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Nanotoxicology
Experimental and Computational Perspectives
 View Online

Issues in Toxicology

Series Editors:
Diana Anderson, University of Bradford, UK
Published on 03 November 2017 on http://pubs.rsc.org | doi:10.1039/9781782623922-FP001

Michael D. Waters, Michael Waters Consulting, USA

Timothy C. Marrs, Edentox Associates, UK

Editorial Advisor:
Alok Dhawan, CSIR-Indian Institute of Toxicology Research, India

Titles in the Series:

1: Hair in Toxicology: An Important Bio-Monitor
2: Male-mediated Developmental Toxicity
3: Cytochrome P450: Role in the Metabolism and Toxicity of Drugs and
other Xenobiotics
4: Bile Acids: Toxicology and Bioactivity
5: The Comet Assay in Toxicology
6: Silver in Healthcare
7: In Silico Toxicology: Principles and Applications
8: Environmental Cardiology
9: Biomarkers and Human Biomonitoring, Volume 1: Ongoing Programs
and Exposures
10: Biomarkers and Human Biomonitoring, Volume 2: Selected
Biomarkers of Current Interest
11: Hormone-Disruptive Chemical Contaminants in Food
.

12: Mammalian Toxicology of Insecticides

13: The Cellular Response to the Genotoxic Insult: The Question of
Threshold for Genotoxic Carcinogens
14: Toxicological Effects of Veterinary Medicinal Products in
Humans: Volume 1
15: Toxicological Effects of Veterinary Medicinal Products in
Humans: Volume 2
16: Aging and Vulnerability to Environmental Chemicals: Age-related
Disorders and their Origins in Environmental Exposures
17: Chemical Toxicity Prediction: Category Formation and Read-Across
18: The Carcinogenicity of Metals: Human Risk Through Occupational
and Environmental Exposure
19: Reducing, Refining and Replacing the Use of Animals in
Toxicity Testing
20: Advances in Dermatological Sciences
21: Metabolic Profiling: Disease and Xenobiotics
22: Manganese in Health and Disease
23: Toxicology, Survival and Health Hazards of Combustion Products
24: Masked Mycotoxins in Food: Formation, Occurrence and
Toxicological Relevance
 View Online

25: Aerobiology: The Toxicology of Airborne Pathogens and Toxins

26: Chemical Warfare Toxicology, Volume 1: Fundamental Aspects
27: Chemical Warfare Toxicology, Volume 2: Management of Poisoning
28: Toxicogenomics in Predictive Carcinogenicity
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29: Human Stem Cell Toxicology

30: The Comet Assay in Toxicology, 2nd edition
31: Computational Systems Pharmacology and Toxicology
32: Ecotoxicology and Genotoxicology: Non-traditional Terrestrial Models
33: Ecotoxicology and Genotoxicology: Non-traditional Aquatic Models
34: Oxidative Stress and Redox Signalling in Parkinson’s Disease
35: Nanotoxicology: Experimental and Computational Perspectives
.

How to obtain future titles on publication:

A standing order plan is available for this series. A standing order will bring
delivery of each new volume immediately on publication.

For further information please contact:

Book Sales Department, Royal Society of Chemistry, Thomas Graham House,
Science Park, Milton Road, Cambridge, CB4 0WF, UK
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Visit our website at www.rsc.org/books
 .
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 View Online

Nanotoxicology
Experimental and Computational
Perspectives
Published on 03 November 2017 on http://pubs.rsc.org | doi:10.1039/9781782623922-FP001

Edited by

Alok Dhawan
CSIR-Indian Institute of Toxicology Research, India
Email: [email protected]

Diana Anderson
University of Bradford, UK
Email: [email protected]

and

Rishi Shanker
CSIR-Indian Institute of Toxicology Research, India
Email: [email protected]
.
Published on 03 November 2017 on http://pubs.rsc.org | doi:10.1039/9781782623922-FP001 View Online

Issues in Toxicology No. 35

Print ISBN: 978-1-78262-158-4

PDF eISBN: 978-1-78262-392-2
EPUB eISBN: 978-1-78801-259-1
ISSN: 1757-7179

A catalogue record for this book is available from the British Library

r The Royal Society of Chemistry 2018

All rights reserved

Apart from fair dealing for the purposes of research for non-commercial purposes or for
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Act 1988 and the Copyright and Related Rights Regulations 2003, this publication may not
be reproduced, stored or transmitted, in any form or by any means, without the prior
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the appropriate Reproduction Rights Organization outside the UK. Enquiries concerning
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Published on 03 November 2017 on http://pubs.rsc.org | doi:10.1039/9781782623922-FP007

Foreword

Over the past 20 years nanotechnology has been considered as the tech-
nology of the 21st century. A huge amount of development has occurred
during this period, not only of new applications and products already on the
market, but also of possibilities that may become available within the next
10 to 15 years. Besides innovation and market demands, products are in-
creasingly directly related to the use or implementation of nanomaterials.
The manufacturing of nanomaterials and the reinforcement of products by
the integration of nanomaterials leads directly to the interaction of nano-
materials with humans and the environment. With this increasing number
of scenarios for human exposure to the newly synthesized nanomaterials,
the evaluation of possible adverse effects of such materials is of utmost
importance. This is the reason for a flood of publications on the newly
created discipline of nanotoxicology, which was named as such in 2004 for
.

the first time. More than 25 000 publications have increased the knowledge
of the biological effects of nanomaterials in various species. However, we
have also realized that many such studies were undertaken in a misleading
sense as most of them have been mechanistic studies, but authors as well as
readers have often used them as toxicological studies. Moreover, as har-
monized test protocols and adapted OECD guidelines have not been avail-
able for nanomaterials in the past 15 years, many results are not reliable and
difficult to interpret and repeat. Therefore, we have to take up the challenge
of establishing such protocols and harmonize methods for a better tox-
icological research approach to nanomaterials in the near future.
But this is not the only challenge. Toxicology itself is being orientated into
a new direction. Animal testing is more and more under suspicion to deliver
false-positive or false-negative results, the existing in vitro methods, on the
other hand, also have many restrictions or shortcomings. Thus, the

Issues in Toxicology No. 35

Nanotoxicology: Experimental and Computational Perspectives
Edited by Alok Dhawan, Diana Anderson and Rishi Shanker
r The Royal Society of Chemistry 2018
Published by the Royal Society of Chemistry, www.rsc.org

vii
 View Online

viii Foreword

development of new testing strategies and new assay platforms is an urgent

necessity.
This book brings together a number of outstanding researchers in this
field to deliver an actual and comprehensive picture of the international
Published on 03 November 2017 on http://pubs.rsc.org | doi:10.1039/9781782623922-FP007

science of nanomaterials toxicology and to present an insight into future

opportunities. Here we find the actual knowledge about characterization of
newly synthesized nanomaterials as one of the major challenges for bio-
logists and toxicologists. Moreover, working with standardized operating
procedures (SOPs) is for many researchers an ‘‘old chestnut’’ and many
funding programmes are looking for the newest and most relevant project.
This neglects the fact that this is merely increasing the knowledge of new
mechanisms, but does not help legal regulation. Here we would need better
and more reliable data, which have probably been produced in round robins,
so we can rely on these during the regulatory steps. We should not forget the
necessity for rules in toxicological studies as otherwise we shall, in the long
term, fail in our attempts to regulate nanomaterials for the growing market.
This book is a helpful and excellent example of how we can proceed into a
future where toxicology is on the right track!

Harald Krug
Empa – Swiss Federal Laboratories for Material Science & Technology
Switzerland
.
Published on 03 November 2017 on http://pubs.rsc.org | doi:10.1039/9781782623922-FP009

Preface

The enabling nature of nanotechnology has infused engineered

nanomaterial-based products in the market worldwide. To date more than
1800 nano-based products in the domains of personal care, health and
fitness, electronics, textiles, sports, ceramics, energy, automotive, medicine,
agriculture and environmental remediation, are already in use. There are
more than 40 different types of engineered nanomaterials (ENMs) being
used in various products, these include ENMs of metals, metal oxides,
carbonaceous materials and composites. These ENMs are being inadver-
tently released into the aerial, terrestrial and the aquatic environment.
The need for test methods, validation of existing protocols, methodologies
and procedures for safety as well as, in vitro and in vivo toxicity assessment of
ENMs has been, and will continue to be the objective of studies in the area of
nanomaterial toxicology. The empowering nature of nanotechnology makes
.

the development and validation of toxicity assessment methodologies all

the more necessary if we are to engineer safe materials at the nanoscale,
exploit nanomaterials in drug delivery, determine the fate of ENMs in the
environment and safely dispose of nanoproducts.
This book, Nanotoxicology: Experimental and Computational Perspectives,
incorporates several comprehensive nanomaterial toxicity protocols, which
will serve as a highly useful and ready resource for research students and
scientists working in regulatory toxicology as well as biomedical, bio-
chemical and pharmaceutical sciences.
The authors have actively contributed to peer-reviewed scientific literature
in the area of nanomaterial toxicology. The book addresses the aforesaid
issues in thirteen chapters, each with a comprehensive discussion of the
methodology. The challenges faced in nanomaterial toxicity assessment
have been addressed in the very first chapter of the book to introduce the
reader to this new component of toxicological sciences.

Issues in Toxicology No. 35

Nanotoxicology: Experimental and Computational Perspectives
Edited by Alok Dhawan, Diana Anderson and Rishi Shanker
r The Royal Society of Chemistry 2018
Published by the Royal Society of Chemistry, www.rsc.org

ix
 View Online

x Preface

The initial chapters of the book describe the protocols for the chemical
synthesis of nanoparticles for a range of applications including usage in
biomedicine. The subsequent chapters describe the in vitro and in vivo
protocols for toxicity assessment of engineered nanomaterials, develop-
Published on 03 November 2017 on http://pubs.rsc.org | doi:10.1039/9781782623922-FP009

ments of alternate test models, emerging systems toxicology approaches,

organ-on-chip systems and needs in clinical toxicology assessment.
A component of the book also explores the need for safe nanoparticles for
biological and therapeutic use and computational approaches for modeling
of interactions of nanoparticles with biomolecules. The third section of the
book explores the paradigm of health hazard and risk assessment of these
novel materials in medicine and the environment. In this book the regu-
latory perspective, based on the risk associated with the application of
nanomaterials in nanomedicine, the status of existing assays and emerging
approaches to frame policy of health risk assesssment have been discussed.
The book also introduces the readership to international guidelines and
recommendations for safety and risk assessment.
This book epitomizes the long-term scientific association that the editors
have enjoyed with the authors and is a culmination of their collaborative
efforts.

Alok Dhawan
Diana Anderson
Rishi Shanker
.
Published on 03 November 2017 on http://pubs.rsc.org | doi:10.1039/9781782623922-FP011

Editor Biographies

Professor Alok Dhawan is currently Director of the CSIR-Indian Institute of

Toxicology Research (CSIR-IITR), Lucknow. He also served as Founding
Director of the Institute of Life Sciences, and the Dean for Planning and
Development at Ahmedabad University, Gujarat. Before joining as Director
of CSIR-IITR, he held a number of different scientific positions, such as –
Scientist, Senior Principal Scientist, Principal Scientist, etc. He obtained his
PhD in Biochemistry from the University of Lucknow, India in 1991. He was
awarded a DSc (Honorary) by the University of Bradford, UK in 2017, and was
a Visiting Scholar at Michigan State University, USA, and BOYSCAST Fellow
at the Universities of Surrey, Wales & Bradford, UK. Professor Dhawan
started the area of nanomaterial toxicology in India and published a guid-
ance document on the safe use of nanomaterials. His group elucidated
the mechanism of toxicity of metal oxide nanoparticles in human and
.

bacterial cells, and his work has been widely cited. He set up a state-of-the-
art nanomaterial toxicology facility at CSIR-IITR as well as at the Institute of
Life Sciences. Professor Dhawan has won several honours and awards in-
cluding the INSA Young Scientist Medal in 1994, CSIR Young Scientist Award
in 1999, the Shakuntala Amir Chand Prize of ICMR in 2002, and the Vigyan
Ratna from the Council of Science and Technology, UP in 2011. His work in
the area of nanomaterial toxicology has won him international accolades
as well, and he was awarded two Indo-UK projects under the prestigious
UK-IERI programme. He was also awarded two European Union Projects
under the FP7 and New INDIGO programmes. He founded the Indian
Nanoscience Society in 2007. In recognition of his work he has been elected
a Fellow of the Royal Society of Chemistry, UK; the National Academy of
Sciences, India; The Academy of Toxicological Sciences, USA; The Academy
of Environmental Biology; The Academy of Science for Animal Welfare;
The Society of Toxicology, India; The Indian Nanoscience Society; and of

Issues in Toxicology No. 35

Nanotoxicology: Experimental and Computational Perspectives
Edited by Alok Dhawan, Diana Anderson and Rishi Shanker
r The Royal Society of Chemistry 2018
Published by the Royal Society of Chemistry, www.rsc.org

xi
 View Online

xii Editor Biographies

The Gujarat Science Academy. He was also Vice President of the Environ-
mental Mutagen Society of India (2006–7), and Member of The National
Academy of Medical Sciences; The United Kingdom Environmental Mutagen
Society; and The Asian Association of Environmental Mutagen Societies,
Published on 03 November 2017 on http://pubs.rsc.org | doi:10.1039/9781782623922-FP011

Japan. He has to his credit over 125 publications in peer-reviewed inter-

national journals, 18 reviews/book chapters, four patents, two copyrights
and has edited two books. He is the Editor-in-Chief of the Journal of Trans-
lational Toxicology published by American Scientific Publishers and serves
on the Editorial Board of Mutagenesis, Nanotoxicology, Mutation Research
Reviews, and other journals of repute.

Professor Diana Anderson currently holds the Established Chair in

Biomedical Sciences at the University of Bradford, UK. She obtained her
first degree at the University of Wales and second degrees in the Faculty
of Medicine, University of Manchester. After tutoring at the University of
Sydney, Australia, she became a research worker in the Department of
Cancer Studies at the University of Leeds and at the Paterson Laboratories,
Christie Hospital, Manchester. In 1974, she was appointed Head of
Mutagenesis Studies at ICI’s Central Toxicology Laboratory. Professor
Anderson joined BIBRA International in 1981 as Head of Genetic and
Reproductive Toxicology and became Assistant Director and Group Forum
Co-ordinator in 1987. In 1992, she became Senior Associate and Co-ordinator
of External Affairs at BIBRA. She has attended various management courses.
She has served on the editorial board of 10 international journals, has over
450 publications, has edited/authored 9 books and guest-edited 9 special
.

issues of 4 international journals. She has been/is Series Editor of books in

Current Toxicology for John Wiley & Sons, and Issues in Toxicology for the Royal
Society of Chemistry. As an active Committee member, she has been Vice-
President of the Institute of Biology and was Chair of the Scientific Committee
of the International Association of Environmental Mutagen Societies. She is
Chief Examiner for the International Diploma in Toxicology under the aegis of
the Royal Society of Biology. As a successful supervisor for 28 PhD, 1 MSc and
2 MPhil students, she is currently supervising another 5 PhDs. She has been
external examiner for 27 PhDs and was External Examiner for the Department
of Genetics at the University of Wales, Swansea. She has been invited to speak
at many international meetings and chair many symposia. New research
laboratories in India and Korea have been established with her help under the
auspices of the British Council and UNIDO. Funded by various international
agencies, scientists from America, Australia, the Czech Republic, Italy, India,
Iran, Korea, Poland, Serbia, Spain and Turkey have received training under
her supervision. She has organized both national and international meetings
and was a member of various national (e.g., MRC Advisory Board and
Veterinary Products Committee) and international committees, including
the European Union Scientific Committee for Animal Nutrition (SCAN). For
2007–10 she won an award as Yorkshire Enterprise Fellow and was nominated
for an Albert Einstein Award In Science in 2016. She is a consultant for many
 View Online

Editor Biographies xiii

international organizations, such as the WHO, NATO, TWAS, UNIDO and

the OECD.

Professor Rishi Shanker currently serves as Consultant to CSIR-Indian In-

Published on 03 November 2017 on http://pubs.rsc.org | doi:10.1039/9781782623922-FP011

stitute of Toxicology Research and Advisor to ABC Genomics (India) Private

Ltd at Biotechnology Park, Lucknow, India. He has served as Professor &
Associate Dean at the School of Arts & Sciences, Ahmedabad University,
Gujarat, India (2014–16). Prior to joining Ahmedabad University, he served
as Chief Scientist and Area Coordinator of the Environmental & Nanoma-
terial Toxicology Groups at CSIR-IITR, Lucknow (2001–13). He also served as
Principal Scientist at the CSIR-National Environmental Engineering Research
Institute, Nagpur and set up a state-of-the-art laboratory in the area of En-
vironmental Biotechnology (1991–2001). He obtained his Masters in Bio-
chemistry from University of Lucknow and a PhD in Environmental
Microbiology & Toxicology from CSIR-IITR & CSJM University (1985). Professor
Shanker’s post-doctoral research addressed methanogen microbiology, deep
subsurface microbiology and protein engineering at the University of London
and Pennsylvania State University, USA (1987–90).
Professor Shanker’s research contributions range from genetically
engineered bacteria for bioremediation and molecular probes for pathogen
detection to alternate models in toxicity assessment of chemicals and
engineered nanomaterials. He has successfully steered over 34 national and
international research projects including the Indo-US program: Common
Agenda for Environment, the Indo-Swiss Program in Biotechnology, the
Indo-German, EU FP7, EU New Indigo & Inno Indigo program and Unilever.
.

He has more than 80 publications and 20 reviews/book chapters to his credit

in aforesaid areas. He has mentored 11 PhD students and 20 research
projects of Masters’ students in life sciences.
His work on ‘pathogen detection and water quality’ received recognition in
form of Vigyan Ratna, conferred by the Government of Uttar Pradesh, India.
He was awarded the Visiting Research Fellowship of the Society for General
Microbiology, UK at the Anaerobic Microbiology Laboratory, Queen Mary
University of London. He has served as Visiting Scientist and Visiting
Professor at the University of Washington, Seattle, USA, the Pasteur Institute &
CEA, France, the and the Pohang University of Science & Technology, Republic
of Korea. He is a Founder Fellow of the Indian Nanoscience Society and a
Fellow of the Society of Toxicology, India.
 .
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Published on 03 November 2017 on http://pubs.rsc.org | doi:10.1039/9781782623922-FP015

Contents

Chapter 1 Nanotoxicology: Challenges for Biologists 1

Ashutosh Kumar, Sanjay Singh, Rishi Shanker and
Alok Dhawan

1.1 Introduction 1
1.2 The Hurdles in Toxicity Evaluation of NMs 2
1.3 ENM Interference with Toxicity Test Methods 4
1.3.1 Interference of NPs with Metabolic Activity
Detection Assays 4
1.3.2 Interference of NPs in Assays for Cell Death
Measurement 5
1.3.3 Interference of ENPs with Immunoassays 5
1.3.4 Interference of ENMs in Assays with
.

Enzymes 6
1.3.5 Interference with Measurement of
Free Radicals Generated due to ENM
Exposure 6
1.3.6 Interference in Cellular Uptake Assays 7
1.3.7 Interference with Cell Culture Media
Components 8
1.3.8 Interference due to Oxidation State Change
in Redox-active ENMs 9
1.3.9 Misinterpretation of TEM Images 9
1.3.10 Interference with the Comet Assay 10
1.3.11 Interference in Micronucleus Assays 11

Issues in Toxicology No. 35

Nanotoxicology: Experimental and Computational Perspectives
Edited by Alok Dhawan, Diana Anderson and Rishi Shanker
r The Royal Society of Chemistry 2018
Published by the Royal Society of Chemistry, www.rsc.org

xv
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