Nanosafety & Risk Assessment

Nanosafety & Risk Assessment ... 3 Key Performance Indicators 5 Measures 3 Strategies Vision Objective Thailand has an effective management system of...

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Nanosafety & Risk Assessment - NANOTEC's best practices for promotion of human health and safety of nanotechnology -

Rawiwan Maniratanachote, Ph.D. [email protected]

National Nanotechnology Center (NANOTEC), NSTDA THAILAND

Technical Workshop for the Asia-Pacific Region on Nanotechnology and Manufactured Nanomaterials: Safety Issues Sirindhorn Science Home, NSTDA, Thailand Science Park, Pathum Thani, Thailand September 10, 2015

Nano Safety and Risk Assessment Laboratory (SRA) SRA conducts the researches to address possible adverse effects of nanomaterials and novel substances on human health and the environment. The methodology for safety investigation are thoroughly implemented case-by-case, using the available guidelines and standards, as well as alternative scientific procedures for the best practice.  Our group is currently focusing on development of reliable in vitro models for determining toxicological effects and pharmacological properties of materials upon their routes of exposure.  SRA also introduces models of zebrafish and microorganisms into our research studies and testing services.

Copyright © 2015 NANOTEC

SRA In vitro models Cells/Tissues

Bacteria

In vivo model Zebrafish

Test end points: toxicity, interactions and compatibility, genetics, cellular processes, molecular mechanisms, pharmacology, and development of embryo

Test substances: nanomaterials, chemicals, cosmetics, food and dietary supplements

Researches on Nanosafety - Our Experiences and Inter-Lab Comparison -

Biological Effects of Nanomaterials Cell viability

Cellular uptake Control

ROS generation

AgNPs

Cell cycle

Activity of CYP enzymes 50 nm

SubG1

• Genotoxicity • Immunotoxicity • Expression of mRNA and protein Copyright © 2015 NANOTEC

Designed experiment for study on biological effects

Appropriate experimental design:

The conceivable interaction of insoluble particles with submersed cells grown at the bottom of a well, filled with an appropriate medium of height h. (A) Previously employed picture, (B) more appropriate concept discussed in this study. The number of particles in (A) and (B) is the same.

96- well

6- well

Petri dish

(0.32 cm2)

(9.5 cm2)

(55 cm2)

Volume adjustment for insoluble materials

• Concentration (w/v) • Particles per area

Wittmaak K (2011) ACS nano 5:3766–3778.

Copyright © 2015 NANOTEC

Toxicological effects of AgNPs in hematological system Objectives: -To determine cytotoxic effects of AgNPs on hematological system using human erythroid cells comparing with cell lines Team :NANOTEC (SRA), Mahidol University (Faculty of Medicine) In vitro models: - K562 and HL60 cell lines - Human primary erythroid cells (Healthy controls) A

B

C

D

E

F

A : undifferentiated blast B : proerythroblast C : basophilic normoblast D : early polychromatic normoblast E : late polychromatic normoblast F : orthochromatic normoblast Copyright © 2015 NANOTEC

Erythroid culture by Fibach’s method Day6

Day10

Day14

I

II

III

IV

V

Day20

I; undifferentiated blast, II; proerythroblast III; basophilic normoblast, IV; polychromatic normoblast, V; orthochromatic normoblast

AgNPs caused significant cytotoxic effects in the primary erythroid cells, where as the immortalized HL60 and K562 cells showed much lower cytotoxic effects. Copyright © 2015 NANOTEC

Hemolysis assay

(A)

AgNPs caused a dosedependent hemolysis of erythroid cells.

(B) Hemoglobin (µg/ml)

0.035

*

0.03 0.025

*

*

0.02 0.015 0.01

0.005 0 Control

TEM analysis

(C) Control

20

50

AgNPs (µg/ml)

100

250

H2O2 (µM)

AgNPs 100 μg/ml

AgNPs damaged the erythroid cell membrane.

Rujanapun N, Aueviriyavit S, Viprakasit V, Maniratanachote R et al. Toxicol In Vitro. (2015) 29:1982-1992.

Copyright © 2015 NANOTEC

Toxicological effects of AgNPs in Caco-2 cells

AgNPs but not AgNPs reduced cell viability in intestinal cell model (Caco-2 cells)

Effects of AgNP exposure on oxidative stress in Caco-2 cells

AgNPs induced biological response via Nrf2/HO-1 activation

*p < 0.05; **p < 0.005 comparing the untreated and NP-exposed cells 15 μg/ml AgNPs (filled circles) and 125 μ g/ml AuNPs(open circles) Copyright © 2015 NANOTEC

Proposed mechanism underlying the cellular responses to AgNPs by Nrf2/HO-1 signaling pathway

Aueviriyavit S, Phummiratch D, and Maniratanachote R. Toxicol Lett. (2014) 224:73-83. Copyright © 2015 NANOTEC

Occupational Health Risk Increase production of nanomaterials in industrial sectors

Increase potential exposure and occupational health risk! Copyright © 2015 NANOTEC

Published recommendations for assessment criteria OSHA Permissible Exposure Limits (PELs)- General Industry

Inert or nuisance dust (an average during normal working hours) • Respirable dust (PM 5) 5 mg/m3 • Total dust 15 mg/m3 PELs are 8-hour time weighted averages (TWAs)

National Institute for Occupational Safety and Health (NIOSH) Recommended limit value • Titanium dioxide (< 100 nm) 0.3 mg/m3, 10 h/day and 40 h/week • Carbon nanotubes and –fibers 0.007 mg/m3 (measured as elementary carbon)

Copyright © 2015 NANOTEC

Safety Investigation of Nanoproducts - Our Experiences and Best Practices -

Cosmetics and Personal Care Nanoproducts Analysis of silver in nanoproducts: 20 items These products claimed to be “nano-silver” Sample digestion

SEM-EDX

GFAAS Ag

20 nm

• By using SEM, silver nanomaterials can be detected in only few products • Total Silver concentration was determined by GFAAS after wet acid digestion Wasukan N, Maniratanachote R et al., submitted Copyright © 2015 NANOTEC

Textiles Silver release from textile nanoproducts into artificial sweat Tested samples Laboratory textiles were prepared by pad-dry-cure method (A0, A1, A2, A3, A4) Six commercial claimed nanosilver shirts were purchased (B, C, D, E, F and G)

Physical-chemical analysis

10 µm

500 nm

Kulthong K, Maniratanachote R et al., (2010) Particle and Fibre Toxicology, 7:8 Copyright © 2015 NANOTEC

Measurement of antibacterial properties

Percent reduction of bacteria Sample

S. aureus

E. coli

A0

-

-

A1

98.04

-

A2

99.02

-

A3

97.30

-

A4

99.83

99.93

B

98.23

-

C

98.56

-

Sample A0

Sample A4

S. aureus

E. coli D

-

-

E

-

28.73

F

99.85

99.80

G

99.99

81.44

Kulthong K, Maniratanachote R et al., (2010) Particle and Fibre Toxicology, 7:8 Copyright © 2015 NANOTEC

Release of silver into artificial sweat Silver released into artificial sweat (mg/kg) Sample

Initial silver content (mg/kg)

A0

AATCC pH4.3

ISO pH 5.5

ISO pH 8.0

EN pH6.5

n.d.

n.d.

n.d.

n.d.

n.d.

A1

36.12 ± 22.42

21.01 ± 4.13

15.53 ± 3.62

34.27 ± 2.88

35.83 ± 19.68

A2

56.57 ± 34.28

33.39 ± 15.80

28.81 ± 10.34

66.54 ± 46.29

77.96 ± 23.80

A3

95.12 ± 33.12

70.15 ± 37.29

72.69 ± 11.99

82.22 ± 26.99

152.20 ± 36.54

A4

425.21 ± 93.73

217.61 ± 81.32

177.13 ± 57.13

268.31 ± 131.15

322.21 ± 87.00

B

n.d.

n.d.

n.d.

n.d.

n.d.

C

n.d.

n.d.

n.d.

n.d.

n.d.

D

n.d.

n.d.

n.d.

n.d.

n.d.

E

15.16 ± 9.90

0.08 ± 0.05

0.01 ± 0.01

0.05 ± 0.30

0.36 ± 0.10

F

1.22 ± 0.87

n.d.

n.d.

n.d.

0.05 ± 0.00

G

0.99 ± 1.53

n.d.

n.d.

n.d.

n.d.

* The amount of silver released was dependent on: Initial amount of silver, Quality of the fabrics, Artificial sweat formulations and pH. * This information might be useful for estimating potential human exposure to silver form textile nanoproducts. Kulthong K, Maniratanachote R et al., (2010) Particle and Fibre Toxicology, 7:8 Copyright © 2015 NANOTEC

Structure of Nanosafety and Ethics Strategic Plan (2012-2016) Vision

Objective

Safe-nano for Thailand’s Sustainable Development

To enhance health and environment safety as well as promote social security via ethical, sustainable and proper engagement in R&D, production, distribution and nanotechnology and nanoproduct usage

3 Key Performance Indicators

Thailand has an effective management system of nanosafety and ethics, with related sectors’ operating within 5 years

Nanoproducts in Thailand’s market have labels displaying nanomaterials components and safety information based on scientific evidence

The public has knowledge, understanding, and awareness of nanosafety and risk and are able to select, store, and handle nanoproducts by themselves

3 Strategies

Establish the knowledge management center to manage the information of nanosafety and ethics and nanoproducts

Develop and reinforce measures as well as mechanisms of monitoring and enforcement

Promote public engagement

5 Measures

Engineering

Enforcement

Economics

Education

Empowerment

Copyright © 2015 NANOTEC

NanoMARKS Flagship

NanoMARKS Flagship Goals: Nano Characterization Laboratory (NCL)

NanoMARKS’ targeting Thai nano-

merchandises with safety, international standard, and quality acceptance from Thai consumers and trading partner countries

Nano Safety and Risk Assessment Laboratory (SRA)

Copyright © 2015 NANOTEC

Nanoproducts in our focus  Health and Cosmetic Manufacturers (Health and Cosmetic products)

 Food and Agricultural Manufacturers

(Agricultural products, Processed foods, Food packages)

 Post-petrochemical Manufacturers

(Textiles, Plastic beads, Synthetic rubbers, Glue, and Coating chemicals)

Copyright © 2015 NANOTEC

NanoMARKS Flagship National Advanced NANO Characterization Center (NANC)

2

Income

Standard

NCL SRA

3

Testing Services

Infra structure

Flagship Targeted Nanoproducts • Cosmetics • Food • Petrochemicals

Collaboration & Training 1

Research

Product labeling

Copyright © 2015 NANOTEC

TRM: Physico-Chemical techniques

57   

59

58

• Size(ISO 22412) • Shape (ISO 16700:2004) • Anti Bacteria Activity (JIS Z 2801: 2006, CLSI M27A)

  

  

60

• Chemical composition • Water & Stain Repellent (AATCC-22) • Protocol for testing products • Bacterial stability test

• Surface Area • Chemical composition • Water & Stain Repellent (AATCC22)

• Protocol for testing products

Size characterization: ISO 22412:2008, ISO 16700:2004, ISO/TS 24597:2011 Functions: ISO 22916: 2011, JIS Z 2801: 2006, AATCC 100, ASTM E 2149, ISO 27447:2009, ASTMG21 : 1992 reapprove 2002, JIS Z 2911 :1992, CLSI M27A Copyright © 2015 NANOTEC

TRM: Safety testing techniques

57

  

59

58

• Skin irritation (OECD439) • Skin corrosion (OECD431) • In vitro cytotoxicity (ISO10993-5)



60

• Genotoxicity (micronucleus) • Toxicokinetics

   

• Phototoxicity (OECD432) • Skin sensitization (h-CLAT) • Genotoxicity (Comet) • Hematocompatibility

 

• Systemic toxicity /Environmental (aquatic) toxicity  Zebrafish embryo Acute toxicity  Teratogenicity

• Carcinogenicity

Biological effects and Biocompatibility test matrix (ISO 10993-1) Copyright © 2015 NANOTEC

Current status Quality test

Safety test

Cosmetics

2015

DLS 100

Genotoxic

2016

SEM

80

60

Quality

40

Sensitization

TEM

20

Standard Safety

0 Phototoxicity

FFF

Skin irritation/ corrosion

ICP

Cytotoxicity

Food

2015

DLS

2017

100 Genotoxic

Ecotoxic

SEM

80

Release kinetics

60 40

20

Kinetic DI

Petrochemicals

Genotoxic

Cytotoxicity

FFF

80 60

ICP

TEM

40

0

Sensitization

FFF

ICP

Phototoxicity HPLC/GC

2018

SEM

20

TEM

0

2015

DLS 100

Cytotoxicity Skin irritation/ corrosion

Our supporting roles • The labs support and promote manufacturer’s capability to compete in national and international markets. • Scientific-based characterization and toxicological testing can aid the manufacturers in the selection of raw materials in order to manufacture safe and high-quality products. • The testing information of the products also help for consumer decisions in product selection.

Copyright © 2015 NANOTEC

Our supporting roles

Finished products and raw materials

• • • •

Skin corrosion/irritation (OECD TG431 and TG439) Cytotoxicity (ISO 10993-5) Release and sustainability (ASTM F619-03) Toxicity and human health effects

Impact value on promoting commercialized Nanotechnology > 1,500 million THB (From a company in 2013 and 2014) Copyright © 2015 NANOTEC

Nanotoxicology

Toxicology Workplace Exposure measurement

Ma terials Sci ence & Technolog y

Universities Genotoxicity

• CU

Thailand Institute of Scientific and Technological Research

• SWU • MU

Measurement and standard

NCL

SRA

• MFU

Ministry of Public Health Department of Labour Protection and Welfare Occupational health

Office of the Consumer Protection Board Product safety Copyright © 2015 NANOTEC

SRA research team

Copyright © 2015 NANOTEC