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

Specific investigations: other studies

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

cytotoxicity

Remarks:

in vitro

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

no guideline followed

Principles of method if other than guideline:

The objective of this study was to evaluate the relative in vitro susceptibility of lens epithelial cells, from different mammalian species, to the test substance toxicity. Lens epithelial cell cultures derived from dogs, primates (monkeys), and mice were exposed in vitro to multiple concentrations of the test substance; a human corneal epithelial cell culture was similarly exposed. Cellular toxicity was measured using a two-color fluorescence cell viability assay.

GLP compliance:

no

Type of method:

in vitro

Endpoint addressed:

other: Cytotoxicity

Specific details on test material used for the study:

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

Species:

other: Tissues from monkeys, dogs and rats and two cell lines (SV-40 Human Corneal Epithelial Cells and Nakano Mouse Lens Epithelial Cell Line)

Dose / conc.:

0 other: µg/mL

Dose / conc.:

0 other: µg/mL

Dose / conc.:

0.001 other: µg/mL

Dose / conc.:

0.01 other: µg/mL

Dose / conc.:

0.1 other: µg/mL

Dose / conc.:

1 other: µg/mL

Dose / conc.:

10 other: µg/mL

Dose / conc.:

100 other: µg/mL

Dose / conc.:

1 000 other: µg/mL

Details on study design:

To determine the cytotoxic effects of the test substance, live/dead assays were conducted on lens epithelial cells (dog, monkey, mouse) and corneal epithelial cells (human; SV-40 HCEC); the time points chosen were 3 hours, 24 Hours, and 48 hours. These time points were chosen because the cells were to be seeded in the absence of serum, and the viability of these cells would decrease after an extended period of time. The cells were seeded in the absence of serum because the presence of serum can lead to inaccurate results using the Cytofluor 2350 system. The assay was completed by trypsinizing one confluent flask of cells (canine lens epithelial cells, primate lens epithelial cells, NK-35 cells, or SV-40 HCEC) and centrifuging the contents for two minutes at high speed. Cells from differing animals were admixed upon trypsinization to minimize variability. The resulting pellet of cells was resuspended in PBS, and the cells were counted using a hemocytometer. The cells were plated at a concentration of approximately 3000 cells/well in a volume of 20 µL on each of three 96 well plates. Various concentrations of the test substance were added at a volume of 200 µL. Concentrations of the test substance tested were 0 pg/mL, 100 pg/mL, 1 ng/mL, 10 ng/mL, 100 ng/mL, 1 µg/mL, 10 µg/mL, 100 µg/mL, and 1 mg/mL. The compound was diluted in vehicle. The vehicle chosen for all lens cells was 10% DMSO in Ham’s F-12 media. Ham’s F-12 was chosen because it contains no phenol red, which can lead to inaccurate results using the Cytofluor 2350 system. Because the SV-40 HCEC toxicity was evaluated over a short (<3 hr) time span, PBS was chosen as the assay media.

Details on results:

Both primary lens epithelial cell cultures (canine and monkey) and the immortalized lens epithelium cell line (mouse, NK-35) demonstrated cytotoxicity when exposed to the high dose of 1 mg/mL of the test substance for 3, 24, or 48 hours. The same results were observed with the HCEC line (SV-40). Cytotoxicity at this concentration was therefore nonspecific with regard to species and cell type. At 1 mg/mL, the decreased calcein fluorescence was interpreted to be indicative of decreased cellular esterase activity (decreased cell viability); the increased ethidium fluorescence was interpreted to be indicative of increased cell membrane damage (i.e.,cell death).
For the remaining in vitro exposure concentrations (100 µg/mL and below), interpretation of the fluorescent patterns was less obvious. Neither the decreased calcein staining nor the increased ethidium staining was likely to have been due to test substance cytotoxicity at dose levels of 100 µg/mL and below, although an equivocal effect was observed in some assays at 100 µg/mL.

Conclusions:

Overall there were no clear differences observed in the susceptibility of the 4 cell culture systems (2 primary lens epithelial cell cultures in canine and monkey, an immortalized lens epithelial cell line in mouse, NK-35, and an immortalized corneal epithelial cell line in humans, SV-40) to test substance induced cytotoxicity.

Executive summary:

An in vitro cytotoxicity study with the test substance was conducted in 2 primary lens epithelial cell cultures (canine and monkey), an immortalized lens epithelial cell line (mouse, NK-35), and an immortalized corneal epithelial cell line (human, SV-40). All cell cultures demonstrated cytotoxicity when exposed to the high exposure concentration of 1 mg/ml of the test substance for 3, 24, and 48 hours. Overall there were no clear differences observed in the susceptibility of the 4 cell culture systems to test substance induced cytotoxicity.

Reference 2

Endpoint:

biochemical or cellular interactions

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

no guideline followed

Principles of method if other than guideline:

The objective of this study was to determine the potential of test substance to alter hepatic cytochrome P-450 (CYP) content in rats and mice fed test substance for 2 weeks. Groups of 5 male and 5 female Crl:CD®(SD)IGS BR rats were exposed to 0 ppm or 20000 ppm test substance, and groups of 5 male and 5 female Crl:CD-1®(ICR) BR mice were exposed to 0 ppm or 7000 ppm test substance. Body weights and food consumption were determined weekly and cage-site examinations to detect abnormal behavior or appearance occurred daily.

GLP compliance:

yes

Type of method:

in vivo

Endpoint addressed:

other: Investigation of Cytochrome P-450 Induction

Specific details on test material used for the study:

Substance name: Famoxadone Technical
Lot #: DPX-JE874-221
Purity: 97.28%

Species:

other: Rat and mouse

Strain:

other: Crl:CD®(SD)IGS BR (rat) and Crl:CD-1®(ICR) BR (mouse)

Sex:

male/female

Route of administration:

oral: feed

Vehicle:

other: Test substance was dissolved in acetone and added to diet

Analytical verification of doses or concentrations:

yes

Duration of treatment / exposure:

14 days

Frequency of treatment:

Daily

Dose / conc.:

20 000 ppm

Remarks:

Rat

Dose / conc.:

7 000 ppm

Remarks:

Mouse

No. of animals per sex per dose:

5

Control animals:

yes, plain diet

Details on results:

Mean Body Weights and Body Weight Gains of Rats: Body weights of male and female rats fed diet containing 20000 ppm test substance were significantly lower when compared to the control group on test days 7 (83% and 88% of control, respectively) and 14 (74% and 85% of control, respectively). Mean body weight gain of rats fed 20000 ppm test substance was significantly lower when compared to the control group over the intervals of test days 0-7 for male and female rats, 7-14 for male rats, and over the entire treatment interval (0-14) for male and female rats. Over treatment interval 0-14, male rats fed 20000 ppm test substance gained a mean of 18.4 g body weight compared to the control group which gained a mean of 107.0 g. Similarly, female rats fed 20000 ppm test substance gained a mean of 12.6 g body weight compared to the control group which gained a mean of 50.0 g.

Food Consumption, Food Efficiency, and Intake of Famoxadone by Rats: Mean food consumption of rats fed 20000 ppm test substance was significantly lower when compared to the control group over the intervals of test days 0-7 for male and female rats (75% and 69% of control, respectively), 7-14 for male rats (73% of control), and over the entire treatment interval (test days 0-14) for male and female rats (74% and 76% of control, respectively). Mean food efficiency of male and female rats fed 20000 ppm test substance was significantly lower when compared to the control group over the intervals of test days 0-7, 7-14, and over the entire treatment interval (test days 0-14). Over the entire treatment interval (test days 0-14), mean food efficiency was decreased to 21% and 32% of control for male and female rats respectively. The decreased food consumption and food efficiency likely contributed to the body weight decrements that were observed.
The mean daily intake values of test substance over the 14-day test period for 20000 ppm males and females were 1540 mg/kg/day and 1543 mg/kg/day, respectively.

Clinical Observations and Mortality in Rats: No test substance-related or biologically significant increases in the incidences of clinical signs of toxicity or mortality occurred in rats fed diet containing 20000 ppm test substance.

Liver Weight and Cytochrome P-450 Measurements in Rats: In male rats, mean absolute and relative liver weights were significantly decreased when compared to the control group (63% and 85% of control, respectively). In female rats, mean relative liver weights were significantly increased when compared to the control group (116% of control). There were no compound-related effects on mean absolute liver weight in female rats fed diet containing 20000 ppm test substance.
Total hepatic cytochrome P-450 content was significantly increased in male and female rats fed 20000 ppm test substance when compared to the control group (138% and 174% of control, respectively). Specific cytochrome P-450 isozymes were also affected by test substance treatment. In male rats, CYP2B1/2, CYP3A, and CYP4A content were increased to 2452%, 228%, and 142% of control, respectively. In female rats, CYP2B1/2, CYP3A, and CYP4A content were increased to 2759%, 363%, and 208% of control, respectively. No alterations were observed in CYP1A1 content in male or female rats fed diet containing 20000 ppm test substance.

Mean Body Weights and Body Weight Gains of Mice: There were no compound-related effects on mean final body weights or body weight gain in male and female mice fed diet containing 7000 ppm test substance.

Food Consumption, Food Efficiency, and Intake of Famoxadone by Mice: Mean food consumption of male mice fed 7000 ppm test substance was significantly higher when compared to the control group over the interval of test days 0-7 (130% of control). This increase was not considered compound-related or biologically significant. There were no compound-related effects on mean food consumption in female mice fed diet containing 7000 ppm test substance. Mean food efficiency was significantly lower when compared to the control group over the entire treatment interval (test days 0-14) in male mice fed 7000 ppm test substance (36% of control), and was unaffected in female mice.
The mean daily intake values of test substance over the 14-day test period for 7000 ppm males and females were 1559 mg/kg/day and 1633 mg/kg/day, respectively.

Clinical Observations and Mortality in Mice: No test substance-related or biologically significant increases in the incidences of clinical signs of toxicity or mortality occurred in mice fed diet containing 7000 ppm test substance.

Liver Weight and Cytochrome P-450 Measurements in Mice: Mean absolute and relative liver weights were significantly increased in male (155% and 161% of control, respectively) and female (174% and 167% of control, respectively) mice when compared to the control group.
Total hepatic cytochrome P-450 content was significantly increased in male and female mice fed 7000 ppm test substance when compared to the control (211% and 260% of control, respectively). Specific cytochrome P-450 isozymes were also affected by famoxadone treatment. In male mice, CYP2B1/2 and CYP4A content were increased to 1379% and 254% of control, respectively. In female mice, CYP2B1/2 and CYP4A content were increased 940% and 401% of control, respectively. No alterations were observed in CYP1A1 or CYP3A content in male or female mice fed diet containing 7000 ppm test substance.

Conclusions:

Rat: Test substance administration decreased absolute and relative liver weights in males, and increased relative liver weights in females. These changes in liver weights were associated with increases in total cytochrome P-450 content, as well as increases in the isozymes CYP2B1/2, CYP3A, and CYP4A for male and female rats. No alterations were observed in CYP1A1 content in either male or female rats.
Mouse: Test substance administration increased absolute and relative liver weights in males and females. These changes in liver weights were associated with increases in total cytochrome P-450 content, as well as increases in the isozymes CYP2B1/2 and CYP4A for male and female mice. No alterations were observed in CYP1A1 or CYP3A content in either male or female mice.

Executive summary:

The objective of this study was to determine the potential of test substance to alter hepatic cytochrome P-450 (CYP) content in rats and mice fed famoxadone for 2 weeks. Groups of 5 male and 5 female Crl:CD®(SD)IGS BR rats were exposed to 0 ppm or 20000 ppm test substance, and groups of 5 male and 5 female Crl:CD-1®(ICR) BR mice were exposed to 0 ppm or 7000 ppm famoxadone. Body weights and food consumption were determined weekly and cage-site examinations to detect abnormal behavior or appearance occurred daily. Approximately 2 weeks after the initiation of test substance administration, rats and mice were euthanized. Livers from each animal were dissected and weighed to correlate with the hepatic cytochrome P-450 data. A portion of the liver was homogenized and hepatic microsomes prepared by differential centrifugation for the evaluation of total cytochrome P-450 content and quantitation of the isozymes CYP1A1, CYP2B1/2, CYP3A, and CYP4A.

The mean daily intake for 20000 ppm male and female rats over the 14-day test period were 1540 mg/kg and 1543 mg/kg, respectively. The mean daily intake for 7000 ppm male and female mice over the 14-day test period were 1559 mg/kg and 1633 mg/kg, respectively.

In rats, test substance administration decreased absolute and relative liver weights in males, and increased relative liver weights in females. These changes in liver weights were associated with increases in total cytochrome P-450 content, as well as increases in the isozymes CYP2B1/2, CYP3A, and CYP4A for male and female rats. No alterations were observed in CYP1A1 content in either male or female rats. The test substance also caused decrements in body weight and body weight gain. These decrements were associated with decreased food consumption and food efficiency.

In mice, test substance administration increased absolute and relative liver weights in males and females. These changes in liver weights were associated with increases in total cytochrome P-450 content, as well as increases in the isozymes CYP2B1/2 and CYP4A for male and female mice. No alterations were observed in CYP1A1 or CYP3A content in either male or female mice. The test substance also decreased food efficiency in male mice over the entire treatment interval. The decreased food efficiency was not associated with altered food consumption, and test substance treatment did not result in alterations in body weight or body weight gain in either male or female mice.

Reference 3

Endpoint:

specific investigations: other studies

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

no guideline followed

Principles of method if other than guideline:

Studies were carried out to address species differences in hemolytic responsiveness to the test substance. A 100 mg/kg dose of [14C-PA]test substance (phenylamino-labeled) was administered to 5 male rats by oral gavage. After 10 hours, erythrocytes (RBCs) were fractionated with toluene, and the intracellular distribution of radioactivity was assessed. An in vitro hepatocyte/RBC co-incubation system was developed to investigate species differences in metabolism-hematotoxicity interactions in rats, cynomolgus monkeys and humans.

GLP compliance:

yes

Type of method:

other: In vitro and in vivo

Endpoint addressed:

basic toxicokinetics

Specific details on test material used for the study:

Substance name: [14C-PA]famoxadone
Specific Activity: 58.820 µCi/mg
Radiochemical Purity: 99.5%

Species:

rat

Strain:

other: Crl:CD®(SD)IGS BR

Sex:

male

Route of administration:

oral: gavage

Vehicle:

other: 1% aqueous sodium carboxymethylcellulose/0.01M ammonium acetate

Analytical verification of doses or concentrations:

yes

Duration of treatment / exposure:

In vivo: Single oral dose
In vitro: 4 hours expsoure

Dose / conc.:

500 other: µM

Remarks:

In vitro

Dose / conc.:

100 mg/kg bw (total dose)

Remarks:

in vivo oral

No. of animals per sex per dose:

5 (in vivo)

Control animals:

no

Details on results:

Intracellular Distribution of Radioactivity in Erythrocytes of Rats Treated with [14C-PA]test substance In Vivo: Erythrocytes (RBCs) were collected from [14C-PA]test substance-treated rats approximately 10 hours after dosing, corresponding to the reported Tmax for radioactivity in these cells. Washed RBCs contained an average of 78446 dpm/g, or approximately 0.035 μCi/g. This equates to a [14C-PA]test substance residue concentration of 12.4 nmol equiv/g, or approximately 12 μM equivalents in the RBCs. The fractionation procedure used in these experiment results in the separation of RBCs into 4 subcellular compartments or layers. The upper toluene layer contains membrane lipids and other non-polar chemical species. The intracellular fluid layer is composed primarily of deproteinated cytosol. Between the toluene and ICF is a stromal interface which contains cytoskeletal and membrane proteins, and amphipathic lipids. The fourth (bottom) layer is composed primarily of precipitated hemoglobin. The majority of radioactivity present in the washed RBCs (68.3% TRR) resided in the hemoglobin fraction. A significant fraction of the total radioactivity was also present in the ICF, which contained 26.1% TRR. The toluene and stroma layers contained 4.1 and 1.5% TRR, respectively. Pilot experiments suggested that some Hb remains in the ICF following the fractionation procedure. This suggests that some of the radioactivity present in the ICF may have been due to the presence of residual Hb rather than free compounds in the cytosol. Overall, the data suggest a high degree of association of [14C-PA]test substance residues with Hb following exposure by oral gavage, which is consistent with the retention of PA-labeled residues in RBCs observed in the previous famoxadone ADME study.

Covalent Binding of Radioactive Residues to Globin in Erythrocytes from Rats treated with [14C-PA]test substance: To determine if the high level of radioactivity associated with Hb in the distribution experiment was due to covalent binding, globin was isolated from RBCs of [14C-PA]test substance-treated rats by acid/acetone precipitation and washed exhaustively with organic solvents to remove unbound radioactivity. The specific activity of the washed globin was 259200 dpm/g, which equates to a binding activity of approximately 41.5 nmol equivalent [14C-PA]test substance residues per gram of globin. The covalently bound radioactivity in this experiment accounted for approximately 64% of the total RBC activity, which was consistent with the results of the RBC distribution experiment. Together, the results of the distribution and covalent binding experiments support the hypothesis that the extended T1/2 of radioactivity in RBCs from [14C-PA]test substance-treated rats observed previously(3) is due to covalent binding of PA-labeled famoxadone metabolites to hemoglobin.
Pilot experiments were carried out to develop methods for identification of the covalently-bound radioactive residues in erythrocyte hemoglobin following exposure to [14C-PA]test substance in vivo. Several different approaches were attempted, including enzymatic digestion and chemical hydrolysis with solvent extraction.
Protease and trypsin digestions: Digestion with a ternary mixture of proteases (pronase E, aminopeptidase M and prolidase, overnight at 37°C, pH ~8) was carried in an attempt to separate the adducted globin into its component amino acids and dipeptides. Although a significant portion of the radioactivity was recovered in solvent extracts of the digest, the radioactivity could not be resolved chromatographically into discrete peaks, and differences between control and treated digests were not evident from LC/MS analysis. Similar problems were encountered with trypsin digestion (overnight at 37°C, pH ~8). Although the tryptic peptides were well resolved by HPLC, the radioactivity associated with the digests eluted as a very broad, low-intensity band. High-resolution time of flight mass spectrometry (LC-ToF-MS) was employed to analyze the trypsin digests. Complete digests were very complex, containing numerous peaks, and analysis did not reveal differences between control and treated globin, which could be interpreted in terms of known or proposed test substance metabolite adducts. Attempts were made to simplify mass spectral analysis by fractionating and analyzing only the part of the digest with associated radioactivity and by collecting and digesting only that part of the globin molecule with bound radioactivity (primarily β-chain). While both of these approaches decreased the complexity of the analysis by reducing the number of peaks in the resulting mass chromatograms, neither approach allowed identification of peptides unique to the treated globin, which could be understood with respect to known or proposed test substance metabolites.
Acid-catalyzed hydrolysis: As an alternative approach to enzymatic digestion, methods for acidcatalyzed globin hydrolysis were examined. The goal of these experiments was to liberate bound test substance metabolites, which could then be extracted into an appropriate solvent and identified by MS. Although the initial experiment (0.1N HCL, 80 °C, 60 minutes) suggested that a significant amount of radioactivity was recovered in solvent extracts, no test substance metabolites could be observed by LC/MS. A repetition of this experiment using a larger globin sample did not result in significant recovery of radioactivity in the solvent extract. Similarly, in an experiment using more vigorous hydrolysis conditions (6 N HCL, 140 °C, 4 hours), only 6% of the adducted radioactivity was liberated.

Extraction and Characterization of Radioactivity in Erythrocytes from [14C-PA]test substance-Treated Rats: In order to characterize the free radioactivity present in RBCs from [14C-PA]test substance-treated rats, pooled RBCs were lysed in hypotonic phosphate buffer and the lysate was extracted extensively with acetonitrile. The organic extract contained less than 5% of the total radioactivity of the washed RBCs, consistent with the high degree of covalent binding of [14C-PA]test substance residues observed in the distribution and covalent binding experiments. Because of the low level of extractable radioactivity, the extract was not analyzed further.

Intracellular Distribution of Radioactivity in Rat Erythrocytes Co-incubated with Freshly-Isolated Hepatocytes in the Presence of [14C-PA]test substance In Vitro: As an in vitro model of the interaction of metabolism and hematotoxicity of test substance, the test substance was incubated with a mixture of freshly-isolated rat hepatocytes and rat RBCs. The intracellular distribution of radioactivity in the isolated RBCs was evaluated following fractionation with toluene. Washed RBCs from this experiment had 14C levels of 140831 dpm/g or approximately 0.063 μCi/g. This equates to a [14C-PA]test substance residue concentration of 153.9 nmol/g, or approximately 154 μM in the RBCs. In contrast to the in vivo distribution pattern for [14C-PA]test substance residues, the toluene fraction and the hemoglobin fraction contained similar amounts of radioactivity (39.9 and 31.0% TRR, respectively). The remainder of the radiolabel was divided approximately evenly between the stroma (16.6% TRR) and the ICF (12.7% TRR). A possible explanation for the difference in erythrocyte 14C distribution pattern between the in vivo and in vitro experiments is that the former situation represents an open system, whereas the latter model is a closed system. In vivo, test substance and its metabolites can diffuse into and out of the RBCs and be excreted from the body. However, in vitro, elimination from the system is not possible, and test substance and its more lipophilic metabolites are likely to partition preferentially into hydrophobic fractions such as the toluene and stroma fractions. In support of this hypothesis, HPLC analysis of the toluene fraction showed that the major UV absorbing component in this fraction coeluted with test substance.

Covalent Binding of Radioactive Residues to Globin in Erythrocytes Co-incubated with Freshly-Isolated Hepatocytes in the Presence of [14C-PA]famoxadone In Vitro: To investigate the capacity of rat hepatocytes to metabolize test substance to products capable of covalent binding to hemoglobin, [14C-PA]test substance was incubated with a mixture of hepatocytes and RBCs, and globin was isolated from the RBCs and analyzed for bound radioactive residues. The specific activity of the washed globin was 68899 dpm/g, which represents a binding activity of approximately 75.4 nmol [14C-PA]test substance residues per gram of globin. The covalently bound radioactivity in this experiment accounted for approximately 63% of the total RBC activity, which was very similar to the proportion of radioactivity bound to globin in rats treated with [14C-PA]test substance in vivo. The higher molar binding activity of [14C-PA]test substance observed in the in vitro experiment may be related in part to the limited capacity for elimination of reactive metabolites in the closed co-incubation system, compared to the whole animal. The results of this experiment demonstrate that rat hepatocytes convert famoxadone to metabolites with hemoglobin binding potential in measurable quantities. This suggests that the hepatocyte-RBC co-incubation system is an appropriate in vitro model for investigating the relationships between metabolism and hematotoxicity of test substance.

The metabolite profiles of [14C-PA]test substance in freshly-isolated rat, cynomolgus monkey, and human hepatocytes were determined after a 4-hour incubation with 500 μM [14C-PA]test substance. In general, the metabolite profile was similar in all species. The following metabolites were detected in rat hepatocyte extracts by LC/MS: IN-KZ007 and a positional isomer of IN-KZ007, IN-KZ532, IN-KZ000 sulfate, IN-KZ007 sulfate, IN-KZ532 sulfate, IN-KZ007 glucuronide, IN-KZ532 glucuronide, two IN-KZ534 monosulfate isomers, IN-KZ534 disulfate, and an unspecified acylated aminophenol sulfate, which was not detected in other species. Cleaved metabolites detected by GC/MS in rat hepatocytes include aniline, 2-aminophenol, 4-aminophenol, and 4-acetamidophenol. All of these metabolites were also identified in monkey and human hepatocytes with the exception of IN-KZ534 disulfate and one of the IN-KZ534 monosulfate in monkey hepatocytes, and IN-KZ000 sulfate and IN-KZ534 sulfates in human hepatocyte extracts. IN-BY759 (4-acetoxyaniline) was not detected in hepatocyte extracts from any species.

Conclusions:

[14C-PA]Test substance was metabolized to 14C-containing metabolites capable of forming covalent adducts with rat hemoglobin, following oral administration, or in a hepatocyte/erythrocyte co-incubation system. These findings are consistent with the selective retention of 14C from [14C-PA]test substance by erythrocytes observed in previous toxicokinetic studies and suggest that the in vitro co-incubation system is a suitable model for investigation of metabolism-hemoglobin binding interactions. Freshly isolated hepatocytes from rat, monkey and human metabolized [14C-PA]test substance by similar pathways including aromatic hydroxylation, oxazolidinedione ring cleavage, sulfation, glucuronidation, and N-acetylation. These results highlight the utility of hepatocyte suspensions for qualitative biotransformation studies and suggest that primates do not produce any unique [14C-PA]test substance metabolites not found in rodents. This suggests that the difference in hemolytic responsiveness between rats and monkeys is due either to enzyme kinetic factors and/or inherent differences in erythrocyte sensitivity to famoxadone metabolites. The inability to identify [14C-PA]test substance metabolites with sufficient hemolytic potential, either as hemoglobin adducts or as hepatocyte metabolites, prevented completion of comparative kinetic and oxidative stress experiments.

Executive summary:

Studies were carried out to address species differences in hemolytic responsiveness to test substance. A 100 mg/kg dose of [14C-PA]test substance (phenylamino-labeled) was administered to 5 male rats by oral gavage. After 10 hours, erythrocytes (RBCs) were fractionated with toluene, and the intracellular distribution of radioactivity was assessed. The percentage of total recoverable radioactivity (%TRR) observed in the toluene (lipophilic), stroma (cytoskeletal), intracellular fluid (ICF), and hemoglobin (Hb) fractions were 4.1%, 1.5%, 26.1% and 68.3%, respectively. Globin from these animals was isolated, extracted exhaustively and assayed to determine the amount of radioactive covalent adducts. The activity of bound [14C-PA]test substance residues was 41.5 nmol equivalents/g globin, accounting for approximately 64% of the total RBC activity. Less than 5% of the total radioactivity of washed RBCs from [14C-PA]test substance-treated rats was extractable into acetonitrile. An in vitro hepatocyte/RBC co-incubation system was developed to investigate species differences in metabolism-hematotoxicity interactions in rats, cynomolgus monkeys and humans. Rat hepatocytes (5 x 106/mL) and RBCs (hematocrit approximately 13%) were co-incubated with 500 μM [14C-PA]test substance for 4 hours and the distribution and covalent binding of radioactivity were assessed. The toluene, stroma, ICF, and Hb fractions contained 39.9, 16.6, 12.6, and 31.0% TRR, respectively. The activity of covalently-bound [14CPA]test substance residues in isolated globin was 75.4 nmol equivalents/g globin, accounting for approximately 63% of the total RBC activity.

The metabolite profiles of [14C-PA]test substance in freshly-isolated rat, cynomolgus monkey, and human hepatocytes were determined after a 4-hour incubation with 500 μM [14C-PA]test substance. In general, the metabolite profile was similar in all species. The following metabolites were detected in rat hepatocyte extracts by LC/MS: IN-KZ007 and a positional isomer of IN-KZ007, IN-KZ532, IN-KZ000 sulfate, IN-KZ007 sulfate, IN-KZ532 sulfate, IN-KZ007 glucuronide, IN-KZ532 glucuronide, two IN-KZ534 monosulfate isomers, IN-KZ534 disulfate, and an unspecified acylated aminophenol sulfate, which was not detected in other species. Cleaved metabolites detected by GC/MS in rat hepatocytes include aniline, 2-aminophenol, 4-aminophenol, and 4-acetamidophenol. All of these metabolites were also identified in monkey and human hepatocytes with the exception of IN-KZ534 disulfate and one of the IN-KZ534 monosulfate in monkey hepatocytes, and IN-KZ000 sulfate and IN-KZ534 sulfates in human hepatocyte extracts. IN-BY759 (4-acetoxyaniline) was not detected in hepatocyte extracts from any species.

Metabolites of [14C-PA]test substance with significant hemolytic potential were not identified in hepatocyte extracts from any species. Likewise, attempts to identify potential hemolytic test substance metabolites adducted to hemoglobin were unsuccessful. For these reasons, further comparative studies on kinetics of hemolytic metabolite formation and erythrocyte sensitivity were not carried out.

Reference 4

Endpoint:

specific investigations: other studies

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

no guideline followed

Principles of method if other than guideline:

The objective of this study was to evaluate the potential for reversal of the red blood cell effects when exposure to test substance ends, and to evaluate the time-course for this reversal. This study evaluated the reversibility of hematology effects produced in female rats by dietary exposure to famoxadone, and evaluated the time-course of this reversibility. Two groups of 10 female rats each were exposed to 0 or 800 ppm famoxadone in the diet, a concentration previously demonstrated to produce mild hemolytic anemia. Body weight, food consumption, and clinical observations were evaluated weekly. Blood was collected from all rats 16 days after initiation of exposure, and every 2 weeks thereafter for evaluation of red blood cell count (RBC), hematocrit (Hct), and hemoglobin concentration (Hgb).

GLP compliance:

yes

Type of method:

in vivo

Endpoint addressed:

other: Reversibility study of red blood cell mass effects in female rats

Specific details on test material used for the study:

Substance name: Famoxadone (DPX-JE874) Technical
Lot #: DPX-JE874-221
Purity: 97.28%

Species:

rat

Strain:

other: Crl:CD®(SD)IGS BR

Sex:

female

Route of administration:

oral: feed

Vehicle:

other: Test substance was dissolved in acetone and added to diet

Analytical verification of doses or concentrations:

yes

Duration of treatment / exposure:

Test Day 0-35 – Test substance administration

Frequency of treatment:

Daily

Post exposure period:

Test Day 35-56 – Recovery period

Dose / conc.:

800 ppm

No. of animals per sex per dose:

10

Control animals:

yes, plain diet

Details on results:

Mean Body Weights and Body Weight Gain: Dietary exposure to 800 ppm test substance produced a compound-related decrease in mean body weight and body weight gain during the exposure phase of the study. Mean body weight in rats exposed to test substance was significantly lower than in controls on all weigh days except test days 0 and 14. On test day 35, the last day of the exposure phase, mean body weight in exposed rats was 90% of control mean body weight. Body weight gain in exposed rats was lower than in controls during the exposure phase over all weekly intervals except test days 7-14; however, the statistical significance was variable. A statistically significant lower body weight gain (61% of control) was observed in rats fed test substance during the exposure phase (test days 0-35).
When exposed rats were fed control diet, there was no statistically significant difference between the groups in weekly body weight gain. The overall body weight gain in the recovery phase (test days 35-56) was 30% higher in the rats previously exposed to test substance than in controls (not statistically significant). However, mean body weight in this group remained significantly below that of controls during the recovery phase. In test substance-exposed rats, mean final body weight (test day 56) was 93% of control and body weight gain over the entire study (test days 0-56) was 78% of control (statistically significant).

Food Consumption, Food Efficiency, and Intake of Famoxadone Technical: Mean food consumption in exposed rats was lower than in controls over all weekly intervals of the exposure phase, and the difference was statistically significant except over the test day 7-14 interval. In the exposed group, overall food consumption during the exposure phase was 86% of control (statistically significant). No statistically significant difference in food consumption was observed in any of the individual weeks, nor in the overall recovery phase. Mean food consumption over the entire study (test days 0-56) was 90% of controls (statistically significant).
Food efficiency results generally paralleled those of body weight gain. During the exposure phase, mean food efficiency in exposed rats was lower than in controls over most weekly intervals (variable statistical significance), and mean food efficiency for the 0-35 test day interval was 71% of controls. No statistically significant differences were observed during the recovery phase in weekly or overall food efficiency. However overall food efficiency in the previously exposed rats was 30% higher than in controls during the recovery phase. Mean food efficiency over the entire study (test days 0-56) was 87% of controls (statistically significant).
Mean daily intake of test substance over the entire exposure phase was 61.6 mg/kg/day, with weekly mean intake values ranging from 48.5 to 70.9 mg/kg/day.

Clinical Observations and Survival: No rats died during the study and no compound-related clinical signs were observed in any rat.

Hematology: Changes in the three hematologic parameters measured (hemoglobin, red blood cell count, and hematocrit) were first detected at the Day 30 sampling time. At this time, means for all three parameters were mildly decreased compared to the control group, indicating decreased red cell mass. For the reversibility phase of the study, all rats were fed control diet starting on Day 35. At the next sampling point (Day 44), mean hemoglobin and hematocrit values in females previously fed 800 ppm were minimally above control group values (statistically significant), although red cell counts were still minimally decreased (statistically significant). On Day 58, after two more weeks on control diet, the mean red blood cell value in females previously fed 800 ppm was similar to the control group mean, while hemoglobin and hematocrit were still minimally increased.

Conclusions:

Under the conditions of this study, dietary exposure to 800 ppm (61.6 mg/kg/day) test substance produced compound-related reductions in red cell mass, body weight, and nutritional parameters. Body weight and nutritional effects were evident within 7 days of exposure, while red cell mass effects were detected following 30 days of exposure. Partial recovery of the red cell mass effects was observed after 9 days of recovery. Full recovery from the red cell mass effects and partial recovery from the body weight and nutritional effects occurred within 23 days of cessation of exposure to test substance. Therefore, the mild anemia produced by dietary exposure to test substance is fully reversible soon after exposure ends.

Executive summary:

This study evaluated the reversibility of hematology effects produced in female rats by dietary exposure to test substance, and evaluated the time-course of this reversibility. Two groups of 10 female rats each were exposed to 0 or 800 ppm famoxadone in the diet, a concentration previously demonstrated to produce mild hemolytic anemia. Body weight, food consumption, and clinical observations were evaluated weekly. Blood was collected from all rats 16 days after initiation of exposure, and every 2 weeks thereafter for evaluation of red blood cell count (RBC), hematocrit (Hct), and hemoglobin concentration (Hgb). Once toxicologically significant reductions in these parameters were observed, the exposed rats were changed to control diet at the next weigh day. Blood continued to be evaluated for RBC, Hct, and Hgb until the reductions in these values were reversed. Rats were then sacrificed without pathologic evaluation.

No statistically significant reduction in red cell mass parameters was observed after 16 days of exposure to test substance. After 30 days of exposure a mild anemia was produced; the three red cell mass parameters were reduced 11-16% below control values (all statistically significant). Rats were started on control diet on test day 35. After 9 days of exposure to control diet (test day 44), mean Hgb and Hct values were slightly higher than control values (statistically significant), but RBC was still minimally decreased (statistically significant). After 23 days of exposure to control diet (test day 58), the anemia was completely resolved, with RBC, Hgb, and Hct values all similar to or greater than control values.

Dietary exposure to 800 ppm test substance produced compound-related lower body weight, body weight gain, food consumption, and food efficiency, all observed by test day 7. Once exposure to test substance ended, no further reductions in these parameters occurred. Partial reversal of these test substance-induced effects was noted, as mean body weight gain and food efficiency values in previously exposed rats were 30% higher than in control rats during the overall (test day 35-56) recovery phase (not statistically significant).

No compound-related effects on mortality or clinical signs of toxicity were produced by dietary exposure to test substance.

Under the conditions of this study, dietary exposure to 800 ppm (equivalent to 61.6 mg/kg/day) test substance produced compound-related reductions in red cell mass, body weight, and nutritional parameters. Full recovery from the red cell mass effects and partial recovery from the body weight and nutritional effects occurred within 23 days of cessation of exposure to test substance.

Additional information

Red blood cell distribution studies

Studies were carried out to address species differences in haemolytic responsiveness to famoxadone.  A 100 mg/kg bw dose of [¹⁴C‑phenylamino]famoxadone was administered to five male rats by oral gavage.  After 10 hours, erythrocytes (RBCs) were fractionated, and the intracellular distribution of radioactivity was assessed.  The percentage of total recoverable radioactivity (% TRR) observed in the lipophilic, cytoskeletal, intracellular fluid (ICF), and haemoglobin (Hb) fractions were 4.1, 1.5, 26.1, and 68.3%, respectively.  Globin from these animals was isolated, extracted, and assayed to determine the amount of radioactive covalent adducts.  The activity of bound [¹⁴C‑PA]famoxadone residues was 41.5 nmol equivalents/g globin, accounting for approximately 64% of the total RBC activity.  Less than 5% of the total radioactivity of washed RBCs from [¹⁴C‑PA]famoxadone‑treated rats was extractable into acetonitrile. 

An in vitro hepatocyte/RBC co‑incubation system was developed to investigate species differences in metabolism‑haematotoxicity interactions in rats, cynomolgus monkeys, and humans.  Rat hepatocytes and RBCs were co‑incubated with 500 mM [¹⁴C‑PA]famoxadone for 4 hours, and the distribution and covalent binding of radioactivity were assessed.  The lipophilic, stroma, ICF, and Hb fractions contained 39.9, 16.6, 12.6, and 31.0% TRR, respectively.  The activity of covalently‑bound [¹⁴C‑PA]famoxadone residues in isolated globin was 75.4 nmol equivalents/g globin, accounting for approximately 63% of the total RBC activity.

The metabolite profiles of [¹⁴C‑PA]famoxadone in freshly‑isolated rat, cynomolgus monkey, and human hepatocytes were determined after a 4‑hour incubation with 500 mM [¹⁴C‑PA]famoxadone.  In general, the metabolite profile was similar in all species.  The following metabolites were detected in rat hepatocyte extracts by LC/MS:  IN‑KZ007 and a positional isomer of IN‑KZ007, IN‑KZ532, IN‑KZ000 sulfate, IN‑KZ007 sulfate, IN‑KZ532 sulfate, IN‑KZ007 glucuronide, IN‑KZ532 glucuronide, two IN‑KZ534 monosulfate isomers, IN‑KZ534 disulfate, and an unspecified acylated aminophenol sulfate, which was not detected in other species.  Cleaved metabolites detected by GC/MS in rat hepatocytes include aniline, 2‑aminophenol, 4‑aminophenol, and 4‑acetamidophenol.  All of these metabolites were also identified in monkey and human hepatocytes with the exception of IN‑KZ534 disulfate and one of the IN‑KZ534 monosulfate in monkey hepatocytes, and IN‑KZ000 sulfate and IN‑KZ534 sulfates in human hepatocyte extracts.  IN‑BY759 (4‑acetoxyaniline, the presumed famoxadone metabolite responsible for the haematology effects observed in treated animals) was not detected in hepatocyte extracts from any species.  Metabolites of [¹⁴C‑PA]famoxadone with significant haemolytic potential were not identified in hepatocyte extracts from any species.  Likewise, attempts to identify potential hemolytic famoxadone metabolites adducted to haemoglobin were unsuccessful. 

Hematology Effects – Reversibilty Study

Two groups of 10 female rats each were exposed to 0 or 800 ppm famoxadone in the diet, a concentration previously demonstrated to produce mild hemolytic anemia.  Under the conditions of this study, dietary exposure to 800 ppm (61.6 mg/kg/day) test substance produced compound-related reductions in red cell mass, body weight, and nutritional parameters. Body weight and nutritional effects were evident within 7 days of exposure, while red cell mass effects were detected following 30 days of exposure. Partial recovery of the red cell mass effects was observed after 9 days of recovery. Full recovery from the red cell mass effects and partial recovery from the body weight and nutritional effects occurred within 23 days of cessation of exposure to test substance. Therefore, the mild anemia produced by dietary exposure to test substance is fully reversible soon after exposure ends.

Cytochrom P-450 Study: 

The objective of this study was to determine the potential of test substance to alter hepatic cytochrome P-450 (CYP) content in rats and mice fed famoxadone for 2 weeks. Groups of 5 male and 5 female Crl:CD®(SD)IGS BR rats were exposed to 0 ppm or 20000 ppm test substance, and groups of 5 male and 5 female Crl:CD-1®(ICR) BR mice were exposed to 0 ppm or 7000 ppm famoxadone. Approximately 2 weeks after the initiation of test substance administration, rats and mice were euthanized. Livers from each animal were dissected and weighed to correlate with the hepatic cytochrome P-450 data. A portion of the liver was homogenized and hepatic microsomes prepared by differential centrifugation for the evaluation of total cytochrome P-450 content and quantitation of the isozymes CYP1A1, CYP2B1/2, CYP3A, and CYP4A.

In rats, test substance administration decreased absolute and relative liver weights in males, and increased relative liver weights in females. These changes in liver weights were associated with increases in total cytochrome P-450 content, as well as increases in the isozymes CYP2B1/2, CYP3A, and CYP4A for male and female rats. No alterations were observed in CYP1A1 content in either male or female rats. The test substance also caused decrements in body weight and body weight gain. These decrements were associated with decreased food consumption and food efficiency.

In mice, test substance administration increased absolute and relative liver weights in males and females. These changes in liver weights were associated with increases in total cytochrome P-450 content, as well as increases in the isozymes CYP2B1/2 and CYP4A for male and female mice. No alterations were observed in CYP1A1 or CYP3A content in either male or female mice. The test substance also decreased food efficiency in male mice over the entire treatment interval. The decreased food efficiency was not associated with altered food consumption, and test substance treatment did not result in alterations in body weight or body weight gain in either male or female mice.

Mechanistic Study in the Eye:

An in vitro cytotoxicity study with the test substance was conducted in 2 primary lens epithelial cell cultures (canine and monkey), an immortalized lens epithelial cell line (mouse, NK-35), and an immortalized corneal epithelial cell line (human, SV-40). All cell cultures demonstrated cytotoxicity when exposed to the high exposure concentration of 1 mg/ml of the test substance for 3, 24, and 48 hours. Overall there were no clear differences observed in the susceptibility of the 4 cell culture systems to test substance induced cytotoxicity.