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Abstract: Polyurethanes (PU) are polymers made with diisocyanates such as MDI (4,4′-methylene diphenyl diisocyanate) and TDI (2,4-toluene diisocyanate and 2,6-toluene diisocyanate). Investigations have been undertaken with MDI and TDI to assess dermal uptake and resulting systemic exposure. Absorption, distribution and excretion of MDI was studied in rats using a single dermal administration of 14C-MDI dissolved in acetone at nominal 165mg/kg body weight and 15mg/kg bw (4.0 and 0.4mg/cm2) and intradermal injection of 14C-MDI dissolved in corn oil at nominal 1.4mg/kgbw. Dermal absorption of 14C-MDI (at both doses) was low; at or below 1% of the applied dose. Considerable amounts of the applied radioactivity were found at the application site which could not be washed off. By intradermal administration of 14C-MDI approximately 66% of applied radioactivity remained at the application site with approximately 26% recovered in excreta, cage wash, tissues and carcass. The absorption, distribution and excretion of 2,4-TDI was studied in rats following a single dermal administration of radiolabelled 14C-2,4-TDI at nominal 350mg/kg body weight (12mg/cm2). Dermal absorption of 14C-2,4-TDI was at or below 1% of the applied dose. Considerable amounts of the applied radioactivity were found at the application site which could not be washed off. In summary the results show that dermal uptake of MDI and TDI is very low. Due to the chemical reactivity of isocyanates it can be expected that small amounts which might be absorbed will react with tissue constituents directly at the exposed skin area, or will be converted to adducts with biomacromolecules or to biologically inactive oligoureas. Overall it is concluded that, following dermal exposure to MDI and TDI, systemic exposures and resulting toxicity, other than the known sensitization, can be expected to be very low. In addition studies were performed with dermal application of unlabelled 2,4 and 2,6 TDI to check the availability and fate of this chemical on rat skin surface and to assess possible tissue damage. These experiments showed that unchanged test material can be detected on rat skin for up to 8h if not washed off. Dermal treatment with 2,4 or 2,6 TDI was associated with irritation with increased severity over a 48h period after washing with a decontaminant solution. [Copyright &y& Elsevier]

The aerosol release during the professional application of two different isocyanate based two component spray systems was identified and the physicochemical properties of the released airborne aerosols were characterized. For this purpose, aerosol release fractions were measured using a mass balance method described by Schwarz and Koch. Besides the release of total aerosol mass special emphasis was directed to the content of free monomeric MDI (4,4'- and 2,4'-diphenylmethane diisocyanate) in three particle size fractions relevant for inhalation uptake: inhalable, thoracic, and respirable size fraction. Two products were investigated: a two component PUR (polyurethane) spray foam (Elastopor) and a polyurea spray coating (Elastocoat). The mass fraction of the applied products released with the overspray as inhalable aerosol is 6.3 × 10-4 (Elastopor) and 4.0 × 10-4 (Elastocoat). Of the released total overspray aerosol 75 or 80% were in the thoracic size range, and 26 or 47% in the respirable regime for the PUR spray foam or the polyurea spray coating, respectively. At the time point of release the content of monomeric MDI in the aerosol corresponds to the composition of the bulk product. However, analysis of air samples indicates that <1% of the spray foam aerosol mass release fraction is attributed to free monomeric 4,4'- and 2,4'-MDI. For the Spray Coating the monomeric MDI fraction is <0.1%. Higher oligomers of MDI and prereacted oligomeric reaction products make up a few percent of the aerosol. This results in a total fraction of 0.0023% (spray foam) and 0.00015% (spray coating), respectively, of the sprayed monomeric MDI that is transferred into an inhalable aged aerosol. This data demonstrates, that during professional spraying only a small fraction of the total applied mass is released as airborne aerosol. The potential distribution of the theoretically inhalable aerosol in the respiratory tract and a low residual monomer content is described, significantly contributing to a refined safety assessment of the spray applications at the workplaces., (© The Author(s) 2019. Published by Oxford University Press on behalf of the British Occupational Hygiene Society.)

There is a continuing interest in determining whether it is possible to identify thresholds for chemical allergy. Here allergic sensitisation of the respiratory tract by chemicals is considered in this context. This is an important occupational health problem, being associated with rhinitis and asthma, and in addition provides toxicologists and risk assessors with a number of challenges. In common with all forms of allergic disease chemical respiratory allergy develops in two phases. In the first (induction) phase exposure to a chemical allergen (by an appropriate route of exposure) causes immunological priming and sensitisation of the respiratory tract. The second (elicitation) phase is triggered if a sensitised subject is exposed subsequently to the same chemical allergen via inhalation. A secondary immune response will be provoked in the respiratory tract resulting in inflammation and the signs and symptoms of a respiratory hypersensitivity reaction. In this article attention has focused on the identification of threshold values during the acquisition of sensitisation. Current mechanistic understanding of allergy is such that it can be assumed that the development of sensitisation (and also the elicitation of an allergic reaction) is a threshold phenomenon; there will be levels of exposure below which sensitisation will not be acquired. That is, all immune responses, including allergic sensitisation, have threshold requirement for the availability of antigen/allergen, below which a response will fail to develop. The issue addressed here is whether there are methods available or clinical/epidemiological data that permit the identification of such thresholds. This document reviews briefly relevant human studies of occupational asthma, and experimental models that have been developed (or are being developed) for the identification and characterisation of chemical respiratory allergens. The main conclusion drawn is that although there is evidence that the acquisition of sensitisation to chemical respiratory allergens is a dose-related phenomenon, and that thresholds exist, it is frequently difficult to define accurate numerical values for threshold exposure levels. Nevertheless, based on occupational exposure data it may sometimes be possible to derive levels of exposure in the workplace, which are safe. An additional observation is the lack currently of suitable experimental methods for both routine hazard characterisation and the measurement of thresholds, and that such methods are still some way off. Given the current trajectory of toxicology, and the move towards the use of non-animal in vitro and/or in silico) methods, there is a need to consider the development of alternative approaches for the identification and characterisation of respiratory sensitisation hazards, and for risk assessment., (Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.)

Several ultraviolet (UV) filters exhibit estrogenic, some also anti-androgenic activity. They are present in waste water treatment plants, surface waters and biosphere including human milk, suggesting potential exposure during development. Developmental toxicity was studied in rats for the UV filters 4-methylbenzylidene camphor (4-MBC, 0.7, 7, 24, 47 mg/kg/day) and 3-benzylidene camphor (3-BC, 0.07, 0.24, 0.7, 2.4, 7 mg/kg/day) administered in chow to the parent generation before mating, during pregnancy and lactation, and to the offspring until adulthood. Neonates exhibited enhanced prostate growth after 4-MBC and altered uterine gene expression after both chemicals. 4-MBC and 3-BC delayed male puberty and affected reproductive organ weights of adult offspring. Effects on the thyroid axis were also noted. Expression and oestrogen sensitivity of oestrogen-regulated genes and nuclear receptor coregulator levels were altered at mRNA and protein levels in adult uterus, prostate and brain regions involved in gonadal control and sexual behaviour. Female sexual behaviour was impaired by both filters; 3-benzylidene camphor caused irregular cycles. Classical endpoints exhibited lowest observed adverse effect levels (LOAELs) and no observed adverse effect levels (NOAELs) of 7/0.7 mg/kg for 4-MBC and 0.24/0.07 mg/kg for 3-BC. Molecular endpoints were affected by the lowest doses studied. Our data indicate that the potential risk posed by endocrine active UV filters warrants further investigations.

Background and Objectives: In previous studies, we found that the ultraviolet filter 4-methyl-benzylidene camphor (4-MBC) exhibits estrogenic activity, is a preferential estrogen receptor (ER)-beta ligand, and interferes with development of female reproductive organs and brain of both sexes in rats. Here, we report effects on male development., Methods: 4-MBC (0.7, 7, 24, 47 mg/kg/day) was administered in chow to the parent generation before mating, during gestation and lactation, and to offspring until adulthood. mRNA was determined in prostate lobes by real-time reverse transcription-polymerase chain reaction and protein was determined by Western blot analysis., Results: 4-MBC delayed male puberty, decreased adult prostate weight, and slightly increased testis weight. Androgen receptor (AR), insulin-like growth factor-1 (IGF-1), ER-alpha, and ER-beta expression in prostate were altered at mRNA and protein levels, with stronger effects in dorsolateral than ventral prostate. To assess sensitivity of target genes to estrogens, offspring were castrated on postnatal day 70, injected with 17beta-estradiol (E(2); 10 or 50 microg/kg, sc) or vehicle on postnatal day 84, and sacrificed 6 hr later. Acute repression of AR and IGF-1 mRNAs by E(2), studied in ventral prostate, was reduced by 4-MBC exposure. This was accompanied by reduced co-repressor N-CoR (nuclear receptor co-repressor) protein in ventral and dorsolateral prostate, whereas steroid receptor coactivator-1 (SRC-1) protein levels were unaffected., Conclusions: Our data indicate that 4-MBC affects development of male reproductive functions and organs, with a lowest observed adverse effect level of 0.7 mg/kg. Nuclear receptor coregulators were revealed as targets for endocrine disruptors, as shown for N-CoR in prostate and SRC-1 in uterus. This may have widespread effects on gene regulation.

Functionally important sites in the predicted first and fourth extracellular linkers of the type IIa Na+/Pi cotransporter (NaPi-IIa) were identified by cysteine scanning mutagenesis (Ehnes et al., 2004). Cysteine substitution or modification with impermeant and permeant methanethiosulfonate (MTS) reagents at certain sites resulted in changes to the steady-state voltage dependency of the cotransport mode (1 mM Pi, 100 mM Na+ at pH 7.4) of the mutants. At Gly-134 (ECL-1) and Met-533 (ECL-4), complementary behavior of the voltage dependency was documented with respect to the effect of cys-substitution and modification. G134C had a weak voltage dependency that became even stronger than that of the wild type (WT) after MTS incubation. M533C showed a WT-like voltage dependency that became markedly weaker after MTS incubation. To elucidate the underlying mechanism, the steady-state and presteady-state kinetics of these mutants were studied in detail. The apparent affinity constants for Pi and Na+ did not show large changes after MTS exposure. However, the dependency on external protons was changed in a complementary manner for each mutant. This suggested that cys substitution at Gly-134 or modification of Cys-533 had induced similar conformational changes to alter the proton modulation of transport kinetics. The changes in steady-state voltage dependency correlated with changes in the kinetics of presteady-state charge movements determined in the absence of Pi, which suggested that voltage-dependent transitions in the transport cycle were altered. The steady-state and presteady-state behavior was simulated using an eight-state kinetic model in which the transition rate constants of the empty carrier and translocation of the fully loaded carrier were found to be critical determinants of the transport kinetics. The simulations predict that cys substitution at Gly-134 or cys modification of Cys-533 alters the preferred orientation of the empty carrier from an inward to outward-facing conformation for hyperpolarizing voltages.

The putative first intracellular and third extracellular linkers are known to play important roles in defining the transport properties of the type IIa Na+-coupled phosphate cotransporter (Kohler, K., I.C. Forster, G. Stange, J. Biber, and H. Murer. 2002b. J. Gen. Physiol. 120:693-705). To investigate whether other stretches that link predicted transmembrane domains are also involved, the substituted cysteine accessibility method (SCAM) was applied to sites in the predicted first and fourth extracellular linkers (ECL-1 and ECL-4). Mutants based on the wild-type (WT) backbone, with substituted novel cysteines, were expressed in Xenopus oocytes, and their function was assayed by isotope uptake and electrophysiology. Functionally important sites were identified in both linkers by exposing cells to membrane permeant and impermeant methanethiosulfonate (MTS) reagents. The cysteine modification reaction rates for sites in ECL-1 were faster than those in ECL-4, which suggested that the latter were less accessible from the extracellular medium. Generally, a finite cotransport activity remained at the end of the modification reaction. The change in activity was due to altered voltage-dependent kinetics of the Pi-dependent current. For example, cys substitution at Gly-134 in ECL-1 resulted in rate-limiting, voltage-independent cotransport activity for V < or = -80 mV, whereas the WT exhibited a linear voltage dependency. After cys modification, this mutant displayed a supralinear voltage dependency in the same voltage range. The opposite behavior was documented for cys substitution at Met-533 in ECL-4. Modification of cysteines at two other sites in ECL-1 (Ile-136 and Phe-137) also resulted in supralinear voltage dependencies for hyperpolarizing potentials. Taken together, these findings suggest that ECL-1 and ECL-4 may not directly form part of the transport pathway, but specific sites in these linkers can interact directly or indirectly with parts of NaPi-IIa that undergo voltage-dependent conformational changes and thereby influence the voltage dependency of cotransport.

Competitive receptor binding assays have been suggested as an in vitro screening tool for assessing the activity of alleged estrogenic substances. In this study, we determined the ability of steroidal and nonsteroidal substances to inhibit the binding of [(3)H]17 beta-estradiol (E2) to the hepatic estrogen receptor (ER) and the plasma sex steroid binding protein (SBP) of the teleost fish, the common carp (Cyprinus carpio). The objectives of the study were (1) to characterize ER binding in the liver cytosol of male and female carp, (2) to establish complete [(3)H]E2 displacement curves from carp ER for a range of natural and xenobiotic substances and to compare the ligand data of carp ER with published data from other vertebrate species to reveal possible species differences, and (3) to determine the interaction of natural and xenobiotic substances with the steroid binding site of SBP in carp plasma. The results indicate the presence of a single class of estrogen binding sites with high affinity and limited capacity in liver cytosol of carp. The various test agents showed partly quantitative differences in their binding affinities, with the xenobiotics generally showing limited ability to displace [(3)H]E2 from the hepatic ER or from plasma SBP of carp. However, we found no evidence that a compound is an ER ligand exclusively in one species. The findings of this study indicate that interspecies extrapolation of steroid receptor binding data is possible on a yes/no basis but not on a quantitative basis., (Copyright 2000 Academic Press.)