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Basic toxicokinetics

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Endpoint:
basic toxicokinetics
Type of information:
experimental study
Adequacy of study:
key study
Study period:
No data.
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: The study was conducted according to guideline and/or standard method but was non-GLP.

Data source

Reference
Reference Type:
publication
Title:
Unnamed
Year:
1995

Materials and methods

Objective of study:
toxicokinetics
Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 417 (Toxicokinetics)
Deviations:
not specified
GLP compliance:
not specified

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
The radiolabelled purity of [ring-U-14C] BTMAC (Chemsyn Science Laboratories, Lenexa, KS, USA) was determined by hplc analysis to be >/= 95%, while the chemical purity of unlabelled BTMAC was 97%.

Unlabelled BTMAC (Aldrich Chemical Co., Milwaukee, WI, USA)
Radiolabelling:
yes
Remarks:
[ring-U-14C] BTMAC

Test animals

Species:
rat
Strain:
Fischer 344
Sex:
male
Details on test animals and environmental conditions:
Male F344 rats, obtained from Charles River Laboratories (Raleigh, NC, USA) were used throughout this work. The age and weight of rats, except where noted, ranged from 2 to 4 months and 18C-295 g respectively.

Administration / exposure

Route of administration:
other: iv, oral gavage or dermal
Vehicle:
water
Details on exposure:
Doses were prepared by diluting [ring-U-I4C] BTMAC (Chemsyn Science Laboratories, Lenexa, KS, USA) with unlabelled BTMAC (Aldrich Chemical Co., Milwaukee, WI, USA) in water to administer the desired dose concentration at 25-50uCi/kg in dose volumes of 1 ml/kg for i.v. injections and 5 ml/kg for gavage. Other individual rats received either single I4C radiolabelled doses of BTMAC administered dermally or unlabelled doses of BTMAC administered by gavage. Dermal doses were prepared by diluting radiolabelled compound with unlabelled compound in ethanol to deliver 63 mg/kg in a dose volume of 30-40ul. The gavage doses (used in the toxicity modulation experiment) were prepared by adding the desired amount of unlabelled
BTMAC to water for administration of a dose volume of 5ml/kg.
Duration and frequency of treatment / exposure:
Various, see study design.
Doses / concentrations
Remarks:
Doses / Concentrations:
Various, see study design.
No. of animals per sex per dose:
various number of males used. See study design.
Control animals:
not specified
Positive control:
No data.
Details on study design:
Disposition
The rates and routes of elimination of BTMAC-derived radioactivity over time were determined by collection and analysis of excreta following p.o. administration of 0.63, 6.3 and 63mg/kg and i.v. administration of 0.63 mg/kg to rats and mice (n = 3 animalslgroup). Animals were individually housed in metabolism cages immediately after dosing for the collection of urine and faeces at time points of 4, 8, 12 and 24h. All animals received food and water ad libitum. The % total dose excreted in urine over time was determined by counting I4C contained in triplicate 20-4 aliquots of each urine sample directly in Ecolume (ICN, Research Products Division, Costa Mesa, CA, USA), in either a model LS 5801 or model 9800 scintillation counter (Beckman Instruments, Inc., Fullerton, CA, USA). The % total dose excreted in faeces was determined after combusting and trapping I4C contained in triplicate 5C-100-mg samples using a model 306 biological oxidizer (Packard Instruments Co., Meriden, CT, USA). Prior to oxidation, faecal samples were air dried, weighed, and ground to a fine powder using a mortar and pestle. The amount of radioactivity contained in each oxidized sample was determined by scintillation counting. The amount of BTMAC-derived radioactivity excreted in expired air was investigated in rats receiving 63 mg/kg by gavage. This was accomplished by placing each rat into an individual glass metabolism cage (Wyse Glass Specialties, Inc., Freeland, MI, USA), attached to a vacuum system that pulled air through the cage at a flow rate of 04-0.6 l/min into two successive cylinders. The first cylinder contained 95% ethanol to trap volatile radioactivity, and the second cylinder contained a 7: 3 mixture of ethylene glycol monomethyl ether and ethanolamine to trap ''C02. The cylinders, each containing 200-400ml of trapping solution, were changed at 4, 8, 12 and 24-h time points. Triplicate 1-ml aliquots of each sample were counted directly in Ecolume in the scintillation counter.

Animals in the previously described treatment groups were killed with C02 at 24 h post-dosing, then necropsied to determine the distribution of BTMAC-derived radioactivity in tissues. Tissues were also collected from rats and mice killed 2 h following p.o. administration of 63 mg/kg and from rats and mice killed 15 min following i.v. administration of 0.63 mg/kg (n = 3/group). Selected tissues were weighed, oxidized (triplicate 50-100-mg samples), and counted in the scintillation counter for the determination of I4C content. Tissue weights were determined gravimetrically, except blood, adipose, skin and muscle, which were estimated to be 8, 11, 16 and 50% of total body weight respectively (Matthews and Anderson 1975, Birnbaum et al. 1980).

BTMAC disposition was also investigated in the rat (n = 6) following dermal administration of 63 mg/kg, to a shaved area of 1 cm2 on the interscapular region of the back. Grooming of the dosing site was prevented by applying a perforated metal cap (Lipshaw Co., Detroit, MI, USA) directly to the skin with cyanoacrylate glue. Urine and faeces were collected at 4,8,12 and 24 h following dose administration. Major tissues were collected, following C02 asphyxiation at the terminal time point (24 h). The dose application site and the metal covering were washed with 70% (v/v) ethanol and the skin of the application site was excised and oxidized. Samples of tissues, ethanol wash, and excreta were analysed for BTMAC-derived radioactivity as previously described.

Biliary excretion of BTMAC-derived radioactivity was investigated in two rats following tail vein injections of 0.63 mg/kg. After anaesthetization with an i.p. injection of 50mg/kg pentobarbital, each rat was opened along a small ventral midline incision to expose the common bile duct. After distal ligation, the duct was cannulated with beveled PE-10 tubing. The cannula was secured with a suture, bile flow was confirmed, and the surgical incision was closed with wound clips. Serial bile samples were collected for 7 h following dose administration. Rats were maintained in an anaesthetized state throughout the experiment with periodic injections of 0.1 ml pentobarbital. Triplicate 10ul aliquots of each bile sample were analysed for I4C content as previously described.

Metabolism
The relative amounts of BTMAC and radiolabelled metabolites in rat and mouse urine, liver, kidney and faeces extracts, and rat plasma, were determined using an hplc system consisting of two model 510 pumps controlled by a model 840 data station, equipped with a model 481 UV detector set at 220nm (Waters Corp., Milford, MA, USA) and a model A-280 radiochemical detector (Radiomatic Instruments and Chemical Co., Meriden, CT, USA). The system utilized a 10-um Waters uBondapak Cis steel column with a linear gradient from 100% buffer composed of 0.05 M potassium phosphate, 0.025 M sodium heptanesulphonate, and 0.0025 M tetramethylammonium chloride (pH = 3.0) to 100% methanol in 15 min at a flow rate of 1-5 ml/min. Ethanol was added to plasma at a 2:1 ratio to precipitate proteins, then plasma as well as urine samples were centrifuged and aliquots (25-50ul) of each were injected directly into the hplc system. Radioactivity was extracted from faeces, liver, kidney and blood samples pooled from rats and mice of selected treatment groups. Liver, kidney and blood samples were homogenized with a Polytron homogenizer (Brinkman) in an equal volume of 1 : 1 (v/v) water and ethanol. Faeces samples were homogenized in a 5:1 volume of 1:1 (v/v) water and ethanol. Homogenates were centrifuged at 10,000 g
for 10 min, supernatants removed, centrifuged for 5 min in the microfuge, filtered (except faecal samples) through Centricon 10 (10000mw cutoff) microconcentrators (Amicon Division of W.R. Grace and Co., Beverly, MA, USA), and analysed by hplc. Precipitated pellets were homogenized and centrifuged twice more for determination of the I4C extraction efficiency.

Modulation of toxicity
Modulation of BTMAC-induced toxicity was investigated in rats (300-350 g) receiving unlabelled doses of 125, 175, 210 and 250mg/kg by gavage. Each rat concurrently received a subcutaneous injection of either saline, neostigmine (0.1 mg/kg), or atropine sulphate (1 mg/kg) in a dose volume of 1 ml/kg (Borchard et al. 1990). In keeping with current standards for the minimization of the use of animals in toxicity testing, only five animals were used per group. Each rat was closely observed for muscarinic type cholinergic symptoms (salivation and chromodacryorrhea), respiratory difficulties, convulsions, and death. Salivation and chromodacryorrhea results were maximized by the use of the following semiquantitative scaling system: 0 = no response, 1 = slight response, and 2 = severe response. These parameters were assessed by a single observer at 60, 120 and 180min following dose administration. Maximal scores depended on survival time. For example, if an animal survived the full 3 h the maximal salivation score would be 6, but if it only survived for 2 h the maximal score is 4. The maximal response per group = 30 (five rats X maximal score of 6). Similar grading systems have been described by Chan and Hayes (1989).

The Reed-Muench method was used to maximize lethality data (Reed and Muench 1938). This method is based on the assumption that an animal that responds to a high dose would also respond to a lower dose and that an animal that does not respond to a high dose would not respond to a lower dose. Therefore, the response of a single animal can, in some cases, be applied to several dose groups enabling a reduction in the number of animals required to obtain meaningful results.

References:
BIRNBAUM, L. S., DECAD, G. M., and MATTHEWS, H. B., 1980, Disposition and excretion of 2,3,7,8-tetrachlorodibenzofuran in the rat. Toxicology and Applied Pharmacology, 55, 342-352.

BORCHARD, R. E., BARKES, C. D., and ELTHERINGTOL, N.,G., 1990, Drug dosage tables. In Drug Dosage in Laboratory Animals. A Handbook, 3rd edn (Caldwell: Telford), pp. 1-512.

CHAN, P. K., and HAYES, A . W., 1989, Principles and methods for acute toxicity and eye irritancy. In Principles and’Methods of Toxicology, 2nd edn, edited by A. W. Hayes (New York: Raven), pp. 169-220.

MATTHEWS, H. B., and ANDERSON, M. W., 1975, The distribution and excretion of 2,4,5,2’,5’-pentachlorobiphenyl in the rat. Drug Metabolism and Disposition, 3, 211-219.

REED,L . J., and MUENCH, H., 1938, A simple method of estimating fifty per cent endpoints. American Journal of Hygiene, 27, 494-497.
Details on dosing and sampling:
Doses were prepared by diluting [ring-U-I4C] BTMAC (Chemsyn Science Laboratories, Lenexa, KS, USA) with unlabelled BTMAC (Aldrich Chemical Co., Milwaukee, WI, USA) in water to administer the desired dose concentration at 25-50uCi/kg in dose volumes of 1 ml/kg for i.v. injections and 5 ml/kg for gavage. Other individual rats received either single I4C radiolabelled doses of BTMAC administered dermally or unlabelled doses of BTMAC administered by gavage. Dermal doses were prepared by diluting radiolabelled compound with unlabelled compound in ethanol to deliver 63 mg/kg in a dose volume of 30-40ul. The gavage doses (used in the toxicity modulation experiment) were prepared by adding the desired amount of unlabelled BTMAC to water for administration of a dose volume of 5ml/kg.

Pharmacokinetics
Pharmacokinetic parameters of BTMAC were determined following p.o. administration of 0.63 and 6.3 mg/kg and i.v. administration of 0.63 mg/kg to rats (3-4 rats/group). Each rat was first anaesthetized with an i.p. injection of ketamine (100mg/kg) and xylazine (3.2mg/kg) in a dose volume of 1 ml/kg, then the right jugular vein was exposed and cannulated with 0.02 X 0.037-inch Silastic medical-grade tubing (Dow Corning Corp., Midland, MI, USA). One end of each cannula, filled with 0.9% saline containing 10 units heparin/ml, was inserted to the heart and anchored to the pectoralis major muscle with a suture. The free end of the cannula was passed under the skin of the neck and exited through a small incision in the interscapular region of the back. After tying off the cannula and closing all surgical incisions, each animal was allowed to recover overnight (16 h). The next morning, BTMAC was administered by gavage or bolus injection directly through the patent cannula and blood (0.3-0.4ml) was collected at 0.08, 0.25, 1, 2, 4, 6, 8, 10, 12 and 24 h using an attached syringe. An equal volume of heparinized saline was injected back into the cannula at each sampled time point. Whole blood was separated into plasma and red blood cell fractions by centrifugation for 5 min in an Eppendorf model 5412 microfuge (Brinkman Instruments, Westbury, NY, USA). Radioactivity in plasma samples was determined by counting triplicate 10-ul aliquots in Ecolume. Total radioactivity/ml blood was determined by oxidizing and counting triplicate 20 ul aliquots as previously described. Data were analyzed using PCNONLIN (SCI Software, Lexington, KY, USA),
Statistics:
Pharmacokinetics
Data were analyzed using PCNONLIN (SCI Software, Lexington, KY, USA).

Results and discussion

Toxicokinetic / pharmacokinetic studies

Details on absorption:
Absorption of the compound through the skin appeared to be linear, but limited, over a course of 24 h following dose administration.
Details on distribution in tissues:
Excretion of BTMAC-derived radioactivity was >90% complete in rat 24h following p.o. administration of the highest dose, with little 14C remaining in either the major tissues or in the GI tract.

I .V. injection resulted in distribution of 14C to all tissues assayed within 15min of dose administration. The highest concentrations of 14C were present in liver and kidney. Only low levels of I4C were detected in brain.
Details on excretion:
Following single dose administration of 0.63, 6.3 and 63 mg/kg of BTMAC by gavage to the rat, elimination of BTMAC-derived radioactivity was rapid. Almost all of the radioactivity was recovered (total dose recovery ranged from 82 to 97%) in urine and faeces within 24 h post-dosing. Excretion of BTMAC-derived radioactivity was >90% complete in rat 24h following p.o. administration of the highest dose, with little 14C remaining in either the major tissues or in the GI tract. The major route of elimination of oral doses by rats was in the faeces. At the low dose (0.63 mg/kg) elimination by rat was similar and favoured faeces over urine by a ratio of approximately 7:1. However, the rat eliminated less BTMAC-derived radioactivity in faeces following p.o. administration of either of the two higher doses. No BTMAC-derived radioactivity was excreted in expired air, either as volatiles or as 14C02, following administration of 63 mg/kg.

Routes of BTMAC-derived radioactivity elimination in rat were dependent upon the route of dose administration. Elimination of the major portion of the dose following p.o. administration of 0.63 mg/kg was in faeces. However, 24 h following i.v. administration of 0.63 mg/kg, > 80% of BTMAC-derived radioactivity was excreted in urine, with no more than 10% of the total dose excreted in faeces. Less than 2% of the total dose of 6.3 mg/kg administered by i.v. injection to the rat was excreted in bile within 7 h, indicating minimal enterohepatic circulation of BTMAC-derived radioactivity. I .V. injection resulted in distribution of 14C to all tissues assayed within 15min of dose administration. The highest concentrations of 14C were present in liver and kidney. Only low levels of I4C were detected in brain. As with p.o. administration elimination of 14C following i.v. administration was rapid, resulting in residual levels (< 3% of the total dose) of radioactivity present in major tissues at 24 h post-dosing. BTMAC was rapidly eliminated from plasma following i.v. injection to rats with cannulated jugular veins. The data best fit a one-component model with an elimination half life of approximately 30min. In contrast, after administration of either 0.63 or 6.3mg/kg by gavage, the half life for the disappearance of BTMAC from plasma was approximately 2 h. With oral administration, the maximum plasma concentration (Cmax) was proportional to dose; however, the maximum concentration was reached at a later time (Tmax) and the estimated bioavailability (F) was greater for the higher dose.

The disposition of BTMAC was also investigated following dermal administration of 63 mg/kg to the rat. Absorption of the compound through the skin appeared to be linear, but limited, over a course of 24 h following dose administration. Only 7 +/- 3% of the total dose was excreted in urine within 24 h, with cumulative 24-h excretion in faeces and 14C tissue distribution accounting for < 5% of the total dose. All remaining 14C was recovered from the site of BTMAC application (total dose recovery = 95-105%).



Metabolite characterisation studies

Metabolites identified:
no
Details on metabolites:
The metabolism of BTMAC in rat was characterized by hplc analysis of urine, plasma, and blood, liver, kidney and faecal extracts. BTMAC-derived radioactivity was readily extracted from rat and mouse blood as evidenced by extraction efficiencies > 95%. Most (75-85%) of the BTMAC-derived radioactivity in rat liver and kidney was also recovered following three successive solvent extractions. Filtration of supernatants from blood, liver and kidney homogenates through microconcentrators containing 10000 mw cutoff filter membranes indicated that little or no BTMAC-derived radioactivity was bound to soluble protein. The amount of BTMAC-derived material that could be extracted from faeces varied greatly between treatment groups. In the rat, > 80% of the total I4C in faeces was extractable following p.0. administration of the high dose (63mg/kg). However, only 15% of the 14C was extractable following p.o. administration of 6.3 mg/kg. Hplc analysis indicated that all 14C extracted from liver, kidney, blood and faeces of rat was composed of unmetabolized BTMAC (RT= 13.4min). Additionally, all 14C detected in rat plasma, obtained 5 and 30 min following i.v. administration of 0.63 mg/kg, consisted of parent BTMAC. Most 14C present in cumulative 24 h rat and mouse urine also consisted of unmetabolized BTMAC. Some, but not all, hplc chromatograms of rat urine contained an unidentified radiolabelled peak eluting at 8.7 min, which accounted for up to 10% to the total 14C excreted in urine.

Any other information on results incl. tables

Table 1 Concentration of BTMAC-derived radioactivity in tissues following i.v. administration of 0.63 mg/kg to the rat

Tissue   0.25 hr  24 hr
 Blood  342 + 51a,b  5 + 1
 Liver  1997 + 226b  22 + 2
 Kidney  1924 + 267b  23 + 3
 Muscle  120 + 5  31 + 4
 Skin  342 + 7  15 + 3
 Adipose  117 + 29  8 + 1
 Brain  30 + 3  2 + 1

a Mean + SD for three animals.

b All extracted 14C consisted of BTMAC

Values are ng equivalents/g tissue.

Results obtained from the toxicity experiments demonstrated that acute toxicity of BTMAC was characterized by severe cholinergic symptoms including salivation, chromodacryorrhea, and sedation. Diarrhea, tremours, clonic convulsions, and respiratory distress were also usually present. The incidence of spontaneous convulsions could be readily increased by handling or otherwise disturbing the animals. Rats that did not survive exhibited a series of regular convulsions, loss of consciousness, rapid heart rate, and shallow respiration with gasping, just before death. Death or survival of each animal was generally determined within 3 h of dose administration.

The effects of atropine and neostigmine on BTMAC-induced salivation and chromodacryorrhea were also examined. The atopine groups were clearly protected against BTMAC-induced salivation and chromodacryorrhea. In contrast, neostigmine appeared to potentiate the salivation response at 125 and 175 mg/kg. At the higher doses, salivation was already near maximum and potentiation of this response by neostigmine was difficult to determine. Neostigmine appeared to potentiate chromodacryorrhea only at the lowest dose, probably because this response was at or near the biological maximum at the next highest dose. In the non-BTMAC treated rat (n = 3) administration of neostigmine resulted in slight but readily detectable salivation and chromodacryorrhea.

The high dose that was used in the toxicity modulation experiments was the reported oral LD50 in rat (DeWitt et al. 1953), therefore some mortality among animals was expected. However, BTMAC appeared to be more toxic than previously reported, since mortality in the present study was near 100% at the high dose. Although these experiments were not designed to be an LD50 study, death was dose related, and, in an effort to ascertain as much information from this work as possible, estimates of toxicity were calculated. Treatments did not appear to alter BTMAC lethality significantly, since there were no left or right shifts in the curves, only minimal differences in slopes were observed, and the LD50’s for each group were essentially identical. Mortality was identical at 11 of 20 animals for each of the three treatment groups (saline-, atropine- , and neostigmine-treated rat).

Reference:

DEWITT, J. B., BELLACK, E., KLINGENSMITCH. W., WARD, J. C., and TREICHLER, R., 1953, Relationship Between Chemical Structure and Toxic Action on Rats. Chemical Biological Research Center, Review No. 5 (National Research Council, Washington), p. 39.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): low bioaccumulation potential based on study results
The limited absorption and rapid elimination of BTMAC should result in little or no bioaccumulation in tissues following repeated exposure to low levels of this compound. This observation suggests that greater human health risks may be associated with acute high level exposure rather than chronic low level exposure. In man, acute exposure of BTMAC is likely to result from dermal contact. Since BTMAC appears to be poorly absorbed from skin, thorough washing following contact with the chemical should minimize the risk of acute poisoning.
Executive summary:

Benzyltrimethylammonium chloride (BTMAC)-derived radioactivity was rapidly eliminated from the F344 rat following p.o. administration of 0.63-63 mg/kg of [ring-U-14C]BTMAC. Greater than 90% of the radioactivity was excreted in urine and faeces within 24-h post-dosing.

2. BTMAC was poorly to moderately absorbed from the GI tract following p.o. administration. The percent of total dose absorbed did not exceed either 40% in the rat.

3. Absorption was linear, but limited, over time following dermal administration of 63 mg/kg to the rat. Less than 10% of the total dose was absorbed from the skin within 24 h of BTMAC application.

4. Metabolism of BTMAC was minimal in the rat. Toxicity (excessive cholinergic stimulation and mortality) appears to be attributable to the parent compound.

5. The limited absorption and rapid elimination of BTMAC should result in little or no bioaccumulation in tissues following repeated exposure to low levels of this compound. The results suggest that greater human health risks may be associated with acute high level

exposure rather than chronic low level exposure.