Fenitrothion is the ISO approved common name for O,O-dimethyl-O-(4-nitro-meta-tolyl) It is a broad-spectrum organophosphorus pesticide. Its toxicity was first evaluated by the 1969 Joint Meeting, which established a temporary ADI of 0–0.001 mg/kg bw on the basis of a NOAEL of 0.25 mg/kg bw per day in a 3-month study in rats (Annex 1, reference 12). In 1974, new data were reviewed, and the ADI was increased to 0–0.005 mg/kg bw on the basis of inhibition of plasma cholinesterase activity observed in a 92-week study in rats (Annex 1, reference 22). This ADI was reaffirmed by the 1977 Joint Meeting (Annex 1, reference 28). However, some of the pivotal studies used to establish the ADI were based on data generated by Industrial Bio-Test Laboratories and had not been validated. Replacement studies or independent validations were requested, but these were not available in time for the 1982 Joint Meeting. Consequently, a temporary, lower ADI of 0–0.001 mg/kg bw was established (Annex 1, reference 38). At the 1984 Joint Meeting, the ADI was increased to 0–0.003 mg/kg bw but was still considered temporary owing to the absence of a suitable study of developmental toxicity (Annex 1, reference 42). In 1986, an acceptable study of developmental toxicity in rats was reviewed, and an ADI of 0–0.003 mg/kg bw was established (Annex 1, reference 47). The most recent review, in 1988, which reflected the new JMPR policy of using inhibition of brain cholinesterase activity (or erythrocyte acetylcholinesterase activity as a surrogate) instead of inhibition of plasma cholinesterase activity as the toxicologically relevant end-point for cholinesterase-inhibiting compounds, included data that had been reviewed previously and increased the ADI to 0–0.005 mg/kg bw on the basis of a NOAEL of 0.5 mg/kg bw per day in a 2-year study of toxicity in rats; this value was supported by a NOAEL of 0.65 mg/kg bw per day in a study of reproductive toxicity in rats (Annex 1, reference 53). Fenitrothion was reviewed by the present Meeting within the periodic review programme of the Codex Committee on Pesticide Residues.

Fenitrothion is presumably rapidly absorbed from the mammalian intestinal tract, as evidenced by the appearance of radiolabel in blood from guinea-pigs and rats given [³²P]fenitrothion orally. The presence of the oxygen analogue was demonstrated in all tissues examined (brain, heart, lung, liver, kidney, spleen, and muscle), and it was detectable in blood 1 min after intravenous injection of fenitrothion. This oxygen analogue (II in Figure 1) is the most important metabolite with respect to toxicity. It is formed in the microsomal fraction of the cell, the main organs responsible for the transformation being the liver and kidney. Fenitrooxon is further metabolized as indicated in Figure 1. The major excretion product found is 3-methyl-4-nitrophenol (VII) which can be oxidized further to 3-carboxyl-4-nitrophenol (VIII). Other metabolites are the demethyl derivatives (V and VI), which, with increasing dose, are excreted in increasing amounts. Nine metabolites have been isolated, most of which have also beeen identified. In vitro, formation of the oxygen analogue (II) depended on the availability of reduced nicotine adenine dinucleotide phosphate (NADPH₂) and oxygen. Liver slices incubated with fenitrothion did not produce measurable amounts of fenitrooxon, while liver homogenates and the supernatant fraction of such homogenates appreciably activated added fenitrothion. No correlation between the toxicity and rate of formation of fenitrooxon could, however, be demonstrated (Miyamoto et al., 1963a; JMPR, 1969; Miyamoto, 1969). No observations were made in these studies on the distribution into fatty tissues, but studies of residues in milk, meat, and fat from cattle indicated the presence of approximately 0.001 mg/kg in these samples (JMPR, 1969; Miyamoto & Sato, 1969).

Like other organophosphorous compounds, fenitrothion acts in the organism as a cholinesterase inhibitor, probably after conversion to fenitrooxon. Some evidence indicates that the acetylcholinesterase inhibiting effect in brain depends more on the rate of penetration than on the rate of oxidation and decomposition of fenitrothion (JMPR, 1969; Miyamoto, 1969).

Female rats were given a single oral dose of 200 mg/kg bw of fenitrothion and/or malathion. The concentration of fenitrothion in the liver was lower in those animals that received both compounds than in those receiving only fenitrothion. However, after an initial decrease, the content of fenitrothion increased in blood and muscle of rats that received malathion as well. The concentration of fenitrooxon, the toxic metabolite of fenitrothion, increased markedly with time in blood and muscle of rats given fenitrothion and malathion in combination, in contrast to rats given only fenitrothion, which showed a rapid peak and decline in fenitrooxon concentrations. The liver fenitrooxon concentrations were not appreciably altered by the combined regimen. These studies suggest the possibility that fenitrothion is potentiated by malathion, but no tests for potentiation were conducted (Hladká et al., 1974; JMPR, 1974).

After oral administration of fenitrothion to mice and subcutaneous administration to rats and guinea-pigs, it was cleared quite rapidly, 90% of the administered dose being recovered within 3 days. Mice metabolized fenitrothion to fenitrooxon, 3-methyl-4-nitrophenol, and demethylated products rapidly (Douch et al., 1968).

Radiolabel from fenitrothion administered daily to goats in the diet for 7 days was detectable at high concentrations in the gastrointestinal tract and at lower concentrations in the tissues on day 8, with negligible quantities by day 25. Fenitrothion was excreted in both urine and faeces. The concentration in milk was relatively low and decreased rapidly when fenitrothion was withdrawn from the diet (Mihara et al., 1978).

[¹⁴C]Fenitrothion administered orally to rats once at 1.5 or 150 mg/kg bw or at 1.5 mg/kg bw after 14 administrations of unlabelled fenitrothion at 1.5 mg/kg bw per day over 14 days was almost completely recovered in the urine and faeces (95–102%) within 7 days. Most of the administered dose was present in urine, where it accounted for 86–101% of the total. The concentrations of residues in tissues were relatively low, the highest being found in liver. After administration of a low dose or repeated doses, 1–16 µg/kg of fenitrothion equivalents were detected, whereas after a high dose the concentration ranged from 170 to 450 µg/kg. Conjugated phenolic compounds, such as the sulfate and glucuronide of 3-methyl-4-nitrophenol, accounted for 54–66% of the administered dose after single or repeated administration of a low dose. At the high dose, the main metabolites were demethylated compounds such as demethylfenitrothion and demethylfenitrooxon (43–58% of the dose). There was no appreciable difference in metabolism between the sexes. Hence, the major biotransformations were demethylation of one of the P–O methyl groups from either the parent or the oxon, cleavage of the P–O–aryl linkage, and conjugation of the resulting phenol, mainly as sulfate but partly as glucuronide (Iba et al., 1990).

In a study of the penetration of fenitrothion and parathion-methyl into brain tissue and the effects on brain acetylcholinesterase, the concentrations of both compounds in the brain peaked 5 min after administration. Inhibition of brain acetylcholinesterase activity was more rapid and extensive after treatment with parathion-methyl than with fenitrothion (Miyamoto, 1964).

The dermal absorption of fenitrothion was examined in rats by applying a low dose (0.73 g/kg bw), a high dose (3.7 g/kg bw), or repeated doses to shaved skin. After a single application, the maximum plasma concentrations were obtained 8 h later. Whole-blood cholinesterase inhibition correlated with the fenitrothion concentration in plasma. Approximately 55% of the low dose remained unabsorbed (in and on the skin) after 24 h. No data on the absorption of the high dose were supplied. This dose produced significant clinical signs and plasma concentrations that were about 10 times higher than that observed with 0.73 g/kg bw at 48 h. Multiple applications resulted in increasing plasma concentrations of fenitrothion and increasing inhibition of cholinesterase activity (Kohli et al., 1974).

A generalized scheme for mammalian metabolism of fenitrothion is shown in Figure 1. This pathway was derived from data given in many of the studies cited in the following section. An overview of data from a number of studies indicated that fenitrothion is relatively well absorbed from the intestinal tract in most species. Activation to the active metabolite, fenitrooxon, occurs in the liver, followed by relatively rapid degradation to inactive metabolites, which are then excreted. Additionally, a direct degradation pathway is observed in rats.

A study of the metabolism of [¹⁴C-ring-methyl]fenitrothion (purity, > 99.8%) was carried out with particular attention to the fate of the phenolic moiety. Male rats were given a single oral dose of 15 mg/kg bw of labelled fenitrothion, and the concentrations were determined in tissues 1 and 24 h later. After 1 h, not more than 25% of the applied dose was present in the gastrointestinal tract, and significant amounts were found in the kidneys (12 mg of fenitrothion equivalents per kg), stomach and intestines (5.4 mg/kg), liver (2.6 mg/kg), blood (2.1 mg/kg), and lungs (1.1 mg/kg). In these tissues, > 80% of the radiolabel represented water-soluble metabolites, except in the stomach and intestines where 68% was fenitrothion. Kidneys also had a high content of 3-methyl-4-nitrophenol (VII; 2.1 mg/kg); in other tissues, this metabolite was present at < 0.2 mg/kg. Little fenitrooxon (II) was found: ­ 0.024 mg/kg in the stomach and intestines, 0.008 mg/kg in the kidneys, and < 0.005 mg/kg in other tissues. Some fenitrothion was detected in fat and pancreas (both 0.5 mg/kg). By 24 h after administration, all tissues contained < 0.1 mg/kg of total ¹⁴C, with the highest concentrations in liver, kidneys, fat,and stomach and intestine, partly as fenitrothion and partly as metabolites. The concentrations of all metabolites in other tissues were < 0.005 mg/kg.

A similar distribution was observed on autoradiograms of mice and rats, shortly and 24 h after application of 15 mg/kg bw of labelled fenitrothion. The concentrations of fenitrothion were determined in the blood of rats, rabbits, mice, and dogs 1, 3, 9, 24, and 48 h after oral administration of 15 mg/kg bw. A maximum was observed at 1 or 3 h, and the concentrations were < 0.01 mg/kg at 24 h. After five male rabbits were fed 0.3 or 10 mg/kg bw per day of fenitrothion for 6 months, neither fenitrothion (limit of detection, 0.005 ppm) nor fenitrooxon (II; 0.001 ppm) was detectable in blood or skeletal muscle. In fat, 0.13 mg/kg of the oxygen analogue was found.

After a single oral dose of 15 mg/kg bw of labelled fenitrothion to mice, rats, rabbits, and dogs, 80–90% of the radiolabelled metabolites were excreted in the urine within 24 h and 5% with the faeces; excretion was nearly complete by 48 h after treatment. Rats did not expire radiolabel. In urine, 18 metabolites were identified; 3-methyl-4-nitrophenol (VII; as the sulfate, free or as glucuronide) represented 50–70% of the metabolites in the rodents and only 35% in dogs, while O-demethylfenitrothion (V) or O-demethylfenitrooxon (VI) represented from 10% of metabolites in rabbits to 55% in dogs. The urine of rabbits and rats also contained small quantities of other metabolites in which the nitro group was reduced to amino fenitrothion (III) (total, about 15%) or had an oxidized ring methyl group (IV, VIII; about 1%). Fenitrothion was not detectable, and traces of fenitrooxon (II; 0.4%) were determined only in rabbit urine. About 5% remained unidentified. The faeces of rats contained the same major metabolites as urine—70% 3-methyl-4-nitrophenol (VII) and 20% O-demethyfenitrothion/oxon (V, VI)—with some fenitrothion (10%) (Miyamoto et al., 1976; JMPR, 1977).

In order to investigate differences in metabolism between [³²P]parathion-methyl and the structurally related fenitrothion (radiochemical purities, > 95%) in male Swiss mice, each compound was administered separately in olive oil by gavage at two different doses. The mice were housed individually in metabolism cages, and urine and faeces were collected for 72 h after dosing. Whereas parathion-methyl at 3 mg/kg bw caused slight signs of intoxication, severe cholinergic symptoms were observed at 17 mg/kg bw. No clinical signs were observed with fenitrothion at the same doses, but when it was given at 200 or 850 mg/kg bw, slight and severe cholinergic signs, respectively, were seen. Fenitrothion was excreted rapidly and relatively completely. Within 24 h, 55% of the highest dose was collected in the urine, and more than 75% of the three lower doses had been excreted. After 72 h, > 90% of all administered doses of fenitrothion had been excreted in the urine or faeces. The initial excretion of parathion-methyl followed a similar pattern, except that excretion slowed at around 24 h, and only 85% of the administered dose had been excreted by 72 h.

The metabolite profile after administration of fenitrothion at low doses differed substantially from that at high (and toxic) doses. At 3 mg/kg bw, > 30% of the urinary metabolites of fenitrothion consisted of dimethylphosphoric acid (X); desmethylphosphate (XII) made up 26% of the metabolites, while desmethylphosphorothioate (XI) made up 21%. At 850 mg/kg bw, desmethyl-phosphorothioate (XI) made up 66% of the urinary metabolites, while desmethylphosphate (XII) made up 17%. Other metabolites were present in negligible quantities after administration of this dose. In comparison, the main urinary metabolite of parathion-methyl was dimethylphosphoric acid, representing > 50% of the urinary metabolites at 3 mg/kg bw and 32% at 17 mg/kg bw. Less than 20% of the urinary metabolites was made up of desmethylphosphorothioate.

Thus, while demethylation appears to be an important step in the metabolism of fenitrothion, it is of lesser importance for parathion-methyl. Additionally, there appears to be a delay in the oxidation of the P=S bond to P=O in fenitrothion in comparison with parathion-methyl, as indicated by the higher proportion of P=O metabolites in the urine of parathion-methyl-treated animals (72% at 3 mg/kg bw) relative to fenitrothion-treated animals (64% at 3 mg/kg bw). This decrease or delay in activation may contribute both to the lower toxicity of fenitrothion in mammals and to the increase in the demethylation metabolism (Hollingworth et al., 1967).

Fenitrothion appears to affect cytochrome P450 enzyme activity in the liver and testes of rats. In the short term (< 72 h), fenitrothion at a dose of 261 mg/kg bw caused a decrease in activity, resulting in reduced concentrations of serum testosterone (25% of normal), which returned to normal within 5 days. Treatment at 5.5 mg/kg bw for 30 days did not change the enzyme activity or the serum concentration of testosterone (Gradowska-Olszewska et al., 1984; Clos et al., 1994).

When fenitrothion was given orally to male Wistar rats for 28 days at a dose of 0, 7.25, or 14.5 mg/kg bw per day, the highest dose increased plasma corticosterone and glucose concentrations by 2.5-fold (p < 0.01) and 30% (not significant), respectively, by week 1 and preceded a significant increase in adrenal weight (35%; p < 0.05) by week 2. However, these changes were transient, and all values had returned to control levels by the end of treatment. A similar trend was observed at the lower dose, but the changes did not achieve significance (Yamamoto et al., 1982).

In order to ascertain why fenitrothion is less toxic to mammals than parathion-methyl, even though they apparently have comparable insecticidal efficacy, the two compounds were incubated with mouse liver slices or liver microsomes in vitro. After incubation, the degree of bovine erythrocyte or fly-head cholinesterase inhibition was increased with parathion-methyl and slightly reduced with fenitrothion relative to the respective parent compounds. Testing for the presence of metabolites arising from metabolism by liver supernatant revealed that the conversion to inactive metabolites was approximately three times faster with fenitrothion than with parathion-methyl. Therefore, the authors suggested that the lesser toxicity of fenitrothion is due to the greater metabolism of fenitrothion and its metabolites in mammalian liver. After incubation of the two compounds with cockroach fat, both had increased cholinesterase inhibition potency (Vardanis & Crawford, 1964).

Numerous studies have been carried out with technical-grade fenitrothion and are summarized in Table 1. The lowest oral LD₅₀ in rats was 240 mg/kg bw, and that in mice was 780 mg/kg bw. The signs of acute intoxication were consistent with cholinesterase inhibition and included inactivity, salivation, dyspnoea, flaccid paralysis, vomiting, piloerection, exophthalmia, and diarrhoea; male animals were generally more sensitive to the acute effects of fenitrothion than female animals.

The acute toxicity of fenitrothion (purity, 96.6%) dissolved in corn oil was assessed in groups of 10 male and 10 female 5-week-old Sprague-Dawley rats who were treated by inhalation for 4 h at a mean concentration of 0 (vehicle control), 0 (air, negative control), 0.004, 0.009, 0.038, 1.0, or 2.2 mg/L in an aerosol apparatus for whole-body exposure. The median aerodynamic diameter of the aerosol particles was 0.59–0.82 µm. Clinical signs and changes in body weight were recorded during exposure and for 14 days thereafter. Plasma, erythrocyte, and brain cholinesterase activities were determined in satellite groups of 10 animals per sex at the two higher concentrations and five of each sex at the three lower concentrations. Blood was collected for monitoring of cholinesterase activity on days 1, 3, 7, and 14 from all groups and from controls and the groups at the two higher concentrations on days 21, 29, 35, and 42. Additional readings of cholinesterase activity were made for the vehicle controls and those at the highest concentration on days 48 and 56. Gross necroscopy was performed on rats that died during the study and on survivors killed at the end of exposure.

One male rat at 2.2 mg/L died on day 6, and rats at 1.0 mg/L had irregular respiration, nasal discharge, decreased spontaneous activity, lachrymation, salivation, and urinary incontinence; muscular fibrillation was observed only in male rats. At 2.2 mg/L, additional clinical signs such as hyperpnoea and intermittent tremors were observed; tonic convulsions, ataxia, and soft faeces were observed only in male rats. The clinical signs began 30 min after exposure and disappeared within 9 days after the end of exposure. A significant decrease in body-weight gain was found at the end of the observation period in males (p < 0.01) and females (p < 0.05) at 2.2 mg/L, by 58% in males and 24% in females relative to that of vehicle controls. The inhibition of cholinesterase activity relative to vehicle controls is shown in Table 2. At day 14 and thereafter, the reductions in plasma cholinesterase activity were not significant, but the significant inhibition of erythrocyte cholinesterase activity observed at 1.0 and 2.2 mg/L on days 1, 7, and 14 was still evident in animals of each sex on days 21 and 28. In brain, significant reductions in cholinesterase activity were observed at 1.0 and 2.2 mg/L on days 42 and 56, respectively. Thus, inhibition of cholinesterase activity was observed for 1–7 days after the beginning of exposure. Specifically, reduced plasma cholinesterase activity in males and females at concentrations ³ 0.009 mg/L, reduced erythrocyte cholinesterase activity in males at ³ 0.038 mg/L and in females at 0.009 mg/L, and reduced brain cholinesterase activity in males and females at 1.0 mg/L. The activity had generally recovered by 3–56 days after exposure, although the brain cholinesterase activity was still low on day 56 in animals at the highest concentration. As no pathomorphological findings were attributed to treatment, the LC₅₀ was estimated to be > 2.2 mg/L for both male and female rats (Kohda et al., 1986).

Table 2 (continued)

* p £ 0.05; ** p £ 0.01 (Student’s t test)

Fenitrothion (purity, 75.8%) diluted to give a 24% (w/v) solution was instilled into the conjunctival sac of the left eye of seven female and two male New Zealand white rabbits in aliquots of 0.1 mL. The right eye served as a control. The treated eyes of two males and one female were flushed with water 45 s after treatment, and those of the remaining rabbits were left unflushed. The treated eyes were examined for signs of irritation 1, 2, 3, 4, and 7 days after treatment. Rabbits whose eyes showed a response on day 7 were held until day 14. Irritation was graded and scored according to the method of Draize and the criteria for eye irritants of the Federal Hazardous Substances Act, USA, 1972. The studies were conducted in accordance with GLP. The rabbits whose eyes were not flushed after treatment showed mild to moderate erythema (score, 1–2; range, 0–4) 1 day after treatment, but no erythema was observed after day 3 (two of six animals), day 4 (two of six animals), or day 7 (two of six animals). No corneal opacity or iritis was observed after treatment. Slight chemosis (score, 1; range, 0–3) was observed on day 1 only in one female rabbit. A moderate to severe discharge (score, 1–3; range, 0–3) was observed in all animals, which had cleared by day 8 after treatment. Rabbits whose eyes were flushed after treatment showed no corneal opacity or iritis. The mild to moderate erythema (score, 1–2; range, 0–4) observed in these animals had improved by day 4 (two of three animals) and day 7 (one of six animals). Slight chemosis (score, 1; range, 0–3) was observed in one female and one male. Under the conditions of this study, diluted fenitrothion was irritating to the eye, although the effect was reversed by day 8 after exposure (Hixson, 1982c).

Technical-grade fenitrothion (purity, 96.5%) was instilled into the conjunctival sac of one eye of each of nine male New Zealand white rabbits in aliquots of 0.1 mL. The treated eyes of three animals were flushed with water 30 s after treatment for 1 min. The other eye served as a control. The treated eyes were examined for signs of irritation at 1, 24, 48, 72, and 96 h and 1 week after treatment. The irritation was graded and scored according to the Draize system. No indication was given about the GLP status of the study. The only evidence of ocular irritation was slight conjunc-tival redness in the unwashed eyes 1 h after application, which had disappeared within 48 h. Under the conditions of this study, fenitrothion was not irritating to the eye (Hara & Suzuki, 1981).

Technical-grade fenitrothion (purity, 97.4%) was instilled into the conjunctival sac of one eye of each of six New Zealand white rabbits. The exact amount instilled and details of the method were not provided, but the investigator stated that the tests were performed in accordance with the protocol of the 1981 Interagency Regulatory Liaison Group, USA. The treated eyes were examined for signs of irritation at 1, 24, 48, and 72 h and at 1, 2, and 3 weeks thereafter. The irritation was graded and scored according to a scale outlined in the 1972 Federal Register of the USA. No evidence of corneal damage or eye irritation was reported other than slight redness of the conjunctivae after 1 h but not after 24 h. Under the conditions of this study, fenitrothion was not irritating to the eye (Thyssen & Lorke, 1982).

Technical-grade fenitrothion (purity, 96.5%) was applied in a volume of 1.5 ml to intact and abraded shaved skin on the backs of six New Zealand white rabbits and covered with gauze and occlusive dressings for 24 h. At that time, the test sites were wiped to remove the material. The animals were observed for skin reactions 24, 48, and 72 h and 1 week after application, and these were graded and scored according to the system of Draize. No indication was given about the GLP status of the study. No evidence of erythema or oedema was observed. Under the conditions of this study, fenitrothion was not irritating to the skin of rabbits (Hara & Suzuki, 1981).

Technical-grade fenitrothion (purity, 97.4%) was applied to skin of the backs of six male and female New Zealand white rabbits for 24 h, according to the 1973 guidelines of the US Department of Agriculture. The exact amount applied and details of the method were not given. The animals were observed for skin reactions at 24 and 72 h. No evidence of erythema or oedema was observed. Under the conditions of this study, fenitrothion was not irritating to the skin of rabbits (Thyssen & Lorke, 1982).

Technical-grade fenitrothion (purity, 75.8%) was diluted with water to a 24% (w/v) solution and applied in aliquots of 0.5 mL under a 2.5-cm gauze patch to the shaved skin of six male and six female New Zealand white rabbits. Four test sites were used on each animal: two with an abraded stratum corneum and the other two intact. The trunks of the animals were wrapped in a plastic sheet and secured with tape. After 24 h, the sites were cleaned with tissue paper soaked in water, and the skin reactions were graded and scored according to the system of Draize at 24 and 72 h. The study was conducted in accordance with GLP, and all phases of the study were reviewed by a quality assurance unit. All rabbits had slight erythema on the abraded sites 24 h after treatment; three had erythema on the two intact sites and two on one intact site. At 72 h, five rabbits had no erythema, but the remaining rabbit had erythema which cleared after an additional 120 h. Oedema was not observed at any stage. A primary irritation score of 0.46 was calculated from the erythema scores on intact and abraded skin. Under the conditions of this study, fenitrothion was mildly irritating to the skin (Hixson, 1982c).

Technical-grade fenitrothion (purity, 97.2%) was prepared as a 1% or 5% solution in corn oil and applied to the shaved dorsal skin of groups of six male Hartley guinea-pigs on alternate days, followed by a challenge 14 days after the last induction dose. Groups of three vehicle control animals received a challenge with corn oil, 0.1% or 5% fenitrothion, or 0.1% 2,4-dinitrochlorobenzene, while groups of five positive control animals were sensitized and then challenged with 2,4-dinitrochlorobenzene. Animals challenged intradermally with 1% fenitrothion had no reaction, whereas five of six of those challenged with the 5% solution had barely perceptible erythema or swelling 24 h after challenge. All three vehicle control animals had similar reactions after intradermal challenge with 5% fenitrothion and only two after challenge with 0.1%. None of the animals responded to a dermal challenge with the 1% or 5% solution. The positive control group showed the expected haemorrhage and swelling 3–4 h after challenge. Fenitrothion did not senisitize the skin of guinea-pigs under these conditions (Kohda et al., 1972).

Technical-grade fenitrothion (purity, 96.7%) in sodium taurocholate was administered to male CD rats by gavage at a dose of 0, 2.5, 5, 10, or 20 mg/kg bw per day for 30 consecutive days. The animals were observed daily for signs of toxicity and morbidity. Four rats from each group were killed 8, 15, 22, and 30 days after the beginning of treatment and 8, 15, 29, 57, and 85 days after termination of treatment, and the clinical chemical parameters determined were: cholinesterase activity in plasma, erythrocytes, and brain, hepatic and renal carboxylesterase activity, serum aspartate aminotransferase, alkaline phosphatase, and amylase activity, and glucose, blood urea nitrogen, creatinine, and protein concentrations.

Eight of 36 rats at the highest dose died during the first week of treatment. The clinical signs consisted of salivation, piloerection, diarrhoea, chromodacryorrhoea, excitability, ataxia, muscle fasciculations, generalized tremors, and convulsions. No significant changes were observed in organ or body weights during the treatment period in any dose group, apart from the hghest dose. At this dose, a significant (p < 0.05) reduction in mean body weight was observed at days 30 (15%), 60 (22%) and 87 (15%). These values had returned to control levels by day 115 (i.e. 85 days after termination of treatment). A significant (p < 0.05), dose-related decrease in plasma cholinesterase activity (by 30–65%) was observed on days 8–30 of treatment in rats at doses ³ 5 mg/kg bw per day, which had returned to control values by day 38 (i.e. 8 days after termination of treatment). Similarly, a significant (p < 0.05), dose-related decrease in erythrocyte cholinesterase activity (by 30–60%) was observed on days 8–30 of treatment at doses ³ 5 mg/kg bw per day, but the activity did not return to control levels until day 45 (i.e. 15 days after termination of treatment). A significant (p < 0.05), dose-related decrease in brain cholinesterase activity (by 60–70%) was observed on days 8–30 of treatment at 10 or 20 mg/kg bw per day, and statistically significant (p < 0.05) reductions were observed at day 30 (by 30%) with 2.5 mg/kg bw per day and at day 22 (by 45%) with 5 mg/kg bw per day. The activities had returned to control values by day 60 (i.e. 30 days after termination of treatment).

Liver carboxylesterase activity was significantly (p < 0.05) decreased (by 50–80%) on days 8–30 at doses ³ 2.5 mg/kg bw per day but had returned to control values by day 45 (15 days after termination of treatment) at all doses except 20 mg/kg bw per day, at which a decrease of 25% was still observed. At this dose, the values had returned to normal by day 87 (57 days after termination of treatment). A significant decrease in renal carboxylesterase activity (by 20–70%) was observed on days 8–30 at doses ³ 5 mg/kg bw per day. Recovery of activity was rapid, and the values were comparable to those of controls by day 38 (8 days after the end of treatment). No significant treatment-related changes were observed in other clinical biochemical parameters. No NOAEL could be identified since a significant reduction in brain cholinesterase activity was observed at the lowest dose on day 30 of treatment (Trottier et al., 1980).

Technical-grade fenitrothion (purity not stated) was administered in the diet to groups of 16 or 17 male Wistar rats at a concentration of 0, 32, 63, 125, 250, or 500 ppm for 90 days. The rats were observed daily for changes in behaviour and body weight, food intake, and the presence of protein or glucose in the urine. At monthly intervals, four rats per group were killed and the following tissues were taken for examination: brain stem, brain cortex, cerebellum, thyroid, heart, lung, liver, kidney, spleen, adrenal, testis, and prostate. The cholinesterase activities in plasma, erythrocytes, brain, liver, and kidney were determined in the same satellite groups. Noradrenaline and 3-hydroxytyramine concentrations were measured in the brain stem, liver, and spleen, although the rationale was not described. The same tissues from rats killed after 90 days and from rats that died during the study were examined histologically. In addition, two groups of rats maintained on diets containing 0 or 500 ppm of fenitrothion were killed after 11 days of treatment, when the clinical cholinergic signs in the treated group were maximal.

One rat at 500 ppm died. The clinical signs at this dose included muscle fasciculations, ataxia, piloerection, and lachrymation. The ophthalmic changes included corneal opacity and corneal and conjunctival bleeding, which had disappeared by day 30. Four rats at 250 ppm showed muscle fasciculation and lachrymation on days 15–18. Animals at the highest dose appeared to have lost weight (estimated from a graph), which was most marked during the first 7 days of treatment. Food intake also appeared to be reduced over this period. No changes in body weight were observed in any other group. There were no changes in organ weights relative to body weight at 250 ppm, but at 500 ppm the weights of the testes and brain appeared to be increased. This result would be expected in view of the body-weight loss. Absolute organ weights were not reported. No significant histological changes were observed. Urinary analysis showed no treatment-related effects on protein or glucose concentrations.

The inhibition of cholinesterase activity observed during the 3 months of treatment is shown in Table 3. Significant inhibition of plasma cholinesterase activity occured at concentrations ³ 125 ppm, erythrocyte cholinesterase activity was inhibited by > 20% at doses ³ 63 ppm, whereas brain cholinesterase activity was inhibited only at 500 ppm. It is noteworthy that cholinergic signs probably due to inhibition of the peripheral nervous system were observed at 250 ppm, a concentration at which no appreciable inhibition of brain cholinesterase activity was seen. The noradrenaline and 3-hydroxytyramine content of the brain stem, spleen, and liver showed no treatment-related change. The NOAEL was 63 ppm, equivalent to 6.3 mg/kg bw per day, on the basis of a significant reduction in erythrocyte cholinesterase activity at 125 ppm, correlated with a corresponding significant reduction in plasma cholinesterase activity (JMPR, 1969; modified with reference to the original report of Misu et al., 1966).

From JMPR (1969), modified after refrence to the original report of Misu et al. (1966)

Fenitrothion (purity, 97.2%), fenitrooxon (purity, 99%), and 3-methyl-4-nitrophenol (purity, 99.5%) were administered separately in the diet to groups of 15 Wistar rats of each sex for 6 months in the following dosing regimens: controls, fenitrothion at 10, 30, or 150 ppm (equal to 0.6, 1.8, or 9.2 mg/kg bw per day for males and 0.6, 2.0, or 11 mg/kg bw per day for females); fenitrooxon at 5, 15, or 50 ppm (equal to 0.3, 0.9, or 3.0 mg/kg bw per day for males and 0.3, 1.0, or 3.7 mg/kg bw per day for females); or 3-methyl-4-nitrophenol at 150, 500, or 1500 ppm (equal to 9.2, 31, or 95 mg/kg bw per day for males and 10, 33, or 100 mg/kg bw per day for females). The animals were observed daily for behavioural changes and deaths. Body weights and food and water consumption were recorded weekly. At weeks 4, 8, 12, and 24, the urine was analysed for glucose, protein, bilirubin, and urobilinogen concentrations and occult blood. At the end of the study, plasma, erythrocyte, and brain cholinesterase activity was determined. The haematological parameters measured were erythrocyte, leukocyte, and thrombocyte counts, differential leukocyte count, haemoglobin, erythrocyte volume fraction, and sedimentation rate. The clinical chemical parameters examined were sodium, potassium, chloride, total protein, albumin, glucose, bilirubin and blood urea nitrogen concentrations and alkaline phosphatase and alanine and aspartate aminotransferase activities. After sacrifice, gross autopsies were performed on all animals. The organs were weighed, and the following tissues were examined histologically: brain, eye, spinal cord, peripheral nerve, heart, lung, spleen, bone marrow, lymph nodes, thymus, oesophagus, stomach, small intestine, large intestine, liver, pancreas, kidney, urinary bladder, testis/ovary, prostate/uterus, pituitary, thyroid, adrenal, and bronchus.

During the study, there were no clinical signs of toxicity. One animal given 15 ppm of fenitrooxon died just before being killed, but the cause was not described. Only the final body weights were presented: no difference in body weights were found between groups. Although a slight decrease in body-weight gain during initial feeding of fenitrothion at 150 ppm was reported, no data were supplied to support this statement. There was no treatment-related change in food or water consumption. Haematological, clinical chemical, and urinary parameters showed no treatment-related changes.

Table 4 shows the observed inhibition of cholinesterase activity. 3-Methyl-4-nitrophenol had no intrinsic inhibitory activity on cholinesterase. With fenitrothion and fenitrooxon, females appeared to be more sensitive to inhibition: Significant (p < 0.01) inhibition of plasma cholinesterase activity was seen in females at all concentrations, although with no clear dose–response relationship, while the inhibition was significant in males only at 150 ppm. In brain and erythrocytes, significant, dose-related inhibition (p < 0.05, p < 0.01) was observed at 30 and 150 ppm in females and at 150 ppm in males. The NOAEL for fenitrothion was 10 ppm, equal to 0.6 mg/kg bw per day, on the basis of significant inhibition of brain and erythrocyte cholinesterase activity at 30 ppm (JMPR, 1974, 1977; modified with reference to the original reports of Kadota et al., 1972b, 1975b).

In a study conducted according to GLP, five groups of 20 Crl(W1)BR rats of each sex were given whole-body exposure to an aerosol of fenitrothion (purity, 94.6%) in a chamber for 90 days. The groups consisted of controls exposed to air, controls exposed to the vehicle (acetone), and groups exposed to fenitrothion at 0.2, 1, or 10 µg/L per day for 6 h/day, 5 days/week for 90 days. The achieved concentrations of fenitrothion in respirable droplets (100% were £ 6 µm) during exposure were close to those targeted. Each group was subdivided, and each received the same exposure: one was used for toxicological assessments during and at the end of the study, and the other was used only for determinations of plasma and erythrocyte cholinesterase activity during the study.

Animals in the first subgroups were examined twice daily for clinical signs, and abnormalities were recorded. Their body weights and food consumption were recorded weekly. All animals underwent an ophthalmoscopic examination before the study, and rats in the first subgroup were examined during week 13. Blood samples were taken from these rats during week 12 and examined to determine erythrocyte volume fraction, haemoglobin, erythrocyte count, mean corpuscular haemoglobin concentration, mean corpuscular volume, and total and differential leukocyte, platelet, and reticulocyte counts. The clinical chemical end-points determined were glucose, total protein, albumin, blood urea nitrogen, bilirubin, creatinine, sodium, potassium, calcium, phosphorus, chloride, cholesterol, and globulin concentrations, the albumin:globulin ratio, and the activities of alanine and aspartate aminotransferases, creatine phosphokinase, and alkaline phosphatase. Plasma and erythrocyte cholinesterase activity was determined in animals in the second subgroup before the study, before exposures 5, 10, 15, 20, 30, 40, and 50, and after exposure 64. Brain cholinesterase activity was determined after exposure 64.

Rats in the first subgroups were killed after 66 exposures and examined macroscopically, and the following organs were weighed; brain, pituitary, thyroids, thymus, heart, lungs, liver, spleen, kidneys, adrenals, testes with epididymides, and ovaries. The following tissues were preserved for histological examination: nasal passages, tongue, pharynx, larynx, trachea (including bifurcation), lungs, lymph nodes (tracheobronchial, mandibular, cervical, mesenteric), salivary gland, thyroids (with parathyroids), mammary gland, thymus, skin, skeletal muscle, spinal column, spinal cord (cervical, mid-thoracic, and lumbar) kidneys, urinary bladder, testes, seminal vesicles, prostate, ovaries, uterus, vagina, brain (medulla/pons, cerebellar, cortex, cerebral cortex), pituitary, eyes, liver, spleen, pancreas, adrenals, oesophagus, stomach, duodenum, jejunum, ileum, caecum, colon, rectum, heart, aorta, sciatic nerve, sternum/ribs, femur with joint, and any gross abnormalities. The lungs from all rats were examined. All tissues from the air and vehicle controls and animals at the highest dose were examined. One female at 10 µg/L per day and 1 male at 1 µg/L per day died during blood sampling; as both animals were from the subgroup not scheduled for autopsy, the carcasses were discarded without further examination. The only clinical sign, which was seen in all treated groups but could not be quantified, was decreased responsiveness to auditory stimulation during exposure to fenitrothion in comparison with control animals. No significant changes in body weight or food consumption were seen during the study. No ophthalmic abnormalities were seen before treatment, but, at 13 weeks, a number of animals had corneal opacity (2/20, 8/20, 6/20, 5/20, and 3/20); the highest frequency was thus seen in vehicle controls, with no association to treatment with fenitrothion. This is an abnormality commonly seen in ageing rats; the increased occurrence may have been related to exposure to the vehicle. Haematological and clinical chemical examination revealed no findings attributable to treatment. The group mean cholinesterase activities relative to those of the vehicle controls after 13 weeks (i.e. 64 exposures) are shown in Table 5, the plasma activites being adjusted for those before exposure. While a clear concentration-related inhibition of plasma cholinesterase activity was observed in females, appreciable inhibition in erythrocytes was observed only at the highest concentration in animals of each sex. No appreciable inhibition of brain cholinesterase activity was observed in males, but a concentration-related inhibition was observed in females. Although the statistical significance of the reduction in cholinesterase activity in blood was tested at both the 1% and 5% levels, the activity in the brain was apparently tested only at the 1% level since a calculation shows that the degree of inhibition observed in females, namely 21%, was clearly significant at the 5% level. There were no significant macroscopic or histological findings, and the organ weights showed no significant treatment-related effects. No NOAEC could be established, on the basis of significant inhibition of cholinesterase activity in brain in females at the lowest concentration. The LOAEC was 0.2 µg/L per day (Coombs et al., 1988).

Technical-grade fenitrothion (purity, 97.2%) was mixed into the diet and fed to groups of 15 male Japanese albino rabbits at a concentration of 0, 300, or 1000 ppm, equivalent to 0, 3, and 10 mg/kg bw per day, for 6 months. The animals were monitored daily for behaviour and weighed weekly during the first month and twice a month thereafter. Blood was collected once at the end of treatment to assess blood chemistry and haematological end-points. Plasma and erythrocyte cholinesterase activity was measured after 1, 2, 3, 4, 6, 10, 13, 18, and 24 weeks of treatment and that in brain at 24 weeks by an electrometric method (change in pH), whereas cholinesterase activity in a homogenate of medial rectus muscle was measured at 24 weeks by a colorimetric assay (Ellman method), and cholinesterase activity was localized histochemically in frozen sections of the muscle. Sections taken from brain, eye (with optic nerve and ocular muscle), heart, spinal cord, sciatic nerve, bronchus, lung, spleen, bone marrow, mesenteric lymph node, thymus, oesophagus, stomach, intestine, liver, pancreas, kidneys, urinary bladder, testes, epididymides, prostate, pituitary, thyroid, and adrenals after the final kill were examined by light microscopy. Transmission electron microscopy was performed only on the medial rectus muscles. Liver, kidney, spleen, lung, brain, heart, adrenal, testis, thyroid, and pituitary were weighed, and their weights were compared with those of controls.

There were no treatment-related deaths, although one rabbit died accidentally (group not reported) during restraint for blood collection. There were no clinical signs or significant changes in body weight and no noteworthy changes in clinical chemical or haematological end-points. The mean inhibition of cholinesterase activity after 24 weeks of treatment was 27% in plasma, 24% in erythrocytes, 9% in brain, and 9% in muscle at 3 mg/kg bw per day and 41% in plasma, 49% in erythrocytes, 32% in brain, and 8% in muscle at 10 mg/kg bw per day. The cholinesterase activity in plasma and erythrocytes was thus similarly reduced at the two doses whereas that in brain was appreciably less inhibited and almost no inhibition was seen in muscle. As might be anticipated from the absence of substantial cholinesterase inhibition in the muscle homogenate, no difference in cholinesterase activity was found in muscle sections taken from control and treated rabbits. No treatment-related histological lesions were observed in any tissue or by electron microscopy in the medial rectus muscles (Miyamoto et al., 1976).

In a study performed in compliance with GLP, technical-grade fenitrothion (purity, 93.7%) was applied to a shaved area between the shoulder and rump of groups of five male and five female New Zealand white rabbits at 0 (distilled water), 3, 10, 50, or 250 mg/kg bw per day for 21 days. At each treatment, the test material was applied for 6 h under an occlusive bandage, and the test site was then washed with non-irritating soap and water. Behaviour and general health were observed twice daily, the test sites were examined for changes before each application, and dermal reactions were scored according to the method of Draize. Body weight and food consumption were measured weekly. At the end of the study, the haematological parameters assessed were: haemoglobin, reticulocyte, erythrocyte, and leukocyte counts, erythrocyte volume fraction, mean corpuscular volume, mean corpuscular haemoglobin, mean corpuscular haemoglobin concentration, differential blood count, thrombocyte count, coagulation time, and platelet count; and the clinical chemical parameters determined were: sodium, potassium, inorganic phosphorus, calcium, chloride, total bilirubin, total protein, blood urea nitrogen, creatinine, serum glucose, total cholesterol, albumin, and globulin concentrations, albumin:globulin ratio, and the activities of alanine and aspartate aminotransferases, alkaline phosphatase, and plasma, erythrocyte, and brain cholinesterase. Treated animals that died or were killed during the study and survivors at the final kill were necropsied. The absolute and relative weights of the heart, liver with drained gall-bladder, kidneys, testes/ovaries, epididymides, and adrenals were determined. A range of organs and tissues were preserved and examined histopathologically.

At 250 mg/kg bw per day, all males and two of five females either died or were killed when moribund. Before death, characteristic signs of poisoning with organophosphates were evident, namely hypoactivity, muscular hypotonia, tremors, bradypnoea, hypothermia, salivation, clonic convulsions, loose or mucous stools, diarrhoea, and a soiled circumanal region. At this dose, reductions in body weight were observed in males at day 7 (6%), day 14 (10%), and day 20 (21%; p < 0.05), and females showed slight reductions at day 14 (5%) and day 20 (6%), although these were not significant. Treated rabbits showed similar food intakes to controls. At 3, 10, and 50 mg/kg bw per day, very slight erythema (score, 1; range, 0–4) was observed in 13/15 males from day 5–7 onwards and in 10/15 females from day 5–9 onwards. Very slight oedema (score, 1; range, 0–4) was observed in 1/15 males on days 8–18 and in 8/15 females from day 13 onwards. The mean dermal irritation scores were 0.2–1.4 in males and 0.2–1.8 in females. Desquamation was observed in 6/15 males and 13/15 females (time of onset not stated). At 250 mg/kg bw per day, very slight erythema (score, 1) was observed in the surviving four males and three females on days 4–6 and, very slight to well defined erythema (score, 1–2) in one male (day 7–10) and two females (days 7–14). Very slight oedema (score, 1) was observed in one male on days 7 and 8 and one female on days 5–7. Very slight to moderate oedema (score, 1–3) was noted in one male on day 8. The mean dermal irritation scores were 0.2–2.7 in males and 0.2–2.4 in females. Desquamation was observed in three males and three females (time of onset not stated).

Although a significant (p < 0.05) increase in mean prothrombin time (13%) and a decrease in mean alanine aminotransferase activity (43%) was seen in males at 50 mg/kg bw per day, there was no evidence of treatment-related haematological or biochemical changes (except cholinesterase activity) at any dose. Significant (p < 0.01) decreases in mean erythrocyte cholinesterase activity were observed in males at 10 and 50 mg/kg bw per day (46% and 49%, respectively), and in females at 10, 50, and 250 mg/kg bw per day (51%, 70%, and 83%, respectively) 2 h after the last dose. After 24 h, significant (p < 0.01) decreases were observed in males at 50 mg/kg bw per day (58%) and in females at 50 and 250 mg/kg bw per day (65% and 79%, respectively). Similarly, significant (p < 0.01) reductions in mean plasma cholinesterase activity in females were observed at 10 and 50 mg/kg bw per day (73% and 89%, respectively) 2 h after final dosing. After 24 h, significant decreases were observed in males at 50 mg/kg bw per day (40%; p < 0.05) and in females at 10, 50, and 250 mg/kg bw per day (41%, 60%, and 94%, respectively; all p < 0.01). The mean brain cholinesterase activity was also significantly reduced in males at 50 mg/kg bw per day (36%; p < 0.01) and in females at 10 (21%; p < 0.05), 50 (30%; p < 0.01), and 250 mg/kg bw per day (79%; p < 0.01) at termination. Cholinesterase activity was not measured in rabbits at 250 mg/kg bw per day which died. An increase (33%; p < 0.05) in the absolute and relative (57%; not significant) weights of the adrenals was observed in females at 250 mg/kg bw per day. No other organs showed any significant difference in absolute or relative weight between treated and control groups. Histological examination of the skin revealed thickening of the epidermis, hyperkeratosis, and inflammatory infiltrates in the corium in males and females at all doses and haemorrhage in the corium at 10, 50, and 250 mg/kg bw per day. At the highest dose, one male and one female had slight necrosis of the liver, and some animals showed changes in the digestive tract and kidney that were considered to be the cause of the deterioration in general condition. The NOAEL was 3 mg/kg bw per day on the basis of inhibition of erythrocyte and brain cholinesterase activity at higher doses (Suetake et al., 1991).

In a study performed according to GLP, technical-grade fenitrothion (purity, 96.8%) was administered in the diet to groups of six male and six female pure-bred beagle dogs at a concentration of 0, 5, 10 or 50 ppm, equal to 0, 0.17, 0.3, or 1.6 mg/kg bw per day for males and 0, 0.15, 0.3, or 1.6 mg/kg bw per day for females, for 12 months. The animals were housed individually in metabolism cages under standard laboratory conditions, and water and food were provided ad libitum. The animals were monitored for deaths and changes in appearance or behaviour twice daily. Detailed examinations were performed once weekly, and all animals were examined by a veterinarian before treatment and at 3, 6, and 12 months. Body weights and food consumption were recorded weekly. All animals underwent an ophthalmological examination before treatment and at 6 and 12 months. Haematological and clinical chemical end-points were measured before treatment and at 4, 8, 13, 17, 26, 39, and 52 weeks. The haematological parameters included erythrocyte volume fraction, haemoglobin, erythrocyte, leukocyte, differential leucocyte, platelet, and reticulocyte counts, mean corpuscular volume, mean corpuscular haemoglobin, and mean corpuscular haemoglobin concentration. The clinical chemical parameters included the activities of lactic dehydrogenase, aspartate and alanine aminotransferases, and alkaline phosphatase, concentrations of glucose, blood urea nitrogen, total bilirubin, cholesterol, albumin, globulin, total protein, creatinine, sodium, potassium, chloride, calcium, and phosphorus, and albumin:globulin ratio. Urine was analysed before treatment and at weeks 4, 13, 26, 39, and 52 for volume, colour, appearance, pH, specific gravity, protein, glucose, ketones, urobilinogen, nitrite, and bilirubin concentrations, occult blood, and microscopic appearance. Plasma and erythrocyte cholinesterase activity was measured before treatment and at weeks 4, 8, 13, 17, 21, 26, 39, and 52. Brain cholinesterase activity was measured at the end of the study. All animals were necropsied grossly at the end of the study. The liver, kidneys, heart, testis with epididymides, brain, ovaries, pituitary, thyroid/parathyroids, and adrenal glands were weighed. Standard tissues were preserved, but a histopathological examination was not performed; the results of a histological assessment of the stored tissues were reported in a revised report (Spicer, 1986).

No deaths or treatment-related clinical signs were observed. On physical examination, some animals, mainly at the intermediate and high doses, were found to have a relatively rapid heart rate, but this was considered to be of little biological significance. There were no significant dose-related changes in body weight or food consumption during the study. Haematology, clinical chemistry, and urinary analysis showed no significant changes, although there was a slight increase in cholesterol concentration in males at the highest dose early in the study. No changes were observed on ophthalmic examination.

The mean inhibition of cholinesterase activity is shown in Table 6. Plasma cholinesterase was significantly reduced in animals of each sex at the highest dose. Erythrocyte cholinesterase activity was reduced in males at he highest dose only. Brain cholinesterase activity was unaffected by treatment. There were no treatment-related changes in organ weights and no macroscopic lesions. Although histological examination of tissues revealed several lesions, all except one appeared to be unrelated to treatment. The exception was that the incidence of haemorrhagic abdominal lymph nodes was increased, especially among females. Whereas only one male had a trace of haemorrhage in the nodes at 50 ppm, two females each at 10 and 50 ppm were affected, the the severity being scored as a trace in one bitch and mild in the other. No controls or animals at 5 ppm were affected. Although a clear dose–response relationship was not evident, this may have been due to the small group size. The investigators indicated that the spontaneous incidence of lesions including haemorrhagic lymph nodes is common among beagles and they are therefore unlikely to be of pathological significance. Although no data were provided for past controls data in the laboratory to support this contention, it is noteworthy that neither the mandibular nor the thoracic lymph nodes showed an increased incidence of haemorrhagic lesions, suggesting that the incidence of abdominal lymph-node lesions was unrelated to treatment. The NOAEL was 50 ppm on the basis of the absence of change in brain cholinesterase activity (JMPR, 1984; modified with reference to the original data of Griggs et al., 1984, and the amended report of Spicer, 1986).

(c) Long-term studies of toxicity and carcinogenicity

Fenitrothion (purity, 97%) was administered in the diet to groups of 50 ICR Swiss mice of each sex at a concentration of 0, 30, 100, or 200 ppm, equal to 0, 3.1, 11, or 22 mg/kg bw per day for males and 0, 3.7, 12, or 24 mg/kg bw per day for females, for 78 weeks. The mice were treated at a concentration of 0, 10, 30, or 100 ppm during the first 2 weeks of the study, before being changed to the indicated doses for the remainder. Animals were observed daily for deaths. Body weight, food consumption, behaviour, and appearance were recorded weekly during weeks 0-–10, every 2 weeks during weeks 11–26, and every 4 weeks from week 27 to the end of the study. Ophthalmoscopic examinations were performed on all mice at weeks 28 and 78. Gross necropsies were performed on mice that died during the study and on all those killed at the end of the study. The weights of the heart, liver, spleen, kidneys, testes with epididymis, and adrenals were recorded and the organ:body weight ratios determined. The following tissues were preserved for histological examination: brain, pituitary, thoracic spinal cord, eye, thyroids, oesophagus, lung, heart, liver, gall-bladder, spleen, kidneys, adrenals, stomach, pancreas, small intestine, large intestine, mesenteric lymph node, urinary bladder, testes with epididymis, seminal vesicles, prostate, ovaries, uterus, skin, bone (sternum), bone marrow (femur), nerve with muscle, and unusual lesions. Tissues collected from 10 mice of each sex in the control and highest-dose groups, and liver, kidney, and any unusual lesions from 10 mice of each sex at 30 and 100 ppm were examined microscopically. Cholinesterase activity and haematological and clinical chemical end-points were not determined.

No behavioural or clinical abnormalities were observed during the study, and no dose-related changes in body weight or food consumption occurred. The results of the ophthalmological examinations were not presented for individual animals; the summary results indicated no treatment-related effects. Gross and histological examination revealed a number of abnormalities, none of which was considered to be related to treatment. A number of neoplasms, including thyroid-cell adenoma and carcinoma, alveolar and bronchiolar adenoma and carcinoma, hepatocellular carcinoma, osteogenic sarcoma, and mammary adenocarcinoma, were found, but these tumours were either isolated or occurred with similar frequency in treated and control groups and were therefore considered unrelated to treatment. A statistically significant decrease in the absolute and relative weights of the heart was found at the highest dose, which is of questionable biological significance. The NOAEL was 200 ppm, equal to 22 mg/kg bw per day, the highest dose tested (Kundzins, 1975, 1979, 1980a).

In a study of carcinogenicity performed in accordance with Guideline 83-5 of the US Environmental Protection Agency and with GLP, technical-grade fenitrothion (purity, 96.7%) was administered to groups of 50 B6C3F₁ mice of each sex in the diet at a concentration of 0, 3, 10, 100, or 1000 ppm, equal to 0.37, 1.4, 13, and 130 mg/kg bw per day for males and 0.46, 1.5, 13, and 140 mg/kg bw per day for females, for 104 weeks. The stability, homogeneity, and concentration of fenitrothion in the feed were acceptable. Subgroups of 10 mice of each sex were taken at weeks 13, 26, 52, and 78 from satellite groups of 50 mice of each sex per group that had been treated similarly to those in the main study for determination of haematological, blood chemical (including cholinesterase activity), and urinary end-points. The doses were selected on the basis of the results of a 4-week study with 0, 1, 3, 10, 30, 100, 300, or 1000 ppm in the diet, which showed significantly reduced cholinesterase activity at 10 ppm and reduced food intake and weight loss in animals of each sex at 1000 ppm. Other findings at 1000 ppm were reduced erythrocyte count, haemoglobin concentration, erythrocyte volume fraction, and alkaline phosphatase activity in females and increased cholesterol concentration in males and females.

Both the main and the satellite groups were observed for clinical signs and deaths daily, and for body weight, food and water consumption, and the results of palpation weekly for the first 14 weeks and every second week thereafter. All animals that survived to term and those that died or were killed in extremis were examined histologically. At week 104, all mice in the main group were killed for histological examination; brain, heart, liver, spleen, kidneys, adrenals, and testes or ovaries were weighed before fixation, and the thyroid and ovaries were weighed after fixation. The following tissues from mice in the satellite groups at week 52 and from all survivors in the main groups at week 104 were preserved in 10% formalin for histological examination: brain (cerebrum and cerebellum), spinal cord, sciatic nerve, eyeball, thyroid (including parathyroid), oesophagus, trachea, lung, heart, liver, gall-bladder, spleen, kidney, adrenal, stomach, pancreas, small intestine (duodenum, jejunum, ileum), large intestine (caecum, colon, rectum), mesenteric lymph node, urinary bladder, prostate, testis, seminal vesicle, epididymides, ovary, uterus, bone marrow, aorta, thymus, salivary gland, mammary gland, vagina, pituitary, muscle, bone (sternum, femur, lumbar vertebrae), skin, and any unusual lesions. Blood and urine were collected from the survivors in the main group at 104 weeks for determination of haematological end-points (erythrocyte volume fraction, haemoglobin, erythrocyte count, mean corpuscular volume, mean corpuscular haemoglobin, mean corpuscular haemoglobin concentration, leukocyte and differential leukocyte counts), clinical chemical end-points (cholinesterase activity, glucose, blood urea nitrogen, total protein, albumin, bilirubin, and cholesterol concentrations, albumin:globulin ratio, and activities of alkaline phosphatase and alanine and aspartate aminotransferases), and urinary end-points (specific gravity, occult blood, pH, and glucose, ketone, protein, bilirubin, and urobilinogen concentrations). Ophthalmoscopic examinations were performed on mice in the main control and 1000-ppm groups at week 104.

Survival in the main and satellite groups was not significantly affected by treatment, and no appreciable differences were reported in gross changes or behaviour. No consistent treatment-related changes in haematological, urinary, or ophthalmic end-points were observed. The treatment-related effects were:

• reductions in mean body weight and body-weight gain at 1000 ppm in animals of each sex: at 1 year, the body weights were 17% lower than those of controls, and after 2 years the difference had increased to 19% for females but had been reduced to 13% for males;
• reduced mean food consumption and water intake at 1000 ppm correlated with body-weight loss;
• alopecia among females at 1000 ppm, which was reduced at the final kill; this observation correlated well with a significantly reduced incidence of hair follicle atrophy among females at 1000 ppm (i.e. 10/36 in controls and 1/43 at 1000 ppm);
• an elevated plasma cholesterol concentration (p < 0.01–0.05) at 100 and 1000 ppm and reduced plasma glucose concentration (p < 0.01–0.05) at 1000 ppm;
• increased weight of the brain, both absolute and relative to body weight in animals of each sex at 1000 ppm, which was significant (p < 0.01) at the final kill, associated with a significant reduction (p < 0.01) in the expected calcification (grade 1) of the brain parenchyma in 2-year-old mice;
• significant reductions (p < 0.01) in cholinesterase activity in plasma, erythrocytes, and brain at 100 and 1000 ppm in animals of each sex (Table 7).

As shown in Table 8, no treatment-related change in the incidence of neoplasms was observed. There was thus no evidence of carcinogenicity in mice treated with fenitrothion at doses that caused a significant reduction in body weight, increased the plasma cholesterol concentration, and increased the absolute and relative weights of the brain in mice of each sex. The NOAEL was 10 ppm, equal to 1.4 mg/kg bw per day, on the basis of significant inhibition of plasma, erythrocyte, and brain cholinesterase activity and elevated plasma cholesterol concentration at 100 ppm (Tamano et al., 1990).

Fenitrothion (purity, 97.2%) was administered in the diet of groups of 15 Wistar rats of each sex at a concentration of 0, 2.5, 5, or 10 ppm, equal to 0, 0.1, 0.3, or 0.5 mg/kg bw per day for males and 0, 0.2, 0.3, or 0.6 mg/kg bw per day for females, for 92 weeks. The rats were housed individually under controlled conditions. Food and water were available ad libitum. Behaviour was observed daily, and body weight, food intake, and water intake were measured weekly. Plasma and erythrocyte cholinesterase activity was measured at week 2, 4, 6, 8, 12, 16, 20, 24, 42, 68, and 92; brain cholinesterase activity was measured at the end of the study. The activity was determined by an electrometric method (change in pH). No gross pathological or histopathological examinations were performed.

No signs of toxicity were observed during the study, and deaths occurred at similar frequency in all groups. There were no significant treatment-related changes in food consumption or body weight. No appreciable inhibition of brain cholinesterase activity was observed at any concentration of fenitrothion tested (Table 9). The NOAEL was 10 ppm, equal to 0.5 mg/kg bw per day, the highest dose tested (Kadota et al., 1974b, 1977, 1980).

Technical-grade fenitrothion (purity, 97%) was administered to Sprague-Dawley rats in the diet at a concentration of 0, 10, 30, or 100 ppm, equal to 0, 0.5 1.5, or 5 mg/kg bw per day for males and 0, 0.6, 1.9, or 6.5 mg/kg bw per day for females, for 104 weeks. Groups of 50 weanling rats of each sex were selected at random from the F_1a litters of treated rats in a three-generation study and were thus exposed to fenitrothion during gestation and lactation; 60 untreated rats of each sex were also selected. Although the rats were distributed randomly by dose, the mean body weights of those at the high dose were approximately 20% lower than those of controls. The animals were housed individually, and food and water were available ad libitum. Body weights, food consumption, appearance and behaviour were recorded weekly until week 26, every second week during weeks 27–52, and every 4 weeks during weeks 53–104, and the presence of tissue masses was recorded at these times. The animals were observed daily for clinical signs. Blood samples were taken from five rats of each sex per group at weeks 13, 52, and 104 and from five rats in the control and highest-dose groups at week 26. Erythrocyte volume fraction, haemoglobin, and erythrocyte, leukocyte, and differential leukocyte counts were determined in all bled animals. Prothrombin time was determined only for controls and rats at 100 ppm. Blood glucose, urea nitrogen, total serum protein, and total serum bilirubin concentrations, alkaline phosphatase, alanine and aspartate aminotransferase activities, and serum protein electrophoresis characteristics were determined in blood samples from fasted animals. Additionally, at weeks 52 and 104, sodium, potassium, chloride, carbon dioxide, calcium, and albumin concentrations were determined. Analysis of urine included specific gravity, pH, glucose, ketone, protein, and bilirubin concentrations and examination of the sediment. Plasma and erythrocyte cholinesterase activities were determined in 10 rats of each sex per group at weeks 0, 2, 4, 8, 13, 26, 52, 78, and 104. At weeks 52 and 104, brain cholinesterase activity was determined in 10 rats of each sex per group. Ophthalmoscopic examinations were performed on all rats at weeks 81 and 104. All animals that died during the study were necropsied grossly. At week 52, 10 rats of each sex per group were killed for interim autopsy. At week 104, all surviving rats were killed. The weights of the heart, liver, spleen, kidneys, and testes with epididymides were recorded before fixation, and the weights of the thyroid and adrenals were recorded after fixation. The brain, pituitary, thoracic spinal cord, eye, thyroid, oesophagus, lung, heart, liver, spleen, kidneys, adrenals, stomach, pancreas, small intestine, large intestine, mesenteric lymph node, urinary bladder, testis with epididymides and seminal vesicles, or ovaries and uterus, skin, costochondral junction, sternal bone marrow, nerve with muscle, and any unusual lesions were preserved in 10% formalin for histological examination.

There were no treatment-related deaths or clinical signs at any time during the study. The mean body weight of the animals at the high dose was 10–20% lower than that of control animals throughout the study, but there was no significant difference in food consumption among groups. Haematological and clinical chemical examinations showed no significant differences among groups, and there were no significant findings in the urine analysis. Dose-related inhibition of plasma, erythrocyte, and brain cholinesterase activity was observed in males and females (Table 10). No significant treatment-related macro- or microscopic findings were made post mortem. A number of neoplasms, including interstitial-cell tumours (Leydig-cell tumours), gliomas phaechromocytomas, and pituitary adenomas were seen, but the occurrences were isolated, and a statistical analysis revealed no difference in tumour incidence between control and treated groups. The NOAEL was 10 ppm, equal to 0.5 mg/kg bw per day, on the basis of marked inhibition of erythrocyte and brain cholinesterase activity at the next higher concentration (JMPR 1974; modified by reference to the original reports of Kundzins, 1974, 1980b; Patterson, 1981).

The available studies of the genotoxicity of fenitrothion are summarized in Table 11.