The analysis of survival and longitudinal data from life-span carcinogenicity bioassay on Sprague-Dowley rats

Carcinogenicity bioassay are among the best instruments to strengthen the evidence on which regulatory agencies vase their decision to classify harmful agents as human carcinogens, so they are fundamental to protect public health. The statistical analysis is fundamental to validate the results from carcinogenicity bioassay. This work aims to propose and illustrate some methodologies for the analysis of non-cancer outcomes, in particular for the analysis of time-to-death and of longitudinal measurements of body weights. The data from an old experiment were used for this purpose: 4 experiments aimed at testing the carcinogenic potential of Coca-Cola on Sprague-Dawley rats of different ages (randomized males and females of 7, 30, 39, 55 weeks of age, and their non-randomized offspring, observed since birth) were re-analysed. Survival analysis aimed to verify the influence of the treatment, controlling for possible differences due to sex, age at beginning of observation and age of the dams at pregnancy. It was performed using Cox proportional hazards models for the rats of second generation, and accelerated failure-times models for those of first generation; the use of frailty terms was evaluated (univariate gamma frailty to account for unobserved heterogeneity applied to data from breeders; shared gamma frailty at the litter level applied to data from offspring). The analysis of longitudinal body weights of the offspring was aimed at verifying the relevance of treatment, controlling for physiological differences due to sex and age of the dams at gestation. It was performed using linear and nonlinear mixed-effects models to handle the hierarchical structure of the data. Linear models were fitted using log-transformation of time and polynomial terms of order 3; nonlinear models consisted of growth models, in particular the Berkey-Reed model, that is usually used to analyse human growth during infancy, was applied

The analysis of longitudinal data from life-span carcinogenicity bioassays on Sprague-Dawley rats

Background and aim of the work: Long Term Carcinogenicity Bioassays (LTCB) are among the best instruments to strengthen the evidence on which regulatory agencies base their decision to classify harmful agents as human carcinogens, so they are fundamental to protect public health. The statistical analysis is essential to validate the results from cancer and non-cancer outcomes in carcinogenicity bioassay. This work proposes and applies some methodologies for the analysis of non-cancer outcomes, such as body weights. Methods: We use data from studies already concluded, evaluated and published: 4 bioassays aimed at testing the carcinogenic potential of Coca-Cola on Sprague-Dawley rats of different ages. The analysis of body weights of the second generation of rats was performed using mixed-effects models: linear models were fitted for nonlinear models we considered human non-linear growth functions. Results: Linear models were fitted using the log-transformation of time and polynomial term of third order for time. Sex and treatment influence body weight, age of dams during gestation doesn’t. Growth models: Jenns-Bayley, Count and 1st order Berkey-Reed growth functions were evaluated; the latter best describes the data. Sex and treatment significantly influence all parameters. The direction, magnitude and significance of the effect variable is substantially similar in all models. The analysis of residuals highlights the same issues for all models: the extreme trends in the last part of life heavily affect the models’ performance. Conclusions: Mixed-effects models allowed to account for the structural effect of covariates that act the same way on all individuals, and to add random effects that introduce a correlation among subjects if clustering happens; nonlinear human growth models added information about the whole growth process, therefore these may be useful methods in studies focused on development and sexual maturation

Hemolymphoreticular Neoplasias from the Ramazzini Institute Long-term Mice and Rat Studies on Aspartame

Background: Haemolymphoreticular neoplasias (HLRNs) from the Ramazzini Institute (RI) carcinogenicity studies on Aspartame (APM) in rats and mice were heterogeneously grouped over the years and different statistical methods were applied. Objective: We report all the detailed HLRN diagnoses of all the RI rats and mice studies on APM and the related statistics. Methods: Histological subtypes and lineage (myeloid or lymphoid) are reported in males (MM) and females (FF) in line with the International Harmonization of Nomenclature and Diagnostic Criteria for Lesions (INHAND) for rodents and the World Health Organization (WHO) Classification of Tumours of Haematopoietic and Lymphoid Tissues. Statistical analyses included Fisher’s Exact test and Cochran-Armitage trend test. Findings: Results from the post-natal bioassay on Sprague-Dawley (SD) rats (BT6008) showed statistically significant increases in lymphomas (all types) (MM, FF), leukemias (all types) (FF), immunoblastic lymphomas (MM, FF), total lymphoid tumours (MM, FF), monocytic leukemia (FF), myeloid leukemia (FF), histiocytic sarcoma (FF), and total myeloid tumours (FF). Results from the prenatal experiment on SD rats (BT6009), showed statistically significant increases in lymphomas (all types) (FF), leukemias (all types) (FF), total lymphoid tumours (FF), myeloid leukemia (FF), and total myeloid tumours (FF). Finally, results from the prenatal bioassay on Swiss mice (BT6010) showed statistically significant increases in leukemias (all types) (MM, FF), lymphoblastic leukemia (MM, FF), monocytic leukemia (MM) and total myeloid tumours (MM). Conclusions: Our analyses, performed in line with international recommended guidelines for statistics and pathology, confirm and reinforce our previous findings of statistically significant increases of HLRNs in rodents exposed to APM

Protocol for a systematic review and meta-analysis of human exposure to pesticide residues in honey and other bees' products

Background: The presence of pesticides in honey and related products is an increasing concern for consumers and producers, although there is lack of data on the current burden of exposure of the general human population through these products. We present a protocol for a systematic review and meta-analysis of contamination to insecticides, herbicides and fungicides of products from honeybees, and an estimation of how much the consumption of these products contributes to the ADI (Acceptable Daily Intake) of selected substances. Objectives: We aim to systematically review and meta-analyse studies on the contamination to plant protection products in honey, royal jelly, beeswax and propolis, applying the Navigation Guide and WHO-ILO systematic review methodology as an organizing framework. Data sources: We will search electronic academic databases for potentially relevant records from PubMed, TOXNET and EMBASE. We will include quantitative studies analysing the contamination from insecticides, herbicides and fungicides in honey, propolis, royal jelly and beeswax. In particular, we will evaluate the presence of the following substances and classes of pesticides: Glyphosate, Chlorpyrifos, pyrethroid and neonicotinoid pesticides, fungicides and acaricides. Study appraisal and synthesis methods: At least two authors will independently screen titles and abstracts at a first stage of review, and full texts at a second stage, of potentially eligible records against the eligibility criteria; data extraction of included studies will then be performed by at least two authors, in blind. At least two authors will assess risk of bias and the quality of evidence, using the most suited tools currently available. The data on prevalence of contaminated samples and concentration of pesticides in the products will be combined using meta-analysis: when more than three studies reporting the necessary measures to fit the models are available, meta-analysis will be performed separately by product and by exposure; otherwise, weighted descriptive analysis will be performed. We will report the results using the Preferred Reporting Items for Systematic Reviews and Meta-Analyses guidelines (PRISMA)

Occurrence of pesticide residues in indoor dust of farmworker households across Europe and Argentina.

Pesticides are widely used as plant protection products (PPPs) in farming systems to preserve crops against pests, weeds, and fungal diseases. Indoor dust can act as a chemical repository revealing occurrence of pesticides in the indoor environment at the time of sampling and the (recent) past. This in turn provides information on the exposure of humans to pesticides in their homes. In the present study, part of the Horizon 2020 funded SPRINT project, the presence of 198 pesticide residues was assessed in 128 indoor dust samples from both conventional and organic farmworker households across Europe, and in Argentina. Mixtures of pesticide residues were found in all dust samples (25-121, min-max; 75, median). Concentrations varied in a wide range (<0.01 ng/g-206 μg/g), with glyphosate and its degradation product AMPA, permethrin, cypermethrin and piperonyl butoxide found in highest levels. Regarding the type of pesticides, insecticides showed significantly higher levels than herbicides and fungicides. Indoor dust samples related to organic farms showed a significantly lower number of residues, total and individual concentrations than those related to conventional farms. Some pesticides found in indoor dust were no longer approved ones (29 %), with acute/chronic hazards to human health (32 %) and with environmental toxicity (21 %)

Pesticide residues with hazard classifications relevant to non-target species including humans are omnipresent in the environment and farmer residences

Intensive and widespread use of pesticides raises serious environmental and human health concerns. The presence and levels of 209 pesticide residues (active substances and transformation products) in 625 environmental samples (201 soil, 193 crop, 20 outdoor air, 115 indoor dust, 58 surface water, and 38 sediment samples) have been studied. The samples were collected during the 2021 growing season, across 10 study sites, covering the main European crops, and conventional and organic farming systems. We profiled the pesticide residues found in the different matrices using existing hazard classifications towards non-target organisms and humans. Combining monitoring data and hazard information, we developed an indicator for the prioritization of pesticides, which can support policy decisions and sustainable pesticide use transitions. Eighty-six percent of the samples had at least one residue above the respective limit of detection. One hundred residues were found in soil, 112 in water, 99 in sediments, 78 in crops, 76 in outdoor air, and 197 in indoor dust. The number, levels, and profile of residues varied between farming systems. Our results show that non-approved compounds still represent a significant part of environmental cocktails and should be accounted for in monitoring programs and risk assessments. The hazard profiles analysis confirms the dominance of compounds of low-moderate hazard and underscores the high hazard of some approved compounds and recurring “no data available” situations. Overall, our results support the idea that risk should be assessed in a mixture context, taking environmentally relevant mixtures into consideration. We have uncovered uncertainties and data gaps that should be addressed, as well as the policy implications at the EU approval status level. Our newly introduced indicator can help identify research priority areas, and act as a reference for targeted scenarios set forth in the Farm to Fork pesticide reduction goals

Collection of human and environmental data on pesticide use in Europe and Argentina: Field study protocol for the SPRINT project

Current farm systems rely on the use of Plant Protection Products (PPP) to secure high productivity and control threats to the quality of the crops. However, PPP use may have considerable impacts on human health and the environment. A study protocol is presented aiming to determine the occurrence and levels of PPP residus in plants (crops), animals (livestock), humans and other non-target species (ecosystem representatives) for exposure modelling and impact assessment. To achieve this, we designed a cross-sectional study to compare conventional and organic farm systems across Europe. Environmental and biological samples were/are being/will be collected during the 2021 growing season, at 10 case study sites in Europe covering a range of climate zones and crops. An additional study site in Argentina will inform the impact of PPP use on growing soybean which is an important European protein-source in animal feed. We will study the impact of PPP mixtures using an integrated risk assessment methodology. The fate of PPP in environmental media (soil, water and air) and in the homes of farmers will be monitored. This will be complemented by biomonitoring to estimate PPP uptake by humans and farm animals (cow, goat, sheep and chicken), and by collection of samples from non-target species (earthworms, fish, aquatic and terrestrial macroinvertebrates, bats, and farm cats). We will use data on PPP residues in environmental and biological matrices to estimate exposures by modelling. These exposure estimates together with health and toxicity data will be used to predict the impact of PPP use on environment, plant, animal and human health. The outcome of this study will then be integrated with socio-economic information leading to an overall assessment used to identify transition pathways towards more sustainable plant protection and inform decision makers, practitioners and other stakeholders regarding farming practices and land use policy

Evaluation of Toxicant-Associated Fatty Liver Disease and Liver Neoplastic Progress in Sprague-Dawley Rats Treated with Low Doses of Aflatoxin B1 Alone or in Combination with Extremely Low Frequency Electromagnetic Fields

The term toxicant-associated fatty liver disease (TAFLD) has been proposed to describe fatty liver diseases connected to toxicants other than alcohol. Aflatoxins are mycotoxins commonly found as contaminants in foods and feeds, which are known liver toxicants and potential candidates as potential causes of TAFLD. Aflatoxin B1 (AFB1) was administered at low doses to Sprague-Dawley (SD) rats, alone or in combination with S-50 Hz an extremely low frequency electromagnetic field (ELFEMF), to study the evolution of TAFLD, preneoplastic and neoplastic lesions of the liver and the potential enhancing effect of lifespan exposure to ELFEMF. Steatosis, inflammation and foci of different types were significantly increased in both aflatoxin-treated males and females, which is consistent with a pattern of TAFLD. A significant increase in adenomas, cystic dilation of biliary ducts, hepatocellular hyperplasia and hypertrophy and oval cell hyperplasia were also observed in treated females only. The administration of low doses of AFB1 caused TAFLD in SD rats, inducing liver lesions encompassing fatty infiltration, foci of different types and adenomas. Furthermore, the pattern of change observed in preneoplastic liver lesions often included liver steatosis and steatohepatitis (TASH). ELFEMF did not result in any enhancing or toxic effect in the liver of SD rats

WHO/ILO work-related burden of disease and injury: Protocol for systematic reviews of occupational exposure to dusts and/or fibres and of the effect of occupational exposure to dusts and/or fibres on pneumoconiosis

BACKGROUND: The World Health Organization (WHO) and the International Labour Organization (ILO) are developing a joint methodology for estimating the national and global work-related burden of disease and injury (WHO/ILO joint methodology), with contributions from a large network of experts. In this paper, we present the protocol for two systematic reviews of parameters for estimating the number of deaths and disability-adjusted life years attributable to pneumoconiosis from occupational exposure to dusts and/or fibres, to inform the development of the WHO/ILO joint methodology. OBJECTIVES: We aim to systematically review studies on occupational exposure to dusts and/or fibres (Systematic Review 1) and systematically review and meta-analyse estimates of the effect of occupational exposure to dusts and/or fibres on pneumoconiosis (Systematic Review 2), applying the Navigation Guide systematic review methodology as an organizing framework. DATA SOURCES: Separately for Systematic Reviews 1 and 2, we will search electronic academic databases for potentially relevant records from published and unpublished studies, including Medline, EMBASE, Web of Science and CISDOC. We will also search electronic grey literature databases, Internet search engines and organizational websites; hand-search reference list of previous systematic reviews and included study records; and consult additional experts. STUDY ELIGIBILITY AND CRITERIA: We will include working-age (≥15 years) study participants in the formal and informal economy in any WHO and/or ILO Member State but exclude children (<15 years) and unpaid domestic workers. Eligible risk factors will be dusts and/or fibres from: (i) asbestos; (ii) silica; and/or (iii) coal (defined as pure coal dust and/or dust from coal mining). Included outcomes will be (i) asbestosis; (ii) silicosis; (iii) coal worker pneumoconiosis; and (iv) unspecified pneumoconiosis. For Systematic Review 1, we will include quantitative prevalence studies of occupational exposure to dusts and/or fibres (i.e. no versus any exposure) stratified by country, sex, age and industrial sector or occupation. For Systematic Review 2, we will include randomized controlled trials, cohort studies, case-control studies and other non-randomized intervention studies with an estimate of any occupational exposure to dusts and/or fibres on the prevalence of, incidence of or mortality due to pneumoconiosis, compared with the theoretical minimum risk exposure level of no exposure. STUDY APPRAISAL AND SYNTHESIS METHODS: At least two review authors will independently screen titles and abstracts against the eligibility criteria at a first stage and full texts of potentially eligible records at a second stage, followed by extraction of data from qualifying studies. At least two review authors will assess risk of bias and the quality of evidence, using the most suited tools currently available. For Systematic Review 2, if feasible, we will combine relative risks using meta-analysis. We will report results using the guidelines for accurate and transparent health estimates reporting (GATHER) for Systematic Review 1 and the preferred reporting items for systematic reviews and meta-analyses guidelines (PRISMA) for Systematic Review 2. PROSPERO REGISTRATION NUMBER: CRD42018084131.status: publishe