Effects of three PBDEs on development,reproduction and population growth rate of the harpacticoid copepod Nitocra spinipes

The current knowledge concerning effects of polybrominated diphenyl ethers (PBDEs) on aquatic organisms is very limited. A full life-cycle (< or =26 days exposure) ecotoxicity test with the particle-feeding copepod Nitocra spinipes was therefore used to study effects of BDE-47, -99 and -100 on larval development rate (LDR) and population growth rate (r(m)). LDR significantly decreased in copepods exposed for 6 days to nominal concentrations > or =0.013 mg/l BDE-47 and > or =0.03 mg/l BDE-99. Large concentration ratios (< or =338) between adult acute and juvenile subchronic endpoints were observed. Exposure over the full life cycle (< or =26 days) showed that r(m) in general was a less sensitive endpoint than LDR. Still, the r(m) in copepods exposed to 0.04 mg/l BDE-47 was significantly reduced compared to the controls (***P<0.001). Partitioning experiments with 14C-BDE-47 and 14C-BDE-99 in the test system showed that the major fractions (approximately 50-80%) were associated to particulate material. Our findings indicate that development and reproduction in N. spinipes are sensitive to the tested PBDEs and that ingestion of particle-adsorbed PBDEs most likely is the predominant route of exposure in N. spinipes. However, to further improve the usefulness of laboratory effect levels of PBDEs and other lipophilic substances for environmental risk assessment, it is important to develop ecotoxicological tools, which can evaluate and rate the toxic contribution from different matrices, such as suspended particles, sediment, food, water etc.     

Distribution of BDE-99 and effects on metamorphosis of BDE-99 and -47 after oral exposure in Xenopus tropicalis

The high concentrations of polybrominated diphenylethers (PBDEs) in the environment have raised the need for generating more information about the impact of these substances on animals. To study the distribution of (14)C-labelled 2,2',4,4',5-pentabromodiphenyl ether ((14)C-BDE-99) in Xenopus tropicalis (West African clawed frog) (14)C-BDE-99 was administered by dietary exposure to tadpoles at stage 54 or to juvenile frogs at stage 66. Whole-body autoradiography and liquid scintillation counting were used to examine the distribution of the substance at different survival times. Further, X. tropicalis tadpoles were dietarily exposed to the PBDE congeners BDE-47 and BDE-99 to study the effects on metamorphosis process. Measurements like body weight, body length, hind limb length and developmental stage as well as histological measurements on thyroid glands were performed after 14 days of exposure. Autoradiograms revealed high concentrations and long term retention of (14)C-BDE-99 in adipose tissue and melanin in frogs exposed both as tadpoles and juveniles. Further, a difference in uptake was recorded between the exposures at stages 54 and 66, implying that the juvenile frogs have higher uptake and more prolonged retention of the chemical than the tadpoles. Hind limb length was reduced in tadpoles dietarily exposed to 1mg/g feed of both BDE congeners. This was associated with reduced body weight and body length for BDE-47, suggesting general toxicity. Tadpoles exposed to BDE-99 also showed lower developmental stage but no effects on body weight or body length, suggesting possible thyroid hormone disruption. Higher concentrations of both congeners caused increased mortality. Thus, it can be concluded that in the present study, BDE-99 was retained for a longer period in the juvenile frogs than in metamorphosing tadpoles and that BDE-99 had an impact on X. tropicalis metamorphosis that might be of thyroid disrupting origin.     

Analysis of polybrominated diphenyl ethers in Swedish human milk. A time-related trend study, 1972-1997

A previously described method for analysis of organochlorine compounds in human milk was adopted for analysis of brominated diphenyl ethers (BDEs) substituted with three to six bromine atoms. Analytes were extracted from human milk with the lipophilic gel Lipidex 5000. Further purifications were performed on partly deactivated aluminum oxide and silica gel columns, followed by gel permeation chromatography. The concentrations of BDEs were determined by gas chromatography/mass spectrometry (GC/MS). The average recoveries of 2,2',4-triBDE (BDE-17), 2,4,4'-triBDE (BDE-28), 2,2',4,4'-tetraBDE (BDE-47), 2,3',4,4'-tetraBDE (BDE-66), 2,2,3,4,4'-pentaBDE (BDE-85), 2,2',4,4',5-pentaBDE (BDE-99), 2,2',4,4',6-pentaBDE (BDE-100), 2,2',4,4',5,5'-hexaBDE (BDE-153), and 2,2',4,4',5,6'-hexaBDE (BDE-154) added to the samples before extraction ranged from 86% to 102%. Pooled samples of breast milk, collected at eight time periods between 1972 and 1997, were analyzed for PBDEs. BDE-47 was the most abundant PBDE congener in all samples. In total, eight PBDE congeners were identified in the milk. The sum of the concentrations of BDE congeners in human milk increased from 0.07 to 4.02 ng/g lipids during the 25-yr period studied.     

Zebrafish (Danio rerio) embryos as a model for testing proteratogens

Zebrafish embryos have been shown to be a useful model for the detection of direct acting teratogens. This communication presents a protocol for a 3-day in vitro zebrafish embryo teratogenicity assay and describes results obtained for 10 proteratogens: 2-acetylaminofluorene, benzo[a]pyrene, aflatoxin B(1), carbamazepine, phenytoin, trimethadione, cyclophosphamide, ifosfamide, tegafur and thio-TEPA. The selection of the test substances accounts for differences in structure, origin, metabolism and water solubility. Apart from 2-acetylaminofluorene, which mainly produces lethal effects, all proteratogens tested were teratogenic in zebrafish embryos exposed for 3 days. The test substances and/or the substance class produced characteristic patterns of fingerprint endpoints. Several substances produced effects that could be identified already at 1 dpf (days post fertilization), whereas the effects of others could only be identified unambiguously after hatching at ≥ 3 dpf. The LC?? and EC?? values were used to calculate the teratogenicity index (TI) for the different substances, and the EC?? values were related to human plasma concentrations. Results lead to the conclusion that zebrafish embryos are able to activate proteratogenic substances without addition of an exogenous metabolic activation system. Moreover, the teratogenic effects were observed at concentrations relevant to human exposure data. Along with other findings, our results indicate that zebrafish embryos are a useful alternative method for traditional teratogenicity testing with mammalian species.     

Inhalation exposure technology used for varying exposure modes and profiles in toxicology studies

Current technology used in inhalation toxicology studies employs various exposure modes and concentration profiles. Inhalation exposure modes typically utilize wholebody techniques, whereas other exposure modes include nose- and head-only exposure systems and, in some cases, whole- or partial-lung exposure systems. The latter two conditions are utilized when safety considerations are warranted by the hazardous nature of the chemical or agent being tested, the test substance may be dermally adsorbed, and/or the costs of the chemical used are of concern. Inhalation exposure studies may span several minutes to 24 h of continuous exposure and from one day to the full life span of the animal being tested. Time-varying profile exposures, on the other hand, are typically used to mimic human exposures to chemical agents and, in some cases, to more accurately extrapolate animal toxicity data in assessing human risk. Automation of inhalation exposure systems has expedited the timely operation of both accurate and repeatable profiles, and it has allowed for reexamination of classical time-weighted average concentration information relating to human health concerns. The toxicological assessment of potential health effects resulting from exposure to airborne substances typically involves thorough characterizing of the test agent via acute, subchronic, and chronic toxicity testing.     

ANALYSIS OF POLYBROMINATED DIPHENYL ETHERS IN SWEDISH HUMAN MILK. A TIME-RELATED TREND STUDY, 1972-1997

A previously described method for analysis of organochlorine compounds in human milk was adopted for analysis of brominated diphenyl ethers ( BDEs) substituted with three to six bromine atom s. Analytes were extracted from hum an milk with the lipophilic gel Lipidex 5000. Further purifications were performed on partly deactivated aluminum oxide and silica gel columns, followed by gel permeation chromatography. The concentrations of BDEs were determined by gas chromatography/mass spectrometry (GC/MS). The average recoveries of 2,2',4-triBDE (BDE-17), 2,4,4'-triBDE (BDE28) , 2,2',4,4'-tetraBDE (BDE-47), 2,3',4,4'-tetraBDE (BDE-66), 2,2',3,4,4'-pentaBDE (BDE-8 5) , 2,2',4,4',5-p entaBDE (BDE-99), 2,2',4,4',6-pentaBDE (BDE-100) , 2,2',4,4',5,5'-hexaBDE (BDE-153), and 2,2',4,4',5,6'-hexaBDE (BDE-154) added to the samples before extraction ranged from 86% to 102% . Pooled samples of breast milk, collected at eight time periods between 1972 and 1997, were analyzed for PBDEs. BDE-47 was the most abundant PBDE congener in all samples. In total, eight PBDE congeners were identified in the milk. The sum of the concentrations of BDE congeners in human milk increased from 0.07 to 4.02 ng/g lipids during the 25-yr period studied.     

The nickel ion bioavailability model of the carcinogenic potential of nickel-containing substances in the lung

The inhalation of nickel-containing dust has been associated with an increased risk of respiratory cancer in workplaces that process and refine sulfidic nickel mattes, where workers are exposed to mixtures of sulfidic, oxidic, water-soluble, and metallic forms of nickel. Because there is great complexity in the physical and chemical properties of nickel species, it is of interest which specific nickel forms are associated with carcinogenic risk. A bioavailability model for tumor induction by nickel has been proposed, based on the results of animal inhalation bioassays conducted on four nickel-containing substances. The nickel ion bioavailability model holds that a nickel-containing substance must release nickel ions that become bioavailable at the nucleus of epithelial respiratory cells for the substance to be carcinogenic, and that the carcinogenic potency of the substance is proportional to the degree to which the nickel ions are bioavailable at that site. This hypothesis updates the nickel ion theory, which holds that exposure to any nickel-containing substance leads to an increased cancer risk. The bioavailability of nickel ions from nickel-containing substances depends on their respiratory toxicity, clearance, intracellular uptake, and both extracellular and intracellular dissolution. Although some data gaps were identified, a weight-of-evidence evaluation indicates that the nickel ion bioavailability model may explain the existing animal and in vitro data better than the nickel ion theory. Epidemiological data are not sufficiently robust for determining which model is most appropriate, but are consistent with the nickel ion bioavailability model. Information on nickel bioavailability should be incorporated into future risk assessments.     

Effects of all-trans-retinoic acid and all-trans-retinoyl glucuronide in two in vitro systems of distinct biological complexity

In vitro systems are widely used to evaluate the embryotoxic potential of retinoids. The effective concentrations of these retinoids, however, are not consistent in the various in vitro systems used in evaluating embryotoxicity. This may be explained by the different level of complexity for each individual system, which may lead to different concentrations of the substances in the target tissues. To verify this hypothesis we have compared two in vitro systems of distinct biological complexity: the rat whole embryo culture system, and the mouse limb bud organ culture system. The lipid soluble, teratogenic retinoid all-trans-retinoic acid (ATRA), and all-trans-retinoyl-beta-D-glucuronide (ATRAG), an endogenous, water-soluble and biologically active retinoid with limited placental transfer, were compared with regard to their embryotoxic potential in vitro. In both in vitro systems, ATRAG showed a lower degree of embryotoxicity than ATRA. In the limb bud organ culture, ATRAG revealed only slightly less toxicity than ATRA, whereas the effective concentrations of the two compounds in the whole embryo culture system differed by almost two orders of magnitude. During incubation with ATRAG, ATRA is generated by hydrolysis and is found in culture media and exposed tissues. The presence of membrane barriers around the developing embryo in the whole embryo culture system possibly prevents the transfer of ATRAG to the embryo and, therefore, its exposure to the active hydrolysis product ATRA. From these results we conclude that analysis of retinoid concentrations in the culture media and in the exposed tissues is essential for the interpretation of results obtained from in vitro toxicity testing.     

The nickel ion bioavailability model of the carcinogenic potential of nickel-containing substances in the lung

The inhalation of nickel-containing dust has been associated with an increased risk of respiratory cancer in workplaces that process and refine sulfidic nickel mattes, where workers are exposed to mixtures of sulfidic, oxidic, water-soluble, and metallic forms of nickel. Because there is great complexity in the physical and chemical properties of nickel species, it is of interest which specific nickel forms are associated with carcinogenic risk. A bioavailability model for tumor induction by nickel has been proposed, based on the results of animal inhalation bioassays conducted on four nickel-containing substances. The nickel ion bioavailability model holds that a nickel-containing substance must release nickel ions that become bioavailable at the nucleus of epithelial respiratory cells for the substance to be carcinogenic, and that the carcinogenic potency of the substance is proportional to the degree to which the nickel ions are bioavailable at that site. This hypothesis updates the nickel ion theory, which holds that exposure to any nickel-containing substance leads to an increased cancer risk. The bioavailability of nickel ions from nickel-containing substances depends on their respiratory toxicity, clearance, intracellular uptake, and both extracellular and intracellular dissolution. Although some data gaps were identified, a weight-of-evidence evaluation indicates that the nickel ion bioavailability model may explain the existing animal and in vitro data better than the nickel ion theory. Epidemiological data are not sufficiently robust for determining which model is most appropriate, but are consistent with the nickel ion bioavailability model. Information on nickel bioavailability should be incorporated into future risk assessments.     

Gestational exposure to BDE-99 produces toxicity through upregulation of CYP isoforms and ROS production in the fetal rat liver

On gestation day (GD) 6 to GD 19, pregnant Sprague Dawley rats were orally exposed to 0, 0.5, 1, and 2 mg/kg/day to one of the most prevalent polybrominated diphenyl ethers congeners found in humans, 2,2',4,4',5-pentaBDE (BDE-99). All dams were euthanized on GD 20, and live fetuses were evaluated for sex, body weight, and external, internal, and skeletal malformations and developmental variations. The liver from one fetus of each litter was excised for the evaluation of oxidative stress markers and the messenger RNA expression of multiple cytochrome P450 (CYP) isoforms. Exposure to BDE-99 during the gestational period produced delayed ossification, slight hypertrophy of the heart, and enlargement of the liver in fetuses. A transplacental effect of BDE-99, evidenced by the activation of nuclear hormones receptors that induce the upregulation of CYP1A1, CYP1A2, CYP2B1, and CYP3A2 isoforms, was also found in fetal liver. These isoforms are correlated with the activity level of the enzyme catalase and the levels of thiobarbituric acid reactive substances. However, teratogenic effects from BDE-99 exposure were not observed. Clear signs of embryo/fetal toxicity, due to a possible hormonal disruption, were evidenced by a large increase in the CYP system and the production of reactive oxygen species in fetal liver.     

Guideline for the out-of-hospital management of human exposures to minimally toxic substances

All substances are capable of producing toxicity, so nothing is completely non-toxic. Minimally toxic substances are those which produce little toxicity, minor self-limited toxicity, or clinically insignificant effects at most doses. Examples include silica gel, A&D ointment, chalk, lipstick, and non-camphor lip balms, watercolors, hand dishwashing detergents, non-salicylate antacids (excluding magnesium or sodium bicarbonate containing products), calamine lotion, clay, crayons, diaper rash creams and ointments, fabric softeners/sheets, glow products, glue (white, arts, and crafts type), household plant food, oral contraceptives, pen ink, pencils, starch/sizing, throat lozenges without local anesthetics, topical antibiotics, topical antifungals, topical steroids, topical steroids with antibiotics, and water-based paints. Minimally toxic exposures have the following characteristics: (1) The information specialist has confidence in the accuracy of the history obtained and the ability to communicate effectively with the caller. (2) The information specialist has confidence in the identity of the product(s) or substance(s) and a reasonable estimation of the maximum amount involved in the exposure. (3) The risks of adverse reactions or expected effects are acceptable to both the information specialist and the caller based on available medical literature and clinical experience. (4) The exposure does not require a healthcare referral since the potential effects are benign and self-limited. However, decisions regarding patient disposition should take into account the patient's intent, symptoms, and social environment. In addition, individual patient circumstances (e.g., pregnancy, pre-existing medical conditions, therapeutic interventions) need to be considered. Minimally toxic exposures may vary in route (dermal, inhalation, ingestion, ocular), chronicity (acute, chronic), and substance composition (single or multi-ingredient, single or multiple product). Future categorization of substances as "minimally toxic" should be based on a process involving review of current knowledge, a thorough analysis of poisoning experience, and prospective validation.     

An exposure system to study the effects of water-soluble gases on PM-induced toxicity

An aerosol generation and exposure system to evaluate the role of water-soluble gases in particulate matter (PM)-induced injury was designed, built, and validated by generating test atmospheres to study the role of hydrogen peroxide in PM-induced toxicity. In this system, particle number concentration, size distribution, hydrogen peroxide concentration, and water concentration can all be varied. An ammonium sulfate aerosol with mass median diameter 0.46 +/- 0.01 microm was used as a model atmospheric aerosol because ammonium sulfate is a major component of the fine aerosol, and the water uptake of ammonium sulfate aerosol is well characterized. The following four test atmospheres were generated: (1) ammonium sulfate aerosol, (2) an aerosol containing hydrogen peroxide and ammonium sulfate, (3) vapor-phase hydrogen peroxide, and (4) particle-free air. All test atmospheres were maintained at a relative humidity of 85%. Particle size distribution, number concentration, total hydrogen peroxide concentration, temperature, and relative humidity were measured continuously in the exposure chamber. The gas-particle partitioning of hydrogen peroxide was calculated using total hydrogen peroxide concentration, the Henry's law constant for hydrogen peroxide in water, and aerosol water content. We found that the aerosol generation system produced stable concentrations throughout the 2-hour exposures.     

Interspecies comparisons in toxicology: the utility and futility of plasma concentrations of the test substance

A classical dilemma in toxicology is how the dose administered relates to the dose delivered to the target site. Plasma concentrations of the test substance may be misleading since the concentration of any given substance in the plasma may not be representative of its concentration in tissues. Furthermore, a given tissue concentration of a xenobiotic can evoke responses which are highly species-dependent. While evaluating toxicity data within one species, plasma concentrations reflect the effects of route of administration, bioavailability, dose level, multiple dosing, age, gender, etc. However, when toxicity data is compared across species, the relevance of plasma concentrations depends on the nature of the toxicity. Reversible, pharmacodynamic effects often correlate with plasma concentrations, although there may be marked interspecies differences in dose-response relationships. Irreversible effects, if pharmacodynamic in origin, often correlate better with the intensity/duration of the pharmacodynamic response, rather than with plasma concentration. On the other hand, irreversible effects, if chemically mediated, may not correlate at all with plasma concentration, the lesions being caused by reactive metabolites of fleeting existence, which rarely survive long enough to leave their site of synthesis. They cannot be measured in the plasma nor predicted from plasma concentrations of the parent xenobiotic. The limitations of plasma concentrations in interpreting the toxicology of substances which are tissue-sequestered, which are subject to pharmacogenetic factors, or which show plasma concentrations that are not proportional to dose are also discussed. Mention is made of possible alternatives to plasma concentrations in assessing exposure in toxicology studies.     

Toxicity of titanium dioxide nanoparticles to rainbow trout (Oncorhynchus mykiss): gill injury, oxidative stress, and other physiological effects

Mammalian and in vitro studies have raised concerns about the toxicity of titanium dioxide nanoparticles (TiO2 NPs), but there are very limited data on ecotoxicity to aquatic life. This paper is an observational study where we aim to describe the toxicity of TiO2 NPs to the main body systems of rainbow trout. Stock solutions of dispersed TiO2 NPs were prepared by sonication without using solvents. A semi-static test system was used to expose rainbow trout to either a freshwater control, 0.1, 0.5, or 1.0 mg l(-1) TiO2 NPs for up to 14 days. Exposure to TiO2 NPs caused some gill pathologies including oedema and thickening of the lamellae. No major haematological or blood disturbances were observed in terms of red and white blood cell counts, haematocrit values, whole blood haemoglobin, and plasma Na+ or K+ concentrations. Tissue metal levels (Na+, K+, Ca2+ and Mn) were generally unaffected. However, some exposure concentration-dependent changes in tissue Cu and Zn levels were observed, especially in the brain. Exposure to TiO2 NPs caused statistically significant decreases in Na+K+-ATPase activity (ANOVA, P<0.05) in the gills and intestine, and a trend of decreasing enzyme activity in the brain (the latter was not statistically significant). Thiobarbituric acid reactive substances (TBARS) showed exposure concentration-dependent and statistically significant (ANOVA or Kruskal-Wallis test, P<0.05) increases (two-fold or more) in the gill, intestine and brain, but not the liver during exposure to TiO2 NPs compared to controls. TiO2 NP exposure caused statistically significant (ANOVA, P<0.05) increases in the total glutathione levels in the gills, but depletion of hepatic glutathione compared to controls. Total glutathione levels in the brain and intestine were unaffected. Liver cells exposed to TiO2 NPs showed minor fatty change and lipidosis, and some hepatocytes showed condensed nuclear bodies (apoptotic bodies). Fish probably ingested water containing TiO2 NPs during exposure (stress-induced drinking) which may have resulted in some areas of erosion on the intestinal epithelium. Overall we conclude that titanium dioxide nanoparticles are not a major ionoregulatory toxicant, or haemolytic, at the concentration and exposure times used. Respiratory distress is a concern and sub-lethal toxicity involves oxidative stress, organ pathologies, and the induction of anti-oxidant defences, such as glutathione.     

Practical considerations for conducting ecotoxicity test methods with manufactured nanomaterials: what have we learnt so far?

This review paper reports the consensus of a technical workshop hosted by the European network, NanoImpactNet (NIN). The workshop aimed to review the collective experience of working at the bench with manufactured nanomaterials (MNMs), and to recommend modifications to existing experimental methods and OECD protocols. Current procedures for cleaning glassware are appropriate for most MNMs, although interference with electrodes may occur. Maintaining exposure is more difficult with MNMs compared to conventional chemicals. A metal salt control is recommended for experiments with metallic MNMs that may release free metal ions. Dispersing agents should be avoided, but if they must be used, then natural or synthetic dispersing agents are possible, and dispersion controls essential. Time constraints and technology gaps indicate that full characterisation of test media during ecotoxicity tests is currently not practical. Details of electron microscopy, dark-field microscopy, a range of spectroscopic methods (EDX, XRD, XANES, EXAFS), light scattering techniques (DLS, SLS) and chromatography are discussed. The development of user-friendly software to predict particle behaviour in test media according to DLVO theory is in progress, and simple optical methods are available to estimate the settling behaviour of suspensions during experiments. However, for soil matrices such simple approaches may not be applicable. Alternatively, a Critical Body Residue approach may be taken in which body concentrations in organisms are related to effects, and toxicity thresholds derived. For microbial assays, the cell wall is a formidable barrier to MNMs and end points that rely on the test substance penetrating the cell may be insensitive. Instead assays based on the cell envelope should be developed for MNMs. In algal growth tests, the abiotic factors that promote particle aggregation in the media (e.g. ionic strength) are also important in providing nutrients, and manipulation of the media to control the dispersion may also inhibit growth. Controls to quantify shading effects, and precise details of lighting regimes, shaking or mixing should be reported in algal tests. Photosynthesis may be more sensitive than traditional growth end points for algae and plants. Tests with invertebrates should consider non-chemical toxicity from particle adherence to the organisms. The use of semi-static exposure methods with fish can reduce the logistical issues of waste water disposal and facilitate aspects of animal husbandry relevant to MMNs. There are concerns that the existing bioaccumulation tests are conceptually flawed for MNMs and that new test(s) are required. In vitro testing strategies, as exemplified by genotoxicity assays, can be modified for MNMs, but the risk of false negatives in some assays is highlighted. In conclusion, most protocols will require some modifications and recommendations are made to aid the researcher at the bench.     

Development of a flow-through system for the fish embryo toxicity test (FET) with the zebrafish (Danio rerio)

The acute fish test is still a mandatory component in chemical hazard and risk assessment. However, one of the objectives of the new European chemicals policy (REACH – Registration, Evaluation, Authorization and Restriction of Chemicals) is to promote non-animal testing. For whole effluent testing in Germany, the fish embryo toxicity test (FET) with the zebrafish (Danio rerio) has been an accepted and mandatory replacement of the fish test since January 2005. For chemical testing, however, further optimization of the FET is required to improve the correlation between the acute fish test and the alternative FET. Since adsorption of the test chemical to surfaces may reduce available exposure concentrations, a flow-through system for the FET using modified commercially available polystyrene 24-well microtiter plates was developed, thus combining the advantages of the standard FET with those of continuous delivery of test substances. The advantages of the design presented include: small test footprint, availability of adequate volumes of test solution for subsequent chemical analysis, and sufficient flow to compensate for effects of non-specific adsorption within 24 h. The flow-through test system can also be utilized to conduct longer-term embryo larval fish tests, thus offering the possibility for teratogenicity testing.     

Effects of two different kinds of silicium dioxide on release of drugs from suppositories: benzydamine hydrochloride.

Silica gel confirmed its function as viscosity agent for lipophylic excipients for suppositories, ensuring homogeneous drug distribution in the suppository mass. The influence on release rate of a water-soluble drug (benzydamine hydrochloride) was clearly different according to type of silica gel. With Aerosil 200 (hydrophylic), after a progressive decrease in release rate at the lowest concentrations, an increase was observed at the highest concentrations, until it reached that of the suppositories without silica gel. With Aerosil R972, release rate decreased progressively with increased silica gel concentration, until release was practically inhibited even at low concentrations.     

The challenge of reproductive and developmental toxicology under REACH.

The European Union's REACH regulation has explicit requirements for reproductive and developmental toxicity data on all substances manufactured in or imported into the European Union at > or = 10 metric tons/year. Meeting the data requirements with whole-animal testing could result in the use of almost 22 million vertebrate animals for the registration of existing chemicals and cost up to several hundred thousand dollars per registered substance. The requirement for financial and animal resources can be reduced by the use of in vitro testing, quantitative structure-activity relationship models, and grouping of related substances. Although REACH strongly encourages these methods of avoiding vertebrate animal testing, it does not appear that in vitro testing or quantitative structure-activity relationship analysis will be able to replace whole-animal reproductive and developmental toxicity testing. Grouping of related compounds offers the possibility, perhaps in conjunction with in vitro testing and structure-activity analysis, of reducing vertebrate animal testing provided there is sufficient information on the related compounds and sufficient reason to believe that the related compounds will have similar toxicological properties. The designation of a substance as a reproductive or developmental toxicant follows criteria that do not consider the dose level of the substance at which reproductive or developmental effects occur, as long as excessive generalized toxicity does not occur. This method of labeling substances without consideration of effective dose level does not provide information on the actual risk of the chemical. Designation of a substance as a reproductive or developmental toxicant may have important implications under REACH and can be expected to result in the need to obtain authorization for marketing of the substance in the European Union.     

Reproductive toxicology studies and immunotherapeutics

There are many ways in which immunotherapeutic agents could potentially cause developmental toxicity. According to the published data in humans and animals, however, this class of drugs does not appear to present any increased risk of reproductive toxicity by comparison with other therapeutic classes. The basic testing strategy outlined in the ICH guidelines is suitable for the preclinical reproductive toxicity assessment of these drugs (except vaccines). Particular consideration needs to be given to the choice of species when testing antibodies or human molecules. Immune-active drugs may have the potential to interfere with the development of the immune system. For this reason, it may be advisable to assess the integrity of the immune system in animals previously exposed to the test substance during development. Such tests can easily be appended to the study design of the pre- and post-natal study. In view of the lack of any provision for post-weaning exposure in the guidelines and the late maturation of the immune system, a separate post-natal study may be considered for drugs that are likely to be prescribed to children.