This document discusses alternative in vitro test methods that can potentially replace animal tests for assessing eye and skin irritation. It provides background on the development of alternative methods and validation process. Two alternative eye irritation tests - Bovine Corneal Opacity and Permeability Assay and Isolated Chicken Eye Assay - have been validated and accepted by OECD for regulatory use. Reconstructed human epidermis models EpiDerm and EpiSkin have also been validated and accepted to classify chemicals for skin irritation hazard. The document outlines several alternative assay methods and their limitations but emphasizes the scientific push for non-animal tests due to ethical concerns and limitations of animal models.
Dear Friends,
This is my 3rd presentation, which will help you to understand the depth knowledge of acute eye irritation/corrosion (OECD-405) study in rabbit.
Dear Friends,
This is my 3rd presentation, which will help you to understand the depth knowledge of acute eye irritation/corrosion (OECD-405) study in rabbit.
toxicology study according to OECD guidelines, organisation for economic co-orporation and developement, jasdeep singh , maharaja ranjit singh punjab technical university bathinda
In this slide contains diabetics, classification, symptoms, complication, invivo and invitro screening models of anti diabetics.
Presented by: GEETHANJALI ADAPALA (Department of pharmacology).
RIPER, anantapur
Dermal Irritation and Dermal Toxicity Studies Dinesh Gangoda
Dermal irritation and Corrosion test guidelines 204.
Dermal irritation is the production of reversible damage of the skin following the application of a test chemical for up to 4 hours.
Corrosive reactions are typified by ulcers, bleeding, bloody scabs, and, by the end of observation at 14 days, by discolouration due to blanching of the skin, complete areas of alopecia, and scars. Histopathology should be considered to evaluate questionable lesions. [1]
Dermal corrosion is the production of irreversible damage of the skin; namely, visible necrosis through the epidermis and into the dermis, following the application of a test chemical for up to four hours.[2]
REFERENCES
OECD/OCDE, Test No. 404: ‘‘Acute Dermal Irritation/Corrosion’’, 28 July 2015 OECD Publishing, peris, Page no, 1- 8.
Robert A., Turner., Screening Methods in Pharmacology; 1st edition; Academic press an imprint of Elsevier, pp, 279- 281.
OECD Guideline for testing of chemicals (1981). ‘‘Repeated Dose Dermal Toxicity’’, 21/28- day Study.
Presentation with talk by Suzan Commandeur for the Science meets Business café of 15 Nov 2012. For more info, see www.sciencemeetsbusiness.nl/smb-cafe-15-nov/
toxicology study according to OECD guidelines, organisation for economic co-orporation and developement, jasdeep singh , maharaja ranjit singh punjab technical university bathinda
In this slide contains diabetics, classification, symptoms, complication, invivo and invitro screening models of anti diabetics.
Presented by: GEETHANJALI ADAPALA (Department of pharmacology).
RIPER, anantapur
Dermal Irritation and Dermal Toxicity Studies Dinesh Gangoda
Dermal irritation and Corrosion test guidelines 204.
Dermal irritation is the production of reversible damage of the skin following the application of a test chemical for up to 4 hours.
Corrosive reactions are typified by ulcers, bleeding, bloody scabs, and, by the end of observation at 14 days, by discolouration due to blanching of the skin, complete areas of alopecia, and scars. Histopathology should be considered to evaluate questionable lesions. [1]
Dermal corrosion is the production of irreversible damage of the skin; namely, visible necrosis through the epidermis and into the dermis, following the application of a test chemical for up to four hours.[2]
REFERENCES
OECD/OCDE, Test No. 404: ‘‘Acute Dermal Irritation/Corrosion’’, 28 July 2015 OECD Publishing, peris, Page no, 1- 8.
Robert A., Turner., Screening Methods in Pharmacology; 1st edition; Academic press an imprint of Elsevier, pp, 279- 281.
OECD Guideline for testing of chemicals (1981). ‘‘Repeated Dose Dermal Toxicity’’, 21/28- day Study.
Presentation with talk by Suzan Commandeur for the Science meets Business café of 15 Nov 2012. For more info, see www.sciencemeetsbusiness.nl/smb-cafe-15-nov/
Don't Miss a Beat: Understanding Continuous, Real Time Physiologic MonitoringInsideScientific
In vivo, preclinical research encompasses numerous study designs with various species and endpoints being monitored. Having access to all available study data allows the scientist to comprehensively understand the study paradigm and make informed research decisions. During Session 3 of our webseries "Biotelemetry For The Life Sciences", presenters discussed the importance of continuous, real-time monitoring in preclinical research. Case studies included using EEG as a biomarker for CNS activity and drug discovery and using telemetry for disease characterizations and and evaluation of vaccines in Biodefense research.
During this exclusive webinar sponsored by Data Sciences International, Steve Fox shares his experience from pharmaceutical development; discussing the importance of continuous EEG monitoring for sleep studies. Anna Honko explains the importance of having access to real-time, continuous data when studying infectious diseases in non-human primates in a Biodefense setting. In addition, Dusty Sarazan reviews how and why continuous, real-time monitoring has become a preferred and essential method for acquiring and studying physiology in today's preclinical research setting.
Key Topics:
EEG as a biomarker for CNS activity and a platform for pre-clincal drug discovery
Sleep/wake patterns and rhythms, and how qEEG signatures allow for accurate clinical predictions of efficacy and CNS adverse event screening
Considering the FDA Animal Rule
Basic disease characterizations and evaluation of vaccines and therapeutics
Non-human primate models of viral biodefense and emerging pathogens
Translating pre-clinical study findings to human, clinical populations
Guest Speakers:
Steve Fox, BS
Associate Principal Scientist,
Merck & Co., Inc.
Anna Honko, PhD
Staff Scientist,
NIH/NIAID Integrated Research Facility
R. Dustan Sarazan, DVM, PhD
Vice President & Chief Scientific Officer, Data Sciences International
Eye irritation has been defined as “the magnitude of any stinging, scratching, burning, or other irritating sensation from the eye”.It is a common problem experienced by people of all ages. Related eye symptoms and signs of irritation are e.g. discomfort, dryness, excess tearing, itching, grating, sandy sensation, smarting, ocular fatigue, pain, scratchiness, soreness, redness, swollen eyelids, and tiredness, etc. These eye symptoms are reported with intensities from severe to less severe. It has been suggested that these eye symptoms are related to different causal mechanisms.
Presentation of the 2014 Notable Social Studies Trade Books for Young People. Books were published in 2013. Presented by Karen Hildebrand, member of the selection committee sponsored by the Children's Book Council and the National Council for Social Studies.
Viral Challenge Studies: An Innovative Way to Speed Up Vaccine Development; A...SGS
In order to effectively fight influenza, the development of new, higher performing and universal vaccines is essential. However, clinical development is a lengthy and very expensive process, making it difficult for researchers to design vaccines for rapidly mutating viruses such as influenza. Assessing efficacy of a new influenza vaccine as early as possible in the development, to make a first selection and an early ‘go – no go’ decision, is key. Viral Challenge studies are an important tool to aid in the swift development of effective influenza vaccines particularly for potential pandemics like the avian influenza (bird flu).
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
Human Clinical Relevance of Developmental and Reproductive Toxicology and Non...Joseph Holson
Presented at Forest Research Institute, May 13, 2004.
Abstract: Experimental animal models are essential to product development and toxicologic screening. The effective use of such models is dependent on the attributes of: validity, sensitivity, reproducibility, and practicability. For the two endpoints of toxicity of most societal concern, developmental effects, and cancer, experience has taught that differences between animals and humans in drug absorption, distribution, metabolism and elimination most often leads to differences in response both qualitatively, and quantitatively. In developmental toxicology, a high degree of concordance between experimental animal results and human outcomes has been demonstrated. Human reproductive outcomes are often concordant with experimental animal data, but this concordance seems to vary more among species as phenotypes diversify with approaching sexual maturity and subsequent reproductive senescence. This increase in phenotypic diversity also presents difficulties in a priori selection of animal models in non-clinical juvenile toxicity testing. Juvenile periods among species can be divided into pre-term neonatal, neonatal, infancy, childhood and adolescence, based on overall central nervous system and reproductive development. However, because physiologic time differs among species, temporality of target-organ maturation should be reconciled with the human pediatric therapeutic scenario prior to animal model selection. The heuristic impact and resultant guidance for proper selection and use of animal models for juvenile toxicity testing will be demonstrated through the use of case studies involving angiotensin-converting enzyme (ACE) inhibitors, quinilones, fluoxetine and isotretinoin.
Overview of 3D-human skin reconstructed models for irritation and corrosion t...An Van Rompay
Overview of 3D-human skin reconstructed models for irritation and corrosion testing
An Van Rompay
VITO NV & CARDAM, Belgium
from: Invitrom meeting, December 2011
Biological considerations of dental materials and cavity preparationIndian dental academy
Indian Dental Academy: will be one of the most relevant and exciting training center with best faculty and flexible training programs for dental professionals who wish to advance in their dental practice,Offers certified courses in Dental implants,Orthodontics,Endodontics,Cosmetic Dentistry, Prosthetic Dentistry, Periodontics and General Dentistry.
OECD Webinar | OECD Alternatives to in vivo eye irritation testing - David Al...OECD Environment
On 27 September 2019, Anne Gourmelon of the Environment Directorate presented an overview of the various alternative test methods developed as OECD Test Guidelines and relevant guidance material to address eye irritation and serious eye damage for hazard classification of chemicals.
Safer cosmetics through in vitro science - XCellR8 presentation to Cosmetics ...Susie Lee-Kilgariff
How to develop safer cosmetics through in vitro science. This presentation was given to the Cosmetics Business Regulatory Summit 2018 by Dr Carol Treasure of XCellR8. It includes case studies of in vitro safety tests for skin sensitisation, advice on testing finished products and progress on a new model to predict mildness to skin.
Safer cosmetics through in vitro science. XCellR8 presentation to Cosmetics B...Dr Carol Barker-Treasure
In vitro methods for key aspects of cosmetic safety, including skin and eye irritation and skin sensitisation, are now in widespread use for regulatory safety testing around the world. In many cases, they are now established as the default rather than the alternative approach, so it’s timely to ask, “where next?”
In vitro technologies offer so much more than basic regulatory compliance, and when used wisely, can provide the competitive edge, from ensuring that formulations are fully optimised before proceeding to human in vivo studies, to providing detailed mechanistic data for claim support purposes. Importantly, they are a powerful tool to support the current move towards exposure-led safety assessments, enhancing the long-term safety of products and limiting the risk of post-market adverse events.
This presentation, first shared at the Cosmetics Business Regulatory Summit 2018, examines case studies from some early-adopters who are taking the application of in vitro data to the next level and considers the feasibility of applying exciting new techniques to the cosmetics of the future.
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Title: Sense of Taste
Presenter: Dr. Faiza, Assistant Professor of Physiology
Qualifications:
MBBS (Best Graduate, AIMC Lahore)
FCPS Physiology
ICMT, CHPE, DHPE (STMU)
MPH (GC University, Faisalabad)
MBA (Virtual University of Pakistan)
Learning Objectives:
Describe the structure and function of taste buds.
Describe the relationship between the taste threshold and taste index of common substances.
Explain the chemical basis and signal transduction of taste perception for each type of primary taste sensation.
Recognize different abnormalities of taste perception and their causes.
Key Topics:
Significance of Taste Sensation:
Differentiation between pleasant and harmful food
Influence on behavior
Selection of food based on metabolic needs
Receptors of Taste:
Taste buds on the tongue
Influence of sense of smell, texture of food, and pain stimulation (e.g., by pepper)
Primary and Secondary Taste Sensations:
Primary taste sensations: Sweet, Sour, Salty, Bitter, Umami
Chemical basis and signal transduction mechanisms for each taste
Taste Threshold and Index:
Taste threshold values for Sweet (sucrose), Salty (NaCl), Sour (HCl), and Bitter (Quinine)
Taste index relationship: Inversely proportional to taste threshold
Taste Blindness:
Inability to taste certain substances, particularly thiourea compounds
Example: Phenylthiocarbamide
Structure and Function of Taste Buds:
Composition: Epithelial cells, Sustentacular/Supporting cells, Taste cells, Basal cells
Features: Taste pores, Taste hairs/microvilli, and Taste nerve fibers
Location of Taste Buds:
Found in papillae of the tongue (Fungiform, Circumvallate, Foliate)
Also present on the palate, tonsillar pillars, epiglottis, and proximal esophagus
Mechanism of Taste Stimulation:
Interaction of taste substances with receptors on microvilli
Signal transduction pathways for Umami, Sweet, Bitter, Sour, and Salty tastes
Taste Sensitivity and Adaptation:
Decrease in sensitivity with age
Rapid adaptation of taste sensation
Role of Saliva in Taste:
Dissolution of tastants to reach receptors
Washing away the stimulus
Taste Preferences and Aversions:
Mechanisms behind taste preference and aversion
Influence of receptors and neural pathways
Impact of Sensory Nerve Damage:
Degeneration of taste buds if the sensory nerve fiber is cut
Abnormalities of Taste Detection:
Conditions: Ageusia, Hypogeusia, Dysgeusia (parageusia)
Causes: Nerve damage, neurological disorders, infections, poor oral hygiene, adverse drug effects, deficiencies, aging, tobacco use, altered neurotransmitter levels
Neurotransmitters and Taste Threshold:
Effects of serotonin (5-HT) and norepinephrine (NE) on taste sensitivity
Supertasters:
25% of the population with heightened sensitivity to taste, especially bitterness
Increased number of fungiform papillae
Tom Selleck Health: A Comprehensive Look at the Iconic Actor’s Wellness Journeygreendigital
Tom Selleck, an enduring figure in Hollywood. has captivated audiences for decades with his rugged charm, iconic moustache. and memorable roles in television and film. From his breakout role as Thomas Magnum in Magnum P.I. to his current portrayal of Frank Reagan in Blue Bloods. Selleck's career has spanned over 50 years. But beyond his professional achievements. fans have often been curious about Tom Selleck Health. especially as he has aged in the public eye.
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Introduction
Many have been interested in Tom Selleck health. not only because of his enduring presence on screen but also because of the challenges. and lifestyle choices he has faced and made over the years. This article delves into the various aspects of Tom Selleck health. exploring his fitness regimen, diet, mental health. and the challenges he has encountered as he ages. We'll look at how he maintains his well-being. the health issues he has faced, and his approach to ageing .
Early Life and Career
Childhood and Athletic Beginnings
Tom Selleck was born on January 29, 1945, in Detroit, Michigan, and grew up in Sherman Oaks, California. From an early age, he was involved in sports, particularly basketball. which played a significant role in his physical development. His athletic pursuits continued into college. where he attended the University of Southern California (USC) on a basketball scholarship. This early involvement in sports laid a strong foundation for his physical health and disciplined lifestyle.
Transition to Acting
Selleck's transition from an athlete to an actor came with its physical demands. His first significant role in "Magnum P.I." required him to perform various stunts and maintain a fit appearance. This role, which he played from 1980 to 1988. necessitated a rigorous fitness routine to meet the show's demands. setting the stage for his long-term commitment to health and wellness.
Fitness Regimen
Workout Routine
Tom Selleck health and fitness regimen has evolved. adapting to his changing roles and age. During his "Magnum, P.I." days. Selleck's workouts were intense and focused on building and maintaining muscle mass. His routine included weightlifting, cardiovascular exercises. and specific training for the stunts he performed on the show.
Selleck adjusted his fitness routine as he aged to suit his body's needs. Today, his workouts focus on maintaining flexibility, strength, and cardiovascular health. He incorporates low-impact exercises such as swimming, walking, and light weightlifting. This balanced approach helps him stay fit without putting undue strain on his joints and muscles.
Importance of Flexibility and Mobility
In recent years, Selleck has emphasized the importance of flexibility and mobility in his fitness regimen. Understanding the natural decline in muscle mass and joint flexibility with age. he includes stretching and yoga in his routine. These practices help prevent injuries, improve posture, and maintain mobilit
Lung Cancer: Artificial Intelligence, Synergetics, Complex System Analysis, S...Oleg Kshivets
RESULTS: Overall life span (LS) was 2252.1±1742.5 days and cumulative 5-year survival (5YS) reached 73.2%, 10 years – 64.8%, 20 years – 42.5%. 513 LCP lived more than 5 years (LS=3124.6±1525.6 days), 148 LCP – more than 10 years (LS=5054.4±1504.1 days).199 LCP died because of LC (LS=562.7±374.5 days). 5YS of LCP after bi/lobectomies was significantly superior in comparison with LCP after pneumonectomies (78.1% vs.63.7%, P=0.00001 by log-rank test). AT significantly improved 5YS (66.3% vs. 34.8%) (P=0.00000 by log-rank test) only for LCP with N1-2. Cox modeling displayed that 5YS of LCP significantly depended on: phase transition (PT) early-invasive LC in terms of synergetics, PT N0—N12, cell ratio factors (ratio between cancer cells- CC and blood cells subpopulations), G1-3, histology, glucose, AT, blood cell circuit, prothrombin index, heparin tolerance, recalcification time (P=0.000-0.038). Neural networks, genetic algorithm selection and bootstrap simulation revealed relationships between 5YS and PT early-invasive LC (rank=1), PT N0—N12 (rank=2), thrombocytes/CC (3), erythrocytes/CC (4), eosinophils/CC (5), healthy cells/CC (6), lymphocytes/CC (7), segmented neutrophils/CC (8), stick neutrophils/CC (9), monocytes/CC (10); leucocytes/CC (11). Correct prediction of 5YS was 100% by neural networks computing (area under ROC curve=1.0; error=0.0).
CONCLUSIONS: 5YS of LCP after radical procedures significantly depended on: 1) PT early-invasive cancer; 2) PT N0--N12; 3) cell ratio factors; 4) blood cell circuit; 5) biochemical factors; 6) hemostasis system; 7) AT; 8) LC characteristics; 9) LC cell dynamics; 10) surgery type: lobectomy/pneumonectomy; 11) anthropometric data. Optimal diagnosis and treatment strategies for LC are: 1) screening and early detection of LC; 2) availability of experienced thoracic surgeons because of complexity of radical procedures; 3) aggressive en block surgery and adequate lymph node dissection for completeness; 4) precise prediction; 5) adjuvant chemoimmunoradiotherapy for LCP with unfavorable prognosis.
These simplified slides by Dr. Sidra Arshad present an overview of the non-respiratory functions of the respiratory tract.
Learning objectives:
1. Enlist the non-respiratory functions of the respiratory tract
2. Briefly explain how these functions are carried out
3. Discuss the significance of dead space
4. Differentiate between minute ventilation and alveolar ventilation
5. Describe the cough and sneeze reflexes
Study Resources:
1. Chapter 39, Guyton and Hall Textbook of Medical Physiology, 14th edition
2. Chapter 34, Ganong’s Review of Medical Physiology, 26th edition
3. Chapter 17, Human Physiology by Lauralee Sherwood, 9th edition
4. Non-respiratory functions of the lungs https://academic.oup.com/bjaed/article/13/3/98/278874
Report Back from SGO 2024: What’s the Latest in Cervical Cancer?bkling
Are you curious about what’s new in cervical cancer research or unsure what the findings mean? Join Dr. Emily Ko, a gynecologic oncologist at Penn Medicine, to learn about the latest updates from the Society of Gynecologic Oncology (SGO) 2024 Annual Meeting on Women’s Cancer. Dr. Ko will discuss what the research presented at the conference means for you and answer your questions about the new developments.
Recomendações da OMS sobre cuidados maternos e neonatais para uma experiência pós-natal positiva.
Em consonância com os ODS – Objetivos do Desenvolvimento Sustentável e a Estratégia Global para a Saúde das Mulheres, Crianças e Adolescentes, e aplicando uma abordagem baseada nos direitos humanos, os esforços de cuidados pós-natais devem expandir-se para além da cobertura e da simples sobrevivência, de modo a incluir cuidados de qualidade.
Estas diretrizes visam melhorar a qualidade dos cuidados pós-natais essenciais e de rotina prestados às mulheres e aos recém-nascidos, com o objetivo final de melhorar a saúde e o bem-estar materno e neonatal.
Uma “experiência pós-natal positiva” é um resultado importante para todas as mulheres que dão à luz e para os seus recém-nascidos, estabelecendo as bases para a melhoria da saúde e do bem-estar a curto e longo prazo. Uma experiência pós-natal positiva é definida como aquela em que as mulheres, pessoas que gestam, os recém-nascidos, os casais, os pais, os cuidadores e as famílias recebem informação consistente, garantia e apoio de profissionais de saúde motivados; e onde um sistema de saúde flexível e com recursos reconheça as necessidades das mulheres e dos bebês e respeite o seu contexto cultural.
Estas diretrizes consolidadas apresentam algumas recomendações novas e já bem fundamentadas sobre cuidados pós-natais de rotina para mulheres e neonatos que recebem cuidados no pós-parto em unidades de saúde ou na comunidade, independentemente dos recursos disponíveis.
É fornecido um conjunto abrangente de recomendações para cuidados durante o período puerperal, com ênfase nos cuidados essenciais que todas as mulheres e recém-nascidos devem receber, e com a devida atenção à qualidade dos cuidados; isto é, a entrega e a experiência do cuidado recebido. Estas diretrizes atualizam e ampliam as recomendações da OMS de 2014 sobre cuidados pós-natais da mãe e do recém-nascido e complementam as atuais diretrizes da OMS sobre a gestão de complicações pós-natais.
O estabelecimento da amamentação e o manejo das principais intercorrências é contemplada.
Recomendamos muito.
Vamos discutir essas recomendações no nosso curso de pós-graduação em Aleitamento no Instituto Ciclos.
Esta publicação só está disponível em inglês até o momento.
Prof. Marcus Renato de Carvalho
www.agostodourado.com
New Drug Discovery and Development .....NEHA GUPTA
The "New Drug Discovery and Development" process involves the identification, design, testing, and manufacturing of novel pharmaceutical compounds with the aim of introducing new and improved treatments for various medical conditions. This comprehensive endeavor encompasses various stages, including target identification, preclinical studies, clinical trials, regulatory approval, and post-market surveillance. It involves multidisciplinary collaboration among scientists, researchers, clinicians, regulatory experts, and pharmaceutical companies to bring innovative therapies to market and address unmet medical needs.
MANAGEMENT OF ATRIOVENTRICULAR CONDUCTION BLOCK.pdfJim Jacob Roy
Cardiac conduction defects can occur due to various causes.
Atrioventricular conduction blocks ( AV blocks ) are classified into 3 types.
This document describes the acute management of AV block.
2. 80,000 chemicals - currently in use
2000 new chemicals - introduced annually (OTA , 1995)
The public and the environment come in contact with
these substances during their manufacture,
distribution, use and disposal.
To safe guard humanity and their surroundings,
toxicological tests are required for which guidelines
are issued by regulatory agencies.
3. The information generated from these test methods
is used for pre-market evaluation, hazard
classification and risk assessment.
The potential adverse effects of chemicals are
currently assessed largely by tests involving
laboratory animals.
4. The scientific community influenced by,
advances in the understanding of molecular and
cellular mechanisms of toxicity.
Desire to develop tests that will be more predictive
of potential chemical toxicity.
There is also great interest to develop tests that
are more cost and time efficient (Stokes & Marafante,
1998).
5. In Vitro studies
Studies which do not use multicellular whole organisms, but
rather microorganisms or material isolated from whole
organisms, or simulations thereof as test systems.
In-vitro versus In-vivo
provides potential to be more rigorously standardized than
in vivo tests.
more reliable since quality data can be generated
less expensive
human cells can be used directly as test systems
offer good experimental control of the cellular doses of
chemicals
6. the production of “reversible damage of the skin following the
application of a test substance for up to 4 hours” (OECD TG 404,
2002).
the potential of a certain substance to cause erythema or
eschar or oedema after a single topical application.
Dermal irritation
7. Eye irritation
The production of changes in the eye following the
application of a test substance to the anterior
surface of the eye, which are fully reversible within
21 days of application (OECD TG 405, 2002).
8. In vivo irritation studies for the eye and skin assess
the short term effects of materials (Gad, 1997).
These tests have been in existence since the 1930's.
These in vivo methods used rabbits for evaluation of
materials for their potential to cause dermal and eye
irritation.
9. Historical Background
Marshall Hall was one of the first to address the issue of
alternatives- 1876.
In 1969, the Fund for the Replacement of Animals in
Medical Experimentation (FRAME) was founded in the UK.
1981- establishment of the John Hopkins Centre for
Alternatives to Animal Testing (CAAT).
The initial efforts of CAAT focused on the establishment
of a firm scientific foundation of alternative testing
methodologies.
10. 1990- The European Centre for the Validation of Alternative
Methods (ECVAM) was established.
During 1999-2001, some of the most promising in vitro methods
were evaluated in prevalidation studies.
ECVAM started skin irritation validation study (SIVS) in Nov.
2003.
In April 2007, ECVAM approved 2 alternative test, EpiSkin and
EpiDerm Skin Irritation Tests as replacements of the in vivo
rabbit skin irritation test.
2008- devlopment of SkinEthic, Human Oral Epithelium model
11. Validation …?
Validation is the process by which the reliability and
relevance of an alternative method are established for a
particular purpose (Balls et al., 1990).
Reliability - establishing the reproducibility of toxicity
hazard predictions within and between laboratories and
overtime (Bruner et al., 1996).
Relevance - establishing the scientific usefulness of
results from an alternative method (Frazier, 1990).
12. If an alternative method is judged both reliable and
relevant at the end of the validation process, then
the new assay should be considered validated.
Once validated the alternative method may be used
routinely in safety assessment process and may be
considered for acceptance by regulatory agencies
(Zeiger and Stokes, 1998).
13. Conventional Method- eye irritation
OECD TG 405 (2002)
OPPTS 870.2400 (EPA, August 1998)
Test system- albino rabbit
The substance to be tested is applied in
a single dose to one of the eyes of the
experimental animal; the untreated eye
serves as the control.
The eyes of the test animals washed
after 24 h following instillation of the
test substance.
14. Dose level:
Liquids - 0.1 ml
solids, pastes, and particulate substances - volume of 0.1 ml
or a weight not more than 100 mg.
Aerosols- test substance administered to the eye in a burst
of about one second, from a distance of 10 cm.
Observations of eye reactions:
examined at 1, 24, 48 and 72 h after application.
Cornea- Opacity
Iris- congestion, swelling, circumcorneal hyperaemia,
hemorrhages.
Conjunctiva- Redness and hyperaema.
15. OECD TG 404 (2002)
OPPTS 870.2500 (EPA, August 1998)
Test system- albino rabbit
The substance to be tested is applied in a single dose to the
skin of animal; untreated skin areas of the test animal serve
as the control.
Dose level:
liquid- 0.5 ml
solid or paste- 0.5 g
exposure period – 4 h
Conventional Method- skin irritation
16. Application of the test substance
The test substance should be applied to a small area
(6 cm2
) of skin and covered with a gauze patch, which
is held in place with non-irritating tape.
Observations of skin reactions
Scored at 60 minutes, and then at 24, 48 and 72 h
after patch removal.
Examined for signs of erythema and oedema
17. Deficiencies of the Draize Skin and Eye
Tests
These are unable to obtain important information
concerning mechanisms of toxicity of test chemicals.
Inadequate objectivity in obtaining irritancy scores.
expenditures related to the large numbers of animals
required and time-consuming evaluation.
Irreproducibility of results
Issue of variability within the test (Davila et al., 1998).
18. Anatomical considerations
Presence of a nictitating membrane
Larger conjunctival sac
Thinner cornea
Less tear production
……………………..the rabbit is generally considered on
overly sensitive model for humans.
19. Organotypic models or tissue and cell culture
systems.
At present there are two OECD TG based on the use
of in vitro eye irritation tests.
1. Bovine Corneal Opacity and Permeability (BCOP) Test
(OECD TG 437) - 7 Sept 2009
2. Isolated Chicken Eye (ICE) Test, (OECD TG 438) -7
Sept 2009
Alternative Assays for Ocular Irritation
20. Neutral Red (NR) release assay (Reader et al., 1990)
NR uptake assay (Jones et al., 1999)
Tissue equivalent assay (TEA) (Southee et al., 1999)
Chinese hamster lung (CHL) cell lines
Bovine corneal opacity and permeability (BCOP) assay
Isolated chicken eye (ICE) test
Hens egg test on the chorio-allontoic membrane (HET-
CAM)
Alternative Assays for Ocular Irritation
21. Bovine corneal opacity and permeability
(BCOP) assay (Gautheron et al., 1992)
First scientifically valid alternative methods to gain
regulatory acceptance for ocular safety testing.
OECD TG 437 (2009)
Not a complete replacement for the rabbit eye test
Recommended for use as part of a tiered testing
strategy for regulatory classification and labeling.
22. Protocol
Eyes are collected from slaughter house
immersed in Hanks’ Balanced Salt Solution
corneas free of defects are dissected
mounted in specially designed corneal holders
Eagle's Minimum Essential Medium (EMEM)
32 ± 1°C for 1 hour
23. test article is applied as a single dose for 10 min
Post exposure period- 4 h
Endpoints Measured
Opacity- opacitometer
Permeability - determined by the amount of sodium
fluorescein dye penetration.
24. Limitations
false positive for alcohols and ketones
does not consider conjunctival and iridal injuries
does not allow for an assessment of systemic toxicity
associated with ocular exposure
25. Isolated Chicken Eye (ICE) Test
Gained regulatory acceptance for ocular safety
testing (OECD TG 438, 2009).
Not a complete replacement for the rabbit eye test
Recommended for use as part of a tiered testing
strategy for regulatory classification and labeling.
26. Protocol
Eyes isolated from chickens (slaughter house)
placed in a superfusion apparatus
(isotonic saline)
test article is applied as a single dose for 10 sec
(Liquids 0.03 mL
Solids 0.03 g)
Corneal reactions are measured up to 4 h post-treatment
Fluorescein retention is evaluated at 30 min post-treatment
27. End points- Corneal swelling, opacity and fluorescein
retention
Limitations:
false positive for alcohols
does not consider conjunctival and iridal injuries
does not allow for assessment of systemic toxicity
associated with ocular exposure
28. Hens egg test on the chorio-allontoic
membrane (HET-CAM)
The CAM of the developing chicken egg is considered
to be a suitable model to study irritation of mucous
tissues (Spielmann et al., 1997).
The CAM of an embryonated hen’s egg is similar to
the vascularized mucosal tissues of the eye.
29. 9 day fertilized hen’s eggs
The CAM (25%) is treated with the test item for 30 sec
test item is washed off
effects are assessed within 5 minutes
Endpoint – Coagulation, hyperaemia and haemorrhage
The time for each reaction recorded in sec to calculate
irritation score
Solids- 0.3g
Liquids- 0.3 ml
30. Human Tissue Models- EpiOcular
EpiOcular is currently under validation as a replacement
to Draize test by ECVAM (ECVAM, 2010)
Water insoluble and soluble materials
End point – cytotoxicity
Human Reconstituted Corneal
Epithelium
MatTek Corp, USA
31. Alternative Assays for Skin Irritation
Number of in vitro alternatives have been proposed
but there are no in vitro tests for replacing the
classical Draize test.
Promising in vitro methods : EpiDerm™, EpiSkin™, the
non-perfused pig ear model.
EpiDerm and EpiSkin are validated for the purpose of
classification and labelling.
32. Reconstructed Human Epidermis (RhE) Test
Method
As a partial replacement test, within a tiered testing
strategy.
It covers the initial step of the inflammatory cascade
(cell damage resulting in localised trauma) that occurs
during irritation in vivo.
Applicable to solids, liquids, semi-solids and waxes.
33. Test system
Non-transformed human-derived epidermal
keratinocytes.
Cultured to form a multilayered, highly differentiated
model of human epidermis
Consists of organized basal, spinous and granular layers,
and a multilayered stratum corneum analogous to those
found in vivo.
Reconstituted Human
Epidermis Model
34. dose :
Liquid test article- 25 μL/cm2
Solid test article- 25 mg/cm2
exposure period - varies between 15 and 60 min
incubation temperature - between 20 and 37°C
controls :
Positive control- 5% aqueous SDS
Negative control- water or phosphate buffered saline (PBS).
35. MTT - cell viability assay
Reduction of yellow 3-(4,5-dimethythiazol)-2,5-diphenyl
tetrazolium bromide (MTT) by mitochondrial succinate
dehydrogenase.
MTT reduced to an insoluble, dark purple formazan.
The cells are then solubilised with an organic solvent (eg.
isopropanol) and the released, formazan reagent is
measured spectrophotometrically.
Reduction of MTT only occur in metabolically active cells.
The level of activity is a measure of the viability of the
cells (Mosmann, 1983)
36. Reconstituted Human Epidermis Models
(RHE)
EpiSkinTM
model (L’Oreal, France)
EpiDermTM
model (MatTek Corp., USA)
SkinEthicTM
model (Skinethic, Nice, France)
37. EpiSkin
Developed by L’Oreal, France and commercially
supplied by SkinEthic Laboratories, France.
ECVAM validated and recognized as a stand alone
method for screening and replacement (ECVAM, 2010)
It has a reconstructed epidermis with a functional
stratum corneum.
Endpoint – Cell viability, Interleukin -1 α release.
38. Exposure period- 15 min.
Post exposure- 42 h
cytotoxicity measurement using
MTT reduction and Interleukin -1 α
assay (Fentem et al., 1998).
Assessment of irritation:
cell viability ≤ 50% after 15 min
exposure
Interleukin -1 α release > 60 pg/mL
Protocol
39. EpiDerm
(Kubilus et al., 1996)
It incorporates normal human keratinocytes cultured
on permeable millipore membranes.
Endpoint – The time taken to reduce cell viability by
50%.
The EpiDerm test do not qualify as a stand-alone
replacement but is recommended for the
identification of irritant chemicals (ECVAM,2009).
40. Predictive capacity of in vitro human skin
models
Assay Sensitivity Specificity
Episkin (MTT endpoint) 75% 81%
Episkin (MTT + IL-1α endpoints) 91% 79%
EpiDerm 57% 84%
Sensitivity - % in vivo irritants correctly identified by
assay
Specificity - % in vivo non-irritants correctly identified
by assay.
(ECVAM, 2007)
41. A great deal of effort has been directed toward
development and validation of alternative methods for
ocular and dermal irritation testing.
Some alternative tests for reduction and refinement have
been validated and accepted by international authorities
(OECD) and can be used for safety assessment.
Compared to the Draize test the in vitro model based on
human cells is expected to predicts the better range of
responses to toxic injury that occur in the human eye and
skin.
Conclusions