PowerPoint Presentation...ACDP Approved list of biological agents – Hazard Groups Safety Office...

07/01/2014

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Safety Office

www.safety.ncl.ac.uk

Safety Office

Genetic Modification Safety

Dr. Samantha Dainty – University Biological Safety Officer

Safety Office


Objectives of Today

What is GM?

Why does GM Biosafety Matter?

What are the implications for you when things go wrong?

Risk Assessment - Risks relating to biological agents and

hazards at work.

How will you identify risks of your project?

How to contain biological agents and hazards.

How will you prevent yourself and co workers becoming

infected?

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What is GM?

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Genetically Modified Organisms

Any technique which alters the genetic material of an

organism using a method that would not occur by natural

mating or recombination

Also gene deletions or insertion of multiple copies of a

gene are GM if brought about by artificial means

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Examples

1. Insertion of nucleic acid, produced outside an organism

into any virus, plasmid or other vector then incorporation into

a host organism in which it does not occur naturally and a

capable of continued propagation.

2. Direct introduction into an organism of heritable genetic

material prepared outside the organsim, including micro-

injection and micro-encapsulation.

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GM Exemptions

Mutagenesis (eg x-rays, chemicals)

Synthetic nucleotides

Self cloning organisms

“Natural” transformation

Hybridoma’s

Humans and human embryos - IVF

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Why does GM-safety matter?

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Safety Law

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GM Safety Law

The project manager/PI has ultimate legal responsibility of

the project and is liable for their projects.

Individuals are also legally responsible for their own actions

HSE will send a team of investigators following GM incidents.

Prosecutions (prison?) and lab closure is a possibility in very

serious cases

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www.safety.ncl.ac.uk 10

What is different in GM?

• The GM process produces a NEW organism with different, potentially unknown properties.

• Thus the route of exposure and virulence may have changed requiring altered containment.

• Therefore it is a legal requirement for all GM work to be i) notified to HSE and ii) can only be carried out in registered GM centres

• Newcastle University's GM Centre Reference number is GM540

• Note: HSE tend not to accept that the new (GM) organism may be less viable or less infective even if it is a deletion transgenic organism.

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University Reputation – public

perception of GMO

We have to protect ourselves/work from this controversy by

demonstrating the highest level of containment of GMO – “zero risk” of

escape……

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Reputation

14 February 1999

IMPERIAL College, London, renowned as one of

Britain’s leading research institutions, was fined

£25,000 and ordered to pay more than £21,000

costs yesterday for exposing staff to a potentially

lethal new virus for which there is no cure.

The college’s “seriously flawed” approach to

health and safety matters raised a distinct

possibility that both hepatitis C and dengue fever

could be released into the open while it was

attempting to create a hybrid from the two.

Neither staff nor members of the public were

adequately protected from the possibility that the

man-made organism could have escaped,

Blackfriars Crown Court in London was told.

Scientists, led by Dr John Monjardino, failed to

use sealed cabinets while studying the virus and

made no emergency plan for dealing with a

spillage. Staff at the college, part of London

University, were not provided with protective

clothing and had to walk through a room used as

an office by other university employees in order

to dispose of contaminated material.

Keith Morton, for the prosecution, said: “They

were creating a hybrid virus for which no vaccine

or treatment exists. Safety measures should have

been of a very high standard to protect staff

and the general public

.

They have shown a disregard for basic measures

to ensure and monitor safety, as a consequence of

which their employees were exposed to a very

real risk of infection.”

Contrary to expected procedures, the Health

and Safety Executive was only notified that the

research had begun when a researcher inquired

about transporting the hybrid virus to Oxford. A

subsequent inspection in December 1998

uncovered the potentially hazardous regime.

While Judge David Martineau acknowledged that

work on finding a vaccine for the two diseases

was very important, there could be no excuse for

such lapses, he said. Hepatitis C is frequently

fatal, while dengue fever causes a severe but non-

fatal reaction.

Imperial College admitted one count of “failing

to apply principles of good microbiological

practices and principles of good occupational

safety and hygiene” under the Genetically

Modified Organisms (Contained Use)

Regulations 1992. It also pleaded guilty to one

charge of breaching the Health and Safety at

Work Act 1974. The college and its safety

advisers were each fined £20,000 in March for

exposing the public to an “unacceptable risk”

from the HIV virus. In 1998 it was fined

£4,500 for exposing a worker to an “animal

allergen”.

College exposes

staff to lethal virus BY HELEN STUDD

24 July 2001

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Severe Unknown Risks

e.g. GM Mousepox-IL4 Virus

Hypervirulent strain of highly pathogenic

GM virus – with no vaccine

Mousepox does not normally infect

humans

GM mousepox infection of workers or

escape from lab?

Smallpox in humans?

Responsible for 300-500 million deaths in

the 20th Century alone!

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Part 1: Risk Assessment of Work

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Genetically Modified Organism

3 components are required to generate a GMO

Gene cDNA

Host

Vector

Genetic material

GM cells

Host

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Risks associated with host

ACDP Approved list of biological agents – Hazard Groups

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Risks associated with the

vector

Plasmids - Can it be transferred to other organisms?

- non mobilisable or mobilisation defective plasmids

- plasmids with a narrow host range

Viral vectors

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Risks with Lentiviruses

Vectors encoding for cDNA or shRNA of choice

The major risks to be considered

● Potential for generation of replication-competent lentivirus

(RCL)

● User infection - potential for oncogenesis

‘Some’ liver tumours have been observed in neo-natal animals

following Lentiviral administration (source SACGM)

‘Next generation’ vector systems

Minimise risk (providing there are no hazards associated with

the insert)

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Risks associated with the

inserted genetic material

● Toxins

● Cytokines

● Allergens

● Hormones

● Oncogenes

● Prions

● Viral genomes

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The resulting GMO Cell lines

• Cancer cell lines – GM may affect:

− Cell phenotype or functions, increased tumourigenicity

− Immune evasion

• Some cell lines already contain viral components: HPV-E6, SV40, adenoviruses

• Know your cell line before transfecting in recombinant components - viable virus?

• Effects of GM modification could be unknown

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The resulting GMO

microorganisms (GMM)

● Increased ability to cause disease in humans/animals/plants

● Inability to treat disease or offer prophylaxis – antibiotic resistance?

● Establishment or dissemination of GMM in the environment

● Natural transfer of genetic material from or to GMOs

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The resulting GMO

Animals

Increased ability to cause disease in humans

Could act as a human disease vector or reservoir

Flying insects have high escape hazard – must be

contained!

Could they also interact with animals in the

environment?

Changes to behaviour e.g. increased

aggressiveness

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RISKS: GM organisms (GMO)

Plants

Resistance to pests

Resistance to herbicides

Mutant pathogenic plant strain

-GM pollen impossible to contain once

released from the lab – result?

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Assign the activity class

• As well as Hazard group rating of host we now also need to consider GM Class: normally

− HG1 - GM Class 1



• BUT an HG1 organism could become a GM class 2 depending on how it’s been modified

• Hazard group rating sets the “base level” then depending on the modification the organism may be elevated to a higher risk group based on the modification

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What determines Activity Class? Safety Office


GM Guidance

SACGM Compendium of guidance

ACDP Approved list of biological agents

ACDP Biological agents: Managing risks in laboratories and healthcare premises

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How does the University

monitor GM?

Project Risk

Assessment

School GM

Chair

University GM

committee

University Biological

Safety Officer

Project Commences

PI obtains

relevant forms

HSE Approval

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Part 3: Containing GMO

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GMO and GMM Containment

GM containment is Vital

Risk to human health and the ENVIRONMENT

What effects will GMO have on wild type species?

What effects will GMO have on local plant life and

wildlife?

How would someone infected with a GMO be treated?

Will vaccines and antibiotics be effective?

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Exposure Routes

Inhalation

Aerosols

Ingestion

Swallowing

Injection

Sharps injuries, animal bites and scratches

Absorption

Intact skin or external mucous membranes

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Containment Laboratories

Containment levels required for general, animal and plant

laboratories

CL1 for low risk work with HG1 biological agents

CL2 for medium risk work with HG2 biological agents

CL3 for high risk work with HG3 biological agents

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Basic Controls for Animals

Containment laboratory, dedicated equipment

and PPE

Access control and locked rooms

Isolators and individually ventilated cages

Home Office licences and DEFRA licenses

for animal welfare

Escape of GM animals unlikely! Security

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Basic Controls for Plants

Containment laboratory, Isolators and

propagators

Dedicated equipment and PPE

Access control and locked rooms

DEFRA licences required for specific plant

pathogens and pests and plants

POLLEN – containment after escape from the

lab is practically impossible!

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Biological Controls help contain

GMO and GMM

Substitution of wild type strains or environments for less harmful

ones

Attenuated strains (eg vaccine strains)

Host range modified mutant strains Reduced replication capacity

Inactivated strains – e.coli K12

Species cannot survive outside of lab environment

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Lentiviruses in GMO work – Integrated

Safety

Each plasmid contains specific

components for replication

Viral genes not expressed in target cells! –

no viral replication!

Lentivirus are Self Inactivating (SIN) by

removing promoters from 3’ of LTR –

transcriptionally inactive. Can’t be

converted into full length RNA

New generations remove additional

components for biosafety -Removal of tat – essential for replication of w.t. virus -Four vector system

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Other controls

● Lab coats

● Gloves

● Respiratory protective equipment

● Eye/face protection

● Microbiological safety cabinets

● Hand washing

● Vaccination

● Health surveillance

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Sharps Controls

Dispose of sharps immediately after use in sharps bin

Take sharps bins to sharps

Dispose of bins on reaching level

Don’t dispose of sharps in ordinary waste bins

Treat all biological materials as potentially hazardous

Don’t dispose of sharps in clinical waste bags

Never resheath needles

Use gloves and never resheath needles

Don’t transfer used sharps to other workers

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Inactivation of GMMs and GMOs

Disinfectants must be suitable for biological agents - No

universal disinfectant

70% ethanol, UV, hyperchlorites, Trigene, Virkon

Narrow or broad spectrum activity

Disinfectants can be harmful – hyperchlorites!

Dilute accurately and discard when inactive

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Sterile Environment?

Disinfectant Action

Is a quick spray of ethanol good enough to protect your work or you from biological agents?

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Autoclaving

100% kill of GMM/GMO is required for disposing of

waste

Autoclaving is most effective method for

inactivating GMO waste

Standard 121°C or 134 °C for 15-30 minutes

Validation of effectiveness using annual

thermocouple testing is required

Do not autoclave GMO containing radioactive or

hazardous chemical substances

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• adopt working procedures which minimise the risk of an accident happening

• the most likely emergency is spillage of a liquid culture or loose samples; leakage from a container

• know what to do in the event of a spillage of your materials

• the problem is greater with larger scale experiments - so be prepared accordingly!

• special situation in centrifuges due to bottle leakage

• BEFORE an emergency occurs…….consider what might happen

Emergency Situations Safety Office


Emergencies: Typical “Lab

made” Spill kit

Have a emergency Spillage standard operating procedure

Autoclave bag Gloves Mask

Lab coat Absorbant towels

Disinfectant Goggles

Absorbant granules SOP

Gloves

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Summary

• All GM work must be in registered facilities and all GM projects properly

risk assessed and CL2, CL3, CL4 projects notified to HSE

• All GM work must be risk assessed taking into account the effects of the

GM on the agents and host organisms used; products expressed

• GMM’s / GMO’s must be contained

• GMM / GMO waste must be 100% inactivated

• Ensure HG of host and final activity class are considered

• Take additional care with mammalian viral vectors with potentially

harmful inserts

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Any questions?

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