1. Ramazzini Days 2016
The Ramazzini Institute integrated project on Glyphosate Simona Panzacchi Cesare Maltoni Cancer Research Center Ramazzini Institute
Goodmorning to everyone…….thx for coming. As my collegue Dr. Manservisi explained you before, the RI developed a new tool, based on its experience, for integrate different in vivo study;
Today Im going to describe you our first proposed plan based on this new protocol regarding the substances glyphosate and its commercial formulation named roundup
Carpi, October 28th 2016

2. Glyphosate
Glyphosate is one of the widest-used herbicides in the world
It is found in over 750 products: intensive farm (especially GMOs incorporating resistance to it), home garden, forestry, urban applications
Acts by inhibiting the EPSPS enzyme (not in mammalian system) => for aromatic aminoacids
1950: discovered by Dr. Martin
1970: identified its herbicidal activity and first sold in 1974 with the name of Roundup
1996: first genetically engineered hebicide-tollerant plants approved
Glyphosate is a widely used herbicide to controls broadleaf weeds and grasses. Glyphosate is also used in forestry, urban, and home applications. The general population is exposed primarily through residence near sprayed areas, home use, and diet, and the level that has been observed is generally low.
It’s use in…..
it was discovered in 1950
More recently it was employed in GMO crops
Since the 1970's the use of glyphosate-based herbicides has increased 100-fold, and continued growth
Analogue of the natural amino acid glycine. Inibithing the development of plants by interfering with the production of essential aminoacid, inibhiting thr enzyme EPSPS

3. Glyphosate use
the glyphosate use during the past years and could be well summarized in this US maps (from 1992 to 2014)
the use of the glyph has become more and more intense
where before we had the lowest levels we now have the highest
The agricultural use of glyphosate has increased sharply from 1990 the increase in the use of glyphosate is especially due to the advent of genetic engineering, which has led to the creation of crops genetically modified for the resistance to glyphosate.
That why this trend seems connected with corn and soybeen crops (yellow and green bars), that are the main GM crops in US

4. Glyphosate ADI and EDCs
Current Acceptable Daily Intake (ADI)
EU/Aus/NZ: 0.3 mg/kg/day
China/Russia: 1 mg/kg/day
USA: 1.75 mg/kg/day
Endocrine Disruptors Chemicals (EDCs)
EFSA: pending (by August 2017)
EPA: “...no convincing evidence…”
Safety treshold for human exposure
In the case of glyphosate, the ADI value differs from country to country. It has been set at 0.3 mg per kg of bodyweight per day (written as 0.3 mg/kg bw/d) in Europe, and 1.75mg/kg bw/d in the USA. The calculation to set the ADI is based on the lowest dose considered non-toxic in animal feeding trials (30mg/kg bw/d) sponsored by industry.
0.3. was established based on a no-observed-adverse-effect level (NOAEL) of 31mg/kg bw per day, the highest dose tested in a 26-month study of toxicity in rats
Moreover there is a debate over the potential endocrine disruptive activity of glyphosate

5. Glyphosate and carcinogenicity
IARC: PROBABLY CARCINOGENIC to humans (Group 2A)
EFSA: unlikely to pose a carcinogenic hazard to humans
EPA/ECHA: pending (evaluation is ongoing)
Echa: european chemical agency
ECHA's Committee for Risk Assessment (RAC) is currently developing its opinion on the classification of the substance, also taking into account information provided during the public consultation on glyphosate during the early summer RAC will adopt the final opinion on harmonised classification for glyphosate by the end of November 2017

6. PROBABLY CARCINOGENIC
IARC classification
PROBABLY CARCINOGENIC
“Limited” evidence in humans (association between glyphosate and lympoma in case-control studies)
“Sufficient” evidence in experimental animals (rare malignant tumors in mice and benign tumors in rats)
“Strong ” evidence of genotoxicity for glyphosate and its formulations
Different criteria

7. IARC classification
Systematic review of all pubblished scientific data, by independent experts with no conflicts of interest
Written guidelines, with role and responsability recognized all over the world
More then 1000 studies reviewed
The iarc classification is based on a ….

8. unlikely to pose a carcinogenic hazard
EFSA classification
unlikely to pose a carcinogenic hazard
“Very limited” evidence in humans (dismiss the association without clear explanation)
Considers rare tumors in the animal studies, as ”occasional observation” reflecting an over-reliance of industries studies despite independent ones
Down-weighted the “strong ” evidence of genotoxicity, confirmed glyphosate induces oxidative stress (not relevant for cancer)

9. EFSA classification
The European CLP Criteria allow for a similar classification of probable carcinogens when there are ‘studies showing a limited evidence of carcinogenicity in humans together with limited evidence of carcinogenicity in experimental animals’
if the efsa had followed clp criteria would come to the same conclusion of the IARC

10. RAMAZZINI INTEGRATED PROJECT
ON GLYPHOSATE
With such a background the RI decided to plan a new comprehensive in vivo study using realistic doses in order to help settle the argument over the herbicide's safety 10

11. Comprehensive study plan
Compound tested: GLYPHOSATE and ROUNDUP (Bioflow): mixture of glyphosate and different surfactant (MON 52276)
Preliminary pilot study (ongoing): 90days, prenatal, glyphosate and Roundup tested
Integrated study
The experiment will be based on the integrated experimental design that my collegue dr manservisi just presented;
It includes a preliminary phase that is currently ongoing where we are evaluating generel developmental and toxicological endpoints.
We are testing and we are plan to test both the glyphosate itself and its commercial formulation: it’s a soluble liquid formulation (MON 52276), which will replace the current ROUNDUP(R)
is the lead formulation of the Glyphosate Task Force submission. It was one of the representative formulations supporting the 2001 Annex inclusion of glyphosate. This formulation is still registered in Europe and its composition has not changed.
Glyphosate, 360 grams acid equivalent per litre, present as potassium salt.

12. PRELIMINARY PILOT STUDY12

13. Experimental plan
Route of administration: test compound administered in drinking water ad libitum
GROUP
COMPOUND
DOSE I
Drinking water
CONTROL  II
Glyphosate
USA ADI (1.75 mg/kg/day)
III
Roundup
Duration of treatment: from prenatal-life (GD6) to sexual maturation (developmental arm) and to 90days post-weaning (toxicological arm)
The schedule of treatment
Starting from the same generation starting from gestational day 6 (GD6) we produce 2 different arm of experiment: a subcronic tox (that is a 90 days of direct exposure of offspring) and a reproductive/developmental tox (that is a 70 days of direct exposure of offspring, that is a sexual maturation of the animals.)
The dams that have generated all the offspring of the 2 arm are sacrificed after weaning; the whole litters are monitored during weaning;

14. Aims of the Preliminary Pilot Study
Assess the analytical method for glyphosate detection in different matrices (water and biological samples)
Gather information on the effects of exposures to low doses of glyphosate
Evaluate non-cancer endpoints (endocrine disruptive activities, intestinal microbiome etc…)
Assess the pre-natal exposure and the fertility status of the treated animals in order to plan the long term study
We are exploring numerous end point, related not only to the toxicity
THE STUDY IS PRESENTLY ONGOING AND we suppouse to have the preliminary results at the end of the year

15. Peculiarites Parameters evaluated Collaborators/partners
Intestinal microbiome
Mount Sinai School of Medicine, USA
Hormonal state
National Institute of Health, Dpt. of Food Safety and Veterinary Public Health (IT)
Glyphosate and/or AMPA detection in biological matrix
University of Bologna, department of Veterinary Medical Science (IT)
Epididimal and testicular sperm count and morphology
University of Bologna, department of Veterinary Medical Science (IT)
Sperm aneuploidy
Department of Environmental and Occupational Health, USA
Micronuclei test
National Institute for Cancer Research, Environmental Cancer Institute, Genoa (IT)
All this work is possible thanks to a complex network of collaboration, which I would like to thanks also in this occasion.

16. LONG TERM INTEGRATED PROJECT
With such a background the RI decided to plan a new comprehensive in vivo study using realistic doses in order to help settle the argument over the herbicide's safety 16

17. Aims of the Integrated Study
The study is a comprehensive investigation of the toxicity of glyphosate and Roundup
The study involves:
CARCINOGENICITY AND CHRONIC TOXICITY
REPRODUCTIVE TOXICITY (multigenerational)
DEVELOPMENTAL TOXICITY
NEUROTOXICITY
The general protocol for this study was plan In order to fill the gaps related to the safety of theese compounds;
We proposed to study the real world level of exposure
Different harms based on our proosed integrated projec, and in particular:

18. Study design
Study plan: for each group long-term toxicity/carcinogenicity bioassay is integrated with satellite groups, where endocrine disruptive activity in different WOS (which reflect the real-life exposures of millions of people) will be evaluated
Number of animals: 430 animals per group. The total number of animals required is 3010
Duration of the project: 5 years (3 years of in life phase)

19. Doses: from general population to NOAEL
GROUP
COMPOUND
DOSE I
Drinking water
CONTROL  II
Glyphosate
Human general population exposure
III
USA (1.75 mg/kg/day) or EU (0.30 mg/kg/day) ADI IV
NOAEL (175 mg/kg/day) V
Roundup VI
VII
NOAEL (175 mg/Kg/day)
The dose group will be 7; the discussion over the dose is still open and will be based of the results of the ongoing pilot study, but in general we would like to study 3 doses of glyphosate (real worldgeneral population , ADI and NOAEL) and 3 doses of roundup + 1 control group
The max dose is about 10-fold less than what caused cancer in other studies, but you have 10-fold more animals

20. END POINTS 20

21. End-points/investigations
Phase
Arm of the study (*)
End-points/investigations
Preliminary pilot study
- Sub-chronic toxicity
(Treatment: from GD 6 to PND 120)
Histopathology
Molecular biology of target tissues
Urine analysis
Biochemical and haematological evaluation
Microbiome investigations
Metabolite detection in blood/serum and urine
Breeding, conception and neonatal parameters
Markers of sexual development
Sperm aneuploidy, sperm parameters
Hormonal state
- Reproductive/Developmental Toxicity
(Treatment: from GD 6 to sexual maturity)
Long term integrated study
- Chronic toxicity/carcinogenicity
(Treatment: from GD 6 to weeks of age)
Mechanistic studies
(different schedule of sacrifices)
- Reproductive/Developmental Toxicity in specific Windows Of Susceptibility (WOS)
- Neurotoxicity
(*) : GD: gestation day; PND :post natal day

22. CONCLUSIONS
In conclusion…except that we have a lot of work to do 22

23. Conclusions
We need a comprehensive and integrated study design for Glyphosate and Roundup to detect all risks related to the exposure of the general population (including embryo and children)
We have to investigate cancer and non-cancer end points in the same time, with also low and realistic doses of exposure

24. Conclusions
Whatever the outcomes of the study, we expect to produce solid and independent results that could be used for risk assessment
Meanwhile……
“In view of the uncertainty, one simply must apply the precautionary principle and strictly limit exposure to this substance so that we don’t damage our health”
Statement issued by Cesare Maltoni

25. “The reward of great men is that, long after they have died, one is not quite sure that they are dead”
Jules Renard,