Safety evaluation of food contact paper and board using chemical tests and in vitro bioassays: role of known and unknown substances.

In vitro toxicological tests have been proposed as an approach to complement the chemical safety assessment of food contact materials, particularly those with a complex or unknown chemical composition such as paper and board. Among the concerns raised regarding the applicability of in vitro tests are the effects of interference of the extractables on the outcome of the cytotoxicity and genotoxicity tests applied and the role of known compounds present in chemically complex materials, such as paper and board, either as constituents or contaminants. To answer these questions, a series of experiments were performed to assess the role of natural substances (wood extracts, resin acids), some additives (diisopropylnaphthalene, phthalates, acrylamide, fluorescent whitening agents) and contaminants (2,4-diaminotoluene, benzo[a]pyrene) in the toxicological profile of paper and board. These substances were individually tested or used to spike actual paper and board extracts. The toxic concentrations of diisopropylnaphthalenes and phthalates were compared with those actually detected in paper and board extracts showing conspicuous toxicity. According to the results of the spiking experiments, the extracts did not affect the toxicity of tested chemicals nor was there any significant metabolic interference in the cases where two compounds were used in tests involving xenobiotic metabolism by the target cells. While the identified substances apparently have a role in the cytotoxicity of some of the project samples, their presence does not explain the total toxicological profile of the extracts. In conclusion, in vitro toxicological testing can have a role in the safety assessment of chemically complex materials in detecting potentially harmful activities not predictable by chemical analysis alone.

Test procedures for obtaining representative extracts suitable for reliable in vitro toxicity assessment of paper and board intended for food contact.

This paper describes the use of a suite of extraction procedures applicable to the assessment of the in vitro toxicity of paper/board samples intended for food-contact applications. The sample is extracted with ethanol, water, or exposed to modified polyphenylene oxide (Tenax) for fatty, non-fatty and dry food applications, respectively. The water extracts are directly suitable for safety assessment using in vitro bioassays. The ethanol extracts of the paper/board and of the exposed Tenax require pre-concentration to give acceptable sensitivity. This is because the in vitro bioassays can tolerate only a small percentage of added organic solvent before the solvent itself inhibits. The extraction procedures have been selected such that they mimic the foreseeable conditions of use with foods and that they are also fully compatible with the battery of in vitro biological assays for the safety assessment of the total migrate. The application of the extraction protocols is illustrated by the results for one of the many paper/board samples provided by the BIOSAFEPAPER project industrial platform members. The assessment indicated that this sample should not be considered as suitable for use with fatty foodstuffs but was suitable for dry and non-fatty foods. Information subsequently received from the manufacturer revealed that this was a non-food-grade product included in the project to test the capabilities of the bioassay procedures. The selection criteria for the test conditions and the suite of methods developed have been prepared in Comité Européen de Normalisation (CEN) format and is currently being progressed by CEN/TC172 as a European Standard.

The BIOSAFEPAPER project for in vitro toxicity assessments: preparation, detailed chemical characterisation and testing of extracts from paper and board samples.

Nineteen food contact papers and boards and one non-food contact board were extracted following test protocols developed within European Union funded project BIOSAFEPAPER. The extraction media were either hot or cold water, 95% ethanol or Tenax, according to the end use of the sample. The extractable dry matter content of the samples varied from 1200 to 11,800 mg/kg (0.8-35.5 mg/dm2). According to GC-MS the main substances extracted into water were pulp-derived natural products such as fatty acids, resin acids, natural wood sterols and alkanols. Substances extracted into ethanol particularly, were diisopropylnaphthalenes, alkanes and phthalic acid esters. The non-food contact board showed the greatest number and highest concentrations of GC-MS detectable compounds. The extracts were subjected to a battery of in vitro toxicity tests measuring both acute and sublethal cytotoxicity and genotoxic effects. None of the water or Tenax extracts was positive in cytotoxicity or genotoxicity assays. The ethanol extract of the non-food contact board gave a positive response in the genotoxicity assays, and all four ethanol extracts gave positive response(s) in the cytotoxicity assays to some extent. These responses could not be pinpointed to any specific compound, although there appeared a correlation between the total amount of extractables and toxicity.

Application of the threshold of toxicological concern (TTC) to the safety evaluation of cosmetic ingredients.

The threshold of toxicological concern (TTC) has been used for the safety assessment of packaging migrants and flavouring agents that occur in food. The approach compares the estimated oral intake with a TTC value derived from chronic oral toxicity data for structurally-related compounds. Application of the TTC approach to cosmetic ingredients and impurities requires consideration of whether route-dependent differences in first-pass metabolism could affect the applicability of TTC values derived from oral data to the topical route. The physicochemical characteristics of the chemical and the pattern of cosmetic use would affect the long-term average internal dose that is compared with the relevant TTC value. Analysis has shown that the oral TTC values are valid for topical exposures and that the relationship between the external topical dose and the internal dose can be taken into account by conservative default adjustment factors. The TTC approach relates to systemic effects, and use of the proposed procedure would not provide an assessment of any local effects at the site of application. Overall the TTC approach provides a useful additional tool for the safety evaluation of cosmetic ingredients and impurities of known chemical structure in the absence of chemical-specific toxicology data.

Safety assessment of food-contact paper and board using a battery of short-term toxicity tests: European union BIOSAFEPAPER project.

An European Union (EU)-funded project QLK1-CT-2001-00930 (BIOSAFEPAPER) involves the development, validation and intercalibration of a short-term battery of toxicological tests for the safety assessment of food-contact paper and board. Dissemination of the results to industry, legislators (e.g. DG Consumer Protection, DG Enterprises, DG Research), standardization bodies such as CEN, and consumers will create an agreed risk evaluation procedure. The project involves pre-normative research in order to establish a set of in-vitro cytotoxicity and genotoxicity tests that will be easily adaptable to food-contact fibre-based materials and have endpoints relevant to consumer safety, including sub-lethal cellular events. These tests will be performed on samples representing actual migration conditions from food-contact paper and board with respect to different foodstuffs, and should form an experimental basis for scientifically sound recommendations for a harmonized system of risk evaluation and product testing.

Steroid activities comparison of natural and food wrap compounds in human breast cancer cell lines.

In this study, we tested and compared the endocrine disruption activities of compounds in materials used to package foods (bisphenol A, bisphenol F, and bisphenol A diglycidylether BADGE) with natural molecules (genistein, apigenin, kaempferol, and tangeretin) in the human breast cancer cell lines MCF-7 (ER(+)) and MDA-MB453 (AR(+); GR(+)). Octylphenol was also chosen as a xenoestrogen reference. Two compounds had no estrogenic activity: BADGE and tangeretin. Genistein was the most active compound in the E-Screen assay with MCF-7, followed by octylphenol, bisphenol F, bisphenol A and apigenin, with kaempferol the least potent. All estrogenic compounds competed with 17beta-estradiol for binding to the MCF-7 ER and their estrogenic effects were abolished in the presence of tamoxifen, an ER antagonist. In MDA-MB453 cells transfected with pMMTVneo-Luc, all compounds had anti-androgenic activities, with octylphenol the most potent. Kaempferol, genistein, and apigenin were more potent anti-androgens than bisphenols A or F. The natural compounds had a biphasic effect on luciferase activity. At high concentrations, genistein (10(-5)M) and apigenin (10(-6)M) acted as GR agonists in transfected MDA-MB453 cells. Furthermore, apigenin, at a concentration of 10(-5)M, may act as a partial androgen receptor (AR) agonist, as nilutamide, an AR antagonist, inhibited its activity by 26%.

Methods of in vitro toxicology.

In vitro methods are common and widely used for screening and ranking chemicals, and have also been taken into account sporadically for risk assessment purposes in the case of food additives. However, the range of food-associated compounds amenable to in vitro toxicology is considered much broader, comprising not only natural ingredients, including those from food preparation, but also compounds formed endogenously after exposure, permissible/authorised chemicals including additives, residues, supplements, chemicals from processing and packaging and contaminants. A major promise of in vitro systems is to obtain mechanism-derived information that is considered pivotal for adequate risk assessment. This paper critically reviews the entire process of risk assessment by in vitro toxicology, encompassing ongoing and future developments, with major emphasis on cytotoxicity, cellular responses, toxicokinetics, modelling, metabolism, cancer-related endpoints, developmental toxicity, prediction of allergenicity, and finally, development and application of biomarkers. It describes in depth the use of in vitro methods in strategies for characterising and predicting hazards to the human. Major weaknesses and strengths of these assay systems are addressed, together with some key issues concerning major research priorities to improve hazard identification and characterisation of food-associated chemicals.

Estrogenic activity and metabolism of n-butyl benzyl phthalate in vitro: identification of the active molecule(s).

Some phthalates are suspected to disrupt the endocrine system, especially by mimicking estrogens. N-butyl benzyl phthalate (BBP) has estrogenic effects in vitro but not in vivo. The aim of this study was to identify the active molecule(s) (parent compound and/or metabolite(s)) involved in the estrogenic activities of BBP. The estrogenic effects of BBP and its in vivo metabolites were assessed using the following tests: E-Screen, ER binding, and PR induction tests on the human breast cancer cell line MCF-7 (ER(+)). BBP, the parent compound, was a partial agonist. It stimulated MCF-7 proliferation in the E-Screen assay and increased cytosolic progesterone receptors (PR) levels in a concentration-dependent manner. No BBP metabolites were active except hippuric acid (HA), which had a weak effect at very high concentrations. BBP and HA stimulatory effects on MCF-7 proliferation were antagonized by tamoxifen. However, no competition was observed between BBP or HA and 17beta-estradiol for binding to the estrogen receptor (ER). BBP metabolism by MCF-7 cells was also investigated. After a 48-h incubation, only 10% of the initial BBP remained in the culture medium, demonstrating that BBP was extensively metabolized by the MCF-7 cells. The radioactivity recovered in the medium was represented by: mono-n-butyl phthalate (MBuP, 25%) and mono-n-benzyl phthalate (MBeP, 48%), phthalic acid (6%), and benzoic acid (3%). Since none of these metabolites had estrogenic activities, this study demonstrates that the parent compound was the active molecule involved in the in vitro estrogenic effects of BBP.

Metabolism of n-butyl benzyl phthalate in the female Wistar rat. Identification of new metabolites.

n-Butyl benzyl phthalate (BBP), a plasticizer used in polyvinylchloride (PVC) and other polymers, has been orally administered to female Wistar rats with four doses (150, 475, 780 and 1500 mg/kg body weight/day) for 3 consecutive days. Metabolites recovered in urines were analysed by gas chromatography-mass spectrometry (GC-MS) after 24, 48 and 72 hours. Six metabolites were identified. Mono-n-butyl phthalate (MBuP) and mono-n-benzyl phthalate (MBeP) represented respectively 29-34% and 7-12% of the total recovered metabolites. Hippuric acid, the main metabolite of benzoic acid, represented the second major metabolite (51-56%). Phthalic acid, benzoic acid and an omega-oxidized metabolite of MBuP were also recovered in urine but in small quantities. BBP was never identified in urines. Total urinary metabolites recovery represented 56% of the dose administered in the first 24 hours. However, total recovery decreased when the dose increases (43% at 780 mg/kg body weight/day, only 30% at 1500 mg/kg body weight/day). Whatever the time was, BBP metabolites recovered in urines were all present and in the same proportions for the two lowest doses. Discrepancy in metabolites quantities expressed as percentages of the dose observed in urine of rat treated with the highest BBP dose disappeared with time as MBuP, MBeP and hippuric acid recovery has significantly increased at day 3. Metabolic profile of BBP in female rats has been established. The aim of the present study is to identify further the active(s) agent(s) involved in the BBP malformations and teratogenic effects.

Difference between guinea pig and rat in the liver peroxisomal response to equivalent plasmatic level of ciprofibrate.

Guinea pig was previously classified as a species nonresponsive to peroxisome proliferators. However, none of the previous reports was based on pharmacokinetic data. Here, after a comparative pharmacokinetic study between the guinea pig and rat, we evaluate the guinea pig liver peroxisomal response to ciprofibrate, a hypolipemic agent and a potent peroxisome proliferator in rat. (1) Pharmacokinetic results show equivalent in guinea pig and rat when guinea pigs are treated with ciprofibrate at 30 mg/kg twice a day and rats are treated at 3 mg/kg once a day. (2) The treatment of guinea pigs at 30 mg/kg twice a day for 2 weeks leads to a significant increase in the liver peroxisomal palmitoyl-CoA oxidase activity (x 1.6) and also in the microsomal omega-laurate hydroxylase activity (x 1.8). These increases are in accordance with the changes in polypeptide patterns of isolated liver peroxisomes as well as in the immunoblotting of acyl-CoA oxidase. It is deduced that a weak, but significant, peroxisome proliferation can occur in guinea pig liver after a ciprofibrate treatment at dosages corresponding to equivalent plasmic concentrations of the drug between guinea pig and rat. (3) The hybridization of guinea pig liver RNA with the rat liver-inducible acyl-CoA oxidase cDNA probe shows a decrease in the corresponding heterologous mRNA content after treatment with ciprofibrate at 30 mg/kg twice a day. This result contrasts with the slight increase observed in immunodetection and in enzymatic assays, suggesting the existence of at least two different acyl-CoA oxidases in guinea pig liver peroxisomes.

In vitro glucuronidation of peroxisomal proliferators: 2-ethylhexanoic acid enantiomers and their structural analogs.

In order to investigate the glucuronidation of 2-ethylhexanoic acid (2-EHA), a metabolite of the plasticizer di-(2-ethylhexyl) adipate, by liver microsomes of several mammalian species including man, a gas chromatography method for the quantification of the corresponding glucuronides was developed. The variation coefficients for intra- and interassay repeatability were less than 3 and 7%, respectively. The rat liver UDP-glucuronosyl-transferase (UGT) presented similar Km and Vmax toward the two enantiomers. The glucuronidation of the racemate 2-EHA or its enantiomers was strongly increased up to six times by treatment of the rats with phenobarbital and, to a lesser extent, by 3-methylcholanthrene. In contrast, the treatment of the rats clofibrate did not modify the activity. The induction was not stereoselective. The Gunn rats, which present a genetic defect in the bilirubin UGT isoforms, were able to glucuronidate the drug as well as the congenic strain. Moreover, the UGT-2B1 isoform, stably expressed in V79 cells, glucuronidated 2-EHA in an appreciable amount. Interspecies comparison indicated that the most active glucuronidation of 2-EHA occurred in the dog and the rat. The lowest activities were observed in the man and the rabbit. In all species considered, except rabbit and guinea pig which glucuronidated the R isomer faster, the R and S enantiomers were glucuronidated to a similar extent. The glucuronidation activity toward compounds chemically related to 2-EHA increased as a function of molecular weight, but was not affected by the position of the methyl or the ethyl moiety on the hydrocarbon chain. A correlation between the glucuronidation rate of 2-EHA and analogs and the activity of PCoA oxidase was observed.

Regio- and stereo-selectivity in the induction of peroxisome proliferation by substituted hexanoic acids.

Quantitative structure-activity relationship is an effective tool in order to predict drug potency. A similar approach is actually developed for peroxisome proliferation induced by substituted carboxylic acids issued from plasticizer metabolism in rats. The study is focused on acids found in rat urine after adipic diester dosings. Size, location of the substituted group and length of the chain have been studied. 3-D structure has also been taken in account for 2-ethyl hexanoic acids. The results obtained so far demonstrate that peroxisome proliferation potencies of the considered acids are modified according structure changes. At this time location of the group along the chain appears to be a predominant factor.

Toxicity of peroxisome proliferators.

Peroxisome proliferators are not a chemical class of compounds. They do not have a similar chemical structure but all induce characteristic effects in the liver of treated rats or mice. They produce within a few days a striking dose-dependent hepatomegaly accompanied by a characteristic proliferation of the peroxisomal and microsomal compartment as assessed morphologically and biochemically. Such effects are not observed in other species including human. In addition, life-long feeding of the susceptible laboratory animals results in the formation of liver tumor. The effects induced in subchronic studies can be reproduced and investigated in cultured hepatocytes, the target cells. The species specificity is observed with all peroxisome proliferators, and by large the effects observed in subchronic studies are reversible. The hepatocarcinogenesis by peroxisome proliferators is not fully understood, because these compounds are not directly genotoxic, but the understanding of their tumor promotor potential has some implications for the toxicological testing and risk assessment.

Metabolism of an imidazole fungicide (prochloraz) in the rat after oral administration.

The metabolic fate and pathway of the imidazole fungicide prochloraz (1-[N-propyl-N-2-(2,4,6-trichlorophenoxy) ethyl carbamoyl] imidazole) were investigated in the rat after administration of oral single doses with radiolabelled molecules. At both dose levels (50 and 250 mg/kg body weight), virtually all of the ingested [14C-phenyl]prochloraz was excreted in the urine or faeces within 96 hr, the bulk of excretion occurring between 24 and 48 hr after dosing. Urinary elimination accounted for 61 and 68% of the respective initial doses. Urinary metabolic products were isolated and identified by thin-layer chromatography, gas chromatography or gas chromatography coupled with mass spectrometry analysis. Prochloraz was completely metabolized with no unchanged compound being excreted in the urine. The main biotransformation products in rat urine were 2,4,6-trichlorophenoxyacetic acid and its corresponding alcohol, the latter as a glucuronic acid conjugate. Ring hydroxylation also occurred, with the hydroxy-2,4,6-trichlorophenoxyethanol and hydroxy-2,4,6-trichlorophenoxyacetic acid metabolites excreted in small amounts in the urine. 2,4,6-Trichlorophenol and unconjugated 2,4,6-trichlorophenoxyethanol were identified as minor urinary metabolites.

Identification of the proximate peroxisome proliferator(s) derived from di (2-ethylhexyl) adipate and species differences in response.

Identification of the proximate peroxisome proliferator(s) derived from di (2-ethylhexyl) adipate (DEHA) has been achieved using primary hepatocyte cultures derived from different species and cyanide-insensitive fatty acyl CoA oxidase (PCO) as a marker enzyme for peroxisome proliferation. In rat and mouse hepatocytes, the parent compound (DEHA) had no effect on peroxisomal beta-oxidation, but primary metabolites of DEHA, mono (2-ethylhexyl) adipate (MEHA) and 2-ethylhexanol (EH), were approximately equipotent in PCO induction (5-fold at 0.5 mM final concentration). The secondary metabolite of DEHA, 2-ethylhexanoic acid (EHA), was in both species the most potent peroxisome proliferator (25- and 9-fold induction in mice and rats, respectively, at 1 mM final concentration). At 2 mM final concentration a tertiary metabolite of DEHA, 2-ethyl-5-hydroxyhexan-1-oic acid, was less effective in mouse and rat hepatocytes at inducing PCO (15- and 5-fold, respectively). 2-Ethyl-5-oxohexan-1-oic acid and 2-ethylhexan-1,6-dioic acid had little effect (2-3-fold in both rat and mouse hepatocytes). Thus, EHA was identified as the proximate peroxisome proliferator of DEHA and mouse hepatocytes were approximately twice as sensitive as rat hepatocytes to peroxisome proliferation due to MEHA, EH and EHA. We investigated further species differences in response to peroxisome proliferators by using guinea pig and marmoset primary hepatocyte culture. None of the chemicals studied stimulated peroxisomal beta-oxidation in these species up to a final concentration of 2 mM. Higher concentrations lead to cytotoxicity. This lack of sensitivity of guinea pig and marmoset hepatocytes is in agreement with previous studies with di (2-ethylhexyl) phthalate metabolites, suggesting the absence of a threat of hepatocarcinogenic damage to these species and confirming that primary hepatocytes cultures are useful models for investigating the phenomenon of peroxisome proliferation.

Peroxisome proliferation due to di (2-ethylhexyl) adipate, 2-ethylhexanol and 2-ethylhexanoic acid.

The dose-response relationships for peroxisome proliferation due to Di (2-ethylhexyl) adipate (DEHA), 2-ethylhexanol (EH), 2-ethylhexanoic acid (EHA) have been investigated in rats and mice. Linear dose-response relationships were observed for induction of cyanide-insensitive palmitoyl CoA oxidation (PCO), used as a enzyme marker of peroxisome proliferation, by DEHA, EH and EHA in both species. Relative liver weights were also increased in a dose related manner. On a molar basis, DEHA was twice as potent as EH or EHA which were equipotent and PCO was stimulated to a greater extent in male mice than in rats or female mice. At doses above 8 mmol/kg/day, EH was toxic to rats (both sexes) and similarly EHA at 13.5 mmol/kg/day lead to the death of female rats. In a attempt to explain the species difference in carcinogenicity of DEHA previously reported, we also used Fischer 344 rats and B6C3F1 mice. DEHA administration (2.5 g/kg/day) to Fischer 344 rats and B6C3F1 mice lead to toxicity in female rats. Relative liver weights were increased in a dose related fashion by DEHA administration to both rats and mice, PCO but not catalase was markedly increased (up to 15 fold in male rats). Light microscopy examination indicated some glycogen loss, a dose related hypertrophy and increased eosinophilia in both rats and mice. Electron microscopy confirmed peroxisome proliferation accompanied by a marked reduction of lipid in the centrilobular hepatocytes. These data suggest EHA to be the proximate peroxisome proliferator derived from DEHA.(ABSTRACT TRUNCATED AT 250 WORDS)

Enantioselectivity in the induction of peroxisome proliferation by 2-ethylhexanoic acid.

The stereoselectivity of the peroxisome proliferation potency of 2-ethylhexanoic acid (2-EHA), a metabolite of the plasticizer di-(2-ethylhexyl) adipate, was investigated in vitro. The enantiomers of 2-EHA were prepared via the semipreparative HPLC resolution of their diastereoisomeric (+)-(R)-1-phenylethylamine derivatives and the subsequent hydrolytic cleavage. Monolayers of hepatocytes were incubated 3 days with solution of (-)-(R), (+)-(S), and (+/-)-2-EHA. The peroxisome proliferation potency was measured by means of determination of the peroxisomal palmitoyl coenzyme A oxidation. The theoretical induction component due to each enantiomer were calculated from the experimental data considering the enantiomeric purities of the acids. The (+)-(S)-enantiomer was found to be the most potent inducer e.g., the eutomer, while the (-)-(R) was the distomer. The eudismic ratio was about 1.6 and the racemic mixture exhibited an intermediary potency. These results, obtained in vitro in conditions avoiding confounding factors such as pharmacokinetics, suggest that the peroxisome proliferation induced by 2-ethylhexanoic acid is a stereoselective phenomenon.

Expression of liver peroxisomal proteins as compared to other organelle marker enzymes in rats treated with hypolipidemic agents.

Peroxisome proliferation induced by 2 hypolipidemic agents (clofibrate and ciprofibrate) was studied in rats by complementary approaches, ie cell fractionation, electron microscopy, marker enzyme activities, immunoblotting and nucleic acid hybridization techniques. Administration of clofibrates for 2 and 52 weeks in doses of 500 ppm and 50 ppm respectively, or ciprofibrate for 2,28 and 52 weeks in doses of 250, 25 and 25 ppm respectively, did not alter the behavior of the peroxisomes after induction as shown by ultracentrifugation profiles. The peroxisome mass was increased as shown by the purification procedure. Specific enzymes (catalase and mostly cyanide insensitive palmitoyl CoA oxidase) were induced. A mechanism of peroxisome biogenesis might have been initiated ie cytosolic factor, ligand-receptor interaction and/or post-translational modification of the import. Increase in marker enzyme activities showed that the peroxisomes are the most responsive organelles in comparison to lysosomes, mitochondria and smooth endoplasmic reticulum (except for cytochrome P-450 LA omega-hydroxylase). Peroxisomal integral membrane proteins appeared to be differently induced: some of them were virtually absent in untreated rat liver but were strongly expressed in treated liver. Induction was sustained for 52 weeks, indicating that there was no compensatory mechanism.

Induction and inhibition of rat liver cytochrome(s) P-450 by an imidazole fungicide (prochloraz).

Prochloraz (1-[N-propyl-N-2(2,4,6-trichlorophenoxy) ethyl carbamoyl] imidazole) is an imidazole molecule widely used as a fungicide. This study reports the in vivo and in vitro effects of this compound on microsomal drug metabolising enzymes from rat liver. In vivo pretreatment of animals (250 mg/kg body wt for 3 days) with prochloraz elicited complex modifications. When animals were sacrificed 24 h after the last dose, an increase in total cytochrome P-450 was observed as well as an increase in catalytic activities towards benzphetamine, alkoxyresorufins and alkoxycoumarins. However, when animals were sacrificed 48 h after the last dose, a lower induction of 7-ethoxyresorufin O-deethylase and a higher induction of 7-pentoxyresorufin O-depentylase and 7-benzoxyresorufin O-debenzylase were found. Such results lead us to consider prochloraz as a "mixed inducer" of the hepatic cytochromes P-450. In vitro experiments were indicating a strong inhibition of 7-alkoxyresorufin O-dealkylase activities by prochloraz. The analysis of the CO-difference spectrum of cytochrome P-450 showed also tight binding of prochloraz to the haemoprotein in animals sacrificed 24 h but not 48 h after the last dose. Furthermore, prochloraz did not induce significantly the microsomal cytochrome P-450 IVA1-dependent 12-hydroxylation of lauric acid.

The metabolism of mono-(2-ethylhexyl) phthalate (MEHP) and liver peroxisome proliferation in the hamster.

This study has investigated the in vivo metabolism of mono-(2-ethylhexyl) phthalate (MEHP), the initial metabolite of di-(2-ethylhexyl) phthalate in mammals, and the hepatic peroxisome proliferation induced by this compound following multiple oral administration to hamsters. Hamsters received [14C]-MEHP, by gavage, at doses of 50 and 500 mg/kg body wt on each of three consecutive days. Urine was collected every 24 hours and metabolite profiles were determined using capillary gas-chromatography. Multiple high doses of MEHP (500 mg/kg) induced a change in the relative proportions of metabolites produced. As previously reported for the rat, metabolites derived from sequential omega- following by beta-oxidation were increased. This increase was correlated with a parallel 3-fold increase in peroxisomal beta-oxidation--a marker for peroxisome proliferation. Hamsters were less responsive than rats to peroxisome proliferation elicited by MEHP. In contrast to the rat, a large proportion of hamster omega-1 oxidation products of MEHP (metabolites 6 and 9, mono (2-ethylhexyl-5-oxohexyl) phthalate and mono (2-ethyl-5-hydroxyhexyl) phthalate, respectively) were found as their glucuronide conjugates. This metabolic species difference may relate to differences in sensitivity to MEHP as a peroxisome proliferator. The relationship between metabolite conjugation, peroxisome proliferation and production of omega-oxidation metabolites is discussed.