Based upon feed consumption of the 1.5, 10, 30, 100, 300, 1000, 7500, and 10,000 ppm DEHP diet, average consumed doses were 0.12, 0.78, 2.4, 7.9, 23, 77, 592, and 775 mg/kg/day for the P0 animals; 0.09, 0.48, 1.4, 4.9, 14, 48, 391; and 543 mg/kg/day for the F1 animals, and 0.1, 0.47, 1.4, 4.8, 14, 46, and 359 mg/kg/day for the F2 animals (there were no F2 animals at 10,000 ppm). P0 food consumption was increased and decreased at certain time points during exposure, but there was no dose- or time-related pattern (i.e., no consistent effect across time and/or dose level; data not shown). In the P0 generation, male body weights were significantly decreased by 5–6% at 10,000 ppm during weeks 21 and 23 and dam body weights at 10,000 ppm were significantly reduced at delivery of each of the three litters. In the F1 generation, body weights of males and females were significantly reduced throughout exposure in the 10,000-ppm dose group. Body weights were also reduced in 7500-ppm adult F1 males and F2 males and females. Food consumption (grams per animal in kilogram) was generally increased throughout exposure in the F1 7500 and 10,000 ppm males and F2 7500 ppm males and females.
In the mated cohorts, the pregnancy index (number of females delivering/number of cohabiting pairs) was unaffected by treatment in the P0 generation, but a clear effect was evident at the 10,000 ppm dose level in the F1 generation (). No litters were produced by the F1 10,000-ppm dose group so exposure continued only for the 1.5-, 10-, 30-, 100-, 300-, 1000-, and 7500-ppm dose groups. In the production of the F2 generation, the pregnancy index of F1 dams was significantly decreased at the 7500 ppm dose. The litter size (number of live pups) produced by the P0 generation was significantly reduced in the 7500-ppm dose group (), but this effect was not consistent across generations or dose dependent. Of the three litters produced by each generation, average pup weights on PND1 (combined male and female) were generally unaffected by treatment below 7500 ppm. Body weights at PND 1 were reduced in the F1 pups at 10,000 ppm and in the F2 pups at 7500 ppm ().
Summary of Pregnancy Indexa from Three Matings of Cohabited Breeding Pairsb from Each Generation Exposed to DEHP
Summary of Average Litter Size, Proportion of Pups Born Alive, and Pup Weights (combined male and female) from the Three Litters Produced by the P0, F1, and F2 Generations
Two P0 10,000 ppm males had a mild testicular lesion with no consequent effects on fertility in this group and one 7500 ppm animal had a prostate lesion. In stark contrast, all the F1 10,000 ppm males had an RTM (), and no F2 litters were produced (). At 7500 ppm, there were significant increases compared with controls in the incidence of RTMs in the F1 and F2 males. RTMs were also observed in the 1000- and 300-ppm groups of the F1 and F2 generations. A single incidence of a seminal vesicle and prostate malformation was observed in the F1 10-ppm group and 30-ppm group groups. A single testis malformation (tunica albuginea aplasia) was noted in the control (1.5 ppm) group in breeding male of the F2 generation but was not considered for the analysis of malformations because it was not typical of the phthalate syndrome. There was a significant increase in total RTMs, consistent with the phthalate syndrome, at and above 300 ppm when malformations over the F1 and F2 generations were combined (). The dose-response curves were similar for the F1 and F2 generations and when the RTM of the two generations were combined (). A Hill model (Graphpad Prism 5.01, San Diego, CA) of the 1.5–10,000 ppm RTM data, which was constrained between 0 and 100% incidence, calculated a half maximal concentration (EC50
) of 2771 ppm (R2
= 0.9139) for the F1 generation, an EC50
of 1480 ppm (R2
= 0.9894) for the F2 generation, and the EC50
for the combined F1 + F2 generation was 2094 ppm (R2
= 0.9564). The roughly twofold difference in EC50
between the F1 and F2 generations is due to the low-incidence observed in the F1 1000-ppm group, and when removed, the F1 EC50
is 1406 ppm (R2
= 0.9564) similar to the F2 generation EC50
. After evaluating various types of BMD models, the Weibull model provided the best fit across the F1, F2, and F1 + F2 data sets (see Supplementary data
). The BMD and BMDL values for the F1, F2, and combined F1 + F2 data were 257 and 169 ppm, 233 and 77 ppm, and 198 and 142 ppm, respectively.
The Incidence of RTMs Observed Grossly Across the F1 and F2 Generations
FIG. 1. Dose response of the RTMs in the F1 (○), F2 (□), and the F1 + F2 combined (Δ) data with a fitted Hill model. The EC50 is 2771 ppm (R2 = 0.9139) for the F1 generation, 1480 ppm (R2 = 0.9894) for the F2 generation, and 2094 ppm (R2 (more ...)
The Monte Carlo simulations demonstrated that when more males per litter were examined using the non-mated cohort, the ability to detect an effect (i.e., statistical power) increased (). Detection was defined as getting a p value of ≤ 0.05 using Fisher’s exact test with the litter as the unit of analysis. If the probability of an abnormality is 20% in each treated pup and 0% in each control pup, this effect can be detected 99.5% of the time when 3 male pups are examined from each of 20 litters per group versus only 37.9% of the time when 1 pup per litter was examined. A lower probability of an abnormality of 10% would be detected only 4.7% of the time when one male pup per litter is examined but detected 66.4 and 86.5% of the time when three or four pups are examined, respectively.
FIG. 3. Power curves for detecting an effect (one-sided Fisher’s exact test p ≤ 0.05) given the effect size (probability of an abnormality in treated pups) and the number of pups examined per litter, using 20 treated litters and 20 control litters. (more ...)
FIG. 2. BMD curves for the F1 (A), F2 (B), and F1 + F2 (C) incidence of litters with a reproductive lesion. A BMD (solid line) and BMDL (dashed line) were calculated using a Weibull model for a 5% response. F1 BMD and BMDL: 257 and 169 ppm; F2 BMD and BMDL: 233 (more ...)