1. The Ramazzini Institute Integrated Experimental Design
Annual Ramazzini Days
The Ramazzini Institute Integrated Experimental Design
Fabiana Manservisi
Cesare Maltoni Cancer Research Center
Carpi, October 28th, 2016

2. Current guidelines
In order to provide you with a background for the explanation of the RI proposal, I’d like to present you several guideline currently used in rodent bioassays, for the assessment of chronic toxicity-carcinogenicity and developmental reproductive toxicity. 2 2

3. Background: chronic toxicity/carcinogenicity
OECD TG 453 “Chronic toxicity/carcinogenicity” (OECD, 2009) : the objective is to characterize both the toxicological response and the carcinogenic potential of a substance.
In utero exposure is not involved
Study is terminated after 2 years (104 weeks) of exposure
NTP 2-year bioassay (NTP, 2011) : the aim is to determine the toxicologic and/or carcinogenic effects of long-term exposure on rats and mice. For some test articles, rats are exposed during the perinatal phase (starting from GD 6).
As regards Chronic toxicity/carcinogenicity, the most recent OECD TG is the 453, which dates back to 2009.
The objective is to characterize both the toxicological response and the carcinogenic potential of a substance.
According to this protocol in utero exposure is not scheduled and animals are sacrificed after 2 years (104 weeks) of exposure.
Secondly we have the NTP TG 2-year bioassay, which dates back to 2011.
The aim is to determine the toxicologic and/or carcinogenic effects of long-term exposure on rats and mice. For some test articles, rats are exposed during the perinatal phase (starting from GD 6).
As in the case above, study is terminated after 2 years of exposure.

4. Background: reproductive/developmental toxicity
OECD TG 416 “two generation toxicity study” (OECD, 2001): rats belonging to F0, F1 and F2 generations are all monitored for reproductive parameters.
High number of animals used (∼2600 rats)
Developmental neurotoxicity and immunotoxicity endpoints are not evaluated
OECD TG 443 “Extended One-generation Reproductive Toxicity Study” (OECD, 2012) : toxicity is tested across life-stages investigating simultaneously parameters for developmental reproductive toxicity, neurotoxicity and immunotoxicity.
F2 generation can be waived
NTP's Modified One-Generation (MOG) Reproduction Study (NTP, 2011): information on the effects of agents on prenatal and postnatal development, and reproduction.
Not included in the regulatory information requirements of REACH
Another important aspect of safatey assessment of chemicals is determining their potential reproductive and developmental toxicity.
OECD TG 416 “two generation toxicity study” was considered “the gold standard” for the assessment of reproductive toxicity. It dates back to Rats belonging to F0, F1 and F2 generations are all monitored for reproductive parameters. Concern is expressed that this TG would lead to a significant number of animals used (more than two thousand). The two-generation toxicity study is also not designed to evaluate developmental neurotoxicity immunotoxicity endpoints, which require standalone studies using more than one thousand animals.
In 2012 the OECD developed a new design termed the “Extended One-generation Reproductive Toxicity Study”. One of the major drivers for this change was the effort to reduce animal use.
The hallmark of this TG is that toxicity is tested across life-stages investigating simultaneously parameters for developmental reproductive toxicity, neurotoxicity and immunotoxicity. Several improvements have been introduced in this new TG.
However, he main objection is connected to the possibility of leaving out the F2 generation thereby running the risk of missing out relevant information in term of protecting the population from potential harmful effect of chemicals.
Another effort in terms of reproductive/developmental toxicity comes from the NTP's Modified One-Generation (MOG) Reproduction Study, whose design provides information on the effects of agents on prenatal and postnatal development, and reproduction. It is not included in the regulatory information requirements of the European directive REACH (registration, evaluation, authorization and restriction of chemicals) to test reproductive toxicity effects of chemicals with production volume higher than 100 tonnes.

5. The RI integrated experimental design
So, Current Guidelines, as planned, are not aimed to monitor simultaneously cancer and non-cancer hazards and risks of exposure on susceptible individuals such as children and the elderly is not always covered.
Due to all these shortcomings and following our experience on long-term rodent bioassays, we started to figure out a new proposal, very simple in the concept, probably not so much in the experimental conducting. Our design integrates traditional cancer guidelines with more recent proposals of OECD and NTP for studying reproductive and developmental toxicity. 5 5

6. Integrated approach
Carcinogenicity, toxicity and reproductive/developmental toxicity endpoints are investigated in a single protocol, with animals of the same generation, exploring windows of susceptibility that are currently not addressed in the other guidelines design.
The important thing here is that traditionally, separate studies are conducted to evaluate these effects.
We propose to adopt an integrate approach according to which carcinogenicity, toxicity and reproductive/developmental toxicity endpoints are investigated in a single protocol, with animals of the same generation, exploring windows of susceptibility that are currently not addressed in the other guidelines design.

7. Toxicity/Carcinogenicity study
The RI protocol includes: prolonged period of exposure/observation of experimental animals, starting exposures from the gestation and continuing through lactation and weaning until at least 130 weeks or longer
To get straight to the heart of the proposal, the design basically includes a Toxicity/Carcinogenicity arm, largely based on the OECD TG 453, modified only for the earlier start and longer duration.
Indeed in standard/typical bioassays animals start exposure at six or eight weeks of age and they are sacrificed after 2-years, which is roughly equivalent to only 65 years in humans. We believe that this period of exposure is too short for a realistic risk assessment, but not only the RI states this: a number of studies demonstrate that extending animal bioassays beyond 2 years and beginning exposure in utero, especially for endocrine-disrupting chemicals that “act” preferentially in early life, would provide more reliable and appropriate indicators of human risk.
Interim kills are also planned ollowing the OECD TG 453, to provide information on progression/regression of non-neoplastic events and neoplastic changes and mechanistic information.

8. Reproductive/Developmental toxicity
The Reproductive/Developmental Toxicity arm mimics human exposure during critical windows of susceptibility (WOS) for the development
For a realistic risk assessment it is also crucial to characterise chemical exposure at the appropriate life stage. Indeed now is well accepted from the entire scientific community that not only the dose makes the poison. Equally important are the time of exposure and the time when effects become visible.
The Reproductive/Developmental Toxicity arm mimics human exposure during critical windows of suceptibility (WOS) for the development
and other non-cancer effects are studied. The possible adverse effects of the substances are studied in prenatal, neonatal, prepubertal, pubertal, adult parous and nulliparous WOS, and these effects are compared between each WOS, or with the possible long-term carcinogenicity effect.
Please, notice that parous animals are mated once adult in order to generate the F2 pups, in order to obtain toxicity information throughout the reproductive cycle.

9. Number of animals
Our integrated experimental protocol requires 1720 animals for each tested compound,
Multiple toxicological endpoints together are investigated, sparing animal lives in accordance with the 3Rs rule
Reduction up to 53% in animal use as compared to using separate test protocols,
* Considering 15pups/litter in F2 generation (OECD TG 443 generates F2 only if triggered, while NTP MOG and RI include F2 generation by default)
Source material
Number of animals
OECD TG 453 (Chronic toxicity/ Carcinogenicity)
480
OECD TG 443 (Extended One-generation Reproductive Toxicity Study)
1760*
NTP MOG (Modified One-generation Study)
3200*
Total Animals: (OECD 453+OECD 443 or NTP MOG)
RI Integrated project
1720*
An additional challenge to moving forward is the use of 3R models (replace,reduce and refine), laid down in the European Directive 63/2010 on the protection of animals used for scientific purposes.
Our integrated experimental protocol requires 1720 animals,
In our protocol we have a reduction up to 53% in animal use as compared to using separate test protocols, indeed up to 3680 animals
are expected to be required to performing these studies separately.

10. Advantages of the RI proposal
Carcinogenicity, toxicity and reproductive/developmental toxicity endpoints are investigated in a single protocol
Windows of susceptibility
Rats from the same generation (opportunity to compare the results among each arm)
Sparing of animal lives in accordance with the 3Rs rule
The costs are lower than the required to conduct several separte studies
Let me summarize what the advantages of the RI proposal are:
Carcinogenicity, toxicity and reproductive/developmental toxicity endpoints are investigated in a single protocol
Different windows of susceptibility related to reproductive/developmental and other non-cancer effects are studied.
We use rats from the same generation, what gives us the opportunity to compare the results among each arm by minimizing variables
By adopting our protocol there is a spare of animal lives in accordance with the 3Rs rule as compared to using separate test protocols
The cost are lower than the required to conduct several separte studies

11. Challenges of the RI proposal
It is a flexible protocol that could lead to uncertainties in terms of how to conduct the test and how to deal with the additional data obtained
A validation is required: we are planning our first integrated project on Glyphosate/Roundup
Labour intensive testing procedure
The RI proposal is also not without some challenges:
It is a flexible protocol which could be tailor made. This is on the one hand an advantage but on the other hand it could lead to uncertainties in terms of how to conduct the test and how to deal with the additional data obtained
The design and protocol requires a validation in order to demonstrate that the combined test is feasible and is at least as good as the separate tests. We are planning our first integrated project on Glyphosate/Roundup. My colleague Dr. Simona panzacchi will give you further details of this right after me.
It’s a labour intensive testing procedure

12. Conclusion
For nearly five decades long-term studies in rodents have been the accepted benchmark for assessing chronic long-term toxic effects, particularly carcinogenicity, of chemicals.
Current set of internationally utilized test methods only capture some of the potential adverse effects.
There is a need to stimulate new adaptive approaches that break down institutional and traditional scientific barriers.
Interdisciplinary and multidisciplinary team science should be stimulated
An overall reduction in the use of resources (animals, costs, time…)
That bring me to the conclusion of my presentation:
For nearly five decades long-term studies in rodents have been the accepted benchmark for assessing chronic long-term toxic effects, particularly carcinogenicity, of chemicals.
The European Food Safety Authority (EFSA) and the World Health Organization (WHO) have pointed out that the current set of internationally utilized test methods capture only some of the potential adverse effects associated with exposures to these agents over the lifetime.
There is a need to stimulate new adaptive approaches that breack down institutional and traditional scientific barriers and
Interdisciplinary and multidisciplinary team science should be stimulate for a more holistic approach.
The benefits of consolidating carcinogenicity and reproductive/developmental toxicity studies into one large study could accomplish an overall reduction in the use of resources (animals, costs, time…)

13. Conclusion Our work was recent published on EHP.
I have to thank Dr. Belpoggi and all the Ramazzini team, in particular Dr. Babot Marquillas. She was a Visiting Researcher from the University of Barcelona A great contribution also came from Dr.Silbergeld and Dr.Huff, our coauthors and fellows of the Collegium Ramazzini, with decades of experience in planning bioassays. We are quite confident that conducting such integrated bioassays could enhance the scientific information available for making sound decisions that protect public health. This is our most important goal and this was also the Maltoni’s vision many years ago, but still extremely topical in our current society.

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