Select agent regulations

Abstract

Several events over the past two decades have changed the way scientists acquire and work with pathogenic microorganisms and toxins in the United States and internationally. The Select Agent Regulations, which were promulgated by CDC and APHIS to address concerns raised by these events, have been in effect for more than 20 years. Knowledge of these and other regulations that will impact the practice of microbial forensics is essential.

Introduction

Several events over the past 23 years have changed the way scientists acquire and work with pathogenic microorganisms and biological toxins in the United States (US) and internationally (Morse, 2015). These events, which have increased national and international awareness of the potential or real threat of terrorism (including bioterrorism), include the unlawful attempt by an individual with ties to white supremacist groups to acquire Yersinia pestis (Carus, 2002); the documented unsuccessful anthrax attack in Kameido, Tokyo (Olsen, 1999, Takahashi et al., 1993, Danzig et al., 2011) and the release of the nerve agent sarin in the subway system of Tokyo in 1995 by the Aum Shinrikyo (Olsen, 1999, Danzig et al., 2011, Kaplan, 2000); the bombing of the Murrah Federal Building in Oklahoma City in 1995 (Smoak and Geiling, 2004); the terrorist attacks using commercial airlines on September 11, 2001 (Smoak and Geiling, 2004); and the 2001 anthrax attacks in the US (Cole, 2003, Decker, 2018). The 2001 anthrax attack was a major impetus in the development of the field of microbial forensics. In an effort to control the misuse of dangerous pathogens and toxins by those wishing to do harm, the US passed several laws (Public Law 104-132, 1996, Public Law 107-188, 2002, Public Law 107-56, 2001) and subsequently promulgated regulations (i.e., Select Agent Regulations) (42 C.F.R, 9 C.F.R, 7 C.F.R) designed to limit unauthorized access to these agents. For a historical perspective on the development of these regulations, the reader is referred to Morse (2015). The creation of an oversight role for the federal government in the area of biological sciences has evoked concern within the scientific community and is considered by some to actually increase societal vulnerability to biological attacks and natural epidemics (Casadevall and Relman, 2010). The purpose of this chapter is to discuss the Select Agent Regulations and other regulations that will impact the practice of microbial forensics.

The select agent regulations

The practice of microbial forensics may necessitate working with evidence containing a select agent or toxin or with the select agents or toxins themselves. Individuals or entities who want to possess (including store), use, or transfer select agents or toxins should review the current requirements of the select agent regulations, which can be found at https://www.selectagents.gov. Entities must register with either the Centers for Disease Control and Prevention (CDC) of the Department of Health and Human Services (DHHS) or the Animal and Plant Health Inspection Service (APHIS) of the US Department of Agriculture (USDA) if they plan to possess, use, or transfer agents or toxins on the HHS or USDA select agent list, respectively. If the select agent or toxin is an overlap agent (i.e., regulated by both HHS and USDA), the entity may register with either HHS or APHIS, but is not required to register with both. Because the registration process, including the requirement for security risk assessments, may be time-consuming, an entity contemplating working with select agents or toxins should initiate the process well ahead of time. While registration is both agent and laboratory specific, the registration can be amended to include additional agents and laboratory spaces. A registration lasts for a maximum of 3 years (but may be granted for a shorter period of time).

Select agents and toxins

The current HHS, USDA, and overlap list of select agents and toxins is shown in Table 28.1 . The list is not static and is reviewed on a biennial basis by the respective select agent programs with advice and input from the Interagency Select Agents and Toxins Technical Advisory Committee (ISATTAC). The review is designed to provide an objective (i.e., quantitative) review in determining whether an agent or toxin should be added, removed, or maintained on the select agent list. Furthermore, agents and toxins can be added to (or deleted from) the list at any time by amending the regulations in accordance with the Administrative Procedure Act (5 U.S.C. § 552) by providing notification of the proposed amendment in the Federal Register and the opportunity for public comment, as illustrated by the addition of Bacillus cereus Biovar anthracis in 2016 (CDC and U.S. Department of Health and Human Services, 2016). Currently, all regulated toxins are on the HHS list. However, these toxins are subject to the regulation only if the aggregate amount of the toxin under the control of a principal investigator, treating physician or veterinarian, or commercial manufacturer or distributor exceeds the amount specified in the regulations (see Table 28.2 ). These de minimis amounts represent an attempt to balance the requirement for regulatory oversight of toxins that have the potential to pose a severe threat to public health and safety with the need for their availability for critical research, forensic, and educational purposes. The de minimis amounts in the regulations are based on the amount a person with a legitimate need, such as a principal investigator, could safely possess without constituting a significant threat to public health and safety.

Table 28.1.

DHHS^a and USDA^b select agents and toxins.

DHHS select agents and toxins

Abrin

Bacillus cereus Biovar anthracisc

Botulinum neurotoxinsc

Botulinum neurotoxin–producing species of Clostridiumc

Conotoxins (Short, paralytic alpha conotoxins containing the following amino acid sequence X1CCX2PACGX3X4X5X6CX7) d

Coxiella burnetii

Crimean-Congo hemorrhagic fever virus

Diacetoxyscirpenol (DAS)

Eastern equine encephalitis viruse

Ebola virusc

Francisella tularensisc

Lassa fever virus

Lujo virus

Marburg virusc

Monkeypox virus

Reconstructed replication competent forms of the 1918 pandemic influenza virus containing any portion of the coding regions of all eight gene segments (reconstructed 1918 influenza virus)

Ricin

Rickettsia prowazekii

SARS coronavirus (SARS-CoV)

Saxitoxin

South American hemorrhagic fever viruses (Chapare, Guanarito, Junin, Machupo, Sabia)

Staphylococcal enterotoxins (subtypes A–E)

T-2 toxin

Tetrodotoxin

Tick-borne encephalitis complex (flavi) viruses (Far Eastern subtype, Siberian subtype, Kyasanur Forest disease virus, Omsk hemorrhagic fever virus)e

Variola major virus (Smallpox virus)c

Variola minor virus (Alastrim)c

Yersinia pestisc

Overlap select sgents and toxins

B. anthracisc

B. anthracis Pasteur strain

Brucella abortus

Brucella melitensis

Brucella suis

Burkholderia mallei (formerly Pseudomonas mallei)c

Burkholderia pseudomallei (Formerly Pseudomonas pseudomallei)c

Hendra virus

Nipah virus

Rift Valley fever virus

Venezuelan equine encephalitis viruse

USDA Veterinary Services (VS) select agents and toxins

African horse sickness virus

African swine fever virus

Avian influenza virus (highly pathogenic)

Classical swine fever viruse

Foot-and-mouth disease virusc,e

Goat pox virus

Lumpy skin disease virus

Mycoplasma capricolum subspecies capripneumoniae (contagious caprine pleuropneumonia)

Mycoplasma mycoides subspecies mycoides small colony (Mmm SC) (contagious bovine pleuropneumonia)

Peste des petits ruminants virus

Rinderpest virusc

Sheep pox virus

Swine vesicular disease viruse

Virulent Newcastle disease virus

USDA Plant Protection and Quarantine (PPQ) select agents and toxins

Peronosclerospora philippinensis (Peronosclerospora sacchari)

Phoma glycinicola (formerly Pyrenochaeta glycines)

Ralstonia solanacearum

Rathayibacter toxicus

Sclerophthora rayssiae

Synchytrium endobioticum

Xanthomonas oryzae

The current list of select agents and toxins can be found at https://www.selectagents.gov/SelectAgentsandToxinsList.html.

^aDHHS regulation 42 C.F.R. Part 73.

^bUSDA regulations 7 C.F.R. Part 331 and 9 C.F.R. Part 121.

^cTier 1 Select Agents.

^dC = Cysteine residues are all present as disulfides, with the first and third cysteine and the second and fourth cysteine forming–specific disulfide bridges. The consensus sequence includes known toxins α-MI and α-GI and α-GIA, Ac1.1a, α-CnlA, α-CnlB; X1 = any amino acid(s) or Des-X; X2 = asparagine or histidine; P = proline; A = alanine; G = glycine; X3 = arginine or lysine; X4 = asparagine, histidine, lysine, arginine, tyrosine, phenylalanine, or tryptophan; X5 = tyrosine, phenylalanine, or tryptophan; X6 = serine, threonine, glutamate, aspartate, glutamine, or asparagine; X7 = any amino acid(s) or Des-X and “Des-X” = “an amino acid does not have to be present at this position.” For example, if a peptide sequence were XCCHPA, then the related peptide CCHPA would be designated as Des-X.

^eNucleic acids from these select agent viruses can produce infectious forms.

Table 28.2.

Permissible toxin amounts.^a

DHHS toxin	Amount (mg)
Abrin	1,000
Botulinum neurotoxins	1
Short, paralytic alpha conotoxins	100
Diacetoxyscirpenol (DAS)	10,000
Ricin	1,000
Saxitoxin	500
Staphylococcal enterotoxins (subtypes A–E)	100
T-2 toxin	10,000
Tetrodotoxin	500

The current permissible toxin amounts can be found at https://www.selectagents.gov/PermissibleToxinAmounts.html.

^aToxins listed in 42 C.F.R. Part 73.3(d) are not regulated if the amount under the control of a principal investigator, treating physician or veterinarian, or commercial manufacturer or distributor does not exceed, at any time, the amounts indicated here.

While some select agent genomes are not covered under the select agent regulations, other native and recombinant nucleic acid molecules are regulated. The following genetic elements, recombinant and/or synthetic nucleic acids, and recombinant and/or synthetic organisms are regulated as select agents: nucleic acids that can produce infectious forms of any of the select agent viruses that are indicated in Table 28.1; recombinant and/or synthetic nucleic acids that encode the toxic form(s) of select agent toxins if the nucleic acids can be expressed in vivo or in vitro or are in a vector or recombinant host genome and can be expressed in vivo or in vitro; and, select agents and toxins that have been genetically modified. For example, positive strand RNA viruses (e.g., Venezuelan equine encephalitis virus) and certain DNA viruses that utilize host polymerases contain nucleic acids that can be used to produce infectious forms of the viruses. Such nucleic acids are subject to the select agent regulations. Any organism that contains regulated recombinant or synthetic nucleic acids are subject to the regulations unless the organisms or nucleic acids are excluded as (i) a nonviable select agent or nontoxic toxin; (ii) an excluded attenuated strain, or (iii) an excluded select toxin modified to be less potent or toxic (see below).

Under the current select agent regulations, the following are examples of genomic materials from select agent viruses that would not be regulated as a select agent: (i) material from regulated genomes that has been rendered noninfectious; (ii) cDNA made from genomes of regulated select agent pathogens; and (iii) complete genomes of single-stranded, negative-strand RNA viruses, double-stranded RNA viruses, and double-stranded DNA viruses that require a unique polymerase (e.g., monkeypox virus). The select agent regulations do not apply to variola major genetic elements because the World Health Organization (WHO) has already placed significant restrictions on the possession, use, and transfer of these materials. Institutions other than the two currently recognized WHO collaborating centers (CDC, Atlanta, GA, and State Research Center of Virology and Biotechnology, Koltsovo, Novosibirsk, Russia) may not possess genetic fragments exceeding 20% of the variola major virus genome (for further information refer to http://www.who.int/csr/disease/smallpox/research/en/). These restrictions are followed by the CDC but are not legally binding. However, 18 U.S.C. § 175c makes it unlawful for any person to knowingly produce, engineer, synthesize, acquire, transfer directly or indirectly, receive, possess, import, export, or use, or possess and threaten to use the variola virus. Genomic material from bacteria or fungi on the select agent list is not regulated as a select agent. Additionally, the nucleic acid sequences of select agent pathogens are not regulated.

In 2006, the National Science Advisory Board on Biosecurity was concerned that advances in technology could lead to the ability to recreate or synthesize select agent viruses. The Board recommended that the government should take steps to increase awareness among providers and users of synthetic genomic materials regarding compliance with the select agent regulations and provide providers with a list of genomic materials explicitly covered by the regulations. In response, HHS developed recommended baseline standards for the gene and genome synthesis industry and other providers of synthetic double-stranded DNA products regarding the screening or orders so that they are filled in compliance with current US regulations (Department of Health and Human Services, 2010). The first de novo synthesis of horsepox virus, a member of the orthopoxvirus group, which includes Variola virus, has heightened concerns about the reemergence of smallpox as a threat to global health security (Koblenz, 2017).

Tier 1 select agents

The select agents and toxins differ significantly in their pathogenicity, toxicity, and ability to be utilized as an agent of bioterrorism, and therefore, the risk that they might pose to human, animal, and plant health varies substantially (National Science Advisory Board for Biosecurity, 2009). Nevertheless, the 2005 Select Agent Final Rule applied the same regulatory controls regardless of the agent, which made it difficult to conduct legitimate research using less pathogenic select agents (Casadevall and Relman, 2010, National Science Advisory Board for Biosecurity, 2009). Therefore, it was recommended by a government advisory board (National Science Advisory Board for Biosecurity, 2009) that the list of select agents be reduced or stratified. Subsequently, Executive Order 13546, which was signed on July 2, 2010, directed the Secretaries of HHS and USDA to designate those select agents that pose the greatest risk of deliberate misuse with the greatest potential for mass casualties or devastating effects to the economy, critical infrastructure, or public confidence as Tier 1 agents and toxins (see organisms indicated by footnote ^c in Table 28.1) and amend their respective regulations to establish security standards specific to these agents and toxins (The President, 2010). A federal panel employed several criteria, as well as information from the intelligence community, to identify 13 agents that were classified as Tier 1 (Bhattacharjee, 2011). The number of Tier 1 agents was subsequently increased to 14 in 2016 by the addition of B. cereus biovar anthracis (CDC and U.S. Department of Health and Human Services, 2016). Currently, only nucleic acids that encode the toxic forms of botulinum neurotoxins, or infectious forms of Foot-and-Mouth Disease virus, are regulated as a Tier 1 agent, which means that all Tier 1 regulatory requirements apply to these nucleic acids (see below).

The determination that the B. anthracis spores used in the 2001 anthrax letters presumably came from a federal laboratory facility (Decker, 2018) was an important factor in designing the security changes needed to address the insider threat. Specific changes in the regulations related to Tier 1 agents are described in Table 28.3 . Specific guidance for these and other requirements of the Select Agent Regulations can be found in guidance documents (Table 28.4 ), which can be found at https://www.selectagents.gov. The National Science Advisory Board for Biosecurity also issued several recommendations related to hiring and employment practices and to fostering an awareness of biosecurity and promoting responsible conduct as an approach to reducing the insider threat (National Science Advisory Board for Biosecurity, 2011).

Table 28.3.

Select agent regulation (42 C.F.R. § 73, 7 C.F.R. § 331, and 9 C.F.R. § 121) requirements for working with tier 1 agents.

Section	Requirement
15(b)	Entities with Tier 1 select agents and toxins must conduct annual insider threat awareness briefings on how to identify and report suspicious behaviors (training).
12(d)	The biosafety plan must include an occupational health program for individuals with access to Tier 1 agents and toxins, and those individuals must be enrolled in the occupational health program (occupational health).
14(b)	Entities with Tier 1 agents must provide the following additional information in the incident response plan: (i) a plan for how the entity will respond to the activation of the alarm system or information on an intruder in the laboratory; (ii) Procedures on how the entity will notify the appropriate federal, state, or local law enforcement agencies of suspicious activity that may be criminal in nature and related to the entity, its personnel, or its select agents or toxins (incident response plan).
11(f)(1); 11(f)(3)	Persons with access to Tier 1 select agents and toxins must have additional preaccess suitability and on-going assessment requirements (security plan).
11(f)(2)	Entities must describe procedures for how an entity's Responsible Official (RO) will coordinate their efforts with the entity's safety and security professionals to ensure security of Tier 1 select agents and toxins and share appropriate relevant information, which may affect the security plan (security plan).
11(f)(4)	Limit access to laboratories and storage facilities outside of normal business hours to only those specifically approved by RO or designee(s) (security plan).
11(f)(iii)	Procedures must be in place for screening visitors, their property, and vehicles at the entry points to the areas registered for Tier 1 select agents and toxins (security plan).
11(f)(iv)	A requirement for three barriers (physical structure that is designed to prevent access to Tier 1 agents by unauthorized persons [security plan]).
11(f)(v)	A requirement for intrusion detection system (security plan).
11(f)(vii)	Entity must describe procedures to ensure that security is maintained in the event of the failure of the access control system due to power disruption (security plan).
11(f)(viii)	Entity must determine the response time for response force (security plan).

Table 28.4.

Guidance documents.^a

Plan and personnel guidance

Select Agents and Toxins Biosafety/Biocontainment Plan Guidance (July 2018)b

Drills and Exercises Guidance (August 2017)

Guidance for Select Agent Regulation Training Requirements (March 2017)

Incident Response Plan Guidance (February 2018)

Information Systems Security Controls Guidance (March 2017)

Occupational Health Program Guidance (June 2016)

Responsible Officials Resource Manual (March 2017)

Security Plan Guidelines (October 2017)

Suitability Assessment Program Guidance (March 2017)

Shipping and storage guidance

Guidance for Completing the Shipper's Declaration for Dangerous Goodsc

Guidance on the Inventory of Select Agents and Toxins (May 2017)

Guidance on the Transfer of Select Agents and Toxins (October 2017)

Select agent and toxin use guidance

Avian Influenza Viruses (February 2018)

Exclusion Guidance Document (March 2017)

Guidance on the Inactivation or Removal of Select Agents and Toxins for Future Use (August 2017)

Guidance on the Regulation of Select Agent and Toxin Nucleic Acidsc

Restricted Experiments Guidance (October 2017)

Select Toxin Guidance (May 2017)

^aGuidance documents are available at www.selectagents.gov/compliance.html.

^bDate of last revision.

^cNo date given for current version.

Restricted experiments

Certain experiments with select agents or toxins are prohibited unless approved by and conducted in accordance with conditions prescribed by the Secretary (HHS or USDA). See, for example, 42 C.F.R. § 73.13 (restricted experiments) and appropriate guidance document (Table 28.4). Restricted experiments are those (i) utilizing recombinant DNA that involve the deliberate transfer of a drug resistance trait to select agents that are not known to acquire the trait naturally, if such acquisition could compromise the use of the drug to control disease agents in humans, veterinary medicine, or agriculture, and (ii) involving deliberate formation of recombinant DNA containing genes for the biosynthesis of select agent toxins lethal for vertebrates at an LD₅₀  < 100 ng/kg body weight. Approval to conduct a “restricted experiment” requires submission of a written request to the Select Agent Program with supporting scientific information and documentation. A written decision granting or denying the request is issued by the responsible Select Agent Program (i.e., CDC or APHIS). A number of requests to introduce drug resistance genes have already been approved. However, unlike excluded attenuated strains of select agents, which once excluded are no longer subject to the regulation (as long as the attenuated strain is not manipulated to restore or enhance its virulence) and can be used by any investigator (see later), each request to conduct a restricted experiment is reviewed separately to ensure that appropriate precautions and containment will be utilized.

Exemptions

The select agent regulatory exemptions provide that individuals or entities that may find themselves in possession of a select agent or toxin are not required to be in compliance with the select agent regulations if they take the specific actions required and/or meet the specific conditions proscribed by the regulations. The current exemptions in the regulations include (i) diagnostic, verification, or proficiency testing specimens in clinical or diagnostic laboratories; (ii) products licensed or otherwise approved for use by the federal government under specific statutes; (iii) investigational products approved by the federal government under specific statutes; and (iv) when either the HHS Secretary or the USDA Secretary grant specific exemptions due to a public health or agricultural emergency, respectively. A few examples of exemptions should suffice. A clinical laboratory that isolates a select agent from a clinical specimen is exempt from requirements of the select agent regulation, provided that (i) within 7 calendar days after identification, the select agent is transferred in accordance with the regulation or destroyed on-site by a recognized sterilization process (Isolation of a Tier 1 select agent or toxin requires the immediate notification of the Select Agent Program by telephone, facsimile, or email) followed within 7 calendar days by the submission of an APHIS/CDC Form 4; (ii) the select agent is secured against theft, loss, or release during the period between identification and transfer or destruction; and (iii) identification of the select agent is reported to the CDC or APHIS and to other appropriate authorities when required by federal, state, or local law. Another example is that select agents or toxins or products containing select agents or toxins are exempt from the select agent regulation provisions if they are cleared, approved, licensed, or registered under certain laws. These laws include the Federal Food, Drug, and Cosmetic Act (21 U.S.C. § 301 et seq.) and Section 351 of the Public Health Service Act pertaining to biological products (42 U.S.C. § 262). Examples of these products are the three Type A botulinum neurotoxins and one Type B botulinum neurotoxin preparations that have been approved for production and distribution on the US market.

Exclusions

There are certain circumstances (i.e., exclusions) under which the Select Agent Regulations do not apply to the possession, use, or transfer of one of the biological agents or toxins listed in the Regulation. These exclusions include (i) any select agent or toxin that is in its naturally occurring environment, provided the select agent or toxin was not intentionally introduced, cultivated, collected, or otherwise extracted from its natural source (for example, B. anthracis spores in the soil of a ranch in Texas would be excluded from the regulation); (ii) nonviable select agents or nonfunctional toxins (for example, the purified B chain of ricin toxin is not subject to regulation); and (iii) HHS toxins under the control of a principle investigator, treating physician or veterinarian, or commercial manufacturer or distributor if the aggregate amount does not, at any time, exceed amounts listed in the regulation (Table 28.2). For example, a single investigator may possess up to 1000 mg of purified ricin and not be subject to the regulation.

Attenuated strains of select agents or attenuated toxins may be excluded from requirements of the regulation based on a determination by either the CDC or the APHIS that it does not pose a severe threat to public health and safety, animal health and animal products, or to plant health and plant products. To apply for an exclusion, an individual or entity must submit a written request and provide supporting scientific data to either the HHS or USDA Select Agent Programs. The submitted documentation is reviewed by ISATTAC with input from appropriate subject-matter experts. Based on this review, a written decision supporting or denying the request is issued by the appropriate Select Agent Program. If granted, the exclusion becomes effective on notification of the applicant. Exclusions are listed on the Internet at https://www.selectagents.gov. The current list of excluded strains of select agent bacteria and viruses is shown in Table 28.5 ; excluded toxins are shown in Table 28.6 . If an excluded strain or toxin is subjected to any manipulation that restores or enhances its virulence or toxicity, the resulting select agent or toxin becomes subject to requirements of the regulations. An example of this type of manipulation is the introduction of the virulence plasmid pXO2 into the excluded Sterne strain of B. anthracis (pXO1⁺ pXO2⁻).

Table 28.5.

Select agent exclusions.

•Avian influenza virus (low pathogenic). Any low pathogenic strains of avian influenza virus, avian paramyxovirus serotype-1 viruses which do not meet the criteria for Newcastle disease virus, including those identified as pigeon paramyxovirus-12 isolated from a nonpoultry species

•Avian influenza virus (highly pathogenic). Recombinant vaccine reference strains of the H5N1 and H5N3 subtypes (effective 05-07-2004)

• Bacillus anthracis strains devoid of both plasmids pX01 and pX02 (effective: 02-27-2003); strains devoid of the plasmid pX02 (e.g., B. anthracis Sterne, pX01+ pX02−) (effective: 02–27-2003)

• Brucella abortus-lacZ ΔnorD ΔznuA (vaccine strain) (effective: 06-02-2011); B. abortus S2308 Δpgm (vaccine strain of S2308) (effective: 08-09-2006); B. abortus strain 19 (vaccine strain) (effective: 06-12-2003); B. abortus strain RB51 (vaccine strain) (effective 05-07-2003)

• Brucella melitensis-lacZ ΔnorD ΔznuA (effective: 07-07-2015); B. melitensis strain 16MΔvjbR (effective: 12-22-2014)

• Burkholderia mallei Δasd strains (ATCC 23344 Δasd; Ivan Δasd; China 5 Δasd; 2002721278 Δasd) (effective: 12-13-2017); B. mallei strain CLH001 ΔtonB Δhcp1 a mutant of B. mallei strain ATCC 23344 (effective: 08-20-2015)

• Burkholderia pseudomallei Δasd strains (DL2 Δasd; DL25 Δasd; DL28 Δasd; MSHR503 Δasd; NAU44A6 Δasd; MSHR840 Δasd; MSHR1655 Δasd; MSHR87 Δasd; MSHR367b Δasd) (effective: 12-13-2017); B. pseudomallei strain 576mn ΔpurM derivative of strain 576a (effective: 08-18-2017); B. pseudomallei strain JW270, a capsular polysaccharide deletion mutant (effective: 07-02-2014); B. pseudomallei strain B0011, a Δasd mutant of strain 1026b (effective: 12-07-2011); B. pseudomallei strain Bp82, a ΔpurM mutant of strain 1026b (effective: 04-14-2010)

• Coxiella burnetii Phase II, Nine Mile Strain, plaque purified clone 4 (effective: 10-15-2003)

•Eastern equine encephalitis (EEE) virus, South American Genotypes (effective: 12-04-2012); EEE virus/Sindbis chimeric construction that includes the structural gene (only) of EEE virus (effective: 05-29-2007)

•Ebola virus, ΔVP30 replication incompetent virus (effective: 01-02-2013)

•Foot-and-mouth disease virus (FMD). FMD-LL3B3D A24 Cruzeiro vaccine strain (effective: 04-30-2018)

• Francisella tularensis subspecies tularensis, SCHU S4ΔclpB strain (effective: 11-10-2014); F. tularensis subspecies novicida (also referred to as Francisella novicida) strains and F. novicida-like strains (effective: 11-10-2014); F. tularensis subspecies novicida strain Utah 112 (ATCC 15482) (effective: 02-27-2003); F. tularensis subspecies holarctica LVS (live vaccine strain; includes NDBR 101 lots, TSI-GSD lots, and ATCC 29684) (effective: 02-27-2003); F. tularensis subspecies tularensis strain B-38 (ATCC 6223) (effective: 02-27-2003)

•Junin virus vaccine strain Candid No. 1 (effective: 02-07-2003)

•Lassa fever virus, Mopeia/Lassa (MOP/LAS) arenavirus construct ML-29 (effective: 03-02-2005)

•Monkeypox virus, West African clade (effective: 12-04-2012)

• Mycoplasma capricolum. All subspecies are excluded except capripneumoniae (contagious caprine pleuropneumonia)

• Mycoplasma mycoides. All subspecies are excluded except mycoides small colony (Mmm SC) (contagious bovine pleuropneumonia)

•Rift Valley fever (RVF) virus live-attenuated vaccine candidate strain ΔNSs-ΔNSm-ZH501 (effective: 03-12-2012); RVF virus vaccine strain MP-12 (effective: 02-07-2003)

•SARS coronavirus, NATtrol-treated SARS-CoV molecular controls (effective: 02-08-2013)

•Venezuelan equine encephalitis (VEE) subtypes ID and IE (effective: 12-04-2012); VEE virus/Sindbis chimeric construction that includes the structural gene (only) of VEE virus (effective 05-29-2007); VRPs constructed using the V3014 derived helper of VEE virus (effective: 12-29-2004); VEE virus vaccine candidate strain V3526 (effective: 05-05-2003); VEE virus vaccine strain TC-83 (effective 02-07-2003)

• Yersinia pestis CO92 triple mutant Δlpp Δmsb Δail (effective: 05-19-2016); Y. pestis strains which are Pgm− due to the deletion of a 102-kb region of the chromosome termed the pgm locus (i.e., Δpgm). Examples are Y. pestis strain EV or various substrains such as EV76 (effective: 03-14-2003); Y. pestis strains (e.g., Tjiwidej S and CDC A1122) devoid of the 75 kb low-calcium response (Lcr) virulence plasmid (effective: 02-27-2003)

Table 28.6.

Select toxin exclusions^a,b.

•Catalytically inactive Botulinum neurotoxin (ciBoNT) B, C, E, F (effective: March 23, 2016)

•Botulinum neurotoxin type C atoxic derivative (BoNT/C ad) (effective: December 23, 2014)

•Fusion proteins of the heavy-chain domain of BoNT/translocation domain of diphtheria toxin (effective: August 28, 2011)

•Recombinant catalytically inactive botulinum A1 holoprotein (ciBoNT/A1 HP) (effective: May 7, 2010)

•Recombinant botulinum neurotoxin purified protein A1 atoxic derivative (BoNT A1 ad) E224A/Y366A (effective: July 22, 2009)

•Recombinant botulinum neurotoxin serotype A (R362A/Y365F) (effective: March 28, 2006)

•Conotoxins (nonshort, paralytic alpha conotoxins (effective: December 4, 2010)

•Staphylococcal enterotoxin B (SEB) single-site mutant Q210A (effective: June 1, 2016)

•Staphylococcal enterotoxin C (SEC) single site mutant N23A (effective: June 1, 2016)

•Staphylococcal enterotoxin A (SEA) triple mutant L48R/D70R/Y92A (effective: January 16, 2014)

•SEB triple mutant L45R/Y89A/Y94A (effective: January 16, 2014)

•SEC double mutant N23A/Y94A (effective: January 16, 2014)

•Anhydrotetrodotoxin, a derivative of wild-type tetrodotoxin (effective: May 15, 2015)

^aNontoxic HHS toxins (section 73.3 (d)) and excluded toxins modified to be less potent or toxic (section 73.3 (e)).

^bAdditional information is available at https://www.selectagents.gov/SelectAgentsandToxinsExclusions.html.

The current Select Agent Regulations address concerns raised by law enforcement agencies related to seizures (i.e., possession) of known select agents or toxins. While the revisions do not authorize the seizure of a select agent or toxin by a federal law enforcement agency, they establish the conditions under which a federal law enforcement agency can conduct certain law enforcement activities (e.g., collecting evidence from a laboratory crime scene) without being in violation of the regulations. Sections 73.3(f) and 73.4(f) of the HHS regulation provide that any known select agent or toxin seized by a federal law enforcement agency will be excluded from the requirements of the regulation during the period between seizure of the agent or toxin and the transfer or destruction of such agent or toxin provided that (i) as soon as practicable, the federal law enforcement agency transfers the seized agent or toxin to an entity eligible to receive such agent or toxin or destroys the agent or toxin by a recognized sterilization or inactivation process; (ii) the federal law enforcement agency safeguards and secures the seized agent or toxin against theft, loss, or release and reports any theft, loss, or release of such agent or toxin; and (iii) the federal law enforcement agency reports the seizure of the select agent or toxin by submitting APHIS/CDC Form 4. If a federal law enforcement agency seizes a suspected select agent or toxin or unknown material, it will be regarded as a specimen presented for diagnosis or verification and, therefore, will not be subject to the regulations until it has been identified as a select agent or toxin.

Transfers

With two exceptions (see later), the transfer of a select agent or toxin must be authorized by the CDC or APHIS before transfer occurs. A transfer may be authorized if the sender (i) has an active and approved certificate of registration at the time of transfer that covers the particular select agent or toxin; (ii) meets the exemption requirements for the particular select agent or toxin (see earlier discussion); or (iii) is transferring the select agent or toxin from outside the US and meets all import requirements. Regulations governing importation of etiologic agents of diseases of humans (42 C.F.R. § 71 Foreign Quarantine. Section 71.54); diseases of livestock, poultry, and other animals (9 C.F.R. §§ 92, 94-96, 122, and 130); and plant pests (7 C.F.R. § 330) have been previously described (Ezzell, 2005). The recipient must also have a certificate of registration that includes the particular select agent or toxin at the time of transfer. To obtain an authorization for transfer, APHIS/CDC Form 2 must be submitted to either the CDC or APHIS. This authorization for transfer is only valid for 30 calendar days after issuance. After an authorized transfer, the recipient must submit a completed APHIS/CDC Form 2 within 2 business days after the select agent or toxin is received. In the event that the select agent or toxin has not been received within 48 h after the expected delivery time or if the package has been damaged to the extent that a release of the select agent or toxin may have occurred, the recipient must notify the CDC or APHIS immediately.

It is also important to note that the sender must comply with all applicable laws concerning the packaging and shipping of hazardous materials. Select agents are not permitted in the US postal system. Select agents are considered hazardous materials and fall under the Hazardous Materials Regulations (HMR) of the US Department of Transportation (DOT). HMR are issued by the Pipeline and Hazardous Materials Safety Administration and govern the interstate transportation of materials by highway, rail, vessel, and air. HMR are the legally enforceable shipping regulations for hazardous materials in the US and are enforced by the Federal Aviation Administration for air transport. For complete information, see 49 C.F.R. §§171-180 at http://www.phmsa.dot.gov/hazmat/regs. The DOT HMR applies to the offering, acceptance, and transportation of hazardous materials to, from, or within the US and to any aircraft of US registry anywhere in air commerce. Select agents, infectious substances, toxins, and other dangerous goods are not allowed in the passenger compartment of aircraft. Transportation of an infectious substance by a federal, state, or local government agency or the military in a vehicle (e.g., automobile, truck, airplane) operated by a government or military employee is not subject to the HMR, which may be important, in certain circumstances, for transporting forensic evidence containing an infectious substance or toxin by law enforcement or military personnel.

The International Civil Aviation Organization (ICAO) Technical Instructions (TI) on the Safe Transport of Dangerous Goods by Air augment the broad principles governing the international transport of hazardous materials by air contained in Annex 18 to the Convention on International Civil Aviation. The DOT HMR authorizes transport in accordance with the ICAO TI as a means of compliance with the HMR. Because both the DOT HMR and the ICAO TI are based on United Nations (UN) recommendations, the DOT allows shippers to follow the ICAO TI for domestic air transport, but additional requirements may exist (see 49 C.F.R. § 171.23, Requirements for specific materials and packages transported under the ICAO TIs, IMDG Code, Transport Canada TDG Regulations, or the IAEA Regulations).

The International Air Transport Association (IATA) was formed to simplify the extensive and complicated requirements for transport by aircraft. IATA is an international trade organization that represents approximately 230 airlines comprising 93% of scheduled international air traffic. The Dangerous Goods Regulations, which IATA publishes every year, is technically not a regulation per se, but a user-friendly guide for the regulatory ICAO TI. Working closely with the UN Committee of Experts and other national authorities, IATA ensures that the rules and regulations governing the transportation of dangerous goods are effective, efficient, and in complete compliance with ICAO and the UN Model Regulations. For more information, see https://www.icao.int/ and http://www.iata.org/index.htm. It is important to remember that under the DRG, carriers can refuse improperly packed packages and international shipments.

The two exceptions concerning the transfer of select agents and toxins mentioned previously are that (i) a select agent or toxin that is in a specimen for proficiency testing may be transferred without prior authorization from the CDC or APHIS provided that the sender reports to the CDC or APHIS the select agent or toxin to be transferred and the name and address of the recipient(s) at least 7 calendar days prior to the transfer and (ii) on a case-by-case basis, the Secretary of HHS or USDA can authorize a transfer of a select agent or toxin, not otherwise eligible for transfer, under conditions prescribed by the secretary.

Specific guidance on the transfer, importation, and shipment of select agents and toxins has been developed and can be accessed at https://www.selectagents.gov/compliance.html and is updated as needed (Table 28.4).

Records

Proper record keeping is important for the forensic analysis of microbial evidence. It is also a requirement under sections 73.17, 121.17, and 331.17 of the Select Agent Regulations. The select agent regulations require an accurate and current inventory for (i) each select agent (including viral genetic elements, recombinant and/or synthetic nucleic acids, and recombinant and/or synthetic organisms) held in long-term storage (placement in a system designed to ensure viability for future use, such as a freezer or other storage container or lyophilized materials); (ii) any animals or plants intentionally or accidently exposed to or infected with a select agent (including number and species, location, and appropriate disposition); and (iii) each toxin held. Specific guidance on the inventory of select agents and toxins is available at https://www.selectagents.gov/compliance.html and updated periodically (Table 28.4). The information in the guidance document is meant to provide additional information to regulated entities in meeting the requirements of the select agent regulations.

Security

The select agent registration process requires each laboratory to develop a written security plan that is based on a site-specific risk assessment. Regulations governing the assessment do not specify who must perform it, meaning that it can be performed by officials for the laboratory itself. Each laboratory must implement a security plan that is sufficient to safeguard select agents against unauthorized access, theft, loss, or release. The assessment must provide protection based on the risk and intended use of the select agent or toxin. It includes four assessments: an agent-specific risk assessment, threat assessment, vulnerability assessment, and graded protection determination. Before being issued a certificate of registration, an entity must comply with all security requirements (and all other provisions of the Select Agent Regulations). To assist entities in developing and implementing the required written security plan, the CDC and APHIS developed a Security Risk Assessment Tool to assist in creating the entity's site-specific risk assessment, which is the core of a robust security plan. The Security Risk Assessment Tool and specific guidance for developing and implementing a security plan in compliance with the select agent regulations are available at https://www.selectagents.gov/compliance.html. The guidance document includes information regarding site-specific risk assessments, planning requirements, access, inventory and audits, and barriers and is updated periodically as needed (Table 28.4).

Biosafety/biocontainment

The Select Agent registration process requires each entity to develop and implement a biosafety/biocontainment plan to ensure biological containment and safe handling of select agents and toxins. The biosafety/biocontainment plan must be based on a site-specific assessment, which provides protection commensurate with the risk of the agent and its intended use. Biosafety procedures (e.g., operational work practices and personal protective equipment) and physical containment features (e.g., facility design and engineering controls) sufficient to contain the agent must be included. The plan must be reviewed, and drills must be conducted at least annually. A guidance document (Table 28.4) is available to assist entities in developing this written plan and can be accessed at https://www.selectagents.gov/compliance.html. The guidance document has links to several valuable resources including the guide Biosafety in Microbiological and Biomedical Laboratories (Chosewood and Wilson, 2007). Entities registered for Tier 1 select agents and toxins also require an occupational health program. Information regarding the occupational health program may be incorporated into the biosafety/biocontainment plan.

Incident response

A written incident response plan must also be developed as part of the registration packet. This plan should be coordinated with all entity-wide plans and must include response procedures for biological containment and security breaches, natural disasters, and other emergencies. Information must include emergency contact information for responsible persons; roles and lines of authority; and emergency evacuation, medical treatment, first aid, and decontamination procedures. The plan must be reviewed, and drills must be conducted at least annually. A guidance document for developing and implementing an incident response plan has been established (Table 28.4) and is available at https://www.selectagents.gov/compliance.html. It contains information regarding requirements, natural disasters, and goals of incident response planning.

Training

The Select Agent Regulations require that each person with approved access to select agents and toxins, or any person who works in or visits areas where select agents and toxins are handled or stored, must be trained in biosafety and security principles and practices before having access. A guidance document (Table 28.4) that includes what types of training are required, training programs, frequency requirements, and maintenance of training records is available at https://www.selectagents.gov/compliance.html.

Summary

The goals of the Select Agent Programs are to regulate the possession, use, and transfer of select agents and toxins that have the potential to pose a severe threat to public health and safety, to animal health or animal products, and to plant health and plant products. Because of the threat these agents pose, they are likely to be used by bioterrorists and other criminals and, therefore, may become the subject of forensic analyses. Performing research of select agents and toxins is also critical for the development of effective medical countermeasures and, ultimately, the development of effective vaccines. The Select Agent Regulation has been in effect for more than 20 years. During this time, hundreds of entities have registered with the CDC or APHIS to possess, use, or transfer select agents and toxins, and thousands of individuals have been approved to work with these agents at registered facilities. The CDC and APHIS have developed a highly integrated regulatory oversight program, characterized by uniform regulations, a common database (the National Select Agent Registry), and a shared common website (https://selectagents.gov).