/biweekly interval body weight gain in this group demonstrated a mixed pattern of higher and lower weight gain (compared to control) over the entire 18 months.
No adverse compound-related differences in mean body weight or body weight gain were observed in either female group exposed to famoxadone. In the 7000 ppm group, mean body weight on test days 91, 273, and 546 was 98%, 96%, and 99% of control, respectively (none statistically significant). Mean body weight gain over test days 0-91, 0-273, and 0-546 was 88%, 83%, and 94% of control, respectively. In the 2000 ppm female group, mean body weight on test days 91, 273, and 546 was 98%, 99%, and 100% of control, respectively (none statistically significant). Mean body weight gain over test days 0-91, 0-273, and 0-546 was 91%, 96%, and 104% of control, respectively. Mean body weight was significantly lower than control in both groups on one (2000 ppm group) and three (7000 ppm group) test days. The differences in mean body weight gain were statistically significant in both groups over test days 0-91 and in the 7000 ppm group over test days 0-273. None of these statistically significant differences was considered adverse as they were not associated with significantly lower mean body weight, they were reversible, and individual weekly/biweekly interval body weight gain over the entire 18 months demonstrated a mixed pattern of higher and lower weight gain (compared to control).

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

No adverse compound-related effects on food consumption were observed in either male group exposed to the test substance. Mean food consumption in both groups was 102%-104% of control (not statistically significant) over test days 0-91, 0-273, and 0-546. Statistically significant increases and decreases in food consumption (relative to control) were observed over some individual weekly/biweekly intervals in both groups. In the 7000 ppm group, mean food consumption was more often significantly increased than decreased, especially over the first year of dietary exposure. This was probably compound related, but was considered an adaptive response to compensate for lower food efficiency in this group and, therefore, was not adverse. In the 2000 ppm group, statistically significant differences were observed over 2 intervals, but were not considered compound related as they were isolated incidences and were opposite effects (one was increased and one was decreased relative to control).

No adverse effects on food consumption were observed in either female group exposed to test substance. Mean food consumption in the 7000 ppm group over test days 0-91, 0-273, and 0-546 was 103%, 110%, and 109% of control, respectively. The 0-273 and 0-546 interval differences were statistically significant. Mean weekly/biweekly food consumption was significantly greater than in control over numerous intervals. These differences were considered compound related but not adverse, as they were considered to be an adaptive response to compensate for lower food efficiency in this group. Mean food consumption over test days 0-7 was significantly below control. It was considered a spurious observation, although it may represent reduced palatability. In the 2000 ppm female group, mean food consumption over test days 0-91, 0-273, and 0-546 was 102%, 103%, and 102% of control, respectively (none statistically significant). A statistically significant increase in mean food consumption was observed over test days 133-147, but was not considered compound related, as it was an isolated incidence.

The mean daily intake of the test substance over the entire exposure period, by male mice in the 2000 and 7000 ppm groups, was 246 and 887 mg/mg/kg/day, respectively. The mean daily intake of famoxadone over the entire exposure period, by female mice in the 2000 and 7000 ppm groups, was 348 and 1298 mg/kg/day, respectively. The differences in these values between males and females are due to differences in body weight gain and variability in food consumption by each sex during the study.

Food efficiency:

effects observed, treatment-related

Description (incidence and severity):

Effects on food efficiency generally reflected effects on body weight gain in both males and females. An adverse, compound-related reduction (compared to control) in mean food efficiency was observed in 7000 ppm males. Weekly mean food efficiency demonstrated statistically significant increases and decreases in food efficiency (relative to control), but food efficiency was more frequently reduced, and mean food efficiency over test days 0-91, 0-273, and 0-546 was 89%, 80%, and 80% of control, respectively. As was observed with mean body weight gain, this difference was not statistically significant during the first 3 months of dietary exposure, but was statistically significant over longer intervals. The reduction in food efficiency was partially compensated for by increased food consumption in the 7000 ppm mice. However, the 7000 ppm male mice were not able to fully compensate for the food efficiency reduction, resulting in lower mean body weight in this group. No compound-related effects on food efficiency were observed in the 2000 ppm male group. A few individual weekly/biweekly intervals demonstrated statistically significant differences but these included both increases and decreases, and were not considered compound related. Mean food efficiency over test days 0-91, 0-273, and 0-546 was 84%, 80%, and 100% of control, respectively (none statistically significant).

No adverse effects on food efficiency were observed in either female group. In the 7000 ppm group, weekly/biweekly mean food efficiency demonstrated statistically significant increases and decreases in food efficiency (relative to control). Mean food efficiency over test days 0-91, 0-273, and 0-546 was 82%, 83%, and 75% of control, respectively. Only the test day 0-273 interval difference was statistically significant. The lower mean food efficiency observed in this group was probably compound related but was not considered adverse as the differences were generally mild, most effects were not statistically significant, and there was no consistent pattern of decreased body weight gain over weekly/biweekly intervals. Furthermore, the mice were able to compensate for the reduced food efficiency by increasing food consumption, such that mean body weights generally did not differ significantly from control. In the 2000 ppm female group, mean food efficiency over test days 0-91, 0-273, and 0-546 was 91%, 100%, and 100% of control, respectively (none statistically significant). Statistically significant increases and decreases in food efficiency (relative to control) were observed over a few weekly/biweekly intervals. These differences were not considered compound related as they included opposite effects, most weekly/biweekly intervals were not statistically significant, and the test day 0-91, 0-273, and 0-546 intervals were not statistically significant.

Haematological findings:

no effects observed

Description (incidence and severity):

There were no statistically or toxicologically significant changes in white blood cell differential counts in mice fed 7000 ppm famoxadone, when compared to controls.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Compound-related, statistically significant weight changes occurred in the liver of both male and female mice. In males and females, mean absolute and relative (to both body weight and brain weight) liver weights were increased compared to controls at 2000 and 7000 ppm. These increases were similar in magnitude between concentration groups and between males and females.

There were no other compound-related organ weight effects. Other statistically significant weight changes occurred at 7000 ppm. These changes included an increase in mean absolute and relative (to both body weight and brain weight) adrenal gland weights in males and spleen weights in females. The increase in adrenal gland (male) weights were not accompanied by any significant microscopic findings and were considered spurious and therefore of no toxicological significance. Increased spleen weights in females at 7000 ppm were due to 2 mice with splenic tumors. These spleens were excessively heavy due to hemangiosarcoma and histiocytic sarcoma. Since there is no tumor effect in this study, the increased spleen weights were also considered spurious and of no toxicological significance.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

The only compound-related gross observation was an increase in number of large livers noted in 2000 and 7000 ppm male and female mice. This finding was attributed to hepatocellular hypertrophy.

There was an increased incidence of kidney discoloration in males at 7000 ppm (4/50, 2/50, and 15/50 in 0, 2000, and 7000 ppm groups, respectively). There were no compound-related microscopic findings observed in the kidneys, nor were any microscopic findings associated with this observation. Therefore, the kidney discoloration was considered spurious and biologically insignificant.

All other gross observations noted in males and females were consistent with commonly observed spontaneous and/or incidental findings in this age and strain of mouse.

One mass was too autolytic for microscopic evaluation. This was a uterine mass noted in a found dead female from the 7000 ppm dose level. The study conclusions were not affected by the inability to microscopically diagnose this mass.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Compound-related effects were noted in the liver of male and female mice at 2000 and 7000 ppm.

An increase in accumulation of a yellow brown pigment was noted within Kupffer cells of males and females at 7000 (statistically significant) and 2000 ppm. A similar pigment within Kupffer cells was described in the previous 18-month mouse study with this test compound. In that study special stains indicated the pigment was primarily lipofuscin. Lipofuscins are a heterogeneous group of compounds produced by progressive peroxidation processes and composed of variable amounts of protein and other end-products of auto-oxidation. Lipofuscin accumulates normally to some degree in various organs. Increases in lipofuscin accumulation in the liver have been noted with and without evidence of overt toxicity; however, the presence of increased lipofuscin suggests that an autocatalytic process of some nature has occurred.

Necrosis of individual hepatocytes was statistically significantly increased at 7000 ppm in males and was also a compound-related finding in females at 7000 ppm and in males and females at 2000 ppm, though not statistically significant. This lesion consisted of two types of cell necrosis. In the first type, individual necrotic hepatocytes were accompanied by an inflammatory cell infiltrate. This lesion is considered classical necrosis, and can be chemically induced. In the second type, the cytoplasm and nucleus of individual hepatocytes had condensed into acidophilic globules in the absence of an inflammatory cell reaction (apoptosis). Apoptosis is considered a natural, physiologic form of cell death essential for normal tissue homeostasis. However, apoptosis can also be a manifestation of toxic damage. Several compounds have been shown to induce apoptosis above normal background levels. Apoptosis was described as a compound-related finding in female mice at 2000 ppm in the first test substance study.

Centrilobular hepatocellular necrosis was statistically significantly increased at 7000 ppm in males and was also a compound-related finding in females at 7000 ppm, though not statistically significant. Centrilobular necrosis is the most common form of hepatocellular necrosis observed in animals exposed to various hepatotoxic agents.

Diffuse fatty change was statistically significantly increased in females at 7000 ppm. Fatty change can be a response to toxic agents, and was, in fact, increased in both males and females at 3500 and 7000 ppm in a 90-day feeding study with this compound.

Intrahepatocellular erythrocytic inclusions were statistically significantly increased in males at 2000 ppm and were also present in females at 2000 ppm and in males and females at 7000 ppm, though not statistically significant. This finding was characterized by randomly distributed hepatocytes containing erythrocytes. Erythrocytes appeared to be free in the hepatocellular cytoplasm and varied in number from a few to too-numerous-to-count. The hepatocyte nucleus was sometimes displaced to the cell periphery. This finding has been observed in treated animals from previous studies at this laboratory in animals dosed with different test substances and has been reported to occur in rats, mice, and humans. The presence of erythrocytes within hepatocytes has been speculated to be associated with hepatocellular hypoxia, chemical-induced hepatotoxicity, or altered hepatocytic function.

Increased mitotic figures were statistically significantly increased in females at 7000 ppm and were also present in males at 7000 ppm and in males and females at 2000 ppm, though not statistically significant. This finding is considered to be compound related and possibly associated with increased cell turnover subsequent to hepatocellular necrosis.

All of the following findings, when taken together, including diffuse fatty change in females, increased pigment (lipofuscin), hepatocellular necrosis, erythrocytic inclusions, and increased mitotic figures in males and females at 2000 and 7000 ppm are suggestive of hepatotoxicity at these concentrations.

Histopathological findings: neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

Hepatocellular hypertrophy was statistically significantly increased at 2000 and 7000 ppm in males and females. In males, the hypertrophy was primarily centrilobular; however, in females it was predominantly diffuse. Hepatocellular hypertrophy was characterized by enlargement of hepatocytes, either primarily around central veins (centrilobular), or primarily distributed throughout the liver lobule (diffuse). The cytoplasm of affected hepatocytes stained more homogeneously than the more granular/vacuolar cytoplasm of unaffected hepatocytes.

Hepatocellular hypertrophy was most likely the result of test substance metabolizing enzyme induction within hepatocytes. This hypertrophy was considered to be a physiologically adaptive response of the liver as a result of exposure to a xenobiotic and not biologically adverse.

The incidence of hepatocellular adenomas was increased in male mice at 2000 ppm (this increase also resulted in an increase in the combined incidence of animals with any hepatocellular neoplasm, diagnosed as “animals with at least one hepatocellular neoplasm”). Incidences of hepatocellular adenomas (combined single and multiple) across male groups were 6/50 (12%), 12/50 (24%), and 1/50 (2%) for dose groups 0, 2000, and 7000 ppm, respectively. Although the incidence of animals with hepatocellular adenomas in 2000 ppm males was outside the historical control range for this laboratory, this increase was not considered to be compound-related for the following reasons:
1. Incidences of neither hepatocellular adenomas nor combined adenomas/carcinomas were statistically significant by either the Fisher’s exact test or the Cochran-Armitage trend test.
2. The increased incidences of these lesions were not dose related, as the incidence of liver tumors in the 7000 ppm group was actually less than in controls. There were no mitigating factors, such as decreased survival or body weight effects, in 7000 ppm males to explain this lack of a dose response. Furthermore, metabolism studies indicate that the internal dose of test substance (as measured by both Cmax and AUC of radioactive equivalents) was significantly increased in mice fed 7000 ppm compared to those fed 2000 ppm. Thus, if the increased incidence of hepatocellular tumors in 2000 ppm males was compound-related, a similar or greater incidence would be expected at 7000 ppm.
3. There was no increase in hepatocellular foci (putative preneoplastic lesions) and no increase in hepatocellular carcinomas in 2000 ppm males compared to controls.
4. There was no statistically significant increase in liver tumors in female mice at any dose.
5. In the previous 18-month mouse study with test substance, liver tumor incidence in male mice fed 2000 ppm was comparable to control groups of both 18-month studies.

All other microscopic observations noted are known to occur spontaneously in mice of this strain and age and were not present in a dose response fashion in either incidence or severity.

Key result

Dose descriptor:

NOAEL

Remarks:

for neoplastic effects

Effect level:

> 7 000 ppm

Key result

Dose descriptor:

LOAEL

Remarks:

for non-neoplastic effects

Effect level:

2 000 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

food consumption and compound intake

food efficiency

histopathology: non-neoplastic

Conclusions:

The test substance is not an oncogen in mice

Executive summary:

An 18-month feeding study was conducted following US EPA guideline OPPTS 870.4200, to evaluate the oncogenicity of test substance when administered to mice in a nutritionally adequate diet. Three groups of 50 male and three groups of 50 female Crl:CD-1®(ICR)BR mice were fed diets containing either 0, 2000, or 7000 ppm test substance for approximately 18 months. Samples of diet were analyzed near the beginning of the study to verify concentration and homogeneity of the test substance. Stability of test substance in diets, under the conditions of the study, had been demonstrated in a previous study. Six times during the study, at approximately 3-month intervals, diet samples were collected from feeders to verify concentrations of the test substance in the feed. Body weight and individual food consumption were measured weekly for the first 3 months, then every other week for the remainder of the study. Clinical signs of toxicity were monitored throughout the study. A differential blood count was conducted on blood smears collected at 18 months from the control and high-dose groups. All mice found dead, sacrificed in extremis, accidentally killed, or sacrificed by design were necropsied. A gross necropsy was performed on all mice and selected tissues were examined microscopically.

The mean daily intake of test substance by male mice in the 2000 and 7000 ppm groups was 246 and 887 mg/kg/day, respectively (test days 0-546). The mean daily intake of test substance by female mice in the 2000 and 7000 ppm groups was 348 and 1298 mg/kg/day, respectively (test days 0-546).

Compound-related reductions (compared to control) in mean body weight, body weight gain, and food efficiency were observed in males fed 7000 ppm of test substance. Effects were mainly observed after the first three months of dietary exposure. No adverse, compound-related effects on body weight, body weight gain, or food efficiency were observed in males fed 2000 ppm, and no adverse effects on food consumption were observed in any male group. No adverse, compound related effects on body weight, body weight gain, or food consumption, were observed in any female group. Mean food efficiency in 7000 ppm females was below control over the 18-month interval, but was not considered adverse as the difference was not statistically significant, and there was no associated adverse reduction in body weight or body weight gain. Mean food consumption in 7000 ppm males and females was generally greater than in control (variable statistical significance). This increase was considered an adaptive response to compensate for the reduction in food efficiency, and was not considered adverse.

No compound-related effects on survival were observed in males or females. The incidence of a few non-specific clinical observations was significantly increased or decreased in male or female mice fed 7000 ppm. However, the differences in incidence were likely due to biological variability and were not considered to be compound-related effects.

There were no compound-related effects on differential blood count observed in males or females.

The test substance was not oncogenic in male or female mice. Dietary exposure of males and females to 2000 and/or 7000 ppm produced microscopic evidence of hepatotoxicity, including diffuse fatty change (females only), increased lipofuscin pigment, individual cell necrosis, erythrocytic inclusions, and increased mitotic figures. Compound-related increases in liver weight (absolute and relative) and hepatocellular hypertrophy were also observed in males and females fed 2000 or 7000 ppm, but these changes were considered to be non-adverse physiologically adaptive responses to exposure to a xenobiotic.

Under the conditions of this study, famoxadone was not an oncogen in male or female mice. The no-observed-effect level (NOEL) was not determined in the current study, based on an increase in microscopic liver lesions indicative of hepatotoxicity in mice exposed to 2000 or 7000 ppm test substance. Compound-related effects on body weight, body weight gain, and food efficiency were also observed in 7000 ppm male mice. The effects observed in this study were generally consistent with those observed in the previously conducted study, and support an overall NOEL of 700 ppm for famoxadone in male and female mice following chronic exposure. This level was equivalent to 95.6 and 130 mg/kg/day in male and female mice, respectively.

Reference 2

Endpoint:

carcinogenicity: oral

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: MAFF Japan NohSan No. 4200

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPP 83-5 (Combined Chronic Toxicity / Carcinogenicity)

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 453 (Combined Chronic Toxicity / Carcinogenicity Studies)

Deviations:

no

GLP compliance:

yes

Specific details on test material used for the study:

Substance name: Famoxadone (DPX-JE874) Technical
Lot #: DPX-JE874-221
Purity: 97.4%

Species:

rat

Strain:

other: Crl:CD®BR

Details on species / strain selection:

The Crl:CD®BR rat was selected on the bases of extensive experience with this strain and its suitability with respect to hardiness, longevity, sensitivity, and low incidence of spontaneous diseases.

Sex:

male/female

Route of administration:

oral: feed

Vehicle:

other: diet

Analytical verification of doses or concentrations:

yes

Duration of treatment / exposure:

Approximately 2 years

Frequency of treatment:

Daily

Dose / conc.:

10 ppm

Remarks:

Equivalent to 0.422 mg/kg in males and 0.528 mg/kg in females

Dose / conc.:

40 ppm

Remarks:

Equivalent to 1.62 mg/kg in males and 2.15 mg/kg in females

Dose / conc.:

200 ppm

Remarks:

Equivalent to 8.37 mg/kg in males and 10.7 mg/kg in females

Dose / conc.:

400 ppm

Remarks:

Equivalent to 16.8 mg/kg in males and 23.0 mg/kg in females

No. of animals per sex per dose:

92

Control animals:

yes, plain diet

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

The incidence of ruffled fur in male rats demonstrated a statistically significant, increasing trend at the 400 ppm concentration. This sign is non-specific and the increased incidence is not indicative of any specific organ toxicity. No other clinical signs of toxicity were increased in incidence in any group due to exposure to the test substance.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

No compound-related effects on survival were produced by dietary exposure to the test substance in male or female rats. The percent survival at the final weigh day was 32, 34, 44, 43 and 31% in male rats and 38, 40, 26, 34, and 37% in female rats at 10, 40, 200, and 400 ppm, respectively

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

In female rats at 400 ppm, statistically significant, compound-related decreases were observed in final body weight (82% of control), overall body weight gain (75% of control; test day 0-713).

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

Food consumption was comparable among all test groups within a sex over the 2 years of dietary exposure. In females, food consumption in the 400 ppm group was statistically significantly lower than controls over the 0-91 test day interval; however, overall food consumption in this group (test days 0-713) was 99% of the control group mean.
The mean daily intake of the test substance in the 10, 40, 200, and 400 ppm groups was 0.422, 1.62，8.37, and 16.8 mg/kg, respectively, in male rats (test day 0-679) and 0.528, 2.15, 10.7, and 23.0 mg/kg, respectively, in female rats (test day 0-713).

Food efficiency:

effects observed, treatment-related

Description (incidence and severity):

In female rats at 400 ppm, statistically significant, compound-related decreases were observed in food efficiency (75% of control).

Ophthalmological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Ophthalmoscopic examinations performed on all surviving rats after approximately 12 months of dietary exposure and near the termination of the study revealed no abnormalities attributed to administration of the test substance. A low incidence of cataracts is was observed and is considered to be a frequent finding in populations of aging laboratory rodents.

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Male and female rats at 400 ppm demonstrated evidence of compound-related hemolysis. Indicators of circulating red cell mass (erythrocyte count, hemoglobin, and/or hematocrit) were decreased at all sampling times over the first 12 or 18 months of the study in males and females, respectively. Although the decreases were of a small magnitude, they were considered to be adverse as they were of sufficient magnitude to stimulate increased erythropoiesis. No compound related effects on hematology were observed in male or female rats at 200 ppm or below.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

No biologically significant effects on clinical chemistry were evident in any group. A few statistically significant changes were observed but were not considered to be toxicologically significant because they were of too small a magnitude to be biologically important, they lacked a monotonic dose-response, and/or they were not biologically relevant.

Urinalysis findings:

effects observed, non-treatment-related

Description (incidence and severity):

No biologically significant effects on urinalysis were evident in any group. A few statistically significant changes were observed but were not considered to be toxicologically significant because they were of too small a magnitude to be biologically important, they lacked a monotonic dose-response, and/or they were not biologically relevant.

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

Rats exposed to the test substance did not demonstrate any compound-related organ weight changes.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Compound-related gross lesions were observed in both the 1-year interim sacrifice and the 2-year study. At 1 year, an individual 400 ppm male rat had a compound-related gross liver lesion; after 2 years, a slight increase in the incidence of large spleens was noted in the 400 ppm males and females.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

At the interim sacrifice, the hemolytic effects observed in male and female rats at 400 ppm were associated with histopathologic changes. These changes included bone marrow mixed hyperplasia (males), increased splenic extramedullary hematopoiesis (males), increased splenic macrophage pigment (males), and increased Hepatic Kupffer cell pigment (females). These lesions, together with the hematological changes, indicate a compound-related hemolysis with a compensatory increase in erythropoiesis. At the 2-year necropsy, a small increase in the incidence of large spleens was observed in 400 ppm males and females. This observation was also considered to be a response to the hemolytic effects. The increases in pigment and the gross and histologic changes are not considered to represent liver or spleen toxicity but are considered to be secondary to the hematological changes. Male and female rats at 400 ppm demonstrated histopathologic evidence of hepatocellular toxicity. I n male rats, this evidence consisted of an increased incidence of focal cystic degeneration (after 1- year only) and focal hepatocellular degeneration. In female rats, the incidence of focal hepatocellular degeneration was increased after 2 years of dietary exposure to the test substance. These lesions were not associated with an increase in mean serum levels of cytoplasmic liver enzymes; this lack of association was attributed to the small size of the liver lesions. A compound-related increase in the incidence, size, and severity of eosinophilic foci of cellular alterations was also observed in male rats at 400 ppm. The foci of hepatocellular degeneration were occasionally associated with or incorporated into these eosinophilic foci. In one 400 ppm male rat the interim sacrifice, the eosinophilic foci were associated with the gross observation at necropsy of dark, diffuse foci. The biological significance of eosinophilic foci is not clear, as evidence suggests many foci of this type do not progress to neoplasms and will regress if exposure to the inciting compound ceases. In this study, there was no evidence of compound-related liver neoplasia.
Both male and female rats at 400 ppm demonstrated compound-related hepatocellular hypertrophy. Hepatocellular hypertrophy is considered a pharmacological response of the liver to exposure to xenobiotics which induce smooth endoplasmic reticulum and its associated enzymes, including metabolizing enzymes. In this study, a small elevation in the level of cytochrome P-450 was measured in female rats at 400 ppm at the 1-year time point; however, no increases in cytochrome P-450 were measured in females after 2 weeks or in male rats at either time point. In female rats at 400 ppm, there was also an increased incidence of apoptosis noted after 2 years. The hypertrophy, apoptosis, and elevated cytochrome P-450 are considered to be adaptive responses and are not considered to be adverse.

Histopathological findings: neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

A statistically significant increase in the incidence of testicular adenomas with an equivocal increase in the size of the adenomas was observed in 400 ppm males, by trend analysis. This lesion was not considered to be compound related because:
• The incidence was within the test facility historical control range for this lesion.
• The difference between the 400 ppm group mean and the historical control data mean was not statistically significant by pair-wise comparison.
• There was no dose-related increase in the incidence of interstitial cell hyperplasia, a lesion considered to precede interstitial cell adenoma development.

Relevance of carcinogenic effects / potential:

The test substance was not an oncogen in male or female rats. A statistically significant increase in the incidence of testicular adenomas with an equivocal increase in the size of the adenomas was observed in 400 ppm males, by trend analysis. This lesion was not considered to be compound related because:
• The incidence was within the test facility historical control range for this lesion.
• The difference between the 400 ppm group mean and the historical control data mean was not statistically significant by pair-wise comparison.
• There was no dose-related increase in the incidence of interstitial cell hyperplasia, a lesion considered to precede interstitial cell adenoma development.

Key result

Dose descriptor:

NOAEL

Remarks:

for neoplastic effects

Effect level:

> 400 ppm

Based on:

test mat.

Sex:

male/female

Remarks on result:

other: The test substance was not an oncogen in male or female rats

Dose descriptor:

NOAEL

Remarks:

for non-neoplastic effects

Effect level:

40 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

haematology

Conclusions:

The test substance is not an oncogen in male or female rats.

Executive summary:

A 2-year feeding study was performed to evaluate the chronic toxicity and oncogenicity of the test substance when administered to rats in the diet according to the guidelines OECD 453 and EPA 83-5. Five groups of 92 male Crl:CD®BR rats and 92 female rats were fed diets containing either 0, 10，40, 200, or 400 ppm for approximately 23 (males) or 24 (females) months. Samples of diet were analyzed near the beginning of the study to verify concentration, homogeneity, and stability of the test substance in the diets. Eight times during the study, at approximately 3-month intervals, samples were collected from feed jars to verify concentrations of the test substance in the feed. Body weight and individual food consumption were measured weekly for the first 3 months and every other week for the remainder of the study. Clinical signs of toxicity were monitored throughout the study.

Ophthalmologic evaluations were performed on all rats before the study start, and on all surviving rats after approximately 1 year (test day 346) and 22 months (test day 666) of dietary exposure. Clinical pathology evaluations were performed on 10 rats per group at approximately 3, 6, 12, 18, and 22 (male) or 23 (female) months after initiation of dietary exposure. After approximately 2 weeks and 12 months of exposure to the test substance, cell proliferation was evaluated in the livers of 5 rats from each group. At each of these time points, 5 additional rats from each group were sacrificed and evaluated for hepatic peroxisomal beta-oxidation activity and hepatic cytochrome P-450 content. An interim sacrifice was conducted after approximately 1 year in which 10 male and 10 female rats from each group were sacrificed and necropsied. All surviving rats were sacrificed and necropsied after approximately 23 (male) or 24 (female) months. All rats sacrificed in Extremis, found dead, or accidentally killed were also necropsied. A gross examination was performed all rats and selected tissues were examined microscopically.

The mean daily intake of the test substance in the 10, 40, 200, and 400 ppm groups was 0.422, 1.62，8.37, and 16.8 mg/kg, respectively, in male rats (test day 0-679) and 0.528, 2.15, 10.7, and 23.0 mg/kg, respectively, in female rats (test day 0-713).

No compound-related effects on body weight, body weight gain, food consumption, or food efficiency were observed in male rats at any concentration.

In female rats at 400 ppm, statistically significant, compound-related decreases were observed in final body weight (82% of control), overall body weight gain (75% of control; test day 0-713), and food efficiency (75% of control). Food consumption was not significantly decreased over the 0-713 test day interval, although food consumption was significantly lower than in controls at earlier time intervals (test day 0-91). No biologically important changes in any of the above parameters were seen in female rats at 200 ppm or below.

Dietary exposure to the test substance did not affect survival in male or female rats. There were no compound-related increases in any clinical signs of toxicity in any group during the study, except for a statistically significant increase in the incidence of ruffled fur in male rats at 400 ppm. This sign is non-specific, and the increased incidence is not indicative of any specific organ toxicity. Ophthalmoscopic examinations performed on all surviving rats after approximately 12 months of dietary exposure and near the termination of the study revealed no abnormalities attributed to administration of the test substance.

Male and female rats at 400 ppm demonstrated evidence of compound-related hemolysis. Indicators of circulating red cell mass (erythrocyte count, hemoglobin, and/or hematocrit) were decreased at all sampling times over the first 12 or 18 months of the study in males and females, respectively. Although the decreases were of a small magnitude, they were considered to be adverse as they were of sufficient magnitude to stimulate increased erythropoiesis (as evidenced by an increase in reticulocytosis and in associated histological changes). No compound related effects on hematology were observed in male or female rats at 200 ppm or below and no biologically significant effects on clinical chemistry or urinalysis were evident in any group. A few statistically significant changes were observed but were not considered to be toxicologically significant because they were of too small a magnitude to be biologically important, they lacked a monotonic dose-response, and/or they were not biologically relevant.

The test substance was not an oncogen in male or female rats. A statistically significant increase in the incidence of testicular adenomas with an equivocal increase in the size of the adenomas was observed in 400 ppm males, by trend analysis. This lesion was not considered to be compound related because:

• The incidence was within the test facility historical control range for this lesion.


• The difference between the 400 ppm group mean and the historical control data mean was not statistically significant by pair-wise comparison.


• There was no dose-related increase in the incidence of interstitial cell hyperplasia, a lesion considered to precede interstitial cell adenoma development.

Rats exposed to the test substance did not demonstrate any compound-related organ weight changes. Compound-related gross and/or non-neoplastic microscopic effects were observed in the liver, spleen, and/or bone marrow of male and female rats at 400 ppm after 1 and/or 2 years.

At the interim sacrifice, the hemolytic effects observed in male and female rats at 400 ppm were associated with histopathologic changes. These changes included bone marrow mixed hyperplasia (males), increased splenic extramedullary hematopoiesis (males), increased splenic macrophage pigment (males), and increased Hepatic Kupffer cell pigment (females). These lesions, together with the hematological changes, indicate a compound-related hemolysis with a compensatory increase in erythropoiesis. At the 2-year necropsy, a small increase in the incidence of large spleens was observed in 400 ppm males and females. This observation was also considered to be a response to the hemolytic effects. The increases in pigment and the gross and histologic changes are not considered to represent liver or spleen toxicity but are considered to be secondary to the hematological changes.

Male and female rats at 400 ppm demonstrated histopathologic evidence of hepatocellular toxicity. In male rats, this evidence consisted of an increased incidence of focal cystic degeneration (after 1-year only) and focal hepatocellular degeneration. In female rats, the incidence of focal hepatocellular degeneration was increased after 2 years of dietary exposure to the test substance. These lesions were not associated with an increase in mean serum levels of cytoplasmic liver enzymes; this lack of association was attributed to the small size of the liver lesions.

A compound-related increase in the incidence, size, and severity of eosinophilic foci of cellular alterations was also observed in male rats at 400 ppm. The foci of hepatocellular degeneration were occasionally associated with or incorporated into these eosinophilic foci. In one 400 ppm male rat the interim sacrifice, the eosinophilic foci were associated with the gross observation at necropsy of dark, diffuse foci. The biological significance of eosinophilic foci is not clear, as evidence suggests many foci of this type do not progress to neoplasms and will regress if exposure to the inciting compound ceases. In this study, there was no evidence of compound-related liver neoplasia.

Both male and female rats at 400 ppm demonstrated compound-related hepatocellular hypertrophy. Hepatocellular hypertrophy is considered a pharmacological response of the liver to exposure to xenobiotics which induce smooth endoplasmic reticulum and its associated enzymes, including metabolizing enzymes. In this study, a small elevation in the level of cytochrome P-450 was measured in female rats at 400 ppm at the 1-year time point; however, no increases in cytochrome P-450 were measured in females after 2 weeks or in male rats at either time point. In female rats at 400 ppm, there was also an increased incidence of apoptosis noted after 2 years. The hypertrophy, apoptosis, and elevated cytochrome P-450 are considered to be adaptive responses and are not considered to be adverse.

A few statistically significant changes were observed in organ weights and in the incidence of neoplastic or non-neoplastic observations. These changes were not considered to be compound related because the incidences lacked a monotonic dose-response, the changes were considered secondary to normal age-related pathology, and/or the incidences fell within the range of test facility historical controls.

The no-observed-effect level (NOEL) for this study is defined as the highest dose at which toxicologically important effects attributable to the test substance were not detected. Thus, for this study, the NOEL is equivalent to the NOEL as defined by the United States Environmental Protection Agency (1985) and the no-observed-adverse-effect level (NOAEL) as defined by the European Union (1994).

Under the conditions of this study, the test substance is not an oncogen in male or female rats. The NOEL is 200 ppm for male and female rats. This level is equivalent to 8.37 and 10.7 mg/kg/day in male and female rats, respectively.

In male rats, the NOEL is based on an increased incidence of microscopic lesions indicative of hepatotoxicity, and hemolysis of sufficient magnitude to produce a decrease in circulating erythrocyte mass with a compensatory increase in erythropoiesis, all observed at 400 ppm. A few other compound-related lesions were observed in the liver, bone marrow, and spleen at 400 ppm but were considered to be either adaptive hepatocellular responses or secondary responses to the hemolysis.

In female rats, the NOEL is based on compound-related decreases in body weight and food efficiency, an increased incidence of microscopic lesions indicative of hepatotoxicity, and hemolysis of sufficient magnitude to produce a decrease in circulating erythrocyte mass with a compensatory increase in erythropoiesis, all observed at 400 ppm. A few other compound-related lesions were observed in the liver at 400 ppm but were considered to be either adaptive hepatocellular responses or secondary responses to the hemolysis.

Reference 3

Endpoint:

carcinogenicity: oral

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: MAFF Japan NohSan No. 4200

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPP 83-2 (Carcinogenicity)

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 451 (Carcinogenicity Studies)

Deviations:

no

GLP compliance:

yes

Specific details on test material used for the study:

Substance name: Famoxadone (DPX-JE874) Technical
Lot #: DPX-JE874-221
Purity: 97.4%

Species:

mouse

Strain:

other: Crl:CD-1®(ICR)BR

Sex:

male/female

Route of administration:

oral: feed

Vehicle:

other: acetone and diet

Analytical verification of doses or concentrations:

yes

Duration of treatment / exposure:

18 months

Frequency of treatment:

Daily

Dose / conc.:

5 ppm

Dose / conc.:

50 ppm

Dose / conc.:

700 ppm

Dose / conc.:

2 000 ppm

No. of animals per sex per dose:

80

Control animals:

yes, plain diet

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

The incidence of a few clinical signs (pallor, stained or wet fur) in male mice demonstrated a statistically significant increasing trend at the 2000 ppm concentration. However, none of these signs was considered to represent compound-related toxicity as there were no relevant associated effects in clinical pathology or histopathology. The increased incidences were considered to be due to biological variability of commonly observed clinical signs in aging laboratory mice. No compound-related clinical signs were observed in female mice.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

No compound-related effects on survival were produced by dietary exposure to the test substance in male or female mice. The percent survival at the final weigh day was 70, 70, 73, 75, and 68% in male mice and 78, 83 , 72, 72, and 72% in female mice at 0, 5, 50, 700, and 2000 ppm, respectively.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

No compound-related effects on body weight or body weight gain were produced in male or female mice by dietary exposure to test substance. Final mean body weights at 2000 ppm were 98 and 102% of controls in male and female mice, respectively. Mean body weight gains in these groups, over the 0-546 test day interval, were 101% of controls for both males and females.

Sporadic occurrences of significantly increased or decreased body weight gain were observed in male and female mice at all concentrations, compared to controls. These differences were statistically significant; however, they were not considered to be compound-related nor of toxicological significance, due to the lack of a time-or a dose-response relationship.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

No toxicologically significant, compound-related effects on food consumption were produced by dietary exposure to test substance. Statistically significant increases in food consumption were observed in 50 ppm female mice over test days 0-91 and 0-546 and in 2000 ppm female mice over test days 0-546. None of these increases were considered toxicologically significant because they only occurred over sporadic time intervals, they did not demonstrate consistent dose-response relationships, and they were not associated with any significant effects on overall mean body weight.
Due to multiple errors in weighing feeders during the 392-406 and 406-420 test day intervals, mean food consumption, food efficiency, and daily intake data for these intervals were not accurate. However, these errors did not significantly affect the data for these parameters over the entire study or over any large interval. Therefore, the errors did not adversely affect the overall interpretation of the effect of dietary exposure to the test substance on these parameters.

The mean daily intakes of the test substance by male mice in the 5, 50, 700, and 2000 ppm groups were 0.701, 6.78, 95.6, and 274 mg/kg, respectively. Mean daily intakes of test substance in female mice in the 5, 50, 700, and 2000 ppm groups were 0.956, 9.84, 130, and 392 mg/kg, respectively. The differences in these values between sexes result from differences in body weight gain and variability in food consumption by each sex during the study.

Food efficiency:

effects observed, non-treatment-related

Description (incidence and severity):

No toxicologically significant, compound-related effects on food efficiency were produced by dietary exposure to test substance. Statistically significant increases in food efficiency were observed in 50 ppm male mice and in 200 ppm female mice over test days 0-91. None of these increases were considered toxicologically significant because they only occurred over sporadic time intervals, they did not demonstrate consistent dose-response relationships, and they were not associated with any significant effects on overall mean body weight.

Ophthalmological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Ophthalmoscopic examinations performed on all surviving mice after approximately 9 months of dietary exposure and near the termination of the study revealed no abnormalities attributed to administration of the test substance. Cataracts (unilateral or bilateral) were observed in 4 mice (2 males, 2 females) at the 9-month exam and in 5 mice (1 male, A females) at the final exam. These lesions were not considered compound-related because there was no dose-response in either sex (only 1 of these lesions was observed in a high-dose animal). A low incidence of cataracts is considered to be a frequent abnormality in aging laboratory rodents. a few other lesions (retinal degeneration, panophthalmitis, corneal opacity, phthisical globe, retinal pallor) were observed in some mice on study. None were considered compound-related because there was no dose-response for any lesion. All were considered to be either normal aging effects or secondary to trauma during orbital sinus bleeding.

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

No toxicologically important hematologic changes occurred during this study. Several statistically significant hematologic changes were observed, but they were not considered to be toxicologically important.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

Toxicologically important changes in plasma protein concentration did not occur during this study. Statistically significantly increased plasma protein concentration in 5 ppm males at the 18-month sampling time was not considered to be compound-related because the mean values did not exhibit a dose-response relationship.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Final body weights determined just prior to necropsy were used in the assessment of organ weight changes. Male kidney weights (absolute and relative to both body and brain weight) and female absolute liver weights were evaluated by nonparametric statistics as Bartlett’s test for homogeneity of variances was significant for these data sets.
Compound-related, statistically significant weight changes occurred in the liver of both male and female mice. These increases were dose-related and were more marked in the females than in the males. In males, mean absolute and relative (to both body weight and brain weight) liver weights were increased as compared to controls at 700 and 2000 ppm. In females, mean absolute liver weights were increased at 50, 700, and 2000 ppm, however, mean relative (to both body weight and brain weight) liver weights were only increased at 700 and 2000 ppm.

Other statistically significant weight changes occurred only at 2000 ppm. These included an increase in mean relative (to brain) testes weights in males and in females, an increase in mean absolute and relative (to both body and brain) kidney weights, and an increase in mean absolute and relative (to brain) spleen weights. The increase in testes (males) and kidney (females) weights were not accompanied by any significant microscopic findings and were considered spurious and therefore of no toxicological significance. In addition to the increase in spleen (females) weights, there was also an increased incidence of large spleens noted at necropsy. However, there were no compound-related microscopic findings or hematology findings to explain the increased spleen weights, thus, they were considered spurious and of no toxicological significance.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

The only compound-related gross observation was an increase in the number of large livers noted in the 2000 ppm male and female mice. This finding was attributable to hepatocellular hypertrophy. Additionally, an increased number of large spleens was noted in the 2000 ppm females.

One mass was unavailable for microscopic evaluation. This was a liver mass noted in a 700 ppm female. The mass was not apparent at trimming. It is not clear whether the mass was lost or whether this was a recording error, however in any case, the study conclusions were not affected by the unavailability of this mass.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Compound-related effects were noted in the liver of male and female mice at 700 and 2000 ppm, and, in females, equivocally at 50 ppm. Further, a compound-related increase in the incidence of amyloidosis was noted in the 2000 ppm females.

Focal hepatocellular necrosis was statistically significantly increased at 2000 ppm in males. Affected areas were generally small and consisted of hepatocytes which had undergone coagulative necrosis. While this change is not uncommon in mice and can result from infectious agents, more frequently the cause is unknown. However, it can also occur as the result of xenobiotic administration.

Diffuse fatty change was statistically significantly increased at 2000 ppm in males, however the increased incidence was slight and minimal in severity. Fatty change can be produced in the liver by a variety of agents, and was, in fact, increased in both males and females at 3500 and 7000 ppm in a 90-day feeding study with this compound. However, taken alone, it is doubtful that an increased incidence of diffuse fatty change this small could be considered biologically adverse.

Apoptosis was statistically significantly increased at 2000 ppm in females. Apoptosis is considered a natural, physiologic form of cell death essential for normal tissue homeostasis, Several compounds have been shown to induce apoptosis above normal background levels.

In this study, the mechanism responsible for accumulation of the lipofuscin pigment is not absolutely clear. While focal hepatocellular necrosis (males) and apoptosis (females) were increased, the incidence of these findings was not commensurate with the degree of lipofuscin accumulation. However, all of the above findings taken together, including diffuse fatty change and focal necrosis in males, apoptosis in females, and increased pigment (Lipofuscin) in both males and females at 2000 ppm are suggestive of slight hepatоtoxicity at this concentration.

Aside from the compound-related lesions noted in the liver, the only other compound-related lesion was a statistically significantly increased incidence of amyloidosis in the 2000 ppm female mice. This was reflected by an increase in the incidence of amyloid in the following organs : stomach, duodenum, jejunum, ileum, cecum, colon, salivary glands, pancreas, heart, kidneys, spleen, mandibular lymph node, mesenteric lymph node, thyroid gland, parathyroid glands, adrenal glands, ovaries, uterus, skin, and exorbital lacrimal glands. The precise pathogenesis of amyloid deposition remains uncertain. Experimental data suggests that the deposition of amyloid can be influenced by factors that influence the immune system. Nevertheless, the increased incidence of amyloidosis is indicative of an altered homeostasis at the 2000 ppm concentration.

Histopathological findings: neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Hepatocellular hypertrophy was statistically significantly increased at 700 and 2000 ppm in both male and female mice. In males, the hypertrophy was primarily centrilobular, with panlobular, or diffuse, hypertrophy present in only 3 animals at 700 ppm. In females, panlobular hypertrophy was predominant at 2000 ppm while centrilobular hypertrophy was predominant at 700 ppm. The presence of 1 mouse with panlobular hypertrophy at 50 ppm in the females suggests that compound-related hypertrophy may be present at this concentration also.

Hepatocellular hypertrophy was characterized by minimal to mild enlargement of hepatocytes, either primarily around central veins (centrilobular), primarily distributed throughout the liver lobule (panlobular). The cytoplasm of affected hepatocytes stained more homogeneously than the more typical granulovacuolar cytoplasm of unaffected hepatocytes.

Hepatocellular hypertrophy was most likely the result of test substance metabolizing enzyme induction within hepatocytes. This was considered to be a pharmacologically adaptive response of the liver as a result of exposure to a xenobiotic and not biologically adverse.

In this study there was no compound-related increase in the incidence of animals with hepatocellular neoplasms and the incidence of eosinophilic foci was slightly less at the high dose (2000 ppm) as compared to the high-intermediate dose (700 ppm). Thus, in the absence of neoplastic effect, the increased incidence of eosinophilic foci was considered a compound related metabolic hepatocellular alteration and not biologically adverse.

The test substance was not carcinogenic in mice of either sex. The incidence of malignant lymphoma in females was elevated in the 2000 ppm group (1/60, 1.7%; 2/60, 3.3%; 2/61, 3.3%; 0/60, 0%; and 6/60, 10%; for the 0, 5, 50, 700, and 2000 ppm groups, respectively). However, the increase was not statistically significant and, was within the historical control values.

Key result

Dose descriptor:

NOAEL

Remarks:

for neoplastic effects

Effect level:

> 2 000 ppm

Key result

Dose descriptor:

NOAEL

Remarks:

for non-neoplastic effects

Effect level:

50 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

Conclusions:

The test substance is not an oncogen in mice

Executive summary:

An 18-month study was performed to evaluate the oncogenicity of the test substance when administered to mice in a nutritionally adequate diet. This study was conducted following OECD guideline 451 and US EPA 83-2. Five groups of 80 male and 5 groups of 80 female mice were fed diets containing either 0, 5, 50, 700, or 2000 ppm test substance for approximately 18 months. Samples of diet were analyzed near the beginning of the study to verify concentration, homogeneity, and stability of test substance in the diets. Six times during the study, at approximately 3-month intervals, samples were collected from feeders to verify concentrations of the test substance in the feed. Body weight and individual food consumption were measured weekly for the first 3 months and every other week for the remainder of the study. Clinical signs of toxicity were monitored throughout the study.

The mean daily intakes of the test substance by male mice in the 5, 50, 700, and 2000 ppm groups were 0.701, 6.78, 95.6, and 274 mg/kg, respectively. Mean daily intakes of test substance in female mice in the 5, 50, 700, and 2000 ppm groups were 0.956, 9.84, 130, and 392 mg/kg, respectively.

No biologically significant, compound-related decreases were seen in body weight, body weight gain, food consumption, or food efficiency in either sex at any concentration. Mean daily food consumption for the 0-546 test day interval was statistically significantly higher than control values in 2000 ppm female mice. This increase was not considered biologically significant as it was not associated with significant differences in body weight or food efficiency.

The incidence of a few nonspecific clinical signs was significantly elevated in male mice at the 2000 ppm concentration. However, none of these signs was considered to represent compound-related toxicity and the increased incidences were considered to be due to biological variability of commonly observed clinical signs of aging laboratory mice.

No compound-related effects on survival were produced by dietary exposure to test substance in male or female mice.

Ophthalmoscopic examinations performed on all surviving mice after approximately 9 months of dietary exposure and near the termination of the study revealed no abnormalities attributed to administration of the test substance.

No compound-related, toxicologically significant alterations in hematology or plasma protein were observed over the course of the study at any dietary concentration of the test substance.

Dietary exposure to test substance produced an increase in mean absolute and relative (to body weight and to brain weight) liver weights in male and female mice at 700 and 2000 ppm and in mean absolute liver weight in female mice at 50 ppm. The increased liver weights were generally associated with an increased incidence or large livers at necropsy (2000 ppm only) and of hepatocellular hypertrophy (700 and 2000 ppm, equivocal in females at 50 ppm). The pattern of hypertrophy was primarily centrilobular in male mice at 700 ppm and above and in female mice at 700 ppm; the 2000 ppm female mice displayed a predominantly panlobular pattern of hypertrophy.

Hepatocellular hypertrophy is often the result of induction of metabolizing enzymes and is not considered an adverse effect. In the 700 and 2000 ppm groups, the increased incidence of hypertrophy was associated with an increase in cytochrome P-450 levels (approximate 2-2.5 times control values) at both the 2-week and 9-month time points. In the 50 ppm female group, the small increase in the incidence of hepatocellular hypertrophy was also associated with an increase in absolute liver weight at necropsy and an increase in cytochrome P-450 levels at the 2-week time point. Induction of cytochrome P-450 is a reversible pharmacologic effect and is not considered to be indicative of target organ toxicity.

A compound-related increase in the incidence of a few other liver lesions was observed in male and/or female mice at 2000 ppm. These lesions included diffuse fatty change ana focal hepatocellular necrosis in males, apoptosis in females, and accumulation of golden yellow pigment (lipofuscin and hemosiderin) in Kupffer cells in both sexes. The pathogenesis of the pigment is not clear. Increased lipofuscin is suggestive of some form of autocatalytic process. Increased hemosiderin is suggestive of red blood cell breakdown. No compound related effects on RBC parameters were evident in this study. The incidence of eosinophilic foci of cellular alteration was also increased in 700 and 2000 ppm male mice, with a flat dose-response at these concentrations. The biological significance of these foci is not clear and they were not associated with an increase in hepatocellular neoplasms; therefore, these foci are considered to be compound-related but not evidence of specific organ toxicity. However, the overall pattern of liver lesions is indicative of hepatotoxicity at 2000 ppm in both sexes.

The incidence of amyloidosis in multiple organs was increased in 2000 ppm female mice. In addition, the number of female mice at this concentration that died or were sacrificed-in-extremis due to amyloidosis was higher than in other female groups. Although the pathogenesis of amyloidosis is unclear, it is indicative of altered homeostasis and is considered to be evidence of compound-related toxicity in female mice at 2000 ppm.

The incidence of malignant lymphomas was elevated in the female 2000 ppm group. However, the increase was not considered to be compound-related as it was not statistically significant and the incidence in the 2000 ppm group fell well within the historical control range for the laboratory.

A few other statistically significant differences were seen in organ weights, gross observations, and/or microscopic observations (non-neoplastic) in male and female mice at 2000 ppm. The organ weight and gross observations were not considered to be toxicologically relevant as they lacked any histologic correlate. The non-neoplastic observations were not considered to be toxicologically important the as increased incidences were of small magnitude, lacked a clear dose-response, and/or were not accompanied by an increase in severity.

No statistically significant increase in liver cell proliferation (measured as BrdU labeling) was observed in male or female mice at either the 2-week or 9-month time point. Mean labeling indices in 2000 ppm males and females were higher than in controls at both time points, due to a few mice in the high-dose groups with elevated labeling indices. However, at each time point, the range of values in the control and high-dose groups overlapped; therefore, it cannot be determined if the elevated values are compound-related in these individual mice or if they represent individual animal variability in the background level.

Dietary exposure to the test substance produced increases in liver beta-oxidation activity in 2000 ppm male and female mice at both the 2-week and 9-month time points, in 700 ppm male mice at the 2-week time point, and in 700 ppm female mice at the 9-month time point. All increases were approximately 1.8-2.3 times control values. The sustained increase in beta-oxidation activity in male and female mice at 2000 ppm is considered biologically significant as it was associated with histopathologic evidence of hepatic toxicity. At 700 ppm, the increase in beta-oxidation activity was not statistically significant at both time points and was not associated with hepatotoxicity. This increased activity was, therefore, considered to be compound-related but not adverse.

Under the conditions of this study, test substance is not an oncogen in mice. The no-observed-effect level (NOEL) was 700 ppm for both male and female mice. This level is equivalent to 95.6 mg/kg/day in male mice and 130 mg/kg/day in female mice. The NOEL is based on an increased incidence of microscopic lesions in the liver indicative of hepatotoxicity in male and female mice, and an increase in amyloidosis in female mice, all at 2000 ppm. The no-observed-effect level (NOEL) for this study is defined as the highest dose at which toxicologically important effects attributable to the test substance were not detected. Thus, for this study, the NOEL is equivalent to the NOEL as defined by the United States Environmental Protection Agency (1985) and the no-observed-adverse-effect level (NOAEL) as defined by the European Union (1994).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Study duration:

chronic

Species:

rat

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

Based on the results of chronic feeding studies in rats and mice, it can be concluded that the test substance does not pose a carcinogenic concern for humans. Therefore, the test substance is not classified for carcinogenicity according to EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008.

Additional information

The 2-year feeding study with famoxadone demonstrated toxicity at the 200 ppm dose level in both males and females.  Effects were limited to clinical signs (males), decreased body weights (females), mild hepatotoxicity, and mild regenerative haemolytic anaemia.  A pathology peer review of the lenses of male rats confirmed that famoxadone did not induce compound-related changes up to and including a dietary concentration of 400 ppm, the highest concentration evaluated in the study.  Famoxadone was not oncogenic.  The NOAEL was 40 ppm in both male and female rats (1.62 and 2.15 mg/kg bw/day, respectively).

In an initial 18-month mouse feeding study, toxicity at a dose level of 700 ppm was observed in both males and females.  Effects were limited to a slight increase in amyloidosis (females) and mild hepatotoxicity.  Famoxadone was not oncogenic in mice.  The NOAEL was 50 ppm in both males and females (6.78 and 9.84 mg/kg bw/day, respectively).

In a second 18-month mouse carcinogenicity study, famoxadone was not oncogenic at the two dietary levels of 2000 and 7000 ppm that were evaluated.  An increase in the incidence of microscopic lesions in the liver, indicative of hepatotoxicity, was observed in male and female mice at both dietary concentrations.  In addition, test substance-related effects on body weight, body weight gain, food efficiency were also observed in the 7000 ppm male mice.  The results of this study were generally consistent with the initial 18-month mouse study.  The LOAEL was considered to be 2000 ppm.