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PEROXISOME PROLIFERATION

DINP is in a class of chemicals known as "peroxisome proliferators" – chemicals that induce an increase in the size and number of a subcellular organelle known as a "peroxisome" in the liver cells of rodents. Many peroxisome proliferators are known to cause liver tumors in rodents. As observed with peroxisome proliferators, including DINP, rats and mice are susceptible to the morphological, biochemical, and carcinogenic effects of peroxisome proliferators, while non-human primates and humans are completely non-responsive or refractory.  

 

Criteria have been established by IARC to make the determination that tumors resultant from peroxisomal proliferation are not relevant to humans (IARC, 1995 at 12-13):

 

(a)       Information is available to exclude mechanisms of carcinogenesis other than those related to peroxisome proliferation.

 

(b)       Peroxisome proliferation (increases in peroxisome volume density or fatty acid b-oxidation activity) and hepatocellular proliferation have been demonstrated under the conditions of the bioassay.

 

(c)       Such effects have not been found in adequately designed and conducted investigations of human groups and systems.

 

The data for DINP meet all of these criteria. With respect to the first criterion, alternative mechanisms of carcinogenicity, IARC relies substantially on the same types of information considered by ILSI, i.e., is there evidence that peroxisomal proliferation does occur in the species which develop cancer, and, can a role for a genotoxic process be ruled out (Klaunig et al, 2003). As described above, DINP does produce tumors in livers of rats and mice (Moore, 1998a; b), and there is clear evidence of peroxisomal proliferation in the livers of both species (Moore, 1998a; b; Smith et al., 2000; Valles et al., 2003; Kaufmann et al., 2002). DINP is not genotoxic. In addition, there is no evidence of pathologic changes in the livers of these species unrelated to peroxisome proliferation which could provide an alternative explanation for tumor formation (Lington et al., 1997; Moore 1998a; b). Further, the electron microscopic evaluation in mice revealed exclusively findings related to peroxisome proliferation; no other degenerative findings on the subcellular level were observed in either sex (Kaufmann et al., 2002).

 

The second criterion requires that peroxisome proliferation and hepatocellular proliferation be demonstrated under the conditions of the bioassay. As indicated above, increases in peroxisomal volume density, fatty acid b-oxidation, and hepatocellular proliferation in livers of rats and mice treated with DINP have been documented (Barber et al., 1987; Moore, 1998a; b; Smith et al., 2000; BASF AG, 2001; Valles et al., 2003; Kaufmann et al., 2002). In the rat study (1998a), the tumors appeared only at the highest dose (1.2% in the diet or approximately 733 mg/kg/day in male rats and 885 mg/kg/day in females). As also documented in the laboratory report describing that study (1998a), DINP also caused significant increases in liver weight, peroxisomal enzyme induction, and enhanced cell replication at that level. An independent study (Smith et al., 2000) confirmed these observations at the same levels in the same strain of rats. Thus the requirement that peroxisomal proliferation be demonstrated under the conditions of the bioassay has clearly been met in rats.

 

In the mouse study, liver tumors were significantly increased in male mice given 4000 or 8000 ppm (approximately 740 and 1560 mg/kg/day) and in female mice given 1500, 4000 or 8000 ppm (approximately 336, 910 and 1888 mg/kg/day) in the diet for two years (1998b). As defined by the study protocol, liver weights, peroxisomal enzyme induction and cell replication were examined in only the high dose group (8000 ppm) and the control, and all of these parameters were significantly elevated in the high dose group from that study (Moore, 1998b). An independent study also measured liver weight increase, peroxisomal enzyme induction, and enhanced cell replication in the same strain of mice treated at 6000 ppm (Smith et al., 2000), and again all of these parameters were significantly elevated with respect to control. To evaluate peroxisome proliferation at the 1500 ppm and 4000 ppm levels, another study was conducted to determine the dose-response relationships for peroxisomal volume density and peroxisomal enzyme induction in mice treated with DINP. The data indicated that both peroxisome volume density and peroxisomal induction were significantly elevated at the tumorigenic doses (Kaufmann et al., 2002). These new data provide direct evidence of peroxisomal proliferation under the conditions of the bioassay in the mouse as well as the rat.  Taken together, these data demonstrate that, at every tumorigenic dose level in both rats and mice, there is a significant increase in peroxisome proliferation. Thus peroxisomal proliferation has been demonstrated under the conditions of the bioassay for DINP, meeting the second IARC criterion.

 

The third criterion requires evidence that peroxisome proliferation effects do not occur in “adequately designed and conducted investigations of human groups or systems.” For this, IARC normally relies on data from studies in primates and/or human hepatocytes in culture. There have been two studies in non-human primates; in one of these DINP had no effects on the liver and showed no other evidence of peroxisome proliferation in marmosets following 90 days of treatment at levels up to 2500 mg/kg/day (Hall et al., 1999). In the other, DINP had no effects on the liver and showed no other evidence of peroxisome proliferation in cynomolgus monkeys following 14 days of treatment at levels up to 500 mg/kg/day (Pugh et al., 2000). Similarly, there was no evidence of peroxisome proliferation in either human hepatocytes (Hasmall et al., 1999; Kamendulis et al., 2002; Shaw et al., 2002) or other primate hepatocytes tested under in vitro conditions (Benford et al., 1986; Hall et al., 1999; Kamendulis et al., 2002). Thus studies from several laboratories using hepatocytes from different individuals or different species of primates have demonstrated that a peroxisome proliferator response is not elicited by DINP in humans and other primates.

 

Additionally, work has shown that the DINP metabolite, MINP, has the ability to bind PPARa (Bility el al, 2004) while PPARa null mice do not exhibit the same effects as wild type controls to DINP (Valles et al, 2003). 

 

In summary, DINP meets all three IARC criteria for identifying a peroxisome proliferator for which observed liver tumors in rodents are not relevant to humans.

An alternative mechanism for phthalate induced liver tumors has been recently proposed that is independent of PPARα activation (Ito et al., 2007). The hypothesis, based on studies using mice without functional PPARα, suggests increased production of reactive oxygen species as a result of increased oxidative stress in mouse hepatocytes due to DEHP exposure. The applicability of the Ito et al., 2007 results is limited in that a number of reports have indicated that PPARα null mice are more vulnerable to tumorigenesis in the absence of any chemical exposure due to fundamental mechanistic differences. As spontaneous tumors are known to occur in the PPARα null mice at 24 months, the utility of this mouse model to understand alternative mechanisms of tumorigenesis that are independent of PPARα is problematic and can not currently be used to assess relevance to humans.  Therefore, the Ito et al., 2007 data are not sufficient to indicate there is a valid alternative mechanism of carcinogenesis other than that related to peroxisomal proliferation indicating that the first IARC criterion is met.

 

ENDOCRINE MODULATION

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 vivo 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.

 

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

 

Taken as a whole, the available data indicate that DINP or MINP do not have significant interactions with the estrogenic or androgenic receptors. 

 

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, the utility of this study for hazard identification and risk assessment is limited by several factors. First, the study utilized only one very high dose of DINP. Second, there were no adverse phenotypic effects reported in the study. Therefore it is unclear if the observed 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. Thus, conclusions regarding reductions in testosterone synthesis are problematic when assayed at this point. 

 

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. The expression patterns of genes associated with steroidogenesis were also examined. An increase in activity in the 750 mg/kg/day male pups was observed with P450scc, a gene coding for the enzymatic cleavage of the alkyl side chain on cholesterol, a precursor step for testosterone synthesis. No changes were observed in expression of genes associated with membrane transport (StAR), testosterone synthesis (3β-HSD), or overall control of male reproductive tract development (SF-1). With the exception of SF-1, these genes are typically strongly down regulated following exposure to chemicals that interfere with testosterone synthesis. GATA-4 and Insl-3 mRNA levels, genes associated with development of the male reproductive tract were seen to increase in male pups exposed prenatally to 750 mg/kg/day DINP. Again, down regulation of these genes is typically observed with anti-androgenic substances that affect male reproductive tract developmental. No marked effect was observed in concentrations of these gene products. Overall the genomic analysis (both the transcriptome and the proteome) is inconsistent with that observed for other low molecular weight phthalates that produce marked antiandrogenic effects. Further no morphological change in the testis was noted. Therefore, no effect on testosterone synthesis, or expression of the genes and proteins associated with testosterone synthesis were observed in this study.

  

Taken as a whole, DINP does not modulate estrogenic or androgenic endocrine systems.

DINP and its major metabolite MINP are 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. DINP and MINP do not interact with the androgen receptor. 

ANTI-ANDROGENICITY

 

A study during the late gestational period (Gray et al., 2000) was conducted with several phthalate esters, 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. In contrast to the effects observed with low molecular weight phthalate estes (BBP and DEHP), DINP produced slight equivocal changes in phenotypic expression of antiandrogenic effects. No effect was observed on anogenital distance or testis weight following DINP treatment. The authors reported a small statistically significant increase in malformations of the genital tract in male rat exposed in utero to DINP (7.7%).  This statistical result is questionable because statistical significance was achieved only by pooling several different effects and treating them as a single effect.  Further, the statistical unit was the pup as opposed to the litter, the commonly accepted statistical unit for developmental toxicity studies. An increase in percentage of males with retained areolas was observed in the DINP dose group at day 13 of age (22% vs. 0% in controls). However, subsequent publications from this lab indicated that retained areolas in control animals ranged as high as 14% (Ostby et al., 2001). The usefulness of these data for hazard and risk assessment is limited as a single high dose was utilized, effects were pooled to achieve statistical significance, and the only clearly statistically significant finding is suspect based on high incidence of this finding in other control groups. The authors, too, questioned the significance of the statistical power of their analysis.

 

Anti-androgenic parameters were also evaluated in a poorly reported study by Hass et al (2003). This study is only available as an abstract. 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, however, incidence was not reported. In contrast to the study of Gray et al., 2000 no malformations of the male reproductive tract were reported. The poor reporting of the study makes it difficult to conclude on the significance of the findings observed at exceedingly high doses. 

 

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. Maternal intake as estimated for both the gestational and lactational phases. The intakes were 30.7 mg/kg.day, gestation, 66.2 mg/kg/day lactation, 400 ppm; 306.7 mg/kg.day, gestation, 656.7 mg/kg/day lactation, 4000 ppm; and 1164.5 mg/kg/day, gestation, 2656.7 mg/kg/day lactation, 20000 ppm. Offspring evaluations included 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 male pups at 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. In summary, no antiandrogenic effects were observed on the developing male reproductive tract in this study. Levels of exposure were similar to and exceeded those utilized in the studies of Gray et al., 2000 and Hass et al., 2003. 

 

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 sacrificed 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, glans penis weights, on serum testosterone levels, 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 antiandrogenicity 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 disrupter as defined by the Weybridge, IPCS and REACH guidance definitions.