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Derivation of Predicted No Effect Concentrations (PNECs) for aquatic systems is the primary deterministic form of hazard extrapolation used in environmental risk assessment. Depending on the data availability, different regulatory jurisdictions apply application factors (AFs) to the most sensitive measured endpoint to derive the PNEC for a chemical. To assess differences in estimated PNEC values, two PNEC determination methodologies were applied to a curated public database using the EnviroTox Platform (www.EnviroToxdatabase.org). PNECs were derived for 3647 compounds using derivation procedures based on example US EPA and a modified European Union chemical registration procedure to allow for comparisons. Ranked probability distributions of PNEC values were developed and 5th percentile values were calculated for the entire dataset and scenarios where full acute or full chronic data sets were available. The lowest PNEC values indicated categorization based on chemical attributes and modes of action would lead to improved extrapolations. Full acute or chronic datasets gave measurably higher 5th percentile PNEC values. Algae were under-represented in available ecotoxicity data but drove PNECs disproportionately. Including algal inhibition studies will be important in understanding chemical hazards. The PNEC derivation logic flows are embedded in the EnviroTox Platform providing transparent and consistent PNEC derivations and PNEC distribution calculations., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

Unique aspects of childhood exposure to products need childs specific exposure data. This study developed a probabilistic exposure model for lotion transfer to diapered skin through normal use of baby wipes in children up to 48 months of age. Monte Carlo simulations used baby wipe diary data from the US, Germany and the UK, body weight data from the US, and lotion transfer data from single and multiple wipes adjusting for separate diaper changes. The models predicted a declining number of wipes used/day with a reduction in lotion transfer as age and body weight increased. Experimental testing on multiple sequential wipes used on an overlapping area showed a reduction in lotion deposition by 23.9% after the first wipe. Overall, the weighted population average over the approximate diapering period of 0-36 months across the three geographies at 50th, 90th, & 95th percentiles, were between 130, 230, 260 mg/kg/day, respectively, and 150, 270, 310 mg/kg/day depending on whether a reduction due to overlap is implemented. The statistical model represents an effective strategy to determine exposure to baby wipes lotion for exposure based risk assessment., (Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.)

High-content data have the potential to inform mechanism of action for toxicants. However, most data to support this notion have been generated in vivo. Because many cell lines and primary cells maintain a differentiated cell phenotype, it is possible that cells grown in culture may also be useful in predictive toxicology via high-content approaches such as whole-genome microarray. We evaluated global changes in gene expression in primary rat hepatocytes exposed to two concentrations of ten hepatotoxicants: acetaminophen (APAP), β-naphthoflavone (BNF), chlorpromazine (CPZ), clofibrate (CLO), bis(2-ethylhexyl)phthalate (DEHP), diisononyl phthalate (DINP), methapyrilene (MP), valproic acid (VPA), phenobarbital (PB) and WY14643 at two separate time points. These compounds were selected to cover a range of mechanisms of toxicity, with some overlap in expected mechanism to address the question of how predictive gene expression analysis is, for a given mode of action. Gene expression microarray analysis was performed on cells after 24h and 48h of exposure to each chemical using Affymetrix microarrays. Cluster analysis suggests that the primary hepatocyte model was capable of responding to these hepatotoxicants, with changes in gene expression that appear to be mode of action-specific. Among the different methods used for analysis of the data, a combination method that used pathways (MOAs) to filter total probesets provided the most robust analysis. The analysis resulted in the phthalates clustering closely together, with the two other peroxisome proliferators, CLO and WY14643, eliciting similar responses at the whole-genome and pathway levels. The Cyp inducers PB, MP, CPZ and BNF also clustered together. VPA and APAP had profiles that were unique. A similar analysis was performed on externally available (TG-GATES) in vivo data for 6 of the chemicals (APAP, CLO, CPZ, MP, MP and WY14643) and compared to the in vitro result. These results indicate that transcription profiling using an in vitro assay may offer pertinent biological data to support predictions of in vivo hepatotoxicity potential., (Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.)

The OECD validation study of the zebrafish embryo acute toxicity test (ZFET) for acute aquatic toxicity testing evaluated the ZFET reproducibility by testing 20 chemicals at 5 different concentrations in 3 independent runs in at least 3 laboratories. Stock solutions and test concentrations were analytically confirmed for 11 chemicals. Newly fertilised zebrafish eggs (20/concentration and control) were exposed for 96h to chemicals. Four apical endpoints were recorded daily as indicators of acute lethality: coagulation of the embryo, lack of somite formation, non-detachment of the tail bud from the yolk sac and lack of heartbeat. Results (LC50 values for 48/96h exposure) show that the ZFET is a robust method with a good intra- and inter-laboratory reproducibility (CV<30%) for most chemicals and laboratories. The reproducibility was lower (CV>30%) for some very toxic or volatile chemicals, and chemicals tested close to their limit of solubility. The ZFET is now available as OECD Test Guideline 236. Considering the high predictive capacity of the ZFET demonstrated by Belanger et al. (2013) in their retrospective analysis of acute fish toxicity and fish embryo acute toxicity data, the ZFET is ready to be considered for acute fish toxicity for regulatory purposes., (Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.)

In the absence of toxicological data on a chemical, the threshold of toxicological concern (TTC) approach provides a system to estimate a conservative exposure below which there is a low probability of risk for adverse health effects. The original toxicology dataset underlying the TTC was based on NOELs from repeat dose studies. Subsequently there have been several efforts to assess whether or not these limits are also protective for reproductive/developmental effects. This work expands the database of chemicals with reproductive and developmental data, presents these data in a comprehensive and transparent format and groups the chemicals according to the TTC "Cramer Class" rules. Distributions of NOAELs from each of these classes were used to assess whether the previously proposed TTC values based on repeat dose data are protective for reproductive/developmental toxicity endpoints as well. The present analysis indicates that, for each Cramer Class, the reproductive and developmental endpoints would be protected at the corresponding general TTC tiers derived by Munro et al. (1996)., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Microbial enzymes have been used in laundry detergent products for several decades. These enzymes have also long been known to have the potential to give rise to occupational type 1 allergic responses. A few cases of allergy among consumers using dusty enzyme detergents were reported in the early 1970s. Encapsulation of the enzymes along with other formula changes were made to ensure that consumer exposure levels were sufficiently low that the likelihood of either the induction of IgE antibody (sensitization) or the elicitation of clinical symptoms be highly improbable. Understanding the consumer exposure to enzymes which are used in laundry and cleaning products is a key step to the risk management process. Validation of the risk assessment conclusions and the risk management process only comes with practical experience and evidence from the marketplace. In the present work, clinical data from a range of sources collected over the past 40 years have been analysed. These include data from peer reviewed literature and enzyme specific IgE antibody test results in detergent manufacturers' employees and from clinical study subjects. In total, enzyme specific IgE antibody data were available on 15,765 individuals. There were 37 individuals with IgE antibody. The majority of these cases were from the 1970s where 23 of 4687 subjects (0.49%) were IgE positive and 15 of the 23 were reported to have symptoms of allergy. The remaining 14 cases were identified post-1977 for a prevalence of 0.126% (14/11,078). No symptoms were reported and no relationship to exposure to laundry and cleaning products was found. There was a significant difference between the pre- and post-1977 cohorts in that the higher rates of sensitization with symptoms were associated with higher exposure to enzyme. The clinical testing revealed that the prevalence of enzyme specific IgE in the population is very rare (0.126% since 1977). This demonstrates that exposure to these strong respiratory allergens via use of laundry and cleaning products does not lead to the development of sensitization and disease. These data confirm that the risk to consumers has been properly assessed and managed and support the concept that thresholds of exposure exist for respiratory allergy. Expansion of enzyme use into new consumer product categories should follow completion of robust risk assessments in order to continue ensuring the safe use of enzymes among consumers.

A reliable in vitro model to determine the potential estrogenic activity of chemicals of interest is still unavailable. To further investigate the usefulness of a human-derived cell line, we determined the transcriptional changes induced by bisphenol A (BPA) in Ishikawa cells at various doses (1 nM, 100 nM, 10 microM, and 100 microM) and time points (8, 24 and 48 h) by comparing the response of approximately 38,500 human genes and ESTs between treatment groups and controls (vehicle-treated). By trend analysis, we determined that the expression of 2794 genes was modified by BPA in a dose- and time-dependent manner (p< or =0.0001). However, the majority of gene expression changes induced in Ishikawa cells were elicited by the highest doses of BPA evaluated (10-100 microM), while the genomic response of the cells exposed to low doses of BPA was essentially negligible. By comparing the Ishikawa cells' response to BPA vs.17 alpha-ethynyl estradiol we determined that the change in the expression of 307 genes was identical in the direction of the change, although the magnitude of the change for some genes was different. Further, the response of Ishikawa cells to high doses of BPA shared similarities to the estrogenic response of the rat uterus, specifically, 362 genes were regulated in a similar manner in vivo as well as in vitro. Gene ontology analysis indicated that BPA results in changes to multiple molecular pathways affecting various biological processes particularly associated with cell organization and biogenesis, regulation of translation, cell proliferation, and intracellular transport; processes also affected by estrogen exposure in the uterus of the rat. These results indicate that Ishikawa cells are capable of generating a biologically relevant estrogenic response after exposure to chemicals with varied estrogenic activity, and offer an in vitro model to assess this mode of action., (Copyright 2010 Elsevier Ireland Ltd. All rights reserved.)

During the safety evaluation process of new drugs and chemicals, a battery of genotoxicity tests is conducted starting with in vitro genotoxicity assays. Obtaining positive results in in vitro genotoxicity tests is not uncommon. Follow-up studies to determine the biological relevance of positive genotoxicity results are costly, time consuming, and utilize animals. More efficient methods, especially for identifying a putative mode of action like an indirect mechanism of genotoxicity (where DNA molecules are not the initial primary targets), would greatly improve the risk assessment for genotoxins. To this end, we are participating in an International Life Sciences Institute (ILSI) project involving studies of gene expression changes caused by model genotoxins. The purpose of the work is to evaluate gene expression tools in general, and specifically for discriminating genotoxins that are direct-acting from indirect-acting. Our lab has evaluated gene expression changes as well as micronuclei (MN) in L5178Y TK(+/-) mouse lymphoma cells treated with six compounds. Direct-acting genotoxins (where DNA is the initial primary target) that were evaluated included the DNA crosslinking agents, mitomycin C (MMC) and cisplatin (CIS), and an alkylating agent, methyl methanesulfonate (MMS). Indirect-acting genotoxins included hydroxyurea (HU), a ribonucleotide reductase inhibitor, taxol (TXL), a microtubule inhibitor, and etoposide (ETOP), a DNA topoisomerase II inhibitor. Microarray gene expression analysis was conducted using Affymetrix mouse oligonucleotide arrays on RNA samples derived from cells which were harvested immediately after the 4 h chemical treatment, and 20 h after the 4 h chemical treatment. The evaluation of these experimental results yields evidence of differentially regulated genes at both 4 and 24 h time points that appear to have discriminating power for direct versus indirect genotoxins, and therefore may serve as a fingerprint for classifying chemicals when their mechanism of action is unknown.

The spontaneous mutant frequency in germ cells isolated from seminiferous tubules of two lambda/lacI transgenic mouse strains, C57BL/6 and B6C3F1 was evaluated. At least 500 000 phage were screened for mutation at lacI for each animal using standardized assay procedures. The germ cell spontaneous lacI mutant frequency was 17.8 +/- 8.1 x 10(-6) in C57BL/6 mice and 17.0 +/- 10.0 x 10(-6) in B6C3F1 mice. The induction of germ cell mutations by three well characterized alkylating agents were also evaluated in C57BL/6 mice on day 3 after a single dose administration. The lacI mutant frequencies were significantly elevated in transgenic mice dosed with ENU at 150 mg/kg (2-fold increase above control) and iPMS at 200 mg/kg (3-fold increase above control) but not in those receiving MMS at 40 mg/kg. These findings suggest that single dose studies using the lambda/lacI transgenic system may be capable of detecting germ mutations induced by chemicals characterized either by point mutations or small, intragenic deletions but not those characterized by a predominance of multi-locus deletions.

Male C57B1/6 lacI transgenic mice were used to evaluate germ cell mutagenesis in vivo as part of a collaborative study. Groups of 10 mice were administered single intraperitoneal doses of ethylnitrosourea (ENU; 150 mg/kg), isopropyl methanesulfonate (IPMS; 200 mg/kg), methyl methanesulfonate (MMS; 40 mg/kg) or vehicle. Epididymal spermatozoa and testes were recovered 3 days later and DNA isolated subsequently from epididymal spermatozoa and seminiferous tubules were analyzed for lacI mutations. The mutant frequency in seminiferous tubules (average +/- SEM) increased significantly compared with untreated controls (7.2 +/- 0.7 x 10(-5) following treatment with ENU (11.7 +/- 0.8 x 10(-5), p = 0.003) or with IPMS (9.6 +/- 0.5 x 10(-5), p = 0.018) but not following treatment with MMS (8.1 +/- 0.8 x 10(-5), p = 0.213). Group mutant frequencies were not determined for epididymal spermatozoa from MMS- or IPMS-treated mice because of poor DNA recoveries. As another indicator of the genotoxicity of these alkylating agents, the frequencies of micronuclei were determined in the peripheral blood 48 h after carcinogen administration in the same transgenic mice. The micronuclei frequencies were elevated significantly (p < 0.05) by each treatment (IPMS: 1.0%; MMS: 0.94%) compared to vehicle controls (0.3%). In a separate experiment, 40 mg/kg ENU was previously found to increase the frequency of micronuclei in peripheral blood of lacI transgenic mice 48 h after treatment (3.2%; Gibson et al., 1995). These results demonstrate that the lacI transgenic mouse male germ cells are sensitive to some, but not all, mutagens under the conditions used in this experiment. Investigation of other experimental designs would offer additional perspective on the usefulness of this transgenic model for routine mutagenicity testing in germ cells.

Experimental features of a positive selection transgenic mouse mutation assay based on a lambda lacZ transgene are considered in detail, with emphasis on results using germ cells as the target tissue. Sources of variability in the experimental protocol that can affect the statistical nature of the observations are examined, with the goal of identifying sources of excess variation in the observed mutant frequencies. The sources include plate-to-plate (within packages), package-to-package (within animals), and animal-to-animal variability. Data from five laboratories are evaluated in detail. Results suggest only scattered patterns of excess variability below the animal-to-animal level, but, generally, significant excess variability at the animal-to-animal level. Using source of variability analyses to guide the choice of statistical methods, control-vs-treatment comparisons are performed for assessing the male germ cell mutagenicity of ethylnitrosourea (ENU), isopropyl methanesulfonate (iPMS), and methyl methanesulfonate (MMS). Results on male germ cell mutagenesis of ethyl methanesulfonate (EMS) and methylnitrosourea (MNU) are also reported.

Studies from our laboratory have demonstrated several advantages of conducting the Syrian hamster embryo (SHE) cell transformation assay at pH 6.70 compared to that done historically at higher pH values (7.10-7.35). These include reduction of the influence of SHE cell isolates and fetal bovine serum lot variability on the assay, an increase in the frequency of chemically induced morphological transformation (MT) compared to controls, and an increased ease in scoring the MT phenotype. The purpose of this paper is to report a comprehensive protocol for conduct of the pH 6.70 SHE transformation assay including experimental procedures, a description of criteria for an acceptable assay and statistical procedures for establishing treatment-related effects. We have also identified several assay parameters in addition to pH which can affect transformation frequencies, particularly the critical role colony number per plate can have on transformation frequency. Control of this parameter, for which details are provided, can greatly increase the reproducibility and predictive value of the assay.

As evidenced by the recent report of the Commission of the European Communities (CEEC) project (Detection of Aneugenic Chemicals-CEEC project, 1993), there currently is a great deal of effort towards developing and validating assays to detect aneuploidy-inducing chemicals. In this report, we describe the utility of the Syrian hamster embryo (SHE) cell transformation assay for detecting carcinogens with known or suspected aneuploidy-inducing activity. The following carcinogens were tested: asbestos, benomyl, cadmium chloride, chloral hydrate, diethylstilbestrol dipropionate, and griseofulvin. Thiabendazole, a noncarcinogen, was also tested. Chemicals of unknown or inconclusive carcinogenicity data, colcemid, diazepam, econazole nitrate, and pyrimethamine were also evaluated. All of the above chemicals except thiabendazole induced a significant increase in morphological transformation (MT) in SHE cells. Based on these results as well as those published in the literature previously, the SHE cell transformation assay appears to have utility for detecting carcinogens with known or suspected aneuploidy-inducing ability.