Paving the way for application of next generation risk assessment to safety decision-making for cosmetic ingredients.

Next generation risk assessment (NGRA) is an exposure-led, hypothesis-driven approach that has the potential to support animal-free safety decision-making. However, significant effort is needed to develop and test the in vitro and in silico (computational) approaches that underpin NGRA to enable confident application in a regulatory context. A workshop was held in Montreal in 2019 to discuss where effort needs to be focussed and to agree on the steps needed to ensure safety decisions made on cosmetic ingredients are robust and protective. Workshop participants explored whether NGRA for cosmetic ingredients can be protective of human health, and reviewed examples of NGRA for cosmetic ingredients. From the limited examples available, it is clear that NGRA is still in its infancy, and further case studies are needed to determine whether safety decisions are sufficiently protective and not overly conservative. Seven areas were identified to help progress application of NGRA, including further investments in case studies that elaborate on scenarios frequently encountered by industry and regulators, including those where a 'high risk' conclusion would be expected. These will provide confidence that the tools and approaches can reliably discern differing levels of risk. Furthermore, frameworks to guide performance and reporting should be developed.

Riparian Forest Cover Modulates Phosphorus Storage and Nitrogen Cycling in Agricultural Stream Sediments.

Watershed land cover affects in-stream water quality and sediment nutrient dynamics. The presence of natural land cover in the riparian zone can reduce the negative effects of agricultural land use on water quality; however, literature evaluating the effects of natural riparian land cover on stream sediment nutrient dynamics is scarce. The objective of this study was to assess if stream sediment phosphorus retention and nitrogen removal varies with riparian forest cover in agricultural watersheds. Stream sediment nutrient dynamics from 28 sites with mixed land cover were sampled three times during the growing season. Phosphorus dynamics and nitrification rates did not change considerably throughout the study period. Sediment total phosphorus concentrations and nitrification rates decreased as riparian forest cover increased likely due to a decline in fine, organic material. Denitrification rates were strongly correlated to surface water nitrate concentrations. Denitrification rate and denitrification enzyme activity decreased with an increase in forest cover during the first sampling period only. The first sampling period coincided with the greatest connectivity between the watershed and in-stream processing, indicating that riparian forest cover indirectly decreased denitrification rates by reducing the concentrations of dissolved nutrients entering the stream. This reduction in load may allow the sediment to maintain greater nitrogen removal efficiency, because bacteria are not saturated with nitrogen. Riparian forest cover also appeared to lessen the effect of agriculture in the watershed by decreasing the amount of fine material in the stream, resulting in reduced phosphorus storage in the stream sediment.

Beyond the Edge: Linking Agricultural Landscapes, Stream Networks, and Best Management Practices.

Despite much research and investment into understanding and managing nutrients across agricultural landscapes, nutrient runoff to freshwater ecosystems is still a major concern. We argue there is currently a disconnect between the management of watershed surfaces (agricultural landscape) and river networks (riverine landscape). These landscapes are commonly managed separately, but there is limited cohesiveness between agricultural landscape-focused research and river science, despite similar end goals. Interdisciplinary research into stream networks that drain agricultural landscapes is expanding but is fraught with problems. Conceptual frameworks are useful tools to order phenomena, reveal patterns and processes, and in interdisciplinary river science, enable the joining of multiple areas of understanding into a single conceptual-empirical structure. We present a framework for the interdisciplinary study and management of agricultural and riverine landscapes. The framework includes components of an ecosystems approach to the study of catchment-stream networks, resilience thinking, and strategic adaptive management. Application of the framework is illustrated through a study of the Fox Basin in Wisconsin, USA. To fully realize the goal of nutrient reduction in the basin, we suggest that greater emphasis is needed on where best management practices (BMPs) are used within the spatial context of the combined watershed-stream network system, including BMPs within the river channel. Targeted placement of BMPs throughout the riverine landscape would increase the overall buffering capacity of the system to nutrient runoff and thus its resilience to current and future disturbances.

Comparison of the skin sensitizing potential of unsaturated compounds as assessed by the murine local lymph node assay (LLNA) and the guinea pig maximization test (GPMT).

The skin sensitization potential of eight unsaturated and one saturated lipid (bio)chemicals was tested in both the LLNA and the GPMT to address the hypothesis that chemicals with unsaturated carbon-carbon double bonds may result in a higher number of unspecific (false positive) results in the LLNA compared to the GPMT. Seven substances (oleic acid, linoleic acid, linolenic acid, undecylenic acid, maleic acid, squalene and octinol) gave clear positive results in the LLNA (stimulation index (SI)> or = 3) and thus would require labelling as skin sensitizer. Fumaric acid and succinic acid gave clearly negative results. In the GPMT, besides some sporadic skin reactions, reproducible skin reactions indicating an allergic response were found in a few animals for four test substances. Based on the GPMT results, only undecylenic acid would have to be classified and labelled as a skin sensitizer according to the European Dangerous Substance Directive (67/548/EEC) (results for linoleic acid were inconclusive), while the other seven test substances would not require labelling. Possible mechanisms for unspecific skin cell stimulation and lymph node responses are discussed. In conclusion, the suitability of the LLNA for unsaturated compounds bearing structural similarity to the tested substances should be carefully considered and the GPMT should remain available as an accepted test method for skin sensitization hazard identification.

Relevance of the rat lung tumor response to particle overload for human risk assessment-Update and interpretation of new data since ILSI 2000.

The relevance of particle-overload related lung tumors in rats for human risk assessment following chronic inhalation exposures to poorly soluble particulates (PSP) has been a controversial issue for more than three decades. In 1998, an ILSI (International Life Sciences) Working Group of health scientists was convened to address this issue of applicability of experimental study findings of lung neoplasms in rats for lifetime-exposed production workers to PSPs. A full consensus view was not reached by the Workshop participants, although it was generally acknowledged that the findings of lung tumors in rats following chronic inhalation, particle-overload PSP exposures occurred only in rats and no other tested species; and that there was an absence of lung cancers in PSP-exposed production workers. Since the publication of the ILSI Workshop report in 2000, there have been important new data published on the human relevance issue. A thorough and comprehensive review of the health effects literature on poorly soluble particles/lung overload was undertaken and published by an ECETOC (European Centre for Ecotoxicology and Toxicology of Chemicals) Task Force in 2013. One of the significant conclusions derived from that technical report was that the rat is unique amongst all species in developing lung tumors under chronic inhalation overload exposures to PSPs. Accordingly, the objective of this review is to provide important insights on the fundamental differences in pulmonary responses between experimentally-exposed rats, other experimental species and occupationally-exposed humans. Briefly, five central factors are described by the following issues. Focusing on these five interrelated/convergent factors clearly demonstrate an inappropriateness in concluding that the findings of lung tumors in rats exposed chronically to high concentrations of PSPs are accurate representations of the risks of lung cancer in PSP-exposed production workers. The most plausible conclusion that can be reached is that results from chronic particle-overload inhalation studies with PSPs in rats have no relevance for determining lung cancer risks in production workers exposed for a working lifetime to these poorly soluble particulate-types.

Inhalation pharmacokinetics of 1,3-butadiene and 1,2-epoxybutene-3 in rats and mice.

Studies were conducted on inhalation pharmacokinetics of 1,3-butadiene and of its primary reactive metabolic intermediate 1,2-epoxybutene-3 in rats (Sprague-Dawley) and mice (B6C3F1). Investigations of inhalation pharmacokinetics of 1,3-butadiene revealed saturation kinetics of 1,3-butadiene metabolism in both species. For rats and mice linear pharmacokinetics apply at exposure concentrations below 1000 ppm 1,3-butadiene; saturation of 1,3-butadiene metabolism is observed at atmospheric concentrations of about 2000 ppm. The estimated maximal metabolic elimination rates were 400 mumole/hr/kg for mice and 200 mumole/hr/kg for rats. This shows that 1,3-butadiene is metabolized by mice at about twice the rate of rats. Investigations of inhalation pharmacokinetics of 1,2-epoxybutene-3 revealed major differences in metabolism of this compound between both species. No indication of saturation kinetics of 1,2-epoxybutene-3 metabolism could be observed in rats up to exposure concentrations of 5000 ppm, whereas in mice the saturation of epoxybutene metabolism became apparent at atmospheric concentrations of about 500 ppm. The estimated maximal metabolic rate for 1,2-epoxybutene-3 was 350 mumole/hr/kg in mice and greater than 2600 mumole/hr/kg in rats. When the animals are exposed to high concentrations of 1,3-butadiene, 1,2-epoxybutene-3 is exhaled by rats and mice. For rats 1,2-epoxybutene-3 concentration in the gas phase of the system reaches a plateau at about 4 ppm. For mice, 1,2-epoxybutene-3 concentration increases with exposure time until, at about 10 ppm, signs of acute toxicity are observed. Under these conditions hepatic nonprotein sulfhydryl compounds are virtually depleted in mice but not in rats.(ABSTRACT TRUNCATED AT 250 WORDS)

Species differences in butadiene metabolism between mouse and rat.

Studies on inhalation pharmacokinetics of 1,3-butadiene were conducted in mice (B6C3F1) and rats (Sprague-Dawley) to investigate the considerable differences in the susceptibility of both species to butadiene-induced carcinogenesis. In rats and mice metabolism of 1,3-butadiene to 1,2-epoxybutene-3 follows saturation kinetics. "Linear" (first-order) pharmacokinetics apply at exposure concentrations below 1000 ppm 1,3-butadiene. Saturation of butadiene metabolism is observed at atmospheric concentrations of about 2000 ppm butadiene. In the lower concentration range where first-order metabolism applies, metabolic clearance of inhaled 1,3-butadiene per kg body weight was 7300 ml (gas volume) x hr-1 for mice and 4500 ml x hr-1 for rats. The calculated maximal metabolic elimination rates (Vmax - conditions) were 400 mumol x hr-1 x kg-1 for mice and 220 mumol x hr-1 x kg-1 for rats. This shows that 1,3-butadiene is metabolized by mice at about twice the rate of rats, under conditions of both low and high exposure concentrations.

Depletion of hepatic non-protein sulfhydryl content during exposure of rats and mice to butadiene.

B6C3F1 mice, Sprague-Dawley and Wistar rats were exposed to 1,3-butadiene in a closed exposure system. Exposure concentrations were kept above 2000 ppm to ensure saturation of butadiene metabolism in both species (Vmax conditions). Hepatic non-protein sulfhydryl (NPSH) content was determined in butadiene-exposed animals (and air-exposed controls) after exposures for 0, 7 and 15 h. Depletion of hepatic NPSH content was different for the species and strains investigated. In mice, hepatic NPSH content declined to about 20% after 7 h and was further depleted to about 4% at 15 h when signs of acute toxicity were observed. After a 7 h exposure of rats to butadiene, hepatic NPSH content was depleted to about 65% (Wistar) or 80% (Sprague-Dawley) of the corresponding controls but remained practically stable after a 15 h exposure to butadiene. The time-courses of depletion by butadiene of hepatic NPSH support previous findings on differences in butadiene metabolism between rats and mice and offer an additional explanation for the considerable species differences observed in the toxicity and carcinogenicity of this compound.

Alkylation of nuclear proteins and DNA after exposure of rats and mice to [1,4-14C]1,3-butadiene.

B6C3F1 mice and Wistar rats were exposed to [1,4-14C]1,3-butadiene in a closed exposure system. Based on body weight, mice metabolized the test compound at about twice the rate, compared to rats. Nucleoproteins and DNA were isolated from the livers of the animals and covalent binding of [14C]-butadiene-derived radioactivity was determined. In both species comparable amounts of radioactivity were covalently bound to liver DNA. Covalent binding to mouse-liver nucleoproteins was twice as high as in rats and thus it paralleled the higher metabolic rate for butadiene in this species.

Validation study of alternatives to the Draize eye irritation test in Germany: Cytotoxicity testing and HET-CAM test with 136 industrial chemicals.

According to OECD guideline 405 revised in 1987 Draize eye tests need not be performed for severely irritating and corrosive chemicals if results from 'well-validated alternative studies' are presented. In 1988 a validation study on alternatives to the Draize eye test was started in Germany to establish 'well-validated alternative methods' for this purpose. During database development, the last stage of the validation programme, 136 chemicals from the German chemical industry were classified in a blind trial with the 3T3 cell neutral red/kenacid blue cytotoxicity assay and the hen's egg chorioallantoic membrane (HET-CAM) test using fertile chicken eggs. The major goal of this stage of validation was to demonstrate the feasibility and limitations of the two alternative methods. Chemicals were, therefore, selected as representatives of chemical structural groups as well as of physicochemical and toxicological properties. In addition, some of the chemicals were chosen because they were of interest to the cosmetic and detergent industries. Draize eye testing data in vivo were provided by industry. In contrast to data from a previous interlaboratory assessment trial, it was impossible to correlate cytotoxicity data to the EEC classification for in vivo eye irritation. However, seven of 10 severely irritating chemicals (EEC labelling R-41) could be identified correctly in the HET-CAM assay, whereas test conditions of the study described here did not allow identification of irritating chemicals (EEC labelling R-36). The HET-CAM test is, therefore, fulfilling the criteria of a 'well-validated alternative method' according to OECD guideline 405 and should be incorporated into eye irritation testing at the earliest possible stage to reduce effectively the suffering of rabbits in the Draize eye test. Although an 80% correct prediction of 'non-labelled' chemicals in the HET-CAM test is encouraging, for safety assessment of non-irritant chemicals, for use as cosmetic formulations, for example, both government and industry will accept an in vitro assay only if its prediction of the absence of irritant properties is 100% correct.

Eye irritation: Reference chemicals data bank.

A list of 55 chemicals has been developed for which comprehensive in vivo rabbit eye irritation data are available. No new in vivo testing has been carried out to qualify a chemical for inclusion in the list. The 55 chemicals selected are available at high and consistent purity and are expected to be stable on storage. They have been tested undiluted in in vivo studies, except those chemicals where high concentrations of the substance could be expected to cause severe effects. The in vivo data have been generated since 1981 in studies carried out according to OECD Test Guideline 405 and following the principles of Good Laboratory Practice. The data were obtained from tests normally using at least three rabbits evaluated at the same time, involving instillation of 0.1 ml (or equivalent weight) into the conjunctival sac, and in which observations were made at least at 24, 48 and 72 hr after instillation. The chemicals represent a range of chemical classes (acetates, acids, alcohols, alkalis, aromatics, hydrocarbons, inorganics and surfactants) and different degrees of irritancy. They are ranked for eye irritation potential on the basis of a 'modified maximum average score'. The reference data bank should be of use in validation tests for promising alternatives to the in vivo rabbit eye irritation test. This is an essential step in the progression to regulatory acceptance of alternative procedures.

Interlaboratory assessment of alternatives to the Draize eye irritation test in Germany.

A national interlaboratory study to validate two alternative methods to the Draize rabbit's eye test, co-ordinated by ZEBET at the German Federal Health Office (BGA), is described. The aim of the study is to classify chemicals according to their irritation potential using the neutral red/kenacid blue (NR/KB) cytotoxicity assay and the hen's egg chorioallantoic membrane (HET-CAM) test. During the last two years 12 toxicology laboratories from industry, universities and other research institutions have tested 32 substances from a variety of chemical classes, characterized by a broad spectrum of locally irritating properties, using the NR/KB cytotoxicity test and the HET-CAM assay. Intra- and interlaboratory reproducibility of the two methods was investigated under standardized conditions. The so-far limited evaluation of the interlaboratory assessment phase of validation indicates that the results of the Draize rabbit's eye test correlate better with the results of the HET-CAM test than with those of the cytotoxicity test as far as false negative results are concerned. However, the intra- and interlaboratory reproducibility of the cytoxicity test is better than that of the HET-CAM test. The validation project has recently entered the stage of database development during which 150 chemicals will be tested in seven laboratories to provide information on whether and to what extent the NR/KB test and the HET-CAM test can replace the Draize rabbit's eye test for the classification and labelling of chemicals with regard to their eye irritation potential.

Effect of proteins on availability of zinc. II. Bioavailability of zinc from casein and whey protein--retention study in young rats.

The availability of zinc from two semi-synthetic diets with isolated whey protein (Wp D) or with isolated casein (Cas D) as protein component (20% W/W) was compared in a 21-day study with growing male rats (initial weight 40 g; 14 animals/group). Zinc concentration in both diets (18 ppm) was adequate to meet the requirements of the animals fed ad libitum. For radiolabeling approximately 13 micrograms 65Zn (= 4 microCi) was given daily by intragastric intubation to each animal. The investigation was designed primarily as a retention study, but also general parameters like weight development, food and water intake, organ weights etc. were registered and the activity of alkaline phosphatase was determined in serum and femur tissue. A significantly higher percentage of 65Zn was retained in the whole body from the Wp D (36.5%) than from the Cas D (31.6%) during the experimental period. The same is valid for the percentage retention of 65Zn in the femur and for the 65Zn concentration in femur and hair as well as for the total zinc concentration (65Zn and non-labeled zinc) of the femur. The other parameters determined were not unequivocally influenced by the protein component of the diet. The study clearly demonstrated that the availability of zinc by the growing rat was better from a diet with whey protein than from one with casein as the protein component. The reason on this phenomenon has to be elucidated by further investigations.

Species differences in butadiene metabolism between mice and rats evaluated by inhalation pharmacokinetics.

Metabolism of 1,3-butadiene to 1,2-epoxybutene-3 in rats follows saturation kinetics. Comparative investigation of inhalation pharmacokinetics in mice also revealed a saturation pattern. For both species "linear" pharmacokinetics apply at exposure concentrations below 1000 ppm 1,3-butadiene; saturation of butadiene metabolism is observed at atmospheric concentrations of about 2000 ppm. For mice metabolic clearance per kg body weight in the lower concentration range where first order metabolism applies was 7300 ml X h-1 (rat: 4500 ml X h-1. Maximal metabolic elimination rate (Vmax) was 400 mumol X h-1 X kg-1 (rat: 220 mumol X h-1 X kg-1. This shows that 1,3-butadiene is metabolized by mice at higher rates compared to rats. Based on these investigations, the metabolic elimination rates of butadiene in both species were calculated for the exposure concentrations applied in two inhalation bioassays with rats and with mice. The results show that the higher rate of butadiene metabolism in mice when compared to rats may only in part be responsible for the considerable difference in the susceptibility of both species to butadiene-induced carcinogenesis.

Inhalation pharmacokinetics of 1,2-epoxybutene-3 reveal species differences between rats and mice sensitive to butadiene-induced carcinogenesis.

Comparative investigations of inhalation pharmacokinetics of 1,2-epoxybutene-3 (vinyl oxirane, the primary reactive intermediate of butadiene) revealed major differences in metabolism of this compound between rats and mice. Whereas in rats no indication of saturation kinetics of epoxybutene metabolism could be observed up to exposure concentrations of 5000 ppm, in mice saturation of epoxybutene metabolism becomes apparent at atmospheric concentrations of about 500 ppm. The estimated maximal metabolic rate (Vmax) in mice for epoxybutene was only 350 mumol X h-1 X kg-1 (rats: greater than 2600 mumol X h-1 X kg-1). In the lower concentration range where first order metabolism applies (up to about 500 ppm) epoxybutene is metabolized by mice at higher rates compared to rats (metabolic clearance per kg body weight, mice: 24,900 ml X h-1, rats: 13,400 ml X h-1). Under these conditions the steady state concentration of epoxybutene in the mouse is about 10 times that in the rat. When mice are exposed to high concentrations of butadiene (greater than 2000 ppm; conditions of saturation of butadiene metabolism; closed exposure system) epoxybutene is exhaled by the animals, and its concentration in the gas phase increases with exposure time. At about 10 ppm epoxybutene signs of acute toxicity are observed. When rats are exposed to butadiene under similar conditions, the epoxybutene concentration reaches a plateau at about 4 ppm. Under these conditions hepatic non-protein sulfhydryl compounds are virtually depleted in mice but not in rats.(ABSTRACT TRUNCATED AT 250 WORDS)

Efficacy of ozone in eradication of Legionella pneumophila from hospital plumbing fixtures.

The effect of ozonation of supply water for one wing of an unoccupied hospital building which had positive cultures for Legionella pneumophila from multiple potable water fixtures was studied in a prospective, controlled fashion. Mean ozone residual concentrations of 0.79 mg/liter eradicated L. pneumophila from the fixtures, but so did nonozonated water in the control wing fixtures. The efficacy of the nonozonated water was most probably due to a mechanical flushing effect and to an unexpected rise in the chlorine content of the supply water. Determination of the in vitro activity of ozone against L. pneumophila did not predict the efficacy of its eradication from water fixtures treated with ozone.