EPA's Safer Choice Checklist for Formulations Containing Microorganisms
(This document is principally derived from "Points to Consider in the Preparation of
TSCA Biotechnology Submissions for Microorganisms," US EPA/OPPT, 6/2/97,
available on the web at http://www.epa.gov/biotech rule/pubs/pdf/ptcbio.pdf. Also
includes points from Environment Canada's "Guidelines for the Notification and Testing
of New Substances," Sec. 4: Technical Information Requirements.)
The following information will help Safer Choice assess the human health and
environmental profile of your product. For certain microorganisms and uses, i.e., those
that have been well characterized and present minimal potential for exposure, Safer
Choice may only need information from Parts I, II, and IV.
I. Manufacture and Use
	Company Status
•	Are you a manufacturer or blender (or both) of microbiological products?
•	If you are a blender, who is your microorganism supplier?
As with chemical toxicological assessments, potential for harm from microorganisms is a
function of both inherent characteristics and dose (i.e., number of cells or colony forming
units (cfus)) to which another living thing is exposed. Information in this section helps
define potential exposure pathways and the magnitude of exposure.
	Uses
•	Describe the intended use(s) of the microorganism (e.g., drain maintenance; fats,
oils, grease (FOG) degradation; hydrocarbon remediation; etc.) or products the
microorganism is intended to produce (e.g., enzymes for detergents). Provide
commercial product name for each use, if available. Also, list past and potential
future uses for the microorganism or microbial product.
•	Provide a general description of the locations of the application, e.g., hazardous
waste sites, grease traps, industrial wastewaters, etc.
•	Describe the method of application or use and quantity, frequency, and duration
of application (e.g., product label instructions); potential for human contact or
unintended release (include, if possible, the number of persons that may be
exposed and the degree of exposure).
•	Describe the mechanisms of dispersal of the microorganism and modes of
interaction with any dispersal agents.
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• Describe the known mode of action in relation to the intended use(s).
•	Describe the enzymes produced by the microorganism(s) for the intended uses,
and whether the enzymes are produced intracellular^ or extracellular by the
microorganism(s)
II. Microorganism Identity
All formulators using microbiological ingredients should know with as much certainty as
possible the identity of the microorganism(s) in their products.
Step one in assessing the potential hazard of a microorganism is to verify its identity.
Since there are no universally applicable methods for identifying microorganisms, expert
judgment and experience must inform this process and guide the pairing of
microorganism with test method, as necessary, to increase identification confidence
levels.
The taxonomy of microorganisms is undergoing rapid change and this makes even
more difficult the complex task of properly identifying microorganisms. EPA experience
has revealed that attempts at shortcuts to microbial identification often yield equivocal
results. Recognizing the difficulties likely to be encountered, and considering the central
role of product identification in permitting a meaningful review, EPA/Safer Choice will
work with applicants to ensure that appropriate identification information is obtained and
provided to the Agency.
The following is general guidance for this process. The applicant may need to expect
that this will be an iterative process, with EPA/Safer Choice supplying suggestions,
where available, for specific methods or approaches tailored to the specific organisms
proposed by the applicant.
	Taxonomic designation, including strain (if possible)
•	If the microorganism is a strain identified by a national service culture collection,
provide appropriate documentation, e.g., collection number and product
information sheet; purchase order; any testing used in identification; etc.
•	If the microorganism is not obtained from a national service culture collection,
verify the taxonomic designation by providing one of the following: 1) a letter from
a source culture collection that describes how the identification was performed,
including available test data and literature references, or 2) results of tests to
determine characteristics (e.g., commercial methods such as Biolog/Microlog,
API 20NE Rapid NFT; research methods such as GC-FAME, 16S rDNA
sequencing; or specific tests unique to the supplier of the identification) and a
statement addressing who performed the tests (i.e., the submitter, a commercial
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service, a consultant, etc.). Also, for species that are difficult to identify, a specific
description of the methods used to distinguish and detect the microorganism.
•	Synonyms, common names, and superseded names.
•	Identification of any patent or application for a patent.
Depending on the species/strain type, the Agency may recommend particular methods
or approaches that have been found to provide species identification with better degrees
of confidence. All test reports should include: confidence levels, name of lab, date of
testing, etc.
Additional Characterization
•	Life cycle
•	Growth characteristics: Generation time, growth temperature (optimum and
range), pH, oxygen requirements, preferred energy and carbon sources.
•	Factors affecting growth, survival, or reproduction (e.g., sporulation, encystment,
other non-vegetative growth stages, ability to exist in the viable but non-culturable
(VNBC) state, auxotrophy, etc.).
•	Resistance to antibiotics and tolerance to metals and pesticides.
III. Potential Human Health and Environmental Effects
Microorganisms, like individual chemicals, vary greatly in the degree to which they have
been characterized toxicologically. Depending on microorganism type and product
application, the following information may be pertinent in developing a human health
and environmental profile and assessing the hazards the microorganism may pose.
Note that in most cases the information listed below (esp. on human health effects) will
be available in microbiology texts or from literature/database sources (see for example:
Current Contents, Medline, Biosis, Science Citation Index, Agricola, Enviroline,
TOXNET, Biotechnology Citation Index, Current Biotechnology Abstracts, Current
Research Information System (CRIS)).
The search should provide information for a thorough overview of the requested
information. If most of this information is available in recent reports, a search of the
literature dating back a number of years may not be necessary. Where recent reports
are unavailable, inconclusive or contradictory, a more extensive search over a longer
time period may be needed. The literature search report should indicate the time period
of the search, the information sources, title of published papers, and search strategy,
including search terms.

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Whenever possible, the information should be provided for the specific organism in your
formulation. Where there is little information available on this organism, information on a
surrogate organism may be substituted (please consult with Safer Choice on the choice
of a suitable surrogate). When there is no relevant information from the scientific
literature or unpublished studies for items pertaining to human health effects or
ecological hazards, laboratory tests may be required.
	Human Health
•	Nature and degree of pathogenicity (including the capacity to act as an
opportunistic pathogen), virulence, or infectivity in humans.
•	Nature and degree of toxigenicity and toxicity (host tissue damage) to humans.
•	Nature and degree of allergenic or immunological responses in humans after
exposure viaingestion, inhalation, or dermal contact.
•	Potential host range of the microorganism(s), infective dose, routes of
transmission, and normal reservoirforthe microorganism(s).
•	Ability to colonize humans or grow at body temperature.
•	Susceptibility to control measures, e.g., antibiotics or disinfectants.
	Ecological
•	Nature and degree of pathogenicity, virulence, or infectivity to mammals, fish,
insects, other invertebrates, and to plants, including host range. Include test data
that supports potential effects.
•	Toxicity of microbially produced toxins and toxigenicity to mammals, fish, insects,
other invertebrates, and to plants.
•	Identification of plant and animal species likely to be exposed and, where
infectivity, pathogenicity, toxicity, or toxigenicity to non-human organisms has
been identified, the identification of the receptor species likely to be exposed.
•	Potential for gene transfer to other microorganisms of traits for pathogenicity,
infectivity, toxicity, toxigenicity to non-human species or of antibiotic resistance.
•	Potential for causing adverse effects on mammals, fish, insects, other
invertebrates, and plants, indirectly through means such as, but not limited to,
changes in the availability of nutrients, changes in the solubility or oxidation states
of metals, creation of anoxic conditions in surface waters, etc.
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•	Involvement in or effects on biogeochemical processes (e.g., effects on nutrient
cycling, particularly C, N, P, and S; effects on primary production (C02 fixation);
utilization of complex substrates, such as cellulose and lignin degradation; effects
on nitrogen fixation and nitrification).
•	Known or predicted effects on other organisms including microorganisms in the
environment, including effects on competitors, prey, hosts, symbionts, predators,
parasites, pathogens, community structure, and species diversity.
•	Known or expected substrate range of degradative gene protein products,
including both contaminant compounds to be bioremediated and environmental
substrates (e.g., lignin) on which gene protein products may also act.
•	Known or expected metabolic pathways of both targetxenobioticcompounds
and other contaminants present during degradation.
•	Nature and degree of toxicity of metabolites (dead-end or intermediate products
resulting from degradation of the target compound) to mammals, fish, insects,
other invertebrates, and to plants.
	Survival and Environmental Fate
•	Natural habitats and prevalence of the microorganism(s) in the environment,
including a description of habitats where the microorganism may persist or
proliferate.
•	Habitat at the potential locations of introduction, and the nature of selection
pressure that may operate on the microorganism at these locations.
•	Survival/persistence in in the environmental media (e.g., water, soil, air) into
which the microorganism(s) is to be introduced, including an estimate of the
quantities of the microorganism in those media at the points of introduction and
the estimated population trends.
•	Survival/persistence in environments other than the intended introduction site
(surface water, ground water, other soils) into which the microorganisms may
disseminate.
•	Known and predicted environmental conditions that may affect survival,
multiplication, etc.
•	Method of detection and detection limits.
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•	Prevalence of natural gene exchange among the microorganism(s) and natural
microbial populations.
IV. Measuring the Health/Environmental Benefits
In determining whether a microorganism has a more positive health and environmental
profile or contributes to a formulation with these characteristics, it is important to
understand the formulatory context, in other words, what were the choices, among
microorganisms or chemical ingredients, in formulating a particular product. Whether a
product receives Safer Choice recognition depends on the microorganism, its
characteristics, product application, and comparison to what might be used in its stead.
Once a more positive profile is established, it is important to ensure that a product
maintains that profile, i.e., that its microorganism make-up remains consistent and free
of harmful contaminants.
	Comparing Ingredients
•	What other microorganisms might be substituted for your microorganism for each
of the uses listed in Part 1MB?
•	Does your product replace a microorganism- or chemical-based product? If so,
what ingredients does the microorganism- or chemical-based product contain?
	Quality Assurance/Quality Control
•	For each production lot or batch, verification of absence of pathogenic
microorganisms as contaminants. Species of concern include not only frank
pathogens, but certain opportunistic microorganisms such as, but not limited to,
Pseudomonas aeruginosa and Burkholderia cepacia.
•	For each production lot or batch, verification of (certificate of analysis)
consistency of total bacterial counts or cell density (cfu/ml); activity of
microorganisms/efficacy of product; concentration of surfactants/emulsifiers; and
concentration of free enzymes; concentration of other ingredients.
•	Shelf-lifeofproduct.
Thank you for your help in understanding and improving the health and environmental
profile of your formulations.
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