ASTM E3214-19

• Abstract:

  SIGNIFICANCE AND USE; 4.1 The technology to engineer industrial microorganisms (IMs) is evolving rapidly and the public, regulatory bodies, and industrial sectors require new tools to help evaluate the products of biotechnology (1)3. In particular, there is a need to clarify the nature and intent of genetic alterations present in many industrial microbial strains (2, 3). ; 4.2 Currently, there is no systematic classification system to help differentiate among the many subtypes of engineered industrial microorganisms (4, 5). In response, a classification system for industrial microorganisms has been developed with the intent of facilitating the commercial use and development of industrial microorganisms and the biotechnology sector in general. ; 4.3 This classification will be applied to all microorganisms for which there is an intended use, broadly referred to as “industrial microorganisms.” This classification covers both viable and non-viable microorganisms, in addition to any product that contains microbial DNA. ; 4.4 This classification is not intended to apply to downstream products of industrial microorganisms that do not contain microbial DNA, for example, highly purified proteins or small molecules produced by industrial microorganisms.; SCOPE; 1.1 This classification applies to all industrial microorganisms, both classically derived and those produced through genetic engineering methods. ; 1.2 The scope of this classification does not include plants and animals. This classification would not be applied to any downstream products derived from industrial microorganisms unless they contain microbial deoxyribonucleic acid (DNA). ; 1.3 This classification includes fields for genotype class, biosafety, mode/intent of use, and the extent of DNA sequence information for a given industrial microbial strain. ; 1.4 Units—The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard. ; 1.5 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety, health, and environmental practices and determine the applicability of regulatory limitations prior to use. ; 1.6 This international standard was developed in accordance with internationally recognized principles on standardization established in the Decision on Principles for the Development of International Standards, Guides and Recommendations issued by the World Trade Organization Technical Barriers to Trade (TBT) Committee.

• Status: Published
• Publication Date: 31-May-2019
• ICS: 07.080 - Biology. Botany. Zoology
• Technical Committee: E62 - Industrial Biotechnology and Synthetic Biology
• Drafting Committee: E62.02 - Best Practices

• Refers: ASTM E3072-18 - Standard Terminology for Industrial Biotechnology
• Effective Date: 01-Jan-2018
• Refers: ASTM E3072-17 - Standard Terminology for Industrial Biotechnology
• Effective Date: 15-Jan-2017

Overview

ASTM E3214-19: Standard Classification for Industrial Microorganisms is an internationally recognized framework developed by ASTM International for the classification of all types of industrial microorganisms (IMs). This standard addresses the rapid advancements in biotechnology and the increasing need for clear differentiation among the various subtypes of both classically derived and genetically engineered industrial microorganisms. ASTM E3214-19 provides a systematic method to classify and evaluate IMs for their commercial, regulatory, and research applications.

The standard is particularly significant in supporting biotechnology industries, regulatory agencies, and product developers to understand and communicate the nature and purpose of genetic alterations present in industrial microbial strains. It also clarifies which products are subject to this classification-specifically, those that include microbial DNA-enhancing transparency and safety in the use of microbial biotechnology.

Key Topics

• Scope of Coverage: Applies to all industrial microorganisms, whether derived by classical or genetic engineering methods. Excludes plants, animals, and downstream products without microbial DNA.

• Four-Field Classification System:

  • Genotype Class (A-D): Differentiates microorganisms based on their genetic composition and modifications.
  • Biosafety Risk Group (1-4): Assigns biosafety risk to microorganisms aligned with international guidelines, considering human health risk and available countermeasures.
  • Mode/Intent of Use: Articulates the primary application area using standardized codes (e.g., food production, agriculture, pharmaceuticals).
  • Extent of Genome Sequencing: Indicates the level of DNA sequencing (from unsequenced to fully population-sequenced).

• Classification Rationale:

  • Class A: Native strains with no intentional modifications.
  • Class B: Strains with intentional genetic changes within natural diversity but no non-native DNA.
  • Class C: Engineered strains containing non-native DNA sequences from other species or synthetic origins.
  • Class D: Strains with non-native DNA engineered to produce novel chemical entities not found in nature.

• Biosafety Considerations: Emphasizes proper risk assessment based on the potential for human disease and the existence, or lack thereof, of therapeutic or preventive interventions.

• Exclusion of Downstream Products: Highly purified products (e.g., proteins, chemicals) derived from IMs but lacking microbial DNA are not included in this classification.

Applications

The ASTM E3214-19 standard provides practical value by offering a common, transparent language for:

• Biotechnology Companies: Streamlining product development, regulatory submission, and communication about genetic alterations and intended uses of microorganisms.
• Regulatory Agencies: Enabling more precise risk assessment, compliant labeling, and relevant oversight for genetically engineered and classically derived strains.
• Quality Assurance & Safety Evaluations: Supporting biosafety assessments, establishing the extent of sequencing and characterizing potential hazards of microbial strains.
• Academic & Industrial Research: Facilitating genetic studies, process development, and comparison among microbial strains across various research and commercial sectors.
• Product Labelling & Transparency: Informing stakeholders and end-users about the nature and application of microorganisms used in products, especially where microbial DNA is present.

The standard is widely utilized in sectors such as agriculture (crop protection, biopesticides), food and beverage manufacturing (yeasts, fermentative bacteria), pharmaceuticals (therapeutic enzyme production), environmental applications (waste bio-treatment), and specialty chemical production.

Related Standards

Organizations implementing ASTM E3214-19 may also find value in references and associated standards, including:

• ASTM E3072: Terminology for Industrial Biotechnology, providing foundational definitions for understanding IM-related terms.
• NIH Guidelines for Research involving Recombinant or Synthetic Nucleic Acid Molecules (2016): For biosafety risk classification alignment.
• European Commission Directive 2001/18/EC: On genetically modified organism release, relevant for regulatory compliance.
• ISO and OECD Biotechnology Documentation: Related to best practices and standards coordination.

Keywords: industrial microorganisms, ASTM E3214-19, microorganism classification, biotechnology, biosafety, genetic engineering, genotype class, microbial DNA, regulatory compliance, risk assessment, industrial biotech standards.

This international standard was developed in accordance with internationally recognized principles on standardization established in the Decision on Principles for the
Development of International Standards, Guides and Recommendations issued by the World Trade Organization Technical Barriers to Trade (TBT) Committee.
Designation:E3214 −19
Standard Classification for
Industrial Microorganisms
This standard is issued under the fixed designation E3214; the number immediately following the designation indicates the year of
original adoption or, in the case of revision, the year of last revision. A number in parentheses indicates the year of last reapproval. A
superscript epsilon (´) indicates an editorial change since the last revision or reapproval.
1. Scope 3.2 Definitions of Terms Specific to This Standard:
3.2.1 genotype, n—portion of genetic material within an
1.1 This classification applies to all industrial
organism that determines the expression of a specific charac-
microorganisms, both classically derived and those produced
teristic (phenotype).
through genetic engineering methods.
3.2.2 mutagenesis, n—addition, deletion, or substitution of
1.2 The scope of this classification does not include plants
at least one base within an organism’s genome.
and animals. This classification would not be applied to any
3.2.2.1 Discussion—Mutagenesis can occur under either
downstream products derived from industrial microorganisms
natural or directed means.
unless they contain microbial deoxyribonucleic acid (DNA).
3.2.3 native deoxyribonucleic acid (DNA), n—DNA se-
1.3 This classification includes fields for genotype class,
quences derived from within the same microbial species
biosafety, mode/intent of use, and the extent of DNAsequence
3.2.4 non-native DNA, n—DNAsequences originating from
information for a given industrial microbial strain.
other microbial species or designed sequences with three or
1.4 Units—The values stated in SI units are to be regarded
more intentional and predesignated DNA base-pair alterations
as standard. No other units of measurement are included in this
across a genome, relative to native DNA.
standard.
3.2.4.1 Discussion—Non-native DNA shall fall outside the
1.5 This standard does not purport to address all of the
observed genomic diversity within a given microbial species.
safety concerns, if any, associated with its use. It is the
DNA base-pair alterations are defined as either base-pair
responsibility of the user of this standard to establish appro-
insertions, or substitutions, but not deletions.
priate safety, health, and environmental practices and deter-
3.2.5 transgenic, adj—organism that contains non-native
mine the applicability of regulatory limitations prior to use.
DNA sequences.
1.6 This international standard was developed in accor-
3.2.5.1 Discussion—Non-native DNAsequences can be de-
dance with internationally recognized principles on standard-
rived from another species or designed de novo.
ization established in the Decision on Principles for the
Development of International Standards, Guides and Recom-
4. Significance and Use
mendations issued by the World Trade Organization Technical
4.1 The technology to engineer industrial microorganisms
Barriers to Trade (TBT) Committee.
(IMs)isevolvingrapidlyandthepublic,regulatorybodies,and
industrial sectors require new tools to help evaluate the
2. Referenced Documents
products of biotechnology (1) . In particular, there is a need to
2.1 ASTM Standards:
clarify the nature and intent of genetic alterations present in
E3072 Terminology for Industrial Biotechnology
many industrial microbial strains (2, 3).
4.2 Currently, there is no systematic classification system to
3. Terminology
help differentiate among the many subtypes of engineered
3.1 Definitions—See Terminology E3072 for industrial bio-
industrial microorganisms (4, 5). In response, a classification
technology terms.
system for industrial microorganisms has been developed with
the intent of facilitating the commercial use and development
of industrial microorganisms and the biotechnology sector in
This classification is under the jurisdiction of ASTM Committee E62 on
general.
Industrial Biotechnology and Synthetic Biology and is the direct responsibility of
Subcommittee E62.02 on Best Practices.
4.3 This classification will be applied to all microorganisms
Current edition approved June 1, 2019. Published July 2019. DOI: 10.1520/
for which there is an intended use, broadly referred to as
E3214.
For referenced ASTM standards, visit the ASTM website, www.astm.org, or
contact ASTM Customer Service at [e-mail]. For Annual Book of ASTM
Standards volume information, refer to the standard’s Document Summary page on The boldface numbers in parentheses refer to a list of references at the end of
the ASTM website. this standard.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
E3214−19
“industrial microorganisms.” This classification covers both classical approaches, for example, forced lateral gene transfer
viable and non-viable microorganisms, in addition to any between similar, but distinct, microbial species under directed
product that contains microbial DNA. conditions. This class also includes strains in which a native
gene is modified by noncoding regulatory elements
4.4 This classification is not intended to apply to down-
[promotors, repressors, ribosome binding site (RBS) regions,
stream products of industrial microorganisms that do not
and so forth] originating from other species or synthetically
contain microbial DNA, for example, highly purified proteins
derived. Class C organisms are comprised of otherwise native
or small molecules produced by industrial microorganisms.
biochemical building blocks and are only distinct from Classes
A and B in that they contain one or more non-native DNA
5. 5. Basis of Classification
sequences.
5.1 This classification consists of four fields that represent
5.2.4 Class D organisms are defined as strains subjected to
genotype class, biosafety risk grouping, mode/intent of use,
intentionalselectionorengineeringwithadditionofnon-native
and the extent of genome sequence information.
DNA enabling the production of non-natural biochemical
5.2 Field 1 refers to genotype class and is assigned to one of
building blocks (for example, non-natural amino acids, chemi-
four categories (A-D). Classes A-C are organisms that only
cally modified nucleic acids and so forth). Class D includes
contain or produce naturally observed biochemicals regardless
organismswithmetabolicpathwaysengineeredtoproducenew
of the origin of DNA sequences contained within the strain.
chemical entities from native precursors (for example, sugars,
Classes A-C are also referred to as Type 1 microorganisms. In
protein, and lipids). Class D organisms typically display
contrast, Class D organisms contain or produce chemical
phenotypes not previously observed in nature. Examples in-
entities not previously observed in nature, for example, non-
clude organisms with express proteins containing non-natural
natural amino acids or pharmaceuticals produced through
amino acids and strains engineered to produce non-natural
engineered biochemical pathways (6). Class D organisms are
pharmaceuticals as an alternative to chemical synthesis. Class
also referred to as Type 2 microorganisms.
Dorganismscontainnon-nativeDNAthatmaybecomposedof
5.2.1 Class A organisms are defined as native strains in
the natural four nucleotide bases or chemical analogs thereof.
which no intentional selection or engineering was applied with
5.3 Field 2 is biosafety risk grouping (1 to 4). Biosafety risk
the intent of creating a new phenotype. ClassAstrains include
groupings (8) take into account the extended impact of a
microorganisms present in the environment, as well as natural
microorganism and the availability of countermeasures such as
isolates and laboratory propagated strains derived from natural
vaccines and antibiotics. A risk grouping level for a microor-
isolates. Class A organisms may be subject to unintentional
ganism is assigned on the following basis:
selection pressures that may lead to spontaneous genetic
5.3.1 Risk Group 1: Not associated with disease in healthy
changes, for example, those driven by changes to the natural
adult humans;
environment or through propagation under laboratory condi-
5.3.2 Risk Group 2: Associated with human disease that is
tions. Examples of Class A strains include wild yeasts used in
rarely serious and for which preventive or therapeutic inter-
brewing, the human microbiome, microorganisms present in
ventions are often available;
the soil, and naturally-occurring pathogens.
5.3.3 Risk Group 3:Associatedwithseriousorlethalhuman
5.2.2 Class B organisms are defined as strains that have
disease for which preventive or therapeutic interventions may
been subjected to deliberate alteration of the organism’s native
be available; and
genome without the introduction of any non-native DNA, with
the intent of producing an altered phenotype. Any form of 5.3.4 Risk Group 4:Microbialagentsthatarelikelytocause
selective pressure or genetic engineering can be applied serious or lethal human disease for which preventive or
provided the genomic sequence of the resulting strains falls therapeutic interventions are not usually available.
within the bounds of the known genetic diversity of the parent
5.3.5 The great majority of industrial microorganisms fall
microbial species. The definition of microbial species is
within Risk Group 1 and a sub-classification of this category
adopted from Parker, Tindall, and Garrity (7). The origin of
will likely be required in a future revision of this standard that
native DNA can be natural or synthetic provided the sequence
also takes into account the extended impact of industrial
is within the observed native genomic diversity as determined
microbial strains beyond any direct effects on human health.
by DNA sequencing.
5.4 Field 3 represents the mode of use and primary appli-
5.2.3 Class C organisms are defined as strains that have
cation of an industrial microbial strain expressed as a code
been subjected to intentional selection or engineering with
containing two alphabetical symbols separated by a period
addition of non-native DNA. For the purposes of this
where the first letter is uppercase (for example, X.y). These
classification, non-native DNA is defined as DNA sequences
codes are listed in Annex A1 and reflect the current modes of
originating from other species or designed DNA sequences
use and application areas for industrial microorganisms cur-
with three or more intentional base-pair alterations relative to
rently in commerce. In cases in which a strain has more than
an otherwise homologous native DNAsequence. By definition,
one end use, only the primary application is cited.
non-native DNA shall fall outside of the observed genomic
diversity of a given microbial species as determined by DNA 5.5 Field 4 describes genomic sequence information and
sequencing. Class C organisms are typically produced through reflects the extent to which genome of a microbial strain or
modern genetic techniques involving recombinant or synthetic culture has been characterized by genetic analysis (that is,
DNA sequences. Class C organisms can also be produced by DNA sequencing) on a scale of 1 to 4. Genomic sequencing
E3214−19
can confirm whether the genome of a microbial strain altered 6.3 Clarified products derived from IMs should not be
by modern biotechnological methods (for example, gene edit- subject to this classification unless the product contains detect-
ing or other targeted techniques) falls within observed natural able genetic material (typically DNA) upon analysis using
genetic variation (that is, native or non-native DNA). The four polymerase chain reaction (PCR) or equivalent analytical
categories within this field are: techniques for the sequencing and identification of genetic
material.
5.5.1 Unsequenced—Little or no DNA sequence
information,
6.4 This classification should be applied by the producer of
5.5.2 Partially Sequenced—Incomplete DNA sequence the industrial microorganism using it as a guide. Full or even
partial d
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