A. Mantovani

Problems in testing and risk assessment of endocrine disrupting chemicals with regard to developmental toxicology.

Endocrine disrupting chemicals (EDCs) may affect mammalian development either indirectly (by impairing implantation, placental development, lactation, etc.) or directly, altering the maturation of target tissues. Current regulatory tests for reproductive/developmental toxicity should be carefully evaluated with regard to risk assessment of EDCs, considering hazard identification (are relevant endpoints being assessed?) and dose-response assessment (are sensitive NOEL/dose-response curves being provided?). Many in vitro and in vivo assays for sex steroid disruption are available; provided that the metabolic capacities of the assays are defined, they could be integrated in a sensitive battery for early detection of steroid-disrupting potentials. The screening battery should address further regulatory in vivo tests (e.g. what specific parameters have to be investigated). As regards dose-response, qualitative differences may be observed between lower and higher exposures, showing primary hormone-related effects and frank embryotoxicity, respectively. Other problems concern (a) the identification of critical developmental windows, according to hormone concentrations and/or receptor levels in the developing target tissues; (b) the potential for interactions between chemicals with common mechanism/target (e.g. xenoestrogens); (c) most important, besides sex steroids more attention should be given to other mechanisms of endocrine disruption, e.g., thyroid effects, which can be highly relevant to prenatal and postnatal development.

Long-term administration of low doses of mycotoxins to poultry. 1. Residues of aflatoxin B1 and its metabolites in broilers and laying hens.

A study was performed to determine aflatoxin residues in tissues and organs of male broilers and hens that had been fed a diet contaminated with 50 micrograms/kg aflatoxin B1 (AFB1). Residue levels of AFB1, aflatoxicol (Ro), aflatoxin M1 (AFM1) and aflatoxin B2a (AFB2a) were determined by an HPLC method and, with the exception of AFB2a, were detected in the liver, kidney and thigh of both male broilers and hens. The highest levels found were for Ro in liver (1.10 and 0.60 micrograms/kg for male broilers and hens, respectively). On the other hand no detectable amounts of aflatoxins were found in any tissue after withdrawal periods of 14 and 33 days for male broilers and laying hens respectively.

4‐hexylresorcinol as inhibitor of shrimp melanosis: Efficacy and residues studies; evaluation of possible toxic effect in a human intestinal in vitro model (caco‐2); preliminary safety assessment

Studies were performed on the efficacy, residues and in vitro enterocyte toxicity of 4-hexylresorcinol (4-HR), which could be utilized as an inhibitor of shrimp melanosis (black spot). Mediterranean sea shrimp (Parapaeneus longirostris) were treated with solutions of 4-HR in sea-water, at three different concentrations, 25, 50 or 100 mg/kg of shrimp, to test its antioxidative property. As a comparison a group of shrimp was treated with sodium metabisulphite (1 g/kg), while another group was left untreated. 4-HR showed a marked ability to inhibit or slow down melanosis (black spot) in shrimp; the most effective concentration was 100 mg/kg within an optimum period of 7 days but with effects up to the tenth day. During the first 5 days, 4-HR residues in the edible part of the shrimp showed a fast decrease in all three groups, going from initial average values of 20 mg/kg at 0 time, to 0.9 in the group treated at 25 mg/kg; from 42 to 1.8 mg/kg in the group at 50 mg/kg and from 85 to 1.9 mg/kg in the group at 100 mg/kg. In vitro studies on enterocyte-like Caco-2 cells did not indicate any cytotoxic effect up to a concentration of 50 micrograms/ml. Moreover, no inhibition of protein synthesis was observed, which lends further support to the absence of significant damage to the intestinal mucosa induced by 4-HR. The available database on 4-HR pharmacology and toxicology is inadequate to determine even a provisional ADI. There is negative evidence of carcinogenesis and no significant untoward effects were observed in humans when it was used as an anthelmintic. However, it is not possible to determine a NOEL for non-genotoxic effects. 4-HR could become an interesting alternative to the use of sulphites to prevent black spot. However, a more complete database is needed to achieve a regulatory evaluation.

Effect of taurine administration on liver lipids in guinea pig

The oral administration of 0.4% taurine in drinking water for 14 consecutive days showed the following hepatic effects in male guinea pig. The percentage of tauro-conjugated biliary bile acids was increased from 17.2–54.2%; the ratio liver weight/body weight was increased, and fatty change was induced. Liver triglyceride concentration was accordingly increased; diglyceride and phosphatidylcholine concentrations were reduced by the treatment, while phosphatidylethanolamine level was not affected. These changes suggest an adverse effect of taurine administration on phosphatidylcholine hepatic synthesis.

In vivo studies on possible adverse effects on reproduction of the fungicide methyl thiophanate

The fungicide methyl thiophanate (MT), widely used to control some of the most common fungal diseases in crops, is metabolized in animals into benzimidazole compounds, including the well‐known reproductive toxicant carbendazim. However, standard toxicological tests did not indicate that MT may cause testicular toxicity and/or embryotoxicity, which are typical effects of many benzimidazoles.

Evaluation of the Placenta: Suggestions for a Greater Role in Developmental Toxicology

Both in human and in rat, two types of placenta are present: the yolk sac (YS) and the chorioallantoic placenta. Histiotrophy, alpha-fetoprotein synthesis and blood cell formation occur in YS of both species. Besides, the midgut, primordial germ cells and possibly immunological structures originate from the YS tissue. The specialised cells of the chorioallantoic placenta attach the embryo to the uterus and form the vascular connections necessary for the nutrient transport. The placenta redirects maternal endocrine, immune and metabolic functions to conceptus advantage. These complex activities are sensitive to direct toxicity. Indirect effects on the placental functions might be elicited by immunomodulators and endocrine disrupters. Some experimental models could be utilised to identify possible toxic effects on placenta. Among the in vitro models the rodent giant yolk sac culture may be used to study the transport of materials, morphological and/or biochemical alterations and biotransformation activity of the visceral YS epithelium. Other in vitro approaches utilise human derived trophoblastic cells and tissues to investigate implantation and perimplantation toxicology. Besides specific studies, in vivo reproductive toxicity tests could pay more attention to the evaluation of placental tissues. Nowadays, some physiologically based pharmacokinetic models for developmental toxicity are also available to describe the disposition of toxic substances and their metabolites during pregnancy in rodents. Thus, more detailed studies on the embryo-foetal placenta may provide an important tool to understand developmental toxicity mechanisms, with particular regard to embryolethality and delayed development.

Long term administration of low doses of mycotoxins in poultry. 2. Residues of ochratoxin A and aflatoxins in broilers and laying hens after combined administration of ochratoxin A and aflatoxin B1

The effects of combined administration of ochratoxin A (OA) and aflatoxin B1 (AFB1) on the occurrence and the levels of residues of mycotoxins in poultry have been investigated. Male broilers and laying hens were fed from 14 days old with standard diets contaminated with 50 micrograms/kg OA and 50 micrograms/kg AFB1. Two groups of broilers and hens were withdrawn from contaminated feed at 37 and 88 days, respectively. At the time of sacrifice no significant lesions were found. Residues were compared with those found after administration of either toxin alone in former trials. Combined treatment resulted in higher content of OA in broiler livers (40 versus 5.0 micrograms/kg) and, to a lesser extent, in kidneys and skin, and of AFB1 in broiler liver and kidney (0.15 versus 0.02 microgram/kg and 0.40 versus 0.05 microgram/kg respectively). Laying hens showed smaller differences (0.20 versus 0.10 microgram/kg in liver and 0.32 versus 0.08 in kidneys). Withdrawal from treatment led to the almost complete disappearance of OA residues in broilers and in hens. These results show a synergistic effect of OA and AFB1, particularly in broilers.

Faecal bacteria of wild ruminants and the alpine marmot

Faecal samples from 60 red deer (Cervus elaphus), 13 roe deer (Capreoluscapreolus), 7 chamois (Rupicapra rupicapra), 41 alpine marmot (Marmota marmota) and soils mixed with deer faeces from the Stelvio National Park were examined forCampylobacter sp. andSalmonella sp. with negative results.The same material, especially deer faeces, was a habitat highly suitable forYersinia sp.:Y. enterocolitica (two biotypes) was isolated twice,Y. kristen-senii (two serotypes) was isolated 19 times,Y. frederiksenii andY. intermedia were isolated once.Antibiotic-resistantEscherichia coli were isolated from 16 specimens from wild ruminants, one from marmot and two from feeding places.

Macro- and microscopic alterations in 2 rabbit skin regions following topically repeated applications of benzoic acid n-alkyl esters

This paper explores the irritancy potential of n‐alkyl benzoate preservatives (methyl benzoate. MB; ethyl benzoate, EB; n‐propyl benzoate, n‐PB; n‐butyl benzoate, n‐BB), allowable in cosmetic formulations, Comparative experiments were carried out involving repeated daily application for all com‐pounds (up to maximum of 6 days) on clipped rabbit skin in 2 distinct regions: the dorsum (0.5 ml per treatment) and the external surface‐ of the‐ outer ear (0.2 ml per treatment). According to the different anatomo‐physiological features, benzoates elicited dissimilar reactions in the dorsum and the external ear. In the first mentioned area, there were widespread inflammatory reactions accompanied, in some cases, by necrotic changes and slight epidermal hyperplasia; on the other hand, hyperplasia was the most prominent and earliest alteration in the car. The irritancy potential was closely related in the duration of treatment as well to the increase in the molecular weight of benzoates.

Preliminary observations on the presence of visna-maedi in Italy

The presence of visna-maedi in Italy is reported for the first time. History, clinical findings and gross and microscopic lesions typical of the disease were observed in two sheep flocks in Central Italy. Affected animals were afebrile, lacked energy, lost condition and had progressively worsening dyspnoea which ended fatally. A few showed locomotor disturbances but remained alert. Anaemia and leukopenia were present. The lungs were affected with fleshy consolidation. Histologically, the pulmonary lesions included bronchiolar epithelial hyperplasia, peribronchiolar lymphoid cuffing, alveolar epithelialisation and very marked interstitial changes.In agar gel diffusion tests on sera from 106 animals from the two flocks, 44 were positive. Furthermore, when the same test was conducted as a preliminary screening procedure on 682 animals randomly chosen from 94 flocks in five Regions, 93 positive animals were revealed in 39 flocks, involving all of the Regions. The infection is likely to be widespread in Italy and to constitute a problem in at least some flocks or areas.