There is growing interest in the use of novel microorganisms created using synthetic biology or other genetic modification techniques for commercial production of chemicals, fuels, enzymes or other specialty products, or for environmental uses such as enhanced nitrogen fixation or bioremediation. As I’ve previously reported in the blog, commercial use of such organisms in the U.S. would potentially fall under the biotechnology regulations of the U.S. Environmental Protection Agency under the Toxic Substances Control Act (TSCA). Earlier entries on the blog have gone into greater detail regarding the threshold questions determining whether a microorganism would be subject to this rule, but briefly, the rule would cover commercial uses (or importation into the U.S.) of a microorganism that contains deliberate combinations of nucleic acids from more than one genus (a so-called “intergeneric organism”) that would be used for a commercial purpose that is not regulated by the Food and Drug Administration or by EPA as a pesticide.
Complying with these regulations can be relatively straightforward with proper planning. Any proposed commercial use or importation of an intergeneric microorganism for a TSCA-regulated purpose would require the submission to EPA of a Microbial Commercial Activity Notice (MCAN) at least 90 days before the proposed activity (other than uses of certain species that qualify for the so-called “tiered” exemptions under the regulations). When prepared properly to include the information that EPA staff wants to see in an MCAN, EPA’s 90-day review period goes smoothly, but if the submission is missing data or other important information, EPA’s review would likely be delayed, possibly by months. In this blog entry, I’ll provide some guidance for how best to prepare an MCAN to meet EPA’s expectations and requirements (note that portions of this guidance are also applicable to preparation of TERAs, the applications needed to seek approval for outdoor testing of modified microorganisms falling under TSCA jurisdiction).
First, it is often useful, especially for first time applicants, to arrange a presubmission consultation with EPA staff before beginning to prepare an MCAN. This can be useful to introduce the company and its commercial plans to the agency’s biotech staff, and to get an early indication of the types of data the agency will want to see and whether there are any particular issues that should be addressed. The biotech staff at EPA is always willing to hold such meetings, which can be done in person or by phone- or video-conferencing, and which can usually be set up with 2-3 weeks’ notice.
Although MCANs must be submitted using EPA’s online CDX server, unlike other EPA submissions there is not a fillable form for MCANs: instead the content of the MCAN is submitted as a narrative document (text, including figures and tables, generally as a PDF document) which may be accompanied by several attachments. It is helpful to begin using a template or format with which EPA is familiar, to help you assemble the needed information and present it in the optimum way for EPA review. There are several different templates that companies have used over the years, but I prefer a template that is based on the outline of information as presented in EPA’s guidance document “Points to Consider in the Preparation of Biotechnology Submissions for Microorganisms“. Among other reasons, I like this template because it has a logical organization of the needed information into three broad topics: biological information about the microorganism that is the subject of the MCAN (corresponding to Sections A and B of the guidance document); health and environmental safety information (Sections C and D); and information about the facility, the fermentation process, and the equipment, facilities and procedures in place to minimize release of the organism to the environment and to minimize worker exposure to the organism (Sections E and F). Here are some tips for assembling the data and information needed for each category.
Biological Information
Taxonomy of the subject microorganism. EPA requires applicants to submit evidence confirming the taxonomy of the starting (“recipient”) species. This can be done by appropriate analysis to compare genetic sequences (e.g. 16S rRNA sequences, whole genome sequencing, etc.) to the reference genotype for the species, but it can also be done by providing documentation from the repository or reagent company from which the starting strain was obtained.
Detailed description of the construction of the microorganism. EPA requires that the MCAN include a complete, detailed description of how the organism was constructed, with details on all intermediate strains and methodologies used, and submission of nucleotide and amino acid sequence information for the modified traits. The level of detail is comparable to what is typical for a scientific publication or a patent application, and EPA usually likes to see construction flowcharts and plasmid maps showing intermediate strains and constructs. The methods used in the construction should be described at some level of detail, although if published methods are used, it would be sufficient to cite the appropriate publication. It is also very important to include sequence information for all introduced genes and their protein products, and the nucleotide sequences should also show the regulatory elements and the flanking sequences at all sites of chromosomal integration. In recent years, EPA has also asked for a chart that summarizes, for each introduced gene, the identity of the gene product, the site(s) of integration, and the identity of all regulatory sequences controlling expression. This can be provided as an attachment to the MCAN, or as a Table within the body of the MCAN.
Biological characterization of the microorganism. EPA’s guidance document asks applicants to provide some data showing the phenotype and biological characteristics of the modified microorganism. In my experience, it is not always necessary for an MCAN to include a great deal of this sort of data, but among useful data would be comparisons of the growth rate of the modified strain to a parental nonmodified strain, and data demonstrating that the strain can fulfil the purpose for which it was engineered (e.g. data showing levels of production of the desired product). Other information, such as resistance to antibiotics, should be provided if known, and in recent years EPA has been asking for information about whether the morphology of the modified strain differs from wild type in any way.
Health and Safety Information
Information on potential health or environmental risks of the proposed use. Unlike in some jurisdictions where submitters are required to perform some form of risk assessment of the microorganism (e.g. as may be needed under the Cartagena Protocol), this is not required under EPA rules. However, the guidance document does call for the applicant to provide whatever information is known about the potential risks of the microorganism to human or animal health, or to the environment. In almost all cases, this requirement can be satisfied by a literature review to determine what is known about the potential pathogenicity, infectivity or environmental effects of the recipient species, or the potential for toxicity or allergenicity of any protein products of the introduced genes in the subject microorganism. In fact, for the familiar, well-studied species that are the most commonly used in industrial biotechnology (e.g. Saccharomyces cerevisiae, E. coli, Trichoderma reesei and others), it is possible to simply cite publicly-available prior MCANs or EPA’s own risk assessments, without the need for any extensive new literature review. It is worth noting that under TSCA, applicants are not required to carry out any specific toxicology or similar testing, as would be common for pharmaceuticals, pesticides or similar products, but if such testing has been done, it would be required to provide it to EPA with the MCAN.
Process Information
Detailed description of the fermentation process. The MCAN must provide a detailed description of how the organism will be scaled up (e.g. from seed cultures) and how fermentations will be carried out, including media composition and growth conditions (e.g. pH, temperature, etc.). It is important to describe and validate the methods by which the microorganism will be inactivated at the end of the process, with EPA usually looking to see proof that the chosen method can reduce microbial numbers by at least 6 logs. Bench-scale experiments are usually sufficient for this purpose. Some information may be required on downstream processing, particularly any stages of the process where live organisms may still be present, but those steps in the process that take place after the organism has been inactivated would generally be outside the purview of EPA’s review.
Detailed description of the production facility, including containment controls, worker protection, relevant SOPs. MCANs will usually include a description of the facility where the commercial use of the organism will take place, including its address, location and some information about the surrounding area (e.g. residential or industrial?). However, an MCAN can be filed before any specific production facility has been chosen, in which case the MCAN can include a description of a generic production process. Importantly, the procedures and controls for containment of the organism within the production facility must be described. These would include procedures for biological containment, physical features of the process such as filters or scrubbers on fermenter vents, provisions for worker protection (such as protective clothing), and standard operating procedures for operations associated with the fermentation or for emergency spill clean-up. EPA’s guidance document includes sample tables which applicants are recommended to use to provide estimates of the potential for worker exposure to the organism and the potential for environmental release of the organism at different stages of the process where such exposures might occur (potential “release points”). In most cases, applicants have a fair amount of leeway in adopting appropriate containment procedures, provided that the process will be carried out in accordance with standard industrial fermentation practice.
Other Features
The MCAN should include ample literature citations, with references included either on a section-by-section basis or in a single bibliography at the end (Section G). As mentioned earlier, it is common for MCANs to include various attachments, which could include PDF copies of important references, lists or content of Standard Operating Procedures, or other supporting materials. Finally, EPA regulations allow submitters to include confidential information in the MCAN, but all such information must be identified in the document, usually with brackets or boxes in the margins surrounding the confidential text, figure or table. The submitter must then submit a duplicate copy of the MCAN with all the confidential information redacted – this “sanitized” copy must retain the formatting and pagination of the confidential version, but with the confidential text either blacked out or whited out. If the MCAN includes confidential information, it is necessary to submit information to validate (or “substantiate”) the reasons why confidentiality is claimed; and here too there are some available templates for how to provide the needed information.
I would be happy to answer any questions you may have on how to prepare and submit an MCAN, as well as to assist your company in the planning and preparation of MCAN submissions.
D. Glass Associates, Inc. is a consulting company specializing in government and regulatory affairs support for renewable fuels and industrial biotechnology. David Glass, Ph.D. is a veteran of over thirty-five years in the biotechnology industry, with expertise in industrial biotechnology regulatory affairs, U.S. and international renewable fuels regulation, patents, technology licensing, and market and technology assessments. In addition to his work as a consultant assisting industrial biotechnology companies prepare for and comply with government regulations, he has served as Director of Regulatory Affairs for Joule Unlimited Technologies and Vice President of Government and Regulatory Affairs for BioTechnica International. Dr. Glass has extensive experience with the biotechnology regulations of the U.S. EPA and other agencies, and has coordinated or assisted in the preparation and submission of 18 successful Microbial Commercial Activity Notices and several other biotechnology submissions in the U.S. and other countries. Dr. Glass holds a B.S. in Biological Sciences from Cornell University and a Ph.D. in Biochemical Sciences from Princeton University.
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