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This read-across case study characterises thirteen, structurally similar carboxylic acids demonstrating the application of in vitro and in silico human-based new approach methods, to determine biological similarity. Based on data from in vivo animal studies, the read-across hypothesis is that all analogues are steatotic and so should be considered hazardous. Transcriptomic analysis to determine differentially expressed genes (DEGs) in hepatocytes served as first tier testing to confirm a common mode-of-action and identify differences in the potency of the analogues. An adverse outcome pathway (AOP) network for hepatic steatosis, informed the design of an in vitro testing battery, targeting AOP relevant MIEs and KEs, and Dempster-Shafer decision theory was used to systematically quantify uncertainty and to define the minimal testing scope. The case study shows that the read-across hypothesis is the critical core to designing a robust, NAM-based testing strategy. By summarising the current mechanistic understanding, an AOP enables the selection of NAMs covering MIEs, early KEs, and late KEs. Experimental coverage of the AOP in this way is vital since MIEs and early KEs alone are not confirmatory of progression to the AO. This strategy exemplifies the workflow previously published by the EUTOXRISK project driving a paradigm shift towards NAM-based NGRA.

To date, toxicologists use in vivo animal studies to estimate a threshold below which the exposure of in vivo humans to a compounds is safe. EU-ToxRisk aims to support the use of new approach methods (NAMs), such as in vitro and in silico models, in risk assessment. Here we describe a proof of concept case study that investigates the toxicity of a group of (un)branched aliphatic carboxylic acids. Five carboxylic acids induce microvesicular liver steatosis in repeated dose toxicity studies (termed in vivo positive), whereas six structurally highly similar compounds do not (in vivo negative). Here we demonstrate the use of NAMs for a category approach comprising ethylbutyric acid and valproic acid, 4-ene valproic acid, 2-ethylhexanoic acid and pivalic acid. All compounds were tested in HepG2 cells. Exposure of HepG2 cells to test compounds resulted in lipid accumulation at subcytotoxic concentrations after 1 and 3 days of treatment. The onset of lipid accumulation was seen at lower concentration with longer exposure periods. A panel of 27 CALUX reporter gene assays and BAC GFP reporter cell lines showed activation of cellular stress pathways in acute exposures (up to 72h). All assays distinguish clearly between in vivo positive and negative compounds. The nominal EC20 values correlate to NOELs of in vivo studies. These data are compared to recent results obtained from repeated exposures of 3D HepaRG cells, PHHs and zebra fish embryos. We also used an in silico model to predict the corresponding intracellular concentration in the in vitro models. These data are included as additional information into the in vitro to in vivo correlations. This exemplary category approach demonstrates that NAMs from the EU-ToxRisk predict the toxicity of compounds after repeated exposure in a qualitative and quantitative manner. The continued development of integrated testing strategies represents the beginning of a paradigm shift, reducing, and ultimately replacing, in vivo studies in human risk assessment.

COLLABORATORS (in alphabetical order of institution, study name, or location) Aichi Cancer Research Institute, Nagoya, Japan: N Hamajima, K Hirose, K Tajima; Albert Einstein College of Medicine, NY, USA: T Rohan; American Cancer Society, GA, USA: EE Calle, CW Jr Heath; Atlanta, Emory University, GA, USA: RJ Coates, JM Liff; Aviano Cancer Center, Pordenone, Italy: R Talamini; Mahidol University, Bangkok, Thailand: N Chantarakul, S Koetsawang, D Rachawat; Breast Tumor Collaborative Study, Johns Hopkins University, MD, USA: A Morabia, L Schuman, W Stewart, M Szklo; University of Queensland, Brisbane, Australia: C Bain, F Schofield, V Siskind; British Columbia Cancer Agency, BC, Canada: P Band, AJ Coldman, RP Gallagher, TG Hislop, P Yang; Cancer Research Center, University of Hawaii, Hawaii, USA: LM Kolonel, AMY Nomura; Canadian Cancer Registries Epidemiology Research Group, Canada: J Hu, KC Johnson, Y Mao; Catalán Institut of Oncology, Barcelona, Spain: S De Sanjosé; Centers for Disease Control & Prevention, GA, USA: N Lee, P Marchbanks, HW Ory, HB Peterson, HG Wilson, PA Wingo; Central Institute of Cancer Research, Berlin, Germany: K Ebeling, D Kunde, P Nishan; Centre for Genetic Epidemiology, University of Melbourne, Melbourne, Australia: JL Hopper; Channing Laboratory, Brigham and Women's Hospital, Harvard Medical School, MA, USA: G Colditz for Nurses' Health Study Research Group; Chennai Cancer Institute, Madras, India: V Gajalakshmi; Chiang Mai University, Chiang Mai, Thailand: N Martin, T Pardthaisong, S Silpisornkosol, C Theetranont; Chulalongkorn University, Bangkok, Thailand: B Boosiri, S Chutivongse, P Jimakorn, P Virutamasen, C Wongsrichanalai; Danish Cancer Society, Aalborg, Denmark: M Ewertz; Department of Medical Epidemiology, Karolinska Institute, Stockholm, Sweden: HO Adami, L Bergkvist, C Magnusson, I Persson; Deutsches Krebsforschungszentrum, Heidelberg, Germany: J Chang-Claude; University of Otago, Dunedin, New Zealand: C Paul, DCG Skegg, GFS Spears; European Institute of Oncology, Milan, Italy: P Boyle, T Evstifeeva; Fred Hutchinson Cancer Research Center, WA, USA: JR Daling, WB Hutchinson, K Malone, EA Noonan, JL Stanford, DB Thomas, NS Weiss, E White; French Multicentre Breast Study, INSERM, Villejuif, France: N Andrieu, A Brêmond, F Clavel, B Gairard, J Lansac, L Piana, R Renaud; Girona Cancer Registry, Girona, Spain: A Izquierdo, P Viladiu; Hospital General de Mexico, Mexico City, Mexico: HR Cuevas, P Ontiveros, A Palet, SB Salazar; Hospital Universitario, Cali, Colombia: N Aristizabal, A Cuadros; Icelandic Cancer Society, Reykjavik, Iceland: L Tryggvadottir, H Tulinius; INSERM, Institut Gustave-Roussey, Villejuif, France: A Bachelot, MG Lê; Institute of Cancer Research, Sutton and London School of Hygiene and Tropical Medicine, UK: J Peto; International Agency for Research in Cancer, Lyon, France: S Franceschi; Israel Chaim Sheba Medical Centre, Tel-Hashomer, Israel: F Lubin, B Modan, E Ron, Y Wax; Kaiser Permanente, CA, USA: GD Friedman, RA Hiatt; Institut universitaire de medecine sociale et preventive, Lausanne, Switzerland: F Levi; Cancer Research UK Genetic Epidemiology Laboratory, Leeds, UK: T Bishop; Institute of Oncology, Ljubljana, Slovenia: K Kosmelj, M Primic-Zakelj, B Ravnihar, J Stare; Loma Linda University, CA, USA: WL Beeson, G Fraser; Cancer Research UK Department of Mathematics, Statistics & Epidemiology, London: RD Bulbrook, J Cuzick, SW Duffy, IS Fentiman, JL Hayward, DY Wang; London School of Hygiene & Tropical Medicine, London, UK: AJ McMichael, K McPherson; Long Island Breast Cancer Study, NY, USA: RL Hanson, MC Leske, MC Mahoney, PC Nasca, AO Varma, AL Weinstein; University Hospital, Lund, Sweden: TR Moller, H Olsson, J Ranstam; Maastricht University, Maastricht, The Netherlands: RA Goldbohm, PA van den Brandt; University of Philippines, Manila, Philippines: RA Apelo, J Baens, JR de la Cruz, B Javier, LB Lacaya, CA Ngelangel; Istituto ‘Mario Negri', Milan, Italy: C La Vecchia, E Negri; Istituto Nazionale Tumori, Divisione di Statistica Medica e Biometria, Milan, Italy: E Marubini; Istituto di Statistica Medica e Biometria, Milan, Italy: M Ferraroni; Montpellier Cancer Centre & INSERM, Montpellier, France: M Gerber, S Richardson, C Segala; Nairobi Centre for Research in Reproduction, Nairobi, Kenya: D Gatei, P Kenya, A Kungu, JG Mati; National Cancer Institute, MD, USA: LA Brinton, R Hoover, C Schairer; National Institute of Child Health & Human Development, MD, USA: R Spirtas; National University of Singapore, Singapore: HP Lee; The Netherlands Cancer Institute, Amsterdam, The Netherlands: MA Rookus, FE van Leeuwen for the Netherlands Oral Contraceptives and Breast Cancer Study Group; New Jersey State Department of Health, NJ, USA: JA Schoenberg; New South Wales Cancer Council, Sydney, Australia: M McCredie; Columbia University School of Public Health, NY, USA: MD Gammon; Ontario Cancer Treatment & Research Foundation, Ontario, Canada: EA Clarke; Department of Public Health & Primary Care, Oxford, UK: L Jones, A Neil, M Vessey, D Yeates; Cancer Research UK Epidemiology Unit, Oxford, UK (Secretariat): P Appleby, E Banks, V Beral, D Bull, B Crossley, A Goodill, J Green, C Hermon, T Key, N Langston, C Lewis, G Reeves; Cancer Research UK/MRC/BHF Clinical Trial Service Unit & Epidemiological Studies Unit, Oxford, UK: R Collins, R Doll, R Peto; Radiation Effects Research Foundation, Hiroshima, Japan: K Mabuchi, D Preston; Royal College of General Practitioners Oral Contraception Study, London, UK: P Hannaford, C Kay; University of Costa Rica, San Jose, Costa Rica: L Rosero-Bixby; Shanghai Cancer Institute, Shanghai, China: YT Gao, F Jin, J-M Yuan; Shanghai Institute of Planned Parenthood Research, Shanghai, China: HY Wei, T Yun, C Zhiheng; Department of Public Health, Sydney, Australia: G Berry, J Cooper Booth, T Jelihovsky, R MacLennan, R Shearman; Tianjin Cancer Institute, Tianjin, China: Q-S Wang; Department of Public Health Sciences, Toronto, Canada: CJ Baines, AB Miller, C Wall; Tromso University, Tromso, Norway: E Lund, H Stalsberg; Vanderbilt University, TN, USA: XO Shu, W Zheng; University of Athens Medical School, Athens, Greece: K Katsouyanni, A Trichopoulou, D Trichopoulos; University of Chile, Santiago, Chile: A Dabancens, L Martinez, R Molina, O Salas; University of Edinburgh, Edinburgh, UK: FE Alexander; University of Minnesota School of Public Health, MN, USA: K Anderson, AR Folsom on behalf of the Iowa Women's Health Study; University of North Carolina at Chapel Hill, School of Public Health, NC, USA: BS Hulka; University of Nottingham, Nottingham, UK: CED Chilvers; University of Southern California, LA, USA: L Bernstein, S Enger, RW Haile, A Paganini-Hill, MC Pike, RK Ross, G Ursin, MC Yu; University of Wisconsin Comprehensive Cancer Center, WI, USA: MP Longnecker, P Newcomb for the 4 State Study; Vasteras, Sweden: L Bergkvist; World Health Organisation, Geneva, Switzerland: A Kalache; World Health Organisation, UNDP/UNFPA/WHO/World Bank Special Programme of Research, Development and Research Training in Human Reproduction, Geneva, Switzerland: TMM Farley, S Holck, O Meirik. Analysis and writing committee: Beral V, Bull D, Doll R, Peto R, Reeves G Steering committee: Skegg D (Chairman), Colditz G, Hulka B, La Vecchia C, Magnusson C, Muller A, Peterson B, Pike M, Thomas D, Van Leeuven M.; International audience; Alcohol and tobacco consumption are closely correlated and published results on their association with breast cancer have not always allowed adequately for confounding between these exposures. Over 80% of the relevant information worldwide on alcohol and tobacco consumption and breast cancer were collated, checked and analysed centrally. Analyses included 58,515 women with invasive breast cancer and 95,067 controls from 53 studies. Relative risks of breast cancer were estimated, after stratifying by study, age, parity and, where appropriate, women's age when their first child was born and consumption of alcohol and tobacco. The average consumption of alcohol reported by controls from developed countries was 6.0 g per day, i.e. about half a unit/drink of alcohol per day, and was greater in ever-smokers than never-smokers, (8.4 g per day and 5.0 g per day, respectively). Compared with women who reported drinking no alcohol, the relative risk of breast cancer was 1.32 (1.19-1.45, P/=45 g per day alcohol. The relative risk of breast cancer increased by 7.1% (95% CI 5.5-8.7%; P

Background Menarche and menopause mark the onset and cessation, respectively, of ovarian activity associated with reproduction, and affect breast cancer risk. Our aim was to assess the strengths of their effects and determine whether they depend on characteristics of the tumours or the affected women.Methods Individual data from 117 epidemiological studies, including 118 964 women with invasive breast cancer and 306 091 without the disease, none of whom had used menopausal hormone therapy, were included in the analyses. We calculated adjusted relative risks (RRs) associated with menarche and menopause for breast cancer overall, and by tumour histology and by oestrogen receptor expression.Findings Breast cancer risk increased by a factor of 1.050 (95% CI 1.044-1.057; p < 0.0001) for every year younger at menarche, and independently by a smaller amount (1.029, 1.025-1.032; p < 0.0001), for every year older at menopause. Premenopausal women had a greater risk of breast cancer than postmenopausal women of an identical age (RR at age 45-54 years 1.43, 1.33-1.52, p < 0.001). All three of these associations were attenuated by increasing adiposity among postmenopausal women, but did not vary materially by women's year of birth, ethnic origin, childbearing history, smoking, alcohol consumption, or hormonal contraceptive use. All three associations were stronger for lobular than for ductal tumours (p < 0.006 for each comparison). The effect of menopause in women of an identical age and trends by age at menopause were stronger for oestrogen receptor-positive disease than for oestrogen receptor-negative disease (p < 0.01 for both comparisons).Interpretation The effects of menarche and menopause on breast cancer risk might not be acting merely by lengthening women's total number of reproductive years. Endogenous ovarian hormones are more relevant for oestrogen receptor-positive disease than for oestrogen receptor-negative disease and for lobular than for ductal tumours.Funding Cancer Research UK.

Background Menarche and menopause mark the onset and cessation, respectively, of ovarian activity associated with reproduction, and affect breast cancer risk. Our aim was to assess the strengths of their effects and determine whether they depend on characteristics of the tumours or the affected women. Methods Individual data from 117 epidemiological studies, including 118 964 women with invasive breast cancer and 306 091 without the disease, none of whom had used menopausal hormone therapy, were included in the analyses. We calculated adjusted relative risks (RRs) associated with menarche and menopause for breast cancer overall, and by tumour histology and by oestrogen receptor expression. Findings Breast cancer risk increased by a factor of 1.050 (95% CI 1.044-1.057; p < 0.0001) for every year younger at menarche, and independently by a smaller amount (1.029, 1.025-1.032; p < 0.0001), for every year older at menopause. Premenopausal women had a greater risk of breast cancer than postmenopausal women of an identical age (RR at age 45-54 years 1.43, 1.33-1.52, p < 0.001). All three of these associations were attenuated by increasing adiposity among postmenopausal women, but did not vary materially by women’s year of birth, ethnic origin, childbearing history, smoking, alcohol consumption, or hormonal contraceptive use. All three associations were stronger for lobular than for ductal tumours (p < 0.006 for each comparison). The effect of menopause in women of an identical age and trends by age at menopause were stronger for oestrogen receptor-positive disease than for oestrogen receptor-negative disease (p < 0.01 for both comparisons). Interpretation The effects of menarche and menopause on breast cancer risk might not be acting merely by lengthening women’s total number of reproductive years. Endogenous ovarian hormones are more relevant for oestrogen receptor-positive disease than for oestrogen receptor-negative disease and for lobular than for ductal tumours. Funding Cancer Research UK.

Background The Collaborative Group on Hormonal Factors in Breast Cancer has brought together and reanalysed the worldwide epidemiological evidence on the relation between breast cancer risk and use of hormonal contraceptives. Methods Individual data on 53297 women with breast cancer and 100 239 women without breast cancer from 54 studies conducted in 25 countries were collected, checked, and analysed centrally. Estimates of the relative risk for breast cancer were obtained by a modification of the Mantel-Haenszel method. All analyses were stratified by study, age at diagnosis, parity, and, where appropriate, the age a woman was when her first child was born, and the age she was when her risk of conception ceased. Findings The results provide strong evidence for two main conclusions. First, while women are taking combined oral contraceptives and in the 10 years after stopping there is a small increase in the relative risk of having breast cancer diagnosed (relative risk [95% CI] in current users 1.24 [1.15-1.33], 2p

The Collaborative Group on Hormonal Factors in Breast Cancer has brought together and reanalysed the worldwide epidemiological evidence on breast cancer risk and use oi hormonal contraceptives. Original data from 54 studies, representing about 90% of the information available on the topic, were collected, checked and analysed centrally. The 54 studies were performed in 26 countries and include a total of 53,297 women with breast cancer and 100,239 women without breast cancer. The studies were varied in their design, setting and timing. Most information came from case-control studies with controls chosen from the general population; most women resided in Europe or North America and most cancers were diagnosed during the 1980s. Overall 41% of the women with breast cancer and 40% of the women without breast cancer had used oral contraceptives at some time: the median age at first use was 26 years, the median duration of use was 3 years, the median year of first use was 1968, the median time since first use was 16 years, and the median time since last use was 9 years. The main findings, summarised elsewhere,I are that there is a small increase in the risk of having breast cancer diagnosed in current users of combined oral contraceptives and in women who had stopped use in the past 10 years but that there is no evidence of an increase in the risk more than 10 years after stopping use. In addition, the cancers diagnosed in women who had used oral contraceptives tended to be less advanced clinically than the cancers diagnosed in women who had not used them. Despite the large number of possibilities investigated, few factors appeared to modify the main findings either in recent or in past users. For recent users who began use before age 20 the relative risks are higher than for recent users who began at older ages. For women whose use of oral contraceptives ceased more than 10 years before there was some suggestion of a reduction in breast cancer risk in certain subgroups, with a deficit of tumors that had spread beyond the breast, especially among women who had used preparations containing the highest doses of oestrogen and progestogen. These findings are unexpected and need to be confirmed. Although these data represent most of the epidemiologi cal evidence on the topic to date, there is still insufficient information to comment reliably about the effects of specific types of oestrogen or of progestogen. What evidence there is suggests, however, no major differences in the effects for specific types of oestrogen or of progestogen and that the pattern of risk associated with use of hormonal contraceptives containing progestogens alone may be similar to that observed for preparations containing both oestrogens and progestogens. On the basis of these results, there is little difference between women who have and have not used combined oral contraceptives in terms of the estimated cumulative number of breast cancers diagnosed during the period from starting use up to 20 rears after stopping. The cancers diagnosed in women who have used oral contraceptives are, however, less advanced clinically than the cancers diag nosed in never users. Further research is needed to establish whether the associations described here are due to earlier diagnosis of breast cancer in women who have used oral contraceptives, to the biological effects of the hormonal contraceptives or to a combination of both. Little information is as yet available about the effects on breast cancer risk of oral contraceptive use that ceased more than 20 years before and as such data accumulate it will be necessary to reexamine the worldwide evidence. RI Ranstam, Jonas/A-4386-2009; Colditz, Graham/A-3963-2009

Alcohol and tobacco consumption are closely correlated and published results on their association with breast cancer have not always allowed adequately for confounding between these exposures. Over 80% of the relevant information worldwide on alcohol and tobacco consumption and breast cancer were collated, checked and analysed centrally. Analyses included 58515 women with invasive breast cancer and 95067 controls from 53 studies. Relative risks of breast cancer were estimated, after stratifying by study, age, parity and, where appropriate, women’s age when their first child was born and consumption of alcohol and tobacco. The average consumption of alcohol reported by controls from developed countries was 6.0 g per day, i.e. about half a unit/drink of alcohol per day, and was greater in ever-smokers than never-smokers, (8.4 g per day and 5.0 g per day, respectively). Compared with women who reported drinking no alcohol, the relative risk of breast cancer was 1.32 (1.19 - 1.45, P < 0.00001) for an intake of 35 - 44 g per day alcohol, and 1.46 (1.33 - 1.61, P < 0.00001) for greater than or equal to 45 g per day alcohol. The relative risk of breast cancer increased by 7.1% (95% CI 5.5-8.7%; P

This paper reports on recent epidemiological observations in western Colombia, which further demonstrate the presence of naturally-occurring goitrogens contaminating water supplies in areas where goitre persists despite prolonged and continuous iodine supplementation. 'Prospective' and 'cross-sectional' studies in 41 localities where the populations have been on a uniform and adequate iodine supplementation for the last 10-20 years indicate that, in the endemia of western Colombia, environmental factors other than nutritional iodine deficiency are responsible for differences in goitre prevalence. Further epidemiological studies to determine the causal factors for the persistence of the endemia established a correlation between the sources of drinking water and goitre prevalence rates. Organic compounds containing sulfur with marked thionamide-like antithyroid activity were isolated from water supplying endemic goitre districts, and results are presented supporting the hypothesis that sedimentary rocks rich in organic matter are the main source of water-borne goitrogens. Bacteriological investigations showed that the presence of Klebsiella pneumoniae in drinking water and bacterial concentration were related significantly with goitre prevalence only in the presence of other variables, particularly the presence of sedimentary rocks. In the light of these epidemiological observations and experimental studies it may be concluded that, at present, endemic goitre in western Colombia is not due to nutritional iodine deficiency, but that water supplies are contaminated with sulfur-bearing organic compounds with thionamide-like antithyroid activity most probably deriving from sedimentary rocks rich in organic matter and that these compounds are the main factor underlying the endemia.

Immunization of pregnant women in New Guinea with three injections of plain toxoid had previously been shown to prevent neonatal tetanus. In the present study antitoxin levels induced by two oil-adjuvant toxoids (one injection), one AlPO(4) toxoid (two injections) and one plain toxoid (three injections) were compared with those induced by the same plain toxoid as used in the earlier study. At term there was no significant difference in the levels for the five toxoids, but those for the plain toxoids later declined rapidly. AlPO(4)-toxoid titres were significantly higher than the titres for the plain toxoids at the end of a year, but lower than the oil-adjuvant titres, which were the highest and most persistent. However, unacceptable side-effects (induced by subsequent lots of oil-adjuvant toxoids) preclude their routine use at present.The results indicate that a maternal antitoxin level at delivery of 0.01 unit/ml is protective. Aluminium-compound toxoid rapidly achieved titres that were better than this for at least a year, with minimal side-effects. Hence such toxoids are recommended for maternal immunization to prevent neonatal tetanus.

Previous papers in this series have shown that plain toxoids induced early primary antitoxin levels in women in New Guinea that were not significantly different from those induced by adsorbed toxoids but that at the end of 1 year the antitoxin levels differed significantly. Protective levels (not less than 0.01 unit/ml) induced by adsorbed toxoids persisted for more than 3 years. Results of laboratory assays of the toxoids reported in this paper show that per total human immunizing dose, the plain toxoids had 72 or less international units (IU) whereas the adsorbed toxoids had approximately 200 IU. The international "unitage" of these toxoids reflected the persistence of the human protective antitoxin level but not the early primary response. The assay results were in agreement with findings of other workers that the mouse as well as the guinea-pig may be satisfactory for potency assay of adsorbed toxoids. The need for determination of the international unitage of tetanus toxoids used in human studies and the confirmation of relationship of this value to persistence of antitoxin levels is emphasized.

A quantitative study was performed to determine the effect of toxoid concentration and aluminium salt concentration on the primary immune response (PIR) and the secondary response induced by tetanus toxoid in human volunteers. Four toxoid preparations having 5-fold differences in toxoid concentration, aluminium salt concentration, or both, were administered to four comparable groups of human volunteers. Antitoxin titres in the serum of each volunteer were determined at intervals. The PIR was found to be a function of the antigen concentration, the mineral concentration, and the interaction of both. The secondary response was a function of the antigen concentration; increase in mineral adjuvant concentration had no significant effect. The data suggested that the higher the post-secondary response, the slower the rate of decline over the ensuing 10 months. The distribution of primary responses at day 28 tended to be bimodal. The response to the best preparation suggested that a single-dose toxoid might be developed to immunize populations that may be difficult to retrieve for multiple injections.