Article | Colbert et al. doses of 12 mg/kg, administered from GD14 through PND3, were associated with a signiﬁcant increase in social play behavior in PND34 offspring. We observed no Vz-medi- ated differences on PND22, indicating that the effect emerged as offspring matured. The increased play on PND34 was more pro- nounced in males than in females. In adult- hood, male offspring produced significantly fewer penile erections, an effect that was even more sensitive than play behavior because a decrease was noted after maternal doses as low as 1.5 mg/kg. Although other researchers have reported that high doses of Vz (200 mg/kg) administered to rat pups on PND2 and PND3 reduced play behavior (Hotchkiss et al. 2003), this study is the ﬁrst to describe play behavior effects near the LOAEL of 11.5 mg/kg/day (U.S. EPA 2000a). Although we did not observe nipple and areola retention in immature male off- spring, visual inspection of the data suggests that there was a dose-related trend. The play behavior procedure used in the present study was more sensitive to low-dose effects than those used in previous investiga- tions, possibly because of methodologic 6 5 n o i ss 4 e s re 3 p s e c 2 n u o P 1 0 60 50 s ion 40 t c ree 30 l a tTo 20 10 0 704 0 3 6 Maternal dose (mg/kg) Figure 3. Mean ± SEM number of pounces per ses- sion. Data have been averaged across sex and postnatal age groups. The 12-mg/kg Vz group pro- duced pounced significantly more than did the control group. *p < 0.05. * * * Control 1.5 3 6 Maternal dose (mg/kg) Figure 4. Mean ± SEM total number of erections during the ex copula penile reflex procedure. All four of the Vz exposure groups produced signifi- cantly fewer erections than did the control group. *p < 0.01. Figure 6. Mean ± SEM total number of seminal emis- sions during the ex copula penile reﬂex procedure. The 12-mg/kg Vz group produced signiﬁcantly more emissions than did the control group. *p = 0.02. 4 6 8 Maternal dose (mg/kg) 10 12 Figure 8. Linear model for ED10 benchmark dose (BMD) value and 95% lower confidence level (BMDL) for total play behavior in the male offspring on PND34. Control 1.5 3 6 Maternal dose (mg/kg) VOLUME 113 | NUMBER 6 | June 2005 • Environmental Health Perspectives differences. In the present study we examined nape contact, pounce, pin, and wrestle, as well as mount behaviors, whereas previous studies examined only pin (Flynn et al. 2001) or pin and chase behaviors (Hotchkiss et al. 2003). Nape contact, a behavior that often initiates a play bout, was greatly affected by perinatal Vz, and this component was not examined in previous studies. Although we hypothesized that perinatal Vz would demas- culinize male offspring and lead to a reduc- tion of play behavior, we actually observed a dose-related increase in play. Exposure to other developmental toxicants such as prena- tal morphine (Hol et al. 1996; Niesink et al. 1999), mycotoxins (Ferguson et al. 1997), or phytoestrogens (Flynn et al. 2000) has been associated with increased play, and, as men- tioned above, social hyperactivity in juvenile rats is linked to aberrant sexual behavior in adults (Gerall et al. 1967). In the present study we also examined play behavior at two different time points, an approach that detected the apparently tran- sient or age-speciﬁc effect of perinatal Vz. In rats, the ontogeny of play is characterized by an inverted U-shaped function that peaks * * Control 1.5 3 6 Maternal dose (mg/kg) Figure 5. Mean ± SEM total number of reﬂex clus- ters during the ex copula penile reﬂex procedure. A cluster of reﬂexes is deﬁned as a rapid sequence, where each behavior occurs within 15 sec of the previous behavior. The 3-, 6-, and 12-mg/kg Vz groups produced significantly fewer clusters of reﬂexes than did the control group. *p < 0.01. * 12 * 12 20 16 s r tse 12 u l c lex 8 f e R 4 0 0.75 s n o i ss0.50 i m e l a n im0.25 e S 0.00 between PND32 and PND40 (Panskeep 1980; Spear and Brake 1983; Ward and Stehm 1991). Male behaviors peak earlier, during PND26–35, with female behaviors peaking during PND36–40 (Meaney and Stewart 1981). The increased play observed in the PND34 exposed males could be inter- preted as a developmental delay. Peripubertal exposure to higher doses of Vz has been shown to delay the age of preputial separation, which is a milestone of puberty in the male rat (Monosson et al. 1999). Typically, as male rats age, they show an increasing preference for female versus male partners, a shift that was not observed in the juvenile Vz males. The behavior of the exposed males actually resembled the female offspring, who per- formed more play on PND34 than on PND22. Future studies should examine addi- tional time points to better characterize the age-dependent nature of the effects of Vz on play. Alternatively, the increased play in the exposed offspring could be due to greater sen- sitivity to social isolation. Because social depri- vation is often viewed as a means of increasing play motivation, this hypothesis could be explored in future studies that compare play after different periods of deprivation. In nor- mal juvenile rats, play solicitation increases after longer periods of deprivation (Thor and Holloway 1983). Finally, it might be the case that we found signiﬁcant effects at lower doses in the present study because the offspring were Polynomial 2 4 6 8 Maternal dose (mg/kg) 10 12 Figure 7. Polynomial model for ED10 benchmark dose (BMD) value and 95% lower conﬁdence level (BMDL) for the total erections per session variable. * 12 * 12 n60 o is55 s s50 e r p45 e s ion40 tec35 r le30 a to25 BMDL BMD T 0 90 Linear n80 o i ss70 e rs60 e p50 y a lp40 l ta30 o T20 BMDL BMD 0 2