Phosalone was evaluated toxicologically by the JMPR in 1972, 1993 and 1997 (Annex 1, references 18, 68 and 80). The 1997 JMPR allocated an ADI of 0–0.02 mg/kg bw. The compound was re-evaluated by the present Meeting to consider the need to establish an acute reference dose (RfD). Other studies to underpin maintenance of the ADI were submitted and were evaluated by the Committee.

Albino Sprague-Dawley-derived rats were exposed to an aerosol of a 90% w/w solution of phosalone (purity, 93.1%) in acetone for 4 h; the mass median aerodynamic diameter was 2.4–2.5 µm. The LC₅₀ was 2.1 mg/l in males and 1.3 mg/l in females (Paul, 1999).

Phosalone (purity, 93.8%) was administered by gavage in corn oil to one male and one female Crl:CD BR rat at a dose of 60 mg/kg bw. The animals were observed at half-hourly intervals up to 3 h after dosing and hourly up to 8 h. Tremor and piloerection were seen 4 h after dosing, but no signs were seen at 24 h. As no clinical signs were seen until 4 h after dosing, a limited ‘functional observational battery’ (FOB) was administered from 5 h after dosing. In this part of the study, groups of three males and three females received a dose of 12 or 80 mg/kg bw in corn oil, and the FOB was administered before treatment and at 5, 6, 7 and 8 h. Body weights were recorded daily, and the animals were killed after 7 days. Animals at 12 mg/kg bw showed no clinical signs, and the results of the FOB were not significantly different from those before dosing. However, at 80 mg/kg bw, chewing movements were seen after 5 h in males and chewing movements, tremor, exophthalmos and reduced rectal temperature were seen in females. At 6 h, the males had tremors and decreased rectal temperature and the females had the same signs as at 5 h. Females showed salivation and decreased arousal at 7 h. The greatest decrease in rectal temperature in animals at 80 mg/kg bw was seen 7 h after dosing in both sexes. At 8 h, females also showed lachrymation. By 24 h, the males had recovered, whereas the females had not and still had hunched posture, lethargy and staining of the fur and appeared thin. It was concluded that peak effect occurred at 6 h. No control group was included. The NOAEL for changes in the FOB in comparison with that before treatment was 12 mg/kg bw (Hughes, 1999a).

Groups of 10 male and 10 female Crl:CD BR rats were given a single oral dose of phosalone (purity, 93.8%) at 0 or 60 mg/kg bw. The animals were observed for clinical signs after treatment, body weights were measured daily, and blood was taken for measurement of plasma and erythrocyte cholinesterase activity before dosing and then in two subgroups of five animals each, one being sampled at 2.5, 6 and 24 h and the other at 4 and 8 h. The animals were killed after 7 days. The brains were removed and cut in half, and one-half was used to estimate brain cholinesterase activity. The method of Ellman et al. (1961) was used to measure cholinesterase activity.

Clinical signs were seen 5 h after dosing, consisting of salivation, lachrymation, noisy respiration, tremors, unsteadiness, coldness, piloerection and wet urogenital region. The signs continued for up to 2 days, except for the staining and wetness of the fur, which continued longer but had disappeared by the end of day 2 in the case of male rats. In the females, clinical signs including piloerection, hunched posture and staining of the fur were seen on day 3 after dosing. Staining of the fur was still seen in one female on the morning of the fifth day after dosing but not in the afternoon. Weight gain was decreased 24 h after dosing in males by comparison with the controls, while females showed weight loss, weight gain then weight in the normal range but below that of controls throughout the rest of the study. Plasma cholinesterase activity was depressed 2.5 h after dosing and fell further up to 6 h. In males, the activity was 12.5% that of concurrent controls at 4 h and 16% that of controls at 6 h. At 24 h, it was still only 46% that of controls. In females, plasma cholinesterase activity was 11% that of controls at 4 h and 10% at 6 h; recovery was not complete at 24 h, the activity being 34% that of controls. Erythrocyte cholinesterase activity was more variable, but it was 66% that of controls at 4 h, 42% at 6 h and 44% at 24 h in males; and 31% that of controls at 2.5 h, 59% at 4 h, 72% at 6 h and 49% at 8 h in females. Brain cholinesterase activity at sacrifice at 7 days was 74% that of concurrent controls in males and 69% in females. As effects were observed in the treated group, no NOAEL could be identified (Hughes, 1999b).

Phosalone (purity, 93.8%) was given by gavage to groups of 10 male and 10 female Crl:CD BR rats at a dose of 0, 10, 25 or 60 mg/kg bw. Clinical signs were monitored throughout the study at least daily. Body weight was recorded before treatment on the day of treatment and then weekly. Food consumption was monitored weekly. The rats were subjected to an FOB before treatment and 6 h and 7 and 14 days after treatment. Satellite groups of five males and five females were treated similarly with phosalone at each dose, and blood was obtained for estimation of cholinesterase activity before dosing and 6 and 24 h and 7 and 14 days after dosing. These animals were killed on day 15, and brain cholinesterase activity was estimated in tissue that had been stored at –70 °C. Cholinesterase was measured by the method of Ellman et al. (1961). All animals were perfused at sacrifice with glutaraldehyde and paraformaldehyde, and samples of brain were taken. Nervous tissues from five animals of each sex at the highest dose and the controls were processed for neuropathological examination. The structures examined were brain (six levels), spinal cord (cervical and lumbar), dorsal root ganglia and dorsal and ventral root fibres as well as sciatic and tibial nerves. Skeletal muscle (gastrocnemius) was also examined.

No deaths were observed. Abnormal clinical signs including jaw clonus, abnormal gait and tremor were seen on the day of treatment with the highest dose in both sexes, but not at lower doses. Decreased weight gain was seen at the highest dose, in males only, during the first week; the weight gain in other groups was comparable to that of controls and was similar in all groups of females. Food consumption was comparable in the various groups, except that males at the high dose in the satellite group had decreased food consumption in the first week. Food conversion efficieny was comparable in all groups.

Changes in the FOB were seen at the highest dose and, with one exception, at 6 h on day 1 only. The signs consisted of tremors, clonus of the jaws, exophthalmos, piloerection, coldness to touch, hunched posture and abnormal gait. Females also showed increased urination. Decreased activity and rearing counts were seen in females at the highest dose. Tremors were seen 14 days after dosing in males only, and only at the highest dose; as such changes were not seen at 7 days, they are of doubtful significance. At 6 h, both males and females at the highest dose showed decreased locomotor activity. Reduced group mean body temperature was observed in both sexes at the highest dose.

Plasma cholinesterase activity was decreased at 6 and 24 h, but not later in the study, in animals of each sex at 60 mg/kg bw, at 6 h in animals of each sex at 25 mg/kg bw and at 24 h at that dose only in males. Plasma cholinesterase activity was diminished at 6 h in animals of each sex at 10 mg/kg. Erythrocyte cholinesterase activity was decreased at 6 h in males at the highest dose (50% that of concurrent controls); in females at that dose, although the activity was only 75% that of concurrent controls, the difference was not statistically significant. At lower doses, no significant depression of erythrocyte cholinesterase activity was observed at 6 h. At 24 h, erythrocytic cholinesterase activity was reduced to 64% that of controls in males and 75% in females at the highest dose, but at no other time or at lower doses. At termination, brain cholinesterase activity was marginally but significantly decreased in animals at the highest dose (82% that of controls in males and 78% in females). Brain cholinesterase activity was not decreased at lower doses. No intergroup differences were found in brain weight, width or length, and no neuropathological findings were present that could be attributed to treatment. The NOAEL was 25 mg/kg bw on the basis of clinical signs and decreased erythrocyte and brain cholinesterase activity at 60 mg/kg bw (Hughes, 1999c).

Groups of 10 Crl: CD BR rats of each sex received diets containing phosalone (purity, 93.8%) at a concentration of 50, 150 or 600 ppm for 13 weeks, corresponding to intakes of 0, 3.9, 12 and 46 mg/kg bw per day for males and 0, 4.4, 13 and 56 mg/kg bw per day for females. Satellite groups of 10 rats of each sex were treated similarly for 4 or 8 weeks, with mean intakes of 0, 5.3, 16 and 64 mg/kg bw per day for males in weeks 1–3 and 0, 3.9, 12 and 48 mg/kg bw per day in weeks 4–7, and equivalent figures for females of 0, 5.4, 16 and 66 mg/kg bw per day in weeks 1–3 and 0, 4.6, 14 and 58 mg/kg bw per day in weeks 4–7. Groups of similar size received the vehicle only and served as controls. The animals were observed at least daily for signs of ill-health. Body weights were recorded at the start of the study and weekly thereafter. Food consumption was monitored weekly. The first group of animals were subjected to an FOB before treatment and after 4, 8 and 13 weeks of treatment. At termination of the study, half the animals in each main group were killed, and the tissues were fixed by whole-body perfusion for neuropathological examination. Sections were taken of brain (forebrain, midbrain, cerebellum/pons and medulla oblongata), eyes, optic nerves and cervical and lumbar spinal cord, Gasserian ganglion, dorsal root ganglia and fibres and ventral root fibres. Sections were also taken of the gastrocnemius muscle and peripheral nerves (sciatic and tibial). Only controls and animals at the highest dietary concentration underwent neuropathological examination. The remaining animals in each group were used for determination of blood and brain cholinesterase activity. In the satellite groups, blood for estimation of cholinesterase activity was taken at week 4 from five animals in each group, and then these animals were killed for estimation of brain cholinesterase activity. In week 8, blood was taken from the remaining satellite animals for determination of cholinesterase activity, and then these animals were killed for brain cholinesterase estimation. Cholinesterase activity was estimated by the method of Ellman et al. (1961). Brain cholinesterase activity was measured in brains that had been stored at –70 °C.

No deaths occurred during the study, and no inter-group differences in clinical signs were detected.. Weight gain was decreased in females at the highest dietary concentration during weeks 0–13 by comparison with concurrent controls, while food consumption was unimpaired; food efficiency was decreased in this group. Body-weight gain, food consumption and food conversion efficiency in the satellite groups were unaffected. Only minor differences were observed between the groups in the FOB, and these were confined to females: at 8 weeks, abnormal gait was seen at the two higher dietary concentrations, and, at 13 weeks, poor grooming and hair loss were seen at the highest concentration. Differences in group mean activity counts and group rearing counts were not observed.

Some inter-group differences in erythrocyte but not plasma cholinesterase activity were seen before dosing. Significant reductions in cholinesterase activity were observed during treatment. Plasma cholinesterase activity was reduced at week 4 in both sexes at all dietary concentrations by comparison with concurrent controls (marginally so in males at the lowest concentration). At week 8 and weeks 13–14, plasma cholinesterase activity was diminished in males at the two higher dietary concentrations and in females at all dietary concentrations. Erythrocyte cholinesterase activity was not reduced at week 4 in males but was diminished in all treated groups of females at that time, by 80% that of concurrent controls at 50 ppm, 67% at 150 ppm and 64% at 600 ppm. At week 8, significant reductions were seen in males at the two higher dietary concentrations (63% and 71% that of concurrent controls, respectively). At week 13, reductions were seen in males at the two higher concentrations (51% and 41% that of concurrent controls, respectively). Reduced activity was observed in all treated groups of females, the value being 63% that of controls at 50 ppm, 64% at 150 ppm and 63% at 600 ppm. Brain cholinesterase activity was reduced in males at the highest dietary concentration at 4 weeks (47% that of concurrent controls) and in females at 150 and 600 ppm (72% and 19%, respectively). At week 8, all treated groups of males showed reduced brain cholinesterase activity, at 75% that of concurrent controls at 50 ppm, 64% at 150 ppm and 28% at 600 ppm. At that time, females showed significant reductions only at the two higher dietary concentrations (70% and 23% that of concurrent controls at 150 and 600 ppm, respectively). At weeks 13–14, significant reductions were seen in both sexes at the two higher dietary concentrations, but the decreases in activity were marginal at 150 ppm. At this time, activities that were 84% and 40% that of concurrent controls were seen in males at 150 and 600 ppm, respectively, and 86% and 23% that of concurrent controls in females at these doses, respectively No differences were seen in brain weight, width or length, and no inter-group differences were observed on neuropathological examination. The NOAEL was 50 ppm, equal to 3.9 mg/kg bw per day, on the basis of effects on brain cholinesterase activity at 150 ppm, as the depressed brain cholinesterase activity in males at 50 ppm at 8 weeks was considered to be of doubtful biological significance, not being observed at any other time nor in females and not being accompanied by clinical effects (Hughes, 1999d).

In an assay for unscheduled DNA synthesis in rat hepatocytes in vitro, phosalone (purity, 93.1%) was added at concentrations of 0.005–5 µg/ml. No effect was seen (Adams & Kirkpatrick, 2000).

Phosalone is an organophosphate ester, and virtually all its toxic actions are mediated by cholinesterase inhibition.

The LD₅₀ in rats treated orally was approximately 150 mg/kg bw, and WHO (1999) has classified phosalone as moderately hazardous.

After preliminary studies had shown that peak effects occur at about 6 h, neurotoxicity was studied in rats given phosalone by gavage at a single dose of 0, 10, 25 or 60 mg/kg bw. No deaths were observed. Abnormal clinical signs were seen on the day of treatment with the highest dose. Changes in the FOB were seen at the highest dose and, with one exception, on day 1 only. The NOAEL was 25 mg/kg bw on the basis of clinical signs and decreased erythrocyte and brain cholinesterase activity at 60 mg/kg bw.

The Meeting also reviewed studies that were not relevant to establishment of an acute RfD but which were nevertheless relevant to continuation of the current ADI. In a study of acute toxicity in rats exposed by inhalation for 4 h, the LC₅₀ was 2.1 mg/l in males and 1.3 mg/l in females. In a 13-week study of neurotoxicity, rats received phosalone in the diet at a concentration of 50, 150 or 600 ppm; satellite groups of rats received phosalone at the same dietary concentrations for 4 or 8 weeks. The NOAEL was 50 ppm, equal to 3.9 mg/kg bw per day, on the basis of reduced brain cholinesterase activity at the next highest dose (150 ppm, equal to 12 mg/kg bw per day). A study of DNA repair in rat hepatocytes in vitro gave negative results. The Meeting concluded that these studies supported continuation of the current ADI.

After considering previous evaluations of phosalone and the new data, the Meeting established an acute RfD of 0.3 mg/kg bw on the basis of the NOAEL of 25 mg/kg bw in the study of acute toxicity in rats treated by gavage and a safety factor of 100.

Adams, K. & Kirkpatrick, D. (2000) Phosalone in vitro DNA repair test using rat hepatocytes. Study No. RNP 659/002569. Huntingdon Life Sciences Ltd, Woolley Road, Alconbury, Cambridgeshire, England. Supplied to WHO by Rhône-Poulenc Agrochimie, Lyon, France. GLP compliant: UK Statutory Instruments 654, 1997; 3106, 1999; OECD ENV/MC/CHEM(98)17; EU Directive 1999/11/EC). Data requirements: USEPA FIFRA 84-2, OECD Guideline 482.

Ellman, G.L., Courtney, D. & Andres, V. (1961) A new and rapid colorimetric determination of acetylcholinesterase activity. Biochem. Pharmacol., 7, 88–95.

Hughes, E.W. (1999a) Phosalone single dose study by oral gavage administration dose range and time to peak effect in rats. Study No. RNP 601/990040, Huntingdon Life Sciences Ltd, Huntingdon, Cambridgeshire, England. Supplied to WHO by Rhône-Poulenc Agrochimie, Lyon, France.

Hughes, E.W. (1999b) Phosalone single dose study by oral gavage administration to assess cholinesterase inhibition in rats. Study No. RNP 600/990041, Huntingdon Life Sciences Ltd, Huntingdon, Cambridgeshire, England. Supplied to WHO by Rhône-Poulenc Agrochimie, Lyon, France. Sponsor did not require GLP compliance.

Hughes, E.W. (1999c) Phosalone neurotoxicity study by a single oral gavage administration to CD rats followed by a 14-day observation period. Study No. RNP 586/992263. Huntingdon Life Sciences Ltd, Huntingdon, Cambridgeshire, England. Supplied to WHO by Rhône-Poulenc Agrochimie, Lyon, France. GLP compliant (UK Statutory Instrument 654, 1997; FIFRA 40 CFR 160; OECD ENV/MC/CHEM(98)17; EU Directives 87/18/EEC and 1999/11/EC). Data requirement EPA FIFRA 81-8.

Hughes, E.W. (1999d) Phosalone 13-week neurotoxicity study in rats by dietary administration. Study No. RNP 587/984768. Huntingdon Life Sciences Ltd, Huntingdon, Cambridgeshire, England. Supplied to WHO by Rhône-Poulenc Agrochimie, Lyon, France. GLP compliant (UK Statutory Instrument 654, 1997; FIFRA 40 CFR 160; OECD ENV/MC/CHEM(98)17; EU Directives 87/18/EEC and 1999/11/EC). Guidelines: EPA FIFRA Pesticide assessment guidelines, subdivision F, Addendum F, Neurotoxicity (March 1991).

Paul, G.R. (1999) Phosalone acute (four-hour) inhalation study in rats. Study No. RNP 600/994096. Huntingdon Life Sciences Ltd, Huntingdon, Cambridgeshire, England. Supplied to WHO by Rhône-Poulenc Agrochimie, Lyon, France. GLP compliant (UK Statutory Instrument 654, 1997; FIFRA 40 CFR 160; OECD ENV/MC/CHEM(98)17; EU Directives 87/18/EEC and 1999/11/EC). Guidelines: EPA OPPTS guideline 870.1300.

WHO (1999) Recommended Classification of Pesticides by Hazard and Guidelines to Classification 1998–1999 (WHO/PCS/98.21/Rev. 1), Geneva, International Programme on Chemical Safety.

    See Also:
       Toxicological Abbreviations
       Phosalone (ICSC)
       Phosalone (WHO Pesticide Residues Series 2)
       Phosalone (WHO Pesticide Residues Series 5)
       Phosalone (Pesticide residues in food: 1976 evaluations)
       Phosalone (Pesticide residues in food: 1993 evaluations Part II Toxicology)
       Phosalone (Pesticide residues in food: 1997 evaluations Part II Toxicological & Environmental)