Effects of perinatal ivermectin exposure on behavioral development of rats

Neurotoxicology and Teratology, Vol. 10, pp. 26%272. ©Pergamon Press plc, 1988. Printed in the U.S.A.

0892-0362/88 $3.00 + .00

Effects of Perinatal Ivermectin Exposure on Behavioral Development of Rats JEAN-MICHEL

POUL

Laboratoire National des Mddicaments Vdtdrinaires, Javend, 35133 Fougkres, France R e c e i v e d 23 J u l y 1987 POUL, J.-M. Effects of perinatal ivermectin exposure on behavioral development of rats. NEUROTOXICOL TERATOL 10(3) 267-272, 1988.--The antiparasitic agent ivermectin was administered to pregnant rats from days 6 to 20 of gestation and 2 to 20 of lactation at 1, 2 or 4 mg/kg/day. Reproductive performance and behavior of offspring in the preweaning period were observed. Ivermectin had no effect on reproductive performance and dams' growth. The highest dose induced 100% pup mortality. Later on, ivermectin at 4 mg/kg was administered only during gestation. At that dose, the drug induced 22% mortality and affected temporarily cliff avoidance, locomotion, negative geotaxis and swimming development. At 2 mg/kg, the drug induced offspring mortality (31%), retarded growth and delayed eye opening, cliff avoidance and surface righting reflex, negative geotaxis, locomotion and swimming development. Ivermectin at 1 mg/kg had no effect on mortality and growth but cliff avoidance and locomotion were retarded. Data suggest that newborn rats were highly susceptible to the neurotoxic action of ivermectin. Whether its effects were prenatal or by acute intoxication via mother's milk are discussed. Ivermectin

Behavioral toxicology

Behavioral teratology

I V E R M E C T I N , a semisynthetic macrocyclic lactone antibiotic, is a broad spectrum antiparasitic agent active against nematodes and arthropods (insects, ticks and mites) [4,5]. It was used primarily in the control of parasitic infections of domestic animals, and is now undergoing clinical trials in humans for the treatment of onchocerciasis, one of the leading causes of blindness in Africa and South America [7, 14, 15]. Because of its high potent antiparasitic activity, therapeutic subcutaneous and oral doses of ivermectin were as low as 0.2 to 0.3 mg/kg in most animal species. After a single administration, high concentrations are maintained in tissues leading to persistent anthelmintic activity for prolonged periods [3]. Ivermectin is believed to act as a GABA-ergic substance, by stimulating the neurotransmitter binding to membrane receptors [11]. In susceptible nematodes and arthropods, the drug induced the blockade of GABA-mediated transmission of nerve signal and the paralysis of parasites. Ivermectin appears to have a wide safety margin in domestic animals (20 times the recommended dose). Higher doses produced central nervous system depression, listlessness, ataxia and recumbency in experimental and target animals [4]. Only a few cases of human overdoses have been reported, indicating local irritation, vomiting, hypotension, hypothermia and transient asthenia and dizziness [8]. Ivermectin was contraindicated to pregnant women although the drug has been found not to be teratogenic in rabbits and rats at doses smaller than 1.5 mg/kg [2]. Abnormalities in fetal development occurred only when the drug was given at or near the maternotoxic dosage [5]. In an early experiment in which ivermectin was incorporated in pregnant rat diet at 25 ppm (about 5 mg/kg body weight) daily from day 2 of gestation, we found that all the newborns died one or two days after parturition. An en-

Rats

Antiparasitic agent

hanced sensitivity of neonatal rats via maternal milk when the blood-brain barrier is incompletely formed has been reported by Robertson (unpublished observation mentioned by Aziz [2]). These data may indicate that neonates are more susceptible than adult rats to the toxic effects of ivermectin. Since ivermectin's main toxic effects involved the central nervous system in mammals with particular sensitivity in the postnatal period, the present experiment was undertaken to study the neurotoxic effects of the antiparasitic drug administered to female rats during gestation and lactation. Reproductive performance, viability, growth rate and behavioral preweaning development of offspring were observed. Postnatal behavior was evaluated by standardized tests of the Cincinnati Psychoteratogenicity screening test battery as proposed by Vorhees [16]. METHOD

Subjects and Treatments Male (340--400 g) and female (230-270 g) Sprague-Dawley rats (Iffa-Credo, France) were used. Animals were housed in an air conditioned room and fed ad lib with U A R A03 rodent diet. Virgin females were mated overnight with males. Vaginal smears were taken in the morning and the presence of sperm was considered as day 0 of gestation. After mating, females were randomly assigned to one of the four treatment groups: control, ivermectin 1 mg/kg, ivermectin 2 mg/kg and ivermectin 4 mg/kg body weight. The drug (Merck Sharp and Dohme BV, Haarlem, Holland) was dissolved in peanut oil and administered daily by stomach tube in a volume of 1 ml/kg from days 6 to 20 of gestation and days 2 to 20 of lactation in the ivermectin 1 and 2 mg/kg groups. Control rats were gavaged with peanut oil. In the

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268

TABLE 1 GROWTHAND FOOD INTAKE OF DAMSDURING GESTATION,REPRODUCTIVE PERFORMANCEAND OFFSPRINGMORTALITY Dams

Control (11) Ivermectin 1 mg/kg (11) Ivermectin 2 mg/kg (11) Ivermectin 4 mg/kg (13)

Growth Between Day 1 to Day 21 of Gestation (g)

Food Intake Between Day 1 to Day 21 of Gestation (g)

Length of Gestation (days)

Total

Males

Females

Ratio of Males to Females

141.3 -+3.9 141.7 -+6.4

482.4 -+13.4 467.7 -+13.0

21.9 -+0.1 21.7 -+0.2

14.5 -+0.5 14.6 -+0.7

7.9 -+0.6 7.9 -+0.5

6.6 -+0.9 6.6 -+0.7

1.19 (87/73) 1.19 (87/73)

147.4 -+5.7

487.7 -+12.7

21.7 -+0.2

14.8 -+0.7

7.4 -+0.6

7.0 -+0.4

1.05 (81/77)

30.7~:

139.2 -+9.9

480.5 -+16.7

21.6 -+0.2

14.7 -+0.4

7.2 -+0.4

7.1 -+0.6

1.01 (93/92)

22.2"~:

Number of Pups per Litter (Day 1)

Offspring Mortality Before Weaning % 0

1.2

100re

Numbers in parentheses indicate number of litters per group. *Offspring mortality in litters treated during gestation only (7 litters). tOffspring mortality in litters treated during gestation and lactation (6 litters). ~tSignificantly different from control at p~<0.05 or beyond.

highest dose group (ivermectin 4 mg/kg), treatment was administered only during gestation in 7/13 females, because ivermectin was shown highly toxic during lactation, inducing the death of all pups some days after parturition in the first litters treated. Dams' body weight and food consumption were recorded at days 0, 5, 10, 12, 15, 18 and 20 of gestation and days 1, 7, 14 and 21 of lactation. On postnatal day 1, offspring were weighed, sexed and reduced to 10 pups, 5 males and 5 females when possible. In each litter, two males and two females were selected by randomization and marked with black ink; developmental measures were recorded on these pups until weaning.

Swimming ontogeny was observed every two days from postnatal days 5 to 20. The three aspects of swimming development, namely direction, head position above water and use of limbs, were scored after an observation period of 5-15 sec, the test pups being totaly immersed except for the nose in warmed water. Rotorod performance was measured on postnatal day 19. Each test rat was given 3 consecutive trials, where it was placed on a 3 cm diameter rod which was gradually accelerated until 25 rpm. The speed at which the rat fell from the wheel was noted. Open field activity was recorded on day 20. The test rat was placed in the center of a circular arena (50 cm diameter) and observed during 5 rain for crossed sectors, rearing, grooming and defecations.

Behavioral Testing Behavioral development of pups was assessed in a battery of tests that were extensively described by Vorhees et al. [17-19]. Physical landmarks observed were pinna detachment, incisor eruption and eye opening beginning on postnatal days 1, 8 and 12, respectively. Cliff avoidance was recorded dally from day 2. Surface righting reflex was measured dally from day 3 as a mean of 3 trials (30 see maximum) per day, until pups could fight themselves in a time <2 sec for two consecutive days. Locomotion was observed from postnatal day 1 until day 12. Pivoting was quantified daily by the number of 90 ° turns and time spent rotating. Forward locomotion was measured by the moving time during a 60 see observation period. Negative geotaxis development was assessed in one trial per day (60 sec maximum) from postnatal days 3 to 12. The latency before the onset of rotation and the time to rotate 180 ° were noted.

Data Analysis Statistical analysis of data was performed by analysis of variance procedures or the nonparametric test of Kruskal and Wallis [13]. The minimum level of change accepted for significance was p ~ 0 . 0 5 . The litter was used as the subject instead of individual pups. In the tables, results are presented as mean and standard deviation. RESULTS

Growth and Reproduction Treatment with ivermectin (1, 2 and 4 mg/kg) from days 6 until 20 of gestation had no effect on growth and food intake of pregnant rats, and no significant differences were found in the length o f gestation, number of pups per litter and sex ratio in the different groups (Table 1). In the litters of rats treated with the highest dose of ivermectin (4 mg/kg) during gestation and from day 2 of lacta-

D E V E L O P M E N T E F F E C T S OF P E R I N A T A L IVERMECTIN

269

TABLE 2 BODY WEIGHT(g) EVOLUTIONOF DAMSAND PUPS DURINGLACTATIONPERIOD Offspring Dams Day 1 Control (11) Ivermectin 1 mg/kg (11) Ivermectin 2 mg/kg (11) Ivermectin 4 mg/kg (13)

Day 7

Males

Day 14

Day 21

Females

Day 1

Day 7

Day 14

Day 21

Day 1

Day 7

17.5 ±0.4 17.1 ___0.4

33.3 ±0.6 32.9 ±0.6

56.2 ---0.8 54.9 ±1.0

6.58 ±0.14 6.56 ±0.14

Day 14

Day 21

17.2 ±0.5 16.9 ---0.3

32.5 ±0.8 32.4 -+0.6

55.3 ±1.1 53.4 ±0.7

15.7 ±0.7

26.3:~ -+2.6

45.5:~ ±3.8

30.0* ±1.3

49.2* ±1.8

302.6 ±6.0 301.6 ±6.9

328.4 ±6.5 326.2 ±6.9

342.3 ±6.6 343.0 ±6.9

326.2 ---6.0 326.3 ±7.2

6.95 ±0.14 6.76 ±0.13

303.3 ±8.2

330.2 -+8.6

331.1 ±10.2

331.0 ±6.8

6.82 16.4 -+0.20 -+0.7

27.4~: ±2.4

50.3 -+3.1

6.44 ±0.19

301.7 ±8.8

317.0 -+9.2

321.7 ±5.0

309.7 ±5.8

6.59* ±0.12

30.8* ±1.0

51.3" ±1.5

6 . 1 - 4 " 14.9" -+0.13 ±0.9

15.6" -+0.9 7.7t¢ +0.3

7.9t~ --0.1

Numbers in parentheses indicate number of litters per group. *Offspring in litters of dams treated during gestation only (7 litters). tOffspring in litters of dams treated during gestation and lactation (6 litters). :~Significantlydifferent from control at p~<0.05 or beyond.

tion, all pups died between postnatal days 3 and 8. If the treatment was discontinued after parturition, the mortality rate of pups in the perinatal period was decreased but still significant (22%). Ivermectin at a dose of 2 mg/kg during gestation and lactation induced 31% mortality before weaning, but a dose of 1 mg/kg had no significant influence on postnatal mortality (Table 1). The body weights of dams and pups before weaning are shown in Table 2. Growth of the mothers was unaffected by ivermectin. In the offspring, the drug induced a significant decrease in body weight of males on postnatal day 14 and of females on postnatal days 14 and 21 in the 2 mg/kg group. Ivermectin at 4 mg/kg administered during gestation and lactation totally inhibited the growth of pups still alive on postnatal day 7. Offspring of dams treated with 1 mg/kg ivermectin and with 4 mg/kg ivermectin prenatally had body weights comparable to those of the controls.

Preweaning Behavioral Development Treatment with ivermectin had no action on pinna detachment and incisor eruption but delayed eye opening at the 2 mg/kg dose (Table 3). Cliff avoidance reflex was altered in all groups and there was a dose-effect relationship between 0 and 2 mg/kg. Prenatal treatment with 4 mg/kg induced an intermediate delay of the appearance of the reflex. Treatment with 2 mg/kg ivermectin had a significant delaying effect on the acquisition of the surface righting reflex (Table 3). The postnatal development of locomotion is presented in Fig. 1. Ivermectin at 2 mg/kg induced a strong inhibition of the number of 90° turns and time spent in pivoting until postnatal day 13. Ivermectin at 1 mg/kg significantly decreased the pivoting activity of pups between days 6 and 10. After prenatal administration of ivermectin at 4 mg/kg, 90° turns were decreased before day 11 and increased at days 12 and 13 from control values. Forward locomotion was significantly reduced until day 11 by 1 mg/kg ivermectin and pre-

TABLE 3 PHYSICAL LANDMARKS,CLIFF AVOIDANCEAND SURFACE RIGHTINGREFLEX DEVELOPMENT*

Pinna detachment Incisor eruption Eyelid opening Cliff avoidance Surface righting

Control (11)

Ivermectin Ivermectin 1 mg/kg 2 mg/kg (11) (11)

Ivermectin 4 mg/kgt (6)

2.6 -+ 0.2

2.8 -+ 0.1

3.0 -+ 0.2

3.1 -+ 0.2

11.6 -+ 0.2 11.6 +- 0.2

11.3 ± 0.2

11.4 -+ 0.2

14.8 -+ 0.2 15.1 ± 0.2

15.7 -+ 0.2~t 14.7 -+ 0.3

7.2 ± 0.2

8.8 ± 0.4:~ 12.5 --. 0.3~

9.2 --- 0.5~:

6.9 -+ 0.3

7.9 ± 0.4

7.5 --- 0.8

9.5 ± 0.3~

Numbers in parentheses indicate number of litters per group. *Values represent mean age in days. tDams were treated during gestation only. ~:Significantlydifferent from control at p~<0.05 or beyond.

natal treatment with 4 mg/kg, and was strongly inhibited by the 2 mg/kg dosage until postnatal day 13. Negative geotaxis reflex development (Fig. 2) was particularly inhibited in the 2 mg/kg ivermectin group which exhibited high latency time before beginning to turn and inability to complete a 180" turn until postnatal day 12. On postnatal day 7, the prenatal 4 mg/kg ivermectin group showed temporary but significant departure from controls for both latency and turning time. Swimming ontogeny was impaired to different degrees by the three doses of ivermectin (Fig. 3). The most severe im-

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IS' PIVOTING

u. •'

~

ge'

m

40.

10' .m

10'

S,

0

2nglkg

• 4uglkg (gest:a~on)

~t --~

-

.

.

.

.

t .

.

.

.

~t,,..\~l..~

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tt .

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IbO"

PIVOTING

~-

40"

o

20. G IS

]

..

~- 10'

' ik

10

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;O'N'ATAL'

'J

1

.

,,

3"

/

a:~l Wkkligg

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FIG. 2. Negative geotaxis reflex development. *Significantly different from control at p~0.05 or beyond.

t

FORWARD L O C O M O T I O N

'

I/ I;

0

I'

;s

lit

2

3

4

5

II

1

li

I)

POSTNATAL DAYS

.10 11

tt

11

13

FIG. 1. Postnatal development of locomotion. *Significantly different from control at p~0.05 or beyond.

3 z q(

2 FiLing

~ -

FIG. 3. Postnatal swimming development. *Significantly different from control a t p ~ 0 . 0 5 or beyond. Scoring: DIRECTION: 0=sank, l=floated, 2=circular swimming, 3=straight swimming. ANGLE: 0=sank or nose below the surface, l = n o s e at surface, 2=nose and top of the head at surface with ears below surface, 3 =ears half above surface, 4=ears above surface. PADDLING: 0=no paddling, l=paddling with all four limbs, 2=paddling with hindlimbs, forelimbs partially inhibited, 3=paddling with hindlimbs only.

o ~1~

I"

POSTNATAL

DAYS

D E V E L O P M E N T E F F E C T S OF P E R I N A T A L IVERMECTIN

271

TABLE 4 PREWEANINGROTORODA N D OPENFIELD PERFORMANCE Openfield Rotorod* Males Females Trial Trial Trial 1 2 3 AmbulationClimbing Rearing GroomingDefecationAmbulation Climbing Rearing Grooming Defecation Control (11) Ivermectin 1 mg/kg (11) Ivermectin 2 mg/kg

12.2 ±0.7 12.1 -+0.8

14.3 ±0.6 14.2 ±1.1

15.3 +-0.5 15.2 ±1.0

71.9 +-3.5 60.5 ±6.9

6.5 -+0.8 5.4 ±1.0

7.5 +-1.3 3.4* ±0.6

3.5 +-0.2 2.8 ±0.2

2.4 +-0.3 1.8 ±0.4

78.8 ±4.9 73.3 ±7.9

7.6 ±0.8 6.7 -1.1

9.1 ± 1.5 7.2 -+1.7

2.4 ±0.2 2.5 -+0.3

3.0 ±0.3 1.9, -+0.4

11.0 +-0.7

12.0 +-0.8

13.2 -+0.8

53.7 +-7.2

2.7 +-I.0

2.3¢ +-1.0

2.0* ±0.3

1.1 +-0.5

62.5 +-10.5

4.1 -+0.6

3.3 -+0.9

1.8 -+0.2

1.2, -+0.3

60.3

5.3

+-6.7

+-0.6

4.6 +-1.4

2.9 ±0.4

2.3 ±0.3

72.3 +-6.7

6.3 +-1.1

5.1 +-1.2

2.4 +-0.2

1.8, +-0.2

(8)

Ivermectin 4 mg/kg

12.2 ±0.5

13.8 -+0.7

13.7 ±0.5

(6)t Numbers in parenthesesindicatenumberof litters per group. *Values represent mean speed (rpm) at which test rats fell from the rod. ~'Damswere treated duringgestation only. ¢Significantlydifferentfrom control at p~<0.05or beyond.

pairment was seen in the 2 mg/kg group where all three aspects of swimming were delayed during the preweaning period and significant differences from control persisted on postnatal day 20 for the head position above water and the use of limbs. The 1 mg/kg ivermectin group had swimming development comparable to control except at postnatal day 5 for the angle parameter. Prenatal ivermectin 4 mg/kg group showed direction and paddling scores at the control level, with temporary delays in swimming angle development on postnatal days 13 and 15. No effects of ivermectin treatment were found in rotorod performance on postnatal day 19 (Table 4) although the 2 mg/kg group showed decreased values from control, but the differences were not statistically significant. Preweaning open field activity was assessed at postnatal day 20 by the number of crossed sections. None of the groups were statistically different from controls, perhaps because of a high level of variability, although the 2 mg/kg ivermectin group seemed to be less active than control. However, differences from control for at least one of the other parameters of open field performance, in each treatment group, i.e., rearing, grooming or defecations, reached the level of significance for males or females (Table 4). DISCUSSION The antiparasitic drug ivermectin (1, 2 and 4 mg/kg/day) was administered orally to female rats during gestation (days 6 to 20) and lactation (days 2 to 20) and effects on preweaning behavioral development of offspring were studied. In these experimental conditions, ivermectin treatment induced 100% pup mortality in the 4 mg/kg group, 31% mortality in the 2 mg/kg group and no significant increase in mortality in the 1 mg/kg group. Ivermectin at 4 mg/kg, administered during gestation only (days 6 to 20), induced 22% mortality in pups during the early postnatal period. There were no significant reductions in food consumption and body weight of female rats treated during gestation and lactation, so ivermectin toxicity in pups did not seem to be the consequence of maternal illness or undernutrition. Postnatal body weight of offspring

was significantly decreased only in the 2 mg/kg ivermectin group, the maximum decrease being 20% of control values. Postnatal behavioral development of pups was impaired by ivermectin. In rats treated during gestation and lactation with 2 mg/kg, eye opening, cliff avoidance reflex, surface righting reflex, pivoting and forward locomotion, negative geotaxis and swimming development were significantly impaired or delayed during the preweaning period. In rats treated during gestation and lactation with 1 mg/kg, cliff avoidance, pivoting activity and forward locomotion were retarded when compared to control performance. In rats treated during gestation only with 4 mg/kg of ivermectin, cliff avoidance, pivoting and forward locomotion, negative geotaxis and some aspects of swimming development were temporarily affected. These results support the evidence that ivermectin has a significant toxic action on the behavior of newborn rats, particularly on motor development and general activity. The no effect level on behavior was less than the dose of 1 mg/kg. This dose produced no significant alteration of reproductive parameters, mortality or growth of dams and pups before weaning. The comparison of percentages of offspring mortality after a 4 mg/kg dose administration during gestation and lactation (100%) or during gestation only (21%) indicates an obvious toxic action of ivermectin during the suckling period. Ivermectin may also be considered a behavioral teratogen since a prenatal treatment with 4 mg/kg induced impairment of behavior in the postnatal period. However, it must be emphasized that after oral or parenteral administration, high concentrations of ivermectin were maintained in body tissues of rats, particularly in fat tissues, for prolonged periods [6]. So, even after a prenatal treatment, newborn rats could be exposed to significant concentrations of the drug via the mother's milk during part of lactation. Pharmacokinetic studies in fetuses and neonates and behavioral studies including cross-fostering procedures are needed to decide whether ivermectin acts via prenatal injury or only by acute intoxication of the pups via their mother's milk. Whatever the period of ivermectin action may be, rat

272

POUL

pups seemed particularly susceptible to its neurotoxic effects. No reports on the level ofivermectin elimination in rat milk were yet available, but in cows, after a therapeutic dose of 0.2 mg/kg, milk residue concentration never exceeded 50 ng/ml (Toutain, personal communication). Ivermectin is believed to potentiate the release and binding of G A B A in nerve synapses, resulting in blockade of postsynaptic transmission of nerve impulses [10,11]. Adverse reactions to ivermectin have been related to central nervous system depression, sedation and ataxia. In mammais, GABA-ergic neurons were found only in the central nervous system and, in adults, ivermectin did not readily cross the blood-brain barrier as demonstrated in the rat [5]. In this study, ivermectin at dose of 4 mg/kg had no effect on growth, food intake, reproductive performance and health of pregnant female rats, but was highly toxic to the suckling rats in the early postnatal period. In newborn rats, the blood-brain barrier was incompletly formed [1] or was permeable to small molecular weight compounds by special-

ized mechanisms [9], increasing the susceptibility of pups to the nervous system toxicity of ivermectin. The decreased permeability of the blood-brain barrier with age might also decrease the toxicity of ivermectin. For example, the above results showed that some behaviors were restored in the late preweaning period as with swimming behavior in the 1 mg/kg group. However, differences between controls and the 2 mg/kg treated group persisted in swimming development and negative geotaxis reflex acquisition throughout the period of evaluation. Complementary studies on long-term effects of perinatal ivermectin treatment are in progress to investigate the possibility of irreversible modification of behavior in adulthood due to ivermectin treatment during development. ACKNOWLEDGEMENTS The author is indebted to Mr. G6rard Jarry for technical assistance and to Mrs. Roselyne Gesbert and Annick Blanchet for preparation of the manuscript.

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