Pirimiphos-methyl

Neurotoxicity

Key value for chemical safety assessment

Effect on neurotoxicity: via oral route

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Effect on neurotoxicity: via inhalation route

Endpoint conclusion

Endpoint conclusion:

no study available

Effect on neurotoxicity: via dermal route

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Acute neurotoxicity study in rats, Nemec 1995a

In an acute neurotoxicity study, performed under GLP and following US EPA 81-8 (1991) and stated to comply with OECD 424 (1997), groups of 17 male and 17 female Sprague Dawley rats received the test substance at concentration of 0, 15, 150 or 1500 mg/kg bw by gavage in corn oil. The functional observation battery (FOB) included observations in the home cage, when handled, in ‘open field’, of sensory responses, neuromuscular performance, locomotor activity and body temperature. Brain cholinesterase assays (Ellman) were performed on 6 brain regions. Histopathological examinations of an extensive range of peripheral and central nervous system tissues were performed on H&E stained sections of control and top dose animals, following in situ perfusion.

There were no deaths during the study. A wide range of clinical signs, including clonic convulsions, were seen in animals administered 1500 mg/kg bw up to day 5, but not subsequently. Performance in most aspects of the FOB on day 1 was significantly altered in the top dose groups. FOB results at subsequent time points and in other groups were similar to controls and/or pre-test findings. Marginal effects on grip strength and rotorod results at 150 mg/kg bw on day 1 and urination marking at day 14 in top dose animals are considered to be within the normal range of variability for the tests. Brain cholinesterase activities were reduced significantly at 1500 mg/kg bw on days 1 and 15 and at 150 mg/kg bw on day 1. Reductions in AChE activity in some brain regions were seen at 15 mg/kg bw in males but not females on day 1. These were 10% or less and are not considered to be adverse. In females on day 15, AChE inhibition in the mid-brain at 15 mg/kg bw is not considered adverse as it is not part of a dose-response, did not achieve statistical significance, was not evident at day 1 and is <10% when an outlying control value (30% greater than the next highest activity) is excluded from the analysis. Erythrocyte cholinesterase activities were reduced in a dose‑related manner on day 1 only. Though a statistically significant reduction in erythrocyte cholinesterase activity is seen in females at 15 mg/kg bw, this finding is not considered adverse as the degree of inhibition is close to the cut-off of 20%, this group had a slightly lower pre-test activity and the control range (1880 - 2332 iU/L - median 2130 iU/L) shows considerable overlap with that for treated animals (1565 - 2315 iU/L - median 1995 iU/L), with only one treated animal outside the control range. The weights of individual brain regions and histopathological findings were similar in all groups.

Viewed as a whole, the findings at 15 mg/kg bw indicate that this dose can be taken as the NOAEL. There was no statistically significant inhibition of either brain or erythrocyte acetylcholinesterase in males at this dose level. In females receiving 15 mg/kg bw a mean inhibition of mid-brain acetylcholinesterase activity by 10% and a mean inhibition of erythrocyte acetylcholinesterase activity by 26% equate closely with the cut-off used as a marker for adversity. When the ranges for controls and 15 mg/kg bw females are compared, there is considerable overlap, with results for an individual animal having a disproportionate influence on the group mean.

As there was a degree of uncertainty around the NOAEL, a benchmark-dose estimation was performed using PROAST version 63.10 with the day 1 (peak response) cholinesterase inhibition data, with a response level of 20 %. There was no dose response for the pre-test data, as would be expected. The erythrocyte cholinesterase response data resulted in a lowest BMDL20 of 23.5 mg/kg bw/day for females and 15.2 mg/kg bw/day for males. For the brain, cholinesterase activity in the cerebellum region was used as it is a significant portion of the brain in terms of mass and it appeared to be the most sensitive region. The data resulted in a lowest BMDL20 of 60.8 mg/kg bw/day for females and 40.9 mg/kg bw/day for males. The BMDU/BMDL ratio for cholinesterase activity in the cerebellum region of the brain is 2.2 for females and 2.1 for males and for erythrocyte cholinesterase activity the ratio is 5.4 for females and 5.8 for males. The BMDU/BMDL ratio indicates that the quality of the data is acceptable. The values obtained based on inhibition of erythrocyte cholinesterase inhibition are consistent with the proposed NOAEL.

The NOAEL for systemic toxicity in this study is considered to be 15 mg/kg bw based on minimal inhibition of erythrocyte and brain acetylcholinesterase activity at this dose level. It is supported by the BMD modelling that gave BMDL values of 23.5 mg/kg bw/day and 15.2 mg/kg bw/day for females and males respectively. There was no evidence of any irreversible neurotoxicity at the highest dose tested (1500 mg/kg bw), and the NOAEL for acute neurotoxicity is considered to be 150 mg/kg bw.

 

Subchronic neurotoxicity study in rats, Nemec 1995b

In a sub-chronic neurotoxicity study, performed under GLP and following US EPA 82-7 (1991) and stated to comply with OECD 424 (1997), groups of 10 male and 10 female Sprague Dawley rats received the test substance at concentration of 0, 3, 30, or 300 ppm in the diet for 92 days.

Animals were observed for clinical signs, body weight, food consumption and in a FOB. The FOB was performed pre-testing and during weeks 4, 8, and 13 (except locomotor activity, weeks 3, 7 and 12). The FOB included observations in the home cage, when handled, in ‘open field’, of sensory responses, neuromuscular performance, locomotor activity and body temperature. Samples for plasma and erythrocyte cholinesterase determinations (Ellman) were obtained from 5 animals/sex/group pre-dosing, and on weeks 3, 7 and 13; brain cholinesterase assays (Ellman) were performed on 6 brain regions from the same animals at terminal sacrifice. Histopathological examinations of an extensive range of peripheral and central nervous system tissues were performed on H&E stained sections of control and top dose animals (5/sex/group), following in situ perfusion.

Homogeneity, stability and achieved concentrations were confirmed as acceptable by chemical analysis, with mean intakes of 0, 0.2, 2.1-2.4 and 21.1-24.7 mg/kg bw/day. There were no deaths during the study. Clinical signs, body weight gains, food consumption and FOB findings were similar in all groups. Apparent increases in grooming and body temperature were seen at 300 ppm when compared to control means, but are not considered adverse as they were not statistically significant and were representative of pre-dosing results. Erythrocyte cholinesterase was decreased significantly at 300 ppm; at 30 ppm the degree of inhibition was <16% and never achieved statistical significance.

Brain cholinesterase activities were reduced significantly at 300 ppm. A marginal effect in some brain regions (<15%) seen in 30 ppm females is not considered adverse as it was not statistically significant and values overlapped control ranges.

The weights of individual brain regions were similar in all groups. The biological significance of sciatic nerve lesions, digestion chambers seen in 2/5 top dose males (versus 0 in controls) and axonal swelling in 1/5 top dose males (versus 0 in controls), is equivocal. These findings are seen routinely in female rats (range 0 - 40%) and in other studies in males (range 0 - 20%). A review of the slides confirmed that the lesions in the test substance treated animals were minimal and consistent with spontaneous findings. Digestion chambers are considered to be produced by Schwann cells repairing myelin and are not indicative of cell death. Neurotoxicants usually produce peripheral neuropathy in distal nerves, but in this study no effects were found in the tibial nerves. The sciatic nerve lesions in top dose males are therefore not considered to represent compound related neuropathy.

The NOAEL for this study is 30 ppm (equal to 2.1-2.4 mg/kg bw/day) based on inhibition of erythrocyte and brain acetylcholinesterase at 300 ppm (21.1-24.7 mg/kg bw/day). There was no evidence of any neuropathological changes. The NOAEL for evidence of neurotoxicity (i.e. effects on the nervous system other than cholinesterase inhibition) was 300 ppm (the highest dose tested).

 

Acute delayed neurotoxicity study in hens, Hakin 1989

In an acute delayed neurotoxicity study, performed under GLP and following OECD (1984), groups of 10 domestic hens were given single oral doses of corn oil, 500 mg/kg bw tri-ortho-cresyl phosphate (TOCP) or 100 mg/kg bw test substance. Groups treated with the test substance were protected with 2-PAM at 50 mg/kg and atropine sulphate at 10 mg/kg. Dose levels were confirmed to be acceptable by chemical analysis. The corn oil dosed control birds remained in good health following dosing. Dosing was followed by a 21 day observation period, including forced activity assessments, when all TOCP treated birds were killed. All negative control birds were re-dosed with corn oil, all test substance treated birds were re‑dosed with 100 mg/kg bw (and protective agents) at the end of Day 21 followed by a 21 day observation period. TOCP treated birds showed no adverse effects immediately following dosing or re-dosing, but all birds subsequently developed clinical signs of neurotoxicity. Almost half the birds treated with the test substance died before the end of the study, but adequate animals survived to permit neurological examination of 10 animals/treatment. Transient clinical signs of toxicity seen up to 10 days post-dosing in surviving birds treated with the test substance included subdued appearance, unsteadiness, leg paralysis and inability to stand; all these birds recovered. There were no clinical signs of delayed neurotoxicity recorded in any of the corn oil dosed control or test substance treated birds following the first dose or the second dose. Birds dosed with the test substance showed very marked fluctuations in body weight gain during the treatment period. Two birds dosed with TOCP were found to have atrophied skeletal muscle of the limbs. One bird dosed with the test substance was found to have multiple dark subscapular areas on the liver. No abnormalities were detected in any other birds examined at termination.

Histopathological examinations, including special staining techniques, were performed on three areas of the brain, three levels of spinal cord, proximal and distal sections of the sciatic nerve and the distal end of the tibial nerve. The examinations revealed a few degenerate axons (maximum grade 2) in the spinal cord and/or peripheral nerve in the majority of birds in the corn oil group and the test substance treated group. In the TOCP treated group there was significant axonal degeneration in the spinal cord in a number of birds and in the peripheral nerve in all birds (maximum grade 4).

Neuropathy target esterase (NTE) and acetylcholinesterase (AChE) activities were investigated in some of the hens (2 corn oil, 1 TOCP, 3 test substance per time point). At 24 and 48 hours after dosing the AChE activity in brain and spinal cord of birds treated with the test substance was inhibited by >75%, whereas that of TOCP-dosed birds was inhibited by <20% compared to the controls. NTE activity in the brain and spinal cord of test substance treated birds at 24 and 48 hours was similar to that of the controls (102% +7%); that of TOCP treated birds was inhibited >80% at both 24 and 48 hours.

The test substance l did not induce acute delayed neurotoxicity in the domestic hen under the conditions of this study.

 

Sub-chronic delayed neurotoxicity study in hens, Roberts 1983

In a sub-chronic delayed neurotoxicity study, performed under GLP and following OECD (1984), groups of 10 adult hens (age 14 months) received 0, 0.5, 1.0, 2.5, 5.0 or 10 mg/kg bw/day of the test substance by gavage in corn oil for 90 days. Ten birds receiving 7.5 mg/kg bw/day TOCP acted as positive controls. Two further groups of 10 birds received 5.0 and 10 mg/kg bw/day test substance for 90 days followed by a recovery period of 90 days. Dose levels were confirmed analytically as being within acceptable variation. All birds were examined daily during the dosing and withdrawal period for signs of ataxia; the text implies some degree of activity / exertion but does not provide details. Histopathological examination (including special staining of axons and myelin) for delayed polyneuropathy was performed on in situ perfused preparations of brain (2 regions), optic nerve, olfactory nerve, 4 levels of spinal cord, dorsal root ganglia, proximal and distal sections of the sciatic nerve and the distal end of the tibial nerve. Nervous tissue specific staining techniques were used in addition to haematoxylin and eosin (H&E). No assays of AChE or NTE were performed.

Mortalities (16/70 of the test substance treated birds) occurred only in those groups where signs of toxicity were exhibited. Dose related signs of toxicity were noted in some birds dosed with the test substance, characterised by varying degrees and combinations of weakness, ruffled feathers, leg stiffness, exaggerated leg movements, unsteadiness or wing drooping. The effects were more frequent and more severe at the high dose levels and did not occur in animals receiving less than 1.0 mg/kg bw. Seven birds in the positive (TOCP) control group showed clinical signs of ataxia. No clinical signs of ataxia were reported for any of the other treatment groups. During the dosing period, treatment related body weight decreases were noted in some test substance treated groups. Gross and histopathological examination of the treated birds revealed no treatment-related changes in the nervous system. One hen treated at 10 mg/kg bw/day had grade 3 lesions of the caudal cervical spinal cord which were above the concurrent control range of grade 2. As no other animals in this group showed similar findings and historic control data are reported to include sporadic incidences of grade 3 lesions, it is considered that the finding in this bird is not indicative of delayed polyneuropathy. Appropriate results were obtained with spinal cord sections from the positive control animals (maximum grade 4) but not peripheral nerves (maximum grade 2).

The study is slightly compromised. The birds used were slightly older than recommended by testing guidelines (8 - 12 months), the positive control results were not conclusive for peripheral nerves and one top dose hen had atrophy in one level of the spinal cord that was in excess of concurrent control findings. However, considering the range of dose levels used, the degree of toxicity produced, and the extent of histopathological investigations, it is concluded that the test substance did not cause delayed neurotoxicity in the domestic hen in this study.

Justification for classification or non-classification

The test substance has been studied for delayed neurotoxicity in hens following single (100 mg/kg bw) and repeat dosing (up to 10 mg/kg bw/day) for 90 days. In neither of the studies was there evidence of pathological lesions typical of delayed neuropathy. Assays for NTE activity were performed in the acute study and found no significant inhibition in treated animals.

Studies of neurotoxicity, including functional observation batteries have been performed in rats following single (15, 150 or 1500 mg/kg bw/day) or 92 days administration (0.2, 2.1-2.4 or 21.1‑24.7 mg/kg bw/day). In neither study was there evidence of functional changes in the absence of significant inhibition of brain acetylcholinesterase activity. In the repeat dose study, axonal swelling and digestion chambers were increased in sciatic nerve sections of some top dose males; these findings are considered to be spontaneous as they were typical of relatively common spontaneous lesions and there were no effects on distal nerves. The NOAELs for these studies are 15 mg/kg bw for a single dose and 2.1 mg/kg bw/day over 90 days.

Overall, there is no evidence that the test substance produces irreversible neuropathology. No changes in behaviour or performance were seen in the absence of significant inhibition of acetylcholinesterase activity.