Comparative analysis of diisononyl phthalate and di(isononyl)cyclohexane-1,2 dicarboxylate plasticizers in regulation of lipid metabolism in 3T3-L1 cells.

Diisononyl phthalate (DINP) and di(isononyl)cyclohexane-1,2-dicarboxylate (DINCH) are plasticizers introduced to replace previously used phthalate plasticizers in polymeric products. Exposure to DINP and DINCH has been shown to impact lipid metabolism. However, there are limited studies that address the mechanisms of toxicity of these two plasticizers. Here, a comparative toxicity analysis has been performed to evaluate the impacts of DINP and DINCH on 3T3-L1 cells. The preadipocyte 3T3-L1 cells were exposed to 1, 10, and 100 µM of DINP or DINCH for 10 days and assessed for lipid accumulation, gene expression, and protein analysis. Lipid staining showed that higher concentrations of DINP and DINCH can induce adipogenesis. The gene expression analysis demonstrated that both DINP and DINCH could alter the expression of lipid-related genes involved in adipogenesis. DINP and DINCH upregulated Pparγ, Pparα, C/EBPα Fabp4, and Fabp5, while both compounds significantly downregulated Fasn and Gata2. Protein analysis showed that both DINP and DINCH repressed the expression of FASN. Additionally, we analyzed an independent transcriptome dataset encompassing temporal data on lipid differentiation within 3T3-L1 cells. Subsequently, we derived a gene set that accurately portrays significant pathways involved in lipid differentiation, which we subsequently subjected to experimental validation through quantitative polymerase chain reaction. In addition, we extended our analysis to encompass a thorough assessment of the expression profiles of this identical gene set across 40 discrete transcriptome datasets that have linked to diverse pathological conditions to foreseen any potential association with DINP and DINCH exposure. Comparative analysis indicated that DINP could be more effective in regulating lipid metabolism.

Effect of subacute and prenatal DINCH plasticizer exposure on rat dams and male offspring hepatic function: The role of PPAR-α.

Plasticizers are compounds added to plastics to modify their physical proprieties. The most well-known class of plasticizers, the phthalates, has been shown to possess antiandrogenic and tumor promoting activities. 1,2-Cyclohexane dicarboxylic acid diisononyl ester (DINCH) was approved for use in food contact containers in 2006 and has been used as a replacement for phthalates in toys and children products. However, we reported previously that the DINCH metabolite MINCH acts on primary rat adipocytes through the peroxisome proliferator activated receptor (PPAR)-α pathway in a manner similar to phthalates. Evidence from our studies, as well as from the current bibliography on DINCH, suggests that the liver might be one of its target organs. In the present study, we collected tissues from dams exposed subacutely and progeny at postnatal day (PND) 3 and 60 exposed in utero to DINCH (1, 10 and 100 mg/kg bw/day). Exposure to DINCH drastically affected liver gene expression in all 3 age groups tested and in particular at the dose of 1 mg/kg bw/day. The PPAR-α pathway along with other metabolic and DNA replication pathways were affected by DINCH. Modifications in PPAR-α and superoxide dismutase (SOD)-1 protein levels were observed in dams at PND21, as well as male progeny at PND3 and 60. No sign of fibrosis or direct liver toxicity was observed after 8 days of stimulus with low doses of DINCH. This study provides evidence that DINCH is not a biologically inert molecule in the rat, and in the liver its actions are mediated, at least in part, by PPAR-α.

Bioavailability of phthalate and DINCH® plasticizers, after oral administration of dust to piglets.

For decades, phthalates have been widely used as plasticizers in a large number of consumer products, leading to a complex exposure to humans via ingestion, inhalation or dermal uptake. Children may have a higher unintended dust intake per day compared to adults. Therefore, dust intake of children could pose a relevant exposure and subsequently a potential health risk. The aim of this study was to determine the relative bioavailability of certain phthalates, such as di(2-ethylhexyl) phthalate (DEHP), di-isononyl phthalate (DINP) and the non-phthalate plasticizer diisononyl 1,2-cyclohexanedicarboxylic acid (DINCH®, Hexamoll®), after ingestion of dust. Seven 5-week-old male piglets were fed five different dust samples collected from daycare centers. Overall, 0.43 g to 0.83 g of dust sieved to 63 µm were administered orally. The piglets' urine was collected over a period of 38 h. The excreted metabolites were quantified using an LC-MS/MS method. The mean uptake rates of the applied doses for DEHP, DINP, and DINCH® were 43% ± 11%, 47% ± 26%, and 9% ± 3.5%, respectively. The metabolites of DEHP and DINP showed maximum concentrations in urine after three to five hours, whereas the metabolites of DINCH®, reached maximum concentrations 24 h post-dose. The oral bioavailability of the investigated plasticizers was higher compared to the bioaccessibility reported from in vitro digestion tests. Furthermore, the bioavailability of DEHP did not vary substantially between the dust samples, whereas a dose-dependent saturation process for DINP was observed. In addition to other intake pathways, dust could be a source of plasticizers in children using the recent intake rates for dust ingestion.

In vitro exposure to the next-generation plasticizer diisononyl cyclohexane-1,2-dicarboxylate (DINCH): cytotoxicity and genotoxicity assessment in human cells.

Plasticizers are currently present in many consumer products, particularly food packaging, children's toys, and medical devices. There are concerns regarding potential leaching to environment or food, thus increasing the risk of human exposure by inhalation, ingestion and/or dermal absorption potentially leading to adverse health consequences. Hexamoll diisononyl cyclohexane-1,2-dicarboxylate (Hexamoll® DINCH®), a non-phthalate plasticizer, has been used as a safer alternative to hazardous phthalates. In contrast to phthalates, evidence indicates that DINCH did not produce endocrine disruption, reproductive dysfunctions, genotoxicity or mutagenicity. However, there are limited data available regarding safety assessment, especially with respect to genotoxicity in human cells. The aim of this study was to assess DINCH cytotoxic and genotoxic effects in human liver and kidney cell lines following several exposure periods. For this purpose, the MTT cell viability, micronucleus, conventional and formamidopyrimidine DNA glycosylase (FPG)-modified comet assays were employed to detect cell death and genotoxicity, respectively. Data demonstrated that DINCH induced cytotoxicity in kidney cells exposed for 48hr, but not in liver cells. No marked chromosomal damage was noted after short-term or longer following treatment of both cell lines. However, DINCH produced oxidative DNA damage in liver cells exposed for 3 h, which decreased after a more prolonged incubation period. The occurrence of oxidative lesions, even transiently, indicates that mutation fixation may occur leading to adverse effects in liver. Therefore, these findings suggest that DINCH may be hazardous to humans and that further investigation is necessary to warrant its safety.

In vitro cytotoxic effects of secondary metabolites of DEHP and its alternative plasticizers DINCH and DINP on a L929 cell line.

BACKGROUND: Phthalic acid esters are widely used to improve the plasticity of PVC in medical devices (MD). The most famous plasticizer is DEHP, whose use in medical devices has been contested by the European authorities since 2008. Several alternative plasticizers are being considered to replace DEHP, such as DEHT, TOTM, DINP or DINCH, but they are also released from the PVC throughout their life cycle and are metabolized in the same way as DEHP. OBJECTIVES: Our study focuses on the in vitro cytotoxicity of two alternative plasticizers (DINCH and DINP) contained in certain medical devices. They are likely to migrate and be transformed in vivo into the primary and secondary metabolites by a metabolism similar to that of DEHP. This preliminary study is the first to assess the in vitro cytotoxicity of oxidized metabolites of DINCH and DINP based on the EN ISO 10-993-5 standards documents. METHODS: We have studied the complete multi-step organic synthesis of secondary metabolites of DINP and DINCH and have performed cytotoxicity tests on L929 murine cells according to the EN ISO 10993-5 standard design for the biocompatibility of a MD. The tested concentrations of obtained metabolites (0.01, 0.05 and 0.1 mg/mL) covered the range likely to be found for DEHP (total metabolism) in biological fluids coming into direct contact with the MD. The concentrations tested in our study were chosen based on a complete transformation of the plasticizers released after direct contact between a MD and the patient's blood. RESULTS: Only 7-oxo-MMeOCH is cytotoxic at the highest concentration (0.1 mg/mL) after 7 days of exposure, just like 5-oxo-MEHP for the same concentration. By contrast, 7-OH-MMeOP, 7-cx-MMeOP, 7-oxo-MMeOP, 7-OH-MMeOCH and 7-cx-MMeOCH were not found to be cytotoxic. CONCLUSION: The known concentrations of these secondary metabolites in urinary samples are in the µg/L range, i.e. about 100-1000 times lower than the concentrations tested in this study. Cytotoxicity is known to be dose-dependent but it is not always the case for endocrine perturbations and the secondary metabolites could induce endocrine perturbations at very low doses.

Hexamoll® DINCH: Lack of in vivo evidence for obesogenic properties.

Hexamoll® DINCH is an important alternative to phthalate plasticizers. Although regulatory reviews have not identified any potential hazards even in sensitive populations, an in vitro study by Campioli et al. (2015) suggested Hexamoll® DINCH might alter fat storage in adipocytes resulting in obesity. To evaluate this hypothesis, data from studies with Hexamoll® DINCH were reviewed for evidence of deposition in fat, changes in body weight, or changes in serum chemistry reflecting altered metabolic status. Body weights of F1 and F2 pups in a two-generation study did not differ from controls even at 1000 mg Hexamoll® DINCH/kg body weight. Mean relative liver weights from the 1000 and 300 mg/kg bw groups were increased, but without histopathologic changes. Triglyceride and cholesterol levels in serum were not affected. In addition, subchronic and chronic studies in rats did not give evidence of an obesogenic effect. Radioactivity from 20 or 1000 mg/kg bw 14C-labelled Hexamoll® DINCH dosed orally remained 2-3 times longer in adipose tissue than in well-perfused tissues; however, levels were 20-500% below other tissues at 1 and 8 h post dosing. Radioactivity concentrations in organs and tissues excluding the GI tract declined rapidly and continuously, and decreased in parallel to the concentration in plasma during the following 20 h. Both, initial and terminal half-lives of radioactivity concentration do not indicate a potential for accumulation. Furthermore, a metabolomic comparison of Hexamoll® DINCH with DEHP and other phthalates shows complete separation of the metabolomic profile of these two chemical classes, meaning that their effects on the body and the body's reaction to the substance are different. Hence, comprehensive in vivo data do not show any evidence of Hexamoll® DINCH altering fat metabolism or having obesogenic properties.

The urinary metabolites of DINCH® have an impact on the activities of the human nuclear receptors ERα, ERß, AR, PPARα and PPARγ.

DINCH® (di-isononyl cyclohexane-1,2-dicarboxylate) is a non-phthalate plasticizer that has been developed to replace phthalate plasticizers such as DEHP (di-2-ethylhexyl phthalate) or DINP (di-isononyl phthalate). DINCH® is metabolized to its corresponding monoester and subsequently to oxidized monoester derivatives. These are conjugated to glucuronic acid and subject to urinary excretion. In contrast to DINCH®, there are almost no toxicological data available regarding its primary and secondary metabolites. The present study aimed at the characterization of potential endocrine properties of DINCH® and five DINCH® metabolites by using reporter gene assays to monitor the activity of the human nuclear receptors ERα, ERß, AR, PPARα and PPARγ in vitro. DINCH® itself did not have any effect on the activity of these receptors whereas DINCH® metabolites were shown to activate all these receptors. In the case of AR, DINCH® metabolites predominantly enhanced dihydrotestosterone-stimulated AR activity. In the H295R steroidogenesis assay, neither DINCH® nor any of its metabolites affected estradiol or testosterone synthesis. In conclusion, primary and secondary DINCH® metabolites exert different effects at the molecular level compared to DINCH® itself. All these in vitro effects of DINCH® metabolites, however, were only observed at high concentrations such as 10 µM or above which is about three orders of magnitude above reported DINCH® metabolite concentrations in human urine. Thus, the in vitro data do not support the notion that DINCH® or any of the investigated metabolites may exert considerable endocrine effects in vivo at relevant human exposure levels.

The flavonoid 6-methoxyflavone allays cisplatin-induced neuropathic allodynia and hypoalgesia.

Chemotherapy-induced peripheral neuropathy (CIPN) is a major dose limiting side-effect of several commonly used chemotherapeutic agents (such as cisplatin) that profoundly impairs patient quality of life. Unfortunately, neither prophylactic strategies nor symptomatic treatments have proven useful in this condition. Flavonoids are found ubiquitously in fruits and vegetables and exert a multiplicity of beneficial effects. In this study, the antinociceptive activity of 6-methoxyflavone (6-MF) was investigated and evaluated in comparison with gabapentin in a rat model of CIPN. The effect on motor balance was also assessed using the rotarod and footprint analysis paradigms. 6-MF possessed both peripheral and central antinociceptive activities against tonic and phasic nociceptive stimuli. Cisplatin administration (3.0mg/kg/week, i.p.) for four consecutive weeks generated temporal mechanical allodynia (decreased paw withdrawal threshold; PWT) and thermal hypoalgesia (increased paw thermal threshold; PTT) in the bilateral hindpaws. Daily treatment with 6-MF (25, 50 and 75mg/kg/day, i.p) for four weeks attenuated the cisplatin-induced expression of nocifensive behaviors observed as a significant increase in PWT and alleviation of PTT during the third and fourth weeks of cisplatin administration. Accordingly, daily gabapentin (75mg/kg, i.p) suppressed the expression of CIPN by normalizing the PWT and hotplate response latency. However, these antinociceptive actions were associated with motor impairment exemplified by a significant decrease in rotarod endurance latency and a deficit in the uniformity of step alternation. In contrast, 6-MF was devoid of these adverse side-effects. These findings suggested that 6-MF afforded desirable neuropathic pain alleviating effects in CIPN and it was devoid of gabapentin-like unwanted motor side-effects.

Additional oxidized and alkyl chain breakdown metabolites of the plasticizer DINCH in urine after oral dosage to human volunteers.

Hexamoll® DINCH® (diisononyl-cyclohexane-1,2-dicarboxylate) is a new high molecular weight plasticizer and a non-aromatic phthalate substitute. In this follow-up study, we further investigated the extensive oxidative metabolism of Hexamoll® DINCH® after oral dosage of 50 mg to three male volunteers (0.552-0.606 mg/kg body weight). Urine samples were consecutively collected over 48 h post-dose. Chemical analysis was carried out by HPLC-MS/MS with labeled internal standards. New metabolites were tentatively identified and quantified via fragmentation analogies and new standard substances. In addition to the five urinary DINCH metabolites previously reported by us, we identified two groups of extensively oxidized metabolites characterized (a) by multiple side chain oxidation and breakdown and (b) by hydroxylation at the cyclohexane ring. The five newly identified carboxylated breakdown metabolites represented in sum 5.12 ± 0.49 % of the applied dose. MCHxCH (cyclohexane-1,2-dicarboxylic acid mono carboxyhexyl ester) was identified as a major metabolite (2.71 ± 0.34 %) and thus represents the second most important specific metabolite of DINCH after OH-MINCH (10.7 ± 2.1 %). Less than 1 % was excreted as ring-hydroxylated metabolites (four metabolites identified). Based upon a new reference standard, we can also update oxo-MINCH to 2.6 % of the applied dose. This follow-up study increases the total amount of the recovered dose from 39.2 to 45.7 % and describes a new major metabolite (MCHxCH) of DINCH that can be used as an additional valuable and specific biomarker to assess DINCH® exposure in future human biomonitoring studies.

Effect of topical neuromodulatory medications on oral and skin keratinocytes.

BACKGROUND: Neuromodulatory medications (NMs), such as amitriptyline, carbamazepine and gabapentin, are used as topical preparations for the management of neuropathic orofacial pain (NOP) and have produced promising preliminary results. The aim of this study was to investigate the effects of three aforementioned NMs on cell lines relevant to the orofacial tissues in vitro as no published studies have examined the effect of these topical NMs. METHODS: Cellular viability was measured using alamarBlue® , testing cumulative and specific time point effects of NMs on human skin keratinocytes and oral keratinocytes. Effects of the NMs on cell counts were investigated by CCK-8 assay. Drug concentrations released from NM orabase pastes after 30-min incubation were measured by high-performance liquid chromatography. Using these clinical concentrations, morphological changes and cytokine expression were investigated using scanning electron microscopy (SEM) and human inflammatory antibody array (AAH), respectively. RESULTS: Cumulative and specific time point viability and cell count methods revealed that amitriptyline caused a significant decrease in cellular viability and counts in both cell lines. Carbamazepine also had significant effects after long-term exposure and at higher concentrations, whilst gabapentin had little demonstrable effect. SEM confirmed the cytotoxicity of amitriptyline, whilst AAH revealed no significant changes in cytokine expression following amitriptyline, carbamazepine or gabapentin exposure compared with control. CONCLUSIONS: The results raise concerns about the safety of topical amitriptyline as it was cytotoxic to skin and oral keratinocytes in both exposure times and concentrations, whilst carbamazepine was cytotoxic only at high concentrations and after longer exposure times and gabapentin had no demonstrable effects.

Urinary concentrations of cyclohexane-1,2-dicarboxylic acid monohydroxy isononyl ester, a metabolite of the non-phthalate plasticizer di(isononyl)cyclohexane-1,2-dicarboxylate (DINCH), and markers of ovarian response among women attending a fertility center.

Di(isononyl)cyclohexane-1,2-dicarboxylate (DINCH), a non-phthalate plasticizer, was introduced commercially in 2002 as an alternative to ortho-phthalate esters because of its favorable toxicological profile. However, the potential health effects from DINCH exposure remain largely unknown. We explored the associations between urinary concentrations of metabolites of DINCH on markers of ovarian response among women undergoing in vitro fertilization (IVF) treatments. Between 2011 and 2015, 113 women enrolled a prospective cohort study at the Massachusetts General Hospital Fertility Center and provided up to two urine samples prior to oocyte retrieval. The urinary concentrations of two DINCH metabolites, cyclohexane-1,2-dicarboxylic acid monohydroxy isononyl ester (MHiNCH) and cyclohexane-1,2-dicarboxylic acid monocarboxyisooctyl ester (MCOCH), were quantified by isotope dilution tandem mass spectrometry. We used generalized linear mixed models to evaluate the association between urinary metabolite concentrations and markers of ovarian response, accounting for multiple IVF cycles per woman via random intercepts. On average, women with detectable urinary MHiNCH concentrations, as compared to those below LOD, had a lower estradiol levels (-325 pmol/l, p=0.09) and number of retrieved oocytes (-1.8, p=0.08), with a stronger association among older women. However, urinary MHiNCH concentrations were unrelated to mature oocyte yield and endometrial wall thickness. In conclusion, we found suggestive negative associations between urinary MHiNCH concentrations and peak estradiol levels and number of total oocyte yields. This is the first study evaluating the effect of DINCH exposure on human reproductive health and raises the need for further experimental and epidemiological studies to better understand the potential effects of this chemical on health.

Urinary toxicokinetics of di-(isononyl)-cyclohexane-1,2-dicarboxylate (DINCH(®)) in humans following single oral administration.

Phthalates such as di-2-ethylhexyl phthalate (DEHP) were restricted due to their toxic mainly reprotoxic effects. Therefore compounds such as di-(isononyl)-cyclohexane-1,2-dicarboxylate (DINCH(®)) substitute these phthalates and the exposure of humanes to substitutes may occur. Here, kinetic data are presented to assess the exposure of humans. Male and female volunteers excreted nearly the complete orally administered dose (1mg/kg b.w. corresponding to the tolerable daily intake of EFSA) of di-(isononyl)-cyclohexane-1,2-dicarboxylate within 70 h. More than 75% were excreted within 24h. Besides the main metabolite cyclohexane-1,2-dicarboxylic acid (CHDA) quantitated after hydrolysis four further metabolites of DINCH(®) are determined. Cyclohexane-1,2-dicarboxylic acid-mono-(7-hydroxy-4-methyl)octyl ester (OH-MINCH) is the main secondary metabolite with about 14% of the administered dose. Differences in excretion of all metabolites between male and females are small. Based on the generated toxicokinetic data exposure of 20 humans is recalculated from their spot urine sample collected in 2014 and the exposure are clearly below the current tolerable daily intake of 1mg/kg b.w.

The publication "Cyclohexane-1,2-dicarboxylic acid diisononyl ester and metabolite effects on rat epididymal stromal vascular fraction differentiation of adipose tissue" by Enrico Campioli, Tam B. Duong, François Deschamps, Vassilios Papadopoulos, Environmental Research 140 (2015), 145-156, merits some critical comments.

In essence, the authors report MINCH, a monoester and minor urinary metabolite (Koch et al., 2013) of a plasticizer marketed by BASF under the brandname Hexamoll(®) DINCH(®), promotes the differentiation of preadipocytes derived from rat epididymal stromal vascular fraction (SVF) to adipocytes. The authors have over-interpreted their in-vitro data and missed important publicly available in-vivo data.

Identification of phototransformation products of the antiepileptic drug gabapentin: Biodegradability and initial assessment of toxicity.

The anticonvulsant drug Gabapentin (GAB) is used for the treatment of various diseases (e.g. epilepsy, bipolar disorder, neuropathic pain) and is being consumed in high amounts. As GAB is not metabolized and shows a weak elimination in sewage treatment plants (STPs), it has been detected in surface water and even in raw potable water. Moreover, the confirmed teratogenic effects of GAB indicate the need for further investigations regarding options for the elimination of GAB in the water cycle. Little is known about the behavior of GAB during treatment with UV light, which is normally used for the disinfection of potable water and discussed for advanced wastewater treatment. In this study, GAB was exposed to polychromatic UV irradiation at different initial concentrations in aqueous solution. Afterwards the structures of the resulting phototransformation products (PTPs) were identified and elucidated by means of high-resolution mass spectrometry. GAB and photolytic mixtures were submitted to the Closed Bottle Test (CBT; OECD 301 D) to assess biodegradability. Furthermore, the toxicity of GAB and its photolytic mixtures was initially addressed on screening level using a modified luminescent bacteria test (LBT) and the umu-test (ISO/FDIS 13829). Environmentally realistic concentrations of GAB were disclosed by predicting STP influent concentrations (24.3 and 23.2 µg L(-1)). GAB with initial concentration of 100 mg L(-1) was eliminated by 80% after 128 min of direct UV irradiation, but just 9% of non-purgeable organic carbon (NPOC) was removed indicating the formation of dead-end transformation products (TPs). Structures of different PTPs were elucidated and several identical PTPs could also be identified at lower initial treatment concentrations (20 mg L(-1), 5 mg L(-1), 1 mg L(-1) and 0.1 mg L(-1)). GAB was classified as not readily biodegradable. Moreover, photo treatment did not result in better biodegradable PTPs. With increasing UV treatment duration, photolytic mixtures of GAB showed an increased inhibition of both, the bacterial luminescence emission as well as the growth in the modified LBT. In the umu-test no significant induction of the umuC gene as an indicator of genotoxicity was observed. Our results show that UV irradiation of GAB containing water would lead to the formation of recalcitrant PTPs. Considering that GAB was found in raw drinking water, the formation of toxic PTPs during drinking water treatment with UV light might be possible. Therefore, further studies should be conducted regarding the fate and effects on human health and the environment of GAB and the PTPs identified within this study.

Toxicity of Hexamoll(®) DINCH(®) following intravenous administration.

Alternative plasticizers to di(2-ethylhexyl) phthalate (DEHP) for blood bags have been sought for many years. Cyclohexane-1,2-dicarboxylic acid, diisononylester (Hexamoll(®) DINCH(®)) is an alternative that has been evaluated in preliminary studies for compatibility and efficacy to preserve whole blood. While Hexamoll(®) DINCH(®) has an extensive database for mammalian toxicity via oral administration, data were needed to evaluate toxicity from intravenous (IV) administration to support the use of the plasticizer Hexamoll(®) DINCH(®) in blood bags. A series of studies was performed by slow IV injection or IV infusion of Hexamoll(®) DINCH(®), a highly viscous, hydrophobic substance, suspended in Intralipid(®) 20% (20% intravenous fat emulsion). Rats were injected once, followed by 14 days of recovery; injected daily for 5 days followed by 5 days of recovery, or infused for 29 days (4h/day) followed by 14 days of recovery. Dose levels were 0, 62, 125, and 250-300mg/kg body weight/day. These dose levels represent the limits of suspension and far exceed any anticipated exposures from migration out of plasticized blood bags. Animals were observed for signs of toxicity; body weight and feed consumption were measured; blood collected for clinical chemistry and hematology; and tissues collected and processed for histopathology. Special emphasis was placed on evaluating endpoints and tissues that are commonly associated with plasticizer exposure in rodents. Urine was collected during the 4-week study to quantify urinary metabolites of Hexamoll(®) DINCH(®). The results of the studies indicate that no substance-related toxicity occurred: no effects on behavior, no effects on organ weight, no effect on serum chemistry including thyroid hormones; and no effect on major organs, especially no testicular toxicity and no indication for peroxisome proliferation in the liver. The only effects seen were petechia and granulomas related to dissipation of suspended Hexamoll(®) DINCH(®) in the aqueous environment of the blood. However, the results of metabolite analyses demonstrate that Hexamoll(®) DINCH(®) was bioavailable. Therefore, based on the lack of Hexamoll(®) DINCH(®)-related systemic toxicity with the exception of the physical limitations, the no-observed-adverse-effect level for parenterally administered Hexamoll(®) DINCH(®) is considered to be 300mg/kg bw/day.