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Capillary gas chromatographic analysis of trichlorfon in dosed feed formulations.

A method was developed for the analysis of trichlorfon as the intact molecule from feed formulations of the chemical. The method involved isolation of trichlorfon by a simple extraction procedure. Subsequent detection and quantitation of the trichlorfon was by gas chromatography with a DB-1 fused-silica wide-bore capillary column and flame photometric detection. Automated on-column injections were performed. The method was linear in the range 5-75 ng injected on-column and had an average recovery of 86%. The limit of detection for trichlorfon in feed was 1 ppm. The applicability of the method was tested by determining the stability of trichlorfon over a 21-day period in a feed blend of trichlorfon at the 50-ppm concentration level. Mass spectrometric analyses were performed to confirm that trichlorfon could be quantitated as the intact molecule.

Animal Feed

Trichlorfon-induced congenital cerebellar hypoplasia in neonatal pigs.

The neuroteratogenicity of trichlorfon was evaluated in 3 groups of pregnant sows (8/group). The treatments were: control (no trichlorfon), trichlorfon (60 mg/kg of body weight) in the feed only on day 55 of gestation, or trichlorfon (60 mg/kg of body weight) in the feed on day 55 and day 70 of gestation. One week after farrowing, all newborn pigs were removed from the sows and were euthanatized. Brain and cerebellum weights of the newborn pigs were recorded. Mean cerebellum weights and cerebellum/total brain weight ratios of the neonatal pigs were 3.780 and 0.106 (group 1, n = 26), 3.183 and 0.098 (group 2, n = 42), and 2.986 and 0.088 (group 3, n = 61). Although trichlorfon interfered with cerebellar development and the severity of the trichlorfon-induced cerebellar hypoplasia was dosage-related, ataxia did not develop in the neonatal pigs.

Animals

Critical tests and safety studies on trichlorfon as an antiparasitic agent in the horse.

Three series of critical tests were completed on a combined total of 46 horses to determine the efficacy of single doses of trichlorfon against bots, ascarids, pinworms, and large strongyles. Different formulations of trichlorfon were administered by tubing intragastrically, mixing with the daily grain ration, injecting intramuscularly, or pouring on the back at dose rates between 20 and 100 mg/kg. Administration by feeding tended to be more efficacious for removal of bots and less toxic to the horese than administration by stomach tube. In many of the tests, trichlorfon was given in the grain ration at the dose rate of 40 mg/kg of body weight, and the aggregate average removals of 2nd and 3rd instars of Gastrophilus intestinalis and Gasterophilus nasalis in the 3 series of tests were between 97 and 100%. Removal of Parascaris equorum was equally efficacious with both the intubation and the grain feeding methods of dosing, and at the dose rate of 40 mg/kg, the aggregate averages were 99 and 100% in the 3 series. Removal of Oxyuris equi was variable--aggregate averages were between 11 (1 infected horse in the initial series) and 96 (5 infected horses in the 3rd series) to 100% (7 infected horses in the 2nd series). Large strongyles, Strongylus vulgaris and Strongylus edentatus were almost completely refractory to the 40-mg/kg dose rate of trichlorfon. Dose rates of 40 mg/kg and less were generally well tolerated by the critical test horses. Higher dose rates (60 and 80 mg/kg) administered by stomach tube induced moderately severe to severe colic and diarrhea, whereas a dose of 80 mg/kg given in the feed resulted in only a transient softening of the feces. Likewise, 5 consecutive doses, 1 week between doses, of a bolus formulation given at the rate of 80 mg/kg to 4 horses were well tolerated. Clinical trials involving a total of 2,294 treatments of trichlorfon at dose rate of 35 to 40 mg/kg in pregnant and nonpregnant mares, stallions, suckling and weanling foals, yearlings, and horses in training on 38 farms in central Kentucky did not cause notable adverse clinical effects.

Administration, Oral

Cytogenetic, genetic, and embryotoxicity studies with dimethyl 2,2,2-trichloro-1-(2,2,2-trichloro-1-hydroxyethoxy)-ethylphosphonat e, a hypothetical impurity in technical grade trichlorfon.

The hemiacetal (CH3O)2P(O)CHOCHOHCCl3)CCl3, a hypothetical contaminant in technical preparations of the organophosphorus pesticide trichlorfon, was tested for cytogenetic, mutagenic, and embryotoxic activity after ip administration to mice of different strains. A single dose of 81 mg/kg (0.2 mmol/kg) caused a significant enhancement in the percentage of chromosome aberrations in bone marrow cells of CFLP mice; a similar effect was induced by an equimolar single dose of chemically pure trichlorfon (51.5 mg/kg). At the same dosage level, the hemiacetal proved to be ineffective in the micronucleus test on fetal blood of DBA and AB Jena/Halle mice. In the dominant lethal mutation assay, a single dose of 81 mg/kg hemiacetal to males resulted in a slight increase in the fetal mortality of DBA mice, whereas AB Jena/Halle mice did not respond under these conditions. Four consecutive doses of 81 mg/kg hemiacetal to pregnant AB Jena/Halle mice at Days 2, 3, 4, and 5 of gestation caused only a very weak embryotoxic effect comparable to that of trichlorfon at equimolar dosage. On the basis of these results the hemiacetal tested may not be considered to represent a potential risk factor in technical grade trichlorfon.

Animals

An acute and subacute neurotoxicity assessment of trichlorfon.

The toxicity of trichlorfon (O,O-dimethyl-2,2,2,-trichloro-1-hydroxyethylphosphonate, Dipterex, Dylox), reported to elicit delayed neurotoxicity in man and chickens, was studied by administering single subcutaneous doses of 100 or 300 mg/kg to adult White Leghorn hens. At 24 h posttreatment, the birds were observed for visible signs of neurotoxicity, were euthanized, and samples of blood plasma, brain, and spinal cord (cervical and thoracic regions) were obtained for quantification of cholinesterase and neurotoxic esterase (NTE) activities. In subacute studies, hens were dosed with trichlorfon (100 mg/kg) every 72 h for a total of six doses. Seventy-two hours after the final dose the hens were euthanized, the brains, spinal cords, and distal sciatic nerves were removed for enzymatic and (or) histological examination. Parallel acute and subacute studies were conducted using diisopropyl phosphorofluoridate (DFP), a known neurotoxic agent, at subcutaneous dosages of 1.0 mg/kg. In the acute studies, both DFP and trichlorfon markedly inhibited tissue cholinesterase activities but only DFP elicited a significant inhibition of NTE. In the subacute studies, DFP produced a characteristic central-peripheral distal axonopathy in the 18-day period of study which was confirmed by clinical and morphological evidence and by marked inhibition of neuronal NTE. Trichlorfon caused little or no obvious neurotoxicity, an observation that was supported by minimal morphological changes and impairment of walking ability and no inhibition of brain or spinal cord NTE.

Animals

Utilization of dichlorvos and trichlorfon in salmonid farming in Norway during 1981-1988.

The main objectives of this investigation were to quantify the use of dichlorvos and trichlorfon in the treatment of salmon lice infestations, to evaluate the prescribing of these drugs, and to estimate possible changes in the salmon lice problem by use of drug statistics. This study has shown that the use of trichlorfon increased from 4.9 tons in 1981 to 28.3 tons in 1985. This figure declined to 3.2 tons in 1988. The use of dichlorvos increased from 0.3 tons in 1986 to 3.2 tons in 1988. The change in the prescribing from trichlorfon to dichlorvos has dramatically reduced the pollution caused by these substances in the marine environment. Moreover, if necessary safety rules are observed, this change reduces the exposure of the workers on fish farms to these drugs, and also reduces the possibilities of intoxications of the fish during the treatment procedure. The sales figures of dichlorvos and trichlorfon, related to the calculated biomass of farmed salmonids in the sea, indicate a dramatic increase in the salmon lice problem.

Agriculture

Cardiovascular effects recorded in horses during anaesthesia after treatment with trichlorfon.

Five horses were anaesthetised twice with thiopentone sodium, guaifenesin and halothane. The second anaesthesia was 16 days after the first and two days following oral administration of trichlorfon. Heart rate, carotid arterial, pulmonary arterial and right atrial pressures, cardiac output and blood temperature were measured every 15 minutes for 120 minutes. Heart rate, carotid arterial pressure and cardiac output were similar on both occasions. Pulmonary arterial and right atrial pressures were highest during anaesthesia after treatment with trichlorfon when compared with values obtained before treatment. Pulmonary vascular resistance was significantly decreased at four measurement times during anaesthesia after treatment with trichlorfon. All cardiovascular measurements were within ranges accepted as normal for halothane anaesthesia in horses. In a second experiment, four ponies were anaesthetised with xylazine and ketamine on two occasions one week apart. Two ponies received trichlorfon two days before the second anaesthesia. Heart rate, arterial pressure and respiratory rate recorded during anaesthesia were not different in ponies after organophosphate treatment. The time to standing after the second anaesthesia was significantly increased in all ponies.

Anesthesia, General

Mutagenic evaluation of trichlorfon using different assay methods with Salmonella typhimurium.

Evaluation of the mutagenicity of trichlorfon pesticide was carried out with strains TA1535, TA100, TA97, TA98 and TA104 of Salmonella typhimurium by means of several assay methods: (i) spot test; (ii) standard plate incorporation test; (iii) plate incorporation test with preincubation; (iv) fluctuation test and (v) fluctuation test with preincubation, with and without post-mitochondrial liver fraction (S9) from Wistar rats pretreated with phenobarbital and 5,6-benzoflavone as a metabolic activation system. Trichlorfon induced base-pair substitution mutations, and its mutagenic activity was decreased by the addition of S9 mix. The fluctuation test and fluctuation test with preincubation were the most sensitive assay methods for detecting the mutagenicity of trichlorfon.

Animals

Efficacy of an oxibendazole-trichlorfon paste formulation against third stage larvae of Gasterophilus intestinalis and its safety in horses.

A paste formulation containing 14.3 per cent of oxibendazole and 44 per cent of trichlorfon was administered to 33 ponies and horses. The dose rate used was equivalent to 10 mg and 30 mg/kg bodyweight, of oxibendazole and trichlorfon respectively. After treatment 25 animals passed between one and 82 third stage larvae of Gasterophilus intestinalis in their faeces. Dosing with 0.2 mg ivermectin/kg bodyweight three weeks later resulted in six animals expelling between one and four bots. The efficacy of the oxibendazole-trichlorfon paste was on average 96.2 per cent. This drug combination given to 52 ponies and horses at the indicated dose rate and to six ponies at twice that dose was tolerated without side effects except transient softening of the faeces in several animals and mild symptoms of colic in two horses.

Administration, Oral

The effects of iodinated glycerol, trichlorfon, and acetaminophen on tumor progression in a Fischer rat leukemia transplant model.

Sixty-day bioassays of iodinated glycerol, trichlorfon, and acetaminophen were conducted using a leukemia transplant model in 6- to 8-week-old F344 rats to investigate the potential of these chemicals to affect tumor progression. The chemicals were administered in the drinking water at doses that approximated those used in previously conducted 2-year carcinogenesis studies. Simultaneous with dose administration, half of a group of young, healthy, syngeneic rats were given subcutaneous transplants of mononuclear cells derived from spleens of leukemic donors. Variables used to quantitate tumor progression included body weight, spleen weight, white blood cell (WBC) and red blood cell (RBC) counts, packed cell volume, hemoglobin concentration, and platelet counts. Iodinated glycerol at 1.25 or 2.5 mg/ml caused a greater increase in leukocytosis in dosed transplant recipients in comparison to that experienced by undosed recipients: trichlorfon at 2.5 or 5.0 mg/ml enhanced splenomegaly and induced greater reductions in RBC parameters in dosed recipients in comparison to that experienced by undosed recipients. Acetaminophen at 3.0 and 6.0 mg/ml resulted in insignificant but dose-related increases in spleen weight and leukocytosis only in the female rat transplant recipients, as was observed in 2-year studies. Based on results from the short-term leukemia transplant model, data from 2-year carcinogenicity studies, and structure-activity considerations, exposure to iodinated glycerol and trichlorfon was more strongly associated with the expression of leukemia than exposure to acetaminophen. The potential carcinogenicity of each of these chemicals should be taken into consideration when calculating estimates of risk and decisions for their use.

Acetaminophen

Direct gas chromatographic analysis of trichlorfon for its decomposition products.

The analysis of trichlorfon by direct gas chromatography is discussed. A procedure is described for the determination of impurities in trichlorfon by using a fused silica capillary column and cold on-column injection. Attempts at quantitating trichlorfon using this system were unsuccessful due to problems with irreproducibility of the peak area. Although no derivatization was performed, there was no decomposition of the analyte as confirmed by mass spectrometry.

Chromatography, Gas

Critical tests of the anthelmintic febantel in the horse: activity of a paste formulation alone or with a trichlorfon paste.

Critical tests were carried out in 10 horses to evaluate the antiparasitic activity of febantel given alone or with trichlorfon. Paste formulations were administered intraorally at dose levels of 6 mg of febantel (active ingredient)/kg and 35 mg of trichlorfon (active ingredient)/kg. In 5 tests with febantel alone, removal of 100% was recorded for mature or immature Parascaris equorum from 2 infected horses. Strongylus vulgaris from 4 infected horses, S edentatus from 5 infected horses, and mature Oxyuris equi from 1 infected horse; and removal of 96% was recorded for small strogyles from 1 horse tested, and bots in 5 infected horses were not affected. In 5 horses treated with both compounds, removal of 100% was recorded for mature P equorum from 2 infected horses, immature P equorum from 1 infected horse, S vulgaris from 5 infected horses, Sedentatus from 5 infected horses, mature O equi from 2 infected horses, immature O equi from 1 horse tested, 2nd Gasterophilus intestin-equi from 1 infected horse, 2nd-instar C nasalis from 1 infected horse, and 3rd-instar C nasalis from 4 infected horses. Removal of 98% was recorded for small strongyles from 1 horse tested, and removal of 65% to 100% for 3rd-instar C intestinalis from 5 infected horses. In the aggregate, removal of 3rd-instar C intestinalis was 99%. Untoward effects of treatment were quite limited. Only a transient softening of feces in 1 of 5 horses given the trichlorfon paste plus the febantel paste was recorded.

Acetanilides

Activity of organophosphorus insecticides in bacterial tests for mutagenicity and DNA repair--direct alkylation versus metabolic activation and breakdown. II. O,O-dimethyl-O-(1,2-dibromo-2,2-dichloroethyl)-phosphate and two O-ether derivatives of trichlorfon.

The following organophosphates were tested for their ability to induce DNA damage in a rec-type repair test with Proteus mirabilis strains PG713 (rec- hcr-) and PG273 (wild-type) and point mutations in the his- strain TA100 of Salmonella typhimurium: O,O-dimethyl-O-(1,2-dibromo-2,2-dichloroethyl)-phosphate (NALED); trichlorfon-O-methyl ether (TCP-O-ME), O,O-dimethyl-(1-methoxy-2,2,2-trichlorethyl)-phosphonate; trichlorfon-O-methyl ether vinyl derivative (TCP-O-MEVD), O,O-dimethyl-(1-methoxy-2,2-dichlorovinyl)-phosphonate. All compounds were negative in the repair test but induced base pair substitutions in S. typhimurium. The mutagenicity of NALED is due to the direct alkylating ability of the parental molecule and to mutagenic metabolites generated by enzymatic splitting of the side chain. Glutathion-dependent enzymes in the S9-mix eliminate the mutagenic activity of NALED completely. Mutation induction by TCP-O-ME and TCP-O-MEVD is predominantly caused by the reactive O-methyl ether configuration of the side chain and is resistant to metabolic inactivation by NADPH- or glutathion-dependent enzymatic pathways in the S9-mix of mice.

Animals

Hepatotoxicity of trichlorfon and dichlorvos in isolated rat hepatocytes.

Hepatotoxicity of organophosphorus insecticides, trichlorofon and dichlorvos, a dechlorinated form of the former, was examined in isolated hepatocytes from untreated control and phenobarbital-pretreated (80 mg/kg, i.p., for 3 days) rats. These compounds produced toxic effects on hepatocytes as evidenced by malondialdehyde production and lactate dehydrogenase leakage in a dose-dependent manner up to the concentration of 2 mM, dichlorvos being more toxic than trichlorfon. Hepatocytes from phenobarbital-pretreated rats were more sensitive to these organophosphates than those from control rats. Dichloroacetaldehyde and dichloroacetic acid, metabolites of dichlorvos, did not injure hepatocytes. The toxic effects of dichlorvos on hepatocytes were enhanced by increasing oxygen concentration during the incubation, or by addition of glycolytic substrates (pyruvate, lactate or fructose) to the incubation mixtures. On the other hand, addition of antioxidants (diethyldithiocarbamate or N,N'-diphenyl-p-phenylenediamine), or cytochrome P-450 inhibitors (SKF-525A or metyrapone) to the incubation mixtures attenuated malondialdehyde production caused by dichlorvos and protected cells from death. Addition of dichlorvos to the incubation mixtures of hepatic microsomes stimulated lipid peroxidation in the presence of NADPH, which was inhibited by further addition of superoxide dismutase but not catalase. These results suggest that hepatotoxicity of trichlorfon and dichlorvos are related to their peroxidative property in microsomes which is accelerated by oxygen.

Animals

Critical tests of morantel-trichlorfon paste formulation against internal parasites of the horse.

Critical tests were completed on six horses to evaluate the antiparasitic activity of a paste formulation mixture of morantel citrate and trichlorfon, administered intraorally at the dose rate of 6 mg morantel base kg-1 and trichlorfon at 30 mg kg-1. Aggregate average removals were: 78% for two horses infected with 2nd instar Gasterophilus intestinalis; 100% for one infected with 2nd instar G. nasalis; 96% for six infected with 3rd instar G. intestinalis; 100% for four infected with 3rd instar G. nasalis; 100% for five infected with Parascaris equorum; 100% for one infected with mature Oxyuris equi; 100% for five infected with Strongylus vulgaris; 72% for five infected with S. edentatus; and partial removal (25%) of Anoplocephala perfoliata infection from one infected animal. Pre- and post-treatment EPG and LPG data indicated a reduction of 97% of the mature small strongyle infections. Evidence of toxicosis was not observed in any of the horses.

Animals

Intermittent chemotherapy with trichlorfon (metrifonate) reverses proteinuria, hematuria, and leukocyturia in urinary schistosomiasis: results of a three-year field study.

Trichlorfon (metrifonate) was given intermittently to 37 schoolboys with urinary schistosomiasis living in a hyperendemic area of the Sudan. Patients were followed up for three years. Initially, 10 mg of trichlorfon/kg of body weight was administered; this dosage was repeated 14 days and 16 months later. Patients still excreting eggs after 24 months received a fourth dose. At month 24, 61% and at month 36, 56% of the patients had no detectable egg excretion; the others showed severe reduction of egg output. The number of ova excreted was always paralleled by a combined scale of hematuria, leukocyturia , and proteinuria, as assessed by urine analysis reagent strips. Quantitative urine analysis at month 36 revealed pathological findings in only eight individuals. Thus, trichlorfon given three or four times in a dose of 10 mg/kg of body weight spaced over a period of two years was highly effective in reducing parasite load and disease in children living under hyperendemic conditions.

Child

The effect of the organophosphate trichlorfon on the neuromuscular blocking activity of atracurium in halothane-anesthetized horses.

To determine whether cholinesterase inhibition by an organophosphate would influence atracurium's neuromuscular blockade, six horses were anesthetized and paralyzed with atracurium (total of five injections per horse) on experimental Day 1, then were given trichlorfon (64 mg/kg per os) 6 days later. On Day 7, horses were anesthetized and paralyzed in the same manner as on experimental Day 1. Blood was taken to measure serum cholinesterase activity prior to anesthesia on Days 1 and 7. No significant difference was noted in atracurium's neuromuscular blocking activity between the 2 experimental days (P less than 0.05), despite Day 7 cholinesterase activity that was 16% of pre-trichlorfon values. For atracurium Injections 1 and 2-5, 85 and 43 micrograms/kg of atracurium, respectively, were required to produce a 95-99% reduction in hoof twitch. The time from injection to maximum twitch reduction was approximately 9 min after Injection 1 and 5 min after subsequent injections. Time from injection to maximum twitch reduction was significantly longer for Injection 1 than Injections 2-5 on both experimental days. The time from maximum twitch reduction until 10% recovery was approximately 8 min, with no significant difference between experimental days. The time for twitch recovery from 10 to 75% was approximately 17 min for all injections. Antagonism of atracurium with edrophonium caused the twitch height to return to pre-atracurium strength in approximately 7 min. Edrophonium caused a significant increase in arterial blood pressure. Heart rate change was variable after edrophonium.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals