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Phthalate Di-isodecyl & Di-isononyl phthalates

5. What health effects can DIDP and DINP cause in laboratory animals?

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    DIDP

    Effects assessment

    Toxicokinetics, metabolism and distribution

    The data available on toxicokinetic suggest that, via gastro-intestinal tract (GIT), absorption of DIDP decreases as dose increases (56% at the low dose of 0.1 mg/kg, 46% at the mid dose of 11.2 mg/kg and 17% at the high dose of 1,000 mg/kg) and seems to be of saturable mechanism; when dose increases, an increasing amount of unabsorbed compound is eliminated. Via the dermal route, the absorption is very low in rats. DIDP showed a very slow excretion, reflecting a slow dermal uptake process. The maximum percentage of absorption may be estimated 4% of applied dose in 7 days by analogy with DINP. In humans, skin absorption is still lower than in rat as indicated by in vitro comparative studies. Inhaled DIDP aerosol seems readily absorbed, thus a 75 % bioavailability seems realistic. In tissues, DIDP is mainly recovered in GIT, liver, kidneys, by the oral or inhalation route, whereas following dermal exposure, muscle and adipose tissues contain most of the dose remaining in the body. Only metabolites of DIDP (the oxidative monoester derivative and phthalic acid) are excreted in urine. In feces, the monoester oxidative derivative, MIDP (monoisodecyl phthalate) as well as DIDP were detected. DIDP is rapidly eliminated and not accumulated in tissues. By the oral and inhalation routes, excretion is shared between urine and faeces. By dermal exposure, only faecal elimination was indicated. In addition, results from a two-generation study suggest a possible transfer of DIDP through the milk when dams are exposed by the oral route.

    Acute toxicity

    Upon single exposure, DIDP has a low acute toxicity by all routes of administration.

    Irritation

    Human or animal data suggest no potential irritant effects on skin, eyes or respiratory system. Sensitisation

    There is no evidence for skin or respiratory sensitisation.

    Repeated dose toxicity

    The liver was identified as a target organ as a result of oral repeated exposure. In rodents, increases in liver weight were accompanied by biochemical evidence of peroxisomal proliferation; thus, a NOAEL of 60 mg/kg/d was identified in rats from a standard 90-day study. Findings in dogs were qualitatively consistent (increases in liver weight and swollen and vacuolated hepatocytes); a NOAEL of 15 mg/kg/d was derived from a 13-week oral dog study, in spite of the large limitations of this study.

    Increases in kidney weights are also observed in repeated dose toxicity tests but in a non- consistent way and with no concurrent histopathological changes. Renal damages are only observed in a two-generation study (about 12 weeks) from 100 - 200 mg/kg/d, but only in male rats; a specific male rat effect is generally assumed.

    In an inhalation study, there was no systemic effect observed and toxicity was limited to local inflammatory changes in the lung.

    Mutagenicity

    DIDP is not mutagenic in vitro in bacterial mutation assays (with and without metabolic activation) and is negative in a mouse lymphoma assay. It is not clastogenic in a mouse micronucleus assay in vivo. This indicates that DIDP is a non-genotoxic agent. Carcinogenicity

    Regarding carcinogenicity, cell transformation tests were conducted on DIDP. One positive result obtained is in accordance with those obtained with well-known peroxisome proliferators. No carcinogenicity long-term study is available for DIDP but an increase in incidence of hepatocellular tumours in rats related to peroxisome proliferation might be anticipated, in regard with the increased incidence in tumour liver cells observed with DEHP and DINP in carcinogenicity studies. Indeed, it is now well-accepted that peroxisome proliferation is specific to rodents. It has been established that peroxisome proliferators exhibit their pleiotropic effects due to activation of PPARα (peroxisome proliferator-activated receptor α) and that PPARα is expressed only at low level in humans, explaining the absence of significant response in humans to the action of peroxisome proliferators. Thus, there is no concern for a potential carcinogenic effect in humans through such a mechanism.

    Toxicity for reproduction

    Regarding toxicity for reproduction, in 42-44 day year old (pubertal) or adult rats there is no indication of organ reproductive effects evidenced by histological observation in repeated dose toxicity studies and a two-generation study. In this two-generation study, a decrease in mean percent normal sperm was observed but of low incidence and only in P1 generation. In pups (F1, F2 and in the cross fostering satellite group) decreases in testes weight and cryptorchidism in F2 high-dose offspring were observed, likely due to the low body weight, since no histopathological damages were observed in adult testes. There were no changes in reproductive indices. From those assays, no adverse effects on fertility may be anticipated.

    Regarding developmental effects, treatment of dams from gd 6-15 did not cause structural malformations but consistently demonstrated skeletal variations (increased fœtal cervical and lumbar ribs) at 1,000 mg/kg/d concurrently with slight signs of maternal toxicity and lead to a NOAEL of 500 mg/kg/d; in a two-generation rat study, body weight decrease was observed in offspring partly related to lactation at the highest dose of 0.8% and leads to a NOAEL of 0.4% (253 to 761 mg/kg/d seeing that received doses are widely dependent on the period considered).

    Developmental toxicity was observed consistently in the two-generation studies, where decrease in survival indices leads to a NOAEL of 0.06% (33 mg/kg/d DIDP).

    A prenatal exposure study in mice conducted at 9,650 mg/kg/d does not affect pregnancy outcome, however, as this test was drawn for screening purpose, it is insufficient to conclude to an absence of effect.

    DIDP 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 DIDP does not increase uterine wet weight or does not give rise to vaginal epithelial cell cornification. In a two-generation study, developmental landmarks (anogenital distance, nipple retention and preputial separation) are not impaired; this suggests a lack of anti-androgenic activities.

    Source & ©: ECB "2003 Risk Assessment Report (RAR 041) on
    Di-"isodecyl" Phthalate (DIDP), Summary of the Report, chapter 4: Human Health

    DINP

    Effects assessment

    Toxicokinetics, metabolism and distribution

    The data available on toxicokinetic suggest that, via gastro-intestinal tract (GIT), absorption of DINP decreases as dose increases (49% at the low dose of 50 mg/kg and 39% at the high dose of 500 mg/kg eliminated in urine); in addition, absorption of the substance seems to be a saturable mechanism. Dermal absorption is slow in rats. The maximum percentage of the applied substance being absorbed in 7 days is less than 4%. In humans skin absorption is still lower than in rat as indicated by in vitro comparative studies. Via inhalation, a bioavailability of 75% may be assumed by analogy with DIDP. In tissues, DINP is mainly recovered in GIT, liver and kidney by oral route whereas following dermal exposure, liver, muscle and adipose tissues contain most of the dose remaining in the body. DINP metabolites were excreted in urine and to a lesser extent in feces. DINP was de-esterified to the monoester which was further metabolised by side-chain oxidation of the ester group or by hydrolysis to phthalic acid. Repeated dosing caused no accumulation of DINP and/or its metabolites in blood and tissue, but resulted in increased formation and elimination of the monoester-oxidation products. DINP is rapidly eliminated. By the oral and dermal routes, excretion is shared between urine and feces. By dermal exposure, biliary excretion is shown.

    Acute toxicity

    Upon single exposure, DINP has a low acute toxicity by all routes of administration.

    Irritation

    Human or animal data suggest no potential irritant effects on skin, eyes or respiratory system.

    Repeated dose toxicity

    The liver is a target for chronic toxicity and a NOAEL of 88 mg/kg/d was assumed for liver effects based on hepatic biochemical changes (increased aspartate-aminotransferase, AST and alanine-aminotransferase, ALT) and on increases of liver weight with histopathological findings. Repeated-dose studies performed to assess the peroxisomal proliferation potential of DINP, reveal biochemical evidence of peroxisome proliferation in rodents. In contrast, there was no evidence of peroxisome proliferation in marmosets or in cynomolgus monkeys; in humans as well, there is no significant response to peroxisome proliferators.

    For kidney effects, a NOAEL of 88 mg/kg/d was determined, based on increased kidney weights. Histologically, there was an increase in frequency/severity of chronic progressive nephropathy at quite low doses, but specifically in males. Histological features are consistent with the specific male rat nephropathy irrelevant to humans, namely alpha 2u globulin nephropathy. It was demonstrated, by immunohistochemical techniques, that exposure to DINP results in a dose dependant alpha 2u globulin accumulation in male rat kidneys and is likely the mechanism for kidney tumours seen only in male rats administered high dietary levels of DINP. In mice, there was also progressive nephropathy observed at very high doses.

    Mutagenicity

    DINP is not mutagenic in vitro in bacterial mutation assays or mammalian gene mutation assay (with and without metabolic activation) and is not clastogenic in one cytogenetic assay in vitro on CHO cells and in one in vivo assay on bone marrow cell of Fisher 344 rats. This suggests that DINP is not genotoxic.

    Carcinogenicity

    In chronic/carcinogenicity studies, DINP was found to induce significant excess of liver neoplasia in rats and mice after oral administration. This is consistent with a peroxisome proliferation mode of action for hepatic tumour induction specific in rodent. It has been established that peroxisome proliferators exhibit their pleiotropic effects due to activation of PPARα (peroxisome proliferator-activated receptor α) and that PPARα is expressed only at low level in humans, explaining the absence of significant response to the action of peroxisome proliferators, thus, there is no concern for a potential carcinogenic effect in humans.

    Regarding MNCL (mononuclear cell leukemia), a clearly increased incidence is observed in the two studies conducted with Fisher rats. However, MNCL, a common neoplasm in the Fischer 344 rats, was categorised by IARC as “an unclassified leukemia with no known human counterpart” and substances which increase MNCL frequency as “not classifiable as to carcinogenicity in humans”. Pertaining to kidney tumours, the species and sex-specific alpha 2u globulin mechanism likely responsible for kidney tumours seen in male rats is not considered as relevant to humans.

    Toxicity for reproduction

    Regarding fertility, no adverse effects are anticipated in adult rats on reproductive organs, in repeated dose toxicity and in one-generation studies. In mice, a very high dose (5,770 mg/kg/d) leads to decrease in testicular weight with abnormal/immature sperm forms and uterus/ovaries atrophy in the 13-week study. In the 104-week chronic study, a NOAEL of 1,500 ppm (276 mg/kg/d) can be assumed for testicular effects, based on decrease in testicular weight (relative and absolute) observed from 742 mg/kg/d. Testicular lesions were not observed in a 13-week gavage study in adult marmosets well as in a 2-week gavage study in prepubertal cynomolgus monkeys. In a two-generation study, no changes in reproductive indices are observed.

    In the developmental studies, visceral (dilated renal pelvis and hydroureter) and skeletal variations (increases in lumbar and cervical ribs) were significantly increased on a litter basis at 1,000 mg/kg/d, leading to a NOAEL of 500 mg/kg/d. In addition, a decrease of mean offspring body weight was observed following parental administration of DINP in the one and two-generation studies from the lowest dose tested (LOAEL of 159 mg/kg/d). In a two-generation study, parental toxicity was limited to lower mean body weight and hepatic changes.

    In regard with offspring survival in rats, a decrease of life birth and survival indices was observed in a one-generation range-finding study at doses higher than 1,000 mg/kg/d.

    DINP is devoid of estrogenic activity in vitro, it does not show 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. However, a recent study in rats (perinatal exposure) indicated that infants male displayed female like areolas/nipples and that incidence of reproductive malformation was slightly but significantly increased (7.7% versus 91% with DEHP).

    Source & ©: ECB "2003 Risk Assessment Report (RAR 046) on
    Di-"isodecyl" Phthalate (DIDP), Summary of the Report, chapter 4: Human Health

    For more information, see the full ECB Risk Assessment Report:
     Chapter 4: Human Health

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