Effect of di(2-ethylhexyl) phthalate (DEHP) on genital organs from juvenile common marmosets: I. Morphological and biochemical investigation in 65-week toxicity study.

Recent studies demonstrated that preadolescent male rats are more sensitive to testicular damage from exposure to DEHP than adults. Male and female marmosets were treated daily with 0, 100, 500, or 2500 mg/kg DEHP by oral gavage for 65 wk from weaning (3 mo of age) to sexual maturity (18 mo). No treatment-related changes were observed in male organ weights, and no microscopic changes were found in male gonads or secondary sex organs. Sperm head counts, zinc levels, glutathione levels, and testicular enzyme activities were comparable between groups. Electron microscopic examination revealed no treatment-related abnormalities in Leydig, Sertoli, or spermatogenic cells. Histochemical examination of the testis after 3beta-hydroxysteroid dehydrogenase (3beta-HSD) staining did not reveal any alterations in steroid synthesis in the Leydig cells. Thus, although marmoset monkeys were treated with 2500 mg/kg DEHP, throughout the pre- and periadolescent period, no histological changes were noted in the testes. For females, increased ovarian and uterine weights and elevated blood estradiol level were observed in higher dosage groups, 500 and 2500 mg/kg. These increased weights were associated with the presence of large corpus luteum, a common finding in older female marmosets. Although an effect on the female ovary cannot be completely ruled out, no abnormal histological changes were observed in the ovaries or uteri in comparison to controls. No increases in hepatic peroxisomal enzyme activities were noted in treated groups; isolated hepatic enzyme activities (P-450 contents, testosterone 6beta-hydroxylase, and lauric acid omega-1omega-hydroxylase activities) were increased in males and/or females of either the mid- or high-dose groups, but no consistent dose-related trend was observed.

Phthalate treatment does not influence levels of IgE or Th2 cytokines in B6C3F1 mice.

Bronchial asthma is mediated, in part, by the immunoregulatory cytokines interleukins 4 and 13 (IL-4 and IL-13). These cytokines stimulate IgE synthesis that in turn is associated with airway hyper-responsiveness. Compounds that stimulate IgE synthesis and elicit bronchial reactivity are generally considered to be respiratory sensitizers. Recently, it has been hypothesized that exposure to phthalates may contribute to childhood asthma. To address this question, di-(2-ethylhexyl) phthalate (DEHP) was tested using a protocol adapted from work by Dearman that involves topical application (and challenge) of test substances to mice followed by measurements of total serum IgE. In addition, auricular lymph nodes were harvested for measurement of IL-4 and IL-13 proteins and their corresponding messenger RNAs. Because skin absorption of high molecular weight phthalates is limited, liver weight increase, a measure of peroxisomal proliferation, was monitored to assure that internal dosing had been achieved. ELISA and RNAse protection assays demonstrated that DEHP treatment did not significantly affect IgE, IL-4, or IL-13 levels. Similarly, IL-4 and IL-13 mRNA levels were not elevated. In contrast, all of these were significantly elevated by trimellitic anhydride (TMA), a respiratory sensitizer used as the positive control in this assay. Liver weights were significantly elevated by DEHP, providing evidence of sufficient percutaneous absorption to induce physiological responses. To extend these observations, three other commercial phthalate ester plasticizers, di-isononyl phthalate (DINP), di-isohexyl phthalate (DIHP), and butyl benzyl phthalate (BBP), were assessed using the same protocol. As above, ELISA and RNAse protection assays showed that IgE, IL-4, and IL-13 proteins, and IL-4 and IL-13 mRNAs in the phthalate-treated animals were all at levels similar to that of control values. The positive control, TMA, produced large, statistically significant increases in all parameters, demonstrating responsiveness of the assay. Another control, dinitrochlorobenzene (DNCB), a contact sensitizer, also responded as expected, producing smaller but statistically significant increases in IgE and in mRNA for IL-4 and IL-13 but not in the levels of these cytokines. In summary, treatment with DEHP, DINP, DIHP, and BBP did not result in significant elevations in total serum IgE, IL-4, or IL-13. As such it is unlikely that these substances would produce antibody-mediated respiratory allergy.

NTP center for the evaluation of risks to human reproduction reports on phthalates: addressing the data gaps.

Between 1998 and 2000 an Expert Panel convened by the National Toxicology Program's Center for the Evaluation of Risks to Human Reproduction (NTP-CERHR) reviewed information related to the developmental and reproductive toxicity of seven phthalate esters; DBP, BBP, DnHP, DEHP, DnOP, DINP, and DIDP. Information on exposures was also considered. The objectives were to determine whether any of these phthalates posed potential human reproductive risks, and if so, to define the circumstances. The Expert Panel also identified some areas of uncertainty. These assessments, ultimately published in 2002, concluded that reproductive risks were minimal to negligible in most cases although some specific uses were considered potentially more problematic. Since the evaluations were completed, numerous studies dealing with both hazard characterization and underlying mechanism have been carried out. Additionally, exposures of the general population have been much better characterized through the use of urinary measurements developed by the Centers for Disease Control (CDC). This additional information makes several important points. First, calculations based on the urinary metabolite measurements indicate that exposures within the general population are at levels similar to or lower than the estimates used by the NTP-CERHR. The demonstration that exposures were not underestimated by the CERHR has removed a substantial portion of the uncertainty. Second, new hazard characterization studies on several phthalates have established NOAELs similar to or higher than those used by the Expert Panel. Thus, these data demonstrate that, to the extent that the rodent data are useful for human health risk assessment, the no effect levels and dose-response relationships are now more precisely defined. In some cases, the no effect levels may be substantially higher than those estimated by the Expert Panel. Third, studies of underlying mechanism and/or hazard characterization studies in other species suggest that primates may be less sensitive than rodents to the reproductive effects of certain phthalates. Finally, the two specific situations that the CERHR identified as potentially problematic, the exposure of young children to DINP through the use of toys or to DEHP from medical devices, have been assessed by the responsible regulatory authorities. The Consumer Product Safety Commission concluded that exposure to DINP from toys was well below effect levels in animals, and, therefore, there was no risk. The Food and Drug Administration estimates of exposures from medical devices indicated that for some limited, intensive medical procedures, DEHP exposures could be similar to or greater than the NOAELs selected by the NTP-CERHR. However, the FDA also acknowledged that more recent information indicates that the NOAELs identified in rodent studies may be substantially higher than values previously proposed by the NTP-CERHR. In summary, much of the uncertainty identified by the CERHR has now been addressed, and the overall conclusions that levels of concern are minimal to negligible in most situations are much better established. The overall objective of this report is to summarize this new research and comment on its relevance to the NTP-CERHR assessments.