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The High Molecular Weight Phthalate Ester (HMWPE) Category consists of phthalate esters with an alkyl carbon backbone with 7 carbon (C7) atoms or greater. The category is formed on the principle that substances of similar structure have similar toxicological properties. The data available on high molecular weight phthalates demonstrate that members of this category have similar biological activities and toxicological properties; verifying the use of read-across data as an appropriate approach to characterize endpoints. DTDP (C13) is a high molecular weight phthalate ester. Where data maybe lacking for DTDP, DINP (C9) and DIDP (C10), which are also high molecular weight phthalate esters, are used as read-across substances to provide toxicological information.  

Additional Toxicological Information Endocrine Effects

 

In Vitro Studies

A series of phthalate esters, including DINP, were screened for estrogenic activity using a recombinant yeast screen (Harris et al., 1997). In the recombinant yeast screen, a gene for a human estrogen receptor was integrated into the main yeast genome and was expressed in a form capable of binding to estrogen response elements, controlling the expression of the reporter gene lac-Z (when receptor is activated, the lac-Z is expressed). DINP was tested at concentrations ranging from 10-3 M to 5.10-7 M. DINP behaved un-reproducibly in the yeast screen. DINP was also tested for the ability to stimulate proliferation of human breast cancer cells (MCF-7 and ZR-75 cells). DINP produced no effects in the MCF-7 assay. In the ZR-75 cells, DINP at concentration of 10-5, 10-6 and 10-7 M induced proliferation to a significantly greater extent than the control, which is in contrast to the findings for this chemical using the yeast screen. It should be noted that these in vitro assays have investigated one mechanism of action only, the ability of phthalates to act as estrogen agonists. More importantly, it should also be noted that these were tests of phthalate diesters. Under in vivo conditions the diesters are metabolized to monoesters which are not estrogen receptor agonists.  The in vitro data need to be evaluated very carefully as the tests may have involved either substances which for all practical purposes do not exist under in vitro conditions or may have employed non-physiological conditions. 

 

The estrogenic activities of DINP were investigated by Zacharewski et al (1998) in vitro using estrogen receptor (ER) competitive ligand-binding and mammalian- and yeast-based gene expression assays.  No significant responses were observed with DINP in any of the in vitro assays. For these in vitro tests, no significant responses were observed with DINP. However, in each of these tests the diester was utilized. Diesters essentially do not exist under in vivo conditions; therefore the results are not relevant to intact animals.  

 

Additionally, there is no indication that MINP, a metabolite of DINP, binds to androgen receptors (McKee et al, 2004). 

 

In Vivo Studies

Uterotrophic assay/vaginal cell cornification assay

In an in vivo study, 20, 200, 2,000 mg/kg/d of DINP was administered by oral gavage once daily for a period of 4 days to ovariectomised Sprague-Dawley rats (10 females per dose, two experiments) (Zacharewski et al., 1998). Ethynyl Estradiol (EE) was used as a positive control. Body weight, uterine wet weight and percentage of vaginal epithelial cell cornification on each day were assessed. DINP did not produce any statistically significant increases in body weight. Additionally, DINP did not produce any reproducible, dose-dependant effect on uterine wet weight relative to vehicle control at any of the dose tested. DINP did not induce a vaginal cornification response at any of the doses tested. Accordingly, it can be concluded that DINP is not estrogenic under in vivo conditions. 

 

Steroidogenesis assay

In a study designed to test effects on testosterone synthesis, 32 pregnant female rats were exposed to either 300 mg/kg-bw DEHP or 750 mg/kg-bw DINP, alone or in combination, from gestation day 7 to gestation day 21 (Borch et al., 2004). The dams were sacrificed on gestation day 21 and the pups were harvested for analysis of testicular testosterone production, testicular testosterone content, plasma testosterone levels, and plasma luteinizing hormone (LH) levels. The results indicate that testicular testosterone production and testicular testosterone content were significantly decreased in the DINP exposed pups while plasma testosterone and plasma LH levels were unaltered. However, no mechanism of toxicity can be determined from this paper since it is limited by several factors. First, the dose was administered via a single oral gavage exposure on each day of testing. This method of administration can result in the overwhelming of normal detoxifying processes which can lead to overt toxicity. Second, there were no adverse phenotypic effects reported in the study. Therefore it is unclear if the decrease in testosterone content is in-fact a toxicologically significant response. Third, while DEHP and DINP alone appeared to induce a decrease in testosterone content, there was no indication of a modulating effect of DINP on DEHP when co-administered. Finally, the authors sampled testosterone levels on gestation day 21, a time point after the developmental surge of testosterone that occurs during gestation day 16-18 in the rat. After gestation day 18, plasma testosterone levels are naturally declining in the fetal rat.

 

Contrasting the work by Borch et al (2003), the effects of developmental exposure to DINP (250 and 750 mg/kg) was examined on gestation day 19.5 in fetal male Sprague Dawley from dams exposed to DINP between gestation days 13.5 – 17.5 (Adammson et al, 2009). No effect on testicular testosterone levels (gd 19.5) were observed with DINP. Gene expression patterns of genes assocaited with steroidogenesis were examined. P450scc was increased at 750 mg/kg/day; however no changes in relative StAR, 3β-HSD or SF-1 mRNA levels were seen in either dose of DINP. GATA-4 and Insl-3 mRNA levels were seen to increase with 750 mg/kg/day. Changes in protein levels were largely inconsistent with the gene expression data. Overall the genomic analysis is inconsistent with that observed for other low molecular weight phthalates and no pathologic change in the testis was noted. Therefore, in utero exposure to DINP did not down-regulate testicular or adrenal steroidogenesis in this study.

 

 

DINP is devoid of estrogenic activity in vitro, it shows no ability of binding to rodent or human estrogen receptors or to induce estrogen receptors-mediated gene expression. In vivo assays demonstrated that DINP does not increase uterine wet weight or does not give rise to vaginal epithelial cell cornification. It is therefore concluded that DTDP is not an endocrine disruptor.

 

Additional Toxicological Information Anti Androgenicity

 

A study during the late gestational period (Gray et al., 2000) was conducted with several phthalates, including DINP. Timed-pregnant rats were gavaged daily with DINP at single dose of 750 mg/kg/d in corn oil as vehicle from gestational day 14 through postnatal day 3. Males with areolas were observed in the DINP dose groups at day 13 of age. Adult males exposed perinatally to DINP (7.7%, p<0.04) had malformations of testis, epididymis, accessory reproductive organs and external genitalia. The usefulness of these data is limited since the study involved only a small number of rats, the low incidence of reported effects was without any dose response, and the reported effects are of unclear significance; the study control values for the relevant effects (areola retention) are reported to be zero, but in a subsequent study, control values are reported as 14% (Ostby et al., 2001). Further complicating the interpretation of the data are questionable statistical methodologies. The authors pooled all of the animal data instead of examining the effects on per litter basis since this was the only way results would have been statistically significant. As infants, the only adverse effect reported in DINP group was retained areolas (22%, reported as statistically significant).  All other endpoints (nipple retention, epididymal agenesis, fluid filled testes, and testes weight) on their own were not significantly different from control values. This type of data manipulation is not routinely performed in toxicological safety evaluations, nor is it considered good statistical practice. Based on the above points the significance of the reported findings is unclear.

 

Anti-androgenic parameters were also tested in a study by Hass et al (2003). Groups of 12 mated female Wistar rats were gavaged from gestation day 7 to PND 17 with 0, 300, 600, 750, or 900 mg/kg/day DINP. Anogenital distance in male pups was significantly decreased at 600, 750 and 900 mg/kg/day. However, birth weights were decreased at the same dose levels and when birth was included as a covariate in the statistical analysis, the anogenital distances were only significantly decreased at 900 mg/kg/day DINP. At doses of 600 mg/kg/day and above, dose-related increases in nipple retention were observed in the male offspring.

 

In contrast to the findings reported by Gray et al., 2000 and Hass et al., 2003, no anti-androgenic effects were observed in male offspring of pregnant rats exposed to higher levels of DINP in the diet (Masutomi, et al., 2003). DINP was administered to Sprague-Dawley rats at concentrations of 400, 4000, and 20,000 ppm from gestational day 15 to PND 10. Offspring were examined in terms of anogenital distances, prepubertal organ weights, onset of puberty, estrous cyclicity, and organ weights and histopathology of endocrine organs at adult stage (week 11) as well as the volume of sexually dimorphic nucleus of the preoptic area (SDN-POA). DINP, at 20,000 ppm (~1165 – 2657 mg/kg/day) did not cause any developmental alterations, other than slight degeneration of Sertoli cells and meiotic spermatocytes noted in the adult stage. DINP did not alter any parameters in the females except for slight ovarian changes in the adult stage (i.e. marginal decrease in the number of corpora lutea). In addition, no change in the volume of the SDN-POA was observed.

 

A study designed similarly to the Hershberger bioassay screen for anti-androgenic chemicals which is currently undergoing validation by OECD (Lee et al, 2007) also tested the antiandrogenic properties of DINP. This assay investigates whether the co-administered chemical treatment interferes with the bioactivity of the endogenously provided testosterone and affects the expected rapid and vigorous re-growth of 5 androgen dependent sex accessory tissues in the young castrated male rat. In accordance with OECD, seven days after surgical castration (removal of testes and epididymides, followed by recovery and growth regression), young male rats were administered 0.4mg/kg/d testosterone propionate (sc) plus an oral gavage dose of a phthalate (DINP) at one of 3 dose levels (20, 100 or 500mg/kd/day). This treatment was repeated for 10 days, after which the animals were killed and target organs weights collected. DINP did not induce consistent changes in the absolute weight of all 5 androgen sensitive tissues (seminal vesicles, ventral prostate, levator anti-bulbocavernous muscle, Cowper’s glands and glans penis). DINP showed significant reductions in seminal vesicle weight at all dose levels, but not in a dose-related manner. DINP did not induce a significant change in Cowper’s gland or glans penis weights or on serum testosterone or LH levels or produce clinical signs of toxicity or mortality. Overall, these data indicate that DINP does not meet the OECD criteria for androgen antagonists as the weights of the sex accessory tissues from the administered groups showed no consistent statistically significant differences from the testosterone-only animals.

 

Collectively, the data for anti-androgenicity of DINP are based on limited study designs with no or only minor effects being observed at very high doses with no dose-response observed. Based on the comprehensive 2-generation reproductive, sub-chronic, and chronic studies it can be concluded that DINP is not an endocrine disruptor as defined by the Weybridge, IPCS and REACH guidance definitions. Since DINP is a surrogate for DTDP, it can be concluded that DTDP will not have antiandrogenic properties.