Involvement of esterases in diazinon resistance and biphasic effects of piperonyl butoxide on diazinon toxicity to Haematobia irritans irritans (Diptera: Muscidae)

Resistance to insecticides remains a major problem for the successful control of the horn fly, Haematobia irritans irritans (L.), one of the most important pests of cattle in many countries including the United States. The organophosphate (OP) insecticide diazinon has been used to control pyrethroid-resistant populations of the horn fly. There are only a few reported cases of horn fly resistance to diazinon in the United States and Mexico. Piperonyl butoxide (PBO) has been used successfully as a synergist of pyrethroid insecticides to control horn flies. PBO-synergized diazinon products are also available for horn fly control in the United States, although PBO is known to inhibit the bio-activation of certain OP insecticides including diazinon. A study was conducted to evaluate the effect of PBO on diazinon toxicity to horn flies using a filter paper bioassay technique. These bioassays in both the susceptible and diazinon-resistant horn fly strains revealed a biphasic effect of PBO on diazinon toxicity to horn flies. PBO inhibited diazinon toxicity when the PBO concentration used was high (5%), and no effect was observed when PBO concentration was intermediate (2%). However, at low concentrations (1% and lower), PBO significantly synergized diazinon toxicity. We demonstrated that enhanced esterase activity was associated with survivability of horn flies exposed to diazinon alone. PBO has been shown to inhibit esterase activity in other insect species. However, results of biochemical assays with esterases from this study suggest that PBO did not have significant effect on the overall esterase activity in the horn fly. The observed synergistic effect of PBO at lower concentrations on diazinon toxicity to horn flies could not be explained by reduced esterase activity due to PBO inhibition. It is likely that PBO synergized diazinon toxicity at lower concentrations by facilitating penetration of diazinon through the cuticle and/or inhibiting the oxidative detoxification of diazinon, and reduced diazinon toxicity at high PBO concentration by inhibiting the bio-activation of diazinon.     

Comparison of catalytic properties and inhibition kinetics of two acetylcholinesterases from a lepidopteran insect

Acetylcholinesterase (AChE) is the primary target of organophosphate (OP) and carbamate (CB) insecticides. Many insect species have been shown to have two different AChE genes. The amino acid identity between the two lepidopteran AChEs is lower than 40%, and potential differences in enzymatic function have not been characterized. In this study, the cDNAs encoding two AChEs (Boma-AChE1 and Boma-AChE2) from Bombyx mandarina were sequenced, and the corresponding proteins were heterologously expressed to compare their enzymatic properties and interactions with insecticides in vitro. Both of these enzymes had high specific activities for acetylthiocholine iodide. Studies on substrate and inhibitor specificities confirmed that both enzymes belong to AChE. Insecticide inhibition assays indicated that Boma-AChE1 was more sensitive than Boma-AChE2 to eight of the nine insecticides tested. However, Boma-AChE2 was more sensitive than Boma-AChE1 to one of the OP insecticides, heptenophos. The results suggested that two AChEs from a lepidopteran insect have distinct catalytic properties and responses to different inhibitors.     

Effects of atropine and pralidoxime pretreatment on serum and cardiac oxidative stress parameters in acute dichlorvos toxicity in rats

Recent several studies have reported that oxidative stress could be an important component of the mechanism of cardiotoxicity due to organophosphate-induced toxicity. The aim of this study is to evaluate the oxidative and antioxidative parameters in cardiac toxicity of organophosphate poisoning, and determine the effects of atropine and pralidoxime on this parameters. The experimental groups were randomly divided into five groups as control (corn oil), dichlorvos (30 mg/kg), atropine (10 mg/kg), pralidoxime (40 mg/kg), and atropine (10 mg/kg) + pralidoxime (40 mg/kg) groups. Serum cholinesterase levels were suppressed with dichlorvos, and these reductions were inhibited with atropine and/or pralidoxime pretreatment. Serum, but not cardiac, total free sulfhydryl groups and paraoxonase activities were significantly increased in the pralidoxime group when compared to the control group. Serum arylesterase activities were elevated in the dichlorvos, atropine, pralidoxime, and atropine + pralidoxime groups when compared to the control group (P < 0.05). Total oxidant status, oxidative stress index, malondialdehyde and catalase activities in serum and cardiac tissues were not markedly different between the groups. No significant changes were also observed with cardiac myeloperoxidase and serum ceruloplasmin activities. In conclusion, these results showed that acute dichlorvos administration did not cause marked cardiac damage, and oxidative stress probably does not play a major role in dichlorvos-induced poisoning. On the other hand, especially pralidoxime treatment markedly increased the serum total free sulfhydryl groups, paraoxonase and arylesterase activities. However, the underlying mechanisms for these changes are not exactly known.     

Synthetic Pyrethroid Insecticides: Some Studies with Locusts

Abstract

Laboratory studies were carried out to determine the course of poisoning and toxicity of some synthetic pyrethroid insecticides (bioresmethrin, cismethrin, cypermethrin, deltamethrin, fenpropathrin, fenvalerate and permethrin) against the desert locust. From doses in excess of the LD₉₉ all the insecticides were quick acting; from doses of about the LD₉₉ or less the insects are rapidly knocked down or show symptoms of poisoning, but may recover. The symptoms which follow treatment by the various insecticides are described. Although these insecticides are all highly toxic to locusts there are many other insecticides which are similarly toxic, can be sprayed at high concentrations and are much cheaper.     

Methylparathion- and carbofuran-induced mitochondrial dysfunction and oxidative stress in Helicoverpa armigera (Noctuidae: Lepidoptera)

The cotton bollworm, Helicoverpa armigera is a polyphagous pest of several crops in Asia, Africa, and the Mediterranean Europe. Organophosphate and carbamate insecticides are used on a large-scale to control Helicoverpa. Therefore, we studied the effect of methylparathion and carbofuran, an organophosphate and carbamate insecticide, respectively, on oxidative phosphorylation and oxidative stress in H. armigera larvae to gain an understanding of the different target sites of these insecticides. It was observed that state III and state IV respiration, respiratory control index (RCI), and P/O ratios were inhibited in a dose-dependent manner by methylparathion and carbofuran under in vitro and in vivo conditions. Methylparathion and carbofuran inhibited complex II by ∼45% and 30%, respectively. Lipid peroxidation, H₂O₂ content, and lactate dehydrogenase (LDH) activity increased and glutathione reductase (GR) activity decreased in a time- and dose-dependent manner in insecticide-fed larvae. However, catalase activity was not affected in insecticide-fed larvae. Larval growth decreased by ∼64% and 67% in larvae fed on diets with 100 μM of methylparathion and carbofuran. The results suggested that both the insecticides impede the mitochondrial respiratory functions and induced lipid peroxidation, H₂O₂, and LDH leak, leading to oxidative stress in cells, which contribute to deleterious effects of these insecticides on the growth of H. armigera larvae, along with their neurotoxic effects.     

Biochemical studies on sheep body louse Bovicola ovis (Schrank) (Phthiraptera: Trichodectidae): comparison of pyrethroid and organophosphate resistant and susceptible strains

Strains of sheep louse Bovicola ovis (Schrank) with various levels of resistance to pyrethroid and one strain with high degree of resistance to organophosphate (OP) insecticides were used to investigate the biochemical mechanisms of insecticide resistance, i.e., enhanced levels of general esterases, specific acetylcholinesterases (AChE), glutathione S-transferase (GST), and mixed function oxidases. Native gel electrophoresis combined with quantitative enzyme assays showed analogous expression profiles of several esterase isozymes in all the strains tested. The determination of the sensitivity of each esterase isozyme to five inhibitors (acetylthiocholine iodide, butyrylthiocholine iodide, paraoxon eserine sulfate, and pCMB) led to the identification of nine esterases in the B. ovis strain. Gel electrophoresis results are supported by enzyme assay studies where, except for the OP resistant strain, no differences in esterase activities were detected in all the pyrethroid resistant and susceptible strains assayed. Statistical analyses demonstrated that some strains have elevated GST activities compared to the susceptible reference strain.     

A review on the mechanisms involved in hyperglycemia induced by organophosphorus pesticides

Organophosphorus (OP) compounds are cholinesterase-inhibiting chemicals used as pesticide. Exposures to OPs cause a significant number of poisonings and deaths each year. One of the reported adverse effects in human exposure to OPs is hyperglycemia. Animal studies have also shown altered glucose homeostasis following acute or chronic exposures to OPs. The objective of this paper is to provide a brief review of the mechanisms involved in the OP-induced glucose homeostasis alteration. To reach this objective, a search of the literature using Medline/Index Medicus, Scopus, and Chemical Abstract were performed, most of relevant citations were studied and summarized. To better understand the nature of glucose homeostasis, the principles of glucose production, metabolism, and its hormonal control have been discussed. Collection of theses studies support the conclusion that hyperglycemia is the outcome of acute or chronic exposure to OPs. OPs can influence body glucose homeostasis by several mechanisms including physiological stress, oxidative stress, inhibition of paraoxonase, nitrosative stress, pancreatitis, inhibition of cholinesterase, stimulation of adrenal gland, and disturbance in metabolism of liver tryptophan.     

Insecticide poisoning: Disruption of a possible autonomic function in pupae of Tenebrio molitor

Poisoning of pupae of Tenebrio molitor by pyrethroid, carbamate, or organophosphate insecticides was monitored by recording pressure in the hemocoel. The pressure was controlled automatically at around a negative 0.5 kPa value and predictable sharp peak increases in pressure of 0.5–1.0 kPa occurred in bursts. The pressure pulses in Tenebrio pupae were thought to represent the activity of an important autonomic function in insects. Insecticide poisoning increased the frequency of the pressure pulses and their amplitudes as high as 6 kPa in a dose-dependent manner. The pattern in pyrethroid-poisoned pupae was distinctly different from that following carbamate or organophosphorus poisoning. At 12–18 hr after treatment, the pattern of pressure bursts in pupae recovering from poisoning began to resemble the pattern before treatment, with peaks of pressure. In pupae not recovering from poisoning, the pattern was distinctly different, with only single peak pulses of pressure. Thus, it was possible to determine the fate of poisoned pupae after 12 hr.     

Can diphenhydramine prevent organophosphate-induced acute pancreatitis? An experimental study in rats

Acute pancreatitis (AP) is a well known complication of organophosphate (OP) poisoning and the true incidence is unknown; but, may be more common than clinically suspected. Previous studies suggest that Diphenhydramine (DPH) may be useful as an alternative or adjunctive therapy in OP poisoning. The aim of this experimental study was to investigate whether DPH could prevent or diminish pancreatic damage caused by OP poisoning as defined by histologic findings, and serum interleukin 10 (IL-10) and tumor necrosis factor alpha (TNF-α) levels. Twenty-four Sprague- Dawley rats were divided into equal three groups. Group 1 did not receive any agent during the experiment. Group 2 received 0.8 g/kg fenthion subcutaneously followed by 3 ml/kg normal saline intramuscularly, 30 min later. Group 3 received 0.8 g/kg fenthion subcutaneously, followed by 30 mg/kg DPH intramuscularly, 30 min later. Twenty-four hours later, pancreatic tissues were excised and blood samples were taken. After blood samples were taken by cardiac puncture, the animals were sacrificed. Treatment with DPH significantly decreased the serum TNF-α and increased the serum IL-10 levels. DPH significantly reduced pancreatic damage, including edema, inflammation, vacuolization and necrosis, as determined by pathologic scoring. The present study show that DPH decreased the severity of OP induced AP in rats. This effects may be related to a decrease of TNF-α level and increase of IL-10 level.     

Comparison of insecticide absorption and detoxification in larvae of the bollworm,Heliothis zea,and the tobacco budworm,H. virescens

Rates and nature of metabolism of several insecticides were compared in larvae of the bollworm, Heliothis zea (Boddie), and the tobacco budworm, H virescens (F.). Both species are resistant to organochlorine compounds. In addition, H. virescens is resistant to carbamate and organophosphate insecticides. All test insecticides were metabolized by larvae of both species under in vivo conditions. In most instances, metabolism was more rapid in H. virescens larvae.A series of in vivo and in vitro assays implicated high levels of NADPH-dependent oxidative enzymes as a major metabolic factor in resistance. Other work demonstrated that H. virescens larvae absorbed insecticides at a slower rate than did H. zea larvae. High resistance to organophosphates present in H. virescens may be due to interaction of factors for increased rate of detoxification and decreased rate of absorption.     

Synergistic and antagonistic effects of atrazine on the toxicity of organophosphorodithioate and organophosphorothioate insecticides to Chironomus tentans (Diptera: Chironomidae)

The acute toxicities of two organophosphorodithioate (dimethoate and disulfoton) and two organophosphorothioate (omethoate and demeton-S-methyl) insecticides were evaluated individually and in binary combination with the herbicide atrazine using fourth-instar larvae of the aquatic midge, Chironomus tentans. Atrazine alone up to 1000 μg/L did not show significant toxicity to the midges in a 48-h bioassay. However, atrazine concentrations as low as 1 μg/L in combination with dimethoate at EC₂₅ (concentration to affect 25% of tested midges), 100 μg/L in combination with disulfoton (EC₂₅), and 10 μg/L in combination with demeton-S-methyl (EC₂₅) significantly enhanced the toxicity of each organophosphate insecticide. In contrast, atrazine concentrations of 10 μg/L and above in combination with omethoate (EC₂₅) significantly decreased the toxicity of the insecticide. Biochemical analysis indicated that increased toxicity of dimethoate, disulfoton, and demeton-S-methyl in binary combination with atrazine correlated to the increased inhibition of acetylcholinesterase. Furthermore, cytochrome P450-dependent O-deethylation activity in the midges exposed to atrazine at 1000 μg/L was 1.5-fold higher than that in the control midges. Thus, atrazine appeared to induce cytochrome P450 monooxygenases in the midges. Elevated cytochrome P450 monooxygenase activity may increase the toxicities of dimethoate, disulfoton, and demeton-S-methyl by enhancing the oxidative activation of dimethoate into omethoate, and disulfoton and demeton-S-methyl into their sulfoxide analogs with increased anticholinesterase activity. In contrast, atrazine reduced the toxicity of omethoate possibly by enhancing the oxidative metabolic detoxification since omethoate does not require oxidative activation.     

Effect of combined exposure of commonly used organophosphate pesticides on lipid peroxidation and antioxidant enzymes in rat tissues

Organophosphate compounds are among the most widely used synthetic chemicals for controlling a wide variety of pests. Organophosphate (OP) poisoning continues to be major cause of morbidity and mortality in the third world countries. Indiscriminate use of these pesticides tends to leave residues on the objects of the environment. Present study is aimed to compare the potential of three commonly used OP pesticides, chlorpyrifos (CPF), methyl parathion (MPT) and malathion (MLT), to generate oxidative stress in rat tissues and to evaluate whether the combined exposure of these pesticides exerts synergistic or antagonistic effects. Results of the present study showed that CPF, MPT and MLT exposure to rats caused accumulation of malondialdehyde (MDA) and 4-hydroxynonanal (4HNE), the two major end products of lipid peroxidation, in liver, kidney, brain and spleen of rats. Combined exposure of these pesticides also resulted in accumulation of MDA and 4HNE in rat tissues but the increase was almost of the same order as observed in rat tissues given these pesticides singly. Exposure with CPF, MPT and MLT singly or in mixture, caused dose-dependent decrease in the activities of antioxidant enzymes namely, catalase (CAT), superoxide dismutase (SOD) and glutathione peroxidase (GPx), in rat tissues when compared with control, and the decrease observed was of the same order in all the groups. Acetylcholinesterase (AChE) activity, an indicator of OP poisoning, was also decreased in rat tissues in dose-dependent manner in CPF, MPT, MLT and mixture treated group. Differential increase in the levels of cytochrome P450 (cyt P450) in hepatic and extra-hepatic tissues of rats given CPF, MPT or MLT singly or in mixture, indicate different rates of metabolism of these pesticides. Results of the present study clearly show that CPF, MPT and MLT exposure singly or in mixture, induced oxidative stress in rat tissues which may be the major contributor of the overall toxicity of the OP pesticides. Combined exposure of these pesticides does not seem to potentiate the toxicity of each other and their toxic effects are not additive.     

Fitness costs associated with insecticide resistance

Insects are exposed to a variety of stress factors in their environment, and, in many cases for insect pests to agriculture, those factors include toxic chemical insecticides. Coping with the toxicity of insecticides can be costly and requires energy and resource allocation for adaptation and survival. Several behavioural, physiological and genetic mechanisms are used by insects to handle toxic insecticides, sometimes leading to resistance by constitutive overexpression of detoxification enzymes or inducing mutations in the target sites. Such actions are costly and may affect reproduction, impair dispersal ability and have several other effects on the insect's fitness. Fitness costs resulting from resistance to insecticides has been reported in many insects from different orders, and several examples are given in this mini‐review. Copyright © 2012 Society of Chemical Industry     

Acetylcholinesterase isozymes of the house fly thorax: In vivo inhibition by organophosphorous insecticides

Polyacrylamide gel electrophoresis of the supernatant fraction of house fly thoracic homogenates demonstrated five electrophoretic bands having cholinesterase activity. The five esteratic bands were considered to be isozymes of acetylcholinesterase based on their staining properties with acetylthiocholine. There appeared to be no visual or densitometric difference in the intensity of staining of the isozymes when acetylthiocholine was compared with butyrylthiocholine as substrate. Selective inhibition of these isozymes in vivo by organophosphate insecticides may contribute only in small part to the mode of action of organophosphate insecticides.     

Kinetic efficiency of mutant carboxylesterases implicated in organophosphate insecticide resistance

Resistance to organophosphorus (OP) insecticides in Lucilia cuprina arises from two mutations in carboxylesterase E3 that enable it to hydrolyse the phosphate ester of various organophosphates, plus the carboxlyester in the leaving group in the case of malathion. These mutations are not found naturally in the orthologous EST23 enzyme in Drosophila melanogaster. We have introduced the two mutations (G137D and W251L) into cloned genes encoding E3 and EST23 from susceptible L. cuprina and D. melanogaster and expressed them in vitro with the baculovirus system. The ability of the resultant enzymes to hydrolyse the phosphate ester of diethyl and dimethyl organophosphates was studied by a novel fluorometric assay, which also provided a sensitive titration technique for the molar amount of esterase regardless of its ability to hydrolyse the fluorogenic substrate used. Malathion carboxylesterase activity was also measured. The G137D mutation markedly enhanced (>30-fold) hydrolysis of both classes of phosphate ester by E3 but only had a similar effect on the hydrolysis of dimethyl organophosphate in EST23. Introduction of the W251L mutation into either gene enhanced dimethyl (23-30-fold) more than diethyl (6-10-fold) organophosphate hydrolysis and slightly improved (2-4-fold) malathion carboxylesterase activity, but only at high substrate concentration.     

Molecular and biochemical mechanisms of organophosphate resistance in laboratory-selected lines of the oriental fruit fly (Bactrocera dorsalis)

Genetic and biochemical factors leading to resistance to various organophosphate (OP) based insecticides were studied in lines selected for OP-resistance in the oriental fruit fly Bactrocera dorsalis. Lines were separately selected for resistance to naled, trichlorfon, fenitrothion, fenthion, formothion, and malathion. Overall, these lines showed increased resistance ratios ranging from 13.7- to 814-fold relative to a susceptible (S) line. Also, in these newly selected lines the same three point mutations in the ace gene, previously identified in resistance studies and designated as I214V, G488S and Q643R, were found. As expected, the enzyme from the resistant lines showed lower overall activity and reduced sensitivity to inhibition by fenitrothion, methyl-paraoxon and paraoxon compared to the wild type acetylcholinesterase (AChE) enzyme. The apparent V_max values for esterase from the resistant lines were 1.2-3.69 times higher than that of the S line. Although only the naled-, trichlorfon- and fenthion-r lines showed lower esterase affinities (based on apparent K_m values) compared with the S line, all of the V_max/K_m ratios were higher in the resistant lines compared to that of the S line. The OP-resistant lines also displayed an overall similar pattern of isozyme expression, except for one additional band found only in the naled-r line and one band that was absent in the trichlorfon-, malathion-, and fenthion-r lines. Our results also show that overall, multiple examples of high OP resistance in selected lines of B. dorsalis exhibiting the same genetic alterations in the ace gene seen previously resulted in different effects on esterase enzyme activity in relation to various OP compounds.     

与昆虫抗药性相关的乙酰胆碱酯酶基因突变研究进展

有机磷和氨基甲酸酯类杀虫剂的广泛使用导致许多害虫产生了明显的抗药性。害虫对这些杀虫剂产生抗性的一个重要原因是其乙酰胆碱酯酶 (AChE)基因发生突变,从而导致AChE敏感度下降。简要概述了AChE基因发生突变的昆虫种类,介绍了AChE基因突变对其结构与功能的影响、变构AChE的特性、AChE基因突变对适合度的影响以及AChE突变不同组合对抗性的影响。这些突变可为设计新颖的反抗性化合物开辟新的途径。     

A beekeeper's perspective on the neonicotinoid ban

Bees and agrochemicals have a long history. For example, the first volume of IBRA's journal Bee World in 1919 contains mention of poisoning of bees by spraying an orchard with lead arsenate. Bees being insects, it is self‐evident that the use of insecticides to control crop pests poses a risk to them. Bee poisoning incidents became a very serious problem in the 1960s and 1970s with spraying of, in particular, oilseed rape with organophosphorus compounds. The introduction of carbamates and then especially synthetic pyrethroids reduced these problems. Data from the Wildlife Incident Investigation Scheme show that in recent years there have been very few poisoning incidents in the United Kingdom that can be attributed to agricultural insecticides. The introduction of neonicotinoid insecticides has, however, been very controversial. Almost as soon as they were introduced in the 1990s, French beekeepers blamed colony losses on imidacloprid used on sunflowers and maize, but restrictions on its use did not lead to a reduction in losses or to a reduction in beekeepers' concerns. Acute pesticide poisoning incidents by neonicotinoids in Germany and Italy in 2008 further sealed their reputation. Despite laboratory evidence showing their harm, field experience remains equivocal, and many commercial beekeepers continue to move their colonies to oilseed rape crops for honey production. The neonicotinoid moratorium has undoubtedly led to the increased use of older insecticides, and the effect of this on bee populations is unknown and unquantified. Many beekeepers are currently confused by the conflicting evidence. © 2016 Society of Chemical Industry     

Dual role of esterases in insecticide resistance in the green rice leafhopper

The role of esterases as related to insecticide resistance was studied in an organophosphorus (OP)-resistant strain of the green rice leafhopper. As judged by p-nitrophenyl acetate hydrolysis, 21, 5, and 74% of the esterase activity was located in nuclei/mitochondria, microsomes, and the soluble fraction, respectively. All the fractions were active in hydrolyzing malathion, paraoxon, and fenvalerate. Hydrolysis of malathion and fenvalerate increased with time while that of paraoxon reached a plateau within 15 min. Since a considerable amount of p-nitrophenol was detected in the paraoxon reaction at 0°C and at zero time, the formation of p-nitrophenol may be due to phosphorylation of the esterases rather than phosphorotriesterase action. The results suggest a dual role for esterases in resistance mechanisms; a catalyst for hydrolysis of malathion and fenvalerate, and a binding protein for the oxygen analogs of other OP insecticides, both of which would protect the intrinsic target, acetylcholinesterase, from inhibition. Chromatofocusing of the soluble fraction resolved five esterase peaks, I–V. These esterases were active toward the three general substrates as well as for the three insecticides tested, except for Peak I in which the overall activity was too low. Thin-layer agar gel electrophoresis showed that the chromatofocusing peaks I–V corresponded to the electrophoretic bands E₁–E₅, some of which were previously shown to be associated with OP resistance. The dual role of these esterases may explain the cross-resistance between malathion and other OP insecticides as well as synergism between OP and carbamate insecticides.     

Avian whole‐brain spheroid cultures: applications in pesticide toxicity

The adult hen is the species of choice when assessing the potential of organophosphate compounds (OPs) to evoke OP‐induced delayed neuropathy (OPIDN). OPIDN has been shown to depend on the irreversible covalent binding of certain OPs to the enzyme neuropathy target esterase (NTE). Levels of this enzyme in human brain are similar to those in avian species. We have previously shown that the hen embryo whole brain spheroids are histotypically similar to adult brain and express measurable levels of acetylcholine esterase (AChE) and NTE, the key enzymes in mechanisms of OP toxicity. Single‐cell suspensions were prepared from meninges‐free 7 day in ovo hen brains. Cells were grown in serum‐free media and incubated on an orbital shaker at 37 °C in 5% carbon dioxide humidified air. Spheroids can be maintained long‐term in culture and allow the effects of repeated dose and recovery studies to be conducted. Hen embryo brain spheroids were exposed to varying concentrations of tri‐o‐cresyl phosphate, leptophos and its metabolite leptophos oxon at day 14 in vitro and the effects on the activity of AChE and NTE measured. Whilst current legislation regarding toxicity testing of OPs requires the use of animals, our in vitro model provides a potential pre‐screen for novel chemical entities and commercial OP mixture variants. Such an approach should refine and reduce the number of animals used in development of new materials. © 2000 Society of Chemical Industry