“Results of rodent carcinogenicity studies on aspartame”

1. “Results of rodent carcinogenicity studies on aspartame” Dr. Morando Soffritti, MD March 16th, 2011 Public Hearing on aspartame European Parliament Brussells Ramazzini Institute Cesare Maltoni Cancer Research Center

2. Aspartame (APM): production and use • 18,000 tons produced as of 2007 • second artificial intense sweetening agent after saccharin • 62% of the intense sweetening agents market • present in more than 6,000 products • hundreds of millions of consumers worldwide • Average daily intake in US and Europe • Projected maximum consumption: 22-34 mg/Kg b.w. • General population: 2-3 mg/Kg b.w. • Children/women of childbearing age: 2.5-5 mg/Kg b.w.

3. APM: hypothetical daily intake Average Daily Intake of Aspartame Substance Quantity/day Concentration of aspartame consumed Diet soda (200 mg/can) 2 cans 400 mg Yogurt (125 mg/yogurt) 2 yogurts 250 mg Diet custard/pudding (75mg/mousse) 1 serving 75 mg Coffee with sweetener (40/mg packet) 4 cups 160 mg Candy/chewing gum (2,5/candy) 10 candies 25 mg Totals 910 mg Equivalent to: Woman 60 kg = 15,1 mg/kg body weight Woman 50 kg = 18,2 mg/kg body weight Child 30 kg = 30,3 mg/kg body weight Child 20 kg = 45,5 mg/kg body weight

4. APM: genotoxicity • Genotoxicity: APM has been shown to be non genotoxic in various tests in vitro and in vivo

5. APM: metabolism and genotoxicity Aspartate Phenylalanine Methanol • metabolized in the GI tract as aspartic acid, phenylalanine and methanol, both in humans and animals • the metabolites, once absorbed, enter the blood circulation • Methanol is not metabolized within the enterocite and rapidly enters the portal circulation and is oxidized in the liver to formaldehyde, which strongly binds to proteins and nucleic acids forming adducts

6. APM: carcinogenicity 1973. Groups of 40 M and 40 F post-weaning Sprague-Dawley rats treated with APM in feed at doses of 0, 1, 2, 4, 6, 8 g/Kg b.w./day for 104 weeks. 60 M and 60 F served as controls. No dose-related increase of brain tumors in treated groups. 1974. Groups of 40 M and 40 F Sprague-Dawley rats treated from fetal life and, after weaning, for 104 weeks with APM in feed at doses of 0, 2, 4 g/Kg b.w./day. 60 M and 60 F served as control. Decrease of brain tumors incidence in treated groups.

7. APM: carcinogenicity 1981. Groups of 86 M and 86 F, 6 week-old Wistar rats treated with APM in feed at doses of 0, 1, 2, 4 g/Kg b.w./day from 6 to 104 weeks of age. No increase of brain tumors observed. 1981. Groups of 36 M and 36 F CD-1 mice treated with APM in feed at doses of 1, 2, 4 g/Kg b.w./day from 6 to 110 weeks of age. 72 males and 72 females served as controls. No carcinogenic effects observed.

8. Motivation to study APM • Given the limited sensitivity of these studies (small number of animals per group, duration of observation 110 weeks); • Given the fact that years before we showed the carcinogenicity of methanol and formaldehyde; • Given the ever-growing use of aspartame throughout the years; • the ERF (now RI) decided in the late 1990s to plan an integrated project that would finally provide an adequate evaluation of the potential carcinogenic effects of aspartame based on: • - total number of animals used • - number of dose levels studied • - conduct of the experiment according to GLP

9. The integrated experimental projectof the Ramazzini Institute on Aspartame started in 1997

10. RI integrated project on APM Animals Experiment Species No. Status first S-D rats 1800 published (2005) second S-D rats 470 published (2007) third S-D rats 429 ongoing (biophase ended) fourth S-D rats 430 ongoing (biophase ended) ongoing (pre-pubblication) fifth Swiss mice 852  Total 3981

11. Materials and conduct Experimental conduct: • Water and food consumption • Body weight • Clinical control • Complete necropsy • Histopathology • Full evaluation of all tissues and organs of each animal of each group • Statistical evaluation • Cochran – Armitage; poly-K test; Cox proportional hazard model

12. Experiments on rats treated with aspartame administered in feed 1. First experiment: • 1800 males and females treated from 6 weeks of age until death; 7 dose level/groups (5000, 2500, 500, 100, 20, 4, 0 mg/Kg b.w.) • Results: significant increase/dose-related of L/L and benign/malignant neoplastic lesions of the renal pelvis in females; significant increase/dose-related of malignant schwannomas of cranial peripheral nerves in males 2.Second experiment: - 470 males and females treated from fetal life until death; 3 dose levels/groups (100, 20, 0 mg/Kg b.w.) - Results: significant increase/dose related of mammary cancers and L/L in females; significant increase of L/L in males

13. Comparison of the cumulative prevalence of hemolymphoreticular neoplasia by age of death Prenatal exposure Start of the treatment 40 35 2000 ppm 30 400 ppm Cumulative 25 0 ppm (control) prevalence 20 (%) 15 10 5 0 0-16 17-32 33-48 49-64 65-80 81-96 97-112 113-128 129-144 145-160 Age at death (weeks) Postnatal exposure

14. III Experiment on Swiss mice

15. TOTAL 444 408 852 Swiss mice experiment: the plan Dose/group, ppm a (mg/Kg b.w.) Age at start Animals 32,000 (3909) 16,000 (1919) 8,000 (987) 2,000 (242) 0 (Control) fetal n. males 83 64 73 122 102 fetal n. females 62 64 62 103 117 Total 145 128 135 225 219 a The treatment lasts for the entire life span

16. Swiss mice experiment: malignant tumors (%) Dose/group, ppm (mg/Kg b.w.) Animals 32,000 (3909) 16,000 (1919) 8,000 (987) 2,000 (242) 0 68.7 60.9 72.6 56.3 56.4 Males (%) 64.5 68.8 64.4 73.8 67.6 Females (%)

17. Swiss mice experiment: Alveolar Bronchiolar Adenomas (A/BA) and Carcinomas (A/BC) in malesa (%) Dose/group, ppm (mg/Kg b.w.) Animals Tumors 0 32,000 (3909) 16,000 (1919) 8,000 (987) 2,000 (242) Males A/BA 7.2 10.9 11.3 8.7 6.8 Males A/BC 13.3* 12.5 11.3 5.8 6.0* Total 20.5# 23.4 22.6 14.6 12.8# a p-values associated with the trend test are near the control incidence * significant (p<0.05) using Cox Regression Model # significant (p<0.05) using Logistic analysis

18. Cumulative prevalence of lung A/BC by age of death 30 APM 32000 ppm APM 16000 ppm APM 8000 ppm 25 APM 2000 ppm control 20 15 Bearing animals % 10 5 0 0 8 16 24 32 40 48 56 64 72 80 88 96 104 112 120 128 136 Age at death (weeks)

19. Swiss mice experiment: Hepatocellular Adenomas (HA)/ Carcinomas (HCC) in malesa, % Dose/group, ppm (mg/Kg b.w.) Animals Tumors 0 32,000 (3909) 16,000 (1919) 8,000 (987) 2,000 (242) Males HA 2.4 9.4 6.5 9.7 7.7 HCC Males 18.1** 15.6* 14.5 11.7 5.1** Total 20.5 25.0# 21.0 21.4 12.8 a p-values associated with the trend test are near the control incidence * significant (p<0.05) using Cox Regression Model ** significant (p<0.01) using Cox Regression Model # significant (p<0.05) using Logistic analysis

20. Cumulative prevalence of HCC by age of death 30 APM 32000 ppm APM 16000 ppm APM 8000 ppm 25 APM 2000 ppm control 20 15 Bearing animals % 10 5 0 0 8 16 24 32 40 48 56 64 72 80 88 96 104 112 120 128 136 Age at death (weeks)

21. Swiss mice experiment: liver hemangioma and hemangiosarcomas (%) Dose/group, ppm (mg/Kg b.w.) Animals Tumors 0 32,000 (3909) 16,000 (1919) 8,000 (987) 2,000 (242) Males Hemangiomas - 1.6 1.6 1.9 1.7 Males Hemangiosarcomas 9.6 7.8 8.1 4.9 4.3 Total 9.6 9.4 9.7 6.8 6.0

22. Summary of the carcinogenic effects of APM in rodents Significantly increasedmalignant tumors Lymph/ leuk KidneysCP Nervous sys.MS Mammary ADC Lung ADC LiverHCC Species Age at start M F M F M F M F M F M F 8 weeks S-D rats +DR +DR +DR fetal S-D rats +DR + +DR S-mice fetal +DR +DR += significantly increased; DR= Dose-related; CP= Carcinomas of the pelvis & ureter; MS= Malignant Schwannomas; ADC= Adenocarcinomas; HCC= Hepatocellular carcinomas;

23. Concluding remarks on the APM project (1/3) • In our experimental conditions APM has been shown to induce a significantly increased incidence of malignant tumors in: • multiple tissues in male and female rats • multiple tissues in male mice • an earlier occurrence in treated animals • an higher incidence and an anticipated onset of cancers when the treatment starts from fetal life The carcinogenic effects of APM were shown also at dose levels to which humans could be exposed

24. Concluding remarks on the APM project (2/3) • Given that APM is completely metabolized in the GIT, it may be concluded that the carcinogenic effects were caused not by itself but rather by its metabolites • The role of methanol inducing malignant neoplasms is reinforced by the following considerations based on experiments performed in our laboratory under the same experimental conditions: • Methanol, which is metabolized to formaldehyde in humans and rats, induces: • - In female Sprague-Dawley rats a significant increase of • hematopoietic neoplasms • - in male mice it cannot be disregarded that the conversion of APM • methanol into formaldehyde adducts in liver cells may results in a • progressive accumulation of adducts, which could explain the • plausibility of the hepatocarcinogenic effects of APM

25. Concluding remarks on the APM project (3/3) • MTBE, which is metabolized to methanol, induces, in female Sprague-Dawley rats, a significant increase of HLRN • Formaldehyde induces, in both male and female Sprague-Dawley rats a significant increase in hematopoietic neoplasms

26. Reactions to the results of the RI studies on APM by the scientific community 1/2 • Supporting evaluations: - Letter to US FDA Commissioners, 2007 signed by 13 scientists of US Academies and Agencies - Editorial on Int. J.Occup.Environ Health, 2007 - Commentary on EHP, 2008 by James Huff et al. - Letter to Cancer Epid. Biomarkers Prev., 2008 by D. Davis - Review article on Environ. Molecular Mutagenesis, 2008 by J. Caldwel et al.

27. Reactions to the results of the RI studies on APM by the scientific community 2/2 • Critical evaluations: - Scientific opinion by EFSA, 2006 - US FDA Statement, 2007 - Review article Critical Reviews on Toxicology, 2007 by B. Magnuson et al. - Letter to Environ. Molecular Mutagenesis, 2009 by J. Goodman et al. - Letter to Environ. Molecular Mutagenesis, 2009 by T. Schoeb et al. - Commentary on Environ Pathobiology, 2009 by T. Schoeb et al. - Scientific Opinion by EFSA, 2009 - Scientific Opinion by EFSA, 2011

28. Final general comments and recommendation 1/2 • Absolute certainty in science is rarely an option; uncertainty is the norm, not the exception and the scientists base their judgement on the weight of evidence • Uncertainty does not mean the science is flawed • Disagreement does not mean that one of the parties is wrong or practicing junk science • Checklists of criteria, while appealing in their convenience, are inadequate tools for assessing many scientific issues, certainly including causation • From: David Michaels, Doubt is their product

29. Final general comments and recommendation 2/2 On the basis of the weight of evidence of our results, APM should be considered a multiple site, trans-species carcinogenic agent in rodents. A re-avaluation of the current regulations on APM remains, in our opinion, urgent.

30. Cesare Maltoni Cancer Research Center Staff Fiorella Belpoggi Luciano Bua Daniela Chiozzotto Luana De Angelis Davide Degli Esposti Laura Falcioni Kathryn Knowles Michelina Lauriola Marco Manservigi Fabiana Manservisi Isabella Manzoli Federica Scagliarini Eva Tibaldi Erica Tommasini