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Turin, Italy M06 Meeting 14-15 June, 2017

WP4

Environment and human safety evaluation of the

coating processes and products

Leader UNIMIB

Partners: UPC, CNR, BIU, CENTI, UL, KLO, MAR, DGA, SYN ,PRJ

https://www.google.co.il/url?sa=i&rct=j&q=&esrc=s&source=images&cd=&cad=rja&uact=8&ved=0ahUKEwidjvmMvLDQAhXC6xoKHbeDAj0QjRwIBw&url=https://spie.org/membership/spie-professional-magazine/spie-professional-archives-and-special-content/2014_jan_archive_spie_pro/horizon2020&psig=AFQjCNH1Fb3xxKgUSP6XWJQwCzyMtBzF8w&ust=1479495426330485


Objectives/Tasks

1. Environment safety assessment of the nano-coating technologies and end products (Task 4.1)

2. Assessment of human nano-toxicology due to exposure of workers and end users to functional NPs and end products (Task 4.2)

3. Estimation of the potential occurrence of bacterial resistance to the new antibacterial products (Task 4.3)

4. Health, safety and environment (HSE) Risk Management of nano-coating processes and end products (Task 4.4)

5. Development of safety related SOPs and information sheets for workers and end-users (Task 4.5)



Gantt WP4

WP/T

No. WPs/Tasks Name

Year 1 Year 2 Year 3

Q1 Q2 Q3 Q4 Q1 Q2 Q3 Q4 Q1 Q2 Q3 Q4

WP4 Environment and human safety evaluation of the

coating processes and products

T4.1 Environment safety assessment of the nano-

coating technologies and end products

D4.1

T4.2

Assessment of human nano-toxicology due

to exposure of workers and end users to

functional NPs and end products

D4.2

T4.3

Estimation of the potential occurrence of

bacterial resistance to the new antibacterial

products

T4.4

Health, safety and environment (HSE) Risk

Management of nano-coating processes and

end products

T4.5

Development of safety related SOPs and

information sheets for workers and end-

users



Concepts of EHS studies in Protect 1- Categorization of the antibacterial NP toxicity in representative in vitro systems and alternative in vivo models 2- Monitoring of the environmental and human exposure to nanos at the workplace and after wearing/leaching of the coated materials (textiles, water membranes) 3- Estimation of the toxicological risk basing on the identified hazards and exposures. 4- Safety data sheets and guidelines



Work Plan for the first year



Task 4.1 Environment safety assessment of the nano-coating

technologies and end products

UNIMIB Atmospheric exposure Aquatic exposure

At the workplace During use

Monitoring of airborne NPs by advanced instruments

FET and/or other standard ecotox test

Set up of environmental and personal monitoring systems for nanos



Work program M1-M12 … after M01 meeting

1- Identification and set up of the environmental monitoring systems for NP release Relevant for worker exposure - NP release at production sites (in collaboration with CNR-ISTEC, WP2) Relevant for consumer and environmental organisms exposure - Airborne NP release from wearing of coated textiles (in collaboration with ETH, Zurich) - NP release in water after washing and leaching of coated textiles and depuration

membranes (in collaboration with AITEX, WP5)

2- Set up of the biological tests for ecotoxicity (FET/FETAX) and human toxicity (see task 4.2) 3-identify the possible exposure scenarios according to the industrial operating pilots – in collaboration with the companies (in collaboration with WP2 (M9-M12) 4-Identify the most promising biocidal NPs to be used for coating and perform antibacterial and toxicity test (M1-M12) 5- Inputs from UL – in connection with SUN and GuideNano projects – to harmonize data collection and methods for exposure, hazard and risk assessment (M6-12)



Biocidal NPs for antibacterial and toxicity tests CuO, ZnO and Zn-doped CuO (from BIU) (Zn-CuO is retained the most efficient) Preliminary studies performed by BIU and UNIMIB. To be refined TiO2@SiO2 (from CNR-ISTEC) and SiO2@TiO2 (from CENTI) (UV-photoactivable promising antibacterial agents). Preliminary studies under way @ BIU-Life and UNIMIB



CuO, ZnO and Zn-CuO NP powders and coated textiles (available from BIU-Chem)

… preliminary environ tox study

Toxicity toward aquatic organisms occured only at very high NP concentrations. Zn-CuO did not induce higher ecotox effects in respect to CuO and ZnO Even in direct contact with coated textiles toxicity is low and comparable among different MeOs

Ecotox effects of single NPs Ecotox effects of coated textiles



Set up of the standard FET (OECD 236/2013) Refinement of the ecotox studies and categorization of the

effects of the different antibacterial nano-metal oxides (M9-M18)



Abrasion test and monitoring of the abraded airborne NPs

SMPS

APS

wZnO

wCuO eZnO

eCuO

Small amount of submicrometer and nm sized particles released



Task 4.2 Assessment of human nano-toxicology due to exposure

of workers and end users to functional NPs and end products

UNIMIB Inhalation toxicology Skin toxicity

Set up and preliminary toxicity test with in vitro

systems

Lung cells (and barriers)

Skin barriers



M1-M12 1- Set up of the biological models to study the toxicity of nano-antibacterials on human cells and tissues - relevant for exposure of workers and consumers (in vitro systems) - Human lung epithelial cells (M1-M6) - Human immune cells (M1-M6) - Human reconstructed skin (M7-M12) - Human alveolar blood barrier (ABB) (M7-M12) 2- Comparative cytotoxic effects of antibacterial nano metal oxides on human lung and immune cells 2a- Cytotoxicity, inflammatory response and mode of action of CuO, ZnO and Zn-CuO on A549 and THP-1 cells – preliminary data (M1-M6) 2b- Cytotoxicity and ROS production of TiO2@SiO2 and SiO2@TiO2 - preliminary data (M1-M6)



Comparative cytotoxic effects of CuO, ZnO and Zn-CuO on lung (A549) and immune (THP-1) cells

From BIU-Chem

… preliminary data……



Cell viability, IC50 (ug/ml)

Refinement of previous studies

Cytotoxicity of Zn-CuO on human cells is comparable to that of ZnO and CuO



Abraded airborne NPs, estimated lung deposition and toxicity for water- and ethanol-based ZnO and CuO Mantecca et al., 2017, Environmental Science and Technology (under review)

The modality of sonochemical coating influences the potential release of airborne NPs. Estimated particle loading to the lung is lower than the tox concentrations, especially for eZnO and eCuO

Cell viability

Calculation by MPPD2 model



Abraded airborne NPs, estimated lung deposition and toxicity for Zn-doped CuO

(M9-M18)



Cytotoxicity and ROS production of TiO2@SiO2 and SiO2@TiO2

OptiMEM medium (1% FBS)

[TiO2]= 750 μg/ml Z-average (nm) SD (nm) PdI SD

A= TiO2SiO2 1/1 1255,667 117,887 0,277 0,049

B= TiOSiO2 1/3 1400,333 144,282 0,394 0,104

C= SiO2:TiO2 1:2 610,067 37,296 0,147 0,064

D= SiO2:TiO2 1:5 408,633 21,669 0,093 0,080

A= TiO2SiO2 1/1

B= TiO2SiO2 1/3 D= SiO2:TiO2 1:5

C= SiO2:TiO2 1:2

Legend [TiO2] ISTEC A= TiO2SiO2 1/1 7,5 g/L ISTEC B= TiOSiO2 1/3 7,5 g/L CENTI C= SiO2:TiO2 1:2 6,7 g/L CENTI D= SiO2:TiO2 1:5 8,3 g/L

DLS analyses in cell culture medium



Intracellular probing of ROS by carboxy-DCFDA after 90 min exposure of A549 cells with TiO2@SiO2 and SiO2@TiO2

(with and without UV photoactivation of NPs for 2,5h) A= TiO2@SiO2 1/1

C= SiO2:@TiO2 1:2 Intracellular ROS - Slight increase after

TiO2@SiO2 exp

- Significant increase after SiO2@TiO2 exp

- No effects of UV-photoactivation



0

20

40

60

80

100

120

UV+ UV - UV+ UV - UV+ UV - UV+ UV -

TiO2SiO2 1/1_A TiO2SiO2 1/3_B SiO2:TiO2 1:2_C SiO2:TiO2 1:5_D

% c

ell

viab

ility

7,5 μg/ml 75 μg/ml 750 μg/ml

A549 cells exposed for 24h to TiO2@SiO2 and SiO2@TiO2 NPs UV+= photoactivation; UV-= no photoactivation

TiO2@SiO2 was not cytotoxic SiO2@TiO2 showed a dose-dependent cytotoxicity

UV-photoactivation did not seem to modulate cytotoxicity. (Check radiation conditions)



Task 4.3 Estimation of the potential occurrence of

bacterial resistance to the new antibacterial products

BIU-Life



Purpose From To What Date

antibacterial and toxicity properties of TiO2@SiO2

and SiO2@TiO2

UNIMIB, BIU-Life

CNR-ISTEC, CENTI

Refinement of the studies and paper

draft (Call conf)

October 2017

NP emission monitoring – parallel activity of WP2,

UNIMIB, CNR-ISTEC

Design of the methods and

instrumentation for NP monitoring at the

workplace

December 2017

Defining methods for risk assessment

UL UNIMIB, CNR-ISTEC

Harmonization of the methodology to

generate exposure and toxicity data

obtain

December 2017

Foreseen Interactions/internal deliverables



Purpose Place Participants Date

Share the research approach and the preliminary results with

the companies. Design the 2nd year safety related activities

involving the companies

UNIMIB, UPC, CNR, BIU, CENTI, UL, KLO, MAR, DGA, SYN ,PRJ

December 2017

Foreseen internal meetings


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