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

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference 1

Reference Type:

study report

Title:

Unnamed

Year:

1995

Report date:

1995

Reference 2

Reference Type:

study report

Title:

Unnamed

Year:

1998

Report date:

1998

Reference 3

Reference Type:

study report

Title:

Unnamed

Year:

1999

Report date:

1999

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 409 (Repeated Dose 90-Day Oral Toxicity Study in Non-Rodents)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OPP 82-1 (90-Day Oral Toxicity)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: MAFF Japan NohSan No. 4200

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

5-methyl-5-(4-phenoxyphenyl)-3-(phenylamino)-1,3-oxazolidine-2,4-dione

EC Number:

603-520-1

Cas Number:

131807-57-3

Molecular formula:

C22H18N2O4

IUPAC Name:

5-methyl-5-(4-phenoxyphenyl)-3-(phenylamino)-1,3-oxazolidine-2,4-dione

Test material form:

solid

Specific details on test material used for the study:

Substance ID: DPX-JE874-221 Technical
Lot #: DPX-JE874-221
Purity: 97.4%

Test animals

Species:

dog

Strain:

Beagle

Details on species / strain selection:

The animal model, the outbred beagle, is recognized as appropriate for toxicity studies and is a widely used strain for which significant historical control data are available.

Sex:

male/female

Administration / exposure

Route of administration:

oral: feed

Details on route of administration:

The oral route of administration was selected because it is the possible route of human exposure.

Vehicle:

other: Feed

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Results from analysis of diet samples submitted for homogeneity indicated a homogeneous distribution of the test article in the diets. The measured concentrations of the test substance in homogeneity diet samples were within ±13% of nominal. The test diets were stable when stored at room temperature for 7 and 14 days, or refrigerated for 14 days. The measured concentrations of test substance in stability diet samples were within ±12% of nominal. Diet samples (days 45 and 90) were within targeted concentrations (±15% of nominal), ranging from 85.8% to 100% of nominal.
Based on analyses of stock test substance samples collected near the beginning and end of the dosing period, the test article was judged to be stable during this study.

Duration of treatment / exposure:

Approximately 90 days

Frequency of treatment:

The test diets were offered for approximately one hour per day for 13 weeks (91 or 92 consecutive days), until the day prior to the scheduled necropsy.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

40 ppm

Remarks:

Equivalent to 1.3 mg/kg bw in males and 1.4 mg/kg bw in females

Doses / concentrations 2

Dose / conc.:

300 ppm

Remarks:

Equivalent to 10.0 mg/kg bw in males and 10.1 mg/kg bw in females

Doses / concentrations 3

Dose / conc.:

600 ppm

Remarks:

The test article was offered at 1000 ppm during weeks 0-4 and the first two days of week 5 (*equivalent to 23.8 mg/kg bw in males and 23.3 mg/kg bw in females) and 600 ppm during the last five days of week 5 and weeks 6-13 (*equivalent to 21.2 mg/kg bw in males and 20.1 mg/kg bw in females).

No. of animals per sex per dose:

4

Control animals:

yes, plain diet

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Test article related clinical signs were limited to the 1000-600 ppm group males and females. The predominant clinical sign was myotonic twitches. This finding was initially observed for all males and females in the 1000-600 ppm group during the fourth week of administration at 1000 ppm (week 3). Ataxia and convulsions were observed for a single female on one occasion. Even after the dietary concentration was lowered to 600 ppm during week 5, myotonic twitches continued to be observed in this group generally through to study termination (week 13). These clinical signs were more frequently observed and were more severe approximately 4-hours after feeding (rarely persisted to the following day). Since the dogs received all exposure to the test article within an approximate 1-hour time interval each day, this 4-hour time of peak effect may correspond to the approximate time of peak blood levels of the test substance. An increased incidence of soft stool in the 1000-600 ppm group, especially during the period of administration at 600 ppm, was attributed to the test article.

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Mean body weight losses and/or reduced mean body weight gains were observed in the 1000-600 ppm group males and females for weeks 0 to 1 through 4 to 5. By the end of this period (week 5)，mean body weights in the 1000-600 ppm group males and females were 17% and 19% lower, respectively, than the control group values. At study termination (week 13)，mean body weights in the 1000-600 ppm group males and females were 11% and 14% lower, respectively, than the control group values.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

Food consumption, evaluated as g/animal/day, was moderately reduced in the 1000-600 ppm group males and females during the first six weeks of test article administration (weeks 0 to 1 through 5 to 6).
The average amounts of test article consumed in the 40, 300 and 1000-600 ppm groups were 1.3, 10.0 and 23.8-21.2 mg/kg/day for males and 1.4, 10.1 and 23.3-20.1 mg/kg/day for females, respectively.

Food efficiency:

effects observed, non-treatment-related

Description (incidence and severity):

Food efficiency values in the 1000-600 ppm group were negative for weeks 0 to 1, 1 to 2 and 2 to 3 for the males and for weeks 0 to 1 and 1 to 2 for the females. In addition, the weeks 2 to 3 and 3 to 4 values for females were markedly lower than the control group values.

Ophthalmological findings:

effects observed, treatment-related

Description (incidence and severity):

At the week 12 ocular examination, bilateral posterior cortical cataracts (graded as slight) were present in two of four males and one of four females in the 300 ppm group and two of four males and two of four females in the 1000-600 ppm group. However, none of these dogs exhibited clinical signs of visual impairment.

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

A test article related effect on red blood cell parameters was apparent in the 1000-600 ppm group males and females at the week 5 and 12 evaluations. Mean red blood cell count, hemoglobin, hematocrit and MCHC (mean corpuscular hemoglobin concentration) were reduced and mean MCV (mean corpuscular volume), reticulocyte count, Heinz bodies count and MCH (mean corpuscular hemoglobin) were increased. The differences from the control group were more pronounced at week 5. In addition, mean platelet counts were increased in the 1000-600 ppm group males and females at weeks 5 and 12. The increases in platelet and reticulocyte counts and MCV were indicative of a physiological response to the lower red blood cell counts.
A few statistically significant changes in red blood cell parameters were observed in the 40 ppm group females and 300 ppm group males and females. In the 300 ppm males and females, these changes were associated with increased pigment (hemosiderin) in hepatic Kupffer cells and/or bone marrow macrophages. However, most values were within the historical control ranges and/or were similar to pretreatment values. Moreover, the reticulocyte counts in the 40 and 300 ppm group males and females were not elevated. Therefore, the differences in red blood cell parameters in the 40 ppm group females and 300 ppm group males and females were not considered to be toxicologically significant since there was no reticulocyte response.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

The only apparent test article related effect on serum chemistry parameters was increased mean potassium in the 1000-600 ppm group males and females at weeks 5 (fasted and nonfasted) and/or 12. This hyperkalemia is the possible cause of the myotonic twitches seen in the 1000-600 ppm group dogs.

Urinalysis findings:

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

Mean absolute and relative testicular and epididymal weights in the 1000-600 group males were lower than the control group values and minimal to mild bilateral seminiferous tubule immaturity was observed microscopically. This is not an uncommon occurrence when body weight gains have been suppressed in dogs as they are reaching sexual maturity. The dogs in this group had statistically significant weight losses early in the study and body weights remained below the control group means for the entire 13-week exposure period. The effect on the testicular and epididymal weights is considered to be secondary to the body weight effect and not a direct toxic effect of the test article.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

An increase in the incidence and severity of dilated renal tubules containing eosinophilic crystalline structures was observed in the 1000-600 ppm female group. This lesion can occasionally be seen in control dogs; therefore, it was considered a test article exacerbation of a background lesion of uncertain biological significance.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Histopathological examination revealed microscopic lesions associated with the lenses of the eyes (7/8 animals), increased pigment (hemosiderin) in the liver and bone marrow (related to the red blood cell reduction) and bilateral seminiferous tubule immaturity (associated with the lower body, testes and epididymal weights). At a concentration of 300 ppm, bilateral posterior cortical cataracts (graded as slight) were observed in 3 of 8 animals. Microscopic changes in the lenses of the eyes were observed for all 8 dogs in the 300 ppm group. In addition, one female in the 40 ppm group had minimal lens changes.

Effect levels

1

Key result

Dose descriptor:

NOAEL

Effect level:

40 ppm

Sex:

male

Basis for effect level:

body weight and weight gain

clinical biochemistry

food consumption and compound intake

haematology

histopathology: non-neoplastic

2

Key result

Dose descriptor:

LOAEL

Effect level:

40 ppm

Based on:

test mat.

Sex:

female

Basis for effect level:

body weight and weight gain

clinical biochemistry

clinical signs

food consumption and compound intake

haematology

histopathology: non-neoplastic

Remarks on result:

other: A NOAEL for female dogs could not be ascertained since one female at 40 ppm exhibited minimal lesions in the lens of the eyes.

Target system / organ toxicity

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

40 ppm

System:

eye

Organ:

lens

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

Applicant's summary and conclusion

Conclusions:

13 week Dog NOAEL (male): 40 ppm (1.3 mg/kg bw)
13 week Dog NOAEL (female): Could not be ascertained

Executive summary:

The toxicologic potential of the test substance was evaluated in this 13-week dietary subchronic toxicity study in outbred beagles according to the guideline OECD 409 and EPA 82-1. Concentrations of 40, 300 and 1000 ppm were selected for the study. The test article was administered in the diet for approximately 1-hour per day for 13 weeks (91 or 92 consecutive days). A concurrent control group received the basal diet on a comparable regimen. The 1000 ppm concentration was lowered to 600 ppm during the sixth week of administration (week 5, day 37) due to myotonic twitches of extended duration and a convulsion observed at the 1000 ppm concentration. Each study group consisted of four males and four females. All animals were observed twice daily for mortality and moribundity. Detailed physical examinations were performed weekly on all dogs. Clinical observations were recorded two to three times daily. Individual body weights were recorded weekly. Food consumption was recorded daily and reported weekly. Clinical pathologic evaluations (hematology, serum chemistry and urinalysis) were conducted once prior to dosing initiation and during the 6th and 13th weeks of dosing (study week numbers 5 and 12, respectively) on fasted animals. In addition, blood was collected from all animals in all groups approximately 4- hours following the feeding period during week 5 (day 36, last day of offering the test article in the diet at 1000 ppm to the high dose group). These samples were analyzed for glucose, phosphorus, chloride, sodium and potassium. Ocular examinations were conducted prior to dosing initiation and during study week 12. A complete necropsy was performed on all dogs euthanized at study termination and selected organs were weighed. Tissues were examined microscopically from all dogs.

All animals survived to the scheduled euthanization. Test article related clinical signs were limited to the 1000-600 ppm group males and females. The predominant clinical sign was myotonic twitches. This finding was initially observed for all males and females in the 1000-600 ppm group during the fourth week of administration at 1000 ppm (week 3). Ataxia and convulsions were observed for a single female on one occasion. Even after the dietary concentration was lowered to 600 ppm during week 5, myotonic twitches continued to be observed in this group generally through to study termination (week 13). These clinical signs were more frequently observed and were more severe approximately 4-hours after feeding (rarely persisted to the following day). Since the dogs received all exposure to the test article within an approximate 1-hour time interval each day, this 4-hour time of peak effect may correspond to the approximate time of peak blood levels of the test substance. An increased incidence of soft stool in the 1000-600 ppm group, especially during the period of administration at 600 ppm, was attributed to the test article.

Mean body weight losses and/or reduced mean body weight gains were observed in the 1000-600 ppm group males and females for weeks 0 to 1 through 4 to 5. By the end of this period (week 5)，mean body weights in the 1000-600 ppm group males and females were 17% and 19% lower, respectively, than the control group values. At study termination (week 13)，mean body weights in the 1000-600 ppm group males and females were 11% and 14% lower, respectively, than the control group values. Food consumption, evaluated as g/animal/day, was moderately reduced in the 1000-600 ppm group males and females during the first six weeks of test article administration (weeks 0 to 1 through 5 to 6). Food efficiency values in the 1000-600 ppm group were negative for weeks 0 to 1, 1 to 2 and 2 to 3 for the males and for weeks 0 to 1 and 1 to 2 for the females. In addition, the weeks 2 to 3 and 3 to 4 values for females were markedly lower than the control group values. The average amounts of test article consumed in the 40, 300 and 1000-600 ppm groups were 1.3, 10.0 and 23.8-21.2 mg/kg/day for males and 1.4, 10.1 and 23.3-20.1 mg/kg/day for females, respectively.

A test article related effect on red blood cell parameters was apparent in the 1000-600 ppm group males and females at the week 5 and 12 evaluations. Mean red blood cell count, hemoglobin, hematocrit and MCHC (mean corpuscular hemoglobin concentration) were reduced and mean MCV (mean corpuscular volume), reticulocyte count, Heinz bodies count and MCH (mean corpuscular hemoglobin) were increased. The differences from the control group were more pronounced at week 5. In addition, mean platelet counts were increased in the 1000-600 ppm group males and females at weeks 5 and 12. The increases in platelet and reticulocyte counts and MCV were indicative of a physiological response to the lower red blood cell counts.

A few statistically significant changes in red blood cell parameters were observed in the 40 ppm group females and 300 ppm group males and females. In the 300 ppm males and females, these changes were associated with increased pigment (hemosiderin) in hepatic Kupffer cells and/or bone marrow macrophages. However, most values were within the WIL Research Laboratories, Inc. historical control ranges and/or were similar to pretreatment values. Moreover, the reticulocyte counts in the 40 and 300 ppm group males and females were not elevated. Therefore, the differences in red blood cell parameters in the 40 ppm group females and 300 ppm group males and females were not considered to be toxicologically significant since there was no reticulocyte response.

The only apparent test article related effect on serum chemistry parameters was increased mean potassium in the 1000-600 ppm group males and females at weeks 5 (fasted and nonfasted) and/or 12. This hyperkalemia is the possible cause of the myotonic twitches seen in the 1000-600 ppm group dogs.

No test article related effects were apparent on urinalysis parameters or at the gross necropsy.

At the week 12 ocular examination, bilateral posterior cortical cataracts (graded as slight) were present in two of four males and one of four females in the 300 ppm group and two of four males and two of four females in the 1000-600 ppm group. However, none of these dogs exhibited clinical signs of visual impairment. At histopathologic examination, these cataracts predominantly consisted of swollen fibers at the Y suture of the posterior lens capsule and were observed in the lenses of the eyes in 4/4 and 3/4 males in the 300 and 1000-600 ppm groups, respectively, and in 1/4, 4/4 and 4/4 females in the 40, 300 and 1000-600 ppm groups, respectively.

Mean absolute and relative testicular and epididymal weights in the 1000-600 group males were lower than the control group values and minimal to mild bilateral seminiferous tubule immaturity was observed microscopically. This is not an uncommon occurrence when body weight gains have been suppressed in dogs as they are reaching sexual maturity. The dogs in this group had statistically significant weight losses early in the study and body weights remained below the control group means for the entire 13-week exposure period. The effect on the testicular and epididymal weights is considered to be secondary to the body weight effect and not a direct toxic effect of the test article.

An increase in the incidence and severity of dilated renal tubules containing eosinophilic crystalline structures was observed in the 1000-600 ppm female group. This lesion can occasionally be seen in control dogs; therefore, it was considered a test article exacerbation of a background lesion of uncertain biological significance.

The test diets were homogeneous and stable at room temperature for 7 and 14 days and under refrigeration for 14 days. The test diets contained the amount of test article specified in the protocol. Analysis of bulk test article samples collected near the beginning and end of the dosing period revealed that the test substance was stable during this study.

In conclusion, a concentration of 1000-600 ppm test substance administered to dogs for 13 weeks produced systemic toxicity consisting of clinical signs of toxicity (primarily myotonic twitches)，inhibition of body weight gain and food consumption, decreased circulating erythrocyte mass, increased serum potassium, bilateral posterior cortical cataracts (4/8 dogs; graded as slight) and lower testes and epididymal weights (secondary to decreased body weight and not a direct toxic effect of the test substance). Histopathological examination revealed microscopic lesions associated with the lenses of the eyes (7/8 animals), increased pigment (hemosiderin) in the liver and bone marrow (related to the red blood cell reduction) and bilateral seminiferous tubule immaturity (associated with the lower body, testes and epididymal weights). At a concentration of 300 ppm, bilateral posterior cortical cataracts (graded as slight) were observed in 3 of 8 animals. Microscopic changes in the lenses of the eyes were observed for all 8 dogs in the 300 ppm group. In addition, one female in the 40 ppm group had minimal lens changes. Mild changes in some red blood cell parameters and increased pigment (hemosiderin) in the liver and/or bone marrow were observed in the 300 ppm group males and females and reductions in red blood cell parameters were seen in the 40 ppm group female dogs. However, the hematology and pigment effects in the 40 and 300 ppm group dogs were not considered to be toxicologically significant as they were of small magnitude, many changes were not statistically significant, most values were within the historical control ranges, and the effects at these concentrations were not associated with elevation in Heinz bodies or reticulocytes.

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)2 and the no-observed-adverse-effect level (NOAEL) as defined by the European Union (1994)3. Under the conditions of this study, the NOEL for this study was 40 ppm for male dogs, based on clinical and microscopic evidence of slight posterior subcortical cataracts observed at 300 ppm. A NOEL for female dogs could not be ascertained since one female at 40 ppm exhibited minimal lesions in the lens of the eyes. Based on the results of this study, concentrations of 10, 20, 40 and 300 ppm were selected for the one year dietary study in dogs with the test substance.