1. Bisphenol A in Russian foods Oleg Sergeyev 1, Denis Feshin 2, Andrey Shelepchikov 2, Efim Brodsky 2, Olga Speranskaya 3 1 Chapaevsk Medical Association, Russia 2 Laboratory of Analytical Ecotoxicology A.N. Severzov Institute of Ecology and Evolution, RAS,Russia 3 Eco-Accord, Russia Funded by IPEN

2. Bisphenol A 1891: monomer synthesized by Russian chemist A.Dianin 1936: Shown to have estrogenic activity (in human – later) – Estrogen receptor binding for BPA shown to be 10,000 to 100,000- fold weaker than to estradiol 1938: Diethylstilbesterol (DES), a synthetic estrogen, found to be more estrogenic than BPA, thus BPA never used pharmaceutically 1940’s - present: BPA used in consumer products (i.e., polycarbonate plastic and epoxy resins)

3. AM Soto et al./Basic Clin Pharmacol Toxicol 102 (2008) 125-133

4. Bisphenol-A Expected 5 million tons will produced in 2010* Used to manufacture: Polycarbonate plastic products Epoxy resins to line metal cans Dental sealants and composites Toys and consumer products *A. Ballesteros-Gomez et al. / J. Chromatogr. A 1216 (2009) 449–469

6. Human Exposure to BPA 2008 CDC report (NHANES data): 93% of general population had detectable urinary levels Primary exposure route considered to be diet. Leaching into foods from polycarbonate plastics and resin liners in food and beverage cans Biological half-life : hours Calafat et al. 2008. Environ Health Perspec 116:39-44. Recent epidemiological studies have suggested some statistically significant associations of BPA exposure (urine concentrations) and health effects (coronary heart disease, reproductive disorders) in adults and behavioural changes in young girls.

7. Bisphenol A as endocrine disruptor Block thyroid hormones? Block synthesis of testosterone? Increased prevalence of breast cancer and prostate cancer? Influence on sexual maturation: - earlier puberty onset among female? - later puberty onset among male? Decreased sperm motility? L.N. Vandenberg et al. / Reproductive Toxicology 24 (2007) 139–177 A. Ballesteros-Gomez et al. / J. Chromatogr. A 1216 (2009) 449–469

8. Bisphenol A: regulation Oral Reference Dose (RfD) US EPA, based on no-observed-adverse- effect level (NOAEL)*; Tolerable Daily Intake (TDI) set by European Food Safety Authority (EFSA) in 2006 opinion and re-confirmed in its 2008 opinion** Both same for BPA - 0.05 mg/kg body weight. This is minimally five hundred-fold above conservative estimates of human exposure, including in bottle-fed infants whether the TDI should be updated (expert meeting November 2010) BPA is permitted for use in food contact plastics in the European Union with a specific migration limit of 0.6 mg/kg food** Russia: maximum permissible level (MPL) for BPA in water – 10 μg/l *Integrated Risk Information System (IRIS), Bisphenol A (CASRN 80-05-7), U.S. Environmental Protection Agency, Washington, DC, 1988. Available on line at http://www.epa.gov/iris/subst/0356.htmhttp://www.epa.gov/iris/subst/0356.htm **EFSA, 2008. Toxicokinetics of Bisphenol A. Scientific Opinion of the Panel on Food additives, Flavourings, Processing aids and Materials in Contact with Food (AFC). Question number EFSAQ- 2008-382. Adopted on 9 July 2008. Available from: http://www.efsa.europa.eu/EFSA/efsa_locale-1178620753812_1211902017492.htm

9. Project “Survey of bisphenol A in Russian foods” International SAICM Implementation Project (ISIP), 2009-2010 Funded by IPEN in December, 2009 Pilot study Great opportunity to know any fact about BPA in Russia, no data before regarding BPA level in foods & biological samples To assess the exposure via foods

10. Study design Samples were collected in three different towns of Russia, 7 samples per site – Moscow – capital of Russia, foods from large supermarket – Samara – large city, 1.3 mln population, foods supermarket – Chapaevsk – small city near Samara, chemical center (organochlorine pesticides), foods from small grocery store and mini-market.

11. Chapaevsk, Russia Chapaevsk  Moscow 524 miles Chapaevsk is 950 Km Southeast of Moscow, 50 km of Samara

12. Khimprom chemical plant Small city with a population of approximately 72,000 Large complex of chemical factories that produced agricultural and industrial organochlorines (OCs) Documented environmental contamination with dioxins, PCBs Documented high exposures among local population Chapaevsk, Russia

13. Chapaevsk, studies history Epidemiological studies since 1998 Cross-sectional study of 2579 adolescents 10- 16 years old (generate data of physical and sexual maturation), 1998-2002 Large prospective cohort study “Russian’s Children Study “ (Children Health) Bio-monitoring of breast milk, urine and serum samples

14. Large prospective cohort study “Russian’s Children Study “ Since 2003 – ongoing, unique study, PI – prof. Russ Hauser, Harvard School of Public Health cohort of 516 families annual examination, interviewing and sample collection (urine) biennial collection of blood samples Boys samples: – analysis for dioxins, PCBs, wide hormone panel in serum, lead in whole blood Maternal sample: – Analysis for dioxins and PCBs in serum

15. RCS: results, findings, papers Burns JS, Williams P, Sergeyev O et al. Predictors of Serum Dioxins and PCBs among Peri-Pubertal Russian Boys. // Environ Health Perspect. – 2009. Vol.117. PP.1593-1599. doi:10.1289/ehp.0800223. http://www.ehponline.org/members/2009/0800223/0800223.pdf http://www.ehponline.org/members/2009/0800223/0800223.pdf Williams PL, Sergeyev O, wt al. Blood Lead Levels and Delayed Onset of Puberty in a Longitudinal Study of Russian Boys // Pediatrics. – 2010. Vol. 125, No.5. PP. 1088- 1096. doi:10.1542/peds.2009-2575 http://pediatrics.aappublications.org/cgi/content/abstract/125/5/e1088 http://pediatrics.aappublications.org/cgi/content/abstract/125/5/e1088 Burns JS et al. Associations of Serum Dioxins and PCBs with Growth among Russian Boys. Submitted to Pediatrics (June 2010) Larisa Altshul et al. Serum concentrations of dioxins and dioxin-like compounds among Russian boys and their mothers: the role of breastfeeding // Organohalogen Compounds. – 2010 http://www.xcdtech.com/dioxin2010/pdf/1376.pdf http://www.xcdtech.com/dioxin2010/pdf/1376.pdf Burns Jane et al. Associations of serum concentrations of chlorinated pesticides with longitudinal growth in a Russian cohort of boys // Organohalogen Compounds. – 2010 http://www.xcdtech.com/dioxin2010/pdf/1211.pdf http://www.xcdtech.com/dioxin2010/pdf/1211.pdf Humblet O., Sergeyev O, et al. Decreasing temporal trend in serial measurements of serum concentrations of dioxins, furans, and PCBs among adult women living in Chapaevsk, Russia. // Environmental Research. In press

16. Chapaevsk, studies history, biomonitoring

17. TEQ 2005 among Chapaevsk women 2000 and 2009

18. Role of breastfeeding, Chapaevsk mothers-sons pairs, n=427

19. Trend of POPs level in Chapaevsk Chapaevsk, 1998, n=40 Chapaevsk, 2007, < 3 км from plant, n=11 Chapaevsk, 2007, > 3 km from plant, n=10 The mean 2005 WHO-TEQ for PCDD/Fs among all donors has declined 4.3 times over the decade of remediation program, from 39.8 pg/g lipids in 1998 to 9.2 pg/g lipids in 2007

20. Development of assay for BPA in beverages and foods Laboratory of Analytical Ecotoxicology A.N. Severzov Institute of Ecology and Evolution, RAS, Russia Bisphenol A determination: by GC-MS analysis after sample spiking by internal standard derivatisation by isopropilchlorformate, organic solvent or solid phase extraction (SPE) and cleaning (if need be)

21. RT:27,12 - 29,73SM:7B 27,528,028,529,029,5 Time (min) 0 20 40 60 80 100 0 20 40 60 80 100 Relative Abundan ce 28,43 29,34 28,59 29,33 29,39 28,98 27,3527,84 27,56 28,09 28,43 28,18 29,31 28,71 27,38 27,6928,86 27,81 29,47 NL: 1,06E4 m/z= 212,50-213,50+ 227,50-228,50 F: MS 100219-31-PF-53 NL: 6,54E4 m/z= 224,50-225,50+ 239,50-240,50 F: MS 100219-31-PF-53

22. Fragment of chromatogram of BPA Fragment of chromatogram of labeled 13 C 12 -BPA

23. Bisphenol A level in beverages ItemBPA level, μg/L MPL for water, μg/L Canadia n level, μg/L ** 1Infant apple juice "Tyoma Tip-Top", since 3 months <0.05*100.57 2Beverage “Grusha"<0.05*100.57 3"Baltika 7", beer<0.05*100.57 4"Agusha", bottled water for infants in plastic 0.11100.57 5Beverage “Schweppes Original Bitter Lemon” 0.44100.57 6Beverage “Pepsi”1.31.3100.57 7Beverage “RedBull”2.81100.57 *Limit of detection - <0,05 μg/L ; average 0.69 μg/L ** Average for 72 drink, ranged 0.032 to 4.5 μg/L ; http://www.hc-sc.gc.ca/fn-an/securit/packag-emball/bpa/index- eng.php 2009 http://www.hc-sc.gc.ca/fn-an/securit/packag-emball/bpa/index- eng.php

24. Item BPA, ng/g Canad., ng/g** 1Liquid infant "Rastishka" Strawbery”<0,1* 2 Infant peaches in banana "HiPP", since 6 months 0.66 3 "Babuskino lukoshko", beef and vegetable marrow pure 1.32 4 "Babuskino lukoshko", peach pure, from 4 months 1.37 5Nutrilon, Infant formula, from the birth2.15 2.88 6Infant formula "Malyutka 1", from the birth3.42 2.88 7Infant formula "Malyutka 1", from the birth6.86.8 2.88 8 Infant chicken and beef pure "Agusha", since 6 months 21.52 9Infant turkey pure "Tyoma", since 6 months24.0 10Infant poultry pure "Tyoma", since 6 months35.22 Bisphenol A level in baby food *LOD - <0.1 ng/g. Average all food - 8.06 ng/g; infant formula – 4.12 ng/g ** Average (2.88) for 21 infant formula, ranged 1.142 to 5.44 ng/g ; http://www.hc-sc.gc.ca/fn- an/securit/packag-emball/bpa/bpa_survey-enquete- eng.php 2008http://www.hc-sc.gc.ca/fn- an/securit/packag-emball/bpa/bpa_survey-enquete- eng.php

25. ItemDescriptionBPA level, ng/g 1"Tushenka govyazhya osobaya" Canned beef 19.39 2"Zelyony Velikan"Canned tomatoes42,9 Bisphenol A in adult foods LOD - <0.1 ng/g

26. Bisphenol A in baby’s dummy ItemDescriptionBPA level, ng/g 1Baby's dummy "Lubby“ (latex) Baby’s dummy 17.04 2Baby's dummy Avent (silicon)Baby’s dummy5.17 LOD - <0.1 ng/g

27. Conclusions 1.Assay using Gas Chromatography-Mass Spectrometry (GC-MS) for bisphenol A analysis was developed with good sensitivity and accuracy 2.21 food samples from 3 different Russia cities was analyzed 3.BPA was determined in 17 of 21 samples (81%) 4.Highest level was determined in canned (tin) food including – infant poultry pure “Tema” (35,22 ng/g) – Infant chicken and beef pure “Agusha” (21.52 ng/g) – Canned tomatoes "Zelyony Velikan (42,9 ng/g) – Canned beef "Tushenka govyazhya osobaya“ (19,39) 5.BPA was also determined in baby’s dummy – Baby's latex dummy "Lubby" (17,04 ng/g) 6.Average Russian level was similar (slightly higher) with published Canadian level for beverages and infant formulas 7.No significant difference between level of different Russian cities

28. Perspectives To calculate daily dietary intake, based on measured concentrations: – For breast-fed babies; – For formula-fed babies using non-PC bottles, – For formula-fed babies using PC bottles; – For adults Assessment of daily human exposure to BPA in the general population by biomonitoring of urinary excretion of BPA metabolites (using samples from Chapaevsk bank of biological samples) – To develop the assay in Russian lab To assess the impact of exposure in epidemiological studies, which can be part of ongoing Chapaevsk study

29. Thank you for attention! September 2010, Conference with international participation dedicated to Centenary of Chapaevsk Health Service contact: olegsergeyev1@yandex.ru

30. Additional slides

31. Consumption of BPA with foods by infant of different ages Daily dietary intakes, based on concentrations measured in food, vary widely, but have been estimated in Europe to be*: – 2.3μg/kg body weight in formula-fed babies (3 months old) using non- PC bottles – 13μg/kg body weight in formula-fed babies (6 months old) using PC bottle and commercial foods 3 months old baby feeding by “NAN” consumes 0.7 μg that equal 0.14 μg/kg body weight 6 months old baby feeding by “NAN” and one pure “Tema” consumes 4.5 μg that equal 0.56 μg/kg body weight *EFSA. http://www.efsa.europa.eu/en/scdocs/scdoc/428.htm