E. Maria Donner

Rationale of genotoxicity testing of nanomaterials: Regulatory requirements and appropriateness of available OECD test guidelines

Abstract The development of an environmental health and safety risk management system for nanoscale particle-types requires a base set of hazard data. Accurate determination of health and environmental risks of nanomaterials is a function of the integration of hazard and exposure datasets. Recently, a nanoparticle risk assessment strategy was promulgated and the components are described in a document entitled “Nanorisk framework” (www.nanoriskframework.com). A major component of the hazard evaluation includes a proposed minimum base set of toxicity studies. Included in the suggested studies were substantial particle characterization, a variety of acute hazard and environmental tests, concomitant with screening-type genotoxicity studies. The implementation of well-accepted genotoxicity assays for testing nanomaterials remains a controversial issue. This is because many of these genotoxicity tests were designed for screening general macroparticle chemicals and might not be suitable for the screening of nanomaterials (even of the same compositional material). Furthermore, no nanoparticle-type positive controls have been established or universally accepted for these tests. Although it is the comparative results of the test material vs. the negative or vehicle control that forms the basis for interpreting the results and potency of test materials in genetic toxicology assays, the lack of a nanoparticle-type positive control questions the suitability of the tests to identify nanomaterials with genotoxic properties. It is also not possible to establish historical positive control ranges that would confirm the sensitivity of the tests. Although several genetic toxicology tests have been validated for chemicals according to the Organisation for Economic Co-operation and Development (OECD) test guidelines, the relevance of these assays for nanoparticulate materials remains to be determined. In an attempt to remedy this issue, the OECD has established current projects designed to evaluate the relevance and reproducibility of safety hazard tests for representative nanomaterials, including genotoxicity assays (i.e., Steering Group 3 – Safety Testing of Representative Nanomaterials). In this article, we discuss our past approaches and experience in conducting genotoxicity assays (1) for a newly developed ultrafine TiO2 particle-type; and (2) in a recent inhalation study, evaluating micronucleus formation in rat erythrocytes following exposures to engineered amorphous nanosilica particles. It seems clear that the development of standardized approaches will be necessary in order to determine whether exposures to specific nanoparticle-types are associated with genotoxic events. The appropriateness of available genotoxicity test systems for nanomaterials requires confirmation and standardization. Accordingly, it seems reasonable to conclude that any specific regulatory testing requirements for nanoparticles would be premature at this time.

Changing the dose metric for inhalation toxicity studies: Short-term study in rats with engineered aerosolized amorphous silica nanoparticles

Inhalation toxicity and exposure assessment studies for nonfibrous particulates have traditionally been conducted using particle mass measurements as the preferred dose metric (i.e., mg or μg/m3). However, currently there is a debate regarding the appropriate dose metric for nanoparticle exposure assessment studies in the workplace. The objectives of this study were to characterize aerosol exposures and toxicity in rats of freshly generated amorphous silica (AS) nanoparticles using particle number dose metrics (3.7 × 107 or 1.8 × 108 particles/cm3) for 1- or 3-day exposures. In addition, the role of particle size (d50 = 37 or 83 nm) on pulmonary toxicity and genotoxicity endpoints was assessed at several postexposure time points. A nanoparticle reactor capable of producing, de novo synthesized, aerosolized amorphous silica nanoparticles for inhalation toxicity studies was developed for this study. SiO2 aerosol nanoparticle synthesis occurred via thermal decomposition of tetraethylorthosilicate (TEOS). The reactor was designed to produce aerosolized nanoparticles at two different particle size ranges, namely d50 = ∼30 nm and d50 = ∼80 nm; at particle concentrations ranging from 107 to 108 particles/cm3. AS particle aerosol concentrations were consistently generated by the reactor. One- or 3-day aerosol exposures produced no significant pulmonary inflammatory, genotoxic, or adverse lung histopathological effects in rats exposed to very high particle numbers corresponding to a range of mass concentrations (1.8 or 86 mg/m3). Although the present study was a short-term effort, the methodology described herein can be utilized for longer-term inhalation toxicity studies in rats such as 28-day or 90-day studies. The expansion of the concept to subchronic studies is practical, due, in part, to the consistency of the nanoparticle generation method.

Genotoxicity of nanomaterials: Refining strategies and tests for hazard identification

A workshop addressing strategies for the genotoxicity assessment of nanomaterials (NMs) was held on October 23, 2010 in Fort Worth Texas, USA. The workshop was organized by the Environmental Mutagen Society and the International Life Sciences Institute (ILSI) Health and Environmental Sciences Institute. The workshop was attended by more than 80 participants from academia, regulatory agencies, and industry from North America, Europe and Japan. A plenary session featured summaries of the current status and issues related to the testing of NMs for genotoxic properties, as well as an update on international activities and regulatory approaches. This was followed by breakout sessions and a plenary session devoted to independent discussions of in vitro assays, in vivo assays, and the need for new assays or new approaches to develop a testing strategy for NMs. Each of the standard assays was critiqued as a resource for evaluation of NMs, and it became apparent that none was appropriate without special considerations or modifications. The need for nanospecific positive controls was questioned, as was the utility of bacterial assays. The latter was thought to increase the importance of including mammalian cell gene mutation assays into the test battery. For in‐vivo testing, to inform the selection of appropriate tests or protocols, it was suggested to run repeated dose studies first to learn about disposition, potential accumulation, and possible tissue damage. It was acknowledged that mechanisms may be at play that a standard genotoxicity battery may not be able to capture. Environ. Mol. Mutagen. 54:229–239, 2013.

Toxicological evaluation of sodium perfluorohexanoate.

Sodium perfluorohexanoate [NaPFHx, F(CF(2))(5)CO(2)Na, CAS#2923-26-4] was evaluated in acute, 90-day subchronic, one-generation reproduction, developmental and in vitro genetic toxicity studies. In the subchronic/one-generation reproduction study, four groups of young adult male and female Crl:CD(SD) rats were administered NaPFHx daily for approximately 90 days by gavage at dosages of 0, 20, 100, or 500 mg/kg. Selected groups of rats were evaluated after 1- and 3-month recovery periods. Rats selected for reproductive evaluations were dosed for approximately 70 days prior to cohabitation, through gestation and lactation, for a total of about 4 months. The subchronic toxicity no observed adverse effect level (NOAEL) was 20mg/(kg day), based on nasal lesions observed at 100 and 500 mg/(kg day). No effects were observed for neurobehavioral endpoints. NaPFHx was a moderate inducer of hepatic peroxisomal beta-oxidation with a no observed effect level (NOEL) of 20 (male rats) and 100mg/(kg day) (female rats). Elevated hepatic beta-oxidation levels were observed following 1-month recovery in male and female rats at 500 mg/(kg day). No NaPFHx-related effects were observed on any reproductive parameters. The P(1) adult rat NOAEL was 20mg/(kg day), based on reduced body weight parameters, whereas the NOAEL for reproductive toxicity was 100 mg/(kg day), based on effects limited to reduced F(1) pup weights. In the developmental study, female rats were dosed via gavage on gestation day (GD) 6-20 with the same doses of NaPFHx administered in the subchronic study. The maternal and developmental toxicity NOAEL was 100 mg/(kg day), based on maternal and fetal body weight effects at 500 mg/(kg day). NaPFHx is therefore concluded not to present a reproductive or developmental hazard. NaPFHx genotoxicity studies showed no mutations in the bacterial reverse mutation (Ames) assay or chromosome aberrations in human lymphocytes treated with NaPFHx in vitro. The lowest NOAEL from all of the studies was 20mg/(kg day) in the subchronic study based on nasal lesions. Benchmark doses (BMDL10) for nasal lesions were 13 and 21 mg/(kg day) for male and female rats, respectively. The relevance of the nasal lesions to humans is not known.

How meaningful are risk determinations in the absence of a complete dataset? Making the case for publishing standardized test guideline and ?no effect? studies for evaluating the safety of nanoparticulates versus spurious ?high effect? results from single investigative studies

Abstract A recent review article critically assessed the effectiveness of published research articles in nanotoxicology to meaningfully address health and safety issues for workers and consumers. The main conclusions were that, based on a number of flaws in study designs, the potential risk from exposures to nanomaterials is highly exaggerated, and that no ‘nano-specific’ adverse effects, different from exposures to bulk particles, have been convincingly demonstrated. In this brief editorial we focus on a related tangential issue which potentially compromises the integrity of basic risk science. We note that some single investigation studies report specious toxicity findings, which make the conclusions more alarming and attractive and publication worthy. In contrast, the standardized, carefully conducted, ‘guideline study results’ are often ignored because they can frequently report no adverse effects; and as a consequence are not considered as novel findings for publication purposes, and therefore they are never considered as newsworthy in the popular press. Yet it is the Organization for Economic Cooperation and Development (OECD) type test guideline studies that are the most reliable for conducting risk assessments. To contrast these styles and approaches, we present the results of a single study which reports high toxicological effects in rats following low-dose, short-term oral exposures to nanoscale titanium dioxide particles concomitant with selective investigative analyses. Alternatively, the findings of OECD test guideline 408, standardized guideline oral toxicity studies conducted for 90 days at much higher doses (1000 mg kg−1) in male and female rats demonstrated no adverse effects following a very thorough and complete clinical chemical, as well as histopathological evaluation of all of the relevant organs in the body. This discrepancy in study findings is not reconciled by the fact that several biokinetic studies in rats and humans demonstrate little or no uptake of nanoscale or pigment-grade TiO2 particles following oral exposures. We conclude that to develop a competent risk assessment profile, results derived from standardized, guideline-type studies, and even ‘no effect’ study findings provide critically useful input for assessing safe levels of exposure; and should, in principle, be readily acceptable for publication in peer-reviewed toxicology journals. This is a necessary prerequisite for developing a complete dataset for risk assessment determinations.

In vitro genotoxicity testing of (1-chloroethenyl)oxirane, a metabolite of β-chloroprene

(1-Chloroethenyl)oxirane (CEO) is a metabolite of beta-chloroprene (2-chloro-1,3-butadiene, CD). The purpose of this study was to evaluate the in vitro mutagenic and clastogenic (chromosome breaking) potential of CEO. For comparative purposes, the study also included an evaluation of the racemic compounds, 3,4-epoxy-1-butene (EB) and 1,2:3,4-diepoxybutane (DEB). Mutagenicity was evaluated in a bacterial reverse mutation test (Ames), using the pre-incubation method in the presence and absence of an exogenous metabolism system (Aroclor)-induced rat liver S9). Four Salmonella typhimurium tester strains, TA97a, TA98, TA100 and TA1535 were used. The exposure concentrations in the sealed incubation vials ranged from 0 to 69 mM for CEO, 0 to 102 mM for EB, and 0 to 83 mM for DEB. All three compounds showed signs of toxicity, with DEB being substantially more toxic than either CEO or EB. Mutagenic activity was observed with all three chemicals in primarily the base pair substitution strains (S. typhimurium TA100 and TA1535), but some activity was also seen in the frameshift elimination strains (S. typhimurium TA97a and TA98). The observed mutagenic responses after exposure with CEO or EB were greater than the observed response for DEB, most likely because of the higher toxicity of DEB. Generally, the mutagenic responses were unchanged in the frameshift strains and base pair substitution strains in the presence of S9 metabolism. In vitro clastogenicity was evaluated using the cytochalasin-B blocked micronucleus test in cultured Chinese hamster V79 cells. The test was conducted without S9 metabolism because of the absence of substantial changes in the Ames test. Exposure concentrations ranged from 0 to 0.943 mM for CEO, 0 to 3.0 mM for EB, and 0 to 0.035 mM for DEB, with the upper exposure concentrations dictated by cytotoxicity. Cytotoxicity, measured as a reduction in the proportion of binucleated cells and altered cell morphology, was observed for CEO at concentrations > or =0.175 mM. Exposure to EB led to a reduced proportion of binucleated cells at concentrations > or =2.0 mM, and cell death was observed after DEB exposure at concentrations > or =0.025 mM. No clastogenicity was observed in the V79 cells when tested up to cytotoxic concentrations of CEO, whereas an elevated frequency of micronuclei was observed after exposure to either EB (> or =1.0 mM) or DEB (> or =0.0125 mM). These results suggest that CEO-induced mutagenicity, but not clastogenicity, may contribute to the observed beta-chloroprene-induced carcinogenicity in the rodent bioassay studies.

Use of genotoxicity information in the development of integrated testing strategies (ITS) for skin sensitization.

Skin sensitization is an end point of concern for various legislation in the EU, including the seventh Amendment to the Cosmetics Directive and Registration Evaluation, Authorisation and Restriction of Chemicals (REACH). Since animal testing is a last resort for REACH or banned (from 2013 onward) for the Cosmetics Directive, the use of intelligent/integrated testing strategies (ITS) as an efficient means of gathering necessary information from alternative sources (e.g., in vitro, (Q)SARs, etc.) is gaining widespread interest. Previous studies have explored correlations between mutagenicity data and skin sensitization data as a means of exploiting information from surrogate end points. The work here compares the underlying chemical mechanisms for mutagenicity and skin sensitization in an effort to evaluate the role mutagenicity information can play as a predictor of skin sensitization potential. The Tissue Metabolism Simulator (TIMES) hybrid expert system was used to compare chemical mechanisms of both end points since it houses a comprehensive set of established structure-activity relationships for both skin sensitization and mutagenicity. The evaluation demonstrated that there is a great deal of overlap between skin sensitization and mutagenicity structural alerts and their underlying chemical mechanisms. The similarities and differences in chemical mechanisms are discussed in light of available experimental data. A number of new alerts for mutagenicity were also postulated for inclusion into TIMES. The results presented show that mutagenicity information can provide useful insights on skin sensitization potential as part of an ITS and should be considered prior to any in vivo skin sensitization testing being initiated.

Is excess male infant mortality from sudden infant death syndrome and other respiratory diseases X‐linked?

Male excess infant mortality is well known but unexplained. In 2004, we reported sudden infant death syndrome (SIDS) and other infant respiratory deaths showed a ~50% male excess in the United States between 1979 and 2002. This study analyses expanded US data from 1968 to 2010 to see whether infant respiratory deaths still show similar ~50% male excess and may be X‐linked.

Toxicological evaluation of 6:2 fluorotelomer alcohol

6:2 fluorotelomer alcohol (6:2 FTOH; CF3[CF2]5[CH2]2OH, CAS# 647-42-7) was evaluated for acute, genetic, and subchronic toxicity using in vitro and in vivo methods. In rats, 6:2 FTOH was considered to be slightly toxic by the oral (LD50=1,750 mg/kg), and dermal (LD50 > 5,000 mg/kg) routes. In rabbits, 6:2 FTOH was not a primary skin or eye irritant, and it did not produce a dermal sensitization response in mice. In a 90-day subchronic study, 6:2 FTOH was administered to rats by oral gavage (0, 5, 25, 125, 250 mg/kg/day). Mortality was observed at 125 and 250 mg/kg/day; deaths occurred after approximately three weeks of dosing and continued sporadically. The NOAEL in the subchronic study was 5mg/kg/day based on hematology and liver effects. 6:2 FTOH was not mutagenic in the bacterial reverse mutation test or in the mouse lymphoma assay and was not clastogenic in a chromosome aberration assay in human lymphocytes. The hazard classification for human health endpoints of 6:2 FTOH according to the United Nations Globally Harmonized System of Classification and Labeling of Chemicals (GHS) is Category 4 for acute oral toxicity based on an LD50 of 1,750 mg/kg. Other acute health endpoints including eye and skin irritation, skin sensitization, as well as genotoxicity, did not meet the criteria for hazard classification. Benchmark Dose Analysis was performed on the most sensitive endpoints from the 90-day oral gavage study and these levels were all above the study NOAEL of 5mg/kg/day. For risk assessment purposes, the recommended point of departure is the more conservative study NOAEL of 5mg/kg/day.

A feasibility study: Can information collected to classify for mutagenicity be informative in predicting carcinogenicity?

Carcinogenicity is a complex endpoint of high concern yet the rodent bioassay still used is costly to run in terms of time, money and animals. Therefore carcinogenicity has been the subject of many different efforts to both develop short-term tests and non-testing approaches capable of predicting genotoxic carcinogenic potential. In our previous publication (Mekenyan et al., 2012) we presented an in vitro-in vivo extrapolation workflow to help investigate the differences between in vitro and in vivo genotoxicity tests. The outcomes facilitated the development of new (Q)SAR models and for directing testing. Here we have refined this workflow by grouping specific tests together on the basis of their ability to detect DNA and/or protein damage at different levels of biological organization. This revised workflow, akin to an Integrated Approach to Testing and Assessment (IATA) informed by mechanistic understanding was helpful in rationalizing inconsistent study outcomes and categorizing a test set of carcinogens with mutagenicity data on the basis of regulatory mutagenicity classifications. Rodent genotoxic carcinogens were found to be correctly predicted with a high sensitivity (90-100%) and a low rate of false positives (3-10%). The insights derived are useful to consider when developing future (non-)testing approaches to address regulatory purposes.