Critical evaluation of key evidence on the human health hazards of exposure to bisphenol A.

Despite the fact that more than 5000 safety-related studies have been published on bisphenol A (BPA), there seems to be no resolution of the apparently deadlocked controversy as to whether exposure of the general population to BPA causes adverse effects due to its estrogenicity. Therefore, the Advisory Committee of the German Society of Toxicology reviewed the background and cutting-edge topics of this BPA controversy. The current tolerable daily intake value (TDI) of 0.05 mg/kg body weight [bw]/day, derived by the European Food Safety Authority (EFSA), is mainly based on body weight changes in two- and three-generation studies in mice and rats. Recently, these studies and the derivation of the TDI have been criticized. After having carefully considered all arguments, the Committee had to conclude that the criticism was scientifically not justified; moreover, recently published additional data further support the reliability of the two- and three-generation studies demonstrating a lack of estrogen-dependent effects at and below doses on which the current TDI is based. A frequently discussed topic is whether doses below 5 mg/kg bw/day may cause adverse health effects in laboratory animals. Meanwhile, it has become clear that positive results from some explorative studies have not been confirmed in subsequent studies with higher numbers of animals or a priori defined hypotheses. Particularly relevant are some recent studies with negative outcomes that addressed effects of BPA on the brain, behavior, and the prostate in rodents for extrapolation to the human situation. The Committee came to the conclusion that rodent data can well be used as a basis for human risk evaluation. Currently published conjectures that rats are insensitive to estrogens compared to humans can be refuted. Data from toxicokinetics studies show that the half-life of BPA in adult human subjects is less than 2 hours and BPA is completely recovered in urine as BPA-conjugates. Tissue deconjugation of BPA-glucuronide and -sulfate may occur. Because of the extremely low quantities, it is only of minor relevance for BPA toxicity. Biomonitoring studies have been used to estimate human BPA exposure and show that the daily intake of BPA is far below the TDI for the general population. Further topics addressed in this article include reasons why some studies on BPA are not reproducible; the relevance of oral versus non-oral exposure routes; the degree to which newborns are at higher systemic BPA exposure; increased BPA exposure by infusions in intensive care units; mechanisms of action other than estrogen receptor activation; and the current regulatory status in Europe, as well as in the USA, Canada, Japan, New Zealand, and Australia. Overall, the Committee concluded that the current TDI for BPA is adequately justified and that the available evidence indicates that BPA exposure represents no noteworthy risk to the health of the human population, including newborns and babies.

Toxicogenomic analysis of N-nitrosomorpholine induced changes in rat liver: comparison of genomic and proteomic responses and anchoring to histopathological parameters.

A common animal model of chemical hepatocarcinogenesis was used to examine the utility of transcriptomic and proteomic data to identify early biomarkers related to chemically induced carcinogenesis. N-nitrosomorpholine, a frequently used genotoxic model carcinogen, was applied via drinking water at 120 mg/L to male Wistar rats for 7 weeks followed by an exposure-free period of 43 weeks. Seven specimens of each treatment group (untreated control and 120 mg/L N-nitrosomorpholine in drinking water) were sacrificed at nine time points during and after N-nitrosomorpholine treatment. Individual samples from the liver were prepared for histological and toxicogenomic analyses. For histological detection of preneoplastic and neoplastic tissue areas, sections were stained using antibodies against the placental form of glutathione-S-transferase (GST-P). Gene and protein expression profiles of liver tissue homogenates were analyzed using RG-U34A Affymetrix rat gene chips and two-dimensional gel electrophoresis-based proteomics, respectively. In order to compare results obtained by histopathology, transcriptomics and proteomics, GST-P-stained liver sections were evaluated morphometrically, which revealed a parallel time course of the area fraction of preneoplastic lesions and gene plus protein expression patterns. On the transcriptional level, an increase of hepatic GST-P expression was detectable as early as 3 weeks after study onset. Comparing deregulated genes and proteins, eight species were identified which showed a corresponding expression profile on both expression levels. Functional analysis suggests that these genes and corresponding proteins may be useful as biomarkers of early hepatocarcinogenesis.

Alternative methods to safety studies in experimental animals: role in the risk assessment of chemicals under the new European Chemicals Legislation (REACH).

During the last two decades, substantial efforts have been made towards the development and international acceptance of alternative methods to safety studies using laboratory animals. In the EU, challenging timelines for phasing out of many standard tests using laboratory animals were established in the seventh Amending Directive 2003/15/EC to Cosmetics Directive 76/768/EEC. In continuation of this policy, the new European Chemicals Legislation (REACH) favours alternative methods to conventional in vivo testing, if validated and appropriate. Even alternative methods in the status of prevalidation or validation, but without scientific or regulatory acceptance may be used under certain conditions. Considerable progress in the establishment of alternative methods has been made in some fields, in particular with respect to methods predicting local toxic effects and genotoxicity. In more complex important fields of safety and risk assessment such as systemic single and repeated dose toxicity, toxicokinetics, sensitisation, reproductive toxicity and carcinogenicity, it is expected that the development and validation of in silico methods, testing batteries (in vitro and in silico) and tiered testing systems will have to overcome many scientific and regulatory obstacles which makes it extremely difficult to predict the outcome and the time needed. The main reasons are the complexity and limited knowledge of the biological processes involved on one hand and the long time frame until validation and regulatory acceptance of an alternative method on the other. New approaches in safety testing and evaluation using "Integrated Testing Strategies" (ITS) (including combinations of existing data, the use of chemical categories/grouping, in vitro tests and QSAR) that have not been validated or not gained wide acceptance in the scientific community and by regulatory authorities will need a thorough justification of their appropriateness for a given purpose. This requires the availability of knowledge and experience of experts in toxicology. The challenging deadlines for phasing out of in vivo tests in the Cosmetics Amending Directive 2003/15/EC appear unrealistic. Likewise, we expect that the application of validated alternative methods will only have a small or moderate impact on the reduction of in vivo tests under the regimen of REACH, provided that at least the same level of protection of human health as in the past is envisaged. As a consequence, under safety aspects, it appears wise to consider established in vivo tests to be indispensable as basic tools for hazard and risk assessment with respect to systemic single and repeated dose toxicity, sensitisation, carcinogenicity and reproductive toxicity, especially regarding quantitative aspects of risk assessment such as NOAELs, LOAELs and health-related limit values derived from them. Based on the overall evaluation in this review, the authors are of the opinion that in the short- and mid-term, the strategy of the development of alternative methods should be more directed towards the refinement or reduction of in vivo tests. The lessons learnt during these efforts will provide a substantial contribution towards the replacement initiatives in the long-term.

The polycyclic musk 7-acetyl-1,1,3,4,4,6-hexamethyl-1,2,3,4-tetrahydronaphthaline lacks liver tumor initiating and promoting activity in rats exposed to human-relevant doses.

7-Acetyl-1,1,3,4,4,6-hexamethyl- 1,2,3,4-tetra-hydronaphthaline (AHTN) is one of the two most widely used fragrances of a group of substances known collectively as the polycyclic musks. In the last few years evidence has been accumulating that AHTN is hepatotoxic when administered at high doses. In the present study the subchronic hepatotoxicity of AHTN administered to rats at doses within the human exposure range was evaluated. For this purpose female and male juvenile Wistar rats were exposed to AHTN (300 microg/kg body weight per day, i.p.) alone or to a single dose of diethylnitrosamine (DEN) (100 mg/kg body weight, i.p.) followed by AHTN (1, 10, 100 or 300 microg/kg body weight per day, i.p.) for 90 days. Thereafter the liver architecture as well as the presence of placental glutathione S-transferase (GST-P)-positive hepatic lesions was assessed. In male animals receiving AHTN alone or in combination with DEN the number of GST-P-positive single hepatocytes was similar to that in untreated rats, while GST-P-positive mini-foci and foci were not observed. In the case of female rats the number of GST-P-positive single hepatocytes and mini-foci in AHTN-treated rats was similar to that in untreated animals, whereas in those animals receiving AHTN either alone or in combination with DEN, GST-P-positive foci could not be detected or were present in a number as similar to that in untreated rats. In conclusion, in the present study it has been shown that AHTN administered over a 90-day period in concentrations similar to those taken up daily by humans does not lead to hepatotoxicity.

Neurotoxicity risk assessment of MPTP (N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) as a synthetic impurity of drugs.

1-Methyl-4-phenyl-1,2,3,6 tetrahydropyridine (MPTP) induces symptoms indistinguishable from those of Parkinson's disease. It selectively destroys dopaminergic neurons in the substantia nigra and the globus pallidus. Death of these same neurons is apparently the cause of idiopathic Parkinson's disease. As phenyl-1,2,3,6 tetrahydropyridine is a commonly encountered subunit in heterocyclic drugs and because MPTP was found as a minor impurity in early batches of a candidate drug at Merck KGaA, it may be assumed that MPTP will also be present as an as yet undiscovered minor impurity in various existing drugs. A neurotoxicity risk assessment on MPTP has been conducted to define the risk of MPTP as an impurity in drugs that are used orally. This risk assessment has shown that compounds containing less than 5.0 p.p.m. MPTP administered orally will not cause a neurotoxicological health risk to patients treated with such a drug.

Genetic toxicology.

Systems for testing genetic toxicology are components of carcinogenic and genetic risk assessment. Present routine genotoxicity-testing is based on at least 20 years of development during which many different test systems have been introduced and used. Today, it is clear that no single test is capable of detecting all genotoxic agents. Therefore, the usual approach is to perform a standard battery of in-vitro and in-vivo tests for genotoxicity. Work-groups of the European Union (EU), the Organization for Economic Co-operation and Development (OECD), and, very recently, the work-group of the International Conference on Harmonization of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH) have defined such standard battery tests. These and some currently used supplementary or confirmatory tests are briefly discussed here. Additional test systems for the assessment of genotoxic and carcinogenic hazard and risk are seriously needed. These tests must be more relevant to man than are current assays and less demanding in respect of cost, time and number of animals. Another aspect for reassessment derives from the actual situation in the pharmaceutical industry. Companies have to prepare for the world economy of the 21st century. Therefore, pharmaceutical research is speeding up tremendously by use of tools such as genomics, combinatorial chemistry, high throughput screening and proteomics. Toxicology and genotoxicology need to re-evaluate their changing environment and must find ways to respond to these needs. In conclusion, genetic toxicology needs to answer questions coming from two major directions: hazard and risk identification and high throughput testing.

Observation of a water-depletion region surrounding loblolly pine roots by magnetic resonance imaging.

Magnetic resonance imaging was used to study sand containing various amounts of water and roots of loblolly pine planted into similar sand. Spin-lattice (T1) relaxation times of sand with water contents ranging from 0 to 25% (wt/wt) ranged from 472 to 1265 ms and increased with water content. Spin-spin (T2) relaxation times ranged from 54 to 76 ms and did not change in a discernible pattern with water content. Based on water content and measured T1 and T2 values, the signal intensity of sand/water images was predicted to increase with water content in a linear fashion, with the slope of the lines increasing with the time of acquisition repetition (TR). Measured signal intensity from images of sand with various water contents was found to follow a similar pattern. This allows interpretation of dark images of sand/water to be regions of low water content, and bright images to have comparatively greater water content. Images of loblolly pine seedling roots planted in identical sand showed the formation of a distinct water-depletion region first around the woody taproot and later showed the region extended and expanded around the lateral roots and clusters of mycorrhizal short roots. This observation strongly suggests that water uptake is occurring through the suberized region of the woody taproot.

Magnetic resonance microscopy of changes in water content in stems of transpiring plants.

Differences in water content and degree of binding in the various stem tissues of Pelargonium hortorum were observed by magnetic resonance imaging. 1H images were obtained with a resolution of 100 microns in the transverse plane and a slice thickness of 1250 microns. It was possible to distinguish the principal tissues of the stem by differences in their proton density or apparent water content and spin lattice relaxation time (T1) or degree of water binding. Measurements were made while the plant was slowly and actively transpiring. In the slowly transpiring plant, T1 of various tissues ranged from an average of 659 to 865 ms with a proton density variation of from 72 to 100%. In the actively transpiring plant, T1 ranged from an average of 511 to 736 ms, and the proton density was reduced, ranging between 62 and 88% of the peak value found in the slowly transpiring plant. The fibrous sheath surrounding the vascular tissue and the epidermal region was found to have the highest spin density and T1. Both tissues are comprised of relatively small thick-walled cells. Cortical and pith parenchyma are composed of larger, thinner-walled cells with numerous intercellular spaces and lower spin density and T1. The differences are attributed to the higher water content by volume in the tissue composed of smaller, more compactly arranged cells. The resolution obtained in this work enables clear definition of tissues in the living plant and quantitative information concerning differences in the distribution and extent of binding of water.

In Vivo Magnetic Resonance Microscopy of Changing Water Content in Pelargonium hortorum Roots.

Magnetic resonance imaging (MRI) was used to nondestructively observe changes in water content in roots of Pelargonium hortorum x Bailey during a period of relatively rapid transpiration. Anatomical regions of the root could be differentiated with a spatial resolution of 0.1 x 0.1 mm. MRI shows great potential for study of plant-water relations.

Direct Observation of Reversible and Irreversible Stomatal Responses of Attached Sunflower Leaves to SO(2).

The effects of SO(2) on stomatal aperture of attached sunflower leaves were observed with a remote-control light microscope system that permitted continuous observation of stomatal responses over periods of several hours. The relationship between actual stomatal aperture and stomatal conductance, measured with a porometer, also was examined on leaves before and after exposure to SO(2).A distinction between uninjured and injured regions was clearly visible on leaves after exposure to 1.5 microliters per liter SO(2) for less than an hour. During the exposure, the mean value of apertures for many stomata, which indicates stomatal conductance and transpiration rate, tended to decrease simultaneously in the uninjured and injured regions. However, the rate of decrease in the injured region was slower than that in the uninjured region because of a transient opening induced by water-soaking in the injured region. The transient opening was less common in stomata near veins and veinlets.There was a good correlation between pore width and stomatal conductance measured with a porometer before exposure to SO(2). This correlation continued in leaves exposed to SO(2) until visible, irreversible injury occurred, but then it disappeared.The results of these experiments indicate the necessity of continuous observation of individual stomata under the microscope to understand the effects of air pollutants such as SO(2) on stomatal behavior.

Dynamic analysis of water stress of sunflower leaves by means of a thermal image processing system.

This paper describes a method for measuring the temperatures of all regions of an intact leaf by using an infrared scanning thermometer at wavelengths between 8 and 14 micrometers combined with a digital image processing system. Pictures obtained every 2 minutes from leaves of sunflower (Helianthus annuus L. cv Large Russian) plants subjected to increasing water stress showed that water deficit develops first at the margins of leaves, accompanied by stomatal closure and increase in temperature. Finally, the temperature of the entire leaf rises 3 to 5 degrees C above that of nonstressed leaves. When transpiration resumed, it did so first at the leaf margins and these proceeded nonuniformly inward.The results of these experiments indicate that there are significant differences in the temperature and water status of different parts of a leaf. This makes it important to determine causes of such behavior and decide in what part of a leaf the temperature and water status should be measured. The thermo-imaging method can be useful in monitoring short term temperature changes occurring in leaves undergoing water, chilling, and other stresses.

Short and long term effects of root and shoot chilling of ransom soybean.

The immediate short term effects on some physiological processes and the long term effects on morphology and reproductive development of root- and shoot-chilled soybeans (Glycine max L. cv Ransom) were studied. Roots or shoots of 16- or 17-day-old plants were chilled at 10 degrees C for one week, and then rewarmed to 25 degrees C. Leaf elongation rate, net CO(2) uptake rate, and stomatal conductance decreased during root or shoot chilling. Root chilling had only temporary effects on water relations, while shoot chilling caused large changes in potentials during chilling. Most processes measured returned to control levels after two days of rewarming. Root-chilled plants harvested 90 days after emergence were similar in morphology and seed weight to controls. Shoot-chilled plants showed a large increase over controls in axillary branch growth, but an early abortion of flowers and a delayed resumption of flowering caused a 78% reduction in seed weight. Root chilling in this study was found to have little or no long term effect on the plants, while shoot chilling caused significant changes in vegetative morphology, and a delay in flowering and subsequent pod filling.

Criteria for the standardization of Salmonella mutagenicity tests: results of a collaborative study. IV. Relationship between the number of his- bacteria plated and number of his+ revertants scored in the Salmonella mutagenicity test.

Five laboratories participated in a joint ring study to investigate the role of bacterial cell number in the Salmonella mutagenicity test. A strictly standardized protocol, using sodium azide and TA 1535, was developed and employed to test the mutagenicity of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) with different dilutions of Salmonella typhimurium TA-100 and TA-1535 cultures. All laboratories detected the mutagenic activity of sodium azide with only a 2-fold variation of test results. For MNNG the interlaboratory variation was approximately 5-fold. Decreasing numbers of test bacteria employed resulted in lower numbers of MNNG-induced revertants in all laboratories. The number of preexisting revertants decreased in direct proportion to the reduced cell content, whereas the number of spontaneous revertants was not as greatly affected. A critical amount of test bacteria was required in order to obtain numbers of induced revertants which were equal to twice the number of spontaneous revertants. Two evaluation parameters which may be employed to describe the mutagenicity of a compound are compared.

Effects of High Atmospheric CO(2) and Sink Size on Rates of Photosynthesis of a Soybean Cultivar.

The effect of sink strength on photosynthetic rates under conditions of long-term exposure to high CO(2) has been investigated in soybean. Soybean plants (Merr. cv. Fiskeby V) were grown in growth chambers containing 350 microliters CO(2) per liter air until pod set. At that time, plants were trimmed to three trifoliolate leaves and either 21 pods (high sink treatment) or 6 pods (low sink treatment). Trimmed plants were either left in 350 microliters CO(2) per liter of air or placed in 1000 microliters CO(2) per liter of air (high CO(2) treatment) until pod maturity. Whole plant net photosynthetic rates of all plants were measured twice weekly, both at 350 microliters CO(2) per liter of air and 1000 microliters CO(2) per liter of air. Plants were also harvested at this time for dry weight measurements. Photosynthetic rates of high sink plants at both measurement CO(2) concentrations were consistently higher than those of low sink plants, and those of plants given the 350 microliter CO(2) per liter of air treatment were higher at both measurement CO(2) concentrations than those of plants given the 1000 microliters CO(2) per liter of air treatment. When plants were measured under treatment CO(2) levels, however, rates were higher in 1,000 microliter plants than 350 microliter CO(2) plants. Dry weights of all plant parts were higher in the 1,000 microliters CO(2) per liter air treatment than in the 350 microliters CO(2) per liter air treatment, and were higher in the low sink than in the high sink treatments.

Effect of abscisic Acid on root hydraulic conductivity.

Reports of the effects of abscisic acid (ABA) on ion and water fluxes have been contradictory. Some of the confusion seems due to the interaction of ion and water transport across membranes. In these experiments root systems were subjected to hydrostatic pressures up to 5.0 bars to enable measurement of root conductance that was independent of measurement of osmotic potentials or ion fluxes.ABA between 5 x 10(-5) molar and 2 x 10(-4) molar resulted in a decrease in the conductance of the soybean root systems as compared with the controls. ABA treatment also eliminated the discontinuity in the Arrhenius plot of total flow versus reciprocal temperature at constant pressure. The results suggest that ABA acts at the membrane that is rate-limiting to water flow directly, or by altering metabolism that in turn affects the membrane.

Effect of temperature on water and ion transport in soybean and broccoli systems.

Steady-state flow rates and exudate osmotic potentials were measured from complete root systems from warm- (28/23 C) or cold-(17/11 C) grown soybean or broccoli (Brassica oleracea) plants at various pressures or different temperatures.In warm-grown soybean roots systems, a break occurred at 14.7 C in the Arrhenius plot of total flow at constant pressure. When plants were grown at lower temperatures, the break point shifted to 8 C. Broccoli, a chilling-resistant species, showed no break for the temperature range used, but cooler growth temperatures decreased the activation energy for water flow through the root system from 18 kilocalories per mole to 9 kilocalories per mole. In both broccoli and soybean, cold-grown plants had lower exudate potentials and greater flow rates at low hydrostatic pressures than the warm-grown plants.These observations indicate that the rate-limiting site for passive water transport is a membrane which may be modified as the plant acclimates to varying growth temperatures. An additional part of the acclimation process is an increase in activity of root ion pumps.

Water Potential and Stomatal Resistance of Sunflower and Soybean Subjected to Water Stress during Various Growth Stages.

Plants of two varieties of soybean (Glycine max (L.) Merr.) and two varieties of sunflower (Helianthus annuus L.) were grown in controlled environments and subjected to water stress at various stages of growth. Leaf resistances and leaf water potentials were measured as stress developed. In soybeans the upper leaf surface had a higher resistance than the lower surface at all leaf water potentials and growth stages. Resistance of the upper surface began to increase at a higher water potential and increased more than the resistance of the lower surface. Resistances returned to prestress values 4 days after rewatering. In sunflowers upper and lower leaf surfaces had similar resistances at all water potentials and growth stages. Leaf resistances were higher in sunflower plants stressed before flowering than in those stressed later. Sunflower plants stressed to -16 bars recovered their prestress leaf resistance and water potential a few days after rewatering, but leaves of sunflower plants stressed to -23 bars died. Leaves of soybean and sunflower plants stressed before flowering suffered less injury than those of older plants and sunflowers stressed after flowering suffered more injury than soybeans.

General model for osmotic and pressure-induced flow in plant roots.

This paper presents a general model to describe coupled solute and water flow through plant roots when they are subjected to osmotic or hydrostatic pressure gradients, or both simultaneously. The model is based on well-established membrane transport equations derived from irreversible thermodynamic considerations. A variety of experimentally observed phenomena such as changes in root resistance with flow rate, apparent negative resistance effects, xylem sap dilution, and apparent non-osmotic water transport can be adequately explained with this model. The model also predicts that an exuding excised root system will be very insensitive to changes in the hydraulic conductivity coefficient. Previous work with a flat membrane of unit surface area and uniform properties is confirmed for a cylindrical coordinate system of nonuniform characteristics.