Assessment strategies and decision criteria for pesticides with endocrine disrupting properties relevant to humans.

There is growing concern that environmental substances with a potential to modulate the hormonal system may have harmful effects on human health. Consequently, a new EU regulation names endocrine disrupting properties as one of the cut-off criteria for the approval of plant protection products, although it currently fails to provide specific science-based measures for the assessment of substances with such properties. Since specific measures are to be presented by the European Commission in 2013 the development of assessment and decision criteria is a key challenge concerning the implementation of this new EU regulation. Proposals of such decision criteria for substances with potential endocrine disrupting properties in human health risk assessment were developed by the German Federal Institute for Risk Assessment (BfR) and discussed at an expert workshop in November 2009. Under consideration of the requirements laid down within the new plant protection product legislation and the scientific discussions during the workshop, a conceptual framework on evaluation of substances for endocrine disrupting properties in a regulatory context is presented in this paper. Central aspects of the framework include assessment of adversity of effects, establishment of a mode/mechanism of action in animals, considerations concerning the relevance of effects to humans and two options for a regulatory decision.

Combined retrospective analysis of 498 rat multi-generation reproductive toxicity studies: on the impact of parameters related to F1 mating and F2 offspring.

The multi-generation reproductive toxicity study (OECD TG 416 and USEPA 870.3800) has been extensively used internationally to assess the adverse effects of substances on reproduction. Recently the necessity of producing a second generation to assess the potential for human health risks has been questioned. The present standardized retrospective analysis of the impact of the second generation on overall study outcome combines earlier analyses and includes 498 rat multi-generation studies representing 438 different tested substances. Detailed assessment of study reports revealed no critical differences in sensitivities between the generations on the basis of a consideration of all endpoints evaluated. This analysis indicates that the second generation mating and offspring will very rarely provide critical information. These findings are consistent with the conclusions of previous retrospective analyses conducted by RIVM, USEPA and PMRA and support adoption of the proposed OECD extended one-generation reproductive toxicity study protocol in regulatory risk assessment testing strategies.

The use of toxicodynamics in risk assessment.

Risk assessment of xenobiotics is a qualitative and quantitative assessment of toxic properties conventionally based on data resulting from tests in animals exposed to the substance. The assessment of dose-effect relationship includes evaluation of exposure at the site of action. More recently, emphasis is put on understanding the relationship between exposure at the site of action and the resulting effect, i.e. toxicodynamic. In this respect, results from genotoxicity studies may be a measure for exposure and at the same time of an effect. Results of toxicodynamic endpoints such as binding to receptors or release of hormones have been used when replacing default values for interspecies extrapolation. It may also be envisaged to use toxicodynamic endpoints in order to get an estimate of intraspecies variability. It was demonstrated that this approach may be helpful only if the relationship between the toxicodynamic endpoint and the definite endpoint is known by using the example of bisphenol A. Whereas there are clear effects of bisphenol A in in vitro and ex vivo studies, the classical two generation study has not been able to detect an effect on reproduction and/or fertility. Looking in the future development of toxicodynamic endpoints, gene profiling and the analysis of proteins ('proteomics') may be helpful tools employed in screening and being related to the mode of action are explored for their suitability in terms of toxicodynamic endpoints.

Exposition to and health effects of residues in human milk.

A great variety of drugs, cosmetics, food ingredients as well as environmental contaminants are secreted with human milk as a result of actual exposure or the accumulated body burden of the mother. Of great concern and least amenable to short-term intervention are persistent substances in the environment with long half-lives in the body due to their lipophilic properties and minimal degradation. Polyhalogenated aromatic hydrocarbons, namely organochlorine pesticides, polychlorinated biphenyls (PCB) and polychlorinated dibenzodioxins (PCDD) and dibenzofurans (PCDF) are fetotoxic, neurotoxic, immunotoxic, some are promoting carcinogens and/or interfere with hormonal receptors. They pass the placenta and equilibrate among the lipid compartments of the body including breast milk lipids. Transplacental exposure is more relevant with regard to physical development and cognitive functioning of the child than postnatal exposure via breastmilk. Restrictions for production, use and release have been successful in decreasing exposure as shown by a downward trend of their contents both in human milk and serum lipids for the last 15 to 20 years. It is difficult to evaluate the potentially late effects of the exposure via breastmilk which is 10 to 100 times higher in industrialised countries than the tolerable daily intake (TDI) of 1 to 4 toxic equivalents (WHO-TEQ) pg/kg/day established in 1998 by WHO for dioxins and dioxin-like PCBs but which lasts for 0.6% of the expected life span only. Carefully conducted long-term follow-up of cohorts with defined exposure levels, with consideration of numerous biological and psychological parameters, is expected to provide the answer.

CYP2E1 expression in bone marrow and its intra- and interspecies variability: approaches for a more reliable extrapolation from one species to another in the risk assessment of chemicals.

When characterizing the health risks for man by exposure to chemicals, species-specific differences have to be taken into consideration, otherwise extrapolation from animal data to the human situation would be inadequate. The site-specific toxicity of chemicals may be explained by the following alternatives: (1) reactive metabolites are generated in the liver and subsequently transported to the target tissue(s); (2) metabolism of the parent compound occurs in the target tissue, a pathway by which the enzymes necessary for activation must be expressed in the target tissue. Cytochrome P450 2E1 (CYP2E1) is an important phase-I enzyme activating several chemicals. In the study described in this paper, myeloid intra- and interspecies variability in the expression of CYP2E1 has been investigated in rats, rabbits and man, because the bone marrow represents an important target organ for toxic effects of several chemicals, e.g. benzene. CYP2E1 at the protein level was detected by Western blotting and enzyme activities were determined by CYP2E1-dependent hydroxylation of chlorzoxazone (CLX). In the bone marrow of Wistar rats, the CLX hydroxylase activities were within the same order of magnitude (range: 0.1-0.4 pmol/mg protein per min) as previously described for mice (range 0.2-0.8 pmol/mg protein per min), whereas the CYP2E1 activities in two strains of rabbits were significantly higher (range: 1.7-4.7 pmol/mg protein per min) than in the rodents (P < 0.05). In human CD34+ bone marrow stem cells, CYP2E1 could also be detected on the protein level by Western blotting. The data demonstrate a presence of CYP2E1 in the bone marrow of all species investigated, thus supporting the hypothesis of CYP2E1-dependent local metabolism of several chemicals as a factor possibly contributing to their myelotoxicity and haematotoxicity. The data show that intraspecies/intrastrain variability of CYP2E1 activity in rodents is small. However, CYP2E1 activity between rodents and a non-rodent species was quite different indicating considerable interspecies variability.

CYP2E1-dependent benzene toxicity: the role of extrahepatic benzene metabolism.

Benzene, a ubiquitous environmental pollutant, is haematotoxic and myelotoxic. As has been shown earlier, cytochrome P450 2E1 (CYP2E1)-dependent metabolism is a prerequisite for the cytotoxic and genotoxic effects of benzene, but which of the benzene metabolites produces toxicity is still unknown. The observed differences between the toxicity of benzene and that of phenol, a major metabolite of benzene, could be explained by alternative hypotheses. That is, whether (1) toxic benzene effects are caused by metabolites not derived from phenol (e.g. benzene epoxide, muconaldehyde). which are formed in the liver and are able to reach the target organ(s); or (2) benzene penetrates into the bone marrow, where local metabolism takes place, whereas phenol does not reach the target tissue because of its polarity. To further investigate hypothesis 2, we used various strains of mice (AKR, B6C3F1, CBA/Ca, CD-1 and C57B1/6), for which different toxic responses have been reported in the haematopoietic system after chronic benzene exposure. In these strains, CYP2E1 expression in bone marrow was investigated and compared with CYP2E1 expression in liver by means of two independent methods. Quantification of CYP2E1-dependent hydroxylation of chlorzoxazone (CLX) by high-performance liquid chromatography (HPLC; functional analysis) was used to characterize specific enzymatic activities. Protein identification was performed by Western blotting using CYP2E1-specific antibodies. In liver microsomes of all strains investigated, considerable amounts of CYP2E1-specific protein and correspondingly high CYP2E1 hydroxylase activities could be detected. No significant differences in CYP2E1-dependent enzyme activities were found between the five strains (range of medians, 4.6 12.0 nmol 6-OH-CLX/[mg protein x min]) in hepatic tissue. In the bone marrow, CYP2E1 could also be detected in all strains investigated. However, chlorzoxazone hydroxylase activities were considerably lower (range of medians, 0.2-0.8x10(-3) nmol 6-OH-CLX/[mg protein x min]) compared with those obtained from liver microsomes. No significant (P>0.05) interstrain differences in CYP2E1 expression in liver and/or bone marrow could be observed in the mouse strains investigated. The data obtained thus far from our investigations suggest that strain-specific differences in the tumour response of the haematopoietic system of mice chronically exposed to benzene cannot be explained by differences in either hepatic or in myeloid CYP2E1-dependent metabolism of benzene.

Reproductive toxicity and tissue concentrations of 3,3',4,4'-tetrachlorobiphenyl (PCB 77) in male adult rats.

1 The aim of this study was to ascertain the reproductive effects of PCB 77 on adult male rats and to determine its concentration in the liver and testis. Adult male rats (n = 15/group) were treated subcutaneously with a single dose of 18 mg/kg bw (PC18) or with 60 mg/kg bw (PC60). The substance was dissolved in a 10 ml volume of peanut oil/kg. Control rats received the same volume of the vehicle. The reproductive effects as well as the concentration of PCB 77 in the liver and testis were investigated 1, 4 and 8 weeks after treatment. 2 In both groups, the daily sperm production (DSP; x10(6)) remained permanently reduced in the PC18 as well as in the PC60 groups throughout the entire investigation period (DSP week 8: control: 31 +/- 7; PC18: 22 +/- 5; PC60: 20 +/- 7). The sperm number (x10(6)) per cauda epididymis was affected only at the 1st and 4th week after treatment (control week 1: 211 +/- 67; PC18 week 1: 135 +/- 62; PC60 week 1: 142 +/- 49). Moreover, a significant increase in the percentage of abnormal sperm was observed 4 weeks following treatment in the PC18 and PC60 groups and 8 weeks after treatment in the PC60 group. Abnormal tails were the most frequent changes observed. 3 The relative testicular and prostata weights (g) were slightly increased in the PC60 group at the 1st and 4th week following treatment (testis weight: control/I: 0.46 +/- 0.02; PC60/I: 0.51 +/- 0.03). 4 The serum testosterone concentrations and effects on testis morphology were not reported. 5 The maximum concentration of PCB 77 was detected in the liver and testis 1 week after treatment. The concentration declined 4 weeks after treatment in both organs, but still a significant amount of PCB 77 was detectable in the liver as well as in the testis 8 weeks after treatment. 6 The results demonstrate that PCB 77 affects sperm variables when applied to adult rats and that the elimination of PCB 77 in the testis parallels that of the liver.

[Polychlorinated biphenyls indoors: attempt at an assessment]. / Polychlorierte Biphenyle in Innenräumen: Versuch einer Bilanz.

The use of products containing PCB in buildings resulted in indoor air contaminations. The search for appropriate measures is hampered by several different uncertainties in the regulatory toxicological process. These refer to problems in exposure assessment including measurement approaches, the toxicological evaluation of health risks including the derivation of a tolerable intake and the use of TCDD toxicity equivalency factors and finally the legal consequences of health risk evaluation. Since no consensus on these aspects has been achieved, the regulatory consequences vary considerably from one German state government to the other. FAO/WHO have not been able to arrive at a satisfactory supranational recommendation of a tolerable intake for PCB.

Tissue distribution, toxicokinetics and induction of hepatic drug metabolizing enzymes in male rats after a single s.c. dose of 3,4,3',4'-tetrachlorobiphenyl (PCB-77).

The tissue distribution and toxicokinetics of PCB-77 after a single s.c. dose of 6 mg/kg body weight was investigated in male adult rats as well as the concurrent induction of hepatic drug-metabolizing enzymes. During five days after treatment, the PCB-77 concentrations on fat weight basis in testis and in whole blood were found to be about in the same range as the concentrations in adipose tissue, whereas in liver fat PCB-77 accumulated. EROD and MROD activities were induced by about 8 and 4 times, respectively, during a period of two weeks after treatment. Concentrations in adipose tissue and in liver as well as the hepatic enzyme activities rapidly declined during the investigation period of eight weeks. An elimination half-life for PCB-77 of approximately 7-9 days was estimated.

Genetically engineered V79 Chinese hamster cells for stable expression of human cytochrome P450IA2.

V79 Chinese hamster cells were genetically engineered for stable expression of human P450IA2. Full length cDNA, encoding human P450IA2, was inserted into an SV40 early promoter containing eukaryotic expression vector and cointroduced with the selection marker neomycin phosphotransferase (conferring resistance to the neomycin derivative G418) into V79 Chinese hamster cells. The recombinant expression vector was introduced into two different V79 sublines, one expressing an endogenous acetyltransferase (V79-NH), the other not (V79-MZ). The presence of human cytochrome CYP1A2 cDNA in the G418 resistant V79 cell clones was confirmed by Southern blotting. The transcription of the cDNA into mRNA was detected by Northern blotting and the translation into an authentic cytochrome P450IA2 protein was shown by Western blotting. The enzymatic activity in these cells was determined by the cytochrome P450IA2-dependent methoxy-, ethoxy-, benzoxy-, and pentoxyresorufin dealkylation activity.

Effect of aciclovir on the development of the chick embryo in ovo.

The effect of aciclovir (ACV) on embryonic development was investigated using the chick embryo in ovo and treatment during organogenesis. ACV 30-1000 micrograms was applied as single doses prior to or after 24 h of incubation into the yolk sac, and ACV 3-100 micrograms after 2, 3 or 4 days of incubation (DI) directly to the embryo. Data were evaluated after a total of 8 days of incubation. (1) A dose-related increase in the rate of abnormal development was found in the surviving embryos. Depending on the route of drug administration a dose of 300, respectively, 5 micrograms ACV/egg had to be applied to induce 50% abnormal development. (2) Gross structural abnormalities of the surviving embryos mainly concerned the beak and the extremities. With the experimental set-up used a different pattern of abnormalities in the survivors after treatment at various stages could not be observed. The results are compared with data obtained with ACV in rodents in our laboratory. It is suggested that chick embryos are also capable of converting ACV into its triphosphate to interfere with DNA metabolism, probably through a chain break mechanism.

Activity of aldrinepoxidase, 7-ethoxycoumarin-O-deethylase and 7-ethoxyresorufin-O-deethylase during the development of chick embryos in ovo.

Since the chick embryo in ovo is susceptible to the action of some agents needing metabolic activation we studied the development of the activity of cytochrome P450-dependent monooxygenases in embryo/fetal tissue. The activities of aldrinepoxidase (AE), 7-ethoxycoumarin-O-deethylase (ECOD) and 7-ethoxyresorufin-O-deethylase (EROD) were measured in whole embryos, liver and yolk sac tissue of the chick embryo during development in ovo from day 4 to day 15 of incubation (DI). In yolk sac tissue enzyme activities could be detected from DI 4 on. While EROD activity was only marginally developed, AE and ECOD activities were more pronounced in the earlier developmental period and showed a clear decrease by the time the liver activities rose. With the methods used AE activity could be measured in the homogenate of the whole embryo proper from DI 4 on while EROD and ECOD activity was not detectable before DI 6 or DI 7, respectively. In liver tissue enzyme activities of the three monooxygenases studied developed to a considerable degree from DI 9 on and tended to exhibit maximum values around DI 11-13. Studies on monooxygenase activities in extra-embryonically located tissues have not been published until now. The importance of the yolk sac as a metabolically relevant organ during embryogenesis is pointed out in this study.

Inhibition of Na,K-ATPase from chick brain by polyamines.

The amounts of the polyamines putrescine, spermine and spermidine as well as the Na,K-ATPase activity have been determined in the developing chick brain. The amounts of spermine and spermidine per gram fresh weight do not change significantly, the amount of putrescine declines until the 17th day of incubation after which an increase takes place. Spermine is able to inhibit the Na,K-ATPase from chick brain competitively. Half maximal inhibition is achieved at 4 X 10(-5) mol/1 spermine. This polyamine functions as an allosteric inhibitor; the Hill coefficient is 2.2 +/- 0.3. A regulatory effect of spermine on the Na,K-ATPase from chick brain is discussed. In contrast to spermine 1 mmol/1 spermidine inhibits the Na,K-ATPase only slightly, while 1 mmol/1 putrescine does not inhibit the Na,K-ATPase at all.