EU REACH regulation changed the way to do chemical risk assessment. All chemicals marketed or manufactured in the EU must have its own dossier. Non standard methods including alternatives to animal testing are accepted.
Half Italian, half English
This presentation provides a knowledge about Toxicology, its types , definition, regulatory guidelines for conducting toxicological studies, OECD guidelines for GLP. This is an assignment in the subject, Pharmacological & Toxicological Screening Methods - II, 2nd Semester, M.Pharm (Pharmacology)
Toxicology is the branch of science that deals with nature, effects, and detection of poison. The degree to which a substance can harm an organism is called toxicity. The types of toxicity depending upon the time of exposure of the toxicant have been described.
This presentation provides a knowledge about Toxicology, its types , definition, regulatory guidelines for conducting toxicological studies, OECD guidelines for GLP. This is an assignment in the subject, Pharmacological & Toxicological Screening Methods - II, 2nd Semester, M.Pharm (Pharmacology)
Toxicology is the branch of science that deals with nature, effects, and detection of poison. The degree to which a substance can harm an organism is called toxicity. The types of toxicity depending upon the time of exposure of the toxicant have been described.
Toxicology testing, also known as safety assessment, or toxicity testing, is conducted to determine the degree to which a substance can damage a living or non-living organism. It is often conducted by researchers using standard test procedures to comply with governing regulations, for example for medicines and pesticides. Much toxicology is considered to be part of the field of preclinical development. Stages of in vitro and in vivo research are conducted to determine safe doses of exposure in humans before a first-in-man study. Toxicology testing may be conducted by the pharmaceutical industry, biotechnology companies or contract research organizations.
Drug development is a high-risk enterprise. The typical new drug takes 10-12 years to get to market and costs up to $500 million. Pharmaceutical companies face continually increasing challenges in drug development— shorter product life cycles, global competition, as well as daunting technical and regulatory hurdles. Meanwhile, as a result of the Human Genome Project and high throughput drug development methods, there are many more drug candidates to test. Thus, there is growing pressure on pharmaceutical and biopharmaceutical companies.
Toxicology testing, also known as safety assessment, or toxicity testing, is conducted to determine the degree to which a substance can damage a living or non-living organism. It is often conducted by researchers using standard test procedures to comply with governing regulations, for example for medicines and pesticides. Much toxicology is considered to be part of the field of preclinical development. Stages of in vitro and in vivo research are conducted to determine safe doses of exposure in humans before a first-in-man study. Toxicology testing may be conducted by the pharmaceutical industry, biotechnology companies or contract research organizations.
Drug development is a high-risk enterprise. The typical new drug takes 10-12 years to get to market and costs up to $500 million. Pharmaceutical companies face continually increasing challenges in drug development— shorter product life cycles, global competition, as well as daunting technical and regulatory hurdles. Meanwhile, as a result of the Human Genome Project and high throughput drug development methods, there are many more drug candidates to test. Thus, there is growing pressure on pharmaceutical and biopharmaceutical companies.
There are tens of thousands of man-made chemicals to which humans are exposed, but only a fraction of these have the extensive in vivo toxicity data used in most traditional risk assessments. This lack of data, coupled with concerns about testing costs and animal use, are driving the development of new methods for assessing the risk of toxicity. These methods include the use of in vitro high-throughput screening assays and computational models.
This presentation by Dr. Richard Judson reviewed a variety of high-throughput, non-animal methods being used at the U.S. EPA to screen chemicals for a variety of toxicity endpoints, including methods for providing mechanistic data like the Adverse Outcome Pathway.
EPA is committed to sound science, and we are proud to have some of the world's best scientists, many of whom are internationally recognized as leaders in their fields. Not only are EPA's scientific experts vital to achieving our mission, but they are dedicated to sharing knowledge and contributing to their the scientific communities, which helps further advance the science that protects human health and the environment. Part of this includes giving presentations to other members of the scientific community. We have posted some of these presentations here so that more people have access.
Learn more about Dr. Richard Judson - https://www.epa.gov/sciencematters/meet-epa-researcher-richard-judson
Learn more about EPA's Chemical Safety Research - https://www.epa.gov/chemical-research
European Commission Joint Research Center presents chemical screening methodology for the impact assessment on criteria to identify endocrine disruptors
A technical meeting on the JRC methodology for evidence screening of chemicals developed in the context of the Impact Assessment on criteria to identify Endocrine Disruptors took place on 6 November 2015 in Brussels.
The aim of this technical meeting was to present to Member States, Members of the European Parliament, countries from outside the EU and stakeholders the methodology developed by the JRC to estimate which chemicals may fall under the different options for criteria to identify endocrine disruptors as outlined in the roadmap. This methodology was developed in the framework of the impact assessment carried out by the European Commission on criteria to identify endocrine disruptors, in the context of the Plant Protection Products Regulation (EC) 1107/2009 and the Biocidal Products Regulation (EU) 528/2012.
Sources
* Methodology for EU EDC screening http://ec.europa.eu/health/endocrine_disruptors/docs/ev_20151106_co01_en.pdf
* Selection of chemical substances to be screened in the context of the impact assessment on criteria to identify endocrine disruptors http://ec.europa.eu/health/endocrine_disruptors/docs/impactassessment_chemicalsubstancesselection_en.pdf
The compound characterization market is growing increasingly profitable and competitive at the same time. In order to develop a new compound product, the testing step is indispensable. Unlike drug discovery, compound testing is not as restrictive, but understanding the main workflow is still necessary to excel in the market. In order to help you improve both the efficiency and safety of compound testing, we developed the protocol to assist you in your findings.
Good Model Organism for Anti Aging TestingWenlan Hu
Drug testing is taking more attention than ever before in a fast growing market for longevity compounds. In order to succeed in a competitive market and develop a pipeline method for quick drug development, we need to understand and choose the most suitable model organism for aging studies. The following content is intended to provide information on how to choose the best model for anti-aging drug testing.
Alternative animal model for compound characterizationWenlan Hu
To pick a model is not easy thing to do, especially when nutraceutical market needs have to be met. Such market requires low budget, fast turn-around rate, and high-quality data at the same time. Traditional models such as mice and cell cultures usually only meet one need at one time. IVB uses C.elegans and zebrafish which lies in the middle of the model spectrum in terms of the three needs to perfectly accommodate all of them at the same time.
How to model longevity and health effect in 3 monthsWenlan Hu
Healthspan analysis is a huge part of longevity study, which is heavily looked at by many aging labs in recent years. The older we grow, our health will be affected accordingly, and this influence what type of compound we use. To better picture what effects compounds brings to us and to extend lifespan as a goal. A three stage compound assessment is developed by IVB to solve this question.
Finding Optimal Compound Dosage for Anti-Aging DrugsWenlan Hu
Anti-aging compound becomes a very integral part of the compound market. However, the lack of experience in this field makes it very hard for testing CROs to fully understand the mechanism of actions as well as efficacy of the compound, particularly the optimal dosage for anti-aging use. In the following slide we are trying to share with you the best way to do testing on the substances that are designed for anti-aging use.
Animal Experimentation for Cosmetics - Resources for Healthy Children www.scribd.com/doc/254613619 - For more information, Please see Organic Edible Schoolyards & Gardening with Children www.scribd.com/doc/254613963 - Gardening with Volcanic Rock Dust www.scribd.com/doc/254613846 - Double Food Production from your School Garden with Organic Tech www.scribd.com/doc/254613765 - Free School Gardening Art Posters www.scribd.com/doc/254613694 - Increase Food Production with Companion Planting in your School Garden www.scribd.com/doc/254609890 - Healthy Foods Dramatically Improves Student Academic Success www.scribd.com/doc/254613619 - City Chickens for your Organic School Garden www.scribd.com/doc/254613553 - Huerto Ecológico, Tecnologías Sostenibles, Agricultura Organica www.scribd.com/doc/254613494 - Simple Square Foot Gardening for Schools - Teacher Guide www.scribd.com/doc/254613410 - Free Organic Gardening Publications www.scribd.com/doc/254609890 ~
Read-across (RAx) is probably the most used strategy to waive new demanding in vivo tests for toxicological assessment of chemicals. It is based on the possibility to translate available information from well-characterized chemicals (source) to the substance for which there is a toxicological data gap (target). In spite of the widespread use, regulatory acceptance is still limited,
New Approach Methods (NAM) may be used to confirm chemical and toxicological similarities and to contribute to the reduction of the uncertainty.
Some slides are in Italian
Summary and description of the principle of investigative toxicology, that is the use of in vitro methods and microphysiological systems for a better prediction of the effect of a drug into the huamn organism
QIVIVE extrapolation requires a precise correlation between exposure and the effective chemical concentration at the site where the MIE occurs.
This work demonstrates that intracellular distribution is not ruled only by physical-chemical parameters, rather it is mainly regulated by specific biological-mediated mechanisms. Substances with
apparent chemical similarity may show different distribution profile, as shown by the intra-nuclear distribution of polyphenols. While our results derive from a limited number of substances applied to
one cell line, it is plausible that using different substances and/or different cell lines would also have shown that intracellular distribution is not directly related to physical-chemical parameters.
Chemical uptake should be specifically measured and simple extrapolations based on physical-chemical properties may provide misleading decision
Testing strategy for the assessment of skin and eye irritation potential of chemicals, mainly in the scope of REACH.
The presentation is partially in Italian
Professional air quality monitoring systems provide immediate, on-site data for analysis, compliance, and decision-making.
Monitor common gases, weather parameters, particulates.
This pdf is about the Schizophrenia.
For more details visit on YouTube; @SELF-EXPLANATORY;
https://www.youtube.com/channel/UCAiarMZDNhe1A3Rnpr_WkzA/videos
Thanks...!
Slide 1: Title Slide
Extrachromosomal Inheritance
Slide 2: Introduction to Extrachromosomal Inheritance
Definition: Extrachromosomal inheritance refers to the transmission of genetic material that is not found within the nucleus.
Key Components: Involves genes located in mitochondria, chloroplasts, and plasmids.
Slide 3: Mitochondrial Inheritance
Mitochondria: Organelles responsible for energy production.
Mitochondrial DNA (mtDNA): Circular DNA molecule found in mitochondria.
Inheritance Pattern: Maternally inherited, meaning it is passed from mothers to all their offspring.
Diseases: Examples include Leber’s hereditary optic neuropathy (LHON) and mitochondrial myopathy.
Slide 4: Chloroplast Inheritance
Chloroplasts: Organelles responsible for photosynthesis in plants.
Chloroplast DNA (cpDNA): Circular DNA molecule found in chloroplasts.
Inheritance Pattern: Often maternally inherited in most plants, but can vary in some species.
Examples: Variegation in plants, where leaf color patterns are determined by chloroplast DNA.
Slide 5: Plasmid Inheritance
Plasmids: Small, circular DNA molecules found in bacteria and some eukaryotes.
Features: Can carry antibiotic resistance genes and can be transferred between cells through processes like conjugation.
Significance: Important in biotechnology for gene cloning and genetic engineering.
Slide 6: Mechanisms of Extrachromosomal Inheritance
Non-Mendelian Patterns: Do not follow Mendel’s laws of inheritance.
Cytoplasmic Segregation: During cell division, organelles like mitochondria and chloroplasts are randomly distributed to daughter cells.
Heteroplasmy: Presence of more than one type of organellar genome within a cell, leading to variation in expression.
Slide 7: Examples of Extrachromosomal Inheritance
Four O’clock Plant (Mirabilis jalapa): Shows variegated leaves due to different cpDNA in leaf cells.
Petite Mutants in Yeast: Result from mutations in mitochondrial DNA affecting respiration.
Slide 8: Importance of Extrachromosomal Inheritance
Evolution: Provides insight into the evolution of eukaryotic cells.
Medicine: Understanding mitochondrial inheritance helps in diagnosing and treating mitochondrial diseases.
Agriculture: Chloroplast inheritance can be used in plant breeding and genetic modification.
Slide 9: Recent Research and Advances
Gene Editing: Techniques like CRISPR-Cas9 are being used to edit mitochondrial and chloroplast DNA.
Therapies: Development of mitochondrial replacement therapy (MRT) for preventing mitochondrial diseases.
Slide 10: Conclusion
Summary: Extrachromosomal inheritance involves the transmission of genetic material outside the nucleus and plays a crucial role in genetics, medicine, and biotechnology.
Future Directions: Continued research and technological advancements hold promise for new treatments and applications.
Slide 11: Questions and Discussion
Invite Audience: Open the floor for any questions or further discussion on the topic.
Seminar of U.V. Spectroscopy by SAMIR PANDASAMIR PANDA
Spectroscopy is a branch of science dealing the study of interaction of electromagnetic radiation with matter.
Ultraviolet-visible spectroscopy refers to absorption spectroscopy or reflect spectroscopy in the UV-VIS spectral region.
Ultraviolet-visible spectroscopy is an analytical method that can measure the amount of light received by the analyte.
Cancer cell metabolism: special Reference to Lactate PathwayAADYARAJPANDEY1
Normal Cell Metabolism:
Cellular respiration describes the series of steps that cells use to break down sugar and other chemicals to get the energy we need to function.
Energy is stored in the bonds of glucose and when glucose is broken down, much of that energy is released.
Cell utilize energy in the form of ATP.
The first step of respiration is called glycolysis. In a series of steps, glycolysis breaks glucose into two smaller molecules - a chemical called pyruvate. A small amount of ATP is formed during this process.
Most healthy cells continue the breakdown in a second process, called the Kreb's cycle. The Kreb's cycle allows cells to “burn” the pyruvates made in glycolysis to get more ATP.
The last step in the breakdown of glucose is called oxidative phosphorylation (Ox-Phos).
It takes place in specialized cell structures called mitochondria. This process produces a large amount of ATP. Importantly, cells need oxygen to complete oxidative phosphorylation.
If a cell completes only glycolysis, only 2 molecules of ATP are made per glucose. However, if the cell completes the entire respiration process (glycolysis - Kreb's - oxidative phosphorylation), about 36 molecules of ATP are created, giving it much more energy to use.
IN CANCER CELL:
Unlike healthy cells that "burn" the entire molecule of sugar to capture a large amount of energy as ATP, cancer cells are wasteful.
Cancer cells only partially break down sugar molecules. They overuse the first step of respiration, glycolysis. They frequently do not complete the second step, oxidative phosphorylation.
This results in only 2 molecules of ATP per each glucose molecule instead of the 36 or so ATPs healthy cells gain. As a result, cancer cells need to use a lot more sugar molecules to get enough energy to survive.
Unlike healthy cells that "burn" the entire molecule of sugar to capture a large amount of energy as ATP, cancer cells are wasteful.
Cancer cells only partially break down sugar molecules. They overuse the first step of respiration, glycolysis. They frequently do not complete the second step, oxidative phosphorylation.
This results in only 2 molecules of ATP per each glucose molecule instead of the 36 or so ATPs healthy cells gain. As a result, cancer cells need to use a lot more sugar molecules to get enough energy to survive.
introduction to WARBERG PHENOMENA:
WARBURG EFFECT Usually, cancer cells are highly glycolytic (glucose addiction) and take up more glucose than do normal cells from outside.
Otto Heinrich Warburg (; 8 October 1883 – 1 August 1970) In 1931 was awarded the Nobel Prize in Physiology for his "discovery of the nature and mode of action of the respiratory enzyme.
WARNBURG EFFECT : cancer cells under aerobic (well-oxygenated) conditions to metabolize glucose to lactate (aerobic glycolysis) is known as the Warburg effect. Warburg made the observation that tumor slices consume glucose and secrete lactate at a higher rate than normal tissues.
A brief information about the SCOP protein database used in bioinformatics.
The Structural Classification of Proteins (SCOP) database is a comprehensive and authoritative resource for the structural and evolutionary relationships of proteins. It provides a detailed and curated classification of protein structures, grouping them into families, superfamilies, and folds based on their structural and sequence similarities.
Richard's aventures in two entangled wonderlandsRichard Gill
Since the loophole-free Bell experiments of 2020 and the Nobel prizes in physics of 2022, critics of Bell's work have retreated to the fortress of super-determinism. Now, super-determinism is a derogatory word - it just means "determinism". Palmer, Hance and Hossenfelder argue that quantum mechanics and determinism are not incompatible, using a sophisticated mathematical construction based on a subtle thinning of allowed states and measurements in quantum mechanics, such that what is left appears to make Bell's argument fail, without altering the empirical predictions of quantum mechanics. I think however that it is a smoke screen, and the slogan "lost in math" comes to my mind. I will discuss some other recent disproofs of Bell's theorem using the language of causality based on causal graphs. Causal thinking is also central to law and justice. I will mention surprising connections to my work on serial killer nurse cases, in particular the Dutch case of Lucia de Berk and the current UK case of Lucy Letby.
(May 29th, 2024) Advancements in Intravital Microscopy- Insights for Preclini...Scintica Instrumentation
Intravital microscopy (IVM) is a powerful tool utilized to study cellular behavior over time and space in vivo. Much of our understanding of cell biology has been accomplished using various in vitro and ex vivo methods; however, these studies do not necessarily reflect the natural dynamics of biological processes. Unlike traditional cell culture or fixed tissue imaging, IVM allows for the ultra-fast high-resolution imaging of cellular processes over time and space and were studied in its natural environment. Real-time visualization of biological processes in the context of an intact organism helps maintain physiological relevance and provide insights into the progression of disease, response to treatments or developmental processes.
In this webinar we give an overview of advanced applications of the IVM system in preclinical research. IVIM technology is a provider of all-in-one intravital microscopy systems and solutions optimized for in vivo imaging of live animal models at sub-micron resolution. The system’s unique features and user-friendly software enables researchers to probe fast dynamic biological processes such as immune cell tracking, cell-cell interaction as well as vascularization and tumor metastasis with exceptional detail. This webinar will also give an overview of IVM being utilized in drug development, offering a view into the intricate interaction between drugs/nanoparticles and tissues in vivo and allows for the evaluation of therapeutic intervention in a variety of tissues and organs. This interdisciplinary collaboration continues to drive the advancements of novel therapeutic strategies.
2. La rivoluzione REACH
Costanza Rovida
Costanza.rovida@chimici.it
Center for Alternatives to Animal Testing – Europe (CAAT-Europe)
University of Konstanz
23 maggio 2014
3.
4.
5. Article 1
Aim and scope
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, as well as the free circulation of
substances on the internal market while enhancing
competitiveness and innovation
6. Article 1
Aim and scope
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, as well as the free circulation of
substances on the internal market while enhancing
competitiveness and innovation
7. List of required
endpoints,
according to
REACH
Regulation
Physico-chemical
information
8. List of required
endpoints,
according to REACH
Regulation
Toxicological
information
9. List of required
endpoints,
according to
REACH
Regulation:
Ecotoxicological
information
14. ECHA Practical Guides
1. How to report in vitro data
2. How to report Robust Study Summary
3. How to report Weight of Evidence
4. How to report waiving
5. How to report (Q)SARs
6. How to report read across and categories
http://echa.europa.eu/doc/press/na_10_16_practical_guides_20100409.pdf
15. Article 1
Aim and scope
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, as well as the free circulation of
substances on the internal market while enhancing
competitiveness and innovation
17. REACH Regulation
ANNEXES VII-X
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
ANNEXES VII-X – Column 2
Specific rules for adaptation from column 1
ANNEX XI
General rules for adaptation of the standard testing regime set out in
annexes VII to X
18. Annex XI
TESTING DOES NOT APPEAR SCIENTIFICALLY NECESSARY
• Use of existing data
• Historical human data
PRO and CONS
Studies are often very poor described
No analytical identification of the substance
No identification of the impurities
……………..
………………
19. Annex XI -Weight of Evidence
There may be sufficient Weight of Evidence from several independent
sources of information leading to the assumption/conclusion that a
substance has or has not a particular dangerous property, while the
information from each single source alone is regarded insufficient to support
this notion.
• newly developed test methods
• not yet included in the test methods referred to in
Article 13(3)
• different studies with the same reliability, no key study,
but consistent to assess the end point
20. Annex XI - (Q)SAR
QUALITATIVE OR QUANTITATIVE
STRUCTURE-ACTIVITY RELATIONSHIP ((Q)SAR)
PRO and CONS
Applicability domain
Fit the purpose
“Poor” training data sets
Difficulties in reporting
……………..
………………
21. Annex XI - Grouping and Read Across
GROUPING OF SUBSTANCES AND READ-ACROSS APPROACH
Two approaches:
GROUPING IN CATEGORIES (HPV-SIDS approach)
chemical similarities
functional groups
physical chemical properties
common environmental fate
common metabolites
READ ACROSS FROM SIMILAR SUBSTANCES
a specific end-point can be assessed with a result on a similar
substance
different end point can be assessed from different substances
22. Annex XI
ALTERNATIVE METHODS (ECVAM activities)
http://tsar.jrc.ec.europa.eu/
Reduction of animals: Sensitisation
Acute oral toxicity
Acute aquatic toxicity
Developing screening metods that can support the reliability of a category
or a read across
Pavia – 7 maggio 2011
23. Annex XI
GROUPING OF SUBSTANCES AND READ-ACROSS APPROACH
Pavia – 7 maggio 2011
Two approaches:
GROUPING IN CATEGORIES (HPV-SIDS approach)
chemical similarities
functional groups
physical chemical properties
common environmental fate
common metabolites
READ ACROSS FROM SIMILAR SUBSTANCES
a specific end-point can be assessed with a result on a similar
substance
different end point can be assessed from different substances
24. Annex XI
SUBSTANCE-TAILORED EXPOSURE-DRIVEN TESTING
Testing in accordance with sections 8.6 and 8.7 of Annex VIII, Annex IX and
Annex X may be omitted, based on the exposure scenario(s) developed in
the Chemical Safety Report
PERFORM a Chemical Safety Assessment complete with Exposure
Scenarios even if the substance is NOT DANGEROUS
CONS: Companies are often not cooperative even if they save money
Pavia – 7 maggio 2011
25. In vitro methods
In vitro toxicity testing is the scientific analysis of the effects of toxic chemical
substances on cultured bacteria or mammalian cells. In vitro (literally 'in
glass') testing methods are employed primarily to identify potentially
hazardous chemicals and/or to confirm the lack of certain toxic properties in
the early stages of the development of potentially useful new substances
such as therapeutic drugs, agricultural chemicals and direct food additives
that may or may not taste good.
In vitro assays for xenobiotic toxicity are recently carefully considered by key
government agencies (e.g. EPA; NIEHS/NTP; FDA), mainly due to a societal
movement to reduce the use of animals in research, and a desire to better
assess human risks. There are substantial activities in using in vitro systems to
advance mechanistic understanding of toxicant activities, and the use of
human cells and tissue to define human-specific toxic effects.
www.answers.com
26. I metodi alternativi: che cosa sono
Principio delle 3R:
Reduction = Riduzione
Refinement = Miglioramento
Replacement = Sostituzione
Russell, W. M. S. and Burch, R. L. 1959. The principles of
humane experimental technique
27. Direttiva EU 63/2010
Sostituisce la vecchia Direttiva EEC 86/609
con una maggiore attenzione al benessere
animale e un esplicito riferimento alle 3R
Maggiore controllo e rigore sugli
esperimenti
28. Element Dir 86/609/EEC Dir 2010/63/EU
Ambito di applicazione Vertebrati. Vertebrati,
cefalopdi, Feti
Compliance with the Three Rs Yes Yes
Project evaluation No Yes
Project authorisation (Yes/No) Yes
Housing and care standards guidelines binding
standards
Main differences between
the current & the new Dir
29. Main differences between
the current & the new Dir
Element Dir 86/609/EEC Dir 2010/63/EU
Project follow-up (Yes) Yes
- Retrospective assessment No Yes
Non-technical summaries No Yes
Severity classification No Yes
Ispezioni periodiche Yes Obbligatorie
33. Confronto metodo in vitro e in vivo
140
120
100
80
60
40
20
0
In vitro Assay Mouse Bioassay
17.2% 52.6% 80.0%
1 7 13 19 25 31 37 43 49 55 61 67 73 79 85 91 97 103 109 115 121 127 133 139 145 151 157
μg STX eq/100 g
correlative sample number
34. In vitro methods
In vitro toxicity testing is the scientific analysis of the effects of toxic chemical
substances on cultured bacteria or mammalian cells. In vitro (literally 'in
glass') testing methods are employed primarily to identify potentially
hazardous chemicals and/or to confirm the lack of certain toxic properties in
the early stages of the development of potentially useful new substances
such as therapeutic drugs, agricultural chemicals and direct food additives
that may or may not taste good.
In vitro assays for xenobiotic toxicity are recently carefully considered by key
government agencies (e.g. EPA; NIEHS/NTP; FDA), mainly due to a societal
movement to reduce the use of animals in research, and a desire to better
assess human risks. There are substantial activities in using in vitro systems to
advance mechanistic understanding of toxicant activities, and the use of
human cells and tissue to define human-specific toxic effects.
www.answers.com
35. IN VITRO METHODS
Validated methods:
Skin corrosion/irritation
Skin absorption
Eye corrosion/irritation (partially)
Screening for genotoxicity and mutagenicity (partially)
Acute Toxicity (partially)
Pre-validation methods:
Sensitisation
Carcinogenic potential
Reproductive toxicity (embryotoxicity)
36. In vitro methods: Skin sensitisation
LOCAL LYMPH NODE ASSAY
Days 1, 2 & 3
Apply Chemical
Day 6 - Inject
3 H-Thymidine
5 hours Later
Remove Lymph
Nodes
Make Cell
Suspension
3
Determine H-Thymidine
Incorporation by Liquid
Scintillation Counting
39. Strategy: sensitisation
Metal salt Metal oxide
Pavia – 7 maggio 2011
MO
Test with OECD TG 428
Skin Absorption ≥ 0.1% Skin Absorption < 0.1%
MX2
LLNA positive
(Strong)
Negative
No classification as skin sensitiser
Positive
Classified as skin sensitiser H317
Read Across
40. Strategy: sensitisation
IN VITRO STUDIES : An example on how to assess doubtful sensitization
results on a basic dye supporting also exposure pattern.
No study available on both Oxalate and Chloride forms of the dye
Demonstration that the two salts are equivalent is missing
There is concern about the skin sensitisation potential
Negative result in an old study (1984)
Positive in a even older human patch test (1966)
• A new study performed on both salts is required
• A new study to confirm the sensitisation property is required
Pavia – 7 maggio 2011
41. Strategy: sensitisation
Pavia – 7 maggio 2011
ITS proposal:
1. Skin absorption assessment: OECD TG 428
2. In vitro sensitisation: U937 method, which passed the pre-validation
and has just started the official validation at
ECVAM (European Centre for Validation of
Alternative Methods)
RATIONALE
Skin Absorption: Only adsorbed substances can cause skin sensitisation
The results can be also exploited as a comparison between
the two salts
Sensitisation: a POSITIVE results is accepted (Annex XI:positive results
from a suitable in vitro test can be accepted)
a NEGATIVE result is also accepted: (peer review literature)
42. Strategy: sensitisationoss
Pavia – 7 maggio 2011
COSTS:
OECD TG 428, in GLP:
2,700 €/per substance
4,320 € for the two salts when tested simultaneously
U937
Performed for free at the University of Milan, as the validating committee
needs new substances to test
The final result will be confidential and will not be published
(LLNA in vivo method):
7,200 €/per substance
10,800 € for both salts when tested simultaneously
43. Strategy: full dossier
In vitro studies to assess the category of nickel compounds and verify the
representative substance to take the existing data in Read Across
BIOACCESSIBILITY BASED READ ACROSS ASSESSMENT OF NICKEL
COMPOUNDS: INHALATION TOXICITY
Sample ID Code(s)2 CAS No.
Ni
Content
(%)3
Interstitial
Bioaccessibility
(% Ni/g sample)4
Pavia – 7 maggio 2011
Lysosomal
Bioaccessibility
(% Ni/g sample)4
Acute Toxicity
(inhalation LC50,
mg/L)5
Ni Sulfate Hexahydrate N58-72 10101-97-0 22 12.80 21.35 2.48
Ni Oxide Green N9/N46 (N126) 1313-99-1 77 (81) 0.10 0.82 >5.08
Ni Sub-Sulfide N129 (N18) 12035-72-2 61 (70) 3.60 26.20 1.14
Ni Hydroxy Carbonate N128 (N109) 12122-15-5 49 (49) 1.65 47.206 >2.09 (F); 0.25 (M)7
Ni Chloride Hexahydrate N98 7791-20-0 25 8.10 25.056 NC
Ni Acetate Tetrahydrate N103 6018-89-9 24 10.90 24.856 NC
Ni Sulfamate
N104 13770-89-3 18 8.60 18.306 NC
Tetrahydrate
Ni Hydroxide N106 12054-48-7 54 0.02 55.80 NC
Ni Oxide Black N105 1313-99-1 75 0.56 24.50 NC
Ni Sulfide N97 16812-54-7 59 1.08 25.95 NC
44. Strategy: full dossier
BIOACCESSIBILITY BASED READ ACROSS ASSESSMENT OF NICKEL
COMPOUNDS: ORAL SYSTEMIC TOXICITY
Sample ID Code1 CAS No.
Ni Content
(%)2
Pavia – 7 maggio 2011
Bioaccessibility (%
Ni content
released)3
In Vivo Acute
Toxicity (oral LD50,
mg/kg/bw)4
Ni Sulfate Hexahydrate N58-72 10101-97-0 23 90.55% 362
Ni Oxide Green N9/N46 1313-99-1 77 0.33% >11000
Ni Oxide Green N112 1313-99-1 81 <LOD5 >11000
Ni Sub-Sulfide N18 12035-72-2 70 22.65% >11000
Ni Chloride Hexahydrate N98 7791 -20-0 25 89.85% 500
Ni Acetate Tetrahydrate N 103 6018-89-9 24 88.50% 550
Ni Hydroxy Carbonate N109 121 22-1 5-5 49 84.30% 2000
Ni Sulfamate
N 104 13770-89-3 18 83.40% 1098
Tetrahydrate
Ni Hydroxide N106 12054-48-7 54 26.30% 5000
Ni Oxide Black N105 131 3-99-1 75 29.60% 9990
Ni Sulfide N97 1681 2-54-7 59 9.75% NC6
Ni Fluoride Tetrahydrate7 N111 13940-83-5 32 82.35% 310
45. European projects
OSIRIS
Optimised Strategies for Risk Assessment of Industrial Chemicals through
Integration of Non-Test and Test Information
http://www.osiris-reach.eu/
Pavia – 7 maggio 2011
46. Improvements
With the first dossiers many data have been generated, that can be used to
improve also the reliability of predictions models in the next years
More in vitro metods will be developed / validated
Multidisciplinary approach within the working groups
Will ECHA be ready?
Pavia – 7 maggio 2011
47. An example for a Strategy
IN VITRO STUDIES : An example on how to assess doubtful sensitization
results on a basic dye supporting also exposure pattern.
No study available on both Oxalate and Chloride forms of the dye
Demonstration that the two salts are equivalent is missing
There is concern about the skin sensitisation potential
Negative result in an old study (1984)
Positive in a even older human patch test (1966)
• A new study performed on both salts is required
• A new study to confirm the sensitisation property is required
48. ITS proposal:
1. Skin absorption assessment: OECD TG 428
2. In vitro sensitisation: THP-1 method, which passed the pre-validation
and has just started the official validation at ECVAM (European Centre
for Validation of Alternative Methods)
RATIONALE
Skin Absorption: Only adsorbed substances can cause skin sensitisation
The results can be also exploited as a comparison between
the two salts
Sensitisation: a POSITIVE results is accepted (Annex XI:positive results
from a suitable in vitro test can be accepted)
a NEGATIVE result is also accepted: (peer review literature)
49. COSTS:
OECD TG 428, in GLP:
2,700 €/per substance
4,320 € for the two salts when tested simultaneously
THP-1 Performed for free at the University of Milan, as the validating
committee needs new substances to test
(LLNA in vivo method):
7,200 €/per substance
10,800 € for both salts when tested simultaneously
50. In vitro methods: Strategy
Existing
data
Weight of
Evidence
GOOD In vitro data
SENSE!
INTELLIGENT = INTEGRATED
Exposure
assessment
Read
across
Categories
In silico
data
53. OECD Member Countries
Countries/Economies Engaged in Working
OECD Relationships with the OECD
54. OECD Methods
428 Skin Absorption: In Vitro Method
430 In Vitro Skin Corrosion: Transcutaneous Electrical Resistance Test (TER)
431 In Vitro Skin Corrosion: Human Skin Model Test
432 In Vitro 3T3 NRU Phototoxicity Test
435 In Vitro Membrane Barrier Test Method for Skin Corrosion
437 Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants
438 Isolated Chicken Eye Test Method for Identifying Ocular Corrosives and Severe Irritants
439 In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method
471 Bacterial Reverse Mutation Test
472 Genetic Toxicology: Escherichia coli, Reverse Assay
473 In Vitro Mammalian Chromosome Aberration Test
474 Mammalian Erythrocyte Micronucleus Test
476 In Vitro Mammalian Cell Gene Mutation Test
477 Genetic Toxicology: Sex-Linked Recessive Lethal Test in Drosophilia melanogaster
479 Genetic Toxicology: In Vitro Sister Chromatid Exchange assay in Mammalian Cells
480 Genetic Toxicology: Saccharomyces cerevisiae, Gene Mutation Assay
481 Genetic Toxicology: Saccharomyces cerevisiae, Mitotic Recombination Assay
482 Genetic Toxicology: DNA Damage and Repair, Unscheduled DNA Synthesis in Mammalian Cells In Vitro
483 Mammalian Spermatagonial Chromosome Aberration Test
487 In Vitro Mammalian Cell Micronucleus Test
55. OECD Methods
428 Skin Absorption: In Vitro Method
430 In Vitro Skin Corrosion: Transcutaneous Electrical Resistance Test (TER)
431 In Vitro Skin Corrosion: Human Skin Model Test
432 In Vitro 3T3 NRU Phototoxicity Test
435 In Vitro Membrane Barrier Test Method for Skin Corrosion
437 Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants
438 Isolated Chicken Eye Test Method for Identifying Ocular Corrosives and Severe Irritants
439 In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method
471 Bacterial Reverse Mutation Test
472 Genetic Toxicology: Escherichia coli, Reverse Assay
473 In Vitro Mammalian Chromosome Aberration Test
474 Mammalian Erythrocyte Micronucleus Test
476 In Vitro Mammalian Cell Gene Mutation Test
477 Genetic Toxicology: Sex-Linked Recessive Lethal Test in Drosophilia melanogaster
479 Genetic Toxicology: In Vitro Sister Chromatid Exchange assay in Mammalian Cells
480 Genetic Toxicology: Saccharomyces cerevisiae, Gene Mutation Assay
481 Genetic Toxicology: Saccharomyces cerevisiae, Mitotic Recombination Assay
482 Genetic Toxicology: DNA Damage and Repair, Unscheduled DNA Synthesis in Mammalian Cells In Vitro
483 Mammalian Spermatagonial Chromosome Aberration Test
487 In Vitro Mammalian Cell Micronucleus Test
57. Un esempio di una strategia
IN VITRO STUDIES : Un esempio di come una strategia in vitro è stata
utilizzata per la valutazione di un risultato dubbio sulla sensitization e per
avere maggiori dati sull'esposizione.
Nessuno studio disponibile su entrambi i Sali in commercio del
colorante (Ossalato e cloruro)
Mancanza di una dimostrazione che i due Sali fossero equivalenti
Dubbio che potesse essere allergenico
Risultato negativo in uno studio molto vecchio (1984)
Risultato positivo in un human patch test ancora più vecchio (1966)
• Necessario uno studio da fare contemporaneamente sui due sali
• Necessario uno studio per decidere sulla skin sensitisation
58. ITS proposal:
1. Skin absorption assessment: OECD TG 428
2. In vitro sensitisation: THP-1 method, che ha già passato la fase di pre-validation
and ed è in corso di validazione ufficiale a ECVAM
(European Centre for Validation of Alternative Methods)
RATIONALE
Skin Absorption: Solo le sostanze che vengono assorbite possono provocare
allergie. Il risultato può essere utilizzato come base per il
confronto tra i due sali
Sensitisation: un risultato POSITIVO è accettato (Annex XI: positive
results from a suitable in vitro test can be accepted)
un risultato NEGATIVO è accettato lo stesso (peer review
literature)
59. Non si conoscono
Sono più difficili da applicare
Spesso sono più costosi
Non si è sicuri che vengano accettati
Si è sempre fatto in un altro modo
Altex 27, 3/10
62. Use of bovine gametes for
reproductive toxicity testing
2 cm
bovine ovaries
In vitro maturation (IVM) of bovine oocytes
bovine oocytes
Endpoint: %
Metaphase II
Chemical
exposure (24h)
during IVM
In vitro fertilisation (IVF) of bovine oocytes
Chemical
exposure (20h)
during IVF
Endpoint:
% 2Pronuclei
IVM, 24 h
63. dall’embrione alle cellule staminali
concepimento blastocisti (5 giorni)
Coltura di cellule
embrionali staminali
68. Report del
National Research
Council of the National
Academies
Pubblicato nel 2007
(Google: "Toxicity Testing in the 21st Century")
69. A New Paradigm: Activation of Toxicity Pathways
Biologic
Inputs
Normal
Biologic
Function
Morbidity
and
Mortality
Cell
Injury
Adaptive Stress
Responses
Early Cellular
Changes
Exposure
Tissue Dose
Biologic Interaction
Perturbation
Low Dose
Higher Dose
Higher yet
(Courtesy of Mel Andersen)
71. Toxicity Pathways
A cellular response pathway that, when sufficiently
perturbed, is expected to result in an adverse health
effect.
Set Chemicals Assays Endpoints Completion Available
ToxCast Phase I 293 ~600 ~1100 2011 Now
ToxCast Phase II 767 ~600 ~1100 03/2013 10/2013
ToxCast Phase IIIa 1001 ~100 ~100 Just starting 2014
E1K (endocrine) 880 ~50 ~120 03/2013 10/2013
Tox21 8,193 ~25 ~50 Ongoing Ongoing
74. “Non sopravvive chi è più forte o
più intelligente, ma chi
reagisce più velocemente ai
cambiamenti”
Charles Darwin
Editor's Notes
Un’importante occasione di lavoro
http://isogenic.info/html/ethical_considerations.html
Russell, W. M. S. and Burch, R. L. 1959. The principles of humane experimental technique Special Edition, Universities Federation for Animal Welfare, Potters Bar, England
L'inizio è filosofico-etico
Differenza tra Direttiva e regolamento
Direttiva non ancora approvata in Italia
28
29
Come si evolvono i metodi alternativi; facciamo un esempio
Azaspiracid AZP, comune in Europa
Non voglio più parlare di 3R o di metodi alternativi, ma, se vogliamo mantenere la A, di metodi avanzati che siano al passo della tossicologia o della ricerca bio-medica del futuro
Si casca sulla predictive capacity
OECD è per tutto, non solo per I metodi alternativi
Le linee guida della szione 4 sono relative alla salute sull'uomo, le 2 per l'ambiente.
Importanti quelle generiche della sezione 0 o 1
34 membri, compresa UE, USA Canada e Giappone. Purtroppo ancora non la Cina
I metodi della sezione 4 sono quelli relativi alla salute umana. Come vedete I numeri sono molto alti. IL metodo 401 è la tossicità acuta orale, ancora su 100 animali, che ovviamente non usa più nessuno
Il problema è che tra I composti che non sono classificati nè negativi, nè positivi, non si può prendere una decisione
Chiaro che non siamo soddisfatti. Ma si è già iniziato a lavorare su due, forse tre piani separati
Reprotoxicity
The reproductive cycle is one of the most complex set of processes that organisms undergo.
We currently still rely on animal tests to predict the potential for chemicals to cause reproductive harm in humans.
(reproduction/fertility, prenatal development, postnatal development), or as a single protocol (two-generation test).
What are equivalent concentrations in vitro and in vivo?