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NAMs reach and reproductive toxicity
- 1. Pomezia - 07 Febbraio 2018 Future role of NAMs (New Approach Methodologies) in the assessment of reproductive toxicity Costanza Rovida– TEAM mastery S.r.l.
- 2. Pomezia - 07 Febbraio 2018 University Konstanz
- 3. Pomezia - 07 Febbraio 2018 REACH and alternative methods • Article 1: – The purpose of this Regulation is to ensure a high level of protection of human health and the environment, including the promotion of alternative methods for assessment of hazards of substances, … • Article 13: – … for human toxicity, information shall be generated whenever possible by means other than vertebrate animal tests, through the use of alternative methods, for example, in vitro methods or qualitative or quantitative structure-activity relationship models or from information from structurally related substances (grouping or read-across). … – Where tests on substances are required to generate information on intrinsic properties of substances, they shall be conducted in accordance with the test methods laid down in a Commission Regulation or in accordance with other international test methods recognised by the Commission or the Agency as being appropriate. … Information on intrinsic properties of substances may be generated in accordance with other test methods provided that the conditions set out in Annex XI are met.
- 4. Pomezia - 07 Febbraio 2018 • ANNEX VI – STEP 2: CONSIDER INFORMATION NEEDS: …These Annexes set out the standard information requirements, but shall be considered in conjunction with Annex XI, which allows variation from the standard approach, where it can be justified… • STEP 4 — GENERATE NEW DATA/PROPOSE TESTING STRATEGY … New tests on vertebrates shall only be conducted or proposed as a last resort when all other data sources have been exhausted REACH ANNEX VI
- 5. Pomezia - 07 Febbraio 2018 … In addition to these specific rules, a registrant may propose to adapt the required standard information set out in column 1 of this Annex according to the general rules contained in Annex XI. … … Before new tests are carried out to determine the properties listed in this Annex, all available in vitro data, in vivo data, historical human data, data from valid (Q)SARs and data from structurally related substances (read-across approach) shall be assessed first. In vivo testing with corrosive substances at concentration/dose levels causing corrosivity shall be avoided. Prior to testing, further guidance on testing strategies should be consulted in addition to this Annex. REACH ANNEX IX and X
- 6. Pomezia - 07 Febbraio 2018 1. TESTING DOES NOT APPEAR SCIENTIFICALLY NECESSARY 1. Use of existing data 2. Weight of evidence 3. Qualitative or Quantitative structure-activity relationship ((Q)SAR) 4. In vitro methods 5. Grouping of substances and read-across approach 2. TESTING IS TECHNICALLY NOT POSSIBLE 3. SUBSTANCE-TAILORED EXPOSURE-DRIVEN TESTING REACH ANNEX XI
- 7. Pomezia - 07 Febbraio 2018 1.4. In vitro methods ……. If the results obtained from the use of such in vitro methods do not indicate a certain dangerous property, the relevant test shall nevertheless be carried out at the appropriate tonnage level to confirm the negative result, unless testing is not required in accordance with Annexes VII to X or the other rules in this Annex. Such confirmation may be waived, if the following conditions are met: (1) results are derived from an in vitro method whose scientific validity has been established by a validation study, according to internationally agreed validation principles; (2) results are adequate for the purpose of classification and labelling and/or risk assessment; and (3) adequate and reliable documentation of the applied method is provided.
- 8. Pomezia - 07 Febbraio 2018 NAMs: Origin Two motivating drivers for the workshop were: •A better understanding of the underlying biology behind how chemicals cause adverse effects to human health; and •New tools and techniques that provide a huge amount of data to be used in solving regulatory issues.
- 9. Pomezia - 07 Febbraio 2018 NAMs: Definition In the ECHA Report, NAMs were taken in a broad context -in silico approaches -in chemico and in vitro assays -information from the exposure of chemicals -high-throughput screening tools -Stem cell biology -high-content methods e.g. genomics, proteomics, metabolomics -toxicokinetic or toxicodynamic knowledge for substances -Any methods to improve understanding of toxic effects
- 10. Pomezia - 07 Febbraio 2018 Validity status of reprotox testing 1)Not robust (about 25% equivocal studies), [Bailey et al., 2005] 2)74 industrial chemicals tested in New Chemical Database: 34 showed effects on offspring, but only 2 chemicals have been classified as developmental toxic [Bremer & Hartung, 2004] 3)55% of positives in screening studies not in multi-generation studies [Bremer & Hartung, 2004] 4)Group size limits statistical power [Hotchkiss 2008] 5)61% inter-species correlation [Hurrt 2003, Bailey 2005] Moreover a)Lack of objectivity of in vivo tests b)Tests at high concentration ALTEX 2018, 35:139-162
- 11. Pomezia - 07 Febbraio 2018 Report from the National Research Council LIMITATIONS OF CURRENT TESTING STRATEGIES -Very long and expensive: Few chemicals are fully charcaterised -Animal-to-Human Extrapolation -Too many false positive -Low-Dose Extrapolation from High- Dose Data -Poor Reproducibility -Limited applicability (Mixtures, nano particles, etc.) 2007: beginning of the Tox21c adventure
- 12. Pomezia - 07 Febbraio 2018 - Read across justification - Mechanism elucidation of specific concerns - Screening (prioritisation or lead selection) - Quick assessment - New cosmetics ingredients NAMs for Reproductive Toxicity: Today
- 13. Pomezia - 07 Febbraio 2018 The ReProTect Project 2005-2010
- 14. Pomezia - 07 Febbraio 2018 Use of bovine gametes for reproductive toxicity testing bovine ovaries 2 cm bovine oocytes Endpoint: % Metaphase II Chemical exposure (24h) during IVM In vitro maturation (IVM) of bovine oocytes Endpoint: % In vitro fertilisation (IVF) of bovine oocytes Chemical exposure (20h) during IVF IVM, 24 h
- 15. Pomezia - 07 Febbraio 2018 https://ecvam-dbalm.jrc.ec.europa.eu/topic-summaries
- 16. Pomezia - 07 Febbraio 2018 Zebrafish Embryo Assay A. Embryos collected after spawning (1 hpf) B. Late blastula to early gastrula period embryos (4–6 hpf) C. Embryo removed from the chorion (24 hpf) D. End of the embryonic period (3dpf) E. Early stage larvae (5dpf) ILAR Journal, 2016, Vol. 57, No. 2
- 17. Pomezia - 07 Febbraio 2018 Horst Spielman – EFSA Symposium 2015
- 18. Pomezia - 07 Febbraio 2018 Induced pluripotent stem cells (iPSC)
- 19. Pomezia - 07 Febbraio 2018 Mini-brains Hogberg et al., Stem Cell Res 2013 Pamies et al., Exp Biol Med 2014 Pamies et al., Altex 2017
- 20. Pomezia - 07 Febbraio 2018 Mini-brains are spontaneously electrophysiologically active
- 21. Pomezia - 07 Febbraio 2018 Zika Virus Infects Human Cortical Neural Progenitors and Attenuates Their Growth Tang, H. et al. Cell Stem Cell 18, 1–4, May 5, 2016 Neural stem cells (NSCs) Ventricular zone (VZ) Subventricular zone (SVZ) Inner and Outer SVZ (ISVZ and OSVZ) Intermediate zone (IZ) Cortical plate (CP) Kelava and Lancaster (2016). Stem Cell Models of Human Brain Development. Cell Stem Cell Toxicology and Applied Pharmacology 354 (2018) 3–6
- 22. Pomezia - 07 Febbraio 2018 Homogeneity vs Complexity (A) Relationship of the complexity of the cells/tissue produced by individual protocols and the homogeneity of the cells/tissues generated (B) Individual regions of cortical tissue from various methods with their relative sizes.
- 23. Pomezia - 07 Febbraio 2018 Annex A: Good Cell culture Practice Annex B: Good Cell culture Practice for stem cells and stem-cell derived models
- 24. Pomezia - 07 Febbraio 2018 a. The limitations of animal tests should be systematically reviewed (EBT). b. The zebrafish teratogenicity assay and other tests should be further assessed and used. c. Human EST (hESC or IPSC) for replacing the 2nd species in reproductive toxicity; d. in silico opportunities: a. systematic and holistic analysis of reproductive toxicity data of adequate quality b. Training sets for discrete reproductive endpoints c. The combined use of multiple types of tools and approaches e. The TTC approaches can be further refined through distinguishing classes of chemicals NAMs for Reproductive Toxicity: the way towards the future
- 25. Pomezia - 07 Febbraio 2018 Not our goal!
- 26. Pomezia - 07 Febbraio 2018
- 27. Pomezia - 07 Febbraio 2018 Microphysylogical Systems (Human on a chip)
- 28. Pomezia - 07 Febbraio 2018
- 29. Pomezia - 07 Febbraio 2018 Summary Traditional approach in vitro methods 3D system Species specific correlation - + / - +++ Reproducibility + / - +++ + Time /money demand - + + + + Applicability domain + (-) - - (+) Test on intact organisms + - + / - Well accepted by Regulators + + / - ? + / - ? Quantity of test item - + + Mechanistic elucidation + / - + + +
- 30. Pomezia - 07 Febbraio 2018 Vision for the future of toxicity testing Altex 31, 3/14
- 31. Pomezia - 07 Febbraio 2018 Vision for the future of toxicity testing Altex 31, 3/14
- 32. Pomezia - 07 Febbraio 2018 No balloon and no aeroplane will ever be practically successful (1902) Heavier-than-air flying machines are impossible (1895) William Thomson, known as Lord Kelvin (1824-1907), President of the Royal Society and inventor of the absolute temperature scale
- 33. Pomezia - 07 Febbraio 2018 Thank you for your attention!!! Questions ?
Editor's Notes
- I will try to explain you why EOGRTS is not always necessary and eventually in the future all in vivo studies will be dismissed
- CAAT Europe is located at the University of Konstanz I Germany. It is within the department of in vitro toxicology in the Faculty of Biology, under the responsibility of Marcel Leist. Thomas Hartung is the Director of CAAT that’ in th eUS
- Let’s first frame the applicability of alternative methods within REACH Any OECD guidelines is immediately applicable
- Annex VI sets the procedure to prepare the registration dossier
- It is not true that in vivo tests are mandatory in REACH
- What’s in Annex XI? It is true that the applicability of these principles in the area of reproductive toxicity is not as straightforward. Now we’re gaining experience with “easier” endpoint, but we’re paving the way also towards other more complex assessments At the moment, Annex XI is mainly applied as WoE when there are only old and non-GLP studies. Please, don’t under estimate exposure. There are many cases when the exposure is really negligible and test should be really waived.
- Please, let me highlight also a common misconception that only in vitro positive results are accepted. This is the case only for non validated methods. I know that in the scope of reproductive toxicity we’re still far from this situation, but keep it in mind
- In 2016, ECHA organised a very important workshop that decided a new way to consider the approach. Two motivating drivers… For the first time they used the terminology “NAMs” to indicate something that is disruptive with the past.
- The new approach is completely new embracing…
- It should be acknowledged that in vivo methods have some limitations. Regarding reproductive toxicity… Also the EOGRTS guideline contains no recommendation for human relevance of the animal data. Human exposure is several order of magnitude lower than doses used in animal studies.
- These doubts are the same that lead to the publication of the very famous book about the toxicology in the 21st century. In 2007 the NIH asked for a report to understand how to change the approach to solve two main problems: Few chemicals have a full toxicological characterization There is concern on some chemicals that do not have explanations What’s written in this book is that the traditional model is based on animals
- Let’s go back to NAMs and reproductive toxicity to understand what is possible today Read across- limits QAAR in a broad range is still limited, but it is very helpful to elucidate specific mechanisms Screening is also very important. It is clear that extensive studies such as the EOGRTS cannot be applied broadly Another area is the assessment during emergences, when there is simply no time to wait for the results of a long tests. This is what happened during the explosion of the Deepwater Horizon, the oil drilling platform. In 8 weeks, some dispersants where tested through the ToxCast program. One was selected and used with no known drawbacks.
- If we focus our attention to the area of reproductive toxicity, we clearly face the complexity of this process. In vitro methods cannot replace the whole cycle, but only step by step. This picture represents the outcome of the EU integrated project ReProTect… Those acronyms refer to accepted tests by ECVAM. I ma not going to talk about endocrine disruptors because it is too complex. Maybe RTC will organise a dedicated workshop in the future.
- I’d like to show you a practical example. We had the suspect that one sample could degrade to form a product which is classified H361FD, toxic for reproduction category 2 so we compared our product with the standard. We used these ReproTEct methods with tests on specific reproductive toxicity on bovine oocytes during the process of in vitro maturation and in vitro fertilisation (bovine oocyte in vitro maturation test, bIVM; The purpose of the oocyte maturation test is to monitor the chemical effects on the oocytes with special reference to the nuclear configuration changes bovine oocyte in vitro fertilisation test, bIVF; The bIVF test mimics the in vivo process of fertilisation and allows to evaluate the effects of chemical exposure both on the sperm (ability to penetrate the oocyte) and on the oocyte (oocyte activation and formation of 2 pronuclei) we also tested in vitro bovine embryo culture test, bIVC during the first 8 days. The bIVC test mimics the period of embryo development during which the embryos freed from the zona start increase in size and prepare for implantation in the uterine wall These tests clearly showed positive results from the standard sample in the IVM and IVF while no effect was recorded for our sample. A pronucleus (plural: pronuclei) is the nucleus of a sperm or an egg cell during the process of fertilization, after the sperm enters the ovum, but before the genetic material of the sperm and egg fuse. Sperm and egg cells are haploid, meaning they carry half the number of chromosomes of somatic cells. The male and female pronuclei don't fuse, although their genetic material does. Instead, their membranes dissolve, leaving no barriers between the male and female chromosomes. Their chromosomes can then combine and become part of a single diploid nucleus in the resulting embryo, containing a full set of chromosomes.
- There are many tests like that. If you need to find them, please visit the EURL ECVAM website, in the DB Alm section Those are listed as INVITTOX protocol. In addition to the ReProTect methods, there are others on Immunotoxicity and developmental neurotoxicity. I cannot talk about all. Just a couple of examples
- hours post-fertilization (hpf) Alternative Models of Developmental and Reproductive Toxicity in Pharmaceutical Risk Assessment and the 3Rs Same principle with drosophila and C-elegans
- Cyprotex: Spontaneously beating 3D cardiac microtissue
- The approach is getting complex and we need a new way to organise the information
- The human brain develops through a series of developmental stages that must occur in a particular sequence and at the right time. The current reliance on DNT testing in rodents to predict effects in humans is called into question. It’s not science fiction and first applications are already available For example, it was used to demonstrate the activity of the Zika virus that affects the embryo neural cells causing immediate death or defective maturation. The possibility to isolate the precise step when the effect occurs is also very helpful to investigate the pharmacological solution. There are also studies that demonstrate the difference between human and rats, the latter resembling in vivo experiments, but different than the experiment performed with human material
- When developing or selectin a methods we should find out the good balance between simplicity and costs SFEB Serum Free Calcium of Embryonic bodies
- EBTC against in vivo methods Zebra Fish
- We are not 70Kg rats, but we’re also not a 70Kg cells. We need to move towards a higher awareness of the mechanism behind a possible effect
- Non sto a entrare nei dettagli dei tipi di cellule che si possono utilizzare
- 6 millions from EPA to develop The primary goal of the new center is to develop a series of 3-D human cell cultures that are heavily wired up with different sensors to record how they respond when exposed to small concentrations of potentially toxic chemicals.
- include: exposure differences between in vitro and in vivo studies, accumulation of the compound in the fetal compartment, maternal metabolism, altered maternal status (e.g., hypoglycemia, hemodynamic changes), effects on placental function, and a sensitive window or other mechanistic aspect missing from the alternative assay No in vitro model is perfect, but given the everincreasing rate of technologic change (see Kurzweil 2005), one can realistically imagine a time in the not-too-distant future when the bulk of safety assessment work will be done in these sorts of constructs, particularly as they represent more and more of the natural complexity of the in vivo situations, and the results from these models will enable scientists to make reliable predictions of what would happen in vivo
- The current approach is first to test unknown chemicals in animal tests. This limits overall throughput and leads to a high rate of false positives and false negatives. Mixtures are hardly ever tested because of the limitation of resources. High costs in combination with a low predictivity lead to many cases of “no testing”. Mechanistic studies are only carried out in few cases of particular interest to identify the factors causing the toxicity. The new approach, suggested here, is based on 21st century in silico and in vitro methods identifying PoT. This will in most cases lead to an amount of data that is sufficient to decide whether a substance is toxic (positive) or non-toxic (negative) for the intended scenario. Only in few cases, when not enough information can be obtained, will animal tests be performed as an additional source of information. Good information can be provided on all chemicals and, due to the high throughput of the approach, also on mixtures.
- Hypothesis driven testing The current approach is first to test unknown chemicals in animal tests. This limits overall throughput and leads to a high rate of false positives and false negatives. Mixtures are hardly ever tested because of the limitation of resources. High costs in combination with a low predictivity lead to many cases of “no testing”. Mechanistic studies are only carried out in few cases of particular interest to identify the factors causing the toxicity. The new approach, suggested here, is based on 21st century in silico and in vitro methods identifying PoT. This will in most cases lead to an amount of data that is sufficient to decide whether a substance is toxic (positive) or non-toxic (negative) for the intended scenario. Only in few cases, when not enough information can be obtained, will animal tests be performed as an additional source of information. Good information can be provided on all chemicals and, due to the high throughput of the approach, also on mixtures.
- 1895, 8 anni prima del volo dei fratelli Wright