750 Main_Biological Hazard Amendment Form

Biological Hazard Amendment Form
Please complete all applicable sections of this form. Any sections that do not apply to the nature of your work can be left blank.

The purpose of this Amendment is to update The Engine Environmental Health and Safety (EHS) team of the additional biological material associated with your work and the risks that they may present to you and those around you. All information must be comprehensive, true, and must be to the best of your knowledge.

Please note, you only need to include additions to your workflows in this Amendment Form. Please leave any section blank that is already captured in your initial EHS Assessment Form. All non-relevant sections may be left blank.
Biological Materials Registration and Risk Assessment

The Biological Project Hazard Assessment is intended to add additional biological material to your company EHS profile. This form may be presented to The Engine Accelerator’s Institutional Biosafety Committee (IBC) for approval, if required by NIH and local guidelines. All information must be true, to the best of your knowledge.

All Laboratory spaces at The Engine are permitted for BSL-2 work. Dedicated Tissue Culture Suites, Class II Type A2 Biosafety Cabinets, and Lentiviral Biosafety Cabinets are provided. Any work with rDNA or work that falls within NIH Guidelines requires Engine EHS review, as well as approval from The Engine's Institutional Biosafety Committee, as needed. For questions, please email labs@engine.xyz.

Relevant Links

Biosafety in Biomedical Laboratories (BMBL)

NIH Guidelines for Research Involving Recombinant or Synthetic Nucleic Acid Molecules

OSHA Bloodborne Pathogens and Needlestick Prevention

Risk Group Assessment Database

Pathogen Safety Data Sheet
Name
EHS Contact or PI Email
example@example.com
Email
Company
Will you be adding additional Tissues, Blood, and/or Body Fluids?

Please complete the table for each tissue, Blood, and/or Body Fluid, as needed

	Tissue Source: (Human, Non-Human Primate, Other Mammalian,Plant, Fungi, etc.)	Tissue Type: (Human Blood, Mouse Ovary, FBS, etc.)	Volume	If Human, Is sample de-identified?	Bloodborne Pathogen Screened?
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If tissue list is larger than the provided table, please upload a complete inventory here:

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Will you be adding additional primary cells or established cell lines?

Please complete the table for each primary cell or established cell line

	Cell Name	Source (vendor, CRO, colleague, etc.)	Biosafety Level	Genetically Modified? (Y/N)	Bloodborne Pathogen Screened?
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If cell list is larger than the provided table, please upload a complete inventory here:

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Will you be adding any bacteria, yeast, fungi, or parasites?

If strain list is larger than the provided table, please upload a complete inventory here:

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Will you be adding any viruses, bacteriophages, or prions?

If strain list is larger than the provided table, please upload a complete inventory here:

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Does your work involve the use of any of the following?

	Yes/No	If yes, explain
Any Select Agent
Any Toxin derived from a living source
Any Cytotoxic Material or Antibody Drug Conjugates

Please refer to the National Select Agents and Toxins list for the current list of regulated agents and toxins

Recombinant DNA Research
Recombinant DNA Research

In the context of the NIH Guidelines, recombinant DNA (rDNA) molecules are defined as either:
- Molecules that are constructed outside living cells by joining natural or synthetic DNA segments to DNA molecules that can replicate in a living cell
- Nucleic Acid molecules that are chemically or by other means synthesized or amplified including those that are chemically or otherwise modified but can base pair with naturally occuring nucleic acid molecules (e.g. synthetic nucleic acids)
- Molecules that results from the replication of those described above
Per our Biosafety Permit through the City of Cambridge, all work with rDNA must only take place in BSL-2 labortory spaces, and be approved by The Engine's Institutional Biosafety Committee, at least annually.

Work that falls under NIH Guidelines III-D requires approval prior to initiation of work.
Will you be adding new research with rDNA as defined by the NIH Guidelines for Research Involving rDNA Molecules?*
Please define the goal of your work with rDNA if it differs from your current project goals (e.g. creation of a new cell line, protein expression, etc.)

Does your new work include the addition of recombinant and/or synthetic nucleic acids in the following?

	Yes/No	If yes, please explain
Cell Cultures
Microorganisms
Viruses
Plants
Fungi
Other

Relevant NIH Section Guidelines

Section of Guidelines	Approval/Review	Example
III-A	NIH Director, RAC, IBC	A drug resistant gene transferred into a (new) microorganis, if aquisition of gene may compromise disease control
III-B	NIH/OSP, IBC	The cloning of toxin molecules with LD50
III-C	IRB, IB	Recombinant or synthetic nucleic acide molecules (or DNA/rDNA derived from rDNA/SNA molecules) transferred into humans
III-D	IBC	Experiments using recombinant DNA/synthetic nucleic acids from Risk Groups 2-4: in host vector systems, in non-pathogenic systems, in whole animals, whole plants. Experiments involving >10L of cell culture. Experiments with Influenza Viruses.
III-E	IBC	Recombinant DNA/synthetic nucleic acid molecules involving no more than 2/3 eukaryotic virus agents, whole plants, arthoropods, or transgenic rodents. Expressions in B strains of E. coli.
III-F	Exempt	Recombinant or synthetic nucleic acid molecules used in a variety of experiments (e.g. synthetic nucleic acid molecules that cannot replicate or generate nucleic acids that can replicate, not found in organisms or viruses, single monoclonal or viral DNA sources, or host DNA transferred to the same host or related species.

Please provide Name(s), Relevant Biosafety Level(s), and NIH Guideline Categorizations of the new work, if applicable.

	Type of Experiment or Manipulation	Biosafety Level	Section(s) of NIH Guidelines
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Please provide information below on the nature and species of sequence(s) added:

	Name of Sequence	Source (species name)	Risk Group	Type of insert (knock-in, knock-out, point mutation, siRNA, transgene, etc,.)	Function/Impact on cell (e.g. cell cycle regulator/oncogenic)	Promoter
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If list of inserted or cloned material is larger than the provided table, please attach a full inventory here:

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Please identify the new/additional cloning/expression/transfection vector(s) that will be used and provide a restriction map of vector if it is available. If commercially available, indicate vendor and catalog number. Include hyperlinks when possible.
Please outline the new/additional method of gene transfer being added, if applicable. Examples include transfection (eukaryotes), transformation (prokaryotes), electroporation, viral transduction.
Does any new sequence encode for a toxin?
If any additional sequence encodes for a toxin, please provide information on these specific sequences. If a toxin has been modified to render it less toxic or nontoxic, please explain and provide a reference that demonstrates this.
Please identify the recipient bacterial strains and/or recipient host cell lines (human, species of animal, plant, etc.)

Does the introduced rDNA to the host cell or viral vector provide any changes to the following? Please refer to the reference table below

	Yes	Specify
Increased Virulence
Overcomes Immunity
Develop Resistance to Drugs
Increase Transmission
Change Tropism
Increase host susceptibility

Modification References:

	Potential Nature of Modification
Increase Virulence	Enhances the harmful consequences of the agent or toxin
Overcomes Immunity	Disrupts immunity or the effectiveness of an immunization against the agent or toxin without clinical or agricultural justification
Develop Resistance to Drugs	Confers to the agent or toxin resistance to clinically or agriculturally useful prophylactic or therapeutic interventions against that agent or toxin or facilitiates their ability to evade detection methodologies
Increase Transmission	Increases the environmental or intrahost stability, transmissibility, or the ability to evade detection methodologies
Change tropism	Alters the host range or tropism of the agent or toxin
Increase host susceptibility	Enhances the susceptibility of a host population to the agent or toxin

If you are adding microorganisms/viruses, please complete the following table

	Microorganism/Virus	Vector	Gene Insert	Gene Source	Description
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If list of viruses and/or microorganisms or is larger than the provided table, please attach a full inventory here:

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Does your new work involve the creation or use of any of the following?

	Yes/No	If yes, explain
Transgenic Plants
Transgenic Fungi
Transgenic Animals (including insects and nematodes)

Gene Editing Technology Overview

Please complete the below section if you are adding additional gene editing technologies such as CRISP, TALENs, zinc fingers, etc.)

Please write N/A in any sections not relevant to your work.
Please describe the additional editing system (CRISPR, TALENs, zinc fingers, etc)
Please indicate the organism to be edited. Indicate whether the work is restricted to cultures, or if it will be done in whole animals and/or plants
If editing whole animals or plants, will germ line cells be targeted?
If yes, please address the potential to create a gene drive. Include any safeguards to contain the gene drive, or safeguards to prevent the creation of a gene drive:
Please describe how the components (e.g. Cas9 and sgRNA) are delivered? Common examples are of plasmid transfection, viral vector, or nanoparticle delivery of purified components.
Are the components (Cas9 and sgRNA) encoded by the same DNA construct or by multiple constructs? Provide the construct information, such as plasmid maps.
Please attach any relevant plasmid maps or other relevant information that could not be included above, as needed:

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Target Gene Function: Provide information on the genes that will be edited

	Gene Name	Original gene Function	Nature of Modification	Anticipated impact on gene function
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If list of target gene functions is larger than the provided table, please attach your list here:

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Is the targeting mechanism specific to animals, humans, or could it affect both? Explain
What is known about off-target effects with the system components that you are using?
Please explain any steps to minimize off-target effects. Examples include, but are not limited to, the use of specific CRISPR design tools, or modified endonucleases with enhanced specificity. Some are listed below for your reference.
Tools for Minimizing Off-Target Effects

CRISPR Design Tools
Endonucleases with Increased Specificity (compared to Cas9)
- CfP1
- fCas9 or fdCas9 (catalytically inactive Cas9 with FokI nuclease)
- Paired Cas9 nickases

Please detail all viral vectors to be used and indicate any safety features that prevent generation of a replication competent product.

	Type of Viral Vector (e.g. lentivirus, adenovirus, AAV)	Expression Plasmid (or transfer vector)	Packaging System (cell line envelope and accessory plasmids; e.g. 293T, pMD2.g, and psPAX2)	Vector Type (i.e. transient ectopic, genomic integration)	Promoter driving transgene expression
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If you are purchasing pre-made viral particles, indicate the vendor and catalog number. Include hyperlinks to products, if possible
Is testing for replication competent virus required? (testing is usually not required for self-inactivating viral vectors)
High Risk Genes

Please select any traits associated with the genes that you are working with that may be considered high risk.

Please reference the NIH Guidelines
Are any of the genes in use considered to be high risk?
Do any of the genes in this research code for production of biological toxins, with an LD50
Do any of the genes involved in this research confer antibiotic resistance that has not occurred naturally or in a medical environment? Example includes the creation and introduction of cephalosporin-resistance cassette to N.gonorrhea when this cassette does not exist in nature. (NIH Guidelines, Section III-A)
Do any of the genes involved in this research broaden the tropism of the agent? (NIH Guidelines, Appendix B-V)
Do any of the genes involved in this research increase virulence? (NIH Guidelines, Section II-A-3)
Are any of your proposed genes well described in the scientific literature as oncogenes?
Will you be silencing or knocking out tumor suppressor genes?
If you answered Yes to any of the above, please explain:

Biological Containment Protocols
Does your new workflow involve culturing anything, or any laboratory work, in volumes >10L?*
If yes, explain:
Please list all associated investigators and personnel conducting the work. If any are new to the project, please note their experience in handling these or related materials
Please identify and discuss the health and safety risks associated with your new work and the use of materials identified*
Describe the signs and symptoms of infection, the mode(s) of transmission, availability of vaccine or therapeutic treatment*
Identify the new procedures that will create the greatest risk of exposure or infection (e.g. generation of aerosols, injection from sharps)
Describe the engineering controls and personal protective equipment (PPE) required to minimize the risk of exposure of laboratory personnel during procedures requiring handling or manipulation of these materials (e.g. biosafety cabinet, gloves, lab coats, safety glasses, centrifuge rotor cups, etc.)
Identify decontamination procedures and disinfectant(s) to be used for work surfaces, instruments, equipment, liquid containing biological materials, and glassware
Identify disposal and/or decontamination procedures for contaminated sharps, solid waste, tissues, pipette tips, etc.

Dual Use Research Concern (DURC)
Dual Use Research Concern

NIH/OSP DURC Reference

Biological research is considered ‘dual-use’ in nature if the methodologies, materials, or results could be used to cause harm. Dual Use Research of Concern (DURC) is a small subset of life sciences research that, based on current understanding, can be reasonably anticipated to provide knowledge, information, products, or technologies that could be directly misapplied to pose a significant threat with broad potential consequences to public health and safety, agricultural crops and other plants, animals, the environment, materiel, or national security.
Agent of Toxin Involved in Project

Verify if this project directly involves nonattenuated forms of 1 or more of the 15 listed agents.

Bacteria:
- Bacillus anthracis
- Burkholderia mallei
- Burkholderia pseudomallei
- Clostridium botulinum (toxin producing strains)
- Francisella tularensis
- Yersinia pestis
Viruses:
- Avian influenza virus (highly pathogenic)
- Ebola virus
- Foot-and-mouth disease virus
- Marburg virus
- Reconstructed 1918 influenza virus
- Rinderpest virus
- Variola major virus
- variola minor virus
Toxin:
- Botulinum neurotoxin (any quantity)
Are any of the agents or toxins listed above involved in your work?*
If you selected yes, please explain why this research cannot be conducted with other agents. Include all categories that apply to the proposed work from the reference below.

Modification References:

	Potential Nature of Modification
Increase Virulence	Enhances the harmful consequences of the agent or toxin
Overcomes Immunity	Disrupts immunity or the effectiveness of an immunization against the agent or toxin without clinical or agricultural justification
Develop Resistance to Drugs	Confers to the agent or toxin resistance to clinically or agriculturally useful prophylactic or therapeutic interventions against that agent or toxin or facilitiates their ability to evade detection methodologies
Increase Transmission	Increases the environmental or intrahost stability, transmissibility, or the ability to evade detection methodologies
Change tropism	Alters the host range or tropism of the agent or toxin
Increase host susceptibility	Enhances the susceptibility of a host population to the agent or toxin

Certification and Signature
Certification and Signature

I agree to abide by the following requirements:
- I will ensure that shop/laboratory personnel have received appropriate information about the shop, physical, chemical, laser, and/or biological hazards of the research outlined in this assessment by making available information regarding precautions to be taken to prevent exposures or release to the laboratory or the environment.
- I am familiar with and will ensure use of appropriate shop and/or laboratory practices and procedures in the conduct of this research.
- I certify that shop and/or laboratory personnel have appropriate technical expertise. I will ensure that shop and/or laboratory personnel know the procedures for dealing with incidents and spills of chemical materials and know the appropriate waste management procedures.
- I will ensure that all shop and/or laboratory personnel have completed all necessary training and that their training records are up to date. If this research changes to involve the use of any organism, microorganism, cell line, viral vector, or any other biological agent that is not explicitly listed above,
- I will submit information pertaining to those new agents to The Engine Accelerator for approval. If this research changes and requires the use or any recombinant DNA technology or other genetic manipulation not explicitly listed above.
- I will submit information pertaining to those new agents to The Engine Accelerator for review.
- If any new project is to be undertaken that is not explicitly listed above, I will submit an additional Safety Assessment Form to The Engine Accelerator EHS team.
- I understand that acceptance of this Assessment Form, as written, does not constitute a requirement for The Engine Accelerator to accept or approve any additional work at any Engine operated facilities.
- I understand that The Engine Institutional Biosafety Committee is required to review each Biological Project Assessment Form and any applicable
As the Principal Investigator, and on behalf of blanks* , I certify that the provided information is accurate and complete.
Principal Investigator Signature*

Clear
Date*

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The Engine Environmental Health and Safety Representative

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Date

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