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

Italian Constitutional Court removes the prohibition on gamete donation in Italy

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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

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-Daw-ley (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 steato-hepatitis (TASH). ELFEMF did not result in any enhancing or toxic effect in the liver of SD rats

ESHRE certification of ART centres for good laboratory and clinical practice

STUDY QUESTION: Three years after the start of the ESHRE ART Centre Certification (ARTCC) programme, what is the current state of the system, in terms of the interest expressed in it and experiences during the assessment of ART services? SUMMARY ANSWER: As of 1 December 2021, 25 European ART centres have been involved in the various stages of certification and the most common recommendations from inspectors were the need for documented training, verification of competencies for all staff members, verification of laboratory and clinical performance indicators, implementation of a quality management system and avoidance of overusing ICSI and add-ons. WHAT IS KNOWN ALREADY: European Union (EU) legislation has included ART activities in the EU Tissue and Cells Directives (EUTCDs). Following inspections by national EUTCD authorities, many details regarding documentation, laboratory environment, handling of reproductive cells and tissues, traceability, coding and patient testing have become standardized. However, the EUTCDs do not cover all ART-specific aspects. For this reason, the ARTCC was established to focus on peculiar areas, including relevant staff qualifications, training, continuing professional development, workload, equipment suitability, (non)-evidence-based laboratory and clinical methods used, treatment approaches according to ESHRE guidelines, recommendations and laboratory and clinical key performance indicators. STUDY DESIGN, SIZE, DURATION: The article reviews the state-of-the-art of the ESHRE certification of ART centres for good clinical and laboratory practice over an initial 3-year period of operation, including the number of ART centres involved in the different stages of certification and the most common recommendations by inspectors. PARTICIPANTS/MATERIALS, SETTING, METHODS: In 2016, the ARTCC working group began to establish a new ESHRE ARTCC programme. Since then, the working group has organized 4 preparatory courses and appointed 37 inspectors (19 clinicians, 17 embryologists and one paramedical). A tool to verify compliance with ESHRE recommendations for good laboratory and clinical practice was developed. The ARTCC has been open for applications since September 2018. In Step 1, the applicant enters basic information about the ART centre, staff and ART activities into the application platform. After review and approval, the applicant is given the opportunity to enter Step 2 and provide detailed online checklists on general, laboratory, clinical services and clinical outcomes. Two inspectors (one clinician and one embryologist) independently evaluate the submitted checklists. The condition to proceed to evaluation is a positive mean score (at least 66%) from each of the four checklists. In Step 3, a live site visit (or virtual owing to the coronavirus disease 2019 (COVID-19) pandemic) is organized and the inspectors prepare a final report with appropriate recommendations. The application may be rejected at any time if the criteria required to advance to the next stage are not met. The ARTCC programme is currently available for European countries listed in ESHRE internal rules, available on the ESHRE website. The certificate is valid for 3 years, after which an application for renewal can be submitted. MAIN RESULTS AND THE ROLE OF CHANCE: Over a 3-year period (until 1 December 2021), 63 ART centres from 25 countries started applying through an online platform. So far, 38 applications did not progress owing to lack of completion of the initial application within a 1-year period or because applications came from non-European countries. Of the remaining 25 applications, 8 centres have been inspected and 7 centres have been certified. The most common recommendations given by inspectors to assessed centres were the need for documented training, verification of competencies, skills and continuing professional development for all staff members, verification of laboratory and clinical performance indicators and implementation of a quality management system. The inspectors identified some recurring areas of medically assisted reproduction that deviate from good practice: the overuse of ICSI, preimplantation genetic testing for aneuploidies, freeze-all and other add-ons. They often reported that the clinical outcomes could not be objectively assessed because of noninclusion of the started cycles or the frequent use of freeze-all cycles. LIMITATIONS, REASONS FOR CAUTION: No major modifications have been made to the application platform and checklists since the early stages of the certification programme. However, in this short time, quite a few changes in clinical practice have occurred, especially concerning the more frequent use of the 'freeze-all' strategy. As a result, problems arose in the evaluation of clinical outcomes. In addition, because of the COVID-19 pandemic, site visits were substituted by the implementation of virtual visits. While this enabled the certification programme to continue, it is possible that certain critical details that would have been noticed during a traditional site visit may have been overlooked. WIDER IMPLICATIONS OF THE FINDINGS: Regular monitoring of the observations of ARTCC inspectors and analysis of their reports is certainly useful to harmonize inspectors' criteria in the assessment process and to identify chronic deficiencies in clinical and laboratory practice. Non-conformities can be addressed by ESHRE through guidelines and recommendations, as well as through discussion with EU institutions and competent authorities

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)

Prognostic significance of germline BRCA mutations in patients with HER2-POSITIVE breast cancer.

Background: HER2-positive breast cancers are rare amongst BRCA mutation carriers. No data exist regarding clinicopathological characteristics and prognosis of this subgroup of patients. Materials and methods: Using a retrospective matched cohort design, we collected data from 700 women who were diagnosed with operable invasive breast cancer from January 2006 to December 2016 and were screened for germline BRCA mutations. Clinicopathological features and survival rates were analyzed by BRCA and HER2 status. Results: One hundred and fifteen HER2-positive/BRCA mutated cases were evaluated in comparison to the three control groups: HER2-positive/BRCA wild type (n = 129), HER2-negative/BRCA mutated (n = 222), HER2-negative/BRCA wild type (n = 234). HER2-positive breast cancers were more likely to have high histologic grade and high proliferation rate than HER2-negative neoplasms, regardless of BRCA mutation status. An interaction between BRCA mutations and HER2-positive status was found to correlate with worse survival after adjusting for prognostic variables (HR = 3.4; 95% CI: 1.3-16.7). Conclusions: Co-occurrence of BRCA mutations and HER2-positive status is a poor prognostic factor in patients with early or locally advanced breast cancer. This finding may be a proof of concept that a combined pharmacological intervention directed to these targets could be synergistic

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