Advancing the 3Rs in regulatory toxicology - Carcinogenicity testing: Scope for harmonisation and advancing the 3Rs in regulated sectors of the European Union.

Different government agencies operating in the European Union regulate different types of chemical products but all require testing for carcinogenicity to support applications for product marketing and commercialisation. A conference was held in Brussels in 2013 where representatives of the pharmaceutical, animal health, chemical and plant protection industries, together with representatives of regulatory agencies, universities and other stakeholders, met under the auspices of The European Partnership for Alternative Approaches to Animal Testing (EPAA) to discuss the varying requirements for carcinogenicity testing, and how these studies might be refined to improve hazard evaluation and risk assessment while implementing principles of the 3Rs (replacement, refinement and reduction in animal studies). While there are some similarities, the regulatory approaches in pharmaceutical, animal health, chemical and plant protection sectors have varying degrees of flexibility in requirements for carcinogenicity testing, to an extent reflecting concerns over the magnitude and duration of human exposure, either directly as in therapeutic exposure to pharmaceuticals, or indirectly through the ingestion of residues of veterinary drugs or plant protection chemicals.

Antibody response against Trichinella spiralis in experimentally infected rats is dose dependent.

Domestic pigs are the main representatives of the domestic cycle of Trichinella spiralis that play a role in transmission to humans. In Europe, backyard pigs of small household farms are the most important risks for humans to obtain trichinellosis. Rats might play a role in the transmission of Trichinella spiralis from domestic to sylvatic animals and vice versa.

Hazardous compounds in tobacco smoke.

Tobacco smoke is a toxic and carcinogenic mixture of more than 5,000 chemicals. The present article provides a list of 98 hazardous smoke components, based on an extensive literature search for known smoke components and their human health inhalation risks. An electronic database of smoke components containing more than 2,200 entries was generated.

Gene expression differences in lungs of mice during secondary immune responses to respiratory syncytial virus infection.

Vaccine-induced immunity has been shown to alter the course of a respiratory syncytial virus (RSV) infection both in murine models and in humans. To elucidate which mechanisms underlie the effect of vaccine-induced immunity on the course of RSV infection, transcription profiles in the lungs of RSV-infected mice were examined by microarray analysis. Three models were used: RSV reinfection as a model for natural immunity, RSV challenge after formalin-inactivated RSV vaccination as a model for vaccine-enhanced disease, and RSV challenge following vaccination with recombinant RSV virus lacking the G gene (DeltaG-RSV) as a model for vaccine-induced immunity.

Toxicity of analytically cleaned pentabromodiphenylether after prolonged exposure in estuarine European flounder (Platichthys flesus), and partial life-cycle exposure in fresh water zebrafish (Danio rerio).

Residues of polybrominated diphenylethers (PBDEs), extensively applied as flame retardants, are widely spread in the aquatic environment and biota. The present study investigates effects of the environmentally relevant lower brominated diphenylethers in two fish species in vivo under controlled laboratory conditions. Euryhaline flounder (Platichthys flesus) and freshwater zebrafish (Danio rerio) were exposed to a range of concentrations of a commercial pentabromodiphenylether mixture, DE-71.

Comparative gene expression profiling in two congenic mouse strains following Bordetella pertussis infection.

Background: Susceptibility to Bordetella pertussis infection varies widely. These differences can partly be explained by genetic host factors. HcB-28 mice are more resistant to B.

Lung response to Bordetella pertussis infection in mice identified by gene-expression profiling.

Host genetics determines the course of Bordetella pertussis infection in mice. Previously, we found four loci, Tlr4 and three novel loci, designated Bps 1-3, that are involved in the control of B. pertussis infection.

Effects of the estrogen agonist 17beta-estradiol and antagonist tamoxifen in a partial life-cycle assay with zebrafish (Danio rerio).

A partial life-cycle assay (PLC) with zebrafish (Danio rerio) was conducted to identify endocrine-disrupting effects of 17beta-estradiol (E2) and tamoxifen (TMX) as reference for estrogen agonist and antagonist activity. Adult zebrafish were exposed for 21 d and offspring for another 42 d, allowing differentiation of gonads in control animals. The assessed end points included reproductive variables (egg production, fertilization, and hatching), gonad differentiation of juveniles, histopathology, and vitellogenin (VTG) expression.

Effects of the antithyroid agent propylthiouracil in a partial life cycle assay with zebrafish.

Some ubiquitous pollutants of the aquatic environment, such as PCBs or other polyhalogenated aromatic hydrocarbons, may disrupt the thyroid hormone system. In a partial life cycle assay with zebrafish (Danio rerio), we studied the effects of the reference compound propylthiouracil (PTU) on reproduction, growth and development, histopathology of some target tissues, and plasma thyroid hormone levels. PTU induced a concentration-dependent increase of egg production with a concomitant decrease of mature oocyte size but had no effect on fertilization rate or hatching.

Dose-dependent effects of UVB-induced skin carcinogenesis in hairless p53 knockout mice.

Exposure to (solar) UVB radiation gives rise to mutations in the p53 tumor suppressor gene that appear to contribute to the earliest steps in the molecular cascade towards human and murine skin cancer. To examine in more detail the role of p53, we studied UVB-induced carcinogenesis in hairless p53 knock-out mice. The early onset of lymphomas as well as early wasting of mice interfered with the development of skin tumors in p53 null-mice.

Long-term exposure to environmental concentrations of the pharmaceutical ethynylestradiol causes reproductive failure in fish.

Heightened concern over endocrine-disrupting chemicals is driven by the hypothesis that they could reduce reproductive success and affect wildlife populations, but there is little evidence for this expectation. The pharmaceutical ethynylestradiol (EE2) is a potent endocrine modulator and is present in the aquatic environment at biologically active concentrations. To investigate impacts on reproductive success and mechanisms of disruption, we exposed breeding populations (n = 12) of zebrafish (Danio rerio) over multiple generations to environmentally relevant concentrations of EE2.

Effects of soy-derived isoflavones and a high-fat diet on spontaneous mammary tumor development in Tg.NK (MMTV/c-neu) mice.

Phytoestrogens such as isoflavonoids and lignans have been postulated as breast cancer protective constituents in soy and whole-grain cereals. We investigated the ability of isoflavones (IFs) and flaxseed to modulate spontaneous mammary tumor development in female heterozygous Tg.NK (MMTV/c-neu) mice.

Comparative toxicological pathology in mammals and fish: some examples with endocrine disrupters.

Toxicologic pathology is a classical discipline in the toxicology arena, and despite various emerging techniques, still is a major cornerstone in the process of hazard identification and risk characterization. Most knowledge derives from laboratory animal studies and, to a lesser extent, human data. Currently interest is growing in applying toxicological pathology for lower animals, in particular fish as being the most developed aquatic genus.

p53 heterozygosity results in an increased 2-acetylaminofluorene-induced urinary bladder but not liver tumor response in DNA repair-deficient Xpa mice.

Both nucleotide excision repair (NER) and the p53 tumor suppressor protein play crucial roles in the prevention of cells becoming cancerous. This is clearly demonstrated by the fact that NER-deficient xeroderma pigmentosum patients and Li-Fraumeni patients who carry a germ-line p53 mutation are highly tumor prone. The NER-deficient Xpa and the p53(+/-) mouse models clearly mimic their human counterparts, because they are both tumor prone as well.

Vitellogenin expression in zebrafish Danio rerio: evaluation by histochemistry, immunohistochemistry, and in situ mRNA hybridisation.

Several histological methods were tested for their potential to detect the in vivo induction of vitellogenin in zebrafish, after exposure to 17beta-oestradiol (E2), and validated by correlating semi-quantitative measurements on digital images to vitellogenin plasma values measured by ELISA and morphological criteria. All methods, except for vitellogenin-specific immunohistochemistry on liver, detected vitellogenin production in male zebrafish at the exposure level of 1 nM E2/l, and correlated well to each other and to ELISA results on plasma, thus indicating their specificity. The level of sensitivity is in the range of the induction of clinical (histopathological) effects, although slightly below the level of sensitivity of the plasma ELISA.

Histopathology as a tool for the evaluation of endocrine disruption in zebrafish (Danio rerio).

The importance of histology as a tool in the evaluation of endocrine disruption in fish depends on the choice and interpretation of appropriate endpoints, as is illustrated by the analysis of the effects of exposure to the estrogen 17beta-estradiol (E2) and the nonaromatizable androgen 17-methyldihydrotestosterone (MDHT). The E2 led to the disappearance of vitellogenic oocytes in the ovary and an increased area of relatively large, eosinophilic cells in the testis, which were identified as spermatogonia under high-power magnification; this was a relative increase, as was shown by histomorphometry, because of a decreased size of spermatogenic cysts and a relative decrease of spermatocyte cysts. The E2 also induced an accumulation of acidophilic fluid in vessels and interstitial spaces, confirmed by immunohistochemistry as vitellogenin, and basophilia in the liver also associated with the production of vitellogenin.

Combined oral benzo[a]pyrene and inhalatory ozone exposure have no effect on lung tumor development in DNA repair-deficient Xpa mice.

There is considerable concern about an enhanced risk of lung tumor development upon exposure of humans to polycyclic aromatic hydrocarbons (PAHs), like benzo[a] pyrene (B[a]P), in combination with induced lung cell proliferation by toxic agents like ozone. We studied this issue in wild-type (WT) C57BL/6 mice, the cancer prone nucleotide excision repair-deficient Xeroderma pigmentosum complementation group A mice (Xpa-/-) and the even more sensitive Xpa-/-/p53+/- mice. The mice were treated with B[a]P through the diet at a dose of 75 p.

Effects of ethynylestradiol on the reproductive physiology in zebrafish (Danio rerio): time dependency and reversibility.

Environmental pollution with natural or synthetic estrogens may pose a serious threat to reproduction of wildlife species. This study describes the effects of 17-alpha-ethynylestradiol (EE2) on fish reproductive organs in a laboratory model. Adult zebrafish were semistatically exposed to nominal concentrations of 0, 10, and 25 ng/L EE2 for 24 d and then transferred to EE2-free medium.