Environmental Protection Agency § 725.421

exemption may submit a Tier II ex- (3) The presence or absence of associ- emption request. The limited reporting ated nucleotide sequences and their as- requirements for the Tier II exemption, sociated functions, where associated including data requirements, are de- nucleotide sequences are those se- scribed in §§ 725.450 and 725.455. quences needed to move genetic mate- (g) EPA review procedures for the rial including linkers, homopolymers, Tier II exemption are set forth in adaptors, transposons, insertion se- § 725.470. quences, and restriction enzyme sites. (h) Subparts A through C of this part (c) Poorly mobilizable. The ability of apply to any submission under this the introduced genetic material to be subpart. transferred and mobilized is inac- tivated, with a resulting frequency of § 725.420 Recipient microorganisms. transfer of less than 10¥8 transfer The following recipient microorga- events per recipient. nisms are eligible for either exemption (d) Free of certain sequences. (1) The under this subpart: introduced genetic material must not (a) Acetobacter aceti. contain a functional portion of any of (b) Aspergillus niger. the -encoding sequences described (c) Aspergillus oryzae. in this paragraph (d). (d) Bacillus licheniformis. (i) For the purposes of this section, a (e) Bacillus subtilis. functional portion of a toxin-encoding (f) acetobutylicum. sequence means any sequence which (g) Escherichia coli K-12. codes for a polypeptide that has one of (h) Penicillium roqueforti. the following effects: (i) Saccharomyces cerevisiae. (A) It directly or indirectly contrib- (j) Saccharomyces uvarum. utes to toxic effects in humans. Di- rectly contributes to toxic effects in § 725.421 Introduced genetic material. humans means those sequences encod- For a new microorganism to qualify ing polypeptides that have direct tox- for either exemption under this sub- icity to target cells. An example of a part, introduced genetic material must sequence which directly contributes to meet all of the criteria listed in this toxic effects in humans is one which section. encodes the portion of diphtheria (a) Limited in size. The introduced ge- toxin, listed in paragraph (d)(2) of this netic material must consist only of the section, capable of interacting with following: elongation factor 2, leading to inhibi- (1) The structural gene(s) of interest. tion of synthesis in target res- (2) The regulatory sequences permit- piratory, heart, kidney, and nerve tis- ting the expression of solely the gene(s) sues. Indirectly contributes to toxic ef- of interest. fects in humans means a sequence (3) Associated nucleotide sequences whose encoded polypeptide is not di- needed to move genetic material, in- rectly toxic to target cells, yet still ad- cluding linkers, homopolymers, adap- versely affects humans. An example of tors, transposons, insertion sequences, a sequence which indirectly contrib- and restriction enzyme sites. utes to toxic effects is the sequence (4) The nucleotide sequences needed which encodes the portion of the botu- for vector transfer. linum toxin, listed in paragraph (d)(3) (5) The nucleotide sequences needed of this section, capable of blocking the for vector maintenance. release of acetylcholine from (b) Well-characterized. For introduced gangliosides. affects genetic material, well-characterized neuromuscular junctions by its block- means that the following have been de- age of acetylcholine release, leading to termined: irreversible relaxation of muscles and (1) The function of all of the products respiratory arrest. expressed from the structural gene(s). (B) It binds a toxin or toxin precursor (2) The function of sequences that to target human cells. participate in the regulation of expres- (C) It facilitates intracellular trans- sion of the structural gene(s). port of a toxin in target human cells.

673

VerDate Mar<15>2010 20:52 Aug 23, 2012 Jkt 226177 PO 00000 Frm 00683 Fmt 8010 Sfmt 8010 Q:\40\40V32.TXT ofr150 PsN: PC150 § 725.421 40 CFR Ch. I (7–1–12 Edition)

(ii) While these are listed Sequence Source Toxin Name (with synonyms in parentheses) in Clostridium caproicum Lysin paragraphs (d)(2) through (d)(7) of this Clostridium chauvoei Delta-toxin section according to the source orga- Clostridium histolyticum Epsilon-toxin nism, it is use of the nucleotide se- Clostridium novyi Gamma-toxin Clostridium oedematiens Delta-toxin quences that encode the toxins that is Theta-toxin (Perfringolysin) being restricted and not the use of the Clostridium septicum Delta-toxin source organisms. The source orga- Clostridium sordellii Lysin nisms are listed to provide specificity Listeria monocytogenes Listeriolysin (A B) in identification of sequences whose Streptococcus pneumoniae Pneumolysin use is restricted. Although similar or Streptococcus pyogene O (SLO) identical sequences may be isolated (5) Sequences for toxins affecting mem- from organisms other than those listed brane function. below in paragraphs (d)(2) through (d)(7) of this section, these comparable Sequence Source Toxin Name toxin sequences, regardless of the orga- Bacillus anthracis Edema factor (Factors I II); nism from which they are derived, Lethal factor (Factors II III) must not be included in the introduced Bacillus cereus (diarrheagenic genetic material. toxin, mouse lethal factor) Bordetella pertussis Adenylate cyclase (Heat-la- (2) Sequences for protein synthesis in- bile factor); Pertussigen hibitor. (, islet acti- vating factor, histamine Sequence Source Toxin Name sensitizing factor, lymphocytosis promoting Corynebacterium diphtheriae factor) & C. ulcerans C2 toxin Pseudomonas aeruginosa A Clostridium difficile Enterotoxin (toxin A) Shigella dysenteriae Shigella toxin (, Clostridium perfringens Beta-toxin; Delta-toxin Shigella dysenteriae type I Escherichia coli & other Heat-labile (LT); toxin, Vero cell toxin) Enterobacteriaceae spp. Heat-stable enterotoxins Abrus precatorius, seeds (STa, ST1 subtypes ST1a Ricinus communis, seeds ST1b; also STb, STII) Legionella pneumophila Cytolysin (3) Sequences for . Vibrio cholerae & Vibrio (choleragen) mimicus Sequence Source Toxin Name (6) Sequences that affect membrane Clostridium botulinum Neurotoxins A, B, C1, D, E, F, G (Botulinum toxins, integrity. botulinal toxins) Clostridium tetani toxin Sequence Source Toxin Name (tetanospasmin) Clostridium bifermentans & Lecithinase Proteus mirabilis other Clostridium spp Staphylococcus aureus Alpha toxin (alpha lysin) Clostridium perfringens Alpha-toxin (phospholipase Yersinia pestis Murine toxin C, lecithinase); Enterotoxin Snake toxins Corynebacterium pyogenes & Cytolysin (phospholipase C), Bungarus caeruleus Caeruleotoxin other Corynebacterium spp. Ovis toxin Bungarus multicinctus Beta- (sphingomyelinase D) (phospholipase) Staphylococcus aureus Beta-lysin (beta toxin) Crotalus spp. Crotoxin (phospholipase) Dendroaspis viridis Neurotoxin (7) Sequences that are general Naja naja varieties Neurotoxin cytotoxins. Notechia scutatus Notexin (phospholipase) Oxyuranus scutellatus Sequence Source Toxin Name toxins Chironex fleckeri Neurotoxin Adenia digitata Modeccin Androctnus australis Neurotoxin Aeromonas hydrophila Aerolysin (beta-lysin, Centruroides sculpturatus Neurotoxin cytotoxic lysin) Clostridium difficile Cytotoxin (toxin B) (4) Sequences for oxygen labile Clostridium perfringens Beta-toxin; Epsilon-toxin; cytolysins. Kappa-toxin Escherichia coli & other Cytotoxin (Shiga-like toxin, Sequence Source Toxin Name Enterobacteriaceae spp. Vero cell toxin) Pseudomonas aeruginosa Proteases Bacillus alve Alveolysin Staphylococcus aureus Gamma lysin (Gamma toxin); Bacillus cereus Cereolysin Enterotoxins (SEA, SEB, Bacillus laterosporus Laterosporolysin SEC, SED SEE); Bacillus thuringiensis Thuringiolysin Pyrogenic A B; Clostridium bifermentans Lysin Toxic shock syndrome tox- Clostridium botulinum Lysin ins (TSST-1)

674

VerDate Mar<15>2010 20:52 Aug 23, 2012 Jkt 226177 PO 00000 Frm 00684 Fmt 8010 Sfmt 8010 Q:\40\40V32.TXT ofr150 PsN: PC150 Environmental Protection Agency § 725.424

Sequence Source Toxin Name (3) The physical containment and control technologies of any facility in Staphylococcus aureus & Leucocidin (, Pseudomonas aeruginosa cytotoxin) which the microorganism will be man- Streptococcus pyogenes Streptolysin S (SLS); ufactured, processed, or used meet the Erythrogenic toxins (scarlet criteria under § 725.422. fever toxins, pyrogenic (4) The manufacturer or importer exotoxins) Yersinia enterocolitica Heat-stable enterotoxins (ST) submits a certification described in paragraph (b) of this section to EPA at § 725.422 Physical containment and least 10 days before commencing initial control technologies. manufacture or import of a new micro- The manufacturer must meet all of organism derived from a recipient the following criteria for physical con- microorganism listed in § 725.420. tainment and control technologies for (5) The manufacturer or importer any facility in which the new micro- complies with the recordkeeping re- organism will be used for a Tier I ex- quirements of § 725.65 and maintains emption; these criteria also serve as records for the initial and subsequent guidance for a Tier II exemption. uses of the new microorganism that (a) Use a structure that is designed verify compliance with the following: and operated to contain the new micro- (i) The certifications made in para- organism. graph (b) of this section. (ii) All the eligibility criteria for the (b) Control access to the structure. Tier I exemption including the criteria (c) Provide written, published, and for the recipient microorganism, the implemented procedures for the safety introduced genetic material, the phys- of personnel and control of hygiene. ical containment and control tech- (d) Use inactivation procedures dem- nologies. onstrated and documented to be effec- (b) Certification. To be eligible for the tive against the new microorganism Tier I exemption under this subpart, contained in liquid and solid wastes the manufacturer or importer must prior to disposal of the wastes. The in- submit to EPA a document signed by a activation procedures must reduce via- responsible company official con- ble microbial populations by at least 6 taining the information listed in this logs in liquid and solid wastes. paragraph. (e) Use features known to be effective (1) Name and address of manufac- in minimizing viable microbial popu- turer or importer. lations in aerosols and exhaust gases (2) Date when manufacture or import released from the structure, and docu- is expected to begin. ment use of such features. (3) The identification (genus, species) (f) Use systems for controlling dis- of the recipient microorganism listed semination of the new microorganism in § 725.420 which is being used to create through other routes, and document the new microorganism which will be use of such features. used under the conditions of the Tier I (g) Have in place emergency clean-up exemption. procedures. (4) Certification of the following: (i) Compliance with the introduced § 725.424 Requirements for the Tier I genetic material criteria described in exemption. § 725.421. (a) Conditions of exemption. The manu- (ii) Compliance with the containment facture or import of a new microorga- requirements described in § 725.422, in- nism for commercial purposes is not cluding the provision in paragraph subject to review under this part if all (a)(3) of this section. of the following conditions are met for (5) The site of waste disposal and the all activities involving the new micro- type of permits for disposal, the permit organism: numbers and the institutions issuing (1) The recipient microorganism is the permits. listed in and meets any requirements (6) The certification statement re- specified in § 725.420. quired in § 725.25(b). Certification of (2) The introduced genetic material submission of test data is not required meets the criteria under § 725.421. for the Tier I exemption.

675

VerDate Mar<15>2010 20:52 Aug 23, 2012 Jkt 226177 PO 00000 Frm 00685 Fmt 8010 Sfmt 8010 Q:\40\40V32.TXT ofr150 PsN: PC150