* The Dow Chemical Company, Midland, Michigan 48674
Primedica, Argus Research Laboratories, Inc., Horsham, Pennsylvania 19044
CVP, Murrysville, Pennsylvania 156680068
§ Dow AgroSciences, LLC, Indianapolis, Indiana 46268
Received October 30, 1998; accepted June 28, 2000
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key Words: chlorpyrifos; developmental neurotoxicity; spatial delayed alternation; motor activity; auditory startle; habituation; learning; memory; safety evaluation..
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The study described in this paper was conducted to collect supplementary information about the potential selective developmental neurotoxicity of chlorpyrifos to be used in risk assessment. This developmental neurotoxicity study expands upon the existing database for chlorpyrifos in that dosing was conducted during both gestation (GDs 621) and the first 10 days of lactation (LDs 110), and that extensive neurobehavioral and neuropathological evaluations were conducted on the offspring.
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Young adult Sprague-Dawley (Crl:CD®BR VAF/plus®) rats were obtained from Charles River Laboratories, Inc., Portage, MI, and bred in-house to provide approximately 20 pregnant dams per dosage level for the developmental neurotoxicity element of the study. An extra (satellite) group of 5 pregnant dams per dosage level was added for the determination of maternal plasma, erythrocyte, and brain ChE inhibition on GD 20 (to document exposure). GD 0 was defined as the day when spermatozoa were observed in a smear of the vaginal contents and/or a copulatory plug observed in situ; and postnatal day 0 (PND 0) was the day of birth. Bred rats were randomly assigned to the various elements of this study.
The test material (CPF) was 99.8% pure and was administered daily to dams by gavage in corn oil, from GD 6 through LD 10, at dosage levels of 0, 0.3 (100 times the chronic RfD), 1.0, and 5.0 mg/kg/day. Dosage volume was 1 ml/kg. The dose was adjusted daily for body-weight changes and given at approximately the same time each day (around 11 A.M.).
Bred females were initially housed singly in stainless steel, wire-bottom cages beginning on GD 0. Beginning no later than GD 20, bred female rats were individually housed in nesting boxes with corn cob bedding (Bed-o'-Cobs, The Andersons Industrial Products Group, Maumee, Ohio). Each dam and litter were housed in a common nesting box during the postpartum period. After weaning, the pups were individually housed in stainless steel, wire-bottom cages. An automatically controlled fluorescent light cycle was maintained at 12-h light:12-h dark, with each dark period beginning at 7 P.M. Rats were given Certified Rodent Diet #5002 (PMI Nutrition, International, St. Louis, MO) available ad libitum from individual feeders. Local water that had been processed by passage through a reverse osmosis membrane was available to the rats ad libitum from an automatic watering system or individual water bottles. Chlorine was added to the processed water as a bacteriostat.
On PND 0, all pups in a litter were weighed (pup body weights were recorded after all pups in a litter were delivered and groomed by the dam). On PND 4, pups to be culled were selected with a table of random units. The remaining pups were individually marked with a tattoo on the paw, litters were reduced to 10 pups each, and one male and/or one female pup from each litter was assigned (when possible) to subsets of rats that were grouped for specific evaluations. Whenever possible, the same number of male and female pups per litter were continued on study. Due to early pup deaths in the high-dosage group, on PND 4, 6 high-dosage litters had fewer than 10 pups. In an attempt to evaluate 20 litters/group with similar maternal and lactational demands in each dosage group, extra pups randomly culled from other high-dosage litters were added to those litters of less than 10 pups. When no other high-dosage pups were available, culled pups from other litters were used as needed to increase the litter size to 10 pups (7 pups from 0.3 mg/kg/day litters and 2 pups from control litters). When pups were taken from other litters and placed with high-dosage litters, their sole purpose was to keep all of the evaluated litters equal in size. These replacement pups were uniquely identified with a tattoo mark on the tail and were not used for any subsequent evaluation.
The time line of the main study activities is presented in Figure 1. The subsets of rats formed for specific evaluations were as follows:
|
Subset 2.
Learning and memory were evaluated by a test of Spatial Delayed Alternation, which was conducted post-weaning (PNDs 22 to 24) and in the same pups as adults (PNDs 61 to 90). Sample size was 8/sex/dosage with only one male or one female pup/litter selected from 16 randomly chosen litters/dosage.
Subset 3.
An automated test of motor activity was conducted on PNDs 13, 17, 21, and subsequently on PND 60. Body temperature was measured on PNDs 21 and 60. Auditory startle was evaluated on PNDs 22 and 61. One male and one female pup from each litter were tested (n = 20/sex/dosage).
Subset 4.
Rats (n = 20/sex/dosage) were evaluated for body weights (PNDs 0, 4, 11, 17, 21, 39, and 65), clinical signs, day of pinna detachment, eye opening, and preputial separation or vaginal opening. On PNDs 65 to 70, 10 rats/sex/dosage (one male or one female/litter) were perfused and their brains weighed. Of these, the brains of 6/sex/dosage were examined neuropathologically and morphometrically.
Evaluation of Dams
Main study.
Dams were examined before the daily treatment, inside and outside the cage, 3 to 4 h after dosing. An individual unaware of treatment group examined the rats for signs of autonomic dysfunction (lacrimation, salivation, palpebral closure, prominence of the eye, piloerection, respiration, urination, and defecation), abnormal postures, abnormal movements, or abnormal behavior patterns. The dams were evaluated for duration of gestation (GD 0 to the time the first pup was delivered), litter sizes (all pups delivered), and pup viability at birth. The dams' behavior was observed daily when the pups were examined during the 22-day postpartum observation period.
Body weights were recorded on GD 0, daily during the dosage period, and on the day sacrificed. Feed consumption values were recorded daily from GD 0 through LD 14, when it was expected that pups would begin to consume solid feed, confounding this parameter.
On LD 21, rats that delivered a litter were sacrificed. A gross necropsy of the thoracic, abdominal, and pelvic viscera was performed and the number and distribution of implantation sites were recorded. Dams with no surviving pups were sacrificed after the last pup was found dead or missing and presumed cannibalized. Rats that did not deliver a litter were sacrificed on GD 25 and examined for gross lesions, pregnancy status and uterine contents. Uteri of apparently non-pregnant rats were stained with 10% ammonium sulfide to confirm the absence of implantation sites (Salewski, 1964).
Satellite study.
On GD 20, 5 dams per dosage level from the ChE-inhibition satellite group were sacrificed via carbon dioxide inhalation 4 to 5 h after dosage, to document exposure. A more extensive study of ChE inhibition and quantitative analysis of chlorpyrifos, its oxon, and trichloropyridinol has been reported elsewhere (Mattsson et al., 2000). Blood was collected from the inferior vena cava, and the brain was removed. The maternal blood (5 ml) was collected into heparinized centrifuge tubes and centrifuged at 2500 g for 5 min, and the plasma was removed and stored undiluted at 70°C. The packed erythrocytes were washed with normal saline; the total volume was brought to 5 ml and re-centrifuged at 2500 g for 5 min. The supernatant was discarded and 500 µl of the packed cells was diluted to 5 ml with 0.1 M sodium phosphate buffer (pH 8.0 with 1% Triton X-100). Maternal brains were homogenized on ice [1 g tissue per 9 ml 0.1 M sodium phosphate buffer (pH 8.0 with 1% Triton X-100)]. The brain, plasma, and erythrocyte samples were frozen at 70°C until analyzed. Cholinesterase activity was measured using a radiometric assay (Johnson and Russell, 1975
) with a final substrate concentration of 1.2 mM. The [3H]acetate produced by the hydrolysis of [3H]acetylcholine was measured using a Wallace 1410 scintillation counter with a counting efficiency of approximately 50%.
Evaluation of Pups
All pups were examined for vital status at birth (liveborn vs. stillborn). Pups that were found dead during the initial examination of the litters were evaluated by removing and immersing the lungs in water. Pups with lungs that sank were considered stillborn; pups with lungs that floated were considered liveborn, and to have died shortly after birth. Each litter was evaluated for viability at least twice each day of the 22-day postpartum period. Dead pups were removed from the nesting box. When not precluded by autolysis or cannibalization by the dam, any pup found dead was necropsied and examined for the cause of death. The pups present in each litter were counted and physical signs (including gross external physical anomalies) were recorded once each day during the 22-day postpartum period. Pup body weight and sex were recorded on PNDs 0, 4, 11, 17, and 21 (all pups on PNDs 0 and 4 were used for body weight measurement). Pups were observed for viability at least twice daily during the postweaning periods. Body weights and clinical observations were also recorded on PNDs 39 and 65. Feed consumption was measured on PNDs 22 to 29, 39 to 46, and 58 to 65.
Learning and memory.
Spatial-delayed alternation was tested in rats in a manner similar to the method used by Stanton et al. (1994). The spatial-delayed alternation apparatus (Coulbourn Instruments, Inc., Allentown, PA) consisted of a Plexiglas® T-maze that included a start box, a runway leading from the start box to the choice-point, and left and right runways extending from the choice-point to the left and right goals (supplied with liquid dippers). Light cream (with sweet condensed milk) or water was used as a reinforcer for pups or adults, respectively. All runways were fitted with hinged tops made of clear Plexiglas®. Automated guillotine-type doors were located at the start box and the left and right sides of the choice-point. The test was divided into discrete trials. The latency and the performance of the rat for each trial were computer-recorded. Pups were tested on PNDs 2224, and the same pups were tested as adults on PNDs 6190.
The spatial-delayed alternation testing was conducted blind to treatment on feed and/or water-deprived rats. Pups were deprived of feed and water, but had access to both in their home cages for 3 h after the end of the last test session of the day, in an attempt to achieve a target of 85% free-feeding weight. At the end of each session, the pups were also eventually fed supplementary light cream to equate the amount of cream received as a reinforcement by all the groups during the test session. They were also supplemented with light cream when they fell below the target weight. Adult rats were deprived of water only, and were given access to it for half an hour, approximately one h after the last test session of the day.
The 3 phases of testing included maze acclimation, acquisition training, and delay testing. Maze acclimation took place on PND 22 and PNDs 61 to 72, and was divided into 2 parts: goal box training and forced runs (12 trials). Goal box training allowed the rat access to the reward in both the right and left arm of the maze. Forced runs allowed the rat to access only one arm of the maze, where the reward was available (either the right or the left).
Acquisition training was comprised of a forced run immediately followed by a choice run (7 blocks of 12 trials on PNDs 23 and 24, and 10 blocks of 6 trials on PNDs 73 to 82). In the forced run, the rat was given access to one arm of the T-maze that was baited. In the choice run, the doors to both arms of the maze were raised simultaneously. To have access to the reward, the rat had to choose the opposite arm from the one previously rewarded in the forced run (spatial alternation). The percent correct was calculated in blocks of 12 consecutive trials, and the learning curve was represented by the average percent correct (groups of 8 rats each) over successive blocks.
Delay testing was conducted under the same conditions as acquisition training, with time delays of various durations added between the forced run and the choice run (4 blocks of 12 trials on PND 24, and 6 blocks of 6 trials on PNDs 85 to 90). Delays alternated every 6 trials. Each animal was evaluated every test day. The percent correct retention could be plotted at each delay ("forgetting" function). The memory component was evaluated by the retention curve and was represented by the slope corresponding to the percent correct retention at the tested delays. The intercept of the slope was an extrapolated value (time zero) and gave information about the non-mnemonic aspects of the task (e.g., changes in motivation, sensory-motor functions, attention, and encoding of information). On PND 24, the delays were set at 5, 15, and 25 s, while they were set at 5, 65, and 125 s for PNDs 85 to 90.
Motor activity.
Motor activity was evaluated on PNDs 13, 17, 21, and 60 by counting beam breaks of a passive infrared sensor mounted outside a stainless-steel wire-bottom cage with Plexiglas® flooring (40.6 x 25.4 x 17.8 cm). Each test session was one h in duration with the number of beam breaks tabulated for each 5-min interval. The apparatus monitored a rack of up to 32 cages and sensors during each session, with each rat tested in the same location on the rack across test sessions. Groups were counterbalanced (sex, dosage group) across testing sessions and cages. Data were collected to demonstrate that the test system was capable of detecting increases or decreases in activity produced by positive control substances. The calibration procedure for the motor activity equipment was performed at least semiannually.
Auditory startle.
The auditory startle test was conducted on PNDs 22 and 61. Animals were evaluated in sets of 4 within a sound-attenuated chamber, and the amplitude and latency of the startle response were measured (Coulbourn Instruments, Inc., Allentown, PA). Each rat was placed inside a cage situated above a platform containing a pressure transducer in its base. The cage had the overall shape of a hexahedron with an 8-cm base, 8-cm height and 16-cm length for younger rats, and with a 9-cm base, 9.5-cm height and 18.5-cm length for older rats. A microcomputer sampled the output of the pressure transducer and controlled the test session. The rats were initially given an adaptation period of five min. During the last minute of this period, ten "blank" trials (no startle auditory stimulus) were given to sample the baseline pressure exerted by the rat. The rats were then presented with 20-ms, 120-dBA bursts of white noise at 10-s intervals for 50 trials. An additional ten blank trials followed. The peak amplitude of each response was recorded, and the average response on baseline trials was subtracted to calculate the peak response. The average peak responses and latencies over 10-trial blocks were compared among the dosage groups. The calibration of auditory signals was performed prior to the start of the study. The calibration of the auditory startle pressure transducers was performed daily prior to usage. Groups were counterbalanced (sex, dosage group) across testing sessions and cages.
Body temperature.
Body temperature was measured on PNDs 21 and 60, immediately following the completion of motor activity testing. The measurement was made with a rectal probe (Physitemp Instruments Model RET-2) and digital thermometer (Physitemp Instruments Model Bat-10 R LOP). The digital thermometer was calibrated daily prior to usage.
Physical signs of maturation.
Pinna unfolding was monitored daily beginning on PND 1 (all rats in all litters) and continued until all pups (100%) had the pinna unfolded. In rats of subset 4, eye opening was monitored beginning on PND 11 and continued until each pup reached the criterion. Male rats were evaluated for the age at preputial separation beginning on PND 38. Female rats were evaluated for the age at vaginal patency beginning on PND 27. A body weight was recorded for the rat on the day the criterion was attained.
Brain weight, histopathology and morphometrics.
At PND 11, 10 male and 10 female pups (one per litter) were euthanized by carbon dioxide overexposure. The head of each pup was severed between the first two cervical vertebrae, the calvaria removed from the top of the skull, and the entire head placed into neutral buffered 10% formalin. After fixation, brains were removed and weighed. Brains from 6/sex/dosage were subsequently examined histopathologically. During PNDs 65 to 70, ten rats/sex/dosage group were randomly selected for brain weight measurements. These grown pups were administered a combination of heparin and an anesthetic (39 mg/kg sodium pentobarbital and 20,000 USP/kg sodium heparin) and perfused in situ with neutral buffered 10% formalin. Of these 10, 6 were randomly selected for neuropathologic evaluation.
In addition to neuropathologic examinations on PND-11 and PND-65 rats, morphometric measurements (6 rats/sex/dosage) were made of the anterior to posterior length of the cerebrum and cerebellum, height of the cerebellum, thickness of the corpus callosum, frontal cortex, parietal cortex, caudate-putamen, hippocampus, and in pups only, the thickness of the external germinal layer of the cerebellum. Linear measurements of the anterior-posterior dimensions of the cerebrum and cerebellum were made with a Vernier caliper. The brains were then weighed and divided into 6 coronal slices. The cuts were made half way between the ventral base of the olfactory bulbs and the optic chiasm, through the optic chiasm, through the infundibulum, through the midbrain just posterior to the mammillary body, through the cerebellum just anterior to its midpoint, and finally through the anterior portion of the medulla. The anterior faces of these coronal brain slices were sectioned (7 µm), paraffin-embedded, and stained with hematoxylin and eosin. After processing, the slides were coded and evaluated, blind to treatment.
Statistical Analyses
Statistical analyses were performed with SAS/STAT© (SAS Institute, 1989) programs or by proprietary programs developed for computer use by the Testing Facility. All tests were 2-tailed. Individual motor activity counts were transformed to square roots. Bartlett's Tests (Sokal and Rohlf, 1969) for homogeneity of variance were run at
= 0.001.
Parametric data were analyzed by factorial analysis of variance (ANOVA, factors of group and sex) or factorial repeated-measure analyses (Rep-ANOVA, factors of group, sex, repeated for time), using the multivariate approach and the Pillai trace statistic (SAS Institute, 1989). The main effects and interactions of interest were: Group: A significant p value indicated that both male and female pups, taken together, differed in some way among the different groups, independent of time. Group x Sex: A significant p value indicated that male and female pups differed among the different groups (independent of time) in the way they reacted to the test compound. Group x Time: A significant p value indicated that both male and female pups, taken together, differed in some way among the different groups at some time interval. Group x Time x Block (motor activity and auditory startle): A significant p value indicated that group effects were different among the different blocks at some time interval. The Group x Sex x Time factor was also added by request, and examined post hoc. A significant p value indicated that group effects were different in males and females at some time interval.
In the event of a statistically significant Group main effect (in the presence or absence of any other statistically significant interaction), one dosage level (beginning with the high-dosage group) was removed, and the same analysis was re-run. If an overall statistically significant effect was present, but disappeared when the high-dosage group data were removed, it was inferred that the statistically significant effect was due to the high-dosage group (Tukey et al., 1985). This approach minimizes the number of comparisons (and, therefore, of Type-I errors) and also of Type-II errors (by maximizing the sample size). The following sequential approach was used in an effort to further reduce Type-I and Type-II errors: If Group x Sex was significant, the same analysis was rerun on separate sexes. If the Group x Time interaction was significant, the data were examined to identify the time period responsible for the statistical significance. The Type I-error rate was set at 0.02 for all primary analyses. Sequential analyses were conducted at
= 0.02, following a statistically significant primary planned analysis (consistent with the recommendations proposed by Tukey et al., 1985). When one male and one female from the same litter were used in a test, the litter was used as the unit for analysis. All p values were reported uncorrected.
Brain morphometric data were analyzed by ANOVA for males and females separately, followed by Dunnett's tests if the ANOVA was statistically significant. This approach was taken because the number of dosage groups examined was not the same, either in males and females, or at the 2 time points analyzed (i.e., male and female pups: morphometric data from control, low-, middle-, and high-dosage groups; male adults: control and high-dosage groups, female adults: control, middle- and high-dosage groups). Such a difference among the number of groups selected for morphometric analysis came from the fact that, at first, the pups from all dosage groups were prepared for morphometric analyses. However, for adults, the stepwise approach suggested in the EPA developmental neurotoxicity guideline (US EPA, 1991) was used, which recommends that control and high-dosage groups be examined first, and then, if evidence of alterations is found, lower dosage groups be examined in sequence. Analysis was performed on all tissues prepared for morphometric analysis. The Type-I error rate was set arbitrarily at 0.05 because this type of analysis has less statistical power than the repeated-measure analyses.
Developmental landmarks were evaluated using the Kruskal-Wallis (Sokal and Rohlf, 1969) (pinna unfolding, vaginal patency, preputial separation, eye opening;
= 0.02). Clinical observations were evaluated using the Chi-square test for proportions (Snedecor and Cochran, 1967
).
More than 200 a priori p values were derived in this study, out of which approximately 40 were statistically significant. These figures provide a context for a better interpretation of the results.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
Table 2 presents ChE activity as a percent of control values, measured in a subgroup of dams sacrificed on GD 20 (satellite study). At 5 mg/kg/day of chlorpyrifos, brain and plasma ChE activity was markedly depressed (approximately 90%) when compared to controls, and RBC ChE levels were depressed almost completely. At 1.0 mg/kg/day, dams' brain ChE activity was depressed approximately 18% relative to controls, while RBC ChE levels were inhibited approximately 84% and plasma levels were depressed approximately 69%. At the low dosage of 0.3 mg/kg/day, there was no indication of effect on brain cholinesterase activity, while plasma and RBC ChE levels were approximately 45% lower than control values.
|
|
When pups' body weights were evaluated over a longer period (i.e., PNDs 065), the analysis showed that they were lower in the high-dosage group in both males and females compared to controls (p < 0.001; F(3, 75) = 14.07) (Fig. 3). A significant Sex x Group effect (p = 0.02; F(3, 71) = 3.52) indicated that males and females were differentially affected by treatment. However, when the data were analyzed separately by sex, the analyses showed that the body weights of both males (p < 0.001; F(3, 73) = 11.47) and females (p < 0.001; F(3, 73) = 8.13) were significantly decreased at the high dosage. A Day x Group significant effect (p < 0.001; F(18, 216) = 2.80) indicated that the difference between groups for both sexes taken together changed as a function of day (e.g., more of a relative effect on PND 65 compared to PND 0). By PND 65, the weights of high-dosage female offspring were comparable to the controls, while the weights in male offspring remained lower.
|
The feed consumption analyses revealed a main Group effect (p < 0.001; F(3, 75) = 8.27) in the high-dosage group without a significant Sex x Group interaction (p = 0.22; F(3, 62) = 1.53). The biological significance of the Day x Sex x Group interaction (p < 0.01; F(6, 124) = 2.86) is unclear, but may reflect a tendency for a small reduction, over time, of the difference in feed consumption between control and high-dosage groups that may be more accentuated in males than in females.
The litter losses/reductions and lower body weights of offspring in the high-dosage group (PNDs 04) corresponded temporally with clinical signs, cannibalization of pups, a trend toward reduced feed consumption, and body weights in the dams. There were no effects of treatment with chlorpyrifos in the 0.3- or 1-mg/kg/day dosage groups.
Developmental Landmarks
In addition to the changes in pups' body weights, delays in some developmental landmarks were present in pups of dams treated with 5 mg/kg/day (Table 4). The only developmental landmark which was identified as statistically significantly different from the controls was delayed vaginal opening (p = 0.01). However, pinna detachment (p = 0.03) and preputial separation (p = 0.05) also appeared to be somewhat delayed, though there were no statistically significant differences. There were no effects at either 0.3 or 1.0 mg/kg/day in any of these measures.
|
|
|
|
|
|
|
|
|
Motor activity.
Litter was used as a factor in the analysis of motor activity. There were no overall differences in motor activity, measured on PNDs 13, 17, 21, and 60, at any dosage level (p = 0.80; F(3, 75) = 0.33). Males did not behave differently from females, as far as the test compound was concerned (p = 0.08; F(3, 73) = 2.33). The Group x Time interaction (p = 0.10; F(9, 225) = 1.65) was not significant, nor were the Group x Time x Block interactions (p = 0.18; F(99, 135) = 1.18), i.e., no effect on habituation. Examination of the data showed that high-dosage pups may have had a decrease in activity at PND 13 in the high-dosage group, relative to controls, and a slight increase in activity on PNDs 17, 21, and 60 (Fig. 8).
Auditory startle.
Litter was used as a factor in the analysis for auditory startle. Evaluation of the auditory startle response on PND 22 and 61 revealed an overall statistically non-significant increase in latency-to-peak response (overall p = 0.03; F(3, 75) = 2.95; Fig. 9), and an overall non-significant decrease in response amplitude (overall p = 0.14; F(3, 75) = 1.87; Fig. 10
). The decreased amplitude was consistent with an increased latency, both were mainly observed in the 5 mg/kg/day dosage group, and the effects may have been real. No effects appeared to be present in pups at 0.3 or 1.0 mg/kg/day. Examination of these same data on PND 61 failed to produce any evidence of an effect on either of these measures among treatment groups. The decrease in startle amplitude and increase in latency in PND-22 pups of the 5 mg/kg/day group were consistent with a developmental delay (Sheets et al., 1988
) also present in other parameters (e.g., body weights) at 5 mg/kg/day. The non-statistically significant interaction Group x Time x Block for amplitude (p = 0.15; F(12, 222) = 1.43) indicated that the distribution of peak response in blocks within the test session over times did not change as a function of treatment, i.e., there were no effects of chlorpyrifos on habituation of the peak response at any time, another primitive form of learning.
Body temperature.
There were no statistically significant differences in body temperature among any of the dosage groups (p = 0.86; F(3, 75) = 0.25).
Pathologic Evaluation
Examination at necropsy of dams treated with CPF, and of the offspring examined on PND 21 or PND 65 did not reveal any gross pathologic changes attributed to treatment (data not shown). Overall terminal body weights on PND 21 or PND 65 were depressed (p = 0.004; F(3, 143) = 4.57). The Sex x Group interaction was also significant (p = 0.01; F(3, 143) = 3.61), as well as the Sex x Group x Time (p = 0.01; F(3, 143) = 4.04) whereas the Group x Time interaction was not significant (p = 0.14; F(3, 143) = 1.88). When male and female data were analyzed separately with all dosage groups, no effects were seen in females at any time. In males, the Group main effect was statistically significant (p = 0.004; F(3, 71) = 4.90). When the male body weights were analyzed separately without the high-dosage group, no statistically significant effects were seen (p = 0.19; F(2, 53) = 1.71). As far as the Sex x Group x Time interaction is concerned, it may have suggested the presence of a Group effect that was more marked in one sex at a time point (i.e., males on PND 65). Overall, the analysis can be interpreted as reflecting a decrease in the terminal body weights in males in the high-dosage group on PNDs 11 and 65. Examination of the data shows that the group effect on body weight may extend to high-dosage females on PND 11. No gross pathologic changes were associated with lower body weights (data not shown). All observations were considered within background variability and were consistent with the ages and strain of rat examined. Likewise, there were no microscopic neuropathologic alterations in any of the brains (data not shown) from any treatment group.
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The only statistically significant effect seen in the middle dosage group (1 mg/kg/day) was recorded as a 4.2% thinner parietal cortex (one of the 8 morphometric measurements) in adult females (PND65). No parietal cortex effects were observed either in adult males, or in male and female pups (PND11) at this dosage. The middle- and high-dosage groups differed by 0.9% and such a difference was considered small enough to question the existence of a dose-response relationship. The data a posteriori, corrected for brain weight, showed a clear loss of dose-response relationship and a loss of statistical significance in the high-dosage group. In the middle-dosage group, there were no behavioral or functional findings, and no inhibition of blood or brain cholinesterase (Mattsson et al., 2000). From the neurogenesis point of view, the rat neocortex, parietal and frontal, develops mainly between GDs 14 and 20 (Bayer et al., 1993
). If treatment affected cortical neurogenesis, it would be expected that parietal and frontal cortices would be similarly affected, and this was not the case in the present study. Also, as Bayer et al. (1993) concluded from their studies, using X-irradiation when an environmental insult occurs during neurogenesis, the neuron precursors are killed and are not replaced. Furthermore, such a loss of neurons is often followed by abnormal cell migration and differentiation. As Bayer et al. (1993) wrote, "Once ectopia has been produced by abnormal migration, it becomes a permanent anatomical feature of the brain." The paper continued, stating that hypoplasia and dysplasia are also permanent phenomena in the developing brain. If such events had taken place in the parietal cortex, they would have been present on PND 11. No gross or microscopic neuropathological alterations were observed, however, whether on PNDs 11 or 65, at any dose. Finally, from a statistical point of view, the multiplicity problem should be recalled in light of the numerous statistical tests performed in this study. As Ware et al. (1992) stated, "In that view, one of every 20 tests will produce a p value smaller than 0.05 merely by chance, and this likelihood must simply be kept in mind in the interpretation of any collection of hypothesis tests and their p values." Similar positions can be found in Tukey (1980) and Wilkinson et al. (1999). For the reasons stated above, the statistically significant middle-dose effect in the parietal cortex of females at PND 65 was isolated, did not support a biologically plausible interpretation, and was, therefore, considered spurious.
Toxicity was limited to the highest dosage level tested, 5 mg CPF/kg/day, and no effects were seen in the pups in the absence of maternal toxicity. At this dosage level, ChE activity of brain erythrocyte and plasma were depressed by 90% when measured in the dams on GD20. Clinical signs of ChE inhibition, reduced body weight, and feed consumption were seen in high-dosage dams primarily at the end of gestation, and during the first few days of lactation. No treatment-related effects (other than ChE inhibition) were seen in the dams at dosages of 0.3 or 1 mg CPF/kg/day. At 5 mg/kg/day, pup survival was reduced during the first 4 postnatal days, when maternal effects were most evident, while pup survival was unaffected from postnatal day 5 throughout the remainder of the study. Pups from this group also exhibited lower body weight, delayed appearance of some developmental landmarks, and on PND 11, decreased weight and linear dimensions of the brain. Such an effect on brain does not, ipso facto, qualify the chemical as a developmental neurotoxicant when it is related to general growth retardation, as Makris et al. (1998) recognize. Slight changes in the auditory startle response were present on PND 22 (non-statistically significant), but were not detected on PND 61. There was some indication that motor activity measured on PND 13 may have been reduced (although no statistically significant differences were found). It would be consistent with the documented decrease in motor activity caused by chlorpyrifos in young rats (Moser et al., 1998). Cognitive functions measured in pups from exposed dams were not affected by CPF. The effects that were noted in the pups at 5 mg/kg/day were considered a result of the lower body weight gain, were viewed as indicative of a transient maturational delay, and were considered secondary to the maternal effects, although some contribution from pup toxicity could not be completely ruled out.
Several lines of evidence support that the effects seen in the pups were secondary to effects on the dam. The fetal effects were observed only at a dosage level that caused clear maternal toxicity and occurred in the same time interval. Absolute brain weight was decreased by 89% when measured on PND 11, although the brain weight relative to body weight was increased, and no effect was seen on brain weight when the pups were 2 months of age. Thus the effects on pup brain weight were transient, and are consistent with what is expected due to decreased nutrition or maternal care. The relative sparing of brain growth has been described by Dobbing and Sands (1971) in undernourished rats produced by creating artificially large (15 or more pups) and small (3 pups) litters. Undernutrition during early development causes slower body growth, slower rate of brain growth to a different degree, and contrary to popular belief, depresses the growth rate of various processes within the brain to the same extent (Peeling and Smart, 1994). At weaning, offspring from these large litters showed approximately a 16% difference in absolute brain weight. This brain weight difference was also transient, but resolved more slowly in undernourished litters than in the present study.
Some more arguments about maternal toxicity have been advanced by Schardein and Scialli (1999) who stated in a review of the present data, "The developmental findings in the high-dose pups in this study are consistent with the decreased pup weight that occurred in parallel with decreased maternal feed consumption and lactation weight gain. When maternal toxicity and developmental toxicity occur at the same dose, it is not axiomatic that the maternal toxicity is the cause of the developmental toxicity; however, in this case, the nature of the developmental effects and the lack of persistent neurologic abnormalities strongly suggest that the pup effects were secondary to maternal toxicity. More directly, no evidence of specific toxicity of chlorpyrifos for the developing nervous system was identified. The maternal and developmental NOAELs in this study were 1 mg/kg/day."
Finally, data from Mattsson et al. (2000) showed that brain ChE was inhibited by approximately 85% and 78% in dams on LDs 1 and 5. In contrast, brain ChE was inhibited by 35% in pups on PND 1, but was comparable to control levels on PND 5. Neonatal mortality was highest between PNDs 1 and 5, and thus, mortality did not correlate with inhibition of neonatal brain ChE, which was normal by PND 5. However, neonatal mortality did occur concurrently with clinical signs of maternal toxicity (muscle fasciculation, hyperpnea, hyperreactivity), and the time when dams' body weight and body weight gains were depressed. All these effects pointed at the dams as responsible for neonatal mortality and slower growth rather than at the pups.
The results of the present study are consistent with the majority of scientific data published on chlorpyrifos. Nostrandt et al. (1997) concluded that in adult rats given a single oral dose of CPF, a depression in brain ChE in excess of 6070% was necessary before any association with behavioral changes could be measured. They observed classical cholinergic signs (miosis and salivation) only in adult rats with >90% inhibition of brain ChE. In the present study, this degree of brain ChE inhibition was seen only in the dams given 5 mg/kg/day. No cholinergic signs were observed in dams given the 0.3- or 1-mg/kg/day dosage, and maternal brain ChE was inhibited by less than 20% at either dose on GD20. Decreased body weight and reduced survival were also observed previously at a 5-mg/kg/day dosage level in pups from the first generation of a 2-generation reproduction study (Breslin et al., 1996), but not in the second generation. Reproductive indices were unaffected. In the present study, no effects were observed in the pups at the lower dosage levels (i.e., 0.3 and 1 mg/kg/day).
In the present study, cognitive functions, such as learning (i.e., rate of acquisition of a discrimination), short-term memory (i.e., retention), habituation (a primitive form of learning; Cabe and Eckerman, 1982) of motor activity and auditory startle amplitude, and, potentially, long-term memory (a posteriori observation) were not affected by chlorpyrifos. The absence of any cognitive effects in the present study was not unexpected in light of data from a previous cognitive study in adult rats administered CPF (Maurissen et al., 2000). These authors demonstrated that in adults there were no differences in retention of previously acquired information at dosages of up to 10 mg/kg/day for 28 days, though brain ChE was inhibited by up to 85% and cholinergic effects, as well as motor slowing, were prominent in the high-dosage group. Stanton et al. (1994) injected PND-21 rats, sc, with CPF, and no effects were observed in rats given 90 mg/kg. Rat pups in the 240-mg/kg group could not be evaluated because of overt toxicity. At 120 mg/kg, brain ChE activity was inhibited by approximately 80%, and was accompanied by a small (<10%) transient effect on acquisition of information on PND 23, but by PND 26 there was no discernible effect on acquisition.
The developmental and cognitive data suggest that the transient changes in brain size have no impact on functional capabilities. A similar conclusion was expressed by Dobbing (1971), who noted there was no evidence that a transient decrease in brain size had any functional significance, even though it has been argued that a temporary retardation of developmental processes could lead to long-term consequences.
The results of the present study contrast with the conclusions from Campbell et al. (1997) who administered CPF sc to rat pups during the early neonatal period (PNDs 14 and 1114) and predicted that the neurochemical changes they reported "can be expected to have a significant impact on nervous system function and may be responsible for neurobehavioral disturbances seen with developmental chlorpyrifos exposure". The present study did not report such findings in the nervous system of rat pups and adults and did not, therefore, confirm such a prediction. The study design and timing of CPF administration made the Campbell study unsuitable for human risk assessment for several reasons: the study was designed to maximize the rate of absorption rather than to simulate environmental exposure (chlorpyrifos sc in DMSO), the route of administration was subcutaneous, and pups were injected (PND 14) when the nervous system development of the rat brain was at a stage corresponding to the 3rd trimester in humans.
Pope and Liu (1997) showed that the increased sensitivity (approximately 6 times) of neonatal rats (relative to adults) to chlorpyrifos is associated with the highest dosage compatible with no mortality (i.e., maximum tolerated dose). At dosage levels that cause no cholinergic effects (e.g., 50% inhibition of brain ChE), the neonate was no more than twice as sensitive as the adult. The authors also concluded that adults may be more sensitive than the pups to sub-lethal alterations following repeated exposures.
Gavage (i.e., bolus dosage leading to high pulses of exposure) is often recommended by governmental agencies for developmental neurotoxicology studies, and was the route of administration used in the present study. However, the same dosage given as a fractionated dosage will most likely provide lower peak concentrations and a less pronounced toxicity profile compared to a bolus administration (Wagner, 1971). Conolly et al. (1999) cautions against use of gavage administration of chemicals in these terms: "Experimental convenience has also been used to justify unrealistic methods of exposure such as corn oil gavage and nasal instillation. Both of these methods of exposure have the potential to deliver chemicals to a target site at a rate that far exceeds anything that would occur in the real world. [...] The relevance of experiments using doses that are multiples of conceivable human exposures and unrealistic routes of exposure is, at most, quite dubious. Mechanisms of action may be elicited under such conditions that would not occur with relevant routes and exposure levels." This admonition appears relevant to our study, because both dose and mode of administration may exacerbate toxic effects. Such a caution is applicable to all studies using a bolus mode of administration of the test compound, such as those using gavage or subcutaneous injection, for example.
In summary, chlorpyrifos did not cause any effects in the offspring when administered to dams during gestation and lactation at dosage levels of 0.3 or 1 mg/kg/day. Administration of chlorpyrifos at a dosage level of 5 mg/kg/day produced clinical evidence of cholinergic toxicity in the dams. Increased pup mortality, decrease in pup body weight, brain size, brain layer thickness, potential transient changes in the startle response, motor activity, pinna detachment, vaginal opening, and preputial separation were seen only at 5 mg/kg/day in the presence of maternal effects, and were consistent with delayed maturation. Cognitive functions (learning, short-term memory, and habituation in 2 different tasks) were not impaired in the pups at any time at any dosage. The no-observed-effect-level (NOEL) for any effects in pups in this study was 1 mg/kg/day. A NOEL was not determined in dams, due to inhibition of plasma and RBC ChE at 0.3 mg/kg/day. This study, using the testing guidelines established by US EPA, indicates that chlorpyrifos is not a selective developmental neurotoxicant in the rat.
![]() |
ACKNOWLEDGMENTS |
---|
![]() |
NOTES |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Breslin, W. J., Liberacki, A. B., Dittenber, D. A., and Quast, J. F. (1996). Evaluation of the developmental and reproductive toxicity of chlorpyrifos in the rat. Fundam. Appl. Toxicol. 29, 119130.[ISI][Medline]
Cabe, P. A., and Eckerman, D. A. (1982). Assessment of learning and memory dysfunction in agent-exposed animals. In Nervous System Toxicology (C. L. Mitchell, Ed.), pp. 138198. Raven Press, New York.
Campbell, C. G., Seidler, F. J., and Slotkin, T. A. (1997). Chlorpyrifos interferes with cell development in rat brain regions. Brain Res. Bull. 43, 179189.[ISI][Medline]
Code of Federal Regulations (CFR) (1985). Title 9: Animals and Animal Products, Subchapter A: Animal Welfare. Office of the Federal Register, Washington, D.C.
Conolly, R. B., Beck, B. D., and Goodman, J. I. (1999). Stimulating research to improve the scientific basis of risk assessment. Toxicol. Sci. 49, 14.
Deacon, M. M., Murray, J. S., Pilny, M. K., Rao, K. S., Dittenber, D. A., Hanley, T. R., Jr., and John, J. A. (1980). Embryotoxicity and fetotoxicity of orally administered chlorpyrifos in mice. Toxicol. Appl. Pharmacol. 54, 3140.[ISI][Medline]
Dobbing, J. (1971). Undernutrition and the developing brain: The use of animal models to elucidate the human problem. In Advances in Experimental Medicine and Biology, Chemistry, and Brain Development (R. Paoletti and A. Davison, Eds.), Vol. 13, pp. 399412. Plenum, New York.
Dobbing, J., and Sands, J. (1971). Vulnerability of developing brain: IX. The effect of nutritional growth retardation on the timing of the brain growth-spurt. Biol. Neonate 19, 363378.[ISI][Medline]
Johnson, C. D., and Russell, R. L. (1975). A rapid, simple radiometric assay for cholinesterase, suitable for multiple determinations. Anal. Biochem. 64, 229238.[ISI][Medline]
Makris, S., Raffaele, K., Sette, W., and Seed, J. (1998). A retrospective analysis of twelve developmental neurotoxicity studies submitted to the US EPA Office of Prevention, Pesticides, and Toxic Substances (p. 46) (OPPTS). US EPA Internal Document.
Mattsson, J. L., Maurissen, J. P., Nolan, R. J., and Brzak, K. A. (2000). Lack of differential sensitivity to cholinesterase inhibition in fetuses and neonates compared to dams treated perinatally with chlorpyrifos. Toxicol. Sci. 53, 438446.
Maurissen, J. P., Shankar, M. R., and Mattsson, J. L. (2000). Chlorpyrifos: Lack of cognitive effects in adult Long-Evans rats. Neurotoxicol. Teratol. 22, 237246.[ISI][Medline]
Moser, V. C., and Padilla, S. (1998). Age- and gender-related differences in the time course of behavioral and biochemical effects produced by oral chlorpyrifos in rats. Toxicol. Appl. Pharmacol. 149, 107119.[ISI][Medline]
Nicholas, K. R., and Hartman, P. E. (1991). Milk secretion in the rat: Progressive changes in milk composition during lactation and weaning and the effect of diet. Comp. Biochem. Physiol. 98A, 535542.[ISI]
Nostrandt, A. C., Padilla, S., and Moser, V. C. (1997). The relationship of oral chlorpyrifos effects on behavior, cholinesterase inhibition, and muscarinic receptor density in the rat. Pharmacol. Biochem. Behav. 58, 1523.[ISI][Medline]
National Research Council (NRC) (1996). Guide for the Care and Use of Laboratory Animals. National Academy Press, Washington, DC.
Peeling, A. N., and Smart, J. L. (1994). Review of literature showing that undernutrition affects the growth rate of all processes in the brain to the same extent. Metabolic Brain Disease 9, 3342.[ISI][Medline]
Pope, C. N., and Liu, J. (1997). Age-related differences in sensitivity to organophosphorus pesticides. Env. Toxicol. Pharmacol. 4, 309314.[ISI]
Salewski, E. (1964). Färbemethode zum makroskopischen Nachweis von Implantationsstellen am Uterus der Ratte. Arch. Pathol. Exp. Pharmakol. 247, 367.
SAS Institute. (1989). SAS/STAT User's Guide, Version 6, 4th ed., Volumes 1 and 2. SAS Institute, Cary, NC.
Schardein, J. L., and Scialli, A. R. (1999). The legislation of toxicologic safety factors: The Food Quality Protection Act with chlorpyrifos as a test case. Reprod. Toxicol. 13, 114.[ISI][Medline]
Sheets, L. P., Dean, K. F., and Reiter, L. W. (1988). Ontogeny of the acoustic startle response and sensitization to background noise in the rat. Behav. Neurosci. 102, 706713.[ISI][Medline]
Snedecor, G. W., and Cochran, W. G. (1967). Statistical Methods, 6th Ed, pp. 240241. Iowa State University Press, Ames, IA.
Sokal, R. R., and Rohlf, F. J. (1969). Biometry, pp. 388-389. W. H. Freeman, San Francisco, CA.
Stanton, M. E., Mundy, W. R., Ward, T., Dulchinos, V., and Barry, C. C. (1994). Time-dependent effects of acute chlorpyrifos administration on spatial delayed alternation and cholinergic neurochemistry in weanling rats. Neurotoxicology 15, 201208.[ISI][Medline]
Tukey, J. W. (1980). We need both exploratory and confirmatory. Am. Statistician 34, 2325.[ISI]
Tukey, J. W., Ciminera, J. L., and Heyse, J. F. (1985). Testing the statistical certainty of a response to increasing doses of a drug. Biometrics 41, 295301.[ISI][Medline]
U.S. Environmental Protection Agency (US EPA) (1989). Federal Insecticide, Fungicide, and Rodenticide Act (FIFRA); Good Laboratory Practice Standards; Final Rule. 40 CFR, Part 160.
U.S. Environmental Protection Agency (US EPA) (1991). Pesticide Assessment Guidelines. Subdivision F, Hazard Evaluation: Human and Domestic Animals, Addendum 10, Neurotoxicity. Health Effects Division, Office of Pesticide Program.
Wagner, J. G. (1971). Biopharmaceutics and Relevant Pharmacokinetics, pp. 149150. Drug Intelligence Publications, Hamilton, IL.
Ware, J. H., Mosteller, F., Delgado, F., Donnelly, C., and Ingelfinger, J. A. (1992). P values. In Medical Uses of Statistics (J. C. Bailar and F. Mosteller, Eds.), pp. 181200. NEJM Books, Boston.
Wilkinson, L., and the Task Force on Statistical Inference. (1999) Statistical methods in psychology journals. Am. Psychol. 54, 594604.[ISI]