The benefits of validation of methods for toxicity testing outweigh its costs

The 4th Annual Forum on Endocrine Disrupters organized by the European Commission brought together authors of this article around the topic: "From bench to validated test guidelines: (pre)validation of test methods". Validation activities are meant to demonstrate the relevance and reliability of methods and approaches used in regulatory safety testing. These activities are essential to facilitate regulatory use, still they are largely underfunded and unattractive to the scientific community. In the last decade, there has been large amounts of funding invested in European research towards the development of approaches that can be used in regulatory decision-making, including for the identification of endocrine disrupters. There is a vast pool of candidate test methods for potential regulatory applications, but most of them will not be used due to the absence of consideration of their relevance and reliability outside the method developer's laboratory. The article explains the reasons why such a gap exists between the outputs of research projects and the uptake in a regulatory context. In parallel, there are also increasing expectations from the regulatory science community that validation becomes more efficient with respect to time and resources. This article shares some of the lessons learned and proposes paths forward for validation of new methods that are not intended as one-to-one replacements of animal studies. This includes submitting only mature methods for validation that were developed following good practices and good documentation, proposing a greater emphasis on well-documented transferability studies, and adopting a cost-sharing model between those who benefit from validated methods.

Validation activities for methods intended to be used to assess chemical safety have a cost but also bring substantial benefits when the validated methods are established as OECD Test Guidelines which results in mutual acceptance of data generated by the methods. The article discusses some of the challenges faced when method validation is underfunded and unattractive for researchers. Proposals are made to improve the current situation, gain efficiency and make validation a shared responsibility.

Cetuximab versus methotrexate in first-line treatment of older, frail patients with inoperable recurrent or metastatic head and neck cancer (ELAN UNFIT): a randomised, open-label, phase 3 trial.

BACKGROUND: At present, there is no established standard treatment for frail older patients with recurrent or metastatic head and neck squamous cell carcinoma. We aimed to compare the efficacy and safety of cetuximab to those of methotrexate (the reference regimen) in this population. METHODS: This randomised, open-label, phase 3 trial was done at 20 hospitals in France. Patients aged 70 years or older, assessed as frail by the ELAN Geriatric Evaluation, with recurrent or metastatic head and neck squamous cell carcinoma in the first-line setting and with an Eastern Cooperative Oncology Group (ECOG) performance status of 0-2 were eligible for inclusion. Patients were randomly assigned (1:1) to receive cetuximab 500 mg/m2 intravenously every 2 weeks or methotrexate 40 mg/m2 intravenously every week, with minimisation by ECOG performance status, type of disease evolution, Charlson Comorbidity Index score, serum albumin concentration, and geriatrician consultation. To avoid deterministic minimisation and assure allocation concealment, patients were allocated with a probability of 0·80 to the treatment that most reduced the imbalance. Treatment was continued until disease progression or unacceptable toxicity, whichever occurred first. The primary endpoint was failure-free survival (defined as the time from randomisation to disease progression, death, discontinuation of treatment, or loss of 2 or more points on the Activities in Daily Living scale, whichever occurred first) and was analysed in the intention-to-treat population. 151 failures expected out of 164 patients were required to detect a hazard ratio (HR) of 0·625 with 0·05 alpha error, with 80% power. A futility interim analysis was planned when approximately 80 failures were observed, based on failure-free survival. Safety analyses included all patients who received at least one dose of the study drug. This study is registered on ClinicalTrials.gov (NCT01884623) and was stopped for futility after the interim analysis. FINDINGS: Between Nov 7, 2013, and April 23, 2018, 82 patients were enrolled (41 to the cetuximab group and 41 to the methotrexate group); 60 (73%) were male, 37 (45%) were aged 80 years or older, 35 (43%) had an ECOG performance status of 2, and 36 (44%) had metastatic disease. Enrolment was stopped for futility at the interim analysis. At the final analysis, median follow-up was 43·3 months (IQR 30·8-52·1). At data cutoff, all 82 patients had failure; failure-free survival did not differ significantly between the groups (median 1·4 months [95% CI 1·0-2·1] in the cetuximab group vs 1·9 months [1·1-2·6] in the methotrexate group; adjusted HR 1·03 [95% CI 0·66-1·61], p=0·89). The frequency of patients who had grade 3 or worse adverse events was 63% (26 of 41) in the cetuximab group and 73% (30 of 41) in the methotrexate group. The most common grade 3-4 adverse events in the cetuximab group were fatigue (four [10%] of 41 patients), lung infection (four [10%]), and rash acneiform (four [10%]), and those in the methotrexate group were fatigue (nine [22%] of 41), increased gamma-glutamyltransferase (seven [17%]), natraemia disorder (four [10%]), anaemia (four [10%]), leukopenia (four [10%]), and neutropenia (four [10%]). The frequency of patients who had serious adverse events was 44% (18 of 41) in the cetuximab group and 39% (16 of 41) in the methotrexate group. Four patients presented with a fatal adverse event in the cetuximab group (sepsis, decreased level of consciousness, pulmonary oedema, and death of unknown cause) as did two patients in the methotrexate group (dyspnoea and death of unknown cause). INTERPRETATION: The study showed no improvement in failure-free survival with cetuximab versus methotrexate. Patients with an ECOG performance status of 2 did not benefit from these systemic therapies. New treatment options including immunotherapy should be explored in frail older patients with recurrent or metastatic head and neck squamous cell carcinoma, after an initial geriatric evaluation, such as the ELAN Geriatric Evaluation. FUNDING: French programme PAIR-VADS 2011 (sponsored by the National Cancer Institute, the Fondation ARC and the Ligue Contre le Cancer), GEMLUC, GEFLUC, and Merck Santé. TRANSLATION: For the French translation of the abstract see Supplementary Materials section.

Review of industry reports on EU priority tobacco additives part A: Main outcomes and conclusions.

The European Union Tobacco Products Directive (EU TPD) mandates enhanced reporting obligations for tobacco manufacturers regarding 15 priority additives. Within the Joint Action on Tobacco Control (JATC), a review panel of independent experts was appointed for the scientific evaluation of the additive reports submitted by a consortium of 12 tobacco manufacturers. As required by the TPD, the reports were evaluated based on their comprehensiveness, methodology and conclusions. In addition, we evaluated the chemical, toxicological, addictive, inhalation facilitating and flavoring properties of the priority additives based on the submitted reports, supplemented by the panel's expert knowledge and some independent literature. The industry concluded that none of the additives is associated with concern. Due to significant methodological limitations, we question the scientific validity of these conclusions and conclude that they are not warranted. Our review demonstrates that many issues regarding toxicity, addictiveness and attractiveness of the additives have not been sufficiently addressed, and therefore concerns remain. For example, menthol facilitates inhalation by activation of the cooling receptor TRPM8. The addition of sorbitol and guar gum leads to a significant increase of aldehydes that may contribute to toxicity and addictiveness. Titanium dioxide particles (aerodynamic diameter <10 µm) are legally classified as carcinogenic when inhaled. For diacetyl no report was provided. Overall, the industry reports were not comprehensive, and the information presented provides an insufficient basis for the regulation of most additives. We, therefore, advise MS to consider alternative approaches such as the precautionary principle.

Review of industry reports on EU priority tobacco additives part B: Methodological limitations.

The Tobacco Products Directive (TPD) defines enhanced reporting obligations applying to 15 priority additives added to cigarettes and roll-your-own tobacco. A consortium of 12 international tobacco companies submitted 14 reports that were reviewed by an independent scientific body within the Joint Action on Tobacco Control (JATC). The reports were evaluated in accordance with the TPD with regard to their comprehensiveness, methodology and conclusions. Here we present their significant identified methodological limitations. The toxicological and chemical evaluation in the industry reports was mainly based on comparative testing, which lacks discriminative power for products with high toxicity and variability, like cigarettes. The literature reviews were biased, the comparative chemical studies did not assess previously identified pyrolysis products, the toxicological evaluation did not include the assessment of inhalation toxicity, and pyrolysis products were not assessed in terms of toxicity, including their genotoxic and carcinogenic potential. For both chemistry and toxicity testing, the statistical approach applied to test the difference between test and additive-free control cigarettes resulted in a high chance of false negatives. The clinical study for inhalation facilitation and nicotine uptake had limitations concerning study design and statistical analysis, while addictiveness was not assessed. Finally, the methodology used to assess characterizing flavors was flawed. In conclusion, there are significant limitations in the methodology applied by the industry. Therefore, the provided reports are of insufficient quality and are clearly not suitable to decide whether a priority additive should be banned in tobacco products according to the TPD.

Lung cancer mortality in the European cohort of titanium dioxide workers: a reanalysis of the exposure-response relationship.

OBJECTIVES: Animal bioassays have demonstrated convincing evidence of the potential carcinogenicity to humans of titanium dioxide (TiO2), but limitations in cohort studies have been identified, among which is the healthy worker survivor effect (HWSE). We aimed to address this bias in a pooled study of four cohorts of TiO2 workers. METHODS: We reanalysed data on respirable TiO2 dust exposure and lung cancer mortality among 7341 male workers employed in TiO2 production in Finland, France, UK and Italy using the parametric g-formula, considering three hypothetical interventions: setting annual exposures at 2.4 (U.S. occupational exposure limit), 0.3 (German limit) and 0 mg/m3 for 25 and 35 years. RESULTS: The HWSE was evidenced. Taking this into account, we observed a positive association between lagged cumulative exposure to TiO2 and lung cancer mortality. The estimated number of lung cancer deaths at each age group decreased across increasingly stringent intervention levels. At age 70 years, the estimated number of lung cancer deaths expected in the cohort after 35-year exposure was 293 for exposure set at 2.4 mg/m3, 235 for exposure set at 0.3 mg/m3, and 211 for exposure set at 0 mg/m3. CONCLUSION: This analysis shows that HWSE can hide an exposure-response relationship. It also shows that TiO2 epidemiological data could demonstrate an exposure-effects relationship if analysed appropriately. More epidemiological studies and similar reanalyses of existing cohort studies are warranted to corroborate the human carcinogenicity of TiO2. This human evidence, when combined with the animal evidence, strengthens the overall evidence of carcinogenicity of TiO2.

How to Differentiate General Toxicity-Related Endocrine Effects from Endocrine Disruption: Systematic Review of Carbon Disulfide Data.

This review provides an overview of the assessment of the endocrine disrupting (ED) properties of carbon disulfide (CS2), following the methodology used at the European level to identify endocrine disruptors. Relevant in vitro, in vivo studies and human data are analyzed. The assessment presented here focuses on one endocrine activity, i.e., thyroid disruption, and two main adverse effects, neurotoxicity and cardiotoxicity. The data available on the different ED or non-ED modes of action (MoA), known to trigger these adverse effects, are described and the strength of evidence of the different MoA is weighted. We conclude that the adverse effects could be due to systemic toxicity rather than endocrine-mediated toxicity. This assessment illustrates the scientific and regulatory challenges in differentiating a specific endocrine disruption from an indirect endocrine effect resulting from a non-ED mediated systemic toxicity. This issue of evaluating the ED properties of highly toxic and reactive substances has been insufficiently developed by European guidance so far and needs to be further addressed. Finally, this example also raises questions about the capacity of the technics available in toxicology to address such a complex issue with certainty.

Weight of epidemiological evidence for titanium dioxide risk assessment: current state and further needs.

We address here the importance of epidemiological evidence in risk assessment and decision-making in Europe. To illustrate this, titanium dioxide (TiO2) was used as a model compound. TiO2 is widely used as an odorless white pigment and opacifying agent. A recent systematic review assessing the weight of evidence on the relationship between exposure to TiO2 (all forms) and cancer in humans questions the assumptions that TiO2 is an inert material of low toxicity. Based on this new data, France submitted a proposal to classify TiO2 as a possible human carcinogen under the European regulation. The European Chemicals Agency Risk assessment committee concluded that TiO2 (all forms) warrants a classification as a suspected human carcinogen via inhalation (Category-2) under the CLP regulation (for Classification, Labeling and Packaging of chemicals). No considerations was given to TiO2 particle size, which may affect human health effects. Consequently, further epidemiological studies are needed to assess possible associations between different physical-chemical characteristics of TiO2 exposures and their impact on human health. This would allow strengthening the evidence on which to build the most appropriate regulation and to guaranty safe use given any exposure route of any TiO2 particle shape or size.

Evidence for Bisphenol B Endocrine Properties: Scientific and Regulatory Perspectives.

BACKGROUND: The substitution of bisphenol A (BPA) by bisphenol B (BPB), a very close structural analog, stresses the need to assess its potential endocrine properties. OBJECTIVE: This analysis aimed to investigate whether BPB has endocrine disruptive properties in humans and in wildlife as defined by the World Health Organization (WHO) definition used in the regulatory field, that is, a) adverse effects, b) endocrine activity, and c) plausible mechanistic links between the observed endocrine activity and adverse effects. METHODS: We conducted a systematic review to identify BPB adverse effects and endocrine activities by focusing on animal models and in vitro mechanistic studies. The results were grouped by modality (estrogenic, androgenic, thyroid hormone, steroidogenesis-related, or other endocrine activities). After critical analysis of results, lines of evidence were built using a weight-of-evidence approach to establish a biologically plausible link. In addition, the ratio of BPA to BPB potency was reported from studies investigating both bisphenols. RESULTS: Among the 36 articles included in the analysis, 3 subchronic studies consistently reported effects of BPB on reproductive function. In rats, the 28-d and 48-week studies showed alteration of spermatogenesis associated with a lower height of the seminiferous tubules, the alteration of several sperm parameters, and a weight loss for the testis, epididymis, and seminal vesicles. In zebrafish, the results of a 21-d reproductive study demonstrated that exposed fish had a lower egg production and a lower hatching rate and viability. The in vitro and in vivo mechanistic data consistently demonstrated BPB's capacity to decrease testosterone production and to exert an estrogenic-like activity similar to or greater than BPA's, both pathways being potentially responsible for spermatogenesis impairment in rats and fish. CONCLUSION: The available in vivo, ex vivo, and in vitro data, although limited, coherently indicates that BPB meets the WHO definition of an endocrine disrupting chemical currently used in a regulatory context. https://doi.org/10.1289/EHP5200.

Approach to the Patient with Recurrent/Metastatic Disease.

OPINION STATEMENT: For most of patients with a recurrent/metastatic (R/M) head and neck squamous cell carcinoma (HNSCC), the treatment remains palliative: The main objective is to reduce the symptoms related to the locoregional relapse, prolong life while maintaining quality of life, which is a big challenge. The systemic treatment needs to be adapted to the performance status, comorbidities, and sequelae of patients. For fit patients, the combination of platinum-based chemotherapy and cetuximab (EXTREME) is the standard of care in first-line treatment since 2008, as no other targeted therapy has been approved in this setting until now. The replacement of 5-FU with a taxane (docetaxel) in the EXTREME regimen has been explored in the large randomized international study TPExtreme which results are awaited in a few months. Depending on the study results on survival, response rate, and tolerance, the TPEx regimen may become a treatment option for patients with R/M HNSCC. Unfit patients are usually treated with platinum-free combinations or with the monotherapies which are recommended in second-line setting (methotrexate, taxanes, cetuximab). However, the irruption of new immunotherapies (e.g., checkpoint inhibitors (CPI)) is changing the guidelines. The tolerance of anti-PD-1 CPI is better than that of chemotherapy, and they seem to be a good option for unfit patients. Anti-PD-1 nivolumab and pembrolizumab are now approved for platinum refractory patients, providing prolonged survival in the case of response, and improvement in quality of life. New options arise in first-line setting with pembrolizumab alone or combined with chemotherapy. Patients with a high PD-L1 biomarker level seem to benefit more from immunotherapy. Other situations (e.g., PD-L1-low, PD-L1-negative, high tumor burden) may more likely to benefit from other combinations, such as cetuximab plus chemotherapy, to avoid local failures and life-threatening fast progression. In terms of perspectives, chemo-free and CPI-free approaches, using other immune oncology agents, should be the next steps.

Treatment of inoperable elderly head and neck cancer patients.

PURPOSE OF REVIEW: Elderly head and neck cancer (HNC) patients are very rarely enrolled in clinical trials, and even more so in dedicated trials in curative or palliative setting. As a result, no standards of treatment exist for this population and thus, adaptation of standard treatments is commonly used. RECENT FINDINGS: The choice between a monotherapy and a platinum-cetuximab combination is based on the performance status, which is not suitable and/or sufficient to evaluate the patient ability to receive a systemic treatment combined or not with radiotherapy. The evaluation of functional age using geriatric assessment is recommended. However, access to comprehensive geriatric assessment is limited in many centers, and the choice of the type of treatment is often not based on objective and reproducible criteria. As a result, fragile elderly HNC patients may be overtreated with a risk of increased toxicity and fit patients proposed for suboptimal treatment with a risk of failure of tumor control. SUMMARY: It is therefore crucial to develop and evaluate customized treatments by enrolling elderly HNC patients in dedicated therapeutics trials, such as the ELAN (Elderly Head and Neck Cancer) studies or new approaches involving promising immunotherapies. To administer the most suitable therapy, a simple and reproducible geriatric assessment could efficiently guide practitioners.

Alteration of mammary gland development by bisphenol a and evidence of a mode of action mediated through endocrine disruption.

The development and function of the mammary gland are endocrine-dependent processes, depending on the stage of development. Foetal and/or postnatal exposure to low doses of BPA alters tissue organisation through epithelial proliferation and stroma-epithelial interactions. BPA also alters the expression of E2-dependent epithelial and stroma transcriptomes. Several signalling pathways are consistent with the observed phenotype: proliferation and apoptosis, a focal adhesion pathway indicating changes in biomechanical properties of the extracellular matrix, and immune function. Some of BPA's effects are reversed by oestrogen and/or GPER inhibitors. BPA also alters the expression of epigenetic marks (EZH2, HOTAIR), which would explain the delayed effect of foetal BPA exposure. In conclusion, experimental evidence shows that pre- or postnatal BPA exposure consistently causes endocrine modifications in the mammary tissue of different animal species, disrupting stromal-epithelial interactions and ultimately increasing its susceptibility to carcinogens. An interspecies comparison highlights why and how these effects apply to humans.

Evidence-based adverse outcome pathway approach for the identification of BPA as en endocrine disruptor in relation to its effect on the estrous cycle.

Proper cyclicity is essential to reach successful optimal fertility. In rats and mice, BPA exposure is repeatedly and reliably reported to show an adverse effect on the estrous cycle after exposures at different life stages. In humans, a possible association between modifications of menstrual cycle characteristics (e.g. length of the cycle, duration of menstrual bleeding) and sub-fecundity or spontaneous abortion has been observed. Alterations of ovarian cyclicity can therefore be definitely considered as an adverse health outcome. As a prerequisite for the EU REACH regulation to identify a substance as an endocrine disruptor and a SVHC,1 the proof has to be established that the substance can have deleterious health effects resulting from an endocrine mode of action. This review provides an overview of the currently available data allowing to conclude that the adverse effects of BPA exposure on ovarian cyclicity is mediated by an endocrine mode of action.

Approach to oligometastatic disease in head and neck cancer, on behalf of the GORTEC.

Median survival for recurrent/metastatic head and neck squamous cell cancer (HNSCC) patients is about 10 months after first-line best systemic treatment. We aimed to assess current approaches of oligometastatic HNSCC patients by the analysis of current concept and published data (1995-2017) in this population. Five-year survival rates are over 20% in selected patients who undergo metastasis-directed therapy by either surgery or stereotactic irradiation. Human papillomavirus(+) HNSCC patients have more disseminated metastases but respond more favorably and also benefit from ablative treatments. Treatments of oligometastases are expanding rapidly. Unmet needs include revised imaging follow-up strategies to detect metastases earlier, identification of predictive noninvasive biomarkers for treatment guidance, assessment and corrections of biases in current studies and randomized clinical trials.

Assessment of the in vitro genotoxicity of TiO2 nanoparticles in a regulatory context.

A review of in vitro genotoxicity studies on titanium dioxide nanoparticles (TiO2-NPs) published between 2010 and 2016 was performed by France in the framework of the CLP Regulation 1272/2008/EC. Neither the few in vivo studies of low quality nor the larger number of acceptable in vitro studies available for genotoxicity allowed France to conclude on the genotoxicity of TiO2-NPs. Based on this work, it was decided to compare the acceptable in vitro studies to understand the reasons for the diverging results observed, such as the materials tested or of the protocols used and their inherent interferences. The systematic review performed on in vitro genotoxicity data for TiO2-NPs was then restricted to studies with the highest level of confidence among studies following OECD guidelines and the largely applied comet assay. Indeed, the aim of this article is to understand why, even if judged of good quality, the 36 publications selected and analyzed did not lead to a clear picture. Some recommendations to be taken into account before performing new in vitro genotoxicity assays for insoluble particles such as TiO2-NPs are proposed. Although secondary genotoxic effects consequent to oxidative stress seem to be the major mechanism responsible for the genotoxicity of TiO2-NPs reported in some studies, primary genotoxic effects cannot be excluded. Further studies are needed to clarify the exact mode of action of TiO2-NPs and to highlight which physicochemical properties lead to their genotoxicity in vitro to ultimately identify a specific combination of parameters that could represent a risk in vivo.

Concerns related to ED-mediated effects of Bisphenol A and their regulatory consideration.

The extensive database on BPA provides strong evidence of its adverse effects on reproductive, neurobehavioural, metabolic functions and mammary gland. Disruption of estrogenic pathway is central in the mediation of these effects although other modes of action may be involved. BPA has a weak affinity for ERα/ß but interaction with extranuclearly located pathways activated by estrogens such as ERRγ and GPER reveals how BPA can act at low doses. The effects are observed later in life after developmental exposure and are associated with pathologies of major societal concern in terms of severity, incidence, impact on quality of life, burden on public health system. The complexity of the dose response raise uncertainties on the possibility to establish safe levels and the scope of ED-mediated effects of BPA may be wider. These concerns fulfill the requirements for ED identification under REACH regulation.

Regulatory identification of BPA as an endocrine disruptor: Context and methodology.

BPA is one of the most investigated substances for its endocrine disruptor (ED) properties and it is at the same time in the center of many ED-related controversies. The analysis on how BPA fits to the regulatory identification as an ED is a challenge in terms of methodology. It is also a great opportunity to test the regulatory framework with a uniquely data-rich substance and learn valuable lessons for future cases. From this extensive database, it was considered important to engage in a detailed analysis so as to provide specific and strong evidences of ED while reflecting accurately the complexity of the response as well the multiplicity of adverse effects. An appropriate delineation of the scope of the analysis was therefore critical. Four effects namely, alterations of estrous cyclicity, mammary gland development, brain development and memory function, and metabolism, were considered to provide solid evidence of ED-mediated effects of BPA.

Comprehensive study of three novel cases of TFEB-amplified renal cell carcinoma and review of the literature: Evidence for a specific entity with poor outcome.

The first case of TFEB-amplified renal cell carcinoma was published in 2014. Since then, 29 additional cases have been described. The prognostic and therapeutic implications of this rare entity remain to be determined. We describe here the clinical, histological, and genetic features of three novel cases, and the first complete literature review. Four tumors were examined from three patients selected from the large collection of genetically characterized renal tumors in our institution. The pathological and immunohistochemical features were centrally reviewed by a uropathologist. Quantitative and structural genomic abnormalities were analyzed using comparative genomic hybridization, fluorescence in situ hybridization, and next generation sequencing. The three cases showed high-level amplification but no translocation of TFEB. Histologically, two tumors showed a papillary or pseudopapillary architecture. They did not show similarities with renal cell carcinoma harboring translocation of TFEB. The tumors were locally advanced high-grade lesions. They exhibited a metastatic course, which was rapidly leading to death in one patient. A second patient developed metastatic disease that did not respond to four lines of targeted treatments. The third patient had a protracted history of pulmonary and cardiac metastases. Complete clinical and biological data were examined and compared to those of the reported cases. Within the classification of renal tumors, TFEB-amplified renal cell carcinoma may constitute a novel entity characterized histologically by high-grade, papillary or pseudopapillary architecture, and necrotic remodeling and clinically by a poor outcome. Its pathogenesis has to be further characterized to develop appropriate targeted therapy.

Use of low density polyethylene membranes for assessment of genotoxicity of PAHs in the Seine River.

The genotoxicity of river water dissolved contaminants is usually estimated after grab sampling of river water. Water contamination can now be obtained with passive samplers that allow a time-integrated sampling of contaminants. Since it was verified that low density polyethylene membranes (LDPE) accumulate labile hydrophobic compounds, their use was proposed as a passive sampler. This study was designed to test the applicability of passive sampling for combined chemical and genotoxicity measurements. The LDPE extracts were tested with the umu test (TA1535/pSK1002 ± S9) and the Ames assay (TA98, TA100 and YG1041 ± S9). We describe here this new protocol and its application in two field studies on four sites of the Seine River. Field LDPE extracts were negative with the YG1041 and TA100 and weakly positive with the TA98 + S9 and Umu test. Concentrations of labile mutagenic PAHs were higher upstream of Paris than downstream of Paris. Improvement of the method is needed to determine the genotoxicity of low concentrations of labile dissolved organic contaminants.