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Basic toxicokinetics

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basic toxicokinetics in vivo
Type of information:
experimental study
Adequacy of study:
supporting study
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: good quality study and detailed reporting but no indication of GLP status.

Data source

Reference Type:

Materials and methods

Objective of study:
GLP compliance:
not specified

Test material

Test material form:
solid - liquid: suspension
migrated information: dispersion
Details on test material:
[U-14C]Chlorendic acid (12 mCi/mmol) was purchased fromPathfinder Laboratories (St. Louis, Mo.). Radiochemical puritywas determined by radio-gas-liquid chromatography on 3% QF-1 byDr. Phillip Albro, Laboratory of Environmental Chemistry, NationalInstitute of Environmental Health Sciences. The radiolabeled compoundwas >99% radiochemically pure. The dose solution was madeup by dissolving [14C]chlorendic acid in a 1:1 mixture of Emulfor,a polyoxyethylated vegetable oil (GAF Corp., New York), and ethanolalong with unlabeled chlorendic acid (K & K Laboratories, Irvine, Calif),>99% pure by nuclear magnetic resonance (NMR), determinedby Dr. Phillip Albro. Distilled water was then added to give a finalchlorendic acid concentration of 3.0 mg per milliliter of the mixtureof Emulfor, ethanol, and water (1:1:8 by volume). [

Test animals

Fischer 344/DuCrj
Details on test animals and environmental conditions:
Male adult Fischer 344 rats weighing 176-215 g were used, housed under a 12-h light cycle for at least 1 wk before use,and fed Purina Rat Chow and offered water ad libitum.

Administration / exposure

Route of administration:
other: intravenous and oral
other: mixture of emulflor (polyoxyethylated vegetable oil), ethanol and water
Details on exposure:
[14C]Chlorendic acid solution was injected iv into the tail vein of rats (3 mg/kg, 7.7 umol/kg, 11 uCi/kg, 1 ml/kg), which were held for 15 min to 7 d, after whichthey were sacrificed by cervical dislocation. For absorption studies, rats received the same dose as in the iv study by oral intubation and were sacrificed by cervical dislocation after 1 d.
Doses / concentrations
Doses / Concentrations:(3 mg/kg, 7.7 umol/kg, 11 uCi/kg, 1 ml/kg. Same dose for intravenous and oral
No. of animals per sex per dose:
3 per time point
Control animals:
Details on study design:
Three animals were exsanguinated by cardiac puncture at each time point, dissected immediately, and the tissues weighed and stored in a freezer until they could be prepared for analysis by oxidation to 14CO2. Recovery of 14CO2 radioactivity was determined and corrected for quenching in a Beckman model LS8100 liquid scintillation system. In each case, a section of the tail injection site with 0.5 cm2 of surrounding tissue was removed and the residual radioactivity determined.When the injection site contained as much as 5% of the [14C] chlorendic acid dose, the animal was discarded and another treated for the respectivetime period. Approximately 5% of the animals were discarded. Mostof the tissue samples were finely minced before oxidation. However,blood (0.2 ml drawn with a heparinized syringe from the heart), adipose tissue (50 mg perirenal), and skin (100 mg portion from theears) were oxidized directly. The skin weight and weight of adipose tissue depots of Fisher 344 rats are 16 and 11% of body weight,respectively (Brinbaum et al., 1980). Estimates of blood volume and muscle weight, 8 and 50% of body weight, respectively, were basedon literature values for rats (Matthews and Anderson, 1975).Animals held for 1 d or longer were housed in individual metabolism cages with food and water ad libitum; feces and urine were collecteddaily. The feces were air-dried, weighed, and ground into a powder with mortar and pestle; two 100-mg samples of each daily collectionwere oxidized. [14C]Chlorendic acid-derived radioactivity in the urine was quantified by determining the volume of each urine collectionand counting two 0.1-ml samples directly into Aquasol. Liquid scintillation counting efficiencies were determined by use of an appropriate [14C]chlorendic acid standard and corrected for quench in all cases.Bile duct cannulation experiments were carried out by first anesthetizing rats with pentobarbital (Matthews and Anderson, 1975). The commonbile duct was cannulated with PE-10 tubing and bile collected at timed intervals for 6 h. Excretion in bile was determined by counting duplicate 10-/ul samples for each time point in 10 ml Aquasol in a liquid scintillation spectrometer.Radioactivity was extracted from 6-g samples of liver, 2 g from each of three 1-d animals, with organic solvents before and after acidhydrolysis (Matthews and Anderson, 1975). Tissue extracts were concentrated to 10 ml by rotary evaporation under vacuum and furtherconcentrated under N2. Concentrated extracts were chromatographed as a band on 20 X 20 cm silica gel G thin-layer plates (Analtech, Inc.,Newark, Del.) for 15 cm. The solvent systems used were (1) n-butanol, acetic acid, and water (12:3:5 by volume) and (2) ethyl acetate andacetic acid (9:1 by volume). An authentic standard of [14C]chlorendic acid (Rf = 0.69 in both solvent systems) was chromatographed onthe same plate with each tissue extract. After chromatography, the silica gel was scraped from the plates in 1-cm bands, placed into liquidscintillation vials, shaken vigorously with 20 ml Aquasol, and counted.Bile samples were analyzed by thin-layer chromatography with the solvent systems described above both before and after hydrolysisin 1 N HCI at 90°C for 1 h. Bile samples were also treated with )3- glucuronidase oraryl sulfatase and then a portion of the unextractedsample was subjected to thin-layer chromatography as described above. Approximately 20 /xl bile containing approximately 30,000cpm was incubated at 37°C for 17 h in 0.1 M acetate buffer, pH 5.0, containing 200 U/ml j3-glucuronidase (bovine liver, type B10, SigmaChemical Co., St. Louis, Mo.) or 30 U/ml aryl sulfatase (abalone entrails, type VIM, Sigma Chemical Co.).Feces were extracted in a Soxhlet apparatus (Matthews and Anderson, 1975) and the extracts analyzed by thin-layer chromatographyas described above. Urine was analyzed after extraction with ether(Matthews and Anderson, 1975). Tissue distribution data were analyzed by a nonlinear regressionanalyses computer program (Morales et al., 1979) based on the exponential decay curves. The number of exponential terms was determinedby best fit. Data are expressed as the mean ± SD, n>3.

Results and discussion

Main ADME resultsopen allclose all
substance absorbed by gastrointestinal tract
initially distributed to the blood, liver, muscle, skin, and kidney and did not accumulate in adipose tissue
not influenced by exposure route
rapid excretion, mainly through feces

Toxicokinetic / pharmacokinetic studies

Details on absorption:
The data showed that orally administered chlorendic acid was absorbed from the gastrointestinal tract
Details on distribution in tissues:
Chlorendic acid-derived radioactivity was initially distributed to the blood, liver, muscle, skin, and kidney and did not accumulate in adipose tissue. Most of the dose was located in the liver.
Details on excretion:
Excretion of [14C] chlorendic acid-derived radioactivity was analyzed by daily collection of urine and feces from individual animals held for 1 d or longer after treatment. The major route of excretion of chlorendic acid was the feces, and approximately 78% of the dose was excreted in the first 24 h. Most of the urinary excretion also occurred within the first 24 h, and less than 0.1% of the dose appeared in the urine on subsequent days. Thus, by the first day, more than 73% of the total dose was recovered in the excreta (Table 2). Since the feces were the major route of elimination, excretion of radioactivity through the bile was studied. 65% of an iv dose of [14C]chlorendic acid derived radioactivity was excreted in the bile within 5 h. This is in close agreement with the fecal excretion data, suggesting that most of the [14C] chlorendic acid-derived radioactivity in bile was excreted in the feces.

Metabolite characterisation studies

Metabolites identified:
Details on metabolites:
The [14C]chlorendic acid-derived radioactivity in the urine, bile, and feces was examined by extraction and thin-layer chromatography.In the urine, 72% of the radioactivity appeared to represent parent compound. The remainder of the radioactivity was released after acidhydrolysis and then cochromatographed with parent compound, suggesting the presence of conjugates. Similar extraction and analysis ofbile collected at time points from 15 min to 5 h indicated that about 20% of the total radioactivity cochromatographed with parent compound.Approximately 25% of the radioactivity extracted from bile chromatographed with an Rf of 0.19 in ethyl acetate and acetic acid(9:1); the remaining radioactivity was at the origin. After acid hydrolysis, all the radioactivity cochromatographed with the parent compound inboth systems (Rf = 0.69), indicating the presence of conjugates. Treatment of unhydrolyzed bile with j3-glucuronidase or aryl sulfatase didnot alter the chromatographic results. Feces were sequentially extracted with hexane, methylene chloride,and acetone before and after acid hydrolysis; only 34% of the radioactivity in feces could be extracted before acid hydrolysis. After acidhydrolysis, 31% was extracted by hexane. Analysis of this extract by thin-layer chromatography in ethyl acetate and acetic acid (9:1) indicatedthat 81% of the radioactivity cochromatographed with the parent compound, 7% had an Rf of 0.19, and the remainder was located atthe origin. The results suggest that most of the radioactivity excreted in bile, and subsequently in feces, represented metabolites of chlorendicacid

Applicant's summary and conclusion

Interpretation of results (migrated information): no bioaccumulation potential based on study resultsThe data showed that orally administered chlorendic acid was absorbed from the gastrointestinal tract. Chlorendic acid-derived radioactivity was initially distributed to the blood, liver, muscle, skin, and kidney and did not accumulate in adipose tissue, as previously observed for dieldrin (IARC, 1974.Distribution to the tissues was apparently not influenced by route of exposure. Most of the dose was located in the liver. Essentially 96% of the tissue burden may be either acid-labile conjugates of Chlorendic acid-derived radioactivity was rapidly excreted, primarily by the feces, with only 3-6% in the urine. Radioactivity in the urine was primarily parent compound; the remainder was most likely conjugates.In contrast, most of the radioactivity in the bile was conjugates of chlorendic acid and only about 20% was parent compound. After acid hydrolysis, extraction of chlorendic acid-derived radioactivity from bile was nearly complete; however, after acid hydrolysis of feces, less than one-third of the chlorendic acid-derived radioactivity could be extracted. Data for control extractions of chlorendic acid-spiked feces showed approximately 90% extraction of chlorendic acid.Chlorendic acid was not stored in any of the tissues examined but was rapidly excreted in the bile; active tubular excretion of this chemical by the kidneys apparently had a relatively minor role in its clearance, in contrast to observations for other organic acids (Pitts, 1979). This result also contrasts with similar studies of the structurally related lipophilic insecticides, aldrin and its metabolite, dieldrin. Dieldrin was shown to be present in the environment and bioaccumulated in adipose tissue, liver, brain, and muscle of mammals, birds, fish and invertebrates (IARC, 1974). It accumulates in the food chain and was detected in human milk and adipose tissue (IARC, 1974). [14C]Aldrin was converted to dieldrin after oral administration to male rats, and the dieldrin was stored in adipose tissue (IARC, 1975).The polarity of chlorendic acid and the ability of the rat to metabolize it and rapidly clear it from the body may explain the lack of storage of this compound or its metabolites in adipose or other lipophilic tissues.Chlorendic acid is an amphipathic molecule with a hydrophilic dicarboxylic acid portion. Apparently the hydrophilic portion of the molecule facilitates its metabolism and excretion.
Executive summary:

The absorption, distribution, and excretion of a highly chlorinated dicarboxylic acid, chlorendic acid, was studied in the male Fischer 344 rat. [14C] Chlorendic acid was absorbed after on oral dose of 7. 7 umol per kilogram of body weight. The distribution in various tissues was similar whether the treatment was by the oral or the intravenous route. The major site of [14C]chlorendic acid deposition was the liver, with smaller amounts found in the blood, muscle, skin, and kidneys. Chlorendic acid-derived radioactivity was excreted primarily through the bile and into the feces. The urine contained less than 6% of the total dose. Within 1 d, more than 75% of the total dose was excreted in the feces, primarily as metabolites. Radioactivity in the liver was also primarily metabolites of chlorendic acid. Thus, chlorendic acid was absorbed, metabolized, and excreted primarily in the feces as metabolites. The rapid metabolism and biliary excretion of chlorendic acid contrast with observations for the closely related lipophilic compounds aldrin and dieldrin.