Pesticide residues in food - 2002 - Joint FAO/WHO Meeting on Pesticide Residues

First draft prepared by
Dr Whang Phang
Health Effects Division, Office of Pesticide Programs,
US Environmental Protection Agency, Washington DC, USA

Questions have been raised about the adequacy of the usual toxicological databases for assessing the safety of pesticides to developing fetuses, infants and children. In recent years, studies of developmental neurotoxicity have been performed with several neurotoxic chemicals. In contrast to other studies of toxicity, those for developmental toxicity comprise a comprehensive examination of neuropathological and neurobehavioural parameters (e.g. with a battery of functional observational tests and tests for motor activity, learning, memory and sensory function) in young animals. The 1999 JMPR agreed that it would be useful to compare the critical NOAELs identified in developmental neurotoxicity studies with those identified from conventional data packages. The results of studies on developmental neurotoxicity were therefore reviewed by the present Meeting. The analysis relied heavily on a document published by the Environmental Protection Agency in the USA (Makris et al., 1998). The objective of the evaluation was to examine the use of such studies in the establishment of acute reference doses (RfDs) and ADIs. Fourteen studies of developmental neurotoxicity were reviewed, and the results of each were compared with those of related studies of prenatal effects on development, reproduction and fertility and of acute and 90-day studies of neurotoxicity in adult rats.

In studies of developmental neurotoxicity based on protocols such as the standard guideline of the Environmental Protection Agency (1998), pregnant rats are given the chemical orally, by gavage or in the diet, from day 6 of gestation through postnatal day 10 (inclusive). These testing days are defined in relation to the day of mating and the day of parturition, designated as gestation and postnatal (lactation) day 0, respectively. The offspring are therefore exposed to the chemical in the maternal circulation or milk, in utero and during early postnatal development for approximately 25 days.

The dams are examined grossly at least once daily before treatment, and detailed clinical observations are made (outside the home cage) on about half of the dams in each group, twice during gestation and twice during lactation. Maternal body weight and food consumption are recorded at least weekly.

The offspring are assessed for evidence of deficits in functional development. Litters are randomly standardized on postnatal day 4 to four pups of each sex per litter, and the pups are assigned for testing. The end-points evaluated between birth and 60 days of age include physical development, reflex ontogeny, motor activity, motor function, sensory function and learning and memory. Daily cage-side observations are conducted, and a group of 10 pups of each sex per group is examined outside the cage on days 4, 11, 21, 35, 45 and 60. The pups are counted and weighed individually at birth, on days 4, 11, 17 and 21 and at least once every 2 weeks thereafter. The age of vaginal opening and preputial separation are recorded. Motor activity is monitored with an automated activity recording apparatus on days 13, 17, 21 and 60 (± 2). The offspring are tested for auditory startle habituation (preferably by prepulse inhibition) and associative learning and memory around the time of weaning (postnatal day 21) and around postnatal day 60. Proper allocation of animals for testing of cognitive functions is crucial. In general, use of separate animals at the two test times is preferred, because initial learning of many tasks may confound assessment of learning at day 60. Flexibility is allowed in the choice of tests for learning and memory, although the guideline provides criteria for selection and some examples of tests that could be used.

On postnatal day 11 and at study termination, six pups of each sex per dose (selected from among the pups of each sex per litter killed on day 11 and pups killed at the end of the study) are subjected to extensive neuropathological examination, including simple morphometric analysis. The brains of the animals are removed and fixed. While immersion perfusion is adequate to preserve brain on day 11, those on day 60 are fixed in situ by transcardial perfusion with paraformaldehyde and gluteraldehyde. The brain weight is recorded both before weaning (day 11) and at the end of the study (day 60). A qualitative neuropathological examination is conducted of control groups and those at the highest dose; if a treatment-related effect is found, the groups at the two lower doses are also examined. Guidance is provided concerning the regions of the brain to be examined and the types of alterations on which to focus, particularly emphasizing structural changes indicative of developmental insult. Simple morphometric analysis of offspring killed on day 11 and at termination consists, at a minimum, of a reliable estimate of the thickness of major layers at representative locations within the neocortex, hippocampus and cerebellum.

The experimental procedures used for the 14 chemicals that were evaluated are summarized in Tables 1–4. Briefly, all studies involved 20 or more pregnant females, and all involved either dietary administration or gavage from day 6 of gestation through at least postnatal day 10. The exception was the study on N,N-diethyl-meta-toluamide, in which the test chemical was administered continuously (in the diet) to dams and to offspring up to 11 months of age. In the studies with dimethoate and malathion, the maternal animals were dosed from day 6 of gestation to postnatal day 10, and the pups were also directly dosed (by gavage) on postnatal days 11–21. In the studies with the remaining 11 chemicals, the pups were not dosed directly, but they were assumed to begin eating the dams’ food during postnatal week 3 (Hanley & Watanabe, 1985; Gerrish et al., 1998). In cases in which the parent compound or metabolites were not transferred in milk, pups would have been exposed postnatally for approximately 7 days if dosing was terminated on day 21. When the chemicals were present in milk, uninterrupted exposure would have occurred until termination of dosing. In the studies with three of the chemicals, developmental neurotoxicity was investigated in a combined protocol, as part of a two-generation study of reproductive toxicity (N,N-diethyl-meta-toluamide and ziram) or a study of prenatal developmental toxicity (N-methyl neodecanamide).

Table 1. Protocols for studies of developmental neurotoxicity: Treatment of parents

Table 2. Protocols for studies of developmental neurotoxicity: Physical examinations of offspring

PND, postnatal day

Table 3. Protocols for studies of developmental neurotoxicity: Neurobehavioural parameters examined in offspring and postnatal day of examination

FOB, battery of functional observational tests

Table 4. Protocols for studies of developmental neurotoxicity: Neuropathological examination of offspring

PND, postnatal day

The parameters examined in offspring and the days of examination are summarized in Tables 2–4. As indicated in these tables, the day and frequency of determination of parameters such as body weight, motor activity, auditory startle and learning and memory were relatively consistent for all 14 chemicals. The days on which nerve tissue was collected were also relatively consistent. Brain weight, microscopic neuropathology, morphometric analysis and observations of sexual maturation were included for essentially all the chemicals. Unfortunately, determinations of neurochemical biomarkers in offspring were not included in the generic protocol and were not performed with many of the chemicals that are known to inhibit cholinesterase activity in the brain, erythrocytes or plasma of offspring. In the studies with chemicals such as chlorpyrifos, malathion and dimethoate, inhibition of cholinesterase activity was examined in a companion study in which brain, erythrocyte and plasma cholinesterase activities were measured in both adult and young rats.

As indicated in the tables summarizing the experimental procedures, the parameters for evaluating maternal toxicity were more limited than those measured in the offspring. Maternal evaluation consisted of clinical observations, body weight, food consumption and a cursory gross pathological examination, and more detailed parameters, such as a battery of functional observational tests and determination of cholinesterase activity or other biomarkers, were seldom included. The available studies of developmental neurotoxicity were conducted according to the guidelines of the Environmental Protection Agency, which require only limited evaluation of maternal animals, and most of the studies were inadequate. The results were therefore difficult to interpret with respect to differential effects of treatment on dams and their offspring.

The results of the 14 studies of developmental neurotoxicity are summarized below and in Tables 5 and 6. The maternal effects of most of the chemicals consisted of decreased body weight and food consumption and certain clinical observations indicative of neurotoxicity, i.e. inhibition of cholinesterase activity and associated clinical signs of toxicity. The effects seen in the maternal animals indicated that the dose was adequate.

Table 5. Results of studies of developmental neurotoxicity: Maternal toxicity

Bw, body weight; PND, postnatal day; GD, gestation day

Table 6. Results of studiesof developmental neurotoxicity: Toxicity in offspring

Results are for both sexes unless otherwise specified

FOB, functional observational battery; wt, weight; PND, postnatal day; bw, body weight

As summarized in Table 6, the 14 pesticides induced changes in nervous system development at at least one of the doses tested. For example, 10/14 pesticides (aldicarb, N,N-diethyl-meta-toluamide, dimethoate, emamectin, fipronil, lindane, malathion, N-methyl neodecanamide, triallate and ziram) caused changes in motor activity, and virtually all caused some effects on physical developmental or on behavioural or functional observations, such as decreased pup body weight, decreased grip strength of hind- or fore-limbs or delayed developmental landmarks. Eight of the chemicals (aldicarb, chlorpyrifos, emamectin fipronil, lindane, malathion, molinate and ziram) changed sensory function or auditory startle habituation. Learning and memory parameters were affected by four of the chemicals (carbofuran, fipronil, molinate and triallate). Morphometric measurements were conducted in studies with 13 pesticides, of which four (carbaryl, chlorpyrifos, malathion and molinate) were associated with morphometric changes that were judged to be related to treatment. A decrease in absolute brain weight was seen with six pesticides (carbofuran, chlorpyrifos, fipronil, emamectin, lindane and molinate). Most of the changes in brain weight were seen on day 11 or 12, although some were also seen on day 60 (with emamectin, fipronil, lindane and molinate). Effects on offspring were often seen in the presence of maternal toxicity.

Groups of 30 presumed pregnant Sprague-Dawley rats received aldicarb by gavage at a dose of 0, 0.05, 0.1 or 0.3 mg/kg bw per day on day 6 of gestation through lactation day 10. The offspring were not given the test material. For F₀ animals (dams), clinical observations, the results of a functional observational battery of tests (FOB), body weights and data on reproduction were recorded. For the offspring (F₁), body weights and emergence of vaginal perforation or balano-preputial separation were recorded; they were observed until approximately day 65 and given neurobehavioural tests (FOB, motor activity, learning and memory test, auditory startle response). Plasma, erythrocyte and brain cholinesterase activities were measured in both dams and pups. On days 11 and 65, 10 pups of each sex from the control group and that at the highest dose were killed for neuropathological assessment.

Maternal toxicity: During treatment, tremors were seen consistently in dams at 0.3 mg/kg bw per day. During FOB measurement on day 6 of gestation, these animals presented clinical signs associated with cholinesterase inhibition: tremor (10/10), lachrymation (7/10), salivation (6/10), hunched posture (9/10), ataxia (9/10), lip smacking(4/10), reduced body temperature and miosis (10/10). The mean number of rears was also reduced significantly (0 < 0.05). Tremors were also seen on days 0–2 of lactation. No clinical signs or changes in FOB parameters were seen at lower doses.

At 0.3 mg/kg bw per day, the mean maternal body weights were also decreased on days 9–20 of gestation. Overall body-weight gain during gestation was reduced (by 17%) relative to that of controls. The body weights of these dams recovered after cessation of treatment. No effects on body weights were seen at lower doses.

Mean maternal plasma and erythrocyte cholinesterase activities were significantly reduced at the highest dose (by 80% and 27%, respectively) on day 7 of gestation and lactation day 7. In dams at 0.1 mg/kg bw per day, a statistically non-significant decrease in cholinesterase activity (by 40%) was seen on day 7 of gestation.

The maternal LOEL was 0.1 mg/kg bw per day on the basis of inhibition of plasma cholinesterase activity (by 40%). At the next highest dose (0.3 mg/kg bw per day), clinical signs associated with cholinesterase inhibition were seen which included tremor, salivation, lachrymation and ataxia. The NOAEL was 0.05 mg/kg bw per day.

Offspring toxicity: A few treatment-related effects on FOB parameters and on motor activity were seen in pups of dams at 0.1 and 0.3 mg/kg bw per day during lactation and after weaning. Decreased motor activity (by 30%) was seen on day 17 in male pups of dams at these doses. On days 35 and 63, male pups of dams at 0.3 mg/kg bw per day had significantly fewer rears in the open field. The number of rears was also reduced in female pups at this dose on day 35. At this time, decreased hind-limb grip strength and splay were seen in female pups of dams at 0.1 and 0.3 mg/kg bw per day. On day 63, increased latency to first step and reduced fore-limb grip strength were seen in males of dams at 0.3 mg/kg bw per day. While many of these measures were seen only on specific days, a number of significant differences were noted in male pups of dams at the highest dose at all times.

Pup body weights adjusted for litter size were significantly decreased at 0.3 mg/kg bw per day on days 1, 4, 7, 11 and 17. Male and female pups of dams at 0.1 mg/kg bw per day also had reduced body weights. The unadjusted body weights of those at 0.1 and 0.3 mg/kg bw per day were consistently decreased throughout the post-weaning period. Significant decreases in pup weight were seen at weeks 1, 2 and 4 for males of dams at 0.1 and 0.3 mg/kg bw per day (by 6–8% and 10–15%, respectively) and in females of dams at 0.3 mg/kg bw per day (by13% to –17%).

No inhibition of plasma, blood or brain cholinesterase activity was seen in offspring, although in some cases fewer than five pups were tested.

The LOAEL for toxicity in offspring was 0.1 mg/kg bw per day on the basis of reduced body weight from birth through post-weaning and reduced motor activity on day 17 in male pups. The NOAEL was 0.05 mg/kg bw per day (Weiler, 1995, 2000).

Groups of 26 pregnant Sprague-Dawley rats were given carbaryl (99.1% active ingredient) by gavage from day 6 of gestation through lactation day 10 at a doses of 0, 0.1, 1 or 10 mg/kg bw per day. An additional six pregnant animals per group dosed at the same levels were used to measure cholinesterase activity, which was determined before dosing (day 6 of gestation) and after dosing at the time of peak effect (1 h after dosing) on days 6, 15 and 20 of gestation and lactation days 4 and 10. FOB measurements were made about 0.5 and 2 h after dosing on the same days as body weight was measured, during dosing (days 0, 6, 9, 12, 15, 18 and 20 of gestation and lactation days 4, 7, 11, 13 and 21). Measures of reproductive performance were evaluated. The offspring were examined for body weight, physical development (tooth eruption and eye opening), FOB assessments (days 4, 7, 11, 13, 17 and 21) and motor activity (days 13, 17 and 21). On lactation day 11, one animal of each sex per litter was killed and its brain was weighed; of these, six of each sex were randomly selected for neuropathological evaluation. The eyes from all treated animals were examined. After lactation day 21, three animals of each sex per litter were separated from the dams and constituted the F₁ adult generation. These animals were evaluated for body weight, physical development (vaginal opening and preputial separation), motor activity (day 60), startle habituation response (days 22 and 60), passive avoidance (day 23) and water maze behaviour (day 60). After completion of the behavioural test (at approximately 10 weeks of age), 12 animals of each sex per group were anaesthetized and perfused for post-mortem examination. Tissues from six animals of each sex in the control group and that at the highest dose were processed for neuropathological evaluation and morphometric measurements; and the eyes from all perfused animals at the two lower doses were examined.

Maternal toxicity: There were no treatment-associated deaths, and no treatment-related clinical signs of toxicity were observed. A statistically significant decrease (by 92%) in body-weight gain was seen in dams at 10 mg/kg bw per day during days 6–9 of gestation. Unfortunately, food consumption was not measured during the study. In the FOB tests, the incidence of dams at 10 mg/kg bw per day with decreased pupil size (pinpoint pupils) was increased on all occasions during dosing. An increased incidence of dams with slight tremors affecting the head, body or limbs was noted at most assessments during dosing. Occasional occurrences of ataxic gait and overall gait incapacity were noted, which were considered to be of toxicological significance.

In dams at 10 mg/kg bw per day, erythrocyte and whole blood cholinesterase activities were statistically significantly decreased (by 28% and 32–34%, respectively) on day 20 of gestation and lactation day 10. Although plasma cholinesterase activity was not statistically significantly altered, the percentage decreases on day 20 of gestation and lactation days 4 and 10 were 32–39%. Brain cholinesterase activity was statistically significantly decreased (by 42%). No treatment-related effects were found at gross necropsy of the F₀ generation.

No effects were observed on maternal performance, including pregnancy rate, gestation index, length of gestation, numbers of live, dead or malformed pups, implantation scars, sex ratio or post-implantation loss. A slight, nonsignificant increase in the number of dead pups was found at 10 mg/kg bw per day, but the value was within the range for other controls of this strain.

The LOAEL for maternal toxicity was 10 mg/kg bw per day on the basis of decreased body-weight gain, alterations in FOB measurements and inhibition of erythrocyte, plasma, whole blood and brain cholinesterase activity. The NOAEL was 1 mg/kg bw per day.

Offspring toxicity: The F₁ generation pups showed no treatment-related effects on weight, survival indices, developmental landmarks (tooth eruption and eye opening), FOB measurements or motor activity. At sacrifice on lactation day 11, no treatment-related effects were found on brain weight or gross or microscopic appearance. Significant differences in morphometric measurements included an increase in line B of the right forebrain and line F of the left cerebellum in male pups of dams at 10 mg/kg bw per day. In the female pups at this dose, line F was significantly decreased through both the right and left cerebellum (by 15% and 22%, respectively).

F₁ generation adults showed no treatment-related effects on clinical condition, body weight, physical development (vaginal opening and preputial separation), motor activity, auditory startle response, passive avoidance or water maze measurements. At sacrifice, no gross or microscopic neuropathological lesions attributable to treatment were observed in the animals that were examined. An increased incidence of retinal fold or rosette was found in pups of dams at 10 mg/kg bw per day (1/12 controls, 4/12 males; 0/12 controls, 2/12 females). The finding was considered not to be of toxicological significance as the incidence was within the range of other control males, occurred at a low rate and was not dose-dependent. The morphometric measurements showed a significant bilateral decrease in line A through the forebrain (7.7–9.8%) and a significant increase in line F through the right cerebellum of males at 10 mg/kg bw per day. Females at this dose showed significant bilateral increases in line F through the cerebellum (7.4–15%). The size and thickness of lobes and of the granule-cell layers of the cerebellum in pups and adults at the highest dose did not differ from those of controls.

The LOAEL for toxicity to offspring was 10 mg/kg bw per day on the basis of a bilateral decrease in the size of the forebrain (line A) in F₁ adult males (7.7–9.8%), a bilateral decrease in the length of the cerebella (line F) in female pups (15–22%) and a bilateral increase in the length of the cerebella (line F) in F₁ female adults (7.4–15%). The NOAEL was 1 mg/kg bw per day. Morphometric assessment could not be conducted on animals at the two lower doses owing to inadequate tissue storage; however, the minimal findings at the LOAEL indicate that effects would be unlikely to occur at 1 mg/kg bw per day (Robinson & Broxup, 1997, 2001a,b).

Groups of 24 impregnated Sprague-Dawley rats were fed diets containing technical-grade carbofuran at a concentration of 0, 20, 75 or 300 ppm, equal to 1.7, 5.0–6.9 and 8.6–31 mg/kg bw per day, from day 6 of gestation and continuing through lactation day 10. Clinical signs and deaths were recorded daily, and body weight and food consumption recorded weekly throughout gestation and lactation. Litters were observed daily for dead pups and were culled to eight pups on postnatal day 4. Pinna detachment, incisor eruption, eye opening, vaginal patency and preputial separation were evaluated in all surviving pups. Motor activity, auditory startle response and swimming, learning and memory ability were measured in one pup of each sex per litter. Other selected pups were killed on postnatal days 11 and 60, and body and brain weights were measured. Brain, spinal cord, sciatic nerve and skeletal muscle from six pups of each sex per group, which were killed on days 11 and 60, were also prepared for microscopic examination.

Maternal toxicity: Dams at 75 ppm had decreased body-weight gain and decreased food consumption on days 6–10 of gestation, and they also lost weight. The body weight of the group at the highest dietary concentration was significantly lower than that of controls on days 10–20 of gestation. The maternal LOAEL was 75 ppm, equal to 5 mg/kg bw per day, and the NOAEL was 20 ppm, equal to 1.7 mg/kg bw per day.

Offspring toxicity: At dietary concentrations >75 ppm, there was increased pup mortality during the first 4 days of lactation, which resulted in highly significantly decreased viability indices in comparison to controls (p < 0.01). Live birth indices were comparable between control and treated groups; although the groups at the two higher dietary concentrations had lower weaning indices than controls, the differences were not statistically significant. Pup body weight was also significantly decreased at birth, by 7–16%, and during the entire lactation period, by 25–38% in the groups at 75 and 300 ppm (p < 0.01). Delayed vaginal patency and preputial separation were seen at 75 and 300 ppm.

Learning and memory development were affected at 75 and 300 ppm, as indicated by delayed development in swimming ability and in Y maze performance; however, no treatment-related effects were seen at gross necropsy in pups examined on day 11 or 60 or subsequently. There were no compound-related effects on absolute or relative brain weight or on the histological appearance of the brain, spinal cord or peripheral nerves of pups killed on days 11 and 60.

The LOAEL for offspring toxicity was 75 ppm, equal to 5 mg/kg bw per day, on the basis of increased pup mortality, decreased body weight, delayed vaginal patency and preputial separation and effects on swimming ability and Y maze performance. The NOAEL was 20 ppm, equal to 1.7 mg/kg bw per day (Ponnock, 1994).

Groups of 25 pregnant Sprague-Dawley rats were given chlorpyrifos (99.8% active ingredient) by gavage from day 6 of gestation through lactation day 11 at a dose of 0, 0.3, 1 or 5 mg/kg bw per day. An additional five pregnant animals per dose were used for evaluation of cholinesterase activity. Dams were examined for body weight, reproductive performance, number of viable pups and post-partum behaviour. During dosing, the dams were observed daily for signs of toxicity to autonomic function. Groups of dams were killed 4–5 h after dosing on day 20 of gestation for analysis of cholinesterase activity in brain, plasma and erythrocytes. Offspring were examined for viability at birth, pup and litter survival, body weight, sex ratio, physical development landmarks (eye opening and pinna detachment), behaviour during nursing and sexual maturation. F₁ generation litters were randomly standardized on lactation day 5 and assigned to one of four subsets for continued observation. The first subset was used on postnatal day 12 to measure the fixed brain weight (10 pups of each sex per dose) and neuropathological evaluations, including morphometrics (six pups of each sex per dose); the remaining 10 pups of each sex per dose were necropsied and examined for gross lesions. In the second subset, eight pups of each sex per dose were selected for evaluation of learning and memory on days 23–25 and 62–92 and were killed on days 97–101, after the last evaluation; the remaining pups were necropsied and examined for gross lesions on day 22. Pups in the third subset were tested for motor activity on days 14, 18, 22 and 61 and for auditory startle habituation on days 23 and 62; all animals were killed on days 63 or 64 after the last evaluation. In the fourth subset, fixed brain weights were determined in 10 pups of each sex per dose, neuropathological examinations were performed on six pups of each sex per dose, and all remaining animals (10 of each sex per dose) were necropsied and examined for gross lesions after sacrifice on days 66–77.

Maternal toxicity: Animals at 5 mg/kg bw per day showed signs of toxicity to autonomic function, manifested at the end of gestation as fasciculations (6/25 treated, 0/25 controls) and during lactation days 1–5 as fasciculations (16/24 treated, 0/25 controls), hyperpnoea (8/24 treated, 0/25 controls) and hyperreactivity (17/24 treated, 2/25 controls). The number of dams that lost all their pups was increased at the highest dose (3/23 treated, 0/25 controls). No significant effects were found on body weight, food consumption or gestation parameters, and there were no unscheduled deaths.

Brain cholinesterase activity was decreased in animals at the highest dose (90% of control value) and the intermediate dose (18% of control value, statistically significant). Cholinesterase activity was also decreased in erythrocytes (41–99%) and plasma (43–92%) in a dose-dependent manner in all treated groups.

A NOAEL for maternal toxicity could not be identified. The LOAEL was 0.3 mg/kg bw per day, on the basis of inhibition of plasma and erythrocyte cholinesterase activity.

Offspring toxicity: In the F₁ generation, the body weights of pups of dams at the highest dose were significantly reduced (by 8–15%) on postnatal days 1 and 5 (before and after culling). The body weights of pups of dams at the highest dose in the fourth subset were also reduced (by 5–19%) between birth and postnatal day 22, and the body-weight gains of these animals were reduced during the same period (by 5–30%). In comparison with controls, terminal body weights were reduced in the pups of dams at the highest dose in the first subset (day 12) (by 17–19%) and in males at this dose in the fourth subset (day 66) (by 10%). The body weights of F₁ generation adult males at the highest dose were decreased on days 22–66 (by 11–17%); F₁ adult females also weighed less than controls on day 22 (decreased by 17%) but were of similar weight on day 66. Body-weight gain was decreased for males at the highest dose on days 22–40 (by 13%) and days 40–66 (by 7%). Food consumption was decreased immediately after weaning (postnatal days 23–30) in males and females at the highest dose (by 13%).

Development (as assessed by pinna unfolding) was delayed by 4 days in animals at the highest dose and by 3.5 days in controls. Sexual maturation was also delayed, as assessed by time to preputial separation (106% of control value) and vaginal patency (103% of control value).

Pup viability was reduced, the number of surviving pups per litter being decreased by 27%, live litter size at culling by 16% and the pup viability index by 29%, and greater percentages of pups were found dead or presumed to have been cannibalized (7.2% treated, 0% controls on day 1; 25% treated, 1.3% controls on days 2–5).

There were no statistically significant differences between groups in average acquisition and delay training, and no differences among groups were found for the retention of information during days 23–25 and 62–92. Motor activity was decreased in male and female pups at the highest dose on day 14 (by 56% in males and 37% in females) and was increased in females at this dose on days 18 and 22 (by 51% on both days). On day 61, motor activity was increased for both sexes (by 16–17%). A statistically significant increase (by 16–25%) was observed in the latency to peak response during auditory startle habituation assessments on day 23 in animals at the highest dose relative to concurrent controls. On day 62, the latency to peak response in animals at the highest dose was 10–12% higher than in controls. The peak response amplitudes in these animals were decreased by 9–29% on days 23 and 62 (not statistically significant).

No gross or microscopic lesions of the nervous system were found in offspring in the first or fourth subset. On day 12, males at the highest dose in the first subset had reduced absolute brain weights in comparison with controls (by 9%), increased relative brain weights (by 13%), reduced anterior-to-posterior length of the cerebellum (by 24%), reduced height of the cerebellum (by 14%), decreased thickness of the parietal cortex (by 6%) and decreased thickness of the hippocampal gyrus (by 9%). Females at this dose had reduced absolute brain weights (by 9%), increased relative brain weights (by 14%), decreased thickness of the parietal cortex (by 6%), decreased width of the caudate putamen (by 10%) and decreased thickness of the hippocampal gyrus (by 12%). Morphometric analysis of F₁ animals in the fourth subset, killed on day 66, revealed significantly decreased parietal cortex size in females at the highest dose (by 5%) and intermediate dose (by 4%) as compared with controls. The statistical results for decreased thickness of the hippocampal gyrus in females at the highest dose (by 7%) were contradictory; the decrease in females at the intermediate dose (by 4%) was not statistically significant. The morphometric data for females at the lowest dose on day 66 were not evaluated. The brain weight of females at the highest dose was similar to that of controls on day 66 (decreased by 0.3%).

The findings in the offspring before weaning could not be definitively attributed to pre- or postnatal exposure, to developmental perturbation or to direct systemic or neurotoxicity. Nevertheless, the adverse findings in adult offspring (around postnatal day 66), i.e., alterations in motor activity, auditory startle response and brain structure (decreased size of the parietal cortex and hippocampal gyrus, in the absence of deficits in brain weight), represent long-term sequelae of exposure to chlorpyrifos during development.

Adverse effects on offspring were identified at 5 mg/kg bw per day; these included a significant, treatment-related decrease in the size of the parietal cortex and possible (although not significant) alterations in the hippocampal gyrus in the brains of female rats on postnatal day 66. The NOAEL was 1 mg/kg bw per day (Hoberman, 1998a,b, 199a,b, 2000).

Groups of F₂ Sprague-Dawley rats, approximately 40 weeks of age, one of each sex from 28 litters in a multigeneration study of reproductive toxicity, were selected for evaluation of developmental neurobehaviour. The parental animals and the offspring received diets containing N,N-diethyl-meta-toluamide at a concentrations of 500, 2000 or 5000 ppm, equivalent to 22, 90 and 220 mg/kg bw per day for males weighing 670 g. Neurotoxicity was evaluated over 8 weeks in FOB tests and tests for grip strength, thermal response, motor activity, M maze, startle reflex and passive avoidance. One male and one female from each of 10 randomly selected litters at each dose were selected for histopathological examination after perfusion in situ.

The mean body weights of both males and females at 2000 and 5000 ppm were decreased (by 10% and 15%, respectively), but food consumption was not reduced. This result could have been affected by the process of selection of the test animals, because in the study of reproductive toxicity the animals at 5000 ppm showed no significant reduction in body weight. Treatment did not affect any of the FOB parameters measured. Males and females at 5000 ppm showed increased horizontal activity during the first 5-min interval in the 40-min session of motor activity measurement. No treatment-related effects were found on grip strength, licking latency to heat stimulus, passive avoidance, M maze trial performance or auditory reflex. Neuropathological evaluation did not show any treatment-related alterations.

The LOAEL for neurotoxicity was 5000 ppm, equivalent to 220 mg/kg bw per day, on the basis of an increase in motor activity at the beginning of the session. The NOAEL was 2000 ppm, equivalent to 90 mg/kg bw per day.

The experimental procedures used in this study did not follow current guidelines; only FOB tests, passive avoidance, M maze and auditory startle habituation were evaluated, and the test animals were perfused in situ for histopathological examination. Nonetheless, the results provide certain insights into the neurobehavioural effects of N,N-diethyl-meta-toluamide and its effects on nervous tissues of offspring exposed before parturition and during lactation and weaning (Schardein, 1990a).

Dimethoate (99.1% active ingredient) was administered to groups of 24 pregnant Crl:CD®BR rats by gavage at a dose of 0, 0.1, 0.5 or 3 mg/kg bw per day from day 6 of gestation through postnatal day 10, and to the offspring on postnatal days 11–21. FOB tests were administered to 10 dams at each dose on days 12 and 18 of gestation and lactation days 4 and 10. The offspring were evaluated in an age-appropriate FOB on days 4, 11, 21, 35, 45 and 60, for automated motor activity on days 13, 17, 22 and 60, for auditory startle response on days 23–24 and 60–61, for assessment of learning and memory (Morris water maze) on days 23–24 and 61–62 (separate groups), for brain weight on days 11, 21 and 65 and for brain histopathology and morphometrics on days 21 and 65. Physical development was assessed from body weight; sexual maturation of females was assessed from age at vaginal opening, and maturation of males was assessed from age at completion of balano-preputial separation.

Maternal toxicity: No treatment-related effects on maternal animals were found. The NOAEL was 3 mg/kg bw per day, the highest dose tested.

Offspring toxicity: Treatment had no effect on body weight, food consumption, clinical signs, auditory startle respone, learning or memory, brain weight or histopathological appearance at any time. No effect was found on litter size or pup weight at birth, but an increase in pup death occurred during early lactation. The number of pup deaths was similar in controls (15 deaths in 10 litters) and groups at the lowest dose (11 deaths in six litters) but was increased at 0.5 mg/kg bw per day (43 deaths in 10 litters, including one total litter loss) and at 3 mg/kg bw per day (89 deaths in 14 litters, including three total litter losses). Decreased activity was measured in the FOB at 3 mg/kg bw per day, and changes in automated motor activity were found, comprising decreased rearing in females at 3 mg/kg bw per day on postnatal day 17 and dose-related increases in horizontal activity in males at 0.5 mg/kg bw per day (by 65%) and 3 mg/kg bw per day (by 120%) on day 17. The LOAEL for offspring toxicity was 0.5 mg/kg bw per day on the basis of increased pup deaths and increased motor activity. The NOAEL was 0.1 mg/kg bw per day (Meyers, 2001a).

In a related study at similar doses, dimethoate significantly inhibited plasma, erythrocyte and brain cholinesterase activity in rat adults and pups (Meyers, 2001b).

Groups of 25 pregnant Sprague-Dawley rats received emamectin (purity, > 97%) by gavage in deionized water at a dose of 0, 0.1, 0.6 or 3.6/2.5 mg/kg bw per day from day 6 of gestation through lactation day 20. Any test animal that had not given birth by day 24 of gestation was killed to determine its pregnancy status. The original dose of 3.6 mg/kg bw per day was reduced to 2.5 mg/kg bw per day between day 17 and day 20 of gestation because of an increased incidence of tremors in pups in a concurrent two-generation study of reproductive toxicity in rats. On postnatal day 4, each litter was reduced to four male and four female pups. On day 11, one male and one female pup from each litter were killed and examined neurohistopathologically. During the pre-weaning period (postnatal days 13, 17 and 21), one male and one female from each litter were selected for neurobehavioural assessment, which consisted of open-field motor activity, auditory startle habituation and passive avoidance tests. After weaning (days 23 and 24), three or four animals of each sex per litter in each group were selected for behavioural assessment, consisting of similar parameters to those tested before weaning plus examination of preputial separation on days 39, 43 and 47 and the presence of vaginal canalization on days 31, 34 and 47. The behaviour of rats after weaning was assessed on day 59 or 66. On day 60, one pup per litter (at least 10 of each sex per dose) was necropsied, and body weight and brain weight were measured. Six pups of each sex per dose were perfused in situ, and the brain, spinal cord, optic and sciatic nerves and skeletal muscle were removed and processed for histological examination.

Maternal toxicity: No treatment-related effects were found on clinical signs, the number of implants per dam, per cent post-implantations per dam, per cent post-implantation survival or body weight. The NOAEL for maternal toxicity was 3.6/2.5 mg/kg bw per day, the highest dose tested.

Offspring toxicity: Decreased pup body weight was seen at the highest dose (by 14–41%) on lactation days 11, 17 and 21. Body-weight gain was decreased for males (by 16%) and females (by 17%) at the highest dose relative to the controls. Increased incidences of head and body tremors, hind-limb extension and splay and unkempt coat were also observed in pups at the highest dose during lactation and after weaning. Males and females at the highest dose also had delayed developmental landmarks, vaginal canalization in females occurring at 3.7 days and preputial separation in males at 3.6 days. Mean horizontal activity was initially increased in both sexes on postnatal day 13 and was subsequently decreased on day 17 in pups at the highest dose. A decrease on postnatal day 17 was also seen in males and females at the intermediate dose. The decreases in females at the intermediate and high doses (by 21% and 41% relative to controls, respectively) were statistically significant and dose-related. On days 22 and 59, the auditory startle reflex was decreased in male and female pups at the highest dose.

There were no treatment-related effects on brain weight or neurohistological findings. The LOAEL for offspring toxicity was 0.6 mg/kg bw per day on the basis of a dose-related decrease in motor activity in females. The NOAEL was 0.1 ng/kg bw per day (Wise, 1993).

Groups of 30 pregnant Sprague-Dawley rats received diets containing fipronil (96.1% active ingredient) at a concentration of 0, 0.5, 10 or 200 ppm, equal to 0.05, 0.9 and 15 mg/kg bw per day, from day 6 of gestation to lactation day 10. The dams were killed after weaning of the last litter. On postnatal day 4, all litters were culled to four pups of each sex per litter. On days 11 and 60, one pup of each sex from each litter was randomly selected for evaluation of effects on the nervous system. Offspring were examined for developmental landmarks, motor activity, auditory startle habituation, swimming development, learning development and memory. At the end of the study, on postnatal day 60, six surviving animals of each sex per dose were randomly selected for neuropathological evaluation.

Maternal toxicity: There was no treatment-related effect on survival or clinical signs of toxicity. Two females at 200 ppm died during lactation, but there was no evidence that the deaths were treatment-related. The mean maternal body weight at 200 ppm was reduced by 16%, 10% and 8.6% in comparison with controls on days 10, 15 and 20 of gestation, respectively. Mean body-weight gain was statistically significantly decreased on days 6–10 of gestation but increased during days 10–15. Statistically significant reductions in mean body weight were seen in dams at 200 ppm on lactation days 0 and 4. Mean body-weight gain was statistically significantly increased on lactation days 4–11. A statistically significant reduction in group mean food consumption was noted at 200 ppm on days 6–10 of gestation, but the value was comparable to that of controls at other intervals. Pregnancy rate and length of gestation were comparable in treated and control groups. There was no treatment-related effect on gross findings at necropsy. The LOAEL for maternal toxicity was 200 ppm, equal to 15 mg/kg bw per day, on the basis of decreased body weight, body-weight gain and food consumption. The NOAEL was 10 ppm, equal to 0.9 mg/kg bw per day.

Offspring toxicity: At 200 ppm, litter size was not affected by treatment, but the live birth index was decreased (not statistically significant). The viability index (survival on postnatal days 0–4) of pups of dams at 200 ppm was significantly decreased (76%; 99% for controls). The weaning index (survival on postnatal days 4–21) was decreased in this group, but the difference was not statistically significant. Pup sex distribution was not affected. Statistically significant decreases in group mean body weight were found for both male (by 9.2–34%) and female offspring (by 8.1–34%) at all intervals during lactation and during various periods after weaning. Statistically significant increases in the mean day of achieving pinna detachment, upper and lower incisor eruption, vaginal patency and preputial separation were noted. Pups at 10 ppm showed a statistically significant decrease in weight and delayed preputial separation. A statistically significant increase in mean motor activity was seen in females on day 17. No treatment-related effects were seen at 0.5 ppm.

The maximum auditory startle response was statistically significantly decreased in males and females at 200 ppm on day 22, but the responses were similar in treated and control groups by postnatal day 60. Swimming direction scores were reduced for males and females at 200 ppm on day 6, although only the scores for males were statistically significant. On day 14, the scores were comparable. The time required to complete a water Y maze was statistically significantly increased for females at 200 ppm on day 24. Statistically significant decreases in absolute brain weights were found for both sexes in 200 ppm on day 11 (by 20% and 11% in males and females, respectively) and day 60 (7% decrease in males and females). Terminal body weights were also decreased in this group on these days. The relative brain weights for both sexes were significantly increased in comparison with controls on day 11; however, on day 60, the values for controls and rats at 200 ppm were comparable. There was no evidence of a treatment-related effect on gross macroscopic or microscopic appearance (including the central and peripheral nervous systems) of pups killed on days 11 and 60. The LOAEL for offspring toxicity was 10 ppm, equal to 0.9 mg/kg bw per day, on the basis of a marginal but statistically significant decrease in group mean pup weight during lactation and a significant increase in time to preputial separation in males. The NOAEL was 0.5 ppm, equal to 0.05 mg/kg bw per day (Mandella, 1995; Bieler, 1998; Mandella & Rodwell, 1998).

Groups of 24 presumed pregnant Hsd Brl Han:Wist (Han Wistar) rats received diets containing lindane (99.78% active ingredient) at a concentration of 0, 10, 50 or 120 ppm from day 6 of gestation through lactation day 10. These concentrations resulted in doses to F₀ maternal animals of 0.8–0.9, 4.2–4.6 and 8–10 mg/kg bw per day, respectively, during gestation and 1.2–1.7, 5.6–8.3 and 14–19 mg/kg bw per day, respectively, during lactation. On postnatal day 4, the litters were culled to five pups of each sex when possible. Developmental neurotoxicity was evaluated in groups of 10 F₁ offspring of each sex in FOB, motor activity, auditory startle response and learning and memory tests and from developmental landmarks such as vaginal perforation and balano-preputial separation; brains were weighed and examined histopathologically on days 11 and 65, including morphometrics.

Maternal toxicity: Small differences in absolute body weights (7–8%) were observed between dams at 120 ppm and controls during gestation and early lactation (through day 11). The body-weight gain of these dams on days 6–20 of gestation was 64–79% (p < 0.01) that of controls. Changes in body weight during lactation were similar in treated and control groups. During gestation, the food consumption of dams at 120 ppm was significantly lower (p < 0.01; 74–92% of control values) than that of the control group during days 10–13, 14–17 and 18–19 of gestation. The food consumption of animals at the two lower dietary concentrations during gestation and that of all treated groups during lactation was similar to that of controls.

Increased reactivity to handling during weeks 2 and 3 of dosing was seen in dams at 120 ppm, and this group also had a greater number of stillborn pups, as indicated by a live birth index of 77%, with 99% for the control group.

The LOAEL for maternal toxicity was 120 ppm, equal to 8 mg/kg bw per day on the basis of decreased body-weight gain, decreased food consumption and increased reactivity to handling. The NOAEL was 50 ppm, equal to 4.2 mg/kg bw per day.

Offspring toxicity: The absolute body weights of male and female pups at 50 and 120 ppm during lactation were 12–18% and 16–20% less than those of controls, respectively, on days 4–11 of lactation, with recovery to < 10% by day 21. Body-weight gain on lactation days 1–4 and 1–11 was 76% and 84%, respectively (p < 0.05 and 0.01), of the control value for males at 50 ppm, 79% and 79%, respectively, for females at 50 ppm, 60% and 73%, respectively, for males at 120 ppm and 63% and 75%, respectively, for females at 120 ppm. The body-weight gain of all treated groups was similar to that of controls on lactation days 11–21. Except for females at 50 and 120 ppm, the body-weight gain was similar for treated and control groups after weaning. Nine litters at 120 ppm either died or were killed when moribund on lactation days 1–4, which resulted in a viability index for the group at 120 ppm of 71%, whereas that for controls was 89%. The mortality rate before day 4 in litters at 50 and 120 ppm that survived to weaning was greater than in controls: three pups in 2/20 control litters, 18 pups in 8/22 litters at 50 ppm and 14 pups in 4/15 litters at 120 ppm. The survival of pups at 10 ppm was not affected at any time. No dose- or treatment-related differences were observed between treated and control groups in the duration of gestation, number of pups per litter on day 1 or per cent male offspring.

A few clinical signs were seen in pups at 120 ppm, which included slower surface righting on day 4. No effects were found on measures of physical or sexual development. Increased motor activity was seen in both sexes at 50 and 120 ppm during lactation. Some decrease in habituation of motor activity was found in females on day 22. While there was no effect on auditory startle reflex amplitude, there was a clear reduction in auditory startle response habituation in both sexes at 120 ppm on days 28 and 60. Slight decreases in absolute, but not relative, brain weights were observed in female pups at 50 and 120 ppm on day 11 (by 9–10%), but the decrease fell to 3–5% by day 65. Brain length and width were similar in treated and control pups. Morphometric brain measurements showed no significant difference in the sizes of the neocortex, hippocampus, corpus callosum or cerebellum on day 11 or 65. There were no histopathological effects on the nervous system.

The LOAEL for offspring toxicity was 50 ppm, equal to 4.2 mg/kg bw per day, on the basis of reduced pup survival, decreased body weights and body-weight gains during lactation, increased motor activity and decreased motor activity habituation. The NOAEL was 10 ppm, equal to 0.8 mg/kg bw per day (Myers, 1999).

Groups of 24 female Crl:CD®BR rats received malathion (96% active ingredient) in corn oil by gavage at a dose of 0, 5, 50 or 150 mg/kg bw per day from day 6 of gestation through postnatal day 10, and the offspring were treated on postnatal days 11–21. FOB tests were administered to 10 dams per dose on days 12 and 18 of gestation and lactation days 4 and 10. The offspring were evaluated in age-appropriate FOB tests on days 4, 11, 21, 35, 45 and 60; for automated motor activity on days 13, 17, 22 and 60; for auditory startle response on days 23–24 and 60–61; for learning and memory (Morris water maze) at days 23–24 and 61–62 (separate groups); for brain weights on days 11, 21 and 65 and for brain histopathology and morphometrics on days 21 and 65. Pup physical development was assessed on the basis of body weight. Sexual maturation of females was assessed from age of vaginal opening, and sexual maturation of males was assessed from age at completion of balano-preputial separation.

Maternal toxicity: No treatment-related maternal deaths occurred before scheduled termination. The clinical signs were limited to transient salivation after dosing (5/24 controls, 4/24 at 5 mg/kg bw per day, 3/24 at 50 mg/kg bw per day and 20/24 at 150 mg/kg bw per day). No other treatment-related effects were observed on cholinergic signs, and there were no effects on maternal body weight, food consumption or reproductive indices. The LOAEL for maternal toxicity was 150 mg/kg bw per day on the basis of an increased incidence of salivation after dosing. The NOAEL was 50 mg/kg bw per day.

Offspring toxicity: Male and female offspring at 5 mg/kg bw per day had an increased auditory startle reflex peak amplitude on postnatal day 23–24. At 50 mg/kg bw per day, there was an increased incidence of slightly flattened gait in males on day 60, and motor activity counts (rearing and ambulatory) were decreased in female pups on postnatal days 17 and 22. At 150 mg/kg bw per day, additional treatment-related findings included increased incidences of whole-body tremors, hypoactivity, prostration, partially closed eyelids and/or abnormal gait on postnatal days 17 and 18, delayed surface righting reflex on postnatal day 11 in female pups, increased incidences of slightly flattened gait in males on postnatal day 60 and increased thickness of the corpus callosum in males and females on postnatal days 63–67. The LOAEL for offspring toxicity was 5 mg/kg bw per day, on the basis of increased auditory startle reflex peak amplitude on postnatal day 23–24. A NOAEL was not identified (Fulcher, 2002).

In a companion study of cholinesterase inhibition, acute or repeated exposure to malathion resulted in statistically and biologically significant decreases in cholinesterase activity in the blood or brain of dams, fetuses, weanling pups and adult male and female rats. In pups, effects on erythrocyte cholinesterase activity were noted at 5 mg/kg bw per day in males and 50 mg/kg bw per day in females after single doses on postnatal day 11, and at 5 mg/kg bw per day in both sexes on postnatal day 21 after 11 repeated doses. After a single dose to young adults, effects on erythrocyte cholinesterase activity were observed at 450 mg/kg bw per day, while after 11 or 14 doses, effects were observed at 50 mg/kg bw per day in young adults and pregnant dams. In pups, brain cholinesterase activity was inhibited (by 44–48%) at 150 mg/kg bw per day after an acute dose on postnatal day 11 or after 11 repeated doses (by 16%) on postnatal day 21. These results indicate that all the behavioural and neuropathological effects observed in dams and offspring in the study of developmental neurotoxicity occurred at doses at which cholinesterase was inhibited (Fulcher, 2001).

Groups of 30 female Alpk:AP_fSD rats received diets containing molinate (96.8% active ingredient) at a concentration of 0, 20, 75 or 300 ppm, equal to 0, 1.8, 6.9 and 26 mg/kg bw per day, respectively, from day 6 of gestation through lactation day 11. On postnatal day 5, the litters were culled to four pups of each sex per litter when possible. Motor activity, memory, learning, auditory startle and FOB tests were conducted on the offspring. On postnatal days 12 and 63, groups of six and eight rats of each sex per dose, respectively, were killed for neuropathological examination, including morphometric analysis.

Maternal toxicity: There were no treatment-related effects on maternal survival or clinical signs of toxicity. The mean body weight af dams at 300 ppm was slightly decreased (93–94% of control value) from day 10 of gestation and throughout lactation (89–95% of control value). The mean body-weight gain of these animals was decreased before dosing on days 1–4 of gestation (88% of control) and during gestation, on days 7–22 (76% of control) and 1–22 (80% of control). Dams at 300 ppm had a negative body-weight gain during the first 3 days of dosing and during lactation days 1–7, and the overall body-weight gain of groups at 75 and 300 ppm was decreased (70% and 73% of control values, respectively). A statistically significant reduction in group mean food consumption was seen at 300 ppm throughout gestation (73–94% of control) and lactation (75–87% of control).

Litter size and the number of pups born live were comparable in all groups, and the mean number of total pups born and the live birth index were unaffected by treatment. The dams at 75 and 300 ppm had lower percentages of litters with all pups born live than the controls. At 300 ppm, an increased number of litters contained small female pups, the mortality rate was slightly higher during postnatal days 1–5, and more pups were missing and presumed dead than in the controls. Whole litter losses occurred in the control group (two litters) and at 300 ppm (four litters) only.

No treatment-related findings were observed in the dams at necropsy (brain weights were not measured). The NOAEL for maternal toxicity was 75 ppm, equal to 6.9 mg/kg bw per day, and the LOAEL was 300 ppm, equal to 26 mg/kg bw per day, on the basis of decreased body weight, body-weight gain and food consumption.

Offspring toxicity: Pups of dams at 300 ppm had an increased mortality rate, and more pups were reported missing and presumed dead than in controls. There were more small pups of each sex at 300 ppm than in the control group. No effect was found on the sex ratio. Decreased body weight was observed for both sexes at 300 ppm (males, 73–84%; females, 72–82% of control values) on days 5–29 of lactation, and the decrease continued after weaning (days 29–63), although the magnitude of the decrease in both sexes (males, 81–88%; females, 84–91% of control values) decreased with time. Decreased body-weight gain was observed mainly during the pre-weaning period for both sexes (64–84% of control value) at 300 ppm. Delayed preputial separation and vaginal opening were found at 300 ppm when compared with the control groups.

On day 23, both sexes showed a significant decrease in startle amplitude at 300 ppm at all five intervals; the females showed a dose-related decrease in startle amplitude, which was statistically significant at all dietary concentrations at three of five intervals. Males at all dietary concentrations and females at 20 and 75 ppm had responses comparable to those of controls on day 61, but females at 300 ppm still had a decreased startle amplitude on day 61. The time to maximum amplitude was increased on day 23 only in males at 300 ppm and only during the second interval. On day 61, females at 300 ppm had an increased time to maximum amplitude during four of the five intervals.

Motor activity was comparable among the female groups, but an effect on this parameter could not be ruled out for males at 300 ppm because of an initial (day 14) decrease and subsequent sustained (days 22 and 60) increases.

Straight-channel swimming time was increased over the control values in both sexes on day 21 but was comparable in all groups at other times. Both sexes at 300 ppm had a lower percentage of successful trials in the Y-shaped water maze, in both initial learning (day 21) and memory (day 24), than controls throughout the test. In the subsequent learning (day 59) and memory (day 62) phases, comparable successes were observed among groups.

A treatment-related decrease in absolute brain weight was found in both sexes at 300 ppm at sacrifice on days 12 and 63. Brain length was decreased in both sexes at 300 ppm on day 12, and the females in this group also had decreased brain width. On day 63, slight decreases in both length and width were observed in both sexes at 300 ppm, but statistical significance was not attained.

There were no treatment-related findings at necropsy on day 12 or day 63, no microscopic abnormalities in the brains of any pup on day 12 and no changes in the central or peripheral nervous system on day 63 that could be attributed to treatment. Morphometric measurements revealed treatment-related changes in the cortex or cerebellum (decreased structural size and decreased thickness of cellular layers) of pups at75 and 300 ppm on day 12 and treatment-related changes in the cortex, hippocampus or cerebellum at 300 ppm on day 63.

A NOAEL for offspring toxicity could not be identified, as a reduction in startle amplitude in the auditory startle test was found in females (day 23) at all dietary concentrations. The LOAEL for offspring toxicity was 20 ppm, equal to 1.8 mg/kg bw per day (lowest dose tested). More effects were found as the dietary concentration increased. At 75 ppm (6.9 mg/kg bw per day), in addition to the reduction in startle amplitude in the auditory test, treatment-related reductions were found in some morphometric measures in areas of the cerebellum of the brain (day 12) in both sexes. At 300 ppm (26 mg/kg bw per day), the following effects were observed: increased mortality; decreased body weight; delayed appearance of developmental landmarks (preputial separation and vaginal opening); increased swimming time in the straight-channel test at day 21 and reduced performance in learning and memory tests on days 21 and 24, respectively; reduced startle amplitude; longer time to maximum amplitude (day 23 or 61); a possible, slight increase in mean motor activity in males; reduced brain weight (both sexes on days 12 and 63), brain length (both sexes on day 12) and brain width (females on day 12) and reductions in several morphometric measures in areas of the cortex, hippocampus and cerebellum (Horner, 1996).

In a combined study of developmental toxicity (embryo and fetal toxicity and teratogenic potential) and developmental neurotoxicity, N-methyl neodecanamide (95.9% active ingredient) was administered by gavage to groups of 25 female Crl:CD BR VAF/Plus (Sprague-Dawley) rats at a dose of 0, 40, 125 or 400 mg/kg bw per day on days 6–15 of presumed gestation for evaluation of developmental toxicity or on day 6 of presumed gestation through day 11 of lactation for evaluation of developmental neurotoxicity.

Maternal toxicity: Dams at 400 mg/kg bw per day showed treatment-related effects on the mortality rate, an increase incidence (p < 0.01) of clinical signs of toxicity (excess salivation, rales, ataxia, urine-stained fur and decreased motor activity), reduced mean body weights on days 9–16 of gestation (by 3.1–5%; p < 0.01 or 0.05), a 21% decrease (p < 0.01) in body-weight gain on days 6–16 of gestation (body-weight gain increased during lactation days 0–22) and decreased feed consumption during days 6–20 (14%, p < 0.01) and 0–20 (8.2–8.8%, p < 0.05) of gestation and lactation days 8–12 (8.2%, p < 0.01).

At 125 mg/kg bw per day, maternal toxicity was characterized by treatment-related clinical signs (excessive salivation and rales) and slight (1.3%) but statistically significant (p < 0.01) reductions in feed consumption during the common treatment interval (days 6–9 of gestation).

The LOAEL for maternal toxicity was 125 mg/kg bw per day, on the basis of clinical signs of toxicity (including salivation, rales, ataxia, urine-stained fur and decreased motor activity) and reduced mean feed consumption. The NOAEL was 40 mg/kg bw per day.

Offspring toxicity: A statistically significant reduction in the mean body weight of surgically delivered male fetuses of dams at 400 mg/kg bw per day (by 4.6%) and of naturally delivered pups of the F₁ generation (by 7.3–12%) was found on days 5–22 of lactation. The brain:body weight ratio was increased (by 13%) in pups of dams at 400 mg/kg bw per day on postnatal day 12. The reductions in body weight were greater in males than in females. Although the body weights during lactation were combined, the post-weaning body weights were found to be particularly reduced in males at the highest dose. These effects persisted long after treatment of dams had been discontinued, although the feed consumption of all groups was comparable to that of controls after weaning.

A slight increase in motor activity was seen in male pups at 400 mg/kg bw per day on day 18, as demonstrated by increased movement. On day 14, the number and time of movements were also increased in females. On days 22 and 61, however, motor activity was comparable to that of controls. The increases in motor activity on days 14 and 18 were considered to represent transient neurotoxicity.

The LOAEL for offspring toxicity was 400 mg/kg bw per day, on the basis of reduced weights of both sexes on days 5–22 and of males on days 1–22 after weaning and a transient increase in motor activity. The NOAEL was 125 mg/kg bw per day (Hoberman, 1993a).

Groups of 21 pregnant Sprague-Dawley rats were given triallate (96.84% active ingredient) in corn oil by gavage at a dose of 10, 30 or 60 mg/kg bw per day from day 6 of gestation through lactation day 20. The control group consisted of 22 pregnant rats which received corn oil. On postnatal day 4, the litters were culled to four pups of each sex. Litters with fewer than eight pups were removed from the study. The pups were given FOB, motor activity, righting reflex, auditory startle habituation and passive avoidance tests. Developmental landmarks such as preputial separation and vaginal opening were also measured. One male and one female from each litter were killed on day 21 and subjected to neurohistopathological examination.

Maternal toxicity: At 60 mg/kg bw per day, the signs of maternal toxicity were limited to reduced body-weight gain on days 6–9 (by 67%, p < 0.01) and 6–12 (by 32%, p < 0.05) of gestation and reduced food consumption on days 6–12 of gestation (by 15%, p < 0.01). No treatment-related effects were seen at 10 or 30 mg/kg bw per day. The LOAEL for maternal toxicity was 60 mg/kg bw per day on the basis of reduced body-weight gain and food consumption. The NOAEL was 30 mg/kg bw per day.

Offspring toxicity: At 60 mg/kg bw per day, several parameters were affected in developing offspring. Body weight was reduced (by 6% in males and 7% in females on postnatal days 17 and 21, p < 0.05 and 0.01) and remained about 4–6% lower than the control value up to day 87. Reduced fore-limb grip strength (by 23%, p < 0.05) was observed, which was possibly associated with the reduced body weight. Increased motor activity was seen on days 17 (by 72% in males, p < 0.01, and 57% in females, p < 0.05), 21 (by 73% in males, p < 0.01 and 54% in females, not significant), 58 (by 21% in males, p < 0.05, and 21% in females, not significant) and 84 (by 7% in males, not significant, and 19% in females, p < 0.05). When the data on motor activity for days 58 and 84 were combined and the litter was used as the statistical unit, the increases were significant for both males (by 15%, p < 0.05) and females (by 21%, p < 0.01). A nonsignificant increase in the number of hyperactive females (4/26 treated and 1/27 control) was also noted and was considered to be of toxicological concern because of the concomitant differences in related observations on motor activity and passive avoidance. No treament-related neuropathological lesions or morphometric changes were found. The LOAEL for offspring toxicity was 60 mg/kg bw per day, on the basis of increased motor activity. The NOAEL was 30 mg/kg bw per day (Lemen et al., 1998).

Ziram (97.8% active ingredient) was evaluated for developmental neurotoxicity within a two-generation study of reproductive toxicity. Groups of 30 male and 30 female Sprague-Dawley CD rats received diets containing the test substance at a concentration of 0, 72, 210 or 540 ppm for two generations. These concentrations resulted in doses to F₁ maternal animals of 5, 13 and 32 mg/kg bw per day during gestation and 11, 30 and 79 mg/kg bw per day during lactation. On postnatal day 4, all litters were culled to four male and four female pups. The developmental neurotoxicity of ziram was evaluated in F₂ offspring on the basis of behavioural alterations, motor activity measures, auditory startle response, learning and memory and the age of sexual maturation (vaginal perforation and balano-preputial separation). Brains were weighed and measured, and the nervous system tissue was examined grossly and histopathologically.

No treatment-related toxicity was observed in dams or offspring at 72 or 210 ppm as compared with controls.

Maternal toxicity: All F₁ dams survived until scheduled sacrifice, and there were no treatment-related clinical signs of toxicity or neurobehavioural alterations. F₁ females at 540 ppm had significantly lower body weights throughout gestation (p < 0.05 or 0.01) and lactation (p < 0.01) as compared with controls, and their body-weight gain was significantly lower (p < 0.01) than that of controls during days 14–20 of gestation. No significant differences were found in body-weight gain during lactation in any treated group as compared with controls. On day 20 of gestation and lactation day 21, the body weights of F₁ animals at 540 ppm were 89% and 93%, respectively, of the control values. Food consumption was significantly (p < 0.05 or p < 0.01) reduced in the group at 540 ppm throughout gestation and lactation. At necropsy, no treatment-related gross or histopathological abnormalities were observed in the dams, and the differences in absolute and relative organ weights as compared with controls were consistent with the reduced body weights of these animals. The LOAEL for maternal toxicity was 540 ppm, equal to 32 mg/kg bw per day, on the basis of reduced body weight or body-weight gain and decreased food consumption during gestation and lactation. The NOAEL was 210 ppm, equal to 13 mg/kg bw per day.

Offspring toxicity: F₂ pups of dams at 540 ppm had lower body weights than controls throughout lactation, and significance was reached on postnatal days 1, 4 (before culling; 92–93%; p < 0.05), 14 and 21 (88–91%; p < 0.01). The mean body weights of F₂ males and females at 540 ppm were also statistically significantly (p < 0.05 or 0.01) lower than those of controls throughout the post-weaning period; however, the final (postnatal day 70) body weights of F₂ males and females were 93% and 96%, respectively, of the control values. The overall body-weight gain of the males at 540 ppm was 94% of the control value, while that of the females was 99% of the control value. The age at sexual maturation of F₂ pups was not affected by treatment.

No clinical signs of neurotoxicity were observed in the F₂ offspring during daily cage-side observation or at detailed physical examination. Motor activity (total or ambulatory counts) was increased at all dietary concentrations, often by two to three times that of controls and in a dose-related manner, in pups of each sex. At 72 ppm, the increase was apparent from day 17 and continued through day 21, while at 210 and 540 ppm they began on day 13 and continued through days 17 and 21. The motor activity counts on day 60 were similar in control and treated rats. The mean peak startle response was decreased (by approximately 30% from control value) in an apparently dose- and treatment-related manner in pups of each sex at 540 ppm on day 22, but this finding was not observed on day 60. The mean latency to peak response, response duration and average response values appeared to be unaffected on days 22 and 60. The results of learning and memory evaluations (in a water T-maze) on days 11 and 70 were similar for control and treated offspring. Brain weight (whole and regional), length and width were not affected on day 11 or 70. Qualitative histopathological evaluation of nervous tissues did not reveal any treatment-related findings.

The LOAEL for offspring toxicity was 72 ppm, equal to 5 mg/kg bw per day, on the basis of increased motor activity on days 17 and 21 in both sexes. The NOAEL was < 72 ppm (5 mg/kg bw per day) (Nemec, 1996).

The developmental neurotoxic potential of a chemical should be evaluated in the context of the entire toxicological database. With this in mind, the results of studies of prenatal developmental toxicity and multigeneration reproductive toxicity and acute and 90-day studies of neurotoxicity were compared with those of studies of developmental neurotoxicity, as all include assessment of effects on animals perinatally and of neurobehavioural or neuropathological effects. The observed effects and the NOAELs and LOAELs in such studies performed in rats with the 14 pesticides are presented in Tables 7–10.

Table 7. Results of studies of prenatal developmental toxicity in rats

All studies conducted by gavage; bw, body weight

Table 8. Results of multigeneration studies of reproductive toxicity in rats

bw, body weight; PND, postnatal day; wt, weight

Table 9. Result of studies of acute neurotoxicity in rats

Test substance administered by gavage unless otherwise specified

bw, body weight; HDT, highest dose tested; FOB, battery of functional observational tests

Table 10. Results of short-term studies of neurotoxicity in rats

As indicated in Tables 7 and 8, the effects commonly seen in studies of prenatal developmental toxicity and multigeneration reproductive toxicity at maternally toxic doses consisted of decreased body weight, body-weight gain and food consumption. In a few cases, when cholinesterase activity was measured, decreased plasma, erythrocyte and/or brain cholinesterase activities were observed. Similarly, the commonest effects on fetuses and offspring were decreased weights. The parameters used to assess maternal toxicity were similar to those used in studies of developmental neurotoxicity, whereas the parameters measured for effects on offspring were far less comprehensive than those used in studies of developmental neurotoxicity.

As shown in Tables 9 and 10, the commonest effects observed in studies of acute neurotoxicity were decreased hind-limb grip strength, loss of righting reflex, tremors, increased landing foot splay, ataxia, decreased activity and changes in motor activity. When inhibition of cholinesterase activity by carbamate and organophosphorus compounds was determined, decreased plasma, erythrocyte or brain cholinesterase activity was also seen at the LOAEL.

Table 11 compares the NOAELs and LOAELs in the five types of study. In most cases, the NOAELs in the studies of developmental neurotoxicity were in the same range or higher than the NOAELs in the other types of studies; the exceptions were in the studies of emamectin and fipronil. JMPR has not evaluated emamectin; the 1997 JMPR assessed the study of developmental neurotoxicity of fipronil in its overall evaluation of this compound (Annex 1, reference 82) but did not base the acute RfD or the ADI on this study.

Table 11. Comparison of NOAELs and LOAELs from studies of developmental toxicity, multigeneration studies of reproductive toxicity and acute and 90-day studies of neurotoxicity in rats

bw, body weight; wt, weight; NA, not available

Studies of developmental neurotoxicity with 14 pesticides which had been evaluated by the Environmental Protection Agency were reviewed. Both generic and chemical-specific experimental study designs were considered. The NOAELs, LOAELs and toxicity end-points of each study and of four related studies (on developmental toxicity, multigeneration reproductive toxicity and acute and short-term neurotoxicity) that had been performed with each chemical were compared.

The comparison showed that, in general, the NOAELs and LOAELs in the studies of developmental neurotoxicity were not significantly lower than those in the four related studies. The Meeting also observed that currently available data indicate that the functional and pathological effects of organophosphorus pesticides in treated animals are not seen at doses lower than those at which cholinesterase inhibition is observed.

The Meeting identified several critical issues and concerns in the conduct of a study of developmental neurotoxicity, including the introduction of artefacts due to stress resulting from direct dosing of pups and bolus (gavage) administration. The Meeting considered that if the toxicological profile of a chemical indicates developmental neurotoxicity, appropriate testing parameters could be incorporated into a multigeneration study of reproductive toxicity.

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       Toxicological Abbreviations