Several previous entries in this blog from 2010 (beginning here), described U.S. Environmental Protection Agency (EPA) regulations under the Toxic Substances Control Act (TSCA) that may cover the use of certain genetically modified microorganisms in biofuel or bio-based chemical production. These regulations specify that some genetically engineered microorganisms intended for use in biofuel production (e.g., as production organisms for biofuel-processing enzymes, or as organisms directly used in the fermentation of biomass to fuel) require notification to EPA prior to commercial use, through the filing of Microbial Commercial Activity Notices (MCANs). In this blog entry, I’ll describe two significant developments under the EPA TSCA biotechnology regulations, both of which have implications for the biofuels industry – one is a proposed expansion of the list of “recipient microorganisms” that are potentially eligible for the tiered exemptions under the rules; and the other is the first use of EPA’s “significant new use” regulations to limit approved uses of a new microorganism covered by an MCAN.
Proposed Revision to List of Exempt Recipient Microorganisms
As described in my earlier blog entries, there are several exemptions from MCAN reporting that are available under the regulations. One category of exemptions are the tiered exemptions that are potentially available for intergeneric microorganisms that might otherwise require MCAN reporting, where well-characterized genetic changes are made to one of several starting organisms (referred to in the regulations as the “recipient organism”). These exemptions were described in detail in one of my 2010 blog posts, and the list of eligible recipient organisms was included in another 2010 post.
To briefly summarize the situation, the TSCA biotechnology rule created Tier I and Tier II exemptions for specific intergeneric microorganisms meeting certain criteria. First, the host (“recipient”) organism must be one that is included on the list found in Section 725.420 of the regulations. This list includes many well-studied species including E. coli K12, Saccharomyces cerevisiae, Bacillus subtilis, and others. Second, the introduced genetic material in the microorganism must be well characterized; must be limited in size to the minimal genetic information needed; must be poorly mobilizable; and must be known to be free of harmful sequences. Third, the regulations specify specific containment and control procedures to minimize the possibility that the engineered microorganism might inadvertently be released. If the applicant can use an intergeneric strain qualifying for the exemption in strict compliance with these procedures, the process is eligible for a “Tier I” exemption and can be used commercially with no prior EPA review or approval, and merely upon 10 days advance notice to EPA. If the applicant intends to use such a microorganism under conditions less strict than these procedures, the applicant can submit a petition for a “Tier II” exemption 45 days before intended manufacture. EPA would approve the Tier II request if it felt that the proposed containment and control procedures, although not identical to the specified procedures, were sufficient for the organism in question.
On September 5, 2012, EPA published a notice in the Federal Register announcing the promulgation of a Proposed Rule that would add two microbial species to the list of recipient organisms potentially eligible for the tiered exemptions. These are Trichoderma reesei and Bacillus amyloliquefaciens. These are both well-studied species that have historically been used quite frequently in industrial fermentations – many previously-filed MCANs covering microorganisms used to produce biofuel-processing enzymes have involved modified strains of T. reesei, while B. amyloliquefaciens has been frequently used as the host organism for production of industrial enzymes in foods, brewing, and other industries. The original biotechnology rule provided a process by which interested parties could file petitions proposing that additional microorganisms be added to the list of species eligible for the tiered exemptions. Using this procedure, Genencor International, Inc. (subsequently supported by the Enzyme Technical Association) and Novozymes North America, Inc., submitted letters of application to EPA requesting that Trichoderma reesei and Bacillus amyloliquefaciens be added as candidate recipient microorganisms for the tiered exemptions. Novozymes’ letter was submitted in 1997 supporting the inclusion of B. amyloliquefaciens, and Genencor’s letter was submitted in 2005 in support of T. reesei.
Upon EPA’s review of these petitions, the agency made a preliminary determination that the information presented was sufficient to support a finding that the requested actions would not present an unreasonable risk of injury to health or the environment. However, in developing its Proposed Rule, EPA decided to somewhat narrow the scope of the exemption. For B. amyloliquefaciens, the exemption would only cover Bacillus amyloliquefaciens subsp. amyloliquefaciens, a subspecies said to encompass essentially all strains that are commonly used industrially. For T. reesei, the exemption would only cover Trichoderma reesei strain QM6a and moreover would apply only if the organism were “used only in submerged standard industrial fermentation operations in which no solid plant material or insoluble substrate is present in the fermentation broth,” and where the fermentation is initiated after specified inactivation procedures have taken place. This usage limitation for T. reesei arises from a concern over a potentially toxic secondary metabolite that has been found to arise when this organism is grown in certain non-standard conditions – this will be described in more detail below.
The September 5 Federal Register notice set a 60-day comment period which expired on November 5, 2012. Under federal rulemaking procedures, EPA must consider any public comments it received during that period and take these into account when publishing a Final Rule. It is not known when EPA might propose a Final Rule, but generally speaking, any final rule would probably take effect 30 or 60 days after its publication in the Federal Register.
Without taking any position on the scientific merits of the proposed revision to the rule, I would simply observe that these are indeed well-characterized microbial species that have been extensively and commonly used in industry for many years. In fact, the idea that these species should be added to the exemption list has been kicking around for some time, as can be seen by the early dates of the Genencor and Novozymes petitions. This seems to me to be a sensible revision to the rule, which would allow the manufacturers of industrial enzymes for many purposes, including biofuel production, to move forward to commercialize new and improved production strains without the need for MCAN filings.
Proposed Significant New Use Rule
The issue mentioned above regarding the potentially toxic metabolite of T. reesei arose during EPA’s review of MCAN J10-0002, submitted by Danisco US, Inc. Specifically, EPA found that this metabolite had only been seen to be synthesized by T. reesei under certain, presumably-uncommon, fermentation conditions. EPA summarized this situation in the Federal Register as follows:
The only health concern associated with T. reesei QM6a is its ability to produce a secondary metabolite called paracelsin, which is a peptaibol. Peptaibols are small linear peptides of 1,000-2,000 daltons characterized by a high content of the non-proteinogenic amino acid [alpha]-amino-isobutyric acid (Aib), with an N-terminus that is typically acetylated, and a C-terminus that is linked to an amino alcohol. … Paracelsin has been shown to have toxicity toward mammalian cells such as hemolytic activity on human erythrocytes and cytotoxicity to rat adrenal medulla PC12 cells.
Paracelsin has not been detected in the use of T. reesei QM6a under submerged standard industrial fermentation operations in which no solid plant material or insoluble substrate is present in the fermentation broth; numerous toxicity studies on enzyme products of T. reesei QM6a have demonstrated a lack of toxicity to laboratory animals. EPA therefore generally expects that paracelsin production will be of insignificant concern with submerged standard industrial fermentation operations in which no solid plant material or insoluble substrate is present in the fermentation broth. … However, under non-standard conditions of fermentation, such as with extended duration of fermentation, or fermentation in the presence of insoluble carbon sources such as cellulose or in the presence of solid plant material, paracelsin may be produced.
It was this concern that led EPA to limit the uses of T. reesei eligible for the tiered exemptions to those taking place in “submerged standard industrial fermentations”. However, this concern also led EPA to take an unusual action with regard to the Danisco MCAN under which the concern was raised. Section 5(a)(2) of TSCA authorizes EPA to determine that a proposed use of a chemical substance is a “significant new use,” a determination EPA must make after considering all relevant health and safety factors, and it generally must formalize this determination through the federal rule-making process. The TSCA biotechnology regulations are based on the premise that EPA can regulate new microorganisms as “new chemicals”, and so it interprets this provision of TSCA to give it the authority to issue Significant New Use Rules (SNURs) for uses of microorganisms that it judges to pose potential risks when used in certain processes or other circumstances. Subpart L of the biotechnology rules in 40 CFR Part 725, specifically §725.980, governs how EPA may issue a SNUR for a new microorganism covered by an MCAN.
Accordingly, on June 13, 2012, EPA published a Federal Register notice proposing a SNUR for the T. reesei strain that is covered by MCAN J10-0002. This notice stated the rationale for the proposed rule as follows:
During review of the specific T. reesei, modified as described in MCAN J-10-2, EPA determined that certain fermentation conditions, other than the typical submerged standard industrial fermentation process for enzyme production described [above], could result in increased exposures thereby constituting a “significant new use.” Specifically, EPA is concerned that where growth on solid plant material or insoluble substrate occurs, T. reesei has been shown to produce a secondary metabolite known as paracelsin, which is associated with a variety of toxic effects to mammalian and bacterial cells. Use of the MCAN microorganism without the specific containment or inactivation controls listed in the MCAN, described [above], may result in adverse human health and environmental effects.
The SNUR itself takes the form of an amendment to the Part 725 rule, adding a new provision to the regulations at Subpart M. Subpart M, which currently has no content, is a placeholder for SNURs that EPA may propose from time to time, and in this case, EPA proposes creating a new §725.1077 that would read as follows:
Sec. 725.1077 Trichoderma reesei (generic).
(a) Microorganism and significant new uses subject to reporting.
(1) The genetically modified microorganism identified generically as Trichoderma reesei (MCAN J-10-2) is subject to reporting under this section for the significant new uses described in paragraph (a)(2) of this section.
(2)(i) The significant new use is any manufacturing, processing, or use of the microorganism other than in a fermentation system that meets all of the following conditions:
(A) Submerged fermentation (i.e., growth of the microorganism occurs beneath the surface of the liquid growth medium).
(B) No solid plant material or insoluble substrate is included with the microorganism for fermentation.
(C) Any fermentation of solid plant material or insoluble substrate, to which fermentation broth is added, is initiated only after the inactivation of the microorganism as delineated in 40 CFR 725.422(d).
Once this rule is finalized, it means that any entity wishing to use T. reesei under the “new use” conditions specified in the rule must first file a Significant New Use Notice (SNUN) with EPA. A SNUN is equivalent to an MCAN (or, for chemical substances, a Premanufacture Notice), and it would allow EPA to review the health and safety implications of the proposed use of the organism under those conditions. In the absence of EPA review of a SNUN, no entity would be allowed to use T. reesei under these conditions.
The Federal Register notice created a brief, one-month, public comment period that expired on July 13, 2012. The status of this proposed rule, and the timing of its possible finalization, are not known. However, this action is noteworthy in that it appears to be the first time EPA has used the “SNUR” process for a microorganism that it reviewed under an MCAN, and it establishes a precedent that EPA may use in the future should analogous situations arise.
D. Glass Associates, Inc. is a consulting company specializing in government and regulatory support for renewable fuels and industrial biotechnology. David Glass, Ph.D. is a veteran of over thirty 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. Dr. Glass also serves as director of regulatory affairs for Joule Unlimited Technologies, Inc. More information on D. Glass Associates’ regulatory affairs consulting capabilities, and copies of some of Dr. Glass’s prior presentations on biofuels and biotechnology regulation, are available at www.slideshare.net/djglass99 and at www.dglassassociates.com. The views expressed in this blog are those of Dr. Glass and D. Glass Associates and do not represent the views of Joule Unlimited Technologies, Inc. or any other organization with which Dr. Glass is affiliated. Please visit our other blog, Biofuel Policy Watch.