Developmental and behavioral effects of prenatal amitraz exposure in rats

Developmental and behavioral effects of prenatal amitraz exposure in rats

Neurotoxicologyand Teratology,VoL 16, No. 1, pp. 65-70, 1994 Copyright©1994ElsevierScienceLtd Printedin the USA.All rightsreserved 0892-0362/94 $6.00 ...

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Neurotoxicologyand Teratology,VoL 16, No. 1, pp. 65-70, 1994 Copyright©1994ElsevierScienceLtd Printedin the USA.All rightsreserved 0892-0362/94 $6.00 + .00


Developmental and Behavioral Effects of Prenatal Amitraz Exposure in Rats J. P A L E R M O - N E T O , *l J. C. F L 6 R I O * A N D M. SAKATEI"

*Applied Pharmacology and Toxicology Laboratory, School o f Veterinary Medicine, University o f Sao Paulo, SP, and ~fClinics Department, Botucatu School o f Veterinary Medicine, SP, Brasil Received 30 N o v e m b e r 1992; Accepted 8 September 1993 PALERMO-NETO, J., J. C. FL6RIO AND M. SAKATE. Developmental and behavioral effects of prenatal amitraz exposure in rats. NEUROTOXICOL TERATOL 16(1) 65-70, 1994.--The effects of prenatal amitraz exposure on physical and behavioral parameters of rats were studied. Pregnant rats were orally gavaged with amitraz (20 mg/Kg) or with distilled water (1.0 ml/Kg) on Days 1, 4, 7, 10, 13, 16, and 19 of pregnancy. After birth, cross-fostering was performed thus generating the following groups: control pups nursed by control dams (CC); control pups nursed by treated dams (CE); treated pups nursed by treated dams (EE) and treated pups nursed by control dams (EC). Results show that compared to pups of Group CC, groups prenatally exposed to amitraz (EC and EE) showed decreased age of vaginal opening. Group EE also showed earlier fur development and a delay in incisor eruption compared to Group CC. The ages of pinna detachment, eye and ear opening, testes descent, and reflex development (surface fighting and startle) were not affected by amitraz exposure. Offspring of group CE also showed earlier fur development. Rats of group EE had higher locomotor activity and rearing frequency and shorter immobility time compared to the rats of group CC when observed in an open-field 30 days after birth but not 60 and 90 days. No significant differences were found in open-field behaviors among the CC, CE, and EC groups. The present findings indicate that prenatal exposure to amitraz caused transient developmental and behavioral changes in the exposed rat offspring and suggest that further investigation of the potential health risk of amitraz exposure to developing human offspring may be warranted. Developmental neurotoxicology


Prenatal intoxication


Further evaluation of animal development often includes careful observations of the day of appearencc of physical landmarks (1). Both evaluations are often used to identify alterations caused by pre- and/or postnatal exposure to toxic compounds (26). Because amitraz crosses the placenta (10), we evaluated the effects of its prenatal exposure on some physical landmarks and behavioral parameters in developing rats. To determine if possible adverse effects were persistent, the animals were also tested as adults for changes in open-field behavior.

AMITRAZ, a formamidine derivative, is widely used in clinical practice for treatment of demodicosis (20,22). It has been reported to block monoamine oxidase (MAO) activity both in the livers and brains of rats and is thus classified as a monoamine oxidase inhibitor (MAOI)-like agent (2,3). Exposure to large doses of amitraz causes sedation, loss of the righting reflex, motor incoordination, and coma (12,17,22). Recently, F16rio et al. (11) determined that acute amitraz administration to adult rats induced many behavioral alterations and increased the levels of noradrenaline and dopamine in different brain regions. During its development, the central nervous system (CNS) can be especially susceptible to the toxic effects of xenobiotics (13,15,26,28,31). Maternal exposure to many xenobiotics during pregnancy a n d / o r lactation has caused developmental neurotoxicity and/or behavioral abnormalities in the offspring that may persist throughout the animals lifetime (5,6).


Animals Genetically similar male and female Wistar rats from our own colony, weighing 250-350 g and about 90 days of age were used. The animals were housed in temperature controlled

~Requests for reprints should be addressed to J. Palermo-Neto, Farmacologia Aplicada e Toxicologia, Faculdade de Medicina Veterin~lriae Zootecnia, Av. Corifeu de Azevedo Marques, 2720, CEP 05340, Sao Paulo, SP, BRASIL. 65

66 (21 °-23°C) and artificially lighted rooms on a 12L : 12D cycle (lights on at 7:00 a.m.) with free access to food and water. The experiments were performed in a different room with the same temperature as the animal colony. The animals were transferred in their home cages 1 h before the experiments. These were performed between 8:00 a.m. and 12:00 noon.

Pesticide Dosing Amitraz (Triatox R, Coopers do Brasil S/A) diluted in distilled water was administered orally to the experimental female rats in doses of 20 mg/Kg (1/40 of the LDs0). In previous pilot work, this was the highest dose that did not reduce the animal's food or water intake or maternal weight gain during pregnancy. Furthermore, it induced no hematological modifications or other clinical and histopathological signs of overt maternal toxicity. These factors were minimized to avoid the confounding effects of maternal toxicity (7).

Pesticide Exposure Fifty nulliparous rats were randomly and equally divided into one experimental and one control group; amitraz was administered to the experimental rats on Days 1, 4, 7, 10, 13, 16, and 19 of pregnancy (Day 0 of pregnancy was the day when spermatozoa, collected daily, were found in vaginal smears). Females of the control group were identically treated but with distilled water (1 ml/kg). It has been reported that amitraz reaches 0 level in plasma within 3 days (16,23). The present treatment was designed to maintain the pesticide within the organism throughout the pregnancy. Experiments were conducted in a blind fashion. Dams were weighed every other day of pregnancy. Only those litters born after a 21-day gestation were used. Within each group, twenty such litters were randomly culled on the day of birth (Postnatal Day 0) to six pups: three males and three females. Although no amitraz is detected in blood 3 days postdosing, and one might expect there to be little or no amitraz transferred to pups postnatally, the actual disposition of this pesticide in the dam's milk is unknown. Therefore, on the day after birth, litters were crossfostered resulting in the following groups of 10 litters each: treated litters nursed by treated dams (Group EE); treated litters nursed by control dams (Group EC); control litters nursed by control dams (Group CC); control litters nursed by treated dams (Group CE). Within each litter, male and female pups were marked for individual identification.

Reproductive Parameters and Maternal Behavior The following reproductive parameters were evaluated: pregnancy duration, number of rat pups alive or dead at birth, presence of malformations, and pup weight immediately after birth. The maternal behavior was studied on Days 1, 5, 10, 15, and 21 after delivery, in dams of all groups, according to a scoring system proposed by S6ndersten and Eneroth (27). The grading system was as follows: 0, absence of nest; 1, presence of nest; 2, all rat pups inside their nest; 3, both'dam and rat pups inside their nest; 4, all rat pups in their nest, being nursed by their respective dams. A high correlation was found between scores of two independent observers (Pearsons's correlation, r = 0.95). A conventional t test was used to analyze the reproductive parameters; maternal behavior scores were treated by Friedman analysis of variance (ANOVA) for nonparametric data, followed by Friedman's multiple comparisons test with controls (24).


Landmark and Reflex Development Within each group, 10 male and 10 female pups from 10 different litters were observed daily for pinna detachment (unfolding of the external ear), development of fur, incisor teeth eruption, ear, eye and vaginal openings, and testes descent. The presence of a startle response (a whole-body startle in response to the clicking of a spring-loaded metal trap, held 2 to 3 cm above and behind the animal) was also evaluated. The method for rapid graphic solutions of time-percentage effect curves was employed to calculate the median effective times (TEs0) and their confidence limits for each developmental landmark assessed in control and treated pups (21). These curves were constructed using the percentage of animals showing each of the above parameters and their time of appearence. Among treatment groups, the TEso values determined for each landmark were compared using Litchfield (21) procedures. The surface righting reflex (the time in seconds spent by an animal to assume a normal ventral position after being placed on its back) was assessed on PNDs 2, 3, 4, and 5. For each treatment group, 10 male and 10 female pups from different litters were used. The body weights of these pups on PNDs 5, 9, 13, 17, 21 (weaning), 30, 60, and 90 were also taken. An ANOVA was used to analyze both pups' weight and surface righting reflex ( a l p h a p < 0.05).

Open-Field Studies Open-field behavior was evaluated on PNDs 30, 60, and 90 using a device based on that described by Broadhurst (4). In short, the device is a round, wooden arena (70 cm in diameter), painted white with the floor divided in 19 parts painted in black. During the experiments a 40-watt white bulb, located 74 cm from the floor provided continuous illumination of the arena. Hand-operated counters and stopwatches were employed to score ambulation frequency (number of floor units entered with both front feet), rearing frequency (number of times the animals stood on hind legs), and immobility time (total seconds of no movement); 10 male and 10 female pups from different litters were used in each group. For open-field observations, each rat was individually placed in the center of the arena and their behavior recorded for 6 min. To minimize the possible influence of circadian changes in rats' open-field behavior, control and experimental animals were alternated, the rats being observed at the same time of the day in each session to obviate possible biasing effects due to odor cues left by previous rats. The open-field was washed with an alcohol-water solution (5070) before placing animals. Because homoscedasticity is necessary for the ANOVA, Bartlet's test (19) was performed. It was concluded that the open-field data were parametric. Thus ANOVA and Duncan's test, were used to analyse the open-field data. Within each group, Student's t test was employed to look for possible differences between male and female open-field data. An alpha level o f p < 0.05 was employed. RESULTS

Reproductive Parameters and Maternal Behavior The length of gestation did not differ between the control and experimental groups (21.8 + 0.6 and 21.6 _+ 0.5 days, respectively). No differences were found between the body weights of the control and experimental dams throughout the pregnancy (data not shown). There was no significant difference between the average of pups alive at birth in the control group (10 + 2) and in the experimental group (9 ± 3). Fur-



thermore, no pups were found dead at birth and there were no differences detected in pup weights immediately after birth. Malformations or other overt signs of pesticide toxicity were also not observed. No differences were found in the maternal behavior of control and experimental rats. Indeed, over all the days of observation and in all groups, the median of the maternal behavior scores was 4. In addition, differences were not detected in pup weights on Days 5, 9, 13, 17, 21 (weaning), 30, 60, and 90 of life (data not shown).

Landmarks and Reflex Development The time-percentage effect curves constructed to study the effects of amitraz exposure during pregnancy on pup development were parallel, thus the data of the different groups were compared. As shown Table 1, amitraz exposure during pregnancy decreased the TEs0 for the development of fur and vaginal opening and increased the TEs0 for incisor eruption (19 < 0.05). Additionally, the TEs0 for the development of fur was smaller for pups in group CE and vaginal opening was premature for pups in Group EC. The TEs0 of the other parameters studied, such as pinna detachment, eye and ear openings, testes descent, and appearence of the startle response were not modified by prenatal amitraz exposure. Table 2 shows that no differences (p < 0.05) were detected among the surface righting reflex data of pups from groups EE, CC, EC, and CE on PND 2 F(3, 36) = 2.15; PND 3,

F(3, 36) = 2.29; PND 4,/7(3, 36).= 2,06; PND 5, F(3, 36) = 1.65.

Open-Field Studies Perinatal amitraz exposure during pregnancy increased locomotion IF(3, 36) = 6.49, p < 0.05] and rearing [/7(3, 36) = 5.34, p < 0.05] frequencies, and decreased immobility time [/7(3, 36) = 7.18, p < 0.05] of treated male (Fig. 1) and female (Fig. 2) offspring compared to controls on PND 30. These effects were not present in 60- and 90-day-old treated offspring. On PND 30, female rats of Group EE had higher activity scores in the open-field than males, F(3, 36) = 6.43, p < 0.05. DISCUSSION Prenatal amitraz exposure in rats changed the time course of development of some physical landmarks (development of fur, incisor eruption, and vaginal opening) and affected the open-field behavior in 30-day-old offspring. Body weights of pregnant dams and their offspring were unaffected by amitraz exposure. Furthermore, no differences were found in the maternal behavior of control and amitraz-treated rats. These findings are important, since undernutrition during pregnancy and lactation as well as alterations on maternal behavior often result in differences in the mean times of maturation of physical features and reflexes (14,25). Offspring of Group EE had increased levels of activity

TABLE 1 EFFECTS OF AMITRAZEXPOSURE (20 mg/kg) DURING PREGNANCYON INDICATIVE LANDMARKSOF PHYSICAL DEVELOPMENTOF RATS Groups* Parameters Development of fur Incisor eruption Ear opening Eye opening Vaginal opening Pinna detachment Testes descent Startle response





6. It (7.0-5.3)

5.3~; (6.2-4.5)

4.9:~ (5.7-4.2)

6.3 (6.8-4.5)

4.8 (5.1-4.5) 13.2 (14.5-12.0) 14.7 (16.0-13.5)

4.9 (5.2-4.6) 13.6 (15.1-12.3) 14.4 (15.8-13.1)

5.3:~ (5.7-4.9) 13.8 (15.4-12.4) 14.8 (16.0-13.7)

5.0 (5.3-4.8) 12.5 (14.0-11.2) 14.1 05.2-13.1)

39.4 (44.9-34.6) 3.2 (3.6-2.8)

39.4 (50.0-31.0) 3.1 (3.4-2.8)

33.0~: (35.0-31.1) 3.1 (3.4-2.8)

35.7~: (40.7-31.3) 3.0 (3.3-2.7)

21.4 (23.3-19.7) 12.9 (13.4-12.4)

22.9 (25.4-20.7) 13.1 (13.5-12.7)

21.9 (23.8-20.2) 12.8 (13.1-12.5)

22.0 (23.7-20.4) 12.5 (12.9-12.1)

*10 male and 10 female pups (from 10 different litters) were used in each group. Treatment was given on Days 1, 4, 7, 10, 13, 16, and 19 of pregnancy; control dams were treated with 1.0 ml/Kg of distilled water. Control pups nursed by control dams (CC); control pups nursed by treated dams (CE); treated pups nursed by treated dams tEE); treated pups nursed by control dams (EC). tMedian effective days (TEs0) plus superior and inferior limits were calculated according to Litchfield (21). ~:p < 0.05 in relation to rats of group CC.




2 3 4 5

18.2 12.8 7.1 6.0

± ± ± ±


20.51" 15.0 10.8 12.8

17.1 8.6 9.6 2.3


+ 18.8 ± 9.4 + 13.8 ± 4.2

18.8 16.7 9.3 6.0


+ 18.2 ± 20.0 ± 14.9 ± 9.6

17.9 8.1 5.5 3.8

± ± + ±

7.4 9.7 6.5 4.4

*10 male and 10 female pups (from 10 different litters) were used in each group. The meaning of the groups is outlined in Table 1.1"time in seconds (Mean ± SD).

within the open-field. L o c o m o t i o n and rearing frequencies measured in the open-field have been used as indexes o f both arousal (9,18) and "emotionality" (29); the decrease or absence o f m o v e m e n t within the apparatus normally indicate a reduction in arousal or an increase in levels o f emotionality (30). In light o f the present findings, it seems reasonable to suggest that amitraz exposure increased arousal a n d / o r decreased emotionality in 30-day-old offspring, i.e., these 30-day-old pups would respond differently to the stresses when exposed to a novel situation. This hypothesis is supported by the findings that open-field changes were not observed in the remaining test sessions on Days 60 and 90 o f age. In this respect, previous studies have shown that habituation to novelty is detected in the open-field (18,30). The effects o f prenatal ami-



traz exposure were similar in male and female offspring. As typically observed (9,29), female rats of group EE had higher activity scores than males. Although the lactational transfer o f amitraz is unlikely due to its short half-life, the apparently accelerated fur development in pups of G r o u p CE indicates that some postnatal damoffspring interaction is differentially affecting this group, a finding that has been described following prenatal exposure o f rats to diphenhydramine (8). Using the present dosing paradigm, one would expect that the chemical would be eliminated from the rat dam before parturition (i.e., 3-day elimination rate). Based on this information, one might anticipate similar developmental and behavioral results from offspring in Groups E C and EE. Per-



t 120




80 w~ r~


20 30

60 130


90 days

l-ICC ~EE [] EC k~CE

lOO ao

c 60 .9


90 days






"~ 20 30


90 days

FIG. 1. Effects of prenatal amitraz exposure on locomotion and rearing frequencies, as well as on immobility time of male rats observed in an open-field, at 30, 60, and 90 days of age. Treatment was given on Days 1, 4, 7, 10, 13, 16, and 19 of pregnancy. Control pups nursed by control dams (CC); control pups nursed by treated dams (CE); treated pups nursed by treated dams (EE); treated pups nursed by control dams (EC). Data are Mean + SD. * p < 0 . 0 5 in relation to rats in Group CC (ANOVA and Duncan's test).



• Locomotion


I / /




1" ,t,,q




~. ~o 2O



i:J YJ





I~:. 60 80 .
















[~EE [ ] EC



2o~ /



90 days

FIG. 2. Effects of prenatal amitraz exposure on locomotion and rearing frequencies, as well as on immobility time of female rats observed in an open-field, at 30, 60, and 90 days of age. The meaning of the groups is outlined in Fig. 1. Data are Mean + SD. *p < 0.05 in relation to rats in Group CC (ANOVA and Duncan's test).

haps the most striking result o f the present study is the finding that whereas G r o u p E E yelded functional abnormalities, G r o u p E C did not. The mechanism o f the effects o f prenatal amitraz on the postnatal dam-offspring interaction is presently u n k n o w n and future studies are needed to determine if prenatal amitraz a n d / o r its metabolities are transferred to suckling neonates in the milk. Alternatively, the role o f amitraz's action on the pregnant dam (e.g., altered endocrine status) in mediating or unmasking the effects o f prenatal exposure in the offspring might be another avenue for investigation. Clearly, m o r e information is needed to interpret these findings. The present data give added support to the notion that nervous tissue, especially the brain, is more sensitive to foreign chemicals and that toxic effects can be manifested as subtle disturbances o f behavior long before classical symptoms o f

poisoning become apparent. In this respect, several papers have discussed the qualitative and quantitative comparability o f human and animal developmental neurotoxicity ( 13,28,31). Amitraz is widely used in the treatment o f demodicosis and the present data raise concerns over the safety o f the use of this treatment during pregnancy. In addition, the present findings p r o m o t e the use o f developmental and behavioral evaluations in animals when assessing the potential developmental neurotoxicity o f these chemicals in humans. ACKNOWLEDGEMENT We want to thank Dr. M. M. Bernardi from the Applied Pharmacology and Toxicology Laboratory, School of Veterinary Medicine, University of Sio Paulo, for the critical revision she kindly made on this manuscript. This work was supported by CNPq (Proc 303355/ 87.5).

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