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Effects of permethrin given before mating on the behavior of F1-generation in mice
NeuroToxicology 27 (2006) 421–428
Effects of permethrin given before mating on the behavior of F1-generation in mice Amina T. Farag a,*, Naglaa F. Goda a, Ayman H. Mansee a, Nasra A. Shaaban b a
Department of Pesticide Chemistry and Toxicology, Faculty of Agriculture, Alexandria University, Alexandria, Egypt b Department of Histology, Faculty of Medicine, Alexandria University, Alexandria, Egypt Received 10 August 2005; accepted 12 December 2005 Available online 19 January 2006
Abstract Permethrin, a type I synthetic pyrethroid insecticide, was evaluated through assessment of the behavioral development of F1 progeny of mice. Groups each of 30 male and 30 female ICR (CD-1) mice, as F0-generation, were given 0, 4.9, 9.8, and 19.6 mg/kg/d permethrin by gavage for 4 weeks before mating. Behavioral endpoints of motor reflexes, motor coordination, and activity were evaluated in F1 progeny. Clinical signs of toxicity including salivation, hyperactivity, and liquid feces which attributed to permethrin were observed in the F0-mice treated with 9.8 and 19.6 mg/kg/d. Reduction of body weight became evident only during gestation and lactation periods for the middle and high dose groups. Significant differences in the development of reflexes, swimming ability, and open field activity were evident in the offspring for the 9.8 and 19.6 mg/kg/d dose groups compared to the control group. These results show that permethrin at dose levels of 9.8 and 19.6 mg/kg/d can induce a significant risk to the offspring following treatment of F0-mice before mating. The NOEL obtained in this study for the effects of permethrin on the development of the F1-progeny is 4.9 mg/kg/d. # 2006 Elsevier Inc. All rights reserved. Keywords: Permethrin; Type I pyrethroid; F1-progeny; Behavioral toxicology; Social interactions; Swimming ability
1. Introduction Synthetic pyrethroid insecticides have been used in agriculture and home formulations for more than 30 years and account for approximately one-fourth of the worldwide insecticide market (Casida and Quistad, 1998). The action of pyrethroid insecticides has been divided into two types (I and II) based on signs and neurophysiologic effects (Laurence and Casida, 1983; Verschoyle and Aldridge, 1980). Generally, the two types can be distinguished structurally by the presence (type II) or absence (type I) of an alpha-cyano substituent. The acute mammalian neurotoxicity of pyrethroids has been well characterized, and several comprehensive reviews of pyrethroid toxicity and actions are available (De Ray, 2001; Kaneko and Miyamoto, 2001; Narahashi, 2001; Shafer et al., 2005; Soderlund et al., 2002). Aspects of
* Corresponding author at: Department of Pesticide Chemistry and Toxicology, Faculty of Agriculture (El-Shatby), University of Alexandria, Egypt. Tel.: +203 5915160; fax: +203 5915160. E-mail address: [email protected] (A.T. Farag). 0161-813X/$ – see front matter # 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.neuro.2005.12.001
open-field behavior and catalepsy (Mandhane and Chopde, 1997), conditioned behavior (Moniz et al., 1994); operant behavior (Stein et al., 1987), schedule-controlled behavior (Peele and Crofton, 1987), motor activity (McDaniel and Moser, 1993), and others can be altered by pyrethroid exposure in adult animals. Neonatal exposure of mice to deltamethrin and bioallethrin increased motor activity in adult age and decreased the density of cortical cholinergic muscarinic receptor (Eriksson and Fredriksson, 1991). Maternal exposure to cyhalothrin during lactation disrupted passive avoidance learning during adulthood (Gomes et al., 1991a), and delayed the descent of the tests in male offspring (Gomes et al., 1991b). Fenvalerate, a type II pyrethroid, was capable of interfering with reproductive parameters of male and female rats (Moniz et al., 1999), and affected the developmental of physical and behavior in infant and adult rats (Moniz et al., 1990). Permethrin [3-(2,2-dichloroethenyl)-2,2-dimethylcylo-propane carboxylic acid (3-phenoxyphenyl) methyl ester], a type I synthetic pyrethroid insecticide, is a mixture of four (1R, S-cis and 1R, S-trans) isomers, only one of which (1R, -cis) has lethal effects in mammals (Casida et al., 1983). It provides
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insecticidal activity for several weeks following a single application and is used to control fleas, flies, mites, and cockroaches (Abou-Donia et al., 2004). Permethrin causes modification of sodium channels leading to prolonged depolarization and repetitive discharges in presynaptic nerve fibers after a single stimulus (Bloomquist, 1996; Narahashi, 1985). This repetitive nerve action is associated with tremor, hyperactivity, ataxia, convulsions, and in some cases paralysis. At low dosage-dependent decrease in locomotor activity and an increase in the amplitude of the startle response to an acoustic stimulus following exposure of rats to repeated doses of permethrin for 30 days (Crofen and Reiter, 1988). Exposure of male mice to Ambush (25.6% permethrin) for 30 min increased activities like chewing (Mitchell et al., 1988). In addition rats exposure to permethrin at two time points on the day of dosing increased aggressive behavior, agitation, resistance to being captured, reactivity to a click stimulus, and induced head and forelimb shaking (McDaniel and Moser, 1993), and disrupted a learning feeding behavior in rats at doses about 20% of LD50 after repeated doses for 30 days (Peele and Crofton, 1987). Furthermore, permethrin can have significant effects on learned behavior, food intake, and decrease in operant response rate in rats after exposure for 20 min (Bloom et al., 1983). Exposure of rats to permethrin for 60 days led to sensorimotor deficits and differential aberrations of the cholinergic system in the CNS (Abou-Donia et al., 2004). There is no information regarding the potential effects of permethrin on the behavior and physical development endpoints on the progeny. The aim of the present study was to evaluate the effects of F0male and female mice exposure to permethrin before mating for 20 days on the development of physical and behavioral aspects in the offsprings at dose levels: one that induces some toxicity on F0-mice, one unable to induce toxicity and an intermediate one. These doses were approximately equal to 1/100, 1/50, and 1/25 of acute oral LD50 for permethrin (40:60 cis/trans) in mice (FAO/WHO, 1999). 2. Material and methods 2.1. Test material Technical grade permethrin (60:40, trans:cis; 94% purity) was kindly donated by El-Watania, Inc., Alexandria, Egypt. 2.2. Test species and husbandry One hundred and twenty Male and 120 female ICR (CD-1) mice, approximately 10 weeks old, were obtained from the High Institute of Public Health, Alexandria University, Alexandria, Egypt. Mice were examined for health status and acclimated to the laboratory environment for 2 weeks prior to use. The animal room was designed to maintain temperature 23 2 8C, relative humidity at approximately 50%, and a 12 h light:12 h dark photoperiod. All animals were housed in stainless-steel cages and given standard diet and water ad libitum throughout the study.
2.3. Breeding and treatment The experimental protocol was approved by the University Animal Care Committee. Male and female mice were divided into treatment groups according to approximately equal mean body weight. Groups each of 30 male and 30 female mice (F0-mice) were given permethrin by gavage at dose levels of 0 (corn oil), 4.9, 9.8, and 19.6 mg/kg/d before mating 5 days/ week for 4 weeks. Solution concentrations were adjusted so that 30 g mouse received 0.2 ml corn oil. After treatment females were mated with males from the same treated groups (one male/one female). The day that a vaginal plug was found was considered day 0 of gestation. 2.4. Clinical observations and body weights F0-permethrin- treated and control mice were observed daily (between 9:00 and 10:00 a.m.) immediately after each permethrin treatment or control solution for the signs of toxicity which including tremors, in coordination, hyperactivity, salivation, paralysis, and other signs were typical for type I of pyrethroids (Bradbury and Coats, 1989). F0-treated females were observed daily for signs of toxicity through the gestation and lactation periods. Each sign was assessed by observing the spontaneous behavior of the mice in the cage. Body weight was measured weekly in the animals of all the study groups throughout the period of the treatment. Maternal body weight was recorded on days 0 and 18 of gestation, and on days 0 and 28 during lactation period. Food and water consumption were recorded daily for males and females throughout the period of the treatment, gestation, and lactation. The day of birth was identified as postnatal day 0 (PND0). Postnatal survival was monitored daily before weaning (day 28), and on day 30 after weaning. 2.5. Offspring studies All the pregnant mice were allowed to give birth (F1generation) and nurture their offspring normally. Parturition day was determined to be PND0. On PND1, all the pups were examined externally and weighed. Pups were weighed at PND1, PND7, PND21 and PND28. 2.5.1. Physical development parameters Each day, beginning on PND1, the pups as mixed sex were observed for the following physical parameters: down appearance, pinna detachment, incisor eruption, development of fur, eye opening, auditive channel opening, testes descent, and vaginal opening. The day of appearance of these landmarks was recorded and pup means were calculated. 2.5.2. Behavioral parameters The following reflexes and behavioral parameters were assessed in all of the pups as mixed sex: surface righting, cliff avoidance, negative geotaxis, swimming behavior, and open field activity. To assess the behavioral development of progeny, pups were observed daily, between 9:00 and 10:00 a.m.,
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separated from the mothers at the moment of observation, and immediately returned to their home cages. Each of behavior endpoints should be evaluated at the specific time of development during the preweaning period. 2.5.2.1. Surface righting reflex. The ability of the young mouse to right itself was determined on three daily trails beginning on postnatal day 3 (PND3–PND7). The pups were placed on their backs on a smooth wooden surface and the time required to right them to a position where all four feet touched the surface was recorded. A criterion of successful righting within 2 s was used (Alder and Zbinden, 1965; Butcher and Vorhees, 1979; Pantaleoni et al., 1988). 2.5.2.2. Negative geotaxis reflex. Negative geotaxis was assessed by recording the time required for neonatal mouse to reorient from a head-down to a head up position on a 258 inclined plane. The plane was made of wood and each test mouse was given one trial on PNDS 10 and 15, with a maximum time allowance of 60 s per trial. Pups sliding off the plane were tested again. The time required to complete a 1808 turn was the dependent measure (Butcher and Vorhees, 1979; Pantaleoni et al., 1988; Wilson and Wurkany, 1965). 2.5.2.3. Cliff avoidance reflex. Beginning on PND4 each pup was placed on a wooden platform elevated 20 cm above a table top. The forepaws and snout of the animal were positioned so that the edge of the platform passed just behind an imaginary line drawn between the eye orbits. The latency required for a retraction of the head completely behind the edge of the platform was daily recorded (Butcher and Vorhees, 1979; Pantaleoni et al., 1988; Wilson and Wurkany, 1965). A criterion for a retraction response of 20 s was used. If the animal was not successful within 60 s the trial was terminated, and a maximum of 60 s was recorded. If the animal fell it was given a second attempt and if it fell again, a latency of 60 s was recorded. All the pups were tested daily on PNDS 4, 6, 8, and 10 regardless of whether they had reached criterion (Adams, 1982).
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2.5.4. Open field activity On PNDS 14 and 21 open fields activity was measured. The pups were placed in the center of a 45-cm Plexiglas circle for three 1-min periods separated by 30 s between each period. The circle was divided into three concentric circles with each quarter divided into six areas. The number of squares crossed by pups per each minutes (entries) was recorded (Butcher and Vorhees, 1979; Pantaleoni et al., 1988; Wilson and Wurkany, 1965). 2.6. Social interaction The social interaction test was carried out in the open field. Observation of the social interaction between pups was done after weaning (day 30 of age) to avoid the effects of separating pups from dams for such a long time. Briefly, F1-mice were housed singly for 5 days prior to testing, being matched to form pairs (from the same treated group) according to their weight. On the day of the social interaction test, the total time in seconds spent by each pair of F1-mice in active social interaction (sniffing, following, grooming, kicking, boxing, biting, and crawling under or over partner) was scored during a 7.5 min test session (File, 1980; File and Hyde, 1979). 2.7. Statistical analysis The mean S.D. of the litter for the behavioral assessment was used as the unit of measurement in the most of the analyses. For surface righting, cliff avoidance, and swimming the percentage of the litter reaching the criterion for the behavioral was used. MANOVA was used to analyze data of control and experimental groups in the comparison according to control and treatment values. ANOVA was employed followed by the Tukey test as post hoc tests compare data presenting interaction; data without interaction were analyzed by a conventional t-test and nonparametric x2-test or Fisher’s least significant difference test (Norusis, 1994). In all the experiments P 0.05 was the criterion for statistical significance. 3. Results
2.5.3. Swimming The ability of the neonatal mouse to swim was assessed in a 30 cm 30 cm plastic tank with the water temperature at 24 8C. On postnatal days 6, 8, 10, 12, 14, and 16 each animal was placed into the tank and swimming behavior was rated for direction (straight = 3, circling = 2, floating = 1). Head angle was rated according to the development of the neonatal mouse to raise head higher with age. The percentage of pups on PND6 was recorded as number of pups with nose and top of the head out of the water = 2; PND10 with ears half out of the water = 3; PND14 with ears out of the water = 4. Limb movement was rated as either 1 = all four limbs used, or 2 = back limbs only used. Latency to being limb movement after being placed in the water was also recorded. The animals were given a maximum of 15 s in the water and were then removed to the warm towel to dry (Butcher and Vorhees, 1979; Pantaleoni et al., 1988; Wilson and Wurkany, 1965).
3.1. Clinical observations There were two, three, two, and one of F0-males died in the groups of control, 4.9, 9.8, and 19.6 mg/kg/d, respectively, during the course of the treatment. Two of F0-females of the treated group 4.9 mg/kg/d died during the course of the treatment. Although there was no death in the pregnant females, two, one, four, and four females died in the control, 4.9, 9.8, and 19.6 mg/kg/d, respectively, during the lactation period. Administration of 4.9 mg/kg/d of permethrin dose did not produce any clinical signs of toxicity in the F0-mice. However, treatment with 9.8 and 19.6 mg/kg/d doses induced signs of toxicity that included hyperactivity, salivation, tremors, and liquid feces; these four signs started to appear in 60% of F0males and 40% of F0-females and in 80% of F0-males and 60% of F0-females in dose groups of 9.8 and 19.6 mg/kg/d,
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in the development of physical features assessment of pinna detachment, primary coat of down hair, incisor eruption, development of fur, eye opening, auditive channel opening, testes descent, and vaginal opening in any of the treated groups compared to the control (negative data not shown here).
respectively. These effects appeared on day 10 of treatment and more pronounced between 3 and 5 h after each oral administration. These responses disappeared in the treated F0-females through both gestation and lactation periods. 3.2. Physical development parameters
3.3. F1-progeny parameters
Body weight and weight gain are presented in Table 1. No differences were noted for F0- male and female body weights during the period of the treatment compared to the control group. Maternal body weights were significantly reduced at the end of the gestation and during lactation periods in the groups treated with 9.8 and 19.6 mg/kg/d compared to the control group. There were no significant changes in the water and food consumption in any of the treated groups for the F0-mice and F1-progeny after weaning throughout the study period. No external abnormalities were observed for the pups in any of the treated groups and no gross difference was found
F1-progeny parameters are presented in Table 2. While the number of live pups was decreased for the middle and high treated groups, the number of dead pups was increased in the same groups. Pups growth retardation was observed in the treated groups of 9.8 and 19.6 mg/kg/d compared to the control group. Starting growth retardation was observed from birth of pups. No statistically significant effects were found in any of the F1-progeny parameters in the treated group of 4.9 mg/kg/d compared to the control group.
Table 1 Body weight and weight gain (g) in F0-mice after exposure to permethrin before mating period Permethrin (mg/kg/d) 0
4.9
9.8
19.6
Before mating (treatment period) Day 1 of treatment Body weight (g) Males Females
39.2 0.2 30.0 0.4
40.5 0.9 29.4 0.4
41.2 0.3 29.9 0.5
40.0 0.7 30.6 0.6
End of treatment Body weight (g) Males Females
42.5 0.5 31.2 0.3
43.5 0.8 30.7 0.5
44.9 0.7 31.4 0.4
43.7 0.2 32.0 0.3
3.3 1.0 1.2 0.7
3.0 0.9 1.3 0.6
3.7 0.8 1.5 0.5
3.7 1.0 1.7 0.9
Body weight gain (g) Males Females Number of females Number of litters
During gestation period 30 26
28 23
30 25
30 18
Maternal body weight (g) Gda 0 18
32.3 0.6 44.2 0.9
31.8 0.7 42.6 0.6
31.2 0.8 36.3 0.6*
31.1 0.4 35.5 0.9*
Maternal weight gain (g)
11.9 1.2
10.8 1.3
5.1 0.9 **
4.4 1.5**
During lactation period Number of dams Ldb 0 28
26 24
23 22
25 21
18 14
Maternal body weight (g) Ldb 0 28
32.1 0.6 34.4 0.4
33.4 0.9 36.2 0.1
26.4 0.7 ** 24.4 0.3 **
25.8 0.5** 23.1 0.4**
Maternal weight gain (g)
2.3 1.9
2.8 0.9
2.0 1.7 **
2.7 2.0**
Data are presented as mean S.D. a Days of gestation. b Days of lactation. * Significantly different from control value at P 0.05. ** Significantly different from control value at P 0.01.
A.T. Farag et al. / NeuroToxicology 27 (2006) 421–428 Table 2 Parameters of mice pups after F0-mice exposure to permethrin before mating Permethrin (mg/kg/d)
Number of litters Number of pupsa Number of live pups (%) a Number of dead pups (%) a
4.9
9.8
19.6
26 210 197 (94)
23 201 183 (91)
25 214 178 (83)*
18 106 62 (58)**
13 (6)
18 (9)
36 (17)*
44 (42)**
1.99 0.5 3.80 0.6 12.11 0.7 14.77 0.9 12.78 0.9
1.90 0.9 3.99 1.0 12.02 0.8 14.30 0.5 12.40 1.0
Body weight (g)/d Developmental days 0 7 21 28 Body weight gain (g)/d
1.29 0.7 * 1.60 0.8 * 6.43 0.9 ** 7.48 0.6 ** 6.19 0.5 **
Table 4 Effects of F0-mice exposure to permethrin before mating on the development of negative geotaxis Reflex in F1-progeny Days
0
1.20 0.1 * 1.44 0.8 * 5.11 0.7 ** 6.36 0.3 ** 5.16 0.8 **
Data are presented as mean S.D. a Data are based on PND0. * Significantly different from control value at P 0.05. ** Significantly different from control value at P 0.01.
3.4. Reflexes of offsprings 3.4.1. Surface righting Development of self-righting reflex of mice pups is presented in Table 3. F0-mice exposed to 4.9 mg/kg/d permethrin before mating did not significantly affect the development of surface righting ability in the 3–7-day-old neonates as reflected by percentage of pups reaching criterion (2 s) compared to the control value. The development of surface righting ability was reduced in the 3–7-day-old neonates in the treated groups of 9.8 and 19.6 mg/kg/d compared to the control group.
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10 15
Permethrin (mg/kg/d) 0 (number of pups = 197)
4.9 (number of pups = 183)
9.8 (number of pups = 178)
19.6 (number of pups = 62)
39.9 10 10.2 5
38.5 10 11.5 9
66.8 12** 32.5 7**
78.9 11** 40.9 9**
Data are presented as mean S.D. of latencies in seconds. ** Significantly different from control value at P 0.01.
group of 4.9 mg/kg/d. Mean latencies (S.D.) to reorient results were statistically different from the control value in the dose groups of 9.8 and 19.6 mg/kg/d compared to the control group. 3.4.3. Cliff avoidance Cliff avoidance in the F1-progeny is shown in Fig. 1. The results of the statistical analysis of acquisition of cliff avoidance in the F1-progeny of treated mice were not significant in the treated group of 4.9 mg/kg/d compared to the control group. Treatment of F0- male and female mice with 9.8 and 19.6 mg/kg/d permethrin before mating significantly slowed the development of the cliff avoidance performance on days 4–10 compared to the control group. There were highly significant lower percentage values in both permethrin exposed groups of 9.8 and 19.6 mg/kg/d on postnatal days 8 and 10.
3.4.2. Negative geotaxis The data of the ability to show geotaxis is presented in Table 4. The ability of days 10 and 15 neonates to show negative geotaxis was not significantly altered in the dose
Table 3 Effects of F0-mice exposure to permethrin before mating on the development of self-righting reflex in F1-progeny Days
Permethrin (mg/kg/d) 0
4.9
9.8
19.6
a.r.c./Na 3 4 5 6 7
(%) 57/198 (29) 120/198 (61) 169/197 (86)b 180/197 (91) 181/197 (92)
44/183 (24) 99/183 (54) 140/183 (77) 166/183 (91) 164/183 (90)
30/179 (17)* 80/179 (45)* 100/178 (56)b,** 107/178 (60)** 109/178 (61)**
10/76 22/76 29/62 30/62 31/62
(13)** (29)** (47)b,** (48)** (50)**
a Number of animals reaching the criterion (2 s)/total number of tested animals. b Number reduced according to the dead number of pups during the test period. * Significantly different from control value at P 0.05. ** Significantly different from control value at P 0.01.
Fig. 1. The effects of F0-mice exposure to permethrin before mating on the developmental cliff avoidance reflex in the F1-progeny. a Number of pups retracted the head completely behind the edge of the platform/total number of pups 100; *P 0.05, **P 0.01.
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Fig. 2. The effect of F0-mice exposure to permethrin before mating on the F1progeny swimming behavior (direction). a Number of pups swimmed in the straight lines/total number of pups 100; *P 0.05, **P 0.01
3.5. Swimming performance Swimming performance is presented in Figs. 2 and 3. (a) Direction. No significant difference in the percentage of pups swimming in straight lines in the treated group of 4.9 mg/kg/d. The percentage of each pup swimming in straight lines was determined and used for statistical analysis. Exposure of F0-mice to permethrin in the dose groups of 9.8 and 19.6 mg/kg/d before mating significantly slowed the development of swimming behavior as reflected in a prolonged tendency to swim in circles rather than in straight lines. The persistence of circular
Fig. 3. The effect of F0-mice exposure to permethrin before mating on the development of swimming behavior in the F1-progeny (head angle criteria was recorded according to development of pup to raise head higher with age. *Total number of pups on PND6 with nose and top of head out of water = 2. **Total number of pups on PND10 with ears half out of water = 3. ***Total number of pups on PND14 with ears out of water = 4; **P 0.01.
Fig. 4. Effect of F0-mice exposure to permethrin before mating on the F1progeny open field activity on postnatal days 14 and 21. a Average number of squares crossed by pups/minute; *P 0.05, **P 0.01.
swimming on days 10 and 12 was significant for the offspring in the F0-mice groups treated with 9.8 and 19.6 mg/kg/d. (b) Head angle. The development of the neonatal mouse to raise its head higher with age when forced to swim was found to be significantly slower for the offspring in the exposure groups of 9.8 and 19.6 mg/kg/d permethrin compared to the control group. The development of the neonatal head angle was not significantly different in the dose group of 4.9 mg/kg/d compared to the control group. (c) Limb movement. The limb movement of the offspring did not significantly differ from control in any of the treated groups on day 16. The pups typically swim with the hind limbs and kept the forelimbs stationary by postnatal day 16 (negative data not shown here).
Fig. 5. Effect of F0-mice exposure to permethrin before mating on the time spent in social interaction of the F1-progeny (30 days age). Data are reported as means S.D., **P 0.01.
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3.6. Open field activity Open field activity is presented in Fig. 4. Open field activity on days 14 and 21 was significantly affected by exposure of F0male and female mice before mating to 9.8 and 19.6 mg/kg/d permethrin for each minute of the open field activity measurements. No significant effects appeared in the treated group of 4.9 mg/kg/d compared to the control. 3.7. Social interaction Fig. 5 shows that exposure to 9.8 and 19.6 mg/kg/d permethrin reduced the time spent in social interaction for F1-progeny, whereas the 4.9 mg/kg/d dose had no effect compared to the control group. 4. Discussion To our knowledge some studies have been reported about the effects of permethrin on the behavior of adult mammals (AbouDonia et al., 2004; Bloom et al., 1983; Crofen and Reiter, 1988; McDaniel and Moser, 1993; Mitchell et al., 1988; Peele and Crofton, 1987) and this is the first study to evaluate the effects on the behavior of F1-progeny following exposure of parents before mating. In the present study, the NOEL obtained developmental effects of permethrin was 4.9 mg/kg/d. This dose was unable to induce maternal or developmental toxicity compared to the control and other treated groups. Conversely, the 9.8 and 19.6 mg/kg/d were maternally and developmentally toxic, with profound decreases in litter size, pup survival, and pup growth (Tables 1 and 2). These maternal and developmental toxicity on F0-mice can be attributed to the penetration and distribution of permethrin in the body fluids including intracellular water as a result of its high lipid solubility (Anadon et al., 1991). Permethrin rapidly enters the CNS after oral administration, and the brain regions contain high levels of the insecticide (Anadon et al., 1991), leading to a prolonged flow of sodium current into the cell, due to a sustained membrane depolarization (Narahashi, 1985; Bloomquist, 1996). These effects may be associated with maternal and developmental toxicity which found in the middle and high dose groups. The data show significant behavior deviations in F1-pups for the 9.8 and 19.6 mg/kg/d dose groups compared to the control group. These deviations, defined by decreases in the performance of reflexes (Tables 3 and 4; Fig. 1), swimming behavior (Figs. 2 and 3), and locomotion frequencies in the open field (Fig. 4). The reduction in the time spent in active social interaction was also recorded in the same dose groups (Fig. 5). Locomotors frequency, measured in the open field, has been used as an index of both arousal (Iviniskis, 1970), and emotionality (Walsh and Cummins, 1976). Therefore, the effects of permethrin on open-field behaviors may result from an impairment of motor activity. Furthermore, the reduction in the time spent in active social interaction which recorded in the 9.8 and 19.6 mg/kg/d dose groups could be due to the alterations in motor activity (File, 1980; File and Hyde, 1979). Reduction of the performance of swimming has been referred
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to as ‘‘behavioral despair’’, which may be considered as acute depressive reaction in response to inescapable swimming (Porsolt et al., 1978, 1979). The development of swimming behavior as reflected in a prolonged tendency to swim in circles rather than in straight lines. Although the data of the present study provide evidence of the effects of permethrin on the developmental of behavior of F1-progeny, these effects cannot be attributed directly to the developmental neurotoxicity of permethrin on F1-pups. Reported studies indicated that 14C-labeled permethrin and its hydrolysis products are excreted from the body in a relatively short time. Four days after treatment, the residues were below 0.01–0.05 ppm permethrin equivalents in blood, bone, brain, heart, kidney, liver, lungs, muscle, spleen, and testes (Anadon et al., 1991; Elliott et al., 1976; Gaughan et al., 1977; FAO/WHO, 1999). The brain regions also contain the higher areas under the tissue concentration–time curve of the metabolites, mainly m-phenoxybenzyl alcohol, indicating a metabolism system may be present; the metabolites could not enter the brain, presumably due to their polarity (Anadon et al., 1991). In the present study, the middle and high doses induced signs of toxicity including salivation, tremors, and hyperactivity. These clinical signs were typical for permethrin (Bradbury and Coats, 1989; FAO/WHO, 1999). Indeed, the effects on body weight became evident only during gestation and lactation periods for the same dose groups. Therefore, these maternal toxicity may have been the trigger for the decreased pup weight gain in the middle and high dose groups which is about half that of control. Then, the neuromuscular parameters evaluated in the offspring could be altered, as a secondary response to such pronounced stunted growth. Technical permethrin 1R, -cis isomer is responsible for the effects, it is only 40% of the applied doses which were actually given to F0-mice in this study. Thus, the 9.8 and 19.6 mg/kg/d were actually given about 3.8 and 7.9 mg/kg/d, respectively. The 3.8 mg/kg/d dose is about 76 times the acceptable daily intake for humans (ADI = 0.05 mg/kg/d for the 40:60 cis:trans mixture of permethrin) (FAO/WHO, 1999). Although 7.9 (19.6 mg/kg/d) is about 152 times the ADI, we are concerned that such an exposure might particularly occur if the appropriate use of this insecticide is not followed. 5. Conclusion The present data demonstrate that permethrin can produce behavioral alterations in F1-mice offspring for F0-mice toxic doses 9.8 and 19.6 mg/kg/d. These behavioral effects are most likely due to maternal toxicity and stunt of growth of the pups rather than the neurotoxicity of permethrin. No evidence of F0mice toxicity and F1-offspring’s development was observed at 4.9 mg/kg/d. References Abou-Donia MB, Deschovskaia A, Goldstein LB, Abdel-Rahman A, Bullman S, Khan WA. Co-exposure to pyridostigmine bromide, DEET, and/or
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permethrin, causes sensorimotor deficit and alterations in brain acetylcholinesterase activity. Pharmacol Biochem Behav 2004;77:253–62. Adams PM. Effects of perinatal chlordiazepoxide exposure on rat preweaning and post weaning behavior. Neurobehav Toxicol Teratol 1982;4:279–82. Alder S, Zbinden G. Methods for the evaluation of physical, neuromuscular, and behavioral development of rats in early postnatal life. In: Wilson JG, Wurkany J, editors. Teratology: principles and techniques. Chicago, IL: University of Chicago Press; 1965. p. 175–85. Anadon A, Martinez-Larranaga MR, Diaz MJ, Bringas P. Toxicokinetics in the rat. Toxicol Appl Pharmacol 1991;110:1–8. Bloom AS, Staatz CG, Dieringer T. Pyrethroid effects on operant responding and feeding. Neurobehav Toxicol Teratol 1983;5:321–4. Bloomquist JR. Ion channels as targets for insecticides. Ann Rev Entomol 1996;41:163–90. Bradbury SP, Coats JR. Comparative toxicology of the pyrethroid insecticides. Rev Environ Contam Toxicol 1989;108:133–77. Butcher RE, Vorhees CV. A preliminary test battery for the investigation of the behavioral teratology of selected psychotropic drugs. Neurobehav Toxicol 1979;1(Suppl):207–12. Casida JE, Quistad GB. Golden age of insecticide research: past, present, or future? Annu Rev Entomol 1998;43:1–16. Casida JE, Gamman DW, Glickman AH, Lawrence LJ. Mechanisms of selective action of pyrethroid insecticides. Annu Rev Pharmacol Toxicol 1983;23:413–38. Crofen KM, Reiter LW. The effects of type I and II pyrethroids on motor activity and acoustic startle response in the rat. Fundam Appl Toxicol 1988;10:624– 34. De Ray. Pyrethroid insecticides: mechanism of toxicity, systemic poisoning syndromes, parethesia, and therapy. In: Krieger R, Doull J, Ecobichon D, editors. Handbook of pesticide toxicology, volume 1: principals. San Diego: Academic Press; 2001. p. 1289–303. Elliott M, Janes NF, Pulman DA, Stevenson JH. Radiosynthesis and metabolism in rats of 1R isomers of the insecticide permethrin. J Agric Food Chem 1976;24:270–6. Eriksson P, Fredriksson A. Neurotoxic effects of two different pyrethroids, bioallethrin and deltamethrin, on immature and adult mice: changes in behavioral and muscarinic receptor variables. Toxicol Appl Pharmacol 1991;108:78–85. FAO/WHO. Joint meeting of FAO Panel of Experts on Pesticide Residues in Food and the Environment and the WHO Core Assessment Group. Toxicological evaluations: Permethrin; 1999. File SE. The use of social interaction as a method for detecting anxiolytic activity of chlordiazepoxide-like drugs. J Neurosci Methods 1980;2:219– 38. File SE, Hyde JR. A test of anxiety that distinguishes between the actions of benzodiazepines and those of minor tranquilizers and stimulants. Pharmacol Biochem Behav 1979;11:65–9. Gaughan LC, Unai T, Casida JE. Permethrin metabolism in rats. J Agric Food Chem 1977;25:9–17. Gomes MS, Bertnardi MM, De Sousa Spinosa H. Effects of prenatal pyrethroid insecticides on sexual development of rats. Vet Hum Toxicol 1991a;33: 427–8. Gomes MS, Bertnardi MM, De Sousa Spinosa H. Pyrethroid insecticides and pregnancy: effects on physical and behavioral development of rats. Vet Hum Toxicol 1991b;33:315–7.
Iviniskis A. A study of vality of open-field measures. Aus J Physiol 1970;22: 175–83. Kaneko H, Miyamoto J. Pyrethroid chemistry and metabolism. In: Krieger R, Doull J, Ecobichon D, editors. Handbook of pesticide toxicology, volume 2: agents. San Diego: Academic Press; 2001. p. 1263–88. Laurence LJ, Casida JE. Pyrethroid toxicology: mouse interacerebral structure– activity relationships. Pestic Biochem Physiol 1983;18:611–5. Mandhane SM, Chopde CT. Neurobehavioral effects of low level fenvalerate exposure in mice. Indian J Exp Biol 1997;35:623–7. McDaniel KL, Moser VC. Utility of neurobehavioral screening battery for differentiating the effects of two pyrethroids, permethrin and cypermethrin. Neurotoxicol Teratol 1993;15:71–83. Mitchell JA, Wilson MC, Kallman MJ. Behavioral effects of phydrin and ambush in male mice. Neurotoxicol Teratol 1988;10:113–9. Moniz AC, Bernardi MM, Spinosa HS, Palermo-Neto J. Effects of exposure to a pyrethroid insecticide during lactation on the behavior of infant and adult rats. Braz J Med Biol Res 1990;23:45–8. Moniz AC, Bernardi MM, Spinosa HS. Effects of pyrethroid type II pesticides on conditioned behaviors of rats. Vet Hum Toxicxol 1994;36:120–4. Moniz AC, Cruz-Casallas PE, Oliveira CA, Lucisano A, Florio JC, Nicolau AA, et al. Perinatal fenvalerate exposure: behavioral and endocrionology changes in male rats. Neurotoxicol Teratol 1999;21:611–8. Narahashi T. Nerve membrane ionic channels as the primary target of pyrethroids. Neurotoxicology 1985;6:3–22. Narahashi T. Neurophysiological effects of insecticides, volume 1: principals. In: Krieger R, Doull J, Ecobichon D, editors. Handbook of pesticide toxicology, San Diego: Academic Press; 2001. p. . p. 335–50. Norusis M. Statistical package for social science, version 6. USA: SPSS Incorporation; 1994. Pantaleoni GC, Fanini D, Sponta AM, Palumbo G, Giorgi R, Adams PM. Effects of maternal exposure to polychlorobiphenyls (PCBs) on F1 generation behavior in the rat. Fund Appl Toxicol 1988;11:440–9. Peele DB, Crofton KM. Pyrethroid effects on schedule controlled behavior: time and dosage relationship. Neurotoxicol Teratol 1987;9:387–94. Porsolt RD, Anton G, Blave N, Jalfre M. Behavioral despair in rats: a new model sensitive to antidepressant treatments. Eur J Pharmacol 1978;379– 91. Porsolt RD, Bertin A, Blavet M, Deniel M, Jalfre M. Immobility induced by forced swimming in rats: effects of agents which modify central catecholamines and serotonin activity. Eur J Pharmacol 1979;57:201–10. Shafer TJ, Meyer AD, Crofton MK. Developmental neurotoxicity of pyrethroid insecticides: critical review and future research needs. Environ Perspect 2005;113:123–36. Soderlund DM, Clark JM, Sheets LP, Mullin LS, Piccirillo VJ, Sargent D, et al. Mechanisms of pyrethroid neurotoxicity: implications for cumulative risk assessment. Toxicology 2002;171:3–59. Stein EA, Washburn M, Walczak C, Bloom AS. Effects of pyrethroid insecticides on operant responding maintained by food. Neurotoxicol Teratol 1987;9:27–31. Verschoyle RD, Aldridge WN. Structure–activity relationships of some pyrethroids in rats. Arch Toxicol 1980;45:325–9. Walsh RN, Cummins RA. The open field test: a critical review. Psychol Bull 1976;3:482–504. Wilson JG, Wurkany J, editors. Teratology: principles and techniques. Chicago, IL: University of Chicago Press; 1965.
Effects of permethrin given before mating on the behavior of F1-generation in mice Amina T. Farag a,*, Naglaa F. Goda a, Ayman H. Mansee a, Nasra A. Shaaban b a
Department of Pesticide Chemistry and Toxicology, Faculty of Agriculture, Alexandria University, Alexandria, Egypt b Department of Histology, Faculty of Medicine, Alexandria University, Alexandria, Egypt Received 10 August 2005; accepted 12 December 2005 Available online 19 January 2006
Abstract Permethrin, a type I synthetic pyrethroid insecticide, was evaluated through assessment of the behavioral development of F1 progeny of mice. Groups each of 30 male and 30 female ICR (CD-1) mice, as F0-generation, were given 0, 4.9, 9.8, and 19.6 mg/kg/d permethrin by gavage for 4 weeks before mating. Behavioral endpoints of motor reflexes, motor coordination, and activity were evaluated in F1 progeny. Clinical signs of toxicity including salivation, hyperactivity, and liquid feces which attributed to permethrin were observed in the F0-mice treated with 9.8 and 19.6 mg/kg/d. Reduction of body weight became evident only during gestation and lactation periods for the middle and high dose groups. Significant differences in the development of reflexes, swimming ability, and open field activity were evident in the offspring for the 9.8 and 19.6 mg/kg/d dose groups compared to the control group. These results show that permethrin at dose levels of 9.8 and 19.6 mg/kg/d can induce a significant risk to the offspring following treatment of F0-mice before mating. The NOEL obtained in this study for the effects of permethrin on the development of the F1-progeny is 4.9 mg/kg/d. # 2006 Elsevier Inc. All rights reserved. Keywords: Permethrin; Type I pyrethroid; F1-progeny; Behavioral toxicology; Social interactions; Swimming ability
1. Introduction Synthetic pyrethroid insecticides have been used in agriculture and home formulations for more than 30 years and account for approximately one-fourth of the worldwide insecticide market (Casida and Quistad, 1998). The action of pyrethroid insecticides has been divided into two types (I and II) based on signs and neurophysiologic effects (Laurence and Casida, 1983; Verschoyle and Aldridge, 1980). Generally, the two types can be distinguished structurally by the presence (type II) or absence (type I) of an alpha-cyano substituent. The acute mammalian neurotoxicity of pyrethroids has been well characterized, and several comprehensive reviews of pyrethroid toxicity and actions are available (De Ray, 2001; Kaneko and Miyamoto, 2001; Narahashi, 2001; Shafer et al., 2005; Soderlund et al., 2002). Aspects of
* Corresponding author at: Department of Pesticide Chemistry and Toxicology, Faculty of Agriculture (El-Shatby), University of Alexandria, Egypt. Tel.: +203 5915160; fax: +203 5915160. E-mail address: [email protected] (A.T. Farag). 0161-813X/$ – see front matter # 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.neuro.2005.12.001
open-field behavior and catalepsy (Mandhane and Chopde, 1997), conditioned behavior (Moniz et al., 1994); operant behavior (Stein et al., 1987), schedule-controlled behavior (Peele and Crofton, 1987), motor activity (McDaniel and Moser, 1993), and others can be altered by pyrethroid exposure in adult animals. Neonatal exposure of mice to deltamethrin and bioallethrin increased motor activity in adult age and decreased the density of cortical cholinergic muscarinic receptor (Eriksson and Fredriksson, 1991). Maternal exposure to cyhalothrin during lactation disrupted passive avoidance learning during adulthood (Gomes et al., 1991a), and delayed the descent of the tests in male offspring (Gomes et al., 1991b). Fenvalerate, a type II pyrethroid, was capable of interfering with reproductive parameters of male and female rats (Moniz et al., 1999), and affected the developmental of physical and behavior in infant and adult rats (Moniz et al., 1990). Permethrin [3-(2,2-dichloroethenyl)-2,2-dimethylcylo-propane carboxylic acid (3-phenoxyphenyl) methyl ester], a type I synthetic pyrethroid insecticide, is a mixture of four (1R, S-cis and 1R, S-trans) isomers, only one of which (1R, -cis) has lethal effects in mammals (Casida et al., 1983). It provides
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insecticidal activity for several weeks following a single application and is used to control fleas, flies, mites, and cockroaches (Abou-Donia et al., 2004). Permethrin causes modification of sodium channels leading to prolonged depolarization and repetitive discharges in presynaptic nerve fibers after a single stimulus (Bloomquist, 1996; Narahashi, 1985). This repetitive nerve action is associated with tremor, hyperactivity, ataxia, convulsions, and in some cases paralysis. At low dosage-dependent decrease in locomotor activity and an increase in the amplitude of the startle response to an acoustic stimulus following exposure of rats to repeated doses of permethrin for 30 days (Crofen and Reiter, 1988). Exposure of male mice to Ambush (25.6% permethrin) for 30 min increased activities like chewing (Mitchell et al., 1988). In addition rats exposure to permethrin at two time points on the day of dosing increased aggressive behavior, agitation, resistance to being captured, reactivity to a click stimulus, and induced head and forelimb shaking (McDaniel and Moser, 1993), and disrupted a learning feeding behavior in rats at doses about 20% of LD50 after repeated doses for 30 days (Peele and Crofton, 1987). Furthermore, permethrin can have significant effects on learned behavior, food intake, and decrease in operant response rate in rats after exposure for 20 min (Bloom et al., 1983). Exposure of rats to permethrin for 60 days led to sensorimotor deficits and differential aberrations of the cholinergic system in the CNS (Abou-Donia et al., 2004). There is no information regarding the potential effects of permethrin on the behavior and physical development endpoints on the progeny. The aim of the present study was to evaluate the effects of F0male and female mice exposure to permethrin before mating for 20 days on the development of physical and behavioral aspects in the offsprings at dose levels: one that induces some toxicity on F0-mice, one unable to induce toxicity and an intermediate one. These doses were approximately equal to 1/100, 1/50, and 1/25 of acute oral LD50 for permethrin (40:60 cis/trans) in mice (FAO/WHO, 1999). 2. Material and methods 2.1. Test material Technical grade permethrin (60:40, trans:cis; 94% purity) was kindly donated by El-Watania, Inc., Alexandria, Egypt. 2.2. Test species and husbandry One hundred and twenty Male and 120 female ICR (CD-1) mice, approximately 10 weeks old, were obtained from the High Institute of Public Health, Alexandria University, Alexandria, Egypt. Mice were examined for health status and acclimated to the laboratory environment for 2 weeks prior to use. The animal room was designed to maintain temperature 23 2 8C, relative humidity at approximately 50%, and a 12 h light:12 h dark photoperiod. All animals were housed in stainless-steel cages and given standard diet and water ad libitum throughout the study.
2.3. Breeding and treatment The experimental protocol was approved by the University Animal Care Committee. Male and female mice were divided into treatment groups according to approximately equal mean body weight. Groups each of 30 male and 30 female mice (F0-mice) were given permethrin by gavage at dose levels of 0 (corn oil), 4.9, 9.8, and 19.6 mg/kg/d before mating 5 days/ week for 4 weeks. Solution concentrations were adjusted so that 30 g mouse received 0.2 ml corn oil. After treatment females were mated with males from the same treated groups (one male/one female). The day that a vaginal plug was found was considered day 0 of gestation. 2.4. Clinical observations and body weights F0-permethrin- treated and control mice were observed daily (between 9:00 and 10:00 a.m.) immediately after each permethrin treatment or control solution for the signs of toxicity which including tremors, in coordination, hyperactivity, salivation, paralysis, and other signs were typical for type I of pyrethroids (Bradbury and Coats, 1989). F0-treated females were observed daily for signs of toxicity through the gestation and lactation periods. Each sign was assessed by observing the spontaneous behavior of the mice in the cage. Body weight was measured weekly in the animals of all the study groups throughout the period of the treatment. Maternal body weight was recorded on days 0 and 18 of gestation, and on days 0 and 28 during lactation period. Food and water consumption were recorded daily for males and females throughout the period of the treatment, gestation, and lactation. The day of birth was identified as postnatal day 0 (PND0). Postnatal survival was monitored daily before weaning (day 28), and on day 30 after weaning. 2.5. Offspring studies All the pregnant mice were allowed to give birth (F1generation) and nurture their offspring normally. Parturition day was determined to be PND0. On PND1, all the pups were examined externally and weighed. Pups were weighed at PND1, PND7, PND21 and PND28. 2.5.1. Physical development parameters Each day, beginning on PND1, the pups as mixed sex were observed for the following physical parameters: down appearance, pinna detachment, incisor eruption, development of fur, eye opening, auditive channel opening, testes descent, and vaginal opening. The day of appearance of these landmarks was recorded and pup means were calculated. 2.5.2. Behavioral parameters The following reflexes and behavioral parameters were assessed in all of the pups as mixed sex: surface righting, cliff avoidance, negative geotaxis, swimming behavior, and open field activity. To assess the behavioral development of progeny, pups were observed daily, between 9:00 and 10:00 a.m.,
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separated from the mothers at the moment of observation, and immediately returned to their home cages. Each of behavior endpoints should be evaluated at the specific time of development during the preweaning period. 2.5.2.1. Surface righting reflex. The ability of the young mouse to right itself was determined on three daily trails beginning on postnatal day 3 (PND3–PND7). The pups were placed on their backs on a smooth wooden surface and the time required to right them to a position where all four feet touched the surface was recorded. A criterion of successful righting within 2 s was used (Alder and Zbinden, 1965; Butcher and Vorhees, 1979; Pantaleoni et al., 1988). 2.5.2.2. Negative geotaxis reflex. Negative geotaxis was assessed by recording the time required for neonatal mouse to reorient from a head-down to a head up position on a 258 inclined plane. The plane was made of wood and each test mouse was given one trial on PNDS 10 and 15, with a maximum time allowance of 60 s per trial. Pups sliding off the plane were tested again. The time required to complete a 1808 turn was the dependent measure (Butcher and Vorhees, 1979; Pantaleoni et al., 1988; Wilson and Wurkany, 1965). 2.5.2.3. Cliff avoidance reflex. Beginning on PND4 each pup was placed on a wooden platform elevated 20 cm above a table top. The forepaws and snout of the animal were positioned so that the edge of the platform passed just behind an imaginary line drawn between the eye orbits. The latency required for a retraction of the head completely behind the edge of the platform was daily recorded (Butcher and Vorhees, 1979; Pantaleoni et al., 1988; Wilson and Wurkany, 1965). A criterion for a retraction response of 20 s was used. If the animal was not successful within 60 s the trial was terminated, and a maximum of 60 s was recorded. If the animal fell it was given a second attempt and if it fell again, a latency of 60 s was recorded. All the pups were tested daily on PNDS 4, 6, 8, and 10 regardless of whether they had reached criterion (Adams, 1982).
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2.5.4. Open field activity On PNDS 14 and 21 open fields activity was measured. The pups were placed in the center of a 45-cm Plexiglas circle for three 1-min periods separated by 30 s between each period. The circle was divided into three concentric circles with each quarter divided into six areas. The number of squares crossed by pups per each minutes (entries) was recorded (Butcher and Vorhees, 1979; Pantaleoni et al., 1988; Wilson and Wurkany, 1965). 2.6. Social interaction The social interaction test was carried out in the open field. Observation of the social interaction between pups was done after weaning (day 30 of age) to avoid the effects of separating pups from dams for such a long time. Briefly, F1-mice were housed singly for 5 days prior to testing, being matched to form pairs (from the same treated group) according to their weight. On the day of the social interaction test, the total time in seconds spent by each pair of F1-mice in active social interaction (sniffing, following, grooming, kicking, boxing, biting, and crawling under or over partner) was scored during a 7.5 min test session (File, 1980; File and Hyde, 1979). 2.7. Statistical analysis The mean S.D. of the litter for the behavioral assessment was used as the unit of measurement in the most of the analyses. For surface righting, cliff avoidance, and swimming the percentage of the litter reaching the criterion for the behavioral was used. MANOVA was used to analyze data of control and experimental groups in the comparison according to control and treatment values. ANOVA was employed followed by the Tukey test as post hoc tests compare data presenting interaction; data without interaction were analyzed by a conventional t-test and nonparametric x2-test or Fisher’s least significant difference test (Norusis, 1994). In all the experiments P 0.05 was the criterion for statistical significance. 3. Results
2.5.3. Swimming The ability of the neonatal mouse to swim was assessed in a 30 cm 30 cm plastic tank with the water temperature at 24 8C. On postnatal days 6, 8, 10, 12, 14, and 16 each animal was placed into the tank and swimming behavior was rated for direction (straight = 3, circling = 2, floating = 1). Head angle was rated according to the development of the neonatal mouse to raise head higher with age. The percentage of pups on PND6 was recorded as number of pups with nose and top of the head out of the water = 2; PND10 with ears half out of the water = 3; PND14 with ears out of the water = 4. Limb movement was rated as either 1 = all four limbs used, or 2 = back limbs only used. Latency to being limb movement after being placed in the water was also recorded. The animals were given a maximum of 15 s in the water and were then removed to the warm towel to dry (Butcher and Vorhees, 1979; Pantaleoni et al., 1988; Wilson and Wurkany, 1965).
3.1. Clinical observations There were two, three, two, and one of F0-males died in the groups of control, 4.9, 9.8, and 19.6 mg/kg/d, respectively, during the course of the treatment. Two of F0-females of the treated group 4.9 mg/kg/d died during the course of the treatment. Although there was no death in the pregnant females, two, one, four, and four females died in the control, 4.9, 9.8, and 19.6 mg/kg/d, respectively, during the lactation period. Administration of 4.9 mg/kg/d of permethrin dose did not produce any clinical signs of toxicity in the F0-mice. However, treatment with 9.8 and 19.6 mg/kg/d doses induced signs of toxicity that included hyperactivity, salivation, tremors, and liquid feces; these four signs started to appear in 60% of F0males and 40% of F0-females and in 80% of F0-males and 60% of F0-females in dose groups of 9.8 and 19.6 mg/kg/d,
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in the development of physical features assessment of pinna detachment, primary coat of down hair, incisor eruption, development of fur, eye opening, auditive channel opening, testes descent, and vaginal opening in any of the treated groups compared to the control (negative data not shown here).
respectively. These effects appeared on day 10 of treatment and more pronounced between 3 and 5 h after each oral administration. These responses disappeared in the treated F0-females through both gestation and lactation periods. 3.2. Physical development parameters
3.3. F1-progeny parameters
Body weight and weight gain are presented in Table 1. No differences were noted for F0- male and female body weights during the period of the treatment compared to the control group. Maternal body weights were significantly reduced at the end of the gestation and during lactation periods in the groups treated with 9.8 and 19.6 mg/kg/d compared to the control group. There were no significant changes in the water and food consumption in any of the treated groups for the F0-mice and F1-progeny after weaning throughout the study period. No external abnormalities were observed for the pups in any of the treated groups and no gross difference was found
F1-progeny parameters are presented in Table 2. While the number of live pups was decreased for the middle and high treated groups, the number of dead pups was increased in the same groups. Pups growth retardation was observed in the treated groups of 9.8 and 19.6 mg/kg/d compared to the control group. Starting growth retardation was observed from birth of pups. No statistically significant effects were found in any of the F1-progeny parameters in the treated group of 4.9 mg/kg/d compared to the control group.
Table 1 Body weight and weight gain (g) in F0-mice after exposure to permethrin before mating period Permethrin (mg/kg/d) 0
4.9
9.8
19.6
Before mating (treatment period) Day 1 of treatment Body weight (g) Males Females
39.2 0.2 30.0 0.4
40.5 0.9 29.4 0.4
41.2 0.3 29.9 0.5
40.0 0.7 30.6 0.6
End of treatment Body weight (g) Males Females
42.5 0.5 31.2 0.3
43.5 0.8 30.7 0.5
44.9 0.7 31.4 0.4
43.7 0.2 32.0 0.3
3.3 1.0 1.2 0.7
3.0 0.9 1.3 0.6
3.7 0.8 1.5 0.5
3.7 1.0 1.7 0.9
Body weight gain (g) Males Females Number of females Number of litters
During gestation period 30 26
28 23
30 25
30 18
Maternal body weight (g) Gda 0 18
32.3 0.6 44.2 0.9
31.8 0.7 42.6 0.6
31.2 0.8 36.3 0.6*
31.1 0.4 35.5 0.9*
Maternal weight gain (g)
11.9 1.2
10.8 1.3
5.1 0.9 **
4.4 1.5**
During lactation period Number of dams Ldb 0 28
26 24
23 22
25 21
18 14
Maternal body weight (g) Ldb 0 28
32.1 0.6 34.4 0.4
33.4 0.9 36.2 0.1
26.4 0.7 ** 24.4 0.3 **
25.8 0.5** 23.1 0.4**
Maternal weight gain (g)
2.3 1.9
2.8 0.9
2.0 1.7 **
2.7 2.0**
Data are presented as mean S.D. a Days of gestation. b Days of lactation. * Significantly different from control value at P 0.05. ** Significantly different from control value at P 0.01.
A.T. Farag et al. / NeuroToxicology 27 (2006) 421–428 Table 2 Parameters of mice pups after F0-mice exposure to permethrin before mating Permethrin (mg/kg/d)
Number of litters Number of pupsa Number of live pups (%) a Number of dead pups (%) a
4.9
9.8
19.6
26 210 197 (94)
23 201 183 (91)
25 214 178 (83)*
18 106 62 (58)**
13 (6)
18 (9)
36 (17)*
44 (42)**
1.99 0.5 3.80 0.6 12.11 0.7 14.77 0.9 12.78 0.9
1.90 0.9 3.99 1.0 12.02 0.8 14.30 0.5 12.40 1.0
Body weight (g)/d Developmental days 0 7 21 28 Body weight gain (g)/d
1.29 0.7 * 1.60 0.8 * 6.43 0.9 ** 7.48 0.6 ** 6.19 0.5 **
Table 4 Effects of F0-mice exposure to permethrin before mating on the development of negative geotaxis Reflex in F1-progeny Days
0
1.20 0.1 * 1.44 0.8 * 5.11 0.7 ** 6.36 0.3 ** 5.16 0.8 **
Data are presented as mean S.D. a Data are based on PND0. * Significantly different from control value at P 0.05. ** Significantly different from control value at P 0.01.
3.4. Reflexes of offsprings 3.4.1. Surface righting Development of self-righting reflex of mice pups is presented in Table 3. F0-mice exposed to 4.9 mg/kg/d permethrin before mating did not significantly affect the development of surface righting ability in the 3–7-day-old neonates as reflected by percentage of pups reaching criterion (2 s) compared to the control value. The development of surface righting ability was reduced in the 3–7-day-old neonates in the treated groups of 9.8 and 19.6 mg/kg/d compared to the control group.
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10 15
Permethrin (mg/kg/d) 0 (number of pups = 197)
4.9 (number of pups = 183)
9.8 (number of pups = 178)
19.6 (number of pups = 62)
39.9 10 10.2 5
38.5 10 11.5 9
66.8 12** 32.5 7**
78.9 11** 40.9 9**
Data are presented as mean S.D. of latencies in seconds. ** Significantly different from control value at P 0.01.
group of 4.9 mg/kg/d. Mean latencies (S.D.) to reorient results were statistically different from the control value in the dose groups of 9.8 and 19.6 mg/kg/d compared to the control group. 3.4.3. Cliff avoidance Cliff avoidance in the F1-progeny is shown in Fig. 1. The results of the statistical analysis of acquisition of cliff avoidance in the F1-progeny of treated mice were not significant in the treated group of 4.9 mg/kg/d compared to the control group. Treatment of F0- male and female mice with 9.8 and 19.6 mg/kg/d permethrin before mating significantly slowed the development of the cliff avoidance performance on days 4–10 compared to the control group. There were highly significant lower percentage values in both permethrin exposed groups of 9.8 and 19.6 mg/kg/d on postnatal days 8 and 10.
3.4.2. Negative geotaxis The data of the ability to show geotaxis is presented in Table 4. The ability of days 10 and 15 neonates to show negative geotaxis was not significantly altered in the dose
Table 3 Effects of F0-mice exposure to permethrin before mating on the development of self-righting reflex in F1-progeny Days
Permethrin (mg/kg/d) 0
4.9
9.8
19.6
a.r.c./Na 3 4 5 6 7
(%) 57/198 (29) 120/198 (61) 169/197 (86)b 180/197 (91) 181/197 (92)
44/183 (24) 99/183 (54) 140/183 (77) 166/183 (91) 164/183 (90)
30/179 (17)* 80/179 (45)* 100/178 (56)b,** 107/178 (60)** 109/178 (61)**
10/76 22/76 29/62 30/62 31/62
(13)** (29)** (47)b,** (48)** (50)**
a Number of animals reaching the criterion (2 s)/total number of tested animals. b Number reduced according to the dead number of pups during the test period. * Significantly different from control value at P 0.05. ** Significantly different from control value at P 0.01.
Fig. 1. The effects of F0-mice exposure to permethrin before mating on the developmental cliff avoidance reflex in the F1-progeny. a Number of pups retracted the head completely behind the edge of the platform/total number of pups 100; *P 0.05, **P 0.01.
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Fig. 2. The effect of F0-mice exposure to permethrin before mating on the F1progeny swimming behavior (direction). a Number of pups swimmed in the straight lines/total number of pups 100; *P 0.05, **P 0.01
3.5. Swimming performance Swimming performance is presented in Figs. 2 and 3. (a) Direction. No significant difference in the percentage of pups swimming in straight lines in the treated group of 4.9 mg/kg/d. The percentage of each pup swimming in straight lines was determined and used for statistical analysis. Exposure of F0-mice to permethrin in the dose groups of 9.8 and 19.6 mg/kg/d before mating significantly slowed the development of swimming behavior as reflected in a prolonged tendency to swim in circles rather than in straight lines. The persistence of circular
Fig. 3. The effect of F0-mice exposure to permethrin before mating on the development of swimming behavior in the F1-progeny (head angle criteria was recorded according to development of pup to raise head higher with age. *Total number of pups on PND6 with nose and top of head out of water = 2. **Total number of pups on PND10 with ears half out of water = 3. ***Total number of pups on PND14 with ears out of water = 4; **P 0.01.
Fig. 4. Effect of F0-mice exposure to permethrin before mating on the F1progeny open field activity on postnatal days 14 and 21. a Average number of squares crossed by pups/minute; *P 0.05, **P 0.01.
swimming on days 10 and 12 was significant for the offspring in the F0-mice groups treated with 9.8 and 19.6 mg/kg/d. (b) Head angle. The development of the neonatal mouse to raise its head higher with age when forced to swim was found to be significantly slower for the offspring in the exposure groups of 9.8 and 19.6 mg/kg/d permethrin compared to the control group. The development of the neonatal head angle was not significantly different in the dose group of 4.9 mg/kg/d compared to the control group. (c) Limb movement. The limb movement of the offspring did not significantly differ from control in any of the treated groups on day 16. The pups typically swim with the hind limbs and kept the forelimbs stationary by postnatal day 16 (negative data not shown here).
Fig. 5. Effect of F0-mice exposure to permethrin before mating on the time spent in social interaction of the F1-progeny (30 days age). Data are reported as means S.D., **P 0.01.
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3.6. Open field activity Open field activity is presented in Fig. 4. Open field activity on days 14 and 21 was significantly affected by exposure of F0male and female mice before mating to 9.8 and 19.6 mg/kg/d permethrin for each minute of the open field activity measurements. No significant effects appeared in the treated group of 4.9 mg/kg/d compared to the control. 3.7. Social interaction Fig. 5 shows that exposure to 9.8 and 19.6 mg/kg/d permethrin reduced the time spent in social interaction for F1-progeny, whereas the 4.9 mg/kg/d dose had no effect compared to the control group. 4. Discussion To our knowledge some studies have been reported about the effects of permethrin on the behavior of adult mammals (AbouDonia et al., 2004; Bloom et al., 1983; Crofen and Reiter, 1988; McDaniel and Moser, 1993; Mitchell et al., 1988; Peele and Crofton, 1987) and this is the first study to evaluate the effects on the behavior of F1-progeny following exposure of parents before mating. In the present study, the NOEL obtained developmental effects of permethrin was 4.9 mg/kg/d. This dose was unable to induce maternal or developmental toxicity compared to the control and other treated groups. Conversely, the 9.8 and 19.6 mg/kg/d were maternally and developmentally toxic, with profound decreases in litter size, pup survival, and pup growth (Tables 1 and 2). These maternal and developmental toxicity on F0-mice can be attributed to the penetration and distribution of permethrin in the body fluids including intracellular water as a result of its high lipid solubility (Anadon et al., 1991). Permethrin rapidly enters the CNS after oral administration, and the brain regions contain high levels of the insecticide (Anadon et al., 1991), leading to a prolonged flow of sodium current into the cell, due to a sustained membrane depolarization (Narahashi, 1985; Bloomquist, 1996). These effects may be associated with maternal and developmental toxicity which found in the middle and high dose groups. The data show significant behavior deviations in F1-pups for the 9.8 and 19.6 mg/kg/d dose groups compared to the control group. These deviations, defined by decreases in the performance of reflexes (Tables 3 and 4; Fig. 1), swimming behavior (Figs. 2 and 3), and locomotion frequencies in the open field (Fig. 4). The reduction in the time spent in active social interaction was also recorded in the same dose groups (Fig. 5). Locomotors frequency, measured in the open field, has been used as an index of both arousal (Iviniskis, 1970), and emotionality (Walsh and Cummins, 1976). Therefore, the effects of permethrin on open-field behaviors may result from an impairment of motor activity. Furthermore, the reduction in the time spent in active social interaction which recorded in the 9.8 and 19.6 mg/kg/d dose groups could be due to the alterations in motor activity (File, 1980; File and Hyde, 1979). Reduction of the performance of swimming has been referred
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to as ‘‘behavioral despair’’, which may be considered as acute depressive reaction in response to inescapable swimming (Porsolt et al., 1978, 1979). The development of swimming behavior as reflected in a prolonged tendency to swim in circles rather than in straight lines. Although the data of the present study provide evidence of the effects of permethrin on the developmental of behavior of F1-progeny, these effects cannot be attributed directly to the developmental neurotoxicity of permethrin on F1-pups. Reported studies indicated that 14C-labeled permethrin and its hydrolysis products are excreted from the body in a relatively short time. Four days after treatment, the residues were below 0.01–0.05 ppm permethrin equivalents in blood, bone, brain, heart, kidney, liver, lungs, muscle, spleen, and testes (Anadon et al., 1991; Elliott et al., 1976; Gaughan et al., 1977; FAO/WHO, 1999). The brain regions also contain the higher areas under the tissue concentration–time curve of the metabolites, mainly m-phenoxybenzyl alcohol, indicating a metabolism system may be present; the metabolites could not enter the brain, presumably due to their polarity (Anadon et al., 1991). In the present study, the middle and high doses induced signs of toxicity including salivation, tremors, and hyperactivity. These clinical signs were typical for permethrin (Bradbury and Coats, 1989; FAO/WHO, 1999). Indeed, the effects on body weight became evident only during gestation and lactation periods for the same dose groups. Therefore, these maternal toxicity may have been the trigger for the decreased pup weight gain in the middle and high dose groups which is about half that of control. Then, the neuromuscular parameters evaluated in the offspring could be altered, as a secondary response to such pronounced stunted growth. Technical permethrin 1R, -cis isomer is responsible for the effects, it is only 40% of the applied doses which were actually given to F0-mice in this study. Thus, the 9.8 and 19.6 mg/kg/d were actually given about 3.8 and 7.9 mg/kg/d, respectively. The 3.8 mg/kg/d dose is about 76 times the acceptable daily intake for humans (ADI = 0.05 mg/kg/d for the 40:60 cis:trans mixture of permethrin) (FAO/WHO, 1999). Although 7.9 (19.6 mg/kg/d) is about 152 times the ADI, we are concerned that such an exposure might particularly occur if the appropriate use of this insecticide is not followed. 5. Conclusion The present data demonstrate that permethrin can produce behavioral alterations in F1-mice offspring for F0-mice toxic doses 9.8 and 19.6 mg/kg/d. These behavioral effects are most likely due to maternal toxicity and stunt of growth of the pups rather than the neurotoxicity of permethrin. No evidence of F0mice toxicity and F1-offspring’s development was observed at 4.9 mg/kg/d. References Abou-Donia MB, Deschovskaia A, Goldstein LB, Abdel-Rahman A, Bullman S, Khan WA. Co-exposure to pyridostigmine bromide, DEET, and/or
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