Utility of in vivo metabolomics to support read-across for UVCB substances under REACH.

Structure-based grouping of chemicals for targeted testing and read-across is an efficient way to reduce resources and animal usage. For substances of unknown or variable composition, complex reaction products, or biological materials (UVCBs), structure-based grouping is virtually impossible. Biology-based approaches such as metabolomics could provide a solution. Here, 15 steam-cracked distillates, registered in the EU through the Lower Olefins Aromatics Reach Consortium (LOA), as well as six of the major substance constituents, were tested in a 14-day rat oral gavage study, in line with the fundamental elements of the OECD 407 guideline, in combination with plasma metabolomics. Beyond signs of clinical toxicity, reduced body weight (gain), and food consumption, pathological investigations demonstrated the liver, thyroid, kidneys (males only), and hematological system to be the target organs. These targets were confirmed by metabolome pattern recognition, with no additional targets being identified. While classical toxicological parameters did not allow for a clear distinction between the substances, univariate and multivariate statistical analysis of the respective metabolomes allowed for the identification of several subclusters of biologically most similar substances. These groups were partly associated with the dominant (> 50%) constituents of these UVCBs, i.e., indene and dicyclopentadiene. Despite minor differences in clustering results based on the two statistical analyses, a proposal can be made for the grouping of these UVCBs. Both analyses correctly clustered the chemically most similar compounds, increasing the confidence that this biological approach may provide a solution for the grouping of UVCBs.

Metabolomics as read-across tool: An example with 3-aminopropanol and 2-aminoethanol.

Read-across and grouping is one of the most commonly used alternative approaches for data gap filling in registrations submitted under the REACH Regulation as defined by the European Chemicals Agency (ECHA) in their 'Read-Across Assessment Framework' (RAAF, 2017). At the same time, the application of read-across is rejected by ECHA frequently due to various reasons. As a major reason hereof, applicants fail to reduce the level of 'remaining uncertainty' intrinsical to every read-across approach compared to testing a substance experimentally. Recently, the use of metabolomics to support read-across cases with biological information has been reported in a case study with phenoxy herbicides (Ravenzwaay et al., 2016). In the present case-study a 'weight-of-evidence' read-across approach from 2-aminoethanol (MEA = 'source') to 3-aminopropanol (3AP = 'target') with metabolomics as 'supporting evidence' reducing the remaining uncertainties is reported. We demonstrate the high structural similarity of the two analogous substances based on the available data and we report how metabolome data add confidence concerning mechanistic similarity in this read-across approach. Finally, the herein described read-across case supported by metabolomics is used to cover the data gaps in repeated dose and reproductive toxicity endpoint of 3AP via weight of evidence for the REACH-registration.

Added value of plasma metabolomics to describe maternal effects in rat maternal and prenatal toxicity studies.

For regulatory purposes prenatal developmental toxicity (OECD No. 414) studies are routinely performed in our laboratories. The suitability of metabolomics as technology to identify maternal toxicity in such studies was investigated. Plasma was sampled from pregnant, non-fasted rats on gestation day 20 before cesarean section. Metabolite profiling was performed by gas- and liquid-chromatography-tandem mass spectrometry techniques. The sensitivity of routinely examined maternal toxicity parameters (OECD No. 414) was compared to those of metabolome analysis. Evaluating 44 studies, the metabolome-derived NOEL was more sensitive in 45% of the cases in detecting maternal toxicity than the maternal NOAEL. Metabolome patterns indicative for liver effects and 4-hydroxyphenylpyruvate dioxygenase (HPPD) enzyme-inhibition were established in pregnant rats based on regulated metabolites using reference compounds. The HPPD inhibition and liver toxicity patterns in pregnant rats were reasonably comparable to the ones established in non-pregnant, fasted rats. Metabolomics is a useful tool for an improved and mechanism-based identification of maternal toxicity in maternal and prenatal toxicity studies. The data suggest that the current classical maternal toxicity parameters may underestimate the extent of effects of compounds on the dams.

Analysis of metabolome changes in the bile acid pool in feces and plasma of antibiotic-treated rats.

The bile acid-liver-gut microbiota axis plays an important role in the host's health. The gut microbiota has an impact on the bile acid pool, but also the bile acids themselves can influence the gut microbiota composition. In this study, six antibiotics from five different classes (i.e. lincosamides, glycopeptides, macrolides, fluoroquinolones, aminoglycosides) were used to modulate microbial communities of Wistar rats to elucidate changes in the bile acid metabolism and to identify key metabolites in the bile acid pool related to gut microbial changes. 20 primary and secondary bile acids were analyzed in plasma and feces of control and treated animals. Antibiotics treatment induced significant changes in primary and secondary bile acids in both matrices. Taurine-conjugated primary bile acids significantly increased in plasma and feces. Contrary, cholic acid and most of the analyzed secondary bile acids significantly decreased in plasma, and cholic acid accumulated in the feces after treatment with all antibiotics but roxithromycin. Despite the different activity spectra of the antibiotics applied against gut microbes, the overall effect on the bile acid pool tended to be similar in both matrices except for streptomycin. These results show that changes in the gut microbial community affect the bile acid pool in plasma and feces and that changes in the bile acid profile can be indicative of alterations of the gut microbiome. Due to the important role of bile acids for the host, changes in the bile acid pool can have severe consequences for the host.

Microbiome-related metabolite changes in gut tissue, cecum content and feces of rats treated with antibiotics.

The metabolic functionality of the gut microbiota contributes to the metabolism and well-being of its host, although detailed insight in the microbiota's metabolism is lacking. Omics technologies could facilitate unraveling metabolism by the gut microbiota. In this study, we performed metabolite profiling of different matrices of the gut, after antibiotic treatment of rats in order to evaluate metabolite changes observed at different dose levels and in different sexes, and to identify the best tissue matrix for further investigations regarding an assessment of metabolic effects of new compounds with antibiotic activity. Three different antibiotics (vancomycin, streptomycin and roxithromycin) were administered orally to rats for 28 days according to the OECD 407 guideline with a subsequent metabolic profiling in feces, cecum content and gut tissue (jejunum, ileum, cecum, colon and rectum). The data were analyzed in the MetaMap®Tox database. Treatment-related effects could be observed in the metabolite profile of feces and cecum content, but not of the different gut tissues. The metabolite profile showed compound specific effects on the microbiome. In line with the activity spectra of the antibiotics tested, vancomycin showed the largest effects, followed by roxithromycin and then by streptomycin for which changes were modest. In general, for all antibiotics the largest changes were observed for the classes of lipids (increase up to 94-fold), bile acids (increase up to 33-fold), amino acids (increase up to 200-fold) and amino acid related (increase up to 348-fold). The most relevant changes in metabolite values were similar in feces and cecum content and among sexes. The results of this targeted analysis indicate that the metabolic profiles of male and female animals in the gut microbiome are comparable. Concluding, taking other samples than feces does not add any extra information. Thus, as a non-invasive sampling method, feces provide a suitable matrix for studies on metabolism by the gut microbiota.

Metabolomics as read-across tool: A case study with phenoxy herbicides.

New technologies, such as metabolomics, can address chemical grouping and read across from a biological perspective. In a virtual case study, we selected MCPP as target substance and MCPA and 2,4-DP as source substances with the goal to waive a 90-day study with MCPP. In order to develop a convincing case to show how biological data can substantiate read across, we used metabolomics on blood samples from the 28-day studies to show the qualitative and quantitative similarity of the substances. The 28-day metabolome evaluation of source substances and the target substance indicate liver and kidneys as target organs. 2,4-DP was identified as the best source substance. Using the information of the 90-day 2,4-DP study, we predicted MCPP's toxicity profile at 2500 ppm: reduced food consumption and body weight gain, liver and kidney weight increases with clinical-pathology changes and a moderate red blood cell parameter reduction. NOEL prediction for MCPP was below that of 2,4-DP (<500 ppm), and similar to that of MCPA (≥150 ppm). Qualitatively, these predictions are comparable to the results of the real MCPP 90-day study in rats (reduced food consumption and body weight gain, weight increases and clinical-pathology changes in liver and kidneys, reduced red blood cells values). Quantitatively, the predicted NOAEL (150 ppm) is similar to the actual study (NOEL = 75 ppm, NOAEL ≤ 500 ppm). Thus, the 90-day rat toxicity study of MCPP could have been waived and substituted by the 90-day results of 2,4-DP by using metabolome data of 28 day studies.

Fluorescence based cell counting in collagen monolayer cultures of primary hepatocytes.

Accurate determination of cell number is essential for the quantitative description of biological processes. The changes should be related to a measurable reference e.g. in the case of cell culture, the viable cell number is a very valuable reference parameter. Indirect methods of cell number/viability measurements may have up to 10 % standard deviation. This can lead to undesirable large deviations in the analysis of "-omics" data as well as time course studies. Such data should be preferably normalized to the exact viable cell number at a given time to allow meaningful interpretation and understanding of the biological processes. Manual counting of cell number is very laborious and not possible in certain experimental setups. We therefore, developed a simple and reliable fluorescence based method with an accuracy of 95-98 % for the determination of the viable cell number in situ. We optimized the seeding cell densities for primary rat hepatocytes for optimal cell adhesion. This will help in efficient use of primary cells which are usually limited in availability. The method will be very useful in the application of "-omics" techniques, especially metabolome analysis where the specific rates of uptake/production of metabolites can be reliably calculated.

The genesis of craniofacial biology as a health science discipline.

The craniofacial complex encapsulates the brain and contains the organs for key functions of the body, including sight, hearing and balance, smell, taste, respiration and mastication. All these systems are intimately integrated within the head. The combination of these diverse systems into a new field was dictated by the dental profession's desire for a research branch of basic science devoted and attuned to its specific needs. The traditional subjects of genetics, embryology, anatomy, physiology, biochemistry, dental materials, odontology, molecular biology and palaeoanthropology pertaining to dentistry have been drawn together by many newly emerging technologies. These new technologies include gene sequencing, CAT scanning, MRI imaging, laser scanning, image analysis, ultrasonography, spectroscopy and visualosonics. A vibrant unitary discipline of investigation, craniofacial biology, has emerged that builds on the original concept of 'oral biology' that began in the 1960s. This paper reviews some of the developments that have led to the genesis of craniofacial biology as a fully-fledged health science discipline of significance in the advancement of clinical dental practice. Some of the key figures and milestones in craniofacial biology are identified.

Occupational physicians' perceptions and impact of 2009 GMC consent guidelines.

BACKGROUND: In 2009, the General Medical Council (GMC) updated its guidance on consent, introducing a new statement that employees should be offered the opportunity to view reports, before the reports are sent to the employer. AIMS: To investigate the effects of this change on the perceptions and practice of occupational physicians. METHODS: A cross-sectional survey of UK occupational physicians via the Society of Occupational Medicine e-newsletter, seeking their opinions of the anticipated and actual effects of the guidance on employers, employees, occupational physicians and occupational health services. RESULTS: Two hundred and ninety-five completed questionnaires were returned (estimated response rate 30%). Respondents included 25% of accredited UK specialists. Some reported improved standards and greater transparency, however the change was generally perceived as unfavourable, with employee and employer losses: 50% reported delay providing timely advice to employers and 35% reported delays in employees returning to work. Significant variation in practice and increased costs were reported, with variable effects on different services. Difficulties in areas such as pensions and health surveillance were reported. Some occupational physicians had moved to instant reporting; others had moved away from this to allow more care with wording of reports. CONCLUSIONS: We found significant variations in practice between occupational physicians and concerns of employee and employer losses as a result of changes to the GMC consent guidance. Clearer guidance on practical implementation was desired. The background ethical reasoning should be stated so that the parameters of the guidance are delineated and its reach should be clarified.

Role of DHEA and growth factors in chromaffin cell proliferation.

Dehydroepiandrostreone (DHEA) is a neuroactive steroid produced by the inner layer of the adrenal cortex close to the adrenomedullary cells. Chromaffin cell growth and proliferation are under the control of insulin-like growth factor II (IGF-II) and basic fibroblast growth factor (bFGF). The aim of the present study was to examine the role of DHEA on chromaffin cell proliferation induced by IGF-II and bFGF. In our model, DHEA significantly decreased IGF-II-induced proliferation by 48.7%, whereas it did not affect the proliferation induced by bFGF. These data suggest that DHEA exerts a paracrine function in the control of chromaffin cell growth.

[Disease Management Program for diabetes mellitus type 2: cooperation or resistance of the general practitioner]. / DMP Diabetes mellitus Typ 2: kooperation oder widerstand der hausärzte?

AIM: of the study was to seize the attitudes of General Practitioners (GPs) towards the disease management program (DMP) for type 2 diabetes implemented in summer 2003 in Germany. Moreover we were interested in the way GPs realise the program in daily practice, e. g. how many patients and which patients they include. METHOD: A postal questionnaire was sent twice to all GPs in the region of Hamburg (n = 1.230), in November 2003 and in December 2004. Response rate without reminder was 20 respective 16 percent. RESULTS: In 2004 81 percent of the GPs taking part in the survey participate in the DMP. These doctors include a third of their patients with type 2 diabetes into the program. 65 percent of the GPs nevertheless do not believe, that the patients will benefit from the program. 47 percent of the participating GPs object to DMP in general. Only 66 percent say they follow the DMP guidelines for pharmacotherapy. Half of the doctors state they actively canvass patients for the program, while one fifth says they advice patients against participation. The GPs participate in first line to supply the demand of the patients and because of the public pressure less because they think the DMP is good in respect of content. In 2003 critics and pessimism regarding benefit for patients were even stronger than in 2004. CONCLUSIONS: GPs participate in the DMP diabetes half-heartedly and with doubts. The results suggest selections in the inclusion of patients. Further research should find out whether patients being likely to profit from the DMP are systematically not included.

[Effectiveness of an outpatient disease management programme for chronic heart insufficiency patients]. / Nutzen eines ambulanten Disease-Management-Programms für Patienten mit chronischer Herzinsuffizienz.

The effectivity of a disease management programme (DMP) for outpatients suffering from chronic heart insufficiency (CHI) in primary care is presented. The programme is predominantly based on a weekly telephone monitoring by a case manager using a standardised questionnaire that scores CHI-relevant information of the patient. If the score exceeds a predefined limit the patient's general practitioner is alarmed. An observational study including a total of 115 patients indicates a significant decline of the hospital admission rate (p < 0.0001), as the primary outcome measure, whereas the total length of hospitalization remained constant. The findings are compared with other studies' results and the aims of a randomised controlled trial on the efficacy of DMP on patients with chronic heart failure are discussed.

C rossmodal agnosia for familiar people as a consequence of right infero polar temporal atrophy.

A 60-year-old, right-handed woman, with no focal brain lesions, suffered from a progressive impairment in recognising people of personal relevance and public figures familiar to her in the premorbid period. The patient did not suffer from general cognitive deterioration. There was no ecological or clear psychometric evidence of visuoperceptual or visuospatial deficits. Her defective person recognition was not overcome by extra-facial (e.g., observing animated people in their usual surroundings) or extra-visual information (e.g., listening to the voice). Moreover, presenting the correct name in the presence of an unrecognised familiar person failed to prompt her familiarity judgement, or retrieval of the relevant biographical knowledge. The patient also had some recognition difficulties with famous buildings and songs as well as with some common objects. It is argued that the patient's difficulty in identifying familiar people was the consequence of progressive loss of stored exemplars of familiar persons and perhaps also of some other "unique items" (famous songs and monuments) in an independent subsystem of semantics that we term "exemplar semantics." We discuss the associative (semantic) nature and specificity of the deficit in person knowledge, the possible top-down negative influences of the loss of exemplars in the person recognition system, and the link between the disorders and the right/left temporal lobe.

Effectiveness of pseudoephedrine plus acetaminophen for treatment of symptoms attributed to the paranasal sinuses associated with the common cold.

BACKGROUND: Little data exist on the cause and treatment of subfacial pain and pressure and other discomfort attributed to the paranasal sinuses that develop early during the course of the common cold. The purpose of this study was to determine the efficacy of the combination of pseudoephedrine hydrochloride with acetaminophen for the treatment of early symptoms during colds, which are attributed by the patient to the sinuses. METHODS: Four hundred thirty subjects (216, pseudoephedrine and acetaminophen recipients; 214, placebo recipients) with cold symptoms of 48 hours or less who reported overall "sinus" symptoms of at least moderate severity were enrolled in this randomized double-blind placebo-controlled 2-dose study. Self-reported symptoms were scored (0 to 4, absent to severe) before and at 2 hours after the first and second doses. The 2 primary were measured 2 hours after the second dose were the overall sinus symptom assessment and a weighted composite assessment of sinus pressure, pain, and congestion (sinus symptoms). RESULTS: Compared with baseline, 2 hours after the second dose, the mean +/- SEM overall sinus symptom assessment score had decreased by 1.30 +/- 0. 06 in the pseudoephedrine and acetaminophen-treated subjects compared with 0.93 +/- 0.06 in the placebo-treated subjects (P< or = .029). The mean +/- SEM weighted average of sinus symptoms 2 hours after the second dose of study medication had decreased by 1.14 +/- 0.06 in the pseudoephedrine and acetaminophen-treated subjects compared with 0.84 +/- 0.06 in the placebo-treated subjects (P< or = .029). Reductions in similar magnitude were also observed for each of the individual sinus symptoms, and headache and rhinorrhea. Nervousness occurred in 4% of the pseudoephedrine and acetaminophen recipients compared with 0% of placebo recipients (P =.007). CONCLUSION: Our results suggest that pseudoephedrine plus acetaminophen is effective for relief of symptoms attributable to the paranasal sinuses that may develop early in the course of a cold. Arch Fam Med. 2000;9:979-985

Paradoxical response to a novel influenza virus vaccine strain: the effect of prior immunization.

BACKGROUND: repeated influenza immunization does not appear to adversely affect the serum antibody response to new influenza strains. OBJECTIVE: to determine whether the immune response to a new influenza strain was inferior in persons previously vaccinated compared with persons not previously vaccinated. DESIGN: randomized, double-blind clinical trial. SETTING: university affiliated community teaching hospital. PATIENTS: 139 healthy adult men and women, mean age 38 years. INTERVENTION: subjects were vaccinated as part of another study. They received influenza vaccines containing influenza strains A/Texas/36/91 (H1N1), A/Nanchang/933/95 (H3N2) and B/Beijing/184/93. One group received a licensed influenza vaccine while the other group received a similar vaccine except the A/Nanchang strain had a diminished potency. MEASUREMENTS: serum hemagglutination inhibition (HAI) antibody titers were determined prior to vaccination and two weeks afterward. If patients had a low postvaccination titer, they were revaccinated and HAI titers were determined two weeks later. RESULTS: 68 adults received the licensed vaccine and 70 received the subpotent vaccine. The groups were similar with regards to baseline characteristics. Those previously vaccinated had significantly lower postvaccination HAI geometric mean titers (GMTs) for all three vaccine strains (A/Texas--127 vs. 359, p < 0.001, A/Nanchang--31 vs. 93, p < 0.001 and B/Beijing--140 vs. 205, p < 0.05). The percentage of subjects with a presumed protective HAI titer of > or =40 was significantly lower among the previously vaccinated groups only for the new influenza strain, A/Nanchang (55% vs. 80%, p < 0.05). For the other two vaccine strains, the percentage with an HAI titer > or =40 was greater than 90% for both groups. CONCLUSIONS: the decrease in serologic response to influenza vaccine among healthy, young adults who were previously vaccinated appears to be unique for this year's influenza vaccine. Further studies are required to determine the frequency and clinical significance of this phenomenon observed in younger healthy adults, and whether it is a general one. Based on its proven efficacy, influenza vaccine should continue to be given on an annual basis to high risk children and adults and to all those 65 years or older.

Facial cellulitis and Pseudomonas luteola bacteremia in an otherwise healthy patient.

Pseudomonas luteola is an aerobic, Gram negative rod, formerly classified as CDC group Ve-1 and Chryseomonas luteola. It is an uncommon clinical isolate. A previously healthy 59-year-old homosexual man with facial cellulitis and Pseudomonas luteola bacteremia is reported. Previously reported cases of P. luteola bacteremia have occurred in association with pancreatic abscess, prosthetic valve endocarditis, cardiac surgery, granulomatous hepatitis, peritonitis, and indwelling vascular catheters. This case suggests that the spectrum of disease caused by this bacteria may continue to expand.

Effectiveness of clemastine fumarate for treatment of rhinorrhea and sneezing associated with the common cold.

Limited data support the use of first-generation antihistamines for treatment of the common cold. The purpose of this study was to test the effectiveness of clemastine fumarate, a first-generation antihistamine, for treatment of sneezing and rhinorrhea associated with naturally occurring common colds. Four hundred three subjects (202 clemastine fumarate recipients and 201 placebo recipients) who reported new onset (< 24 hours) of cold symptoms that included rhinorrhea or sneezing were studied. At baseline (day 1), the mean symptom-severity scores +/- SEM for the clemastine fumarate and placebo groups were not significantly different. The mean rhinorrhea-severity score +/- SEM was not different on day 2; however, on day 3, the mean rhinorrhea-severity score +/- SEM was 1.02 +/- 0.07 for the clemastine fumarate group and 1.39 +/- 0.07 for the placebo group (P < .001). This treatment effect persisted on day 4. A significant effect on sneezing was noted on days 2-4. Sedation occurred in 14% of the clemastine fumarate-treated subjects and 1.5% of the placebo-treated subjects (P < .0001).

Human immunodeficiency virus type 1 pol gene mutations in an AIDS patient treated with multiple antiretroviral drugs.

Multiple mutations were found in the human immunodeficiency virus pol gene following treatment of an AIDS patient with antiretroviral drugs. After approximately 2.5 years of monthly alternating therapy with 3'-azido-3'-deoxythymidine (AZT) and 2',3'-dideoxycytidine (ddC), most of the pol sequences amplified from the patient's peripheral blood mononuclear cell DNA contained known AZT resistance mutations at codons 41, 67, and 215 and a putative ddC resistance mutation at codon 69 as well as other novel mutations. These mutations persisted for 6 months after the patient was switched to 2',3'-dideoxyinosine monotherapy. Mutations known to be associated with 2',3'-dideoxyinosine resistance did not occur during this time. Antiviral susceptibility testing of point mutants, introduced into the genetic background of laboratory strain NL4-3, showed that the codon 41 mutation antagonized ddC resistance when present with the codon 69 mutation. However, this antagonism was not found with a chimeric mutant containing the patient's pol gene sequence from codons 25 to 218, implying that other mutations compensated for the antagonism. Thus, alternating therapy with AZT and ddC resulted in the selection of viruses resistant to both drugs.

Anti-rhinoviral activity of recombinant and hybrid species of interferon alpha.

To define further differences in antiviral activity as well as to identify candidate interferons for study in the prevention of rhinovirus colds, the antiviral activities of nine species of recombinant interferon alpha (IFN-alpha A, IFN-alpha B, IFN-alpha C, IFN-alpha D, IFN-alpha J, [Ser-116]IFN-alpha J1, IFN-alpha K, IFN-alpha J/C(Fnu4HI), and IFN-alpha A/D(BglII)) were evaluated against rhinovirus types 39 (RV 39) and 1A (RV 1A). WI-38 cells were exposed to various concentrations of each interferon and were then infected with RV 39, RV 1A, or VSV. Efficacy was determined by protection from cytopathic effect using a tetrazolium dye assay. The 50% inhibitory concentrations ranged from 4 +/- 3 pg/ml for IFN-alpha C to > 3000 pg/ml for IFN-alpha D against RV 39, and from 6 +/- 4 pg/ml for IFN-alpha J/C(Fnu4HI) to > 3000 pg/ml for IFN-alpha D against RV 1A. IFN-alpha J/C(Fnu4HI), [Ser-116]IFN-alpha J1, and IFN-alpha C were the most active of the interferons, and were all more active than IFN-alpha A, against RV 39, RV 1A, and VSV. These interferons warrant further study against rhinoviruses and other viruses.