A retrospective study on EU harmonised classifications for carcinogenicity to guide future research.

Under European Regulation (EC) No 1272/2008 on the classification, labelling and packaging of substances and mixtures (CLP), chemicals can be classified as carcinogenic if they are considered to induce tumours, increase tumour incidence and/or malignancy, or shorten the time to tumour occurrence. Cancer classifications are divided into different hazard categories: Carc. 1A (known human carcinogen), Carc. 1B (presumed human carcinogen), Carc. 2 (suspected human carcinogen), and chemicals not classified for carcinogenicity. Selecting which classification is appropriate can be challenging, as judgements need to be made both on the existing hazard data and on its relevance to humans. One aspect to be considered in defining human relevance is a chemical's mode of action (MoA); the series of necessary key events that lead from an exposure to the adverse effect (in this case, tumours). This work aims to identify and discuss some of the features that have led ECHA's Committee for Risk Assessment (RAC) to decide upon harmonised cancer classifications for chemicals, and to prioritise future research on MoA and/or human relevance. RAC bases its decisions on cancer classification on both the weight-of-evidence (WoE) and strength-of-evidence (SoE) of this particular activity. Multiple factors contribute, including the species in which tumours are seen, and the relevance of the MoA to human health.

Pathway-based predictive approaches for non-animal assessment of acute inhalation toxicity.

New approaches are needed to assess the effects of inhaled substances on human health. These approaches will be based on mechanisms of toxicity, an understanding of dosimetry, and the use of in silico modeling and in vitro test methods. In order to accelerate wider implementation of such approaches, development of adverse outcome pathways (AOPs) can help identify and address gaps in our understanding of relevant parameters for model input and mechanisms, and optimize non-animal approaches that can be used to investigate key events of toxicity. This paper describes the AOPs and the toolbox of in vitro and in silico models that can be used to assess the key events leading to toxicity following inhalation exposure. Because the optimal testing strategy will vary depending on the substance of interest, here we present a decision tree approach to identify an appropriate non-animal integrated testing strategy that incorporates consideration of a substance's physicochemical properties, relevant mechanisms of toxicity, and available in silico models and in vitro test methods. This decision tree can facilitate standardization of the testing approaches. Case study examples are presented to provide a basis for proof-of-concept testing to illustrate the utility of non-animal approaches to inform hazard identification and risk assessment of humans exposed to inhaled substances.

Tiered application of the neutral red release and EpiOcular™ assays for evaluating the eye irritation potential of agrochemical formulations.

Agrochemical formulations have been underrepresented in validation efforts for implementing alternative eye irritation approaches but represent a significant opportunity to reduce animal testing. This study assesses the utility of the neutral red release assay (NRR) and EpiOcular™ assay (EO) for predicting the eye irritation potential of 64 agrochemical formulations relative to Draize data. In the NRR, formulations with an NRR50 value ≤ 50 mg/mL were categorized as UN GHS Cat 1 and those >250 mg/mL were classified as UN GHS Non Classified (NC). The accuracy, sensitivity, and specificity were 78, 85 and 76% and 73, 85 and 61% for identifying UN GHS 1 and NC formulations, respectively. Specificity was poor for formulations with NRR50 > 50 to ≤250 mg/mL. The EO (ET-40 method) was explored to differentiate formulations that were UN GHS 1/2 and UN GHS NC. The EO resulted in accuracy, sensitivity, and specificity of 65%, 58% and 75% for identifying UN GHS NC formulations. To improve the overall performance, the assays were implemented using a tiered-approach where the NRR was run as a first-tier followed by the EO. The tiered-approach resulted in improved accuracy (75%) and balanced sensitivity (73%) and specificity (77%) for distinguishing between irritating and non-irritating agrochemical formulations.

Application of the KeratinoSens™ assay for assessing the skin sensitization potential of agrochemical active ingredients and formulations.

Assessment of skin sensitization potential is an important component of the safety evaluation process for agrochemical products. Recently, non-animal approaches including the KeratinoSens™ assay have been developed for predicting skin sensitization potential. Assessing the utility of the KeratinoSens™ assay for use with multi-component mixtures such as agrochemical formulations has not been previously evaluated and is a significant need. This study was undertaken to evaluate the KeratinoSens™ assay prediction potential for agrochemical formulations. The assay was conducted for 8 agrochemical active ingredients (AIs) including 3 sensitizers (acetochlor, meptyldinocap, triclopyr), 5 non-sensitizers (aminopyralid, clopyralid, florasulam, methoxyfenozide, oxyfluorfen) and 10 formulations for which in vivo sensitization data were available. The KeratinoSens™ correctly predicted the sensitization potential of all the AIs. For agrochemical formulations it was necessary to modify the standard assay procedure whereby the formulation was assumed to have a common molecular weight. The resultant approach correctly predicted the sensitization potential for 3 of 4 sensitizing formulations and all 6 non-sensitizing formulations when compared to in vivo data. Only the meptyldinocap-containing formulation was misclassified, as a result of high cytotoxicity. These results demonstrate the promising utility of the KeratinoSens™ assay for evaluating the skin sensitization potential of agrochemical AIs and formulations.

Mechanism of 1,3-dichloropropene-induced rat liver carcinogenesis.

1,3-Dichloropropene (1,3-D) is a soil fumigant used primarily for preplanting control of parasitic nematodes. In a previous chronic dietary exposure study, 1,3-D induced an increased incidence of hepatocellular adenomas in male rats at a dose of 25 mg/kg/day. Although the mechanism for tumor induction in the rat liver by 1,3-D has not been specifically elucidated, available data suggested that the observed liver tumorigenesis was through a nongenotoxic mode of action at the tumor promotion stage. Fischer 344 rats containing preneoplastic lesions were treated (via gavage) with 25 mg/kg/day 1,3-D or 80 mg/kg/day phenobarbital (PB) for 30 days and 60 days, or for 30 days followed by a 30-day recovery period (no compound exposure). Following treatment, placental form glutathione S-transferase (GSTP) positive and GSTP-negative liver focal lesions were quantitated as to size and number. 1,3-D treatment had no effect on GSTP-positive foci number or relative size but significantly increased the number, labeling index, and relative size of GSTP-negative focal lesions (identified by H and E staining) after 30 and 60 days of treatment. Following the 30-day recovery period, the number, labeling index, and relative size of the GSTP-negative lesions in 1,3-D-treated animals returned to control levels. As expected, PB treatment produced an increase in number and relative size of the GSTP-positive lesions. The results of this study are consistent with 1,3-D inducing liver carcinogenesis through a nongenotoxic mode of action by functioning as a tumor promoter specifically through induction of a non-GSTP staining focal hepatocyte population.

Retrospective evaluation of the impact of functional immunotoxicity testing on pesticide hazard identification and risk assessment.

Conduct of a T-cell-dependent antibody response (TDAR) assay in rodents according to Environmental Protection Agency (EPA) Test Guideline OPPTS 870.7800 is now required for chemical pesticide active ingredients registered in the United States. To assess potential regulatory impact, a retrospective analysis was developed using TDAR tests conducted on 78 pesticide chemicals from 46 separate chemical classes. The objective of the retrospective analysis was to examine the frequency of positive responses and determine the potential for the TDAR to yield lower endpoints than those utilized to calculate reference doses (RfDs). A reduction in the TDAR response was observed at only the high-dose level in five studies, while it was unaltered in the remaining studies. Importantly, for all 78 pesticide chemicals, the TDAR no-observed-adverse-effect levels (TDAR NOAELs) were greater than the NOAELS currently in use as risk assessment endpoints. The TDAR NOAELs were higher than the current EPA-selected endpoints for the chronic RfD, short-term, intermediate and long-term exposure scenarios by 3-27,000, 3-1,688, 3-1,688 and 4.9-1,688 times, respectively. Based on this analysis, conduct of the TDAR assay had minimal impact on hazard identification and did not impact human health risk assessments for the pesticides included in this evaluation. These data strongly support employment of alternative approaches including initial weight-of-evidence analysis for immunotoxic potential prior to conducting functional immunotoxicity testing for pesticide active ingredients.

Chlorpyrifos: weight of evidence evaluation of potential interaction with the estrogen, androgen, or thyroid pathways.

Chlorpyrifos was selected for EPA's Endocrine Disruptor Screening Program (EDSP) based on widespread use and potential for human and environmental exposures. The purpose of the program is to screen chemicals for their potential to interact with the estrogen, androgen, or thyroid pathways. A battery of 11 assays was completed for chlorpyrifos in accordance with test guidelines developed for EDSP Tier 1 screening. To determine potential endocrine activity, a weight-of-evidence (WoE) evaluation was completed for chlorpyrifos, which included the integration of EDSP assay results with data from regulatory guideline studies and the published literature. This WoE approach was based on the OECD conceptual framework for testing and assessment of potential endocrine-disrupting chemicals and consisted of a systematic evaluation of data, progressing from simple to complex across multiple levels of biological organization. The conclusion of the WoE evaluation is that chlorpyrifos demonstrates no potential to interact with the estrogen, androgen, or thyroid pathways at doses below the dose levels that inhibit cholinesterase. Therefore, regulatory exposure limits for chlorpyrifos, which are based on cholinesterase inhibition, are sufficient to protect against potential endocrine alterations. Based on the results of this WoE evaluation, there is no scientific justification for pursuing additional endocrine testing for chlorpyrifos.

Acute toxicity testing of chemicals-Opportunities to avoid redundant testing and use alternative approaches.

Assessment of the acute systemic oral, dermal, and inhalation toxicities, skin and eye irritancy, and skin sensitisation potential of chemicals is required under regulatory schemes worldwide. In vivo studies conducted to assess these endpoints can sometimes be associated with substantial adverse effects in the test animals, and their use should always be scientifically justified. It has been argued that while information obtained from such acute tests provides data needed to meet classification and labelling regulations, it is of limited value for hazard and risk assessments. Inconsistent application of in vitro replacements, protocol requirements across regions, and bridging principles also contribute to unnecessary and redundant animal testing. Assessment of data from acute oral and dermal toxicity testing demonstrates that acute dermal testing rarely provides value for hazard assessment purposes when an acute oral study has been conducted. Options to waive requirements for acute oral and inhalation toxicity testing should be employed to avoid unnecessary in vivo studies. In vitro irritation models should receive wider adoption and be used to meet regulatory needs. Global requirements for sensitisation testing need continued harmonisation for both substance and mixture assessments. This paper highlights where alternative approaches or elimination of tests can reduce and refine animal use for acute toxicity requirements.

Downregulation of PCNA potentiates p21-mediated growth inhibition in response to hyperoxia.

Prolonged exposure to hyperoxia inhibits cell proliferation in G1 via increased expression of p21. While p21 inhibits proliferating cell nuclear antigen (PCNA)-dependent DNA synthesis, it can also directly lower PCNA abundance; however, it is unclear whether loss of PCNA contributes to growth arrest. Here, we investigate how PCNA loss affects ability of p21 to exert G1 growth arrest of lung epithelial cells exposed to hyperoxia. In A549 cells that express p21 and growth arrest in G1 during hyperoxia, small interfering RNA (siRNA) knockdown of p21 led to G1 checkpoint bypass, increased cell death, and restoration of PCNA expression. Conditional overexpression of the PCNA binding domain of p21 in H1299 cells that do not normally express p21, or exposure to hyperoxia, caused a time-dependent loss of PCNA. Titrating PCNA levels using siRNA to approximate the low amount observed in cells expressing p21 resulted in S phase arrest. While lowering PCNA by itself caused S phase arrest, the combination of hyperoxia and siRNA against PCNA dramatically reduced PCNA abundance resulting in G1 arrest. G1 growth arrest was markedly enhanced upon the addition of p21 to these cells. Our findings suggest a model in which reducing expression of the abundant protein PCNA allows the less abundant protein p21 to be more effective at suppressing the processivity functions of remaining PCNA, thereby fully exerting the G1 checkpoint. Given that high p21 expression is often associated with lower PCNA abundance, our findings are suggestive of a global growth inhibitory mechanism involving p21-mediated PCNA suppression.

p21(Cip1/Waf1/Sdi1) protects against hyperoxia by maintaining expression of Bcl-X(L).

p21(Cip1/WAF1/Sdi1) is a major transcriptional target of p53 that promotes survival of cells exposed to continuous oxidative stress caused by hyperoxia. Because p21 can protect against genotoxic stress by reducing p53-dependent transcription of the proapoptotic proteins PUMA and Bax, the current study uses genetically modified lines of HCT116 colon carcinoma cells to investigate whether p21-mediated protection against hyperoxia involves attenuation of the p53 apoptotic pathway. Hyperoxia stimulated p53-dependent expression of p21 and Bax. Genetic ablation of p21 increased cell death, and loss of Bax or PUMA increased cell survival. Unlike damage caused by adriamycin, whereby p21 sensitivity could be rescued by removal of p53, PUMA, or Bax, increased sensitivity of p21-deficient cells to hyperoxia could not be rescued by additional loss of these genes. Instead, expression of the antiapoptotic protein Bcl-X(L) declined in p21-deficient cells exposed to hyperoxia, but when genetically restored, increased their survival. Conversely, siRNA knockdown of Bcl-X(L) in parental HCT116 cells increased hyperoxia-induced cell death. These findings reveal that p21-mediated protection against hyperoxia does not involve attenuation of p53-dependent apoptosis, but rather functions to maintain Bcl-X(L) expression during periods of persistent oxidative stress.

p21Cip1 protection against hyperoxia requires Bcl-XL and is uncoupled from its ability to suppress growth.

The cyclin-dependent kinase inhibitor p21Cip1/Waf1/Sdi1 protects the lung against hyperoxia, but the mechanism of protection remains unclear because loss of p21 does not lead to aberrant cell proliferation. Because some members of the Bcl-2 gene family have been implicated in hyperoxia-induced cell death, the current study investigated their expression as well as p21-dependent growth suppression and cytoprotection. Conditional overexpression of full-length p21, its amino-terminal cyclin-binding (p211-82NLS) domain or its carboxy-terminal PCNA-binding (p2176-164) domain inhibited growth of human lung adenocarcinoma H1299 cells, but only the full-length protein was cytoprotective. Low levels of p21 inhibited cell proliferation, whereas higher levels were required for protection. Expression of the anti-apoptotic protein Bcl-XL declined during hyperoxia but was maintained in cells expressing p21. RNA interference (RNAi) knockdown of Bcl-XL enhanced hyperoxic death of cells expressing p21, whereas overexpression of Bcl-XL increased cell survival. Consistent with growth suppression and cytoprotection requiring different levels of p21, hyperoxia inhibited PCNA expression in p21+/+ and p21+/- mice but not in p21-/- mice. In contrast, p21 was haplo-insufficient for maintaining expression of Bcl-XL and protection against hyperoxia. Taken together, these data show that p21-mediated cytoprotection against hyperoxia involves regulation of Bcl-XL and is uncoupled from its ability to inhibit proliferation.

p21(Cip1/WAF1/Sdi1) does not affect expression of base excision DNA repair enzymes during chronic oxidative stress.

Exposure to chronic oxidative stress during elevated oxygen (hyperoxia) damages DNA and inhibits cell proliferation in G(1) through induction of the cyclin-dependent kinase inhibitor p21. Cells that fail to express p21 growth-arrest in S phase. The observation that growth arrest in G(1) is associated with reduced DNA damage and enhanced survival suggests that p21 may affect expression of base excision repair (BER) enzymes used to repair oxidized DNA. This hypothesis was tested in p21 wild-type and p21-deficient mice and human lung adenocarcinoma H1299 cells with tetracycline-on regulated expression of p21. The mRNA levels of Ogg1, Tdg, Udg, Mpg, Nth1, and Mgmt remained constant during 3 days of hyperoxia. The expression of Ogg1, Nth1, and APE protein also remained unchanged. Although hyperoxia increased p21, its absence did not significantly affect expression of these repair enzymes. These findings reveal that hyperoxia induces p21 without significantly altering BER enzyme expression. This suggests that p21 may protect oxidized cells by affecting the activity of BER enzymes and/or through other mechanisms, such as apoptosis.

Loss of Gadd45a does not modify the pulmonary response to oxidative stress.

It is well established that exposure to high levels of oxygen (hyperoxia) injures and kills microvascular endothelial and alveolar type I epithelial cells. In contrast, significant death of airway and type II epithelial cells is not observed at mortality, suggesting that these cell types may express genes that protect against oxidative stress and damage. During a search for genes induced by hyperoxia, we previously reported that airway and alveolar type II epithelial cells uniquely express the growth arrest and DNA damage (Gadd)45a gene. Because Gadd45a has been implicated in protection against genotoxic stress, adult Gadd45a (+/+) and Gadd45a (-/-) mice were exposed to hyperoxia to investigate whether it protected epithelial cells against oxidative stress. During hyperoxia, Gadd45a deficiency did not affect loss of airway epithelial expression of Clara cell secretory protein or type II epithelial cell expression of pro-surfactant protein C. Likewise, Gadd45a deficiency did not alter recruitment of inflammatory cells, edema, or overall mortality. Consistent with Gadd45a not affecting the oxidative stress response, p21(Cip1/WAF1) and heme oxygenase-1 were comparably induced in Gadd45a (+/+) and Gadd45a (-/-) mice. Additionally, Gadd45a deficiency did not affect oxidative DNA damage or apoptosis as assessed by oxidized guanine and terminal deoxyneucleotidyl transferase-mediated dUTP nick-end labeling staining. Overexpression of Gadd45a in human lung adenocarcinoma cells did not affect viability or survival during exposure, whereas it was protective against UV-radiation. We conclude that increased tolerance of airway and type II epithelial cells to hyperoxia is not attributed solely to expression of Gadd45a.