Article | Perinatal vinclozolin alters sex differentiation E3 responses were not signiﬁcantly different. Surprisingly, there was a signiﬁcant increase in seminal emissions [F(4,40) = 7.37, p < 0.01; Figure 6] as the 12-mg/kg group expelled more often than did any of the other groups. This effect was unanticipated because rats do not usually emit seminal ﬂuid during the ex copula procedure. Benchmark dose modeling. We performed benchmark dose calculations on the total erec- tions per session and the total play behavior in the male offspring on PND34. These two variables were selected because they are the best overall measures of erectile function and play, and consequently, they generalize more readily to humans. A polynomial model pro- vided the best description of the erection data. The corresponding ED10 benchmark for erec- tions was 1.23 mg/kg with a 95% lower bound of 0.84 mg/kg (Figure 7). The ED01 benchmark was 0.11 mg/kg with a lower bound of 0.08 mg/kg. The linear model pro- vided the best description of the play data. The ED10 associated with total play in the male offspring on PND34 was 1.33 mg/kg with a lower bound of 0.77 mg/kg (Figure 8). The ED01 for total play was 0.13 mg/kg with a lower bound of 0.08 mg/kg. Discussion The results of this study clearly demonstrate that social and reproductive behaviors in the rat are disrupted by exposure to low doses of Vz during the perinatal period. Maternal Nape contact and pounce variables made the greatest contribution to the significant exposure-related effects on total play behaviors. For nape contacts, there was a signiﬁcant expo- sure × PND interaction [F (1,21) = 5.51, p = 0.03]. We also examined the number of nape contacts separately for PND22 and PND34. As with the total play behavior vari- able, there was a signiﬁcant main effect of sex on PND22 [males > females; F (1,21) = 11.13, p < 0.01] and a signiﬁcant main effect of expo- sure on PND34 [F (1,37) = 16.09, p < 0.001]. Probe tests indicated that the male 12-mg/kg Vz group produced significantly more nape contacts than did the 0- and 3-mg/kg groups on PND34 (Figure 2). For the pounce vari- able, there was a significant main effect of exposure [F (1,21) = 6.44, p = 0.02]. Data were averaged across sex and age, and probe tests indicated that the 12-mg/kg group pounced more than did controls (Figure 3). There were no exposure-related differences for pin, wrestle, or mount behaviors. Penile reflex. We found a significant exposure-related decline in total erections per session [F (4,40) = 4.62, p < 0.01; Figure 4] as each of the Vz groups produced signiﬁcantly fewer erections than controls. The decline in total erections was due primarily to a dose- related decline of E1 or low-intensity erections [F (4,40) = 10.07, p < 0.01] as well as the num- ber of reflex clusters per session [F (4,40) = 3.23, p = 0.02; Figure 5]. The latency to the ﬁrst penile reﬂex and the frequency of E2 and 0 mg/kg 3 mg/kg 6 mg/kg 12 mg/kg 80 n70 o i ss 60 e s r pe50 l a to40 T 30 20 70 60 ts50 c a tn40 o c pe30 a N20 10 0 B B probe tests were used to make pairwise com- parisons. We considered p < 0.05 statistically signiﬁcant. For the penile reﬂex procedure, we tested each animal on 2 consecutive days, and the data from the two sessions were averaged before analysis. If an animal was inactive dur- ing one of the sessions, that session was dropped. If an animal was inactive during both sessions, that animal was dropped from the analysis. Only 2 of 47 animals were inac- tive during both sessions. The averaged behavior was analyzed with one-way ANOVA according to exposure level. Because the penile erection data showed a clear dose–response relationship with evidence that even the lowest dose of Vz disrupted the behavior, we further examined the data with Benchmark Dose Modeling Software (BMDS; version 1.3.2; U.S.EPA National Center for Environmental Assessment, Washington, DC). BMDS is a useful alternative to the no observed adverse effect level (NOAEL) approach because it uses the entire dose–response relationship and does not involve extrapolations far below exper- imental observations. We used the BMDS con- tinuous model to calculate benchmark doses that represented the model-estimated control mean, minus proportional deviations equivalent to a 10% (ED10) or 1% (ED01) decrement in behavior. BMDS also provided a 95% lower bound that can be divided by a standard uncer- tainty factor to calculate a reference dose or generate a margin of exposure. Results Maternal and postpartum data. We found no evidence of gross maternal or neonatal toxicity (Table 1), nor were there any exposure-related changes in maternal body weight, pup body weight, or AGD (males, Table 2; females, Table 3). The percentage of male pups that possessed at least one visible areola on PND12 (control, 18%; 1.5 mg/kg, 50%; 3 mg/kg, 44%; 6 mg/kg, 45%; 12 mg/kg, 33%) was not signiﬁcant (p = 0.367). Play behavior. For this procedure, the pri- mary dependent variable was the total number of play behaviors per session. Although we found no signiﬁcant main effects of the expo- sure, sex, or PND factors on total play behav- iors, there was a signiﬁcant exposure × PND interaction [F (1,21) = 7.72, p = 0.01]. We also examined the total number of behaviors sepa- rately for PND22 and PND34. There was a significant effect of sex on PND22 [males > females; F (1,21) = 9.91, p < 0.01] and a signif- icant effect of exposure on PND34 [F (1,37) = 16.38, p < 0.001]. Probe tests revealed that the male 12-mg/kg and 6-mg/kg Vz groups pro- duced signiﬁcantly more play behaviors than the did controls on PND34 (Figure 1A). There were no differences between the female exposure groups (Figure 1B). Environmental Health Perspectives • VOLUME 113 | NUMBER 6 | June 2005 703 80 n70 o i ss60 e s r pe50 l a to40 T 30 20 70 60 ts50 c a tn40 o c e30 p a N20 10 0 A A 0 mg/kg 3 mg/kg 6 mg/kg 12 mg/kg PND22 PND34 PND22 PND34 Figure 1. Mean ± SEM total number of play behaviors per session on PND22 and PND34 for male offspring (A) and their female littermates (B). Males exposed to 6 mg/kg or 12 mg/kg Vz performed significantly more behaviors on PND34 than did same-sex controls; females were not signiﬁcantly different on either day. PND22 PND34 PND22 PND34 Figure 2. Mean ± SEM number of nape contacts per session on PND22 and PND34 for male offspring (A) and their female littermates (B). The male 12-mg/kg Vz group performed signiﬁcantly more nape contacts on PND34 than did controls or the 3-mg/kg group.