21st Century Approaches for Evaluating Exposures, Biological Activity, and Risks of Complex Substances: Workshop highlights.

The June 2019 workshop 21st Century Approaches for Evaluating Exposures, Biological Activity, and Risks of Complex Substances, co-organised by the International Council of Chemical Association's Long-Range Research Initiative and the European Commission's Joint Research Centre, is summarised. Focus was the need for improved approaches to evaluate the safety of complex substances. Approximately 10% and 20% of substances registered under the EU chemicals legislation are 'multi-constituent substances' and 'substances of unknown or variable compositions, complex reaction products and biological substances' (UVCBs), respectively, and UVCBs comprise approximately 25% of the U.S. Toxic Substances Control Act Inventory. Workshop participants were asked to consider how the full promise of new approach methodologies (NAMs) could be brought to bear to evaluate complex substances. Sessions focused on using NAMs for screening, biological profiling, and in complex risk evaluations; improving read-across approaches employing new data streams; and methods to evaluate exposure and dosimetry. The workshop concluded with facilitated discussions to explore actionable steps forward. Given the diversity of complex substances, no single 'correct' approach was seen as workable. The path forward should focus on 'learning by doing' by developing and openly sharing NAM-based fit-for-purpose case examples for evaluating biological activity, exposures and risks of complex substances.

Different effects of an N-phenylimide herbicide on heme biosynthesis between human and rat erythroid cells.

Rat developmental toxicity including embryolethality and teratogenicity (mainly ventricular septal defects and wavy ribs) were produced by S-53482, an N-phenylimide herbicide that inhibits protoporphyrinogen oxidase (PPO) common to chlorophyll and heme biosynthesis. The sequence of key biological events in the mode of action has been elucidated as follows: inhibition of PPO interferes with normal heme synthesis, which causes loss of blood cells leading to fetal anemia, embryolethality and the development of malformations. In this study we investigated whether the rat is a relevant model for the assessment of the human hazard of the herbicide. To study effects on heme biosynthesis, human erythroleukemia, human cord blood, and rat erythroleukemia cells were treated with the herbicide during red cell differentiation. Protoporphyrin IX, a marker of PPO inhibition, and heme were determined. We investigated whether synchronous maturation of primitive erythropoiesis, which can contribute to massive losses of embryonic blood, occurs in rats. The population of primitive erythroblasts was observed on gestational days 11 through 14. Heme production was suppressed in rat erythroid cells. In contrast, heme reduction was not seen in both human erythroid cells when PPO was inhibited. Rats underwent synchronous maturation in primitive erythropoiesis. Our results combined with epidemiological findings that patients with deficient PPO are not anemic led us to conclude that human erythroblasts are resistant to the herbicide. It is suggested that the rat would be an inappropriate model for assessing the developmental toxicity of S-53482 in humans as rats are specifically sensitive to PPO inhibition by the herbicide.

Maternal exposure to anti-androgenic compounds, vinclozolin, flutamide and procymidone, has no effects on spermatogenesis and DNA methylation in male rats of subsequent generations.

To verify whether anti-androgens cause transgenerational effects on spermatogenesis and DNA methylation in rats, gravid Crl:CD(SD) female rats (4 or 5/group, gestational day (GD) 0=day sperm detected) were intraperitoneally treated with anti-androgenic compounds, such as vinclozolin (100 mg/kg/day), procymidone (100 mg/kg/day), or flutamide (10 mg/kg/day), from GD 8 to GD 15. Testes were collected from F1 male pups at postnatal day (PND) 6 for DNA methylation analysis of the region (210 bp including 7 CpG sites) within the lysophospholipase gene by bisulfite DNA sequencing method. F0 and F1 males underwent the sperm analysis (count, motility and morphology), followed by DNA methylation analysis of the sperm. Remaining F1 males were cohabited with untreated-females to obtain F2 male pups for subsequent DNA methylation analysis of the testes at PND 6. These analyses showed no effects on spermatogenesis and fertility in F1 males of any treatment group. DNA methylation status in testes (F1 and F2 pups at PND 6) or sperms (F1 males at 13 weeks old) of the treatment groups were comparable to the control at all observation points, although DNA methylation rates in testes were slightly lower than those in sperm. In F0 males, no abnormalities in the spermatogenesis, fertility and DNA methylation status of sperm were observed. No transgenerational abnormalities of spermatogenesis and DNA methylation status caused by anti-androgenic compounds were observed.

Toward the Development and Application of an Environmental Risk Assessment Framework for Microplastic.

Emissions of plastic waste to the environment and the subsequent degradation into microplastic particles that have the potential to interact with biological organisms represent a concern for global society. Current understanding of the potential impacts on aquatic and terrestrial population stability and ecosystem structure and function associated with emissions of microplastic particles is limited and insufficient to fully assess environmental risks. Multistakeholder discussions can provide an important element in helping to identify and prioritize key knowledge gaps in assessing potential risks. In the present review, we summarize multistakeholder discussions from a 1-d International Council of Chemical Associations-sponsored symposium, which involved 39 scientists from 8 countries with representatives from academia, industry, and government. Participants were asked to consider the following: discuss the scientific merits and limitations of applying a proposed conceptual environmental risk assessment (ERA) framework for microplastic particles and identify and prioritize major research needs in applying ERA tools for microplastic particles. Multistakeholder consensus was obtained with respect to the interpretation of the current state of the science related to effects and exposure to microplastic particles, which implies that it is unlikely that the presence of microplastic in the environment currently represents a risk. However, the quality and quantity of existing data require substantial improvement before conclusions regarding the potential risks and impacts of microplastic particles can be fully assessed. Research that directly addresses the development and application of methods that strengthen the quality of data should thus be given the highest priority. Activities aimed at supporting the development of and access to standardized reference material were identified as a key research need. Environ Toxicol Chem 2019;38:2087-2100. © 2019 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals, Inc. on behalf of SETAC.

Effect of simultaneous exposure to mixture of two skin sensitizers on skin sensitization response in guinea pigs and mice.

Skin sensitization resulting in allergic contact dermatitis is a common occupational health issue. In this study, the effect of mixing two skin sensitizers on the skin sensitization response was investigated. Skin sensitizers are generally classified into T helper type 1 (Th1) or T helper type 2 (Th2), depending on the induced cytokine profile. Dinitrochlorobenzene (DNCB) and oxazolone (Oxa) are Th1 skin sensitizers and phthalic anhydride (PA) and toluene diisocyanate (TDI) are Th2 skin sensitizers. We investigated the effect on skin sensitization response to mixtures of three pairs of these sensitizers: DNCB and Oxa, DNCB and PA, and PA and TDI, using guinea pig maximization test and mouse ear swelling test. In guinea pigs sensitized with the mixture of DNCB and Oxa or PA and TDI, there were changes of skin sensitization response to DNCB and Oxa, and that to PA. On the other hand, there was no mixture effect in guinea pigs sensitized with the mixture of DNCB and PA. The skin sensitization responses were decreased in mice sensitized with the mixtures of DNCB and Oxa or PA and TDI, whereas the mixture effect was not observed in mice sensitized with the mixture of DNCB and PA. The present findings revealed that mixture effect on the skin sensitization response was observed after simultaneous exposure to two skin sensitizers, and the effect was determined by combinations of mixed skin sensitizers.

Historical control data on developmental toxicity studies in rodents.

Historical control data on rodent developmental toxicity studies, performed between 1994 and 2010, were obtained from 19 laboratories in Japan, including 10 pharmaceutical and chemical companies and nine contract research organizations. Rats, mice, and hamsters were used for developmental toxicity studies. Data included maternal reproductive findings at terminal cesarean sections and fetal findings including the spontaneous incidences of external, visceral, and skeletal anomalies. No noticeable differences were observed in maternal reproductive data between laboratories. Inter-laboratory variations in the incidences of fetuses with anomalies appeared to be due to differences in the selection of observation parameters, observation criteria, classification of the findings, and terminology of fetal alterations. Historical control data are useful for the appropriate interpretation of experimental results and evaluation of the effects of chemical on reproductive and developmental toxicities.

Maternal exposure to procymidone has no effects on fetal external genitalia development in male rabbit fetuses in a modified developmental toxicity study.

This study was conducted to evaluate the effects of procymidone (PCM) on development of male rabbit fetal external genitalia. PCM was administered once daily by gavage at dose levels of 0 (control) and 125mg/kg/day to pregnant rabbits from gestation day 6 through 28 and fetal external genitalia was observed in detail. This treatment period covered the critical stage of sexual differentiation of fetal external genitalia in rabbits. In the maternal animals, food consumption was reduced in the PCM group. There were no effects of PCM on maternal caesarean sectioning data or fetal external observations. In fetal external genitalia observations, there were no significant differences between the control and PCM treatment group in any of the following parameters: ano-genital distance (AGD), phallus boundary-genital distance, diameter of preputial lamella, ventral gap of preputial lamella, or ventral gap to diameter ration of preputial lamella, though severe feminization such as decreasing of AGD and hypospadias in male rat offspring at the dose level of 125 mg/kg of PCM were reported. These results suggest that PCM has no effect on fetal external genitalia development in male rabbit fetuses, and species difference of developmental effects of PCM on sexual differentiation exists.

New method for detecting antiandrogenic effects through the measurement of external genitalia in rabbits.

The aim of the present study was to develop a quantitative evaluation method for detecting antiandrogenic activity of chemicals in rabbits that are regularly used for developmental toxicity studies. Kbl: New Zealand White rabbits (n = 8-9) were injected intramuscularly with an antiandrogen, cyproterone acetate (CA; 10 mg/kg body weight [BW]/day), on gestation days (GD) 13-24. On GD 29, live fetuses were obtained by cesarean section and sexed by examination of the internal genitalia. The external genitalia were evaluated in cross-section measurements of the phallus by both diameter and width of the ventral gap of the preputial lamella with a micrometer under a stereoscopic microscope. The diameters of the preputial lamella were 1015 +/- 83.5, 856 +/- 64.0, and 865 +/- 72.6 microm in control males, control females, and CA-treated males, respectively. The ventral gaps of the preputial lamella were 26 +/- 8.2, 437 +/- 72.3, and 318 +/- 59.4 microm in the control males, control females, and CA-treated males, respectively. There were statistically significant differences in both parameters between control males and control females or CA-treated males. The lower fetal BW in CA-treated males did not disturb the detection of the feminization of the ventral gap of the preputial lamella; however, the diameter of the preputial lamella might be influenced by fetal BW because no difference in the relative diameter of the preputial lamella was found between control males and CA-treated males. These results demonstrated that this approach could detect the antiandrogen activity of CA quantitatively by feminization of male external genitalia in rabbit fetuses.