PLATING EFFICIENCY TESTING FLUORESCENT RED … · This study will follow the procedures described in SOP HEL11T001 for Clonogenic - PE ... Cells that reach 50-75% of confluence in

Plating Efficiency Page 1 of 16 GLP study Study code: GLP12PE001 Date printed: 29-Feb-12

PLATING EFFICIENCY TESTING FLUORESCENT RED

SILICA NANOSPHERES (50nm)

STUDY PLAN

Test facility name: Sponsor name:

Health Effects Laboratory European Commission DG RTD

NILU-Norwegian Institute for Air Research Rue de Champ de Mars 21

PO Box 100 BE-1049 Brussels, Belgium

2027 Kjeller

Plating Efficiency Page 2 of 16 GLP study Study code: GLP12PE001 Date printed: 29-Feb-12 NORWAY

STUDY SPECIFIC INFORMATION

TEST SUBSTANCE SPONSOR CODE NILU CODE

Fluorescent red silica

nanospheres

C-SIO-R0.050

Silica-NP50

HEAD OF

LABORATORY

STUDY DIRECTOR SPONSOR

Name:

Maria Dusinska

Tel: +47 6389 8000

Fax: +47 6389 8050

Email: [email protected]

Address: Health Effects Laboratory

NILU-Norwegian Institute for Air Research PO Box 100 2027 Kjeller NORWAY

Name:

Lise Fjellsbø

Tel: +47 6389 8086

Fax: +47 6389 8050

Email: [email protected]

Address: Health Effects Laboratory

NILU-Norwegian Institute for Air Research PO Box 100 2027 Kjeller NORWAY

Name of contact person:

Mr. Jürgen Buesing

Tel:

Fax:

Email:

[email protected]

Name of Institute: European

Commission/ DG RTD/

Address: Rue de Champ de Mars 21 BE-1049 Brussels, Belgium

Date:

Signature:

Date:

Signature:

Date: N/A

Signature: N/A

QA

Name: Evy Sivesind

Address: Health Effects Laboratory, NILU-Norwegian Institute for Air Research, PO Box

100 2027 Kjeller, NORWAY. Tel: +47 6389 8000. Email: [email protected]

IMPORTANT DATES AND VENUE

Proposed experimental dates Start: Week 5, 2012

End: Week 7, 2012

Proposed report dates to sponsor Draft report: 13.02.2012

Final report: 20.02.2012

Test site NILU, Kjeller, HEL

mailto:[email protected]



TEST SUBSTANCE INFORMATION

Name: Fluorescent red silica nanospheres

Formula: SIO2-Rhodamine B

NILU code:

Molecular weight: -

Appearance: Spherical NPs in red fluid

Size

Shape

Surface area

CAS: -

Batch No: CM5050

Expiry date: -

Quantity received:

Purity: -

Sterility:

Solvent:

Solubility in water: -

Solubility in other solvents: -

Stability in water (or other solvents):

Stable in Aqueous buffers, organic solvents

Not stable in Hydrofluoric acid, strong bases,

e.g. 6MNaOH

Density: 1.8g/cm3

Storage conditions: Store at 4ºC – DO NOT FREEZE

Methodology for concentration analyses: -

Technical data sheet: Available in Annex1

Certificate of analyses: Available in Annex2


Contents

1 NATURE AND PURPOSE OF THE STUDY ........................................................................................... 7

2 TEST SYSTEM ................................................................................................................................... 7

2.1 Introduction ............................................................................................................................. 7

2.2 Scientific background .............................................................................................................. 7

2.3 Justification of the test system ................................................................................................ 7

3 TEST SUBSTANCE ............................................................................................................................. 7

3.1 IDENTIFICATION ....................................................................................................................... 7

3.2 REGISTRATION ......................................................................................................................... 8

3.3 SAFETY PRECAUTIONS ............................................................................................................. 8

3.4 DISPERSION PROTOCOL FOR THE PREPARATION OF TEST SUBSTANCE .................................. 8

4 REFERENCE SUBSTANCES ................................................................................................................ 8

4.1 POSITIVE AND NEGATIVE REFERENCE SUBSTANCES ............................................................... 8

5 TEST DESCRIPTION ........................................................................................................................... 8

5.1 GUIDELINES ............................................................................................................................. 8

5.2 EXPERIMENTAL DESIGN ........................................................................................................... 9

5.3 PROTOCOL AND METHOD ....................................................................................................... 9

5.3.1 Cell lines ........................................................................................................................... 9

5.3.2 Media, culture conditions & stocks ................................................................................. 9

5.3.3 Preparation of cultures .................................................................................................... 9

5.3.4 Exposure conditions ........................................................................................................ 9

5.3.5 Measurement of cytotoxicity and viability ...................................................................... 9

5.4 DATA RECORDING ................................................................................................................. 10

5.5 EVALUATION/ANALYSIS ......................................................................................................... 10

5.6 ACCEPTANCE CRITERIA OF THE STUDY .................................................................................. 10

5.7 INTERPRETATION OF RESULTS............................................................................................... 10

6 CRITICAL PHASES ........................................................................................................................... 11

7 STANDARD OPERATING PROCEDURES CONCERNING THE STUDY ................................................ 11

8 RECORDS ........................................................................................................................................ 11

8.1 REPORT .................................................................................................................................. 11

8.2 ARCHIVES ............................................................................................................................... 12

9 QUALITY ASSURANCE .................................................................................................................... 12

Plating Efficiency Page 6 of 16 GLP study Study code: GLP12PE001 Date printed: 29-Feb-12 10 STUDY PLAN DISTRIBUTION ...................................................................................................... 12

11 REFERENCES .............................................................................................................................. 13

12 CHRONOLOGICAL PLANNING .................................................................................................... 14

13 ANNEX 1: SAFETY DATA SHEET .................................................................................................. 15

14 ANNEX 2: CERTIFICATE OF ANALYSIS ........................................................................................ 16


1 NATURE AND PURPOSE OF THE STUDY

The objective of this study is to evaluate cytotoxic potential of the test substance on

mammalian cells in vitro by determination of plating efficiency of cells.

2 TEST SYSTEM

2.1 Introduction

Cytotoxicity of cells can be determined by different endpoints at the membrane level (trypan

blue excretion, neutral red uptake) or by metabolic pathways (MTT assay), but the most

reliable endpoint is measurement of survival/cell death as it gives information on overall

toxicity. Mammalian cells in culture, especially stable cell lines, are normally used either in

suspension or growing attached to the surface. Plating efficiency assay (clonogenic, colony

formation assay), as well as proliferation assay (relative cell growth), give a quantitative

measure of overall toxicity based upon survival and/or cell death as endpoints.

2.2 Scientific background

Plating efficiency assay is based on plating cells in small inoculums on Petri dishes or 6 well-

plates. This assay gives information both on cytotoxicity as well as on viability of the cells.

The assay is performed on mammalian cells growing in monolayer attached to a surface.

Normally, only a hundred or a few hundred cells are inoculated. Each viable cell grows and

forms a colony. After a suitable incubation time (approx. 5-8 days), colonies are stained and

counted manually. Plating efficiency is calculated as % of colonies from the total number of

seeded cells. Cytotoxicity is determined by measuring cloning efficiency (number of cell

colonies) after treatment relative to cloning efficiency (number of colonies) of untreated

control cells.

Justification of the test systemThis test method based on quantitative measurement of

colony formation is scientifically accepted as one of the most reliable assays for mammalian

cytotoxicity testing and is used both in research as well as in regulatory toxicity testing of

chemical compounds. Plating efficiency has been validated as part of mutation assay (Chu et

al., 1968; Aaron et al., 1994; Abbondandolo et al., 1977) is also implemented as

cytotoxicity/viability test in OECD test guideline 476 for determination of mutant frequency.

3 TEST SUBSTANCE

3.1 IDENTIFICATION

TEST

SUBSTANCE

SPONSOR

CODE

NILU

CODE

Fluorescent red

silica nanospheres

C-SIO-R0.050

Silica-NP50


3.2 REGISTRATION

Receipt and usage of the test substance stock will be registered in HEL11P002cFORM,

placed in a binder in the laboratory during study, and archived after study.

3.3 SAFETY PRECAUTIONS

Nanoparticles are considered toxic. Although these particles are in suspension, all work will

be performed in fume hood while wearing double set of gloves. Work with nanoparticles must

follow SOP HEL11S004.

3.4 DISPERSION PROTOCOL FOR THE PREPARATION OF TEST SUBSTANCE

Fluorescent red silica nanospheres (50 nm, Corpuscular) are provided as a suspension in

water, thus no further dispersion is needed. They are stored at 4ºC and need vortex-shaking of

the vials for a few minutes immediately before use. The test substance is then diluted from

the stock solution of 25mg/ml, using in cell culture medium, to obtain a solution equivalent to

a working solution of 75µg/cm2. Nanomaterial will be characterized in the media before and

after the treatment. Then further dilutions are made from the working solution to obtain the

full range of dilutions: 0.12, 0.6, 3, 15 and 75 µg/cm2.

4 CONTROL SUBSTANCES

4.1 POSITIVE AND NEGATIVE REFERENCE AND CONTROL SUBSTANCES

Concurrent negative (untreated) and positive controls are included in each experiment. The

Silica-NP50 stock is dissolved in water by manufacturer (only negligible amount of this stock

was used). The culture medium Dulbecco Minimal essential medium (DMEM) is therefore

used as vehicle during exposure.

Negative control: As negative control (NC) cells incubated in DMEM media are used.

Positive control: Positive control (PC) is toxic compound methyl methanesulfonate (MMS).

Chemical Source Solvent Stock

conc .

Final

conc.

Cell

MMS Sigma,

Cat.no 129925

DMEM 1M 0.1mM Cos-1

5 TEST DESCRIPTION

5.1 GUIDELINES

This study will follow the procedures described in SOP HEL11T001 for Clonogenic - PE

which is based on OECD TG 476 for the testing of chemicals.


5.2 EXPERIMENTAL DESIGN

Mammalian Cos-1 cells in monolayer culture are exposed to five analyzable concentrations

(0.12 - 75 µg/cm2) of the test substance, in addition to controls, for 24 hrs. After the treatment,

cells from each treatment group are trypsinized, diluted and plated in small inoculums to

determine colony formation. Cytotoxicity is measured by the relative plating efficiency

(survival by counting number of colonies) of the cultured cells 8-10 days after plating. The

experiment will be performed twice.

5.3 PROTOCOL AND METHOD

5.3.1 Cell lines

Cos-1 cells (monkey kidney fibroblast cells) were obtained from the European Collection of

Cell Cultures (ECACC, Catalogue No. 88031701, Lot 05/J/031). Cells from the stock are

thawed and sub-cultured 2-4 times before used in experiments as described in SOP

HEL11C001 and HEL11C002. Cos-1 cells have a high cloning efficiency by 70-80% and are

sensitive to chemical toxins.

5.3.2 Media, culture conditions & stocks

Cells are cultivated in DMEM with 10% of fetal calf serum (FCS) and 1%

Penicillin/Streptomycin and are incubated in culture dishes or flasks in humidified atmosphere

in 37oC, 5 % of CO2 as described in SOP HEL11C002 for cultivating cells.

5.3.3 Preparation of cultures

After thawing, cells are incubated in culture medium at 37°C in humidified atmosphere of 5

% CO2. Fresh culture (1-4 weeks after thawing) should be used. Ideally, cells are trypsinized

twice before starting experiment. Cells that reach 50-75% of confluence in monolayer are

suitable for the experiment.

5.3.4 Exposure conditions

Concentration range should be established with regards to expected cytotoxicity, solubility in

the test system and changes in pH or osmolarity. Control and 5 concentrations will be used:

0.12, 0.6, 3, 15 and 75 µg/cm2. Proliferating cells are exposed to the test substance for 24h at

37°C in humidified atmosphere of 5 % CO2.

5.3.5 Measurement of cytotoxicity and viability

At the end of the exposure period, cells are washed and sub-cultured to determine survival

rate. Cytotoxicity will be determined by the relative cloning efficiency (survival) of the cells.

Clonogenic efficiency is initiated immediately after the treatment period. Cells in small

inoculums are cultivated in growth medium for 5-8 days. By this time, each viable cell will

create a colony, and after staining the cells with Methylene blue they can be counted

(manually or using counter) to determine the cell death/survival based on number of colonies

compared to number of inoculated cells.


5.4 DATA RECORDING

Raw data obtained during experiments will be recorded on the specific form

(HEL11T001FORMa).

Excel HEL11T001FORMb will be used for calculations of results.

5.5 EVALUATION/ANALYSIS

Plating efficiency (PE) (viability) is expressed as number of colonies (in %) from all seeded

cells following formula:

PE (%) = (Colonies Counted / Cells Inoculated) x 100

Cytotoxicity is determined by expressing the PE of treated cells relatively to PE of control

cells, where PE of control cells is set to 100%. A test substance is classified as cytotoxic if the

cell viability is reduced by at least 20% compared to control. Individual data are provided for

each treatment group for each experiment. Additionally, data are summarized in tabular form.

5.6 ACCEPTANCE CRITERIA OF THE STUDY

The experiment is considered valid when the following criteria are met:

Before inoculation: Cells should have viability of >85 % measured by Countess

Negative control: Plating efficiency of untreated cells should not vary more than ±15%

from plating efficiency characterized for the particular cell culture.

Positive control should have plating efficiency less than 60% compared to negative

control, thus inducing 60 % cell death.

5.7 INTERPRETATION OF RESULTS

The results are considered positive, and thus the test compound is classified as cytotoxic,

when following criteria are met:

cell viability is reduced by at least 20% compared to viability of the control cells.

a dose related response is observed

or reproducible results, coefficient variation (CV) should be < 30% for the mean of

replicate measurements

Biological significance of the results will be considered first. Statistical methods may be used

as an aid in demonstrating significance of the test results. Statistical significance will not be

the only determining factor for cytotoxicity. A test substance for which the results do not

meet the above criteria, is considered non-cytotoxic.

Positive results in an in vitro mammalian cytotoxicity test indicate that the test substance

induces cytotoxic effect in the cultured mammalian cells used. A positive concentration

response relationship that is reproducible is most meaningful. Negative results indicate that,

under the test conditions, the test substance does not induce cytotoxicity in the cultured

mammalian cells used.


6 CRITICAL PHASES

Sterility and avoiding contamination

Treatment of cells (preparation of stock concentration and dilutions for exposure)

Cell counting, preparation of cell suspension, dilution in steps and plating cells in

small inoculums and equal spreading on the dish.

Scoring of number of colonies

7 STANDARD OPERATING PROCEDURES CONCERNING THE

STUDY

SOP HEL11C003 Freezing and thawing of adherent cells

SOP HEL11C005 Exposure of cells to test substance

SOP HEL11C009 Cell counting

SOP HEL11C010 Cultivation of Cos-1 cells

SOP HEL11T001 Plating efficiency

SOP HEL11Q006 Verification of accuracy of pipetting volumes

SOP HEL11E007 Waterbath Grant

SOP HEL11E018 Incubator Galaxy Mini

SOP HEL11E020 Sterile Laminar Hood

SOP HEL11E022 Cell Counter Countess

SOP HEL11E043 Autoclave

SOP HEL11E044 Pipettes

SOP HEL11G007 Receipt, identification, handling and storage of test- and reference

substances

SOP HEL11G008 Recording system for raw data

SOP HEL11S001 Safety routines for laboratory work

SOP HEL11S003 Work with acute and chronic toxic substances

And NILU handbook for ESH

8 RECORDS

8.1 REPORT

The Study Director will issue an audited draft report for the Sponsor’s review and comment

before the report will be finalized. The report will contain details of the test substance, test

systems, test facilities, test concentrations, test conditions, methods used for generation of

data and the interpretation of the data.


8.2 ARCHIVES

The study dossier (study plan, amendments and deviations, draft report, copy of final report

and attached documents, the original raw data, copy of the product registration forms) will be

retained in archives at NILU for 10 years after issue of the final report. After this time, the

Sponsor will be contacted and his advice sought to return the study file and materials to the

Sponsor, retain at NILU or store at a location designated by the Sponsor. If requested, NILU

will continue to retain the study file and materials or help the Sponsor to find another location

for archiving. The Sponsor will be notified of the financial implications of each of these

options at that time.

9 QUALITY ASSURANCE

The following will be inspected or audited in relation to the study:

Study plan

The critical phases (at least one of the aspects for each of these critical phases will be

controlled by QA)

The report and study data will be audited before issue of the draft report to the

Sponsor for review and comments.

The study will be conducted in compliance with the GLP standards as set forth in the OECD

series on Principles of Good Laboratory Practice and compliance monitoring, Number 1,

ENV/MC/CHEM (98)17 (as revised in 1997) + The Application of the Principles of GLP to

in vitro Studies – ENV/JM/MONO (2004)26.

10 STUDY PLAN DISTRIBUTION

Distributed to:

Sponsor: 1 study plan (copy)

Study Director: 1 study plan (original)

Responsible Study Personnel: 1 study plan (one copy for each of them)

QA 1 study plan (copy)


11 REFERENCES

Aaron, C.S., Bolcsfoldi, G., Glatt, H.R., Moore, M., Nishi, Y., Stankowski, L., Theiss, J. and

Thompson, E. (1994). Mammalian Cell Gene Mutation Assays Working Group Report.

Report of the International Workshop on Standardisation of Genotoxicity Test Procedures.

Mutation Res., 312, 235-239.

Abbondandolo, A., Bonatti, S., Corti, G., Fiorio, R., Loprieno, N. and Mazzaccaro, A. (1977).

Induction of 6-Thioguanine-Resistant Mutants in V79 Chinese Hamster Cells by Mouse-Liver

Microsome-Activated Dimethylnitrosamine. Mutation Res., 46, 365-373.

Chu, E.H.Y. and Malling, H.V. (1968). Mammalian Cell Genetics. II. Chemical Induction of

Specific Locus Mutations in Chinese Hamster Cells In Vitro, Proc. Natl. Acad. Sci., USA, 61,

1306-1312.

Dušinská M., Slameňová D.: Use of mammalian cells in screening for chemical carcinogens,

Biologické‚ listy, 47, 264-278, 1982.

Dušinská M., Slameňová D., Nádaská M.: Effect of silver compounds on in vitro cultrured

mammalian cells. I. Cytotoxic effects of diaminosilver tetraborate on hamster cells, Biologia, 45,

3, 201-209, 1990.

Krahn, D.F., Barsky, F.C., McCooey, K.T. (1982). CHO/HGPRT Mutation Assay: Evaluation

of Gases and Volatile Liquids. In: Tice, R.R., Costa, D.L., Schaich, K.M. (eds.) Genotoxic

Effects of Airborne Agents. New York, Plenum, pp. 91-103.

Li, A.P., Gupta, R.S., Heflich, R.H. and Wasson, J. S. (1988). A Review and Analysis of the

Chinese Hamster Ovary/Hypoxanthine Guanine Phosphoribosyl Transferase System to

Determine the Mutagenicity of Chemical Agents: A Report of Phase III of the U.S.

Environmental Protection Agency Gene-tox Program. Mutation Res., 196, 17-36.

Li, A.P., Carver, J.H., Choy, W.N., Hsie, A.W., Gupta, R.S., Loveday, K.S., O’Neill, J.P.,

Riddle, J.C., Stankowski, L.F. Jr. and Yang, L.L. (1987). A Guide for the Performance of the

Chinese Hamster Ovary Cell/Hypoxanthine-Guanine Phosphoribosyl Transferase Gene

Mutation Assay. Mutation Res., 189, 135-141.

Slameňová D., Dušinská M., Bohušová T., Gábelová A.: Differences between survival,

mutagenicity and DNA replication in MMS- and MNU- treated V79 hamster cells. Mutation

Research, 228, 97, 1990.


Slameňová D., Dušinská M., Gábelová A., Bohušová T., Oravec C.: Differential effects of

theophylline on MMS- and MNNG-induced mutagenesis in V79 hamster cells. Mutation

Research, 271, 128-129, 1992.

Slameňová D., Dušinská M., Gábelová A., Horvátová E., Oravec C., Chalupa I., Szabová H.:

Assessment of toxicity, clastogenicity, mutagenicity and transforming activity of pentoxifylline

in mammalian cells cultured in vitro. Mutation Research, 332, 275-285, 1994.

12 CHRONOLOGICAL PLANNING

After sub-culturingV79 cells cells in monolayer ( 75% confluent) they areseeded for experiment.

Day 1 Day 3 Day 8-10

100-500 cells/dish6 wells-dishes per sample

Cell cultivation Treatment 3h Evaluation of colonies

Each plate representsone concentrations (sample),e.c. Positive and negative control, 3 concentrations, optionallywith and without S9 fraction.After the treatmentcells are trypsinised, suspensionof 1000 cells/ml preparedand from 100-500 cells/dishinocullated.

1x10 cells/ml

1x10 cells/ml

5

3

Staining withmetylene blue (1%)

1x10 cells/ml4


13 ANNEX 1: TECHNICAL DATA SHEET

PRODUCT

TECHNICAL DATA

SHEET

CORPUSCULAR, INC 3590 RT 9, STE 107 COLD SPRING, NY 10516 USA

Ph: +1 845 208 7027 Fax:+1 845 208 7030

______________________________________________________________________________

Product Line C-SiO-R0.05

Catalog No 141311-10

Size 49nm

Surface Chemistry Plain, OH

Solid Content 25mgml

Quantity 10ml

Media Composition DI Water

Buffer, Preservatives None

Particle Size Uniformity PI 0.175 (spec<0.25)

Particles Shape Spherical

Particle Density 1.8g/cm3

Particle Porosity Non-porous

Particles Stability -Stable in Aqueous buffers, organic solvents

Particles Stability -Not stable n Hydrofluoric acid, strong bases, e.g. 6MNaOH

Product Form Aqueous suspension

Volume of One Particle 6.16E-17cm3

Mass of One Particle 1.23E-16g

Surface Area of One Particle 7.54E-11cm2

Number of Particle/g of solid 8.12E+15

Measured Surface Area/g of solid Not measured

Binding Capacity N/A

Charge Density 5.5 OH/nm2

Parking Area (Å2) N/A

pH 7.0

______________________________________________________________________________

Store at 4ºC – DO NOT FREEZE

For research and development use only


14 ANNEX 2: CERTIFICATE OF ANALYSIS

CERTIFICATE OF ANALYSIS

CORPUSCULAR, INC 3590 RT 9, STE 107 COLD SPRING, NY 10516 USA Ph: +1 845 208 7027 Fax:+1 845 208 7030

Date of Issue September 29, 2009

Product

Catalog number

Product ID

Batch number

Date of manufacture

Fluorescent red silica nanospheres

141311-10

C-SIO-R0.050

CM5050

09/2009

Solid concentration, mg/ml

Media-Preservatives

49

DI water-None

Mean particle size, nm

Polydispersty index

49nm (Brookhaven ZETAPALS)

0.136

Particle composition

Surface chemistry

Charge Density (µeq/g)

Parking area (Å2)

Surface area, m2/g

pH

SIO2-Rhodamine B

Plain

N/A

N/A

N/A

Not measured

Store at 4ºC – DO NOT FREEZE

For research and development use only

Documents

PLATING EFFICIENCY TESTING FLUORESCENT RED … · This study will follow the procedures described in SOP HEL11T001 for Clonogenic - PE ... Cells that reach 50-75% of confluence in