Design for the Environment Program 

Master Criteria for Safer Ingredients 

(formerly the General Screen for Safer Ingredients) 

Version 2.0 

July 2010 

Office of Pollution Prevention & Toxics 

U.S. Environmental Protection Agency 
 

 

 

 

U.S. EPA Design for the Environment Program Version 2.0 

Master Criteria July 2010 

Table of Contents 

1 Introduction..........................................................................................................................................2
 

2 General Requirements ........................................................................................................................3 

3 Terms ..................................................................................................................................................4
 

4 Preferences .........................................................................................................................................6
 

5 Attributes of Concern for All Chemicals ..............................................................................................7
 

5.1 Acute Mammalian Toxicity .....................................................................................................7
 

5.2 Carcinogenicity.......................................................................................................................8
 

5.3 Genetic Toxicity....................................................................................................................10
 

5.4 Neurotoxicity.........................................................................................................................12
 

5.5 Repeated Dose Toxicity .......................................................................................................13
 

5.6 Reproductive and Developmental Toxicity...........................................................................15
 

5.7 Respiratory Sensitization......................................................................................................17
 

5.8 Skin Sensitization.................................................................................................................18
 

5.9 Environmental Toxicity and Fate..........................................................................................19
 

5.10 Eutrophication ......................................................................................................................20
 

6 Test Methods.....................................................................................................................................22
 

6.1 Acute Mammalian Toxicity ...................................................................................................22
 

6.2 Carcinogenicity.....................................................................................................................22
 

6.3 Genetic Toxicity....................................................................................................................22
 

6.4 Neurotoxicity.........................................................................................................................22
 

6.5 Repeated Dose Toxicity .......................................................................................................23
 

6.6 Reproductive and Developmental Toxicity...........................................................................23
 

6.7 Skin Sensitization.................................................................................................................24
 

6.8 Environmental Toxicity and Fate..........................................................................................24
 

6.8.1 Acute Aquatic Toxicity.............................................................................................24
 

6.8.2 Persistence (measured as biodegradation) ............................................................25
 

6.8.3 Bioaccumulation......................................................................................................25
 

References..................................................................................................................................................26
 

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U.S. EPA Design for the Environment Program Version 2.0 

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Introduction 

Purpose 

The Design for the Environment (DfE) Master Criteria for Safer Ingredients (Master Criteria) are 

comprehensive, science-based criteria designed to ensure that the safest possible ingredients are used in 

DfE-labeled products. DfE evaluates every ingredient in a formulation within its functional class context 

and based on its key, distinguishing human health and environmental characteristics. In this way, 

potential product ingredients can be viewed as part of a continuum of improved or safer ingredient 

choices. These criteria also enhance the transparency of the DfE Program. 

The Master Criteria make it possible to draw a line demarcating the greener or “low-concern” end of the 

continuum of chemical safety. To define low concern, DfE uses toxicological thresholds established by 

highly respected health and environmental protection authorities, including the United Nation’s Globally 

Harmonized System (GHS) for the Classification and Labeling of Hazard Substances and the U.S. EPA’s 

New Chemicals Program. For functional classes where no low-concern ingredients currently exist, DfE 

works with its stakeholders to carefully modify the Master Criteria in a way that allows for ingredient 

choices while ensuring the safest possible ingredients in that functional class. These criteria were 

designed for use in distinguishing safer chemicals for the DfE Program. 

DfE product review is chemistry and toxicology intensive, calling on the extensive expertise of the EPA’s 

Office of Pollution Prevention and Toxics. The Office’s depth of expertise helps ensure that chemicals 

are fully and accurately characterized based on the best available information. Information for the review 

is drawn from peer-reviewed literature, primary source materials, hazardous chemical lists, Agency 

databases, and predictive tools which estimate potential human health and environmental concerns 

based on a chemical’s structural and/or biological similarity to known chemicals of concern. EPA will 

consider all sources of developing information, such as the Endocrine Disruptor Screening Program or 

enhancements to estimation models such as EPI SuiteTM that occur over time. 

Development 

The Master Criteria were developed by the Environmental Protection Agency’s DfE Program. The 

contents of these criteria, including definitions and toxicological preferences, were developed to facilitate 

use of safer chemistry under the DfE Program. 

Scope 

In DfE’s Safer Product Labeling Program, the Master Criteria serves as the primary tool to advance Green 

Chemistry in product formulation and to implement Informed Substitution, the reasoned transition from a 

chemical of particular concern to safer chemicals or non-chemical alternatives. DfE will use the Master 

Criteria to review all product ingredients (and their chemical components). Where DfE has developed 

more customized criteria for specific functional classes of chemicals, e.g., surfactants, solvents, etc., 

those criteria will apply. All chemicals in products designed for direct release to the environment (e.g. 

graffiti removers and marine cleaners) must also pass the DfE Criteria for Environmental Toxicity and 

Fate for Chemicals in Direct Release Products. 

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2 General Requirements 

2.1	 Data for all relevant routes of exposure will be evaluated. 

2.2	 The GHS criteria and data evaluation approach and EPA risk assessment guidance will inform 

professional judgment in the review of both no observed adverse effect levels/concentrations 

(NOAEL/NOAEC) and lowest observed adverse effect levels/concentrations (LOAEL/LOAEC). 

NOAEL/NOAEC and LOAEL/LOAEC values are preferred over no observed effect 

levels/concentrations (NOEL/NOEC) and lowest observed effect levels/concentrations 

(LOEL/LOEC). In reviews that include conflicting data, a weight of evidence approach will 

determine a pass or fail. 

2.3	 Use of existing data should follow the EPA HPV Challenge Program and OECD HPV Programme 

data adequacy guidelines: http://www.epa.gov/HPV/pubs/general/datadfin.htm. 

2.4 EPA will perform an additional in-depth review of a chemical under certain conditions. Conflicting 

data on a chemical, detection in bio- or environmental monitoring studies, or presence on a 

flagging list will trigger such a review. The additional review will apply GHS criteria and other 

criteria explained in this document. 

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3 Terms 

3.1 Acute aquatic toxicity is the intrinsic property of a substance to be injurious to an organism in a 

short-term exposure to that substance. (GHS) 

3.2 Acute mammalian toxicity refers to those adverse effects occurring following oral or dermal 

administration of a single dose of a substance, or multiple doses given within 24 hours, or an 

inhalation exposure of 4 hours. (GHS) 

3.3 Attribute: The general property of the chemical that is being evaluated (i.e. acute mammalian 

toxicity, biodegradability). 

3.4 Bioaccumulation is a process in which a chemical substance is absorbed in an organism by all 

routes of exposure as occurs in the natural environment, i.e., dietary and ambient environment 

sources. Bioaccumulation is the net result of competing processes of chemical uptake into the 

organism at the respiratory surface and from the diet and chemical elimination from the organism 

including respiratory exchange, fecal egestion, metabolic biotransformation of the parent 

compound and growth dilution. [1] 

3.5 Biodegradation is a process in which the destruction of the chemical is accomplished by the 

action of a living organism. (Handbook of Property Estimation Methods for Chemicals, 2000) 

3.6 Carcinogen denotes a chemical substance or mixture of chemical substances which induces 

cancer or increases its incidence. (GHS) 

3.7 A chemical is identified by its Chemical Abstract Service (CAS) number. 

3.8 Chronic aquatic toxicity is the potential or actual properties of a substance to cause adverse 

effects to aquatic organisms during exposures which are determined in relation to the life cycle of 

the organism. (GHS) 

3.9 Criteria: Endpoints and cutoffs for attribute information. Example: oral acute mammalian toxicity 

LD50 must be > 50 mg/kg. Data quality requirements (including acceptable test methods and 

information sources) are developed for all criteria. 

3.10 Dermal sensitizer: A substance that will induce an allergic response following skin contact 

(GHS) 

3.11 Developmental toxicity: Adverse effects in the developing organism that may result from 

exposure prior to conception (either parent), during prenatal development, or postnatally to the 

time of sexual maturation. Adverse developmental effects may be detected at any point in the 

lifespan of the organism. The major manifestations of developmental toxicity include: (1) death of 

the developing organism, (2) structural abnormality, (3) altered growth, and (4) functional 

deficiency. (EPA Risk Assessment Guidelines [2]) 

3.12	 An endocrine disruptor is an external agent that interferes in some way with the role of natural 

hormones in the body. An agent might disrupt the endocrine system by affecting any of the 

various stages of hormone production and activity, such as by preventing the synthesis of 

hormones, by directly binding to hormone receptors, or by interfering with the natural breakdown 

of hormones. (EPA) [3] 

3.13	 Flagging list: A publicly available list of chemicals that may have potential hazard concerns as 

identified by the authors of that list. 

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3.14 Genotoxicity: The more general terms genotoxic and genotoxicity apply to agents or processes 

which alter the structure, information content, or segregation of DNA, including those which cause 

DNA damage by interfering with normal replication processes, or which in a non-physiological 

manner (temporarily) alter its replication. Genotoxicity test results are usually taken as indicators 

for mutagenic effects. (GHS) 

3.15	 An ingredient may be one chemical or a blend of multiple chemicals that are intentionally added. 

3.16 Inorganic phosphate: This category includes all inorganic soluble forms of phosphates, such as, 

phosphoric acid [PO4H3 or OP(=O)(O)O] and its salts or phosphate salts, pyrophosphates, 

polyphosphates, and organic and inorganic forms of phosphorous that can be oxidized to 

phosphates rapidly. Inorganic forms of phosphonic acid (H2PO3 or OP(=O)O) are not included in 

this category because monopotassium phosphonic acid [13977-65-6] has been shown not to be 

an algal nutrient, not to be a replacement for phosphate in algal growth medium, and not to cause 

exponential growth of green algae. (US EPA New Chemicals) [Note: Inorganic forms of 

phosphonic acid are not typically used in cleaning products.] 

3.17 LOAEL: Lowest Observed Adverse Effect Level 

3.18 Mutagen: The term mutagenic and mutagen will be used for agents giving rise to an increased 

occurrence of mutations in populations of cells and/or organisms. (GHS) 

3.19 Neurotoxicity: An adverse change in the structure or function of the central and/or peripheral 

nervous system following exposure to a chemical, physical, or biological agent. (US EPA Risk 

Assessment Guidelines) 

3.20 NOAEL: No Observed Adverse Effect Level 

3.21 Persistence: The length of time the chemical can exist in the environment before being 

destroyed (i.e., transformed) by natural processes. (EPA PBT Final Rule [4]) 

3.22 Reproductive toxicity: The occurrence of biologically adverse effects on the reproductive 

systems of females or males that may result from exposure to environmental agents. The toxicity 

may be expressed as alterations to the female or male reproductive organs, the related endocrine 

system, or pregnancy outcomes. The manifestation of such toxicity may include, but not be 

limited to, adverse effects on onset of puberty, gamete production and transport, reproductive 

cycle normality, sexual behavior, fertility, gestation, parturition, lactation, developmental toxicity, 

premature reproductive senescence, or modifications in other functions that are dependent on the 

integrity of the reproductive systems. (US EPA Risk Assessment Guidelines [5]) 

3.23 Respiratory sensitizer: A substance that will induce hypersensitivity of the airways following 

inhalation of the substance. (GHS) 

3.24 Suitable analog: Suitable analogs will be based on a chemically (e.g., based on chemical 

structure) or biologically (e.g., based on metabolic breakdown, or likely mechanistic/mode of 

action considerations) similar chemical. Guidance for identifying a suitable analog can be found 

in OECD Series on Testing and Assessment No. 80 Guidance on Grouping of Chemicals [6]. 

The analog used must be appropriate for the attribute being evaluated. 

3.25 Weight-of-evidence: For the purposes of this document, weight-of-evidence refers to the 

process of considering the strengths and weaknesses of various pieces of information in reaching 

and supporting a conclusion concerning a property of the substance. (ECHA [7]) 

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4 Preferences 

4.1 When data are developed to meet the requirements for Repeated Dose Toxicity, EPA requests 

that a functional observational battery, such as OPPTS 870.6200: Neurotoxicity Screening 

Battery [8], be added to the test method to provide neurotoxicity information. 

4.2 Data for the evaluation of chemicals under these criteria are preferred in the following order: 1) 

measured data on the specific chemical, 2) measured data from a suitable analog, 3) estimated 

data from appropriate models. Data requirements specific to each attribute are outlined in 

Section 4. The majority of measured data are expected to be from laboratory experiments. 

However, any available human data will be considered, e.g. Human Repeat Insult Patch Tests. 

Human data may require appropriate review for ethical treatment of the subjects. 

4.3 The links and references in this document are current as of the publication date of these Criteria. 

The reviewer must use the most recent version of each authoritative list, EPA data interpretation 

guidance, and test protocol when reviewing a chemical against these criteria. In the case where 

a GHS reference in this document is superseded by a more recent version, EPA may choose to 

update these Criteria to incorporate that newer version. EPA will consider all sources of 

developing information, such as the EPA Endocrine Disruptor Screening Program1

 [9] or 

enhancements to estimation models such as EPI SuiteTM [10] that occur over time. 

1

 The Agency does not consider endocrine disruption to be an adverse endpoint per se, but as a step that 

could lead to toxic outcomes, such as cancer or adverse reproductive effects....”[3. USEPA, 

Special Report on Environmental Endocrine Disruption: An Effects Assessment and Analysis. , in Risk 

Assessment Forum. 1997: Washington DC. 

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5 Attributes of Concern for All Chemicals 

Each attribute listed below applies to all chemicals not covered by specific functional class criteria. 

5.1 ACUTE MAMMALIAN TOXICITY 

Criteria 

Applying GHS criteria [11], a chemical does not pass the Criteria if the median lethal dose or 

concentration is less than or equal to those values listed in Table 1a. For inhalation studies, exposure 

duration should be at least four hours; the thresholds for inhalation are the same for exposures greater 

than four hours. Exposures of less than four hours will be evaluated on a case-by-case basis. 

Table 1a – GHS Thresholds 

Route (units) Median Lethal 

Dose/Concentration 

Oral LD50 (mg/kg bw) 2,000 

Dermal LD50 (mg/kg bw) 2,000 

Inhalation, gas LC50 (ppmV) 20,000 

Inhalation, vapor LC50 (mg/L) 20 

Inhalation, dust/mist/fumes LC50 (mg/L) 5 

Additionally, a chemical does not pass the Criteria if it carries one of the following EU Risk Phrases, which 

align with the GHS thresholds in Table 1a: 

Table 1b – Acute Toxicity Risk Phrases 

R20 Harmful by inhalation 

R21 Harmful in contact with skin 

R22 Harmful if swallowed 

R23 Toxic by inhalation 

R24 Toxic in contact with skin 

R25 Toxic if swallowed 

R26 Very toxic by inhalation 

R27 Very toxic in contact with skin 

R28 Very toxic if swallowed 

And all combination risk phrases containing one or more of the 

above. 

Data Requirements 

All available data, measured and/or estimated, for the chemical and/or a suitable analog will be reviewed 

against the criteria using a weight-of-evidence approach. 

Sources for Data Interpretation 

– GHS Ch 3.1 Acute Toxicity [11]. 

– EU Dangerous Substances Directive, http://ecb.jrc.ec.europa.eu/documentation/. To access 

the list of substances carrying Risk Phrases, click on “CLASSIFICATION-LABELLING”, then 

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“DIRECTIVE 67-548-EEC”, then “ANNEX I OF DIRECTIVE 67-548-EEC”, and then either of 

the files listed as: “Annex I of Directive 67548EEC”. [12]; and 

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5.2 CARCINOGENICITY 

Criteria 

Chemicals considered carcinogens according to the authoritative lists in Table 2 do not pass the Criteria. 

Chemicals not on those authoritative lists, but that are known or presumed human carcinogens (Category 

1), or suspected human carcinogens (Category 2) under GHS [13], do not pass the Criteria. 

Table 2 – Authoritative Lists and GHS Criteria 

Authoritative Body Does not pass DfE Criteria 

National Toxicology Program 

(NTP) 

Known to be Human Carcinogen 

Reasonably Anticipated to be Human Carcinogen 

U.S. Environmental Protection 

Agency (EPA) 

(2005/1999) Carcinogenic to humans, Likely to be carcinogenic to 

humans, or Suggestive evidence of carcinogenic potential2 

(1996) Known/Likely 

(1986) Group A – Human Carcinogen, Group B – Probable human 

carcinogen, or Group C – Possible human carcinogen2 

International Agency for 

Research on Cancer (IARC) 

Group 1 – Carcinogenic to humans 

Group 2A – Probably carcinogenic to humans 

Group 2B – Possibly carcinogenic to humans2 

EU CMR List [14] 

Category 1 – Known to be carcinogenic to humans 

Category 2 – Should be regarded as if carcinogenic to humans 

Category 3 – Cause for concern for humans owing to possible 

carcinogenic effects 

EU Risk Phrases [14] 

R45: May cause cancer 

R49: May cause cancer by inhalation 

R40: Limited evidence of a carcinogenic effect 

And all combination risk phrases containing one or more of the above. 

Globally Harmonized System 

(GHS) [13] 

Category 1A – Known to have carcinogenic potential for humans 

Category 1B – Presumed to have carcinogenic potential for humans 

Category 2 – Suspected human carcinogens 

Data Requirements 

All available data will be evaluated. Measured and/or estimated data, for the chemical and/or a suitable 

analog will be reviewed against GHS criteria using a weight-of-evidence approach. 

 Flagging Lists 

All relevant data and information used to place a chemical on the following flagging lists will be 

considered when reviewing a listed chemical against the carcinogenicity criteria. 

1. Substances prioritized for testing for endocrine disruption by the European Commission as 

Category 1 or 2 [15, 16], 

2. Substances prioritized for testing for endocrine disruption by the US EPA Endocrine Disruptor 

Screening Program [9], 

2 Chemicals listed as “possibly carcinogenic to humans” are evaluated largely on animal studies. DfE will consider 

appropriate data that show cancer concerns are not relevant to humans, e.g., because of an animal specific tissue 

effect or mode of action. If the data demonstrate that cancer concerns are not relevant to humans, that chemical can 

be considered under the DfE Criteria. 

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3. Substances listed on the State of California Environmental Protection Agency, Office of 

Environmental Health Hazard Assessment (OEHHA) California Proposition 65 (Safe Drinking 

Water and Toxic Enforcement Act Of 1986) as Known to the State to Cause Cancer [17]. 

Sources for Data Interpretation 

– EU Dangerous Substances Directive, http://ecb.jrc.ec.europa.eu/documentation/. To access 

the list of substances carrying Risk Phrases, click on “CLASSIFICATION-LABELLING”, then 

“DIRECTIVE 67-548-EEC”, then “ANNEX I OF DIRECTIVE 67-548-EEC”, and then either of 

the files listed as: “Annex I of Directive 67548EEC”. [12]; 

– EU Dangerous Preparations Directive Article 6 and Annex II (1999/45/EC and subsequent 

updates/amendments) [18-20]; 

–	 GHS Ch 3.6 Carcinogenicity [13]; 

–	 Section 2, Hazard Assessment in Guidelines for Carcinogen Risk Assessment (Risk 

Assessment Forum) (EPA 2005), 

http://oaspub.epa.gov/eims/eimscomm.getfile?p_download_id=439797 [21] and 

–	 The following link can be used to identify substances prioritized for testing for endocrine 

disruption by the European Commission: 

http://ec.europa.eu/environment/endocrine/strategy/substances_en.htm#priority_list. To 

download the list of substances, see the zipped file under the heading “Priority List” [15]. 

–	 The following report describes the process used to develop the endocrine disrupters priority 

list: http://ec.europa.eu/environment/endocrine/documents/final_report_2007.pdf [16]. 

–	 EPA Endocrine Disruptors Screening Program, available at: http://www.epa.gov/endo/ [9]. 

–	 Supplemental Guidance for Assessing Susceptibility from Early-Life Exposure to 

Carcinogens, available at: http://cfpub.epa.gov/ncea/CFM/recordisplay.cfm?deid=160003 

[22]. 

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5.3 GENETIC TOXICITY 

Criteria 

Chemicals considered mutagens or genetic toxicants according to the authoritative lists in Table 3 do not 

pass the Criteria. Chemicals not on those authoritative lists, but which are classifiable as to their potential 

to cause genetic toxicity according to GHS [23] do not pass the Criteria. Effects to be considered include 

heritable germ cell mutagenicity (including gene mutation and chromosome mutation), germ cell genetic 

toxicity, and somatic cell mutagenicity or genetic toxicity. 

Table 3 – Authoritative Lists and GHS Criteria 

Authoritative Body Does not pass DfE Criteria 

EU CMR List [14] 

Category 1 – Substances known to be mutagenic to humans 

Category 2 – Substances which should be regarded as if they 

are mutagenic to humans 

Category 3 – Substances which cause concern for human owing 

to possible mutagenic effects3 

EU Risk Phrases [14] 

R46: May cause heritable genetic damage 

R68: Possible risk of irreversible effects 

And all combination risk phrases containing one or more of the above. 

Globally Harmonized System (GHS) 

[23] 

Category 1A – Chemicals known to induce heritable mutations 

in germ cells of humans 

Category 1B – Chemicals which should be regarded as if they 

induce heritable mutations in the germ cells of humans 

Category 2 – Chemicals which cause concern for humans owing 

to the possibility that they may induce heritable mutations in 

the germ cells of humans 

Data Requirements 

All available data, including in vivo, in vitro, and epidemiological studies, will be evaluated. Measured 

and/or estimated data, for the chemical and/or a suitable analog will be reviewed against GHS criteria 

using a weight-of-evidence approach. 

Sources for Data Interpretation 

–	 EU Dangerous Substances Directive, http://ecb.jrc.ec.europa.eu/documentation/. To access 

the list of substances carrying Risk Phrases, click on “CLASSIFICATION-LABELLING”, then 

“DIRECTIVE 67-548-EEC”, then “ANNEX I OF DIRECTIVE 67-548-EEC”, and then either of 

the files listed as: “Annex I of Directive 67548EEC”; [12] 

–	 EU Dangerous Preparations Directive Article 6 and Annex II (1999/45/EC and subsequent 

updates/amendments) [18-20]; and 

–	 GHS Ch 3.5 Germ Cell Mutagenicity [23]. 

3

 Per EU guidance, chemicals classified as Category 3 substances may be placed in that category based on positive 

results in assays showing (a) mutagenic effects or (b) other cellular interaction relevant to mutagenicity. If a chemical 

is classified in Category 3(b) only and that classification appears overly conservative, then the submitter may request 

EPA expert review. In such as case, if EPA determines the data do not support a concern for possible mutagenic 

effects, then the chemical will pass the criteria. 

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5.4 NEUROTOXICITY 

Criteria 

Chemicals that are considered neurotoxicants under GHS [24] (see GHS guidance values in Table 4) do 

not pass the screen. Neurotoxicity is evaluated using the GHS chapter called Specific Target Organ 

Toxicity Repeated Exposure. 

Table 4 – GHS Guidance Values 

Route of Administration (units) Guidance Value* 

Oral (mg/kg-bw/day) 100 

Dermal (mg/kg-bw/day) 200 

Inhalation (gas) (ppm/6h/day) 250 

Inhalation (vapor) (mg/L/6h/day) 1.0 

Inhalation (dust/mist) (mg/L/6h/day) 0.2 

*The doses provided are for 90-day studies. Guidance values are tripled for chemicals evaluated in 28-day studies. 

Data Requirements 

All available data, measured and/or estimated, for the chemical and/or a suitable analog will be reviewed 

against the criteria using a weight-of-evidence approach. 

Sources for Data Interpretation 

– Section 3, Hazard Characterization in Guidelines for Neurotoxicity Risk Assessment [25]; and 

– GHS Ch. 3.9 Specific Target Organ Toxicity Repeated Exposure [24]. 

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5.5 REPEATED DOSE TOXICITY 

Criteria 

Chemicals that are considered repeated dose (systemic) toxicants under GHS [24] (see GHS guidance 

values in Table 5a) do not pass the Criteria. Repeated dose toxicity is evaluated using the GHS chapter 

called Specific Target Organ Toxicity Repeated Exposure. 

Table 5a – GHS Guidance Values 

Route of Administration (units) Guidance Value* 

Oral (mg/kg-bw/day) 100 

Dermal (mg/kg-bw/day) 200 

Inhalation (gas) (ppm/6h/day) 250 

Inhalation (vapor) (mg/L/6h/day) 1.0 

Inhalation (dust/mist/fume) (mg/L/6h/day) 0.2 

*The doses provided are for 90-day studies. Guidance values are tripled for chemicals evaluated 

in 28-day studies and similarly modified for studies of longer durations. 

Additionally, a chemical does not pass the Criteria if it carries one of the following EU Risk Phrases: 

Table 5b – Repeated Dose Toxicity Authoritative Lists 

Authoritative Body Does not pass DfE Criteria 

EU Risk Phrases [14] 

R33: Danger of cumulative effects (repeated exposure) 

R39: Danger of very serious irreversible effects (single 

exposure) 

R48: Danger of serious damage to health by prolonged 

exposure (repeated exposure) 

R68: Possible risk of irreversible effects (single exposure) 

And all combination risk phrases containing one or more of the above. 

Data Requirements 

All available data, measured and/or estimated, for the chemical and/or a suitable analog will be reviewed 

against the criteria using a weight-of-evidence approach. 

Flagging Lists 

All relevant data and information used to place a chemical on the following flagging lists will be 

considered when reviewing a listed chemical against the repeated dose toxicity criteria. 

1. Substances prioritized for testing for endocrine disruption by the European Commission as 

Category 1 or 2 [15, 16], 

2. Substances prioritized for testing for endocrine disruption by the US EPA Endocrine Disruptor 

Screening Program [9], 

Sources for Data Interpretation 

–	 GHS Ch 3.9 Specific Target Organ Toxicity Repeated Exposure [24], 

–	 The following link can be used to identify substances prioritized for testing for endocrine 

disruption by the European Commission: 

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http://ec.europa.eu/environment/endocrine/strategy/substances_en.htm#priority_list. To 

download the list of substances, see the zipped file under the heading “Priority List”. [15] 

–	 The following report describes the process used to develop the endocrine disrupters priority 

list: http://ec.europa.eu/environment/endocrine/documents/final_report_2007.pdf[16]. 

–	 EPA Endocrine Disruptors Screening Program, available at: http://www.epa.gov/endo/ [9]. 

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5.6 REPRODUCTIVE AND DEVELOPMENTAL TOXICITY 

Criteria 

Chemicals that are considered reproductive or developmental toxicants under GHS criteria [26] and 

demonstrate adverse effects at doses at or below the values in Table 6a do not pass the Criteria. 

Table 6a – TSCA 8(e) Guidance Values [27] 

Route of Administration 

(units) Guidance Value 

Oral (mg/kg-bw/day) 250 

Dermal (mg/kg-bw/day) 500 

Inhalation (gas) 

(ppm/6h/day) 250 

Inhalation (vapor) 

(mg/L/6h/day) 2.5 

Inhalation (dust/mist) 

(mg/L/6h/day) 0.5 

Additionally, a chemical does not pass the Criteria if it carries one of the following EU Risk Phrases: 

Table 6b – Reproductive and Developmental Toxicity Authoritative Lists 

Authoritative Body Does not pass DfE Criteria 

EU CMR List [14] 4 

Category 1 – Known to impair fertility in humans or known to 

cause developmental toxicity in humans 

Category 2 – Should be regarded as if they impair fertility in 

humans or cause developmental toxicity to humans 

Category 3 – Cause concern for human fertility or possible 

developmental toxic effects 

EU Risk Phrases [14] 4 

R60: May impair fertility 

R61: May cause harm to the unborn child 

R62: Possible risk of impaired fertility 

R63: Possible risk of harm to the unborn child 

R64: May cause harm to breastfed babies 

And all combination risk phrases containing one or more of the above. 

Data Requirements 

All available data, measured and/or estimated, for the chemical and/or a suitable analog will be reviewed 

against the criteria using a weight-of-evidence approach. Following the approach in the SIDS Dossier 

[28], both fertility and developmental effects should be considered. 

4 The EU classification criteria do not currently consider a limit dose above which an adverse effect would not trigger 

classification. EPA will consider evidence demonstrating that a chemical carrying a reproductive/developmental 

toxicity risk phrase or listed as toxic to reproduction (in Table 5a) did not cause an adverse effect below the TSCA 

8(e) Guidance Values listed in Table 5b. Such a chemical may be determined, upon EPA review, to pass the DfE 

criteria for reproductive/developmental toxicity. 

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Flagging Lists 

All relevant data and information used to place a chemical on the following flagging lists will be 

considered when reviewing a listed chemical against the reproductive and developmental toxicity 

criteria. 

1. Substances prioritized for testing for endocrine disruption by the European Commission as 

Category 1 or 2 [15, 16] 

2. Substances prioritized for testing for endocrine disruption by the US EPA Endocrine Disruptor 

Screening Program [9] 

3. Substances listed on the State of California Environmental Protection Agency, Office of 

Environmental Health Hazard Assessment (OEHHA) California Proposition 65 (Safe Drinking 

Water and Toxic Enforcement Act Of 1986) as Known to the State to Cause Reproductive 

Toxicity [17]. 

Sources for Data Interpretation 

–	 EU Dangerous Substances Directive, http://ecb.jrc.ec.europa.eu/documentation/. To access 

the list of substances carrying Risk Phrases, click on “CLASSIFICATION-LABELLING”, then 

“DIRECTIVE 67-548-EEC”, then “ANNEX I OF DIRECTIVE 67-548-EEC”, and then either of 

the files listed as: “Annex I of Directive 67548EEC” [12]; 

–	 EU Dangerous Preparations Directive Article 6 and Annex II (1999/45/EC and subsequent 

updates/amendments) [18-20]; 

–	 GHS Ch 3.7 Reproductive Toxicity [26] 

–	 Part A, Section 3, Hazard Characterization in Guidelines for Reproductive Toxicity Risk 

Assessment (EPA 1998), http://www.epa.gov/ncea/raf/pdfs/repro51.pdf [5]. 

–	 Part A, Section 3, Hazard Characterization in Guidelines for Developmental Toxicity Risk 

Assessment (EPA 1991), http://www.epa.gov/NCEA/raf/pdfs/devtox.pdf [2]. 

–	 The following link can be used to identify substances prioritized for testing for endocrine 

disruption by the European Commission: 

http://ec.europa.eu/environment/endocrine/strategy/substances_en.htm#priority_list. To 

download the list of substances, see the zipped file under the heading “Priority List”. [15] 

–	 The following report describes the process used to develop the endocrine disrupters priority 

list: http://ec.europa.eu/environment/endocrine/documents/final_report_2007.pdf [16] 

– EPA Endocrine Disruptors Screening Program, available at: http://www.epa.gov/endo/ [9]. 

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July 2010 

5.7 RESPIRATORY SENSITIZATION 

Criteria 

Chemicals considered respiratory sensitizers according to the authoritative list in Table 7 do not pass the 

Criteria. Chemicals not on that authoritative list, but that are classified under GHS do not pass the 

Criteria. 

Table 7 – Authoritative Lists and GHS Criteria 

Authoritative Body Does not pass DfE Criteria 

EU Risk Phrase [14] R42: May cause sensitization by inhalation 

Globally Harmonized System (GHS) [29] 

Category 1A – high frequency of occurrence or 

sensitization rate in humans 

Category 1B – low to moderate frequency of 

occurrence or sensitization rate in humans 

Data Requirements 

Acknowledging that recognized animal models for the testing of respiratory hypersensitivity are not 

available at present, data on respiratory sensitization will normally be based on human evidence; all 

available data will be reviewed. EPA will search public literature and EPA-confidential data to support the 

review. Chemicals associated with hypersensitivity after appropriate clinical testing may not pass the 

criteria. See GHS guidance [29] for further details. 

Flagging Lists 

Chemicals designated as sensitizer-induced asthmagens (“Rs” or “Rrs”) on the specified flagging list 

below require review using all relevant and available data to support GHS classification: 

1. Association of Occupational and Environmental Clinics (AOEC) Exposure Code List [30]. 

Sources for Data Interpretation 

–	 EU Dangerous Substances Directive, http://ecb.jrc.ec.europa.eu/documentation/. To access 

the list of substances carrying Risk Phrases, click on “CLASSIFICATION-LABELLING”, then 

“DIRECTIVE 67-548-EEC”, then “ANNEX I OF DIRECTIVE 67-548-EEC”, and then either of 

the files listed as: “Annex I of Directive 67548EEC”. [12]. 

–	 EU Dangerous Preparations Directive Article 6 and Annex II (1999/45/EC and subsequent 

updates/amendments) [18-20]. 

–	 GHS Ch 3.4 Respiratory and Skin Sensitization [29]. 

–	 Association of Occupational and Environmental Clinics Exposure Code List, available from: 

http://www.aoecdata.org/Default.aspx [30]. 

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5.8 SKIN SENSITIZATION 

Criteria 

Chemicals considered skin sensitizers according to the authoritative list in Table 8 do not pass the 

Criteria. Chemicals not on that authoritative list, but that are classified under GHS do not pass the 

Criteria. 

Table 8 – Authoritative Lists and GHS Criteria 

Authoritative Body Does not pass DfE Criteria 

EU Risk Phrase [14] R43: May cause sensitization by skin contact 

Globally Harmonized System (GHS) [29] 

Category 1A – high frequency of occurrence in 

humans and/or a high potency in animals 

Category 1B – low to moderate frequency of 

occurrence in humans and/or a low to moderate 

potency in animals 

Data Requirements 

All available data, including in vivo, in vitro, and epidemiological studies, will be evaluated. Data from 

estimation models may be considered as part of the weight-of-evidence. 

Sources for Data Interpretation 

–	 EU Dangerous Substances Directive, http://ecb.jrc.ec.europa.eu/documentation/. To access 

the list of substances carrying Risk Phrases, click on “CLASSIFICATION-LABELLING”, then 

“DIRECTIVE 67-548-EEC”, then “ANNEX I OF DIRECTIVE 67-548-EEC”, and then either of 

the files listed as: “Annex I of Directive 67548EEC” [12]. 

–	 EU Dangerous Preparations Directive Article 6 and Annex II (1999/45/EC and subsequent 

updates/amendments) [18-20]. 

–	 GHS Ch 3.4 Respiratory and Skin Sensitization [29]. 

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U.S. EPA Design for the Environment Program 

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5.9 ENVIRONMENTAL TOXICITY AND FATE (ET&F) 

Criteria 

If a chemical is an acute aquatic toxicant (i.e., L/E/IC50 < 100 ppm), then it must biodegrade rapidly and 

not be bioaccumulative (see Table 12, lines 1-3). If a chemical has low aquatic toxicity (Table 12, line 4), 

then its half-life must be less than 60 days. 

Table 12 – Environmental Toxicity and Fate 

Acute Aquatic Toxicity 

Value (L/E/IC50)5,6,7 

Persistence 

(Measured in terms of level of 

biodegradation) 

Bioaccumulation 

Potential 

1 If ≤1 ppm... ...then may be acceptable if the chemical 

meets the 10-day window as measured in a 

ready biodegradation test without 

degradation products of concern8 

... 

...and BCF/BAF <1000. 

2 If >1 ppm and ≤10 ppm... ...then the chemical must meet the 10-day 

window as measured in a ready 

biodegradation test without degradation 

products of concern8 ... 

3 If >10 ppm and <100 ppm... ...then the chemical must reach the pass 

level within 28 days as measured in a ready 

biodegradation test without degradation 

products of concern8 

... 

4 If ≥100 ppm... ...then the chemical need not reach the 

pass level within 28 days as measured in a 

ready biodegradation test if there are no 

degradation products of concern8

 and its 

half-life < 60 days... 

Data Requirements 

Acute aquatic toxicity: Measured data are preferred. ECOSAR estimations may be used along with 

data from a suitable analog(s). Data, whether measured or from analogs, are required for each of the 

following groups of organisms: algae, aquatic invertebrates and fish (all fresh water). If only estimated 

data are available, the use of estimated data may be acceptable in combination with EPA expert 

review. Data for marine species may be added when available. 

5

 In general, there is a predictable relationship between acute aquatic toxicity and chronic aquatic toxicity for organic 

chemicals, i.e., chemicals that have high acute aquatic toxicity may also have high chronic aquatic toxicity at low 

concentrations [Rand, G.M., ed. Fundamentals of Aquatic Toxicology. 2nd ed. 1995, Taylor & Francis: Washington, 

DC.]. Since acute aquatic toxicity data are more readily available, the DfE Screens use these data to screen 

chemicals that may be toxic to aquatic life. Where measured chronic toxicity data is available, it will be assessed with 

other data and applied in the screen based on the relationship between acute and chronic aquatic toxicity. 

6

 A case-by-case approach focusing on rate of biodegradation and degradation products of concern will be 

implemented for chemicals toxic to aquatic organisms at ≤ 1ppm. 7

 For determining the aquatic toxicity of substances that are not toxic at their solubility limit, see ECOSAR Technical 

Reference Manual Figure 9, p 17 (http://www.epa.gov/oppt/newchems/tools/ecosartechfinal.pdf); When a chemical 

may have effects at saturation as determined using the guidance in the ECOSAR manual, a weight-of-evidence 

approach in combination with US EPA expert review will be used. EPA may require additional testing including but 

not limited to solubility testing, chronic aquatic toxicity testing, or acute aquatic toxicity testing of analogs. 8

 Degradation products of concern are compounds with high acute aquatic toxicity (L/E/IC50 ≤ 10ppm) which 

mineralize <60% in 28 days. 

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Bioaccumulation potential: Measured data are preferred. Data from a suitable analog is 

acceptable, and EPI SuiteTM estimations (from the most current version) may be used when those 

data are unavailable. Results from both the BAF and BCF models should be considered. An 

estimated BAF is preferred to an estimated BCF for compounds where log Kow > 5. 

Persistence (measured as level of biodegradation): 

Measured data are preferred. In the case where measured data are unavailable, data from estimation 

models or a suitable analog will be accepted as follows: 

(1) If acute aquatic toxicity ≤ 1ppm: Biodegradability must be measured for the chemical or for a 

suitable analog. Biodegradability predictions from estimation models, such as EPI SuiteTM, will be 

used only to support weight-of-evidence. 

(2) If acute aquatic toxicity > 1ppm and ≤ 10ppm: Biodegradability must be measured for the 

chemical or for a suitable analog. Biodegradability predictions from estimation models, such as 

EPI SuiteTM, will be used only to support weight-of-evidence. 

(3) If acute aquatic toxicity > 10ppm and < 100ppm: Biodegradability must be measured for the 

chemical or for a suitable analog. Biodegradability predictions from estimation models, such as 

EPI SuiteTM, will be used only to support weight-of-evidence. 

(4) If acute aquatic toxicity ≥ 100pm: Biodegradability for the chemical or for a suitable analog are 

preferred. Biodegradability predictions from estimation models, such as EPI SuiteTM (the most 

current version), may be acceptable. 

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U.S. EPA Design for the Environment Program 

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5.10 EUTROPHICATION 

Criteria 

The total level of phosphorus in the cleaning product will be limited to a maximum level of 0.5 weight % in 

the cleaning product as sold (measured as elemental phosphorus). Inorganic phosphates, as defined by 

the US EPA New Chemicals Program [31], cannot make up any portion of the 0.5 weight % of 

phosphorus. 

Note: Inorganic phosphates as defined by the US EPA New Chemicals Program [31] will not be allowed 

in DfE-labeled products. Eutrophication is a priority concern for EPA scientists, and inorganic phosphates 

can contribute to eutrophication of fresh water and estuarial ecosystems [31]. Given that the majority of 

DfE-labeled cleaning products are disposed of down the drain, the likelihood of release to wastewater 

streams and eventually, water bodies, is high. EPA acknowledges contributions to the phosphorus load 

from cleaners are relatively small in comparison with other sources9

 and that phosphorus overload may 

not be an issue in all regions. The EPA policy is consistent with a commitment to reduce contributions of 

phosphorus, particularly inorganic phosphates, regardless of concentration. 

9

 See, for example, 32. Detailed Assessment of Phosphorus Sources in Minnesota Watersheds. 2004, 

Prepared by Barr Engineering Company for Minnesota Pollution Control Agency. 23 February 2010. 

http://www.pca.state.mn.us/hot/legislature/reports/phosphorus-report.html. 

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6 Test Methods 

The test methods in this section should be used to develop data for conducting chemical reviews based 

on the criteria in Section 4. 

6.1 Acute Mammalian Toxicity – Test Methods for GHS Review 

–	 OPPTS Harmonized Guideline 870.1100: Acute oral toxicity [33]; 

–	 OPPTS Harmonized Guideline 870.1200: Acute dermal toxicity [34] 

–	 OPPTS Harmonized Guideline 870.1300: Acute inhalation toxicity [35]; 

–	 OECD Test Guideline 420: Acute Oral Toxicity-Fixed Dose Method [36]; 

–	 OECD Test Guideline 423: Acute Oral Toxicity – Acute Toxic Class Method [37]; 

–	 OECD Test Guideline 425: Acute Oral Toxicity – Up-and-Down Procedure [38]; 

–	 OECD Test Guideline 402: Acute Dermal Toxicity [39]; and 

–	 OECD Test Guideline 403: Acute Inhalation Toxicity [40]. 

6.2 Carcinogenicity – Test Methods for GHS Review 

–	 OECD Test Guideline 451: Carcinogenicity Studies [41]; 

–	 OECD Test Guideline 453: Combined Chronic Toxicity/Carcinogenicity Studies [42]; 

–	 OPPTS Harmonized Guidelines 870.4200: Carcinogenicity [43]; 

–	 OPPTS Harmonized Guidelines 870.4300: Combined chronic toxicity/carcinogenicity [44] and 

–	 NTP 2 Year Study Protocol: “Specifications for the conduct of studies to evaluate the toxic 

and carcinogenic potential of chemical, biological and physical agents in laboratory animals 

for the National Toxicology Program” [45]. 

6.3 Genetic Toxicity – Test Methods for GHS Review 

Per GHS [23], results from multiple, acceptable test methods must be used in conjunction for 

evaluation of genetic toxicity. 

–	 OECD Test Guideline 471 (OPPTS 870.5100): Bacterial Reverse Mutation Test [46, 47]; 

–	 OECD Test Guideline 473 (OPPTS 870.5375): In vitro Mammalian Chromosome Aberration 

Test [48, 49]; 

–	 OECD Test Guideline 474 (OPPTS 870.5395): Mammalian Erythrocyte Micronucleus Test 

[50, 51]; 

–	 OECD Test Guideline 475 (OPPTS 870.5385): Mammalian Bone Marrow Chromosome 

Aberration Test [52, 53]; 

–	 OECD Test Guideline 476 (OPPTS 870.5300): In vitro Mammalian Cell Gene Mutation Test 

[54, 55]; and 

–	 OECD Test Guideline 483 (OPPTS 870.5380): Mammalian Spermatogonial Chromosome 

Aberration Test [56, 57]; 

–	 OECD Test Guideline 486: Unscheduled DNA Synthesis (UDS) Test with Mammalian Liver 

Cells in vivo [58]. This guideline does NOT substitute in the necessary minimum set for 

either the gene mutation or the chromosome aberration test. 

6.4 Neurotoxicity – Preferred Test Methods for GHS Review 

– OECD Test Guideline 424: Neurotoxicity Study in Rodents [59] and 

– OPPTS Harmonized Guideline 870.6200: Neurotoxicity screening battery [8]. 

Neurotoxicity – Additional Test Methods for GHS Review 

Additional evidence from OECD Test Guideline 426: Developmental Neurotoxicity Study [60] and 

OPPTS Harmonized Guideline: 870.6300 Developmental neurotoxicity study [61] can be used to 

screen chemicals for neurotoxicity. 

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6.5 Repeated Dose Toxicity – Preferred Test Methods for GHS Review 

– OECD Test Guideline 408: Repeated Dose 90-Day Oral Toxicity Study in Rodents [62] 

– OECD Test Guideline 409: Repeated Dose 90-Day Oral Toxicity Study in Non-Rodents [63] 

– OECD Test Guideline 411: Subchronic Dermal Toxicity: 90-day Study [64] 

– OECD Test Guideline 413: Subchronic Inhalation Toxicity: 90-day Study [65] 

– OPPTS Harmonized Guideline 870.3100: 90-Day oral toxicity in rodents [66] 

– OPPTS Harmonized Guideline 870.3150: 90-Day oral toxicity in nonrodents [67] 

– OPPTS Harmonized Guideline 870.3250: 90-Day dermal toxicity [68] 

– OPPTS Harmonized Guideline 870.3465: 90-Day inhalation toxicity [69] 

Repeated Dose Toxicity – Acceptable Test Methods for GHS Review 

–	 OECD Test Guideline 407: Repeated Dose 28-day Oral Toxicity Study in Rodents [70] 

–	 OECD Test Guideline 410: Repeated Dose Dermal Toxicity: 28-day Study [71] 

– OECD Test Guideline 412: Repeated Dose Inhalation Toxicity: 28-day Study [72] 

– OECD Test Guideline 422: Combined Repeated Dose Toxicity Study with the 

Reproduction/Developmental Toxicity Screening Test [73] 

– OPPTS Harmonized Guideline 870.3050: Repeated dose 28-day oral toxicity study in rodents 

[74] 

– OPPTS Harmonized Guideline 870.3200: 28-Day dermal toxicity [75] 

6.6 Reproductive and Developmental Toxicity – Test Methods for GHS Review 

Fertility test methods, preferred 

– OECD Test Guideline 415: One-Generation Reproduction Toxicity Study [76] and 

– OECD Test Guideline 416: Two-Generation Reproduction Toxicity Study [77]. 

Fertility test methods, acceptable 

The following test methods may be used to identify reproductive toxicity, per GHS [26]: 

– OPPTS Harmonized Guideline 870.3800: Reproduction and fertility effects [78]; 

– OECD Test Guideline 421: Reproduction/Developmental Toxicity Screening Test [79]; 

– OECD Test Guideline 422: Combined Repeated Dose Toxicity Study with the 

Reproduction/Developmental Toxicity Screening Test [73]; 

– OPPTS Harmonized Guideline 870.3550: Reproduction/developmental toxicity screening 

test [80] 

– OPPTS Harmonized Guideline 870.3650: Combined repeated dose toxicity study with the 

reproduction/developmental toxicity screening test [81]. 

Developmental toxicity test methods, preferred 

– OECD Test Guideline 414: Prenatal Developmental Toxicity Study [82] 

Developmental toxicity test methods, acceptable 

The following test methods may be used to identify developmental toxicity, per GHS [26]: 

– OPPTS Harmonized Guideline 870.3800: Reproduction and fertility effects [78]; 

– OECD Test Guideline 421: Reproduction/Developmental Toxicity Screening Test [79]; 

– OECD Test Guideline 422: Combined Repeated Dose Toxicity Study with the 

Reproduction/Developmental Toxicity Screening Test [73]; 

– OPPTS Harmonized Guideline 870.3550: Reproduction/developmental toxicity screening 

test [80]; and 

23 
 

 

 

 

U.S. EPA Design for the Environment Program Version 2.0 

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–	 OPPTS Harmonized Guideline 870.3650: Combined repeated dose toxicity study with the 

reproduction/developmental toxicity screening test [81]. 

6.7 Skin Sensitization – Preferred Test Methods for GHS Review 

– OECD Test Guideline 406: Skin Sensitization [83] 

– OECD Test Guideline 429: Skin Sensitization: Local Lymph Node Assay [84] 

– OPPTS Harmonized Guideline 870.2600: Skin Sensitization [85] 

6.8 Environmental Fate and Toxicity 

6.8.1 Test Methods, Acute Aquatic Toxicity 

A baseline data set is required that includes test data in algae, aquatic invertebrates and fish. 

Additional aquatic toxicity data in other species or in marine species will also be reviewed if 

available. 

Preferred Test Methods for Fish 

– OECD Test Guideline 203: Fish, Acute Toxicity Test [86] and 

– OPPTS Harmonized Guideline 850.1075: Fish acute toxicity test, freshwater and 

marine[87] . 

NOTE – EPA may request that the test be carried out using semi-static renewal or a flowthrough system with mean measured concentration. Any new testing should be done in 

consultation with EPA. 

Preferred Test Methods for Aquatic Invertebrates 

– OECD Test Guideline 202, Part 1, Daphnia sp., Acute Immobilisation Test [88]; 

– OPPTS Harmonized Guideline 850.1010: Aquatic invertebrate acute toxicity test, 

freshwater daphnids[89]; and 

– OPPTS Harmonized Guideline 850.1035: Mysid acute toxicity test[90]. 

NOTE – EPA may request that the test be carried out using semi-static renewal or a flowthrough system with mean measured concentration. Any new testing should be done in 

consultation with EPA. A 96-hour Mysid shrimp acute toxicity test can be used in place of a 

daphnid acute toxicity test when the latter is not available. 

Preferred Test Methods for Algae 

– OECD Test Guideline 201, Alga, Growth Inhibition Test (and biomass) [91] and 

– OPPTS Harmonized Guideline 850.5400: Algal toxicity, Tiers I and II (including growth 

inhibition and biomass) [92]. 

Alternative Test Methods, Acute Aquatic Toxicity 

The following test methods may be considered, when relevant: 

– OPPTS Harmonized Guideline 850.1085: Fish acute toxicity mitigated by humic acid [93]; 

– OPPTS Harmonized Guideline 850.1025: Oyster acute toxicity test (shell deposition) [94]; 

– OPPTS Harmonized Guideline 850.1045: Penaeid acute toxicity test [95]; 

– OPPTS Harmonized Guideline 850.1055: Bivalve acute toxicity test (embryo larval) [96]; 

– OPPTS Harmonized Guideline 850.4400: Aquatic plant toxicity test using Lemna spp. 

Tiers I and II [97]. 

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6.8.2 Test Methods, Persistence (measured as biodegradation) 

Data from experimental methods are generally preferred over estimations of persistence. For the 

purposes of screening safer chemicals in Table 12, rows 1-3, ready biodegradation tests are 

preferred. It is noted that simulation tests are likely to better describe the biodegradability of a 

chemical in specific environmental conditions, and these tests can provide information to evaluate 

the half-life of a chemical that is aquatically toxic at ≥100 ppm. Simulation tests may also 

contribute useful information in a weight-of-evidence evaluation for chemicals aquatically toxic < 

100 ppm. 

Preferred Test Methods for Persistence 

– OECD Test Guideline 301: Ready Biodegradability (sections A-F) [98]; 

– OECD Test Guideline 310: Ready Biodegradability – CO2 in sealed vessels [99]; and 

– OPPTS Harmonized Guideline 835.3110: Ready biodegradability [100]. 

– For chemicals where acute aquatic toxicity ≥100 ppm (i.e., line 4, Table 12), if the 

compound degrades by more than 40% in 28 days during one of the Ready 

Biodegradability tests specified above or by more than 60% in one of the Inherent 

Biodegradability tests detailed in OECD Test Guidelines 302 (A-C) [16-18] then the halflife of a chemical is likely to be less than 60 days [101]. 

– Simulation tests may also be used to determine the half-life of a chemical and may be 

useful in a weight-of-evidence evaluation for chemicals aquatically toxic at < 100ppm. 

o	 OECD Test Guideline 303A (OPPTS 835.3240): Aerobic Sewage Treatment: 

Activated Sludge Units [102, 103], 

o	 OECD Test Guideline 309 (OPPTS Harmonized Guideline 835.3190): Aerobic 

Mineralization in Surface Water - Simulation Biodegradation Test [104, 105], 

o	 OECD Test Guideline 314: Simulation Tests to Assess the Biodegradability of 

Chemicals Discharged in Wastewater (Note: TG 314 uses elements of OECD TG 

301, 303A, 309, 310, and 311) [106], 

o	 OPPTS Harmonized Guideline 835.3280–Simulation Tests to Assess the Primary 

and Ultimate Biodegradability of Chemicals Discharged to Wastewater [107], 

o	 OPPTS Harmonized Guideline 835.3170 - Shake Flask Die-Away Test [108], and 

o	 OPPTS Harmonized Guideline 835.3180 - Sediment/Water Microcosm 

Biodegradation Test [109]. 

Other Methods of Degradation 

On a case-by-case basis, DfE will consider other routes of degradation in the environment, 

such as hydrolysis or photolysis, and degradation in other relevant media, for example, soil or 

sediment. In evaluating such degradation studies, DfE will consider the relevance of that 

degradation pathway to the chemical in question as well as the significance of the 

degradation. 

6.8.3 Test Methods, Bioaccumulation 

A field-measured BAF (located in the literature) is the most preferred data for indicating 

bioaccumulation. 

Alternative Test Methods for Bioaccumulation 

When a field-measured BAF is not available, the following test methods may be used: 

– OECD Test Guideline 305: Bioconcentration: Flow-through Fish Test[110]; 

– OPPTS Harmonized Guideline 850.1710: Oyster BCF[111]; 

– OPPTS Harmonized Guideline 850.1730: Fish BCF[112]; 

Modeled data from sources such as EPI SuiteTM [10] are acceptable when data are 

unavailable.
 

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References 

1. Arnot, J.A. and F.A. Gobas, A review of bioconcentration factor (BCF) and 

bioaccumulation factor (BAF) assessments for organic chemicals in aquatic organisms. 

Environmental Reviews, 2006. 14: p. 257-297. 

2. USEPA, Guidelines for Developmental Toxicity Risk Assessment. Federal Register, 

1991. 56(234): p. 63798-63826. 

3. USEPA, Special Report on Environmental Endocrine Disruption: An Effects Assessment 

and Analysis. , in Risk Assessment Forum. 1997: Washington DC. 

4. EPA. Persistent Bioaccumulative Toxic (PBT) Chemicals Final Rule 1999 [cited 2010 

April 20]; Available from: http://www.epa.gov/fedrgstr/EPA-WASTE/1999/October/Day29/f28169.htm. 

5. USEPA, Guidelines for Reproductive Toxicity Risk Assessment. Federal Register, 1996. 

61(212): p. 56274-56322. 

6. OECD, OECD Series on Testing and Assessment No 80.: Guidance on Grouping of 

Chemicals. 2007. 

7. ECHA. Practical guide 2: How to report weight of evidence. 2010 [cited 2010 April 21]; 

Available from: 

http://echa.europa.eu/doc/publications/practical_guides/pg_report_weight_of_evidence.p 

df. 

8. USEPA, Health Effects Test Guidelines: OPPTS 870.6200: Neurotoxicity Screening 

Battery. 1998. 

9. USEPA. Endocrine Disruptor Screening Program. October 21, 2009 [cited 2009 October 

22, 2009]; Available from: http://www.epa.gov/endo/. 

10. USEPA. EPI Suite TM. 2008 June 2008]; Available from: 

http://epa.gov/opptintr/exposure/pubs/episuite.htm. 

11. GHS, Acute Toxicity. 2009, United Nations. 

12. ECB, Directive 67-548-EEC. 2007. 

13. GHS, Carcinogenicity. 2009, United Nations. 

14. ECB, Annex I of Directive 67-548-EEC. 2007. 

15. EuropeanCommission. Endocrine Disrupters Website. June 2009]; Available from: 

http://ec.europa.eu/environment/endocrine/strategy/substances_en.htm#priority_list. 

16. Peterson, G., D. Rasmussen, and K. Gustavson. Study on Enhancing the Endocrine 

Disruptor Priority List with a Focus on Low Production Volume Chemicals. 2007 June 26, 

2009; May 2007:[Available from: 

http://ec.europa.eu/environment/endocrine/documents/final_report_2007.pdf. 

17. State of California Environmental Protection Agency. California Proposition 65 (Safe 

Drinking Water and Toxic Enforcement Act Of 1986) - Chemicals Known to the State to 

Cause Cancer or Reproductive Toxicity. September 10, 2009 October 22, 2009]. 

18. Council Directive 1999/45/EC, E. Commission, Editor. 1999: Official Journal of the 

European Union. 

19. Council Directive 2001/60/EC, E. Commission, Editor. 2001: Official Journal of the 

European Union. 

20. Council Directive 2006/8/EC, E. Commission, Editor. 2006: Official Journal of the 

European Union. 

21. USEPA, Guidelines for Carcinogen Risk Assessment. USEPA Risk Assessment Forum, 

2005. 

22. Barton, H., et al. Supplemental Guidance for Assessing Susceptibility from Early-Life 

Exposure to Carcinogens 2005 [cited 2009 December 22, 2009]; Available from: 

http://cfpub.epa.gov/ncea/CFM/recordisplay.cfm?deid=160003. 

23. GHS, Germ Cell Mutagenicity. 2009, United Nations. 

24. GHS, Specific Target Organ Systemic Toxicity Repeated Exposure. 2009, United 

Nations. 

25. USEPA, Guidelines for Neurotoxicity Risk Assessment. Federal Register, 1998. 63(93): 

p. 26926-26954. 

26. GHS, Reproductive Toxicity. 2009, United Nations. 

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U.S. EPA Design for the Environment Program Version 2.0 

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27. USEPA. Methodology for Risk-Based Prioritization under ChAMP. 2009 [cited 2010 

February 4]; Available from: 

http://www.epa.gov/oppt/existingchemicals/pubs/RBPMethodology_Web_April%202009.p 

df. 

28. OECD, Manual for the Investigation of HPV Chemicals, Annex 1 Guidance for completing 

a SIDS Dossier 2007. 

29. GHS, Respiratory or Skin Sensitization. 2009, United Nations. 

30. Association of Occupational and Environmental Clinics. Exposure Code List. 2009 

October 22, 2009]; Available from: http://www.aoecdata.org/Default.aspx. 

31. USEPA. U.S. EPA New Chemicals Program Chemical Categories - Inorganic 

Phosphates. 2002 October 22, 2009]; Available from: 

http://www.epa.gov/oppt/newchems/pubs/cat02.htm#Phosphates,%20Inorganic. 

32. Detailed Assessment of Phosphorus Sources in Minnesota Watersheds. 2004, Prepared 

by Barr Engineering Company for Minnesota Pollution Control Agency. 

33. USEPA, Health Effects Test Guidelines: OPPTS 870.1100: Acute Oral Toxicity. 1998. 

34. USEPA, Health Effects Test Guidelines: OPPTS 870.1200: Acute Dermal Toxicity. 1998. 

35. USEPA, Health Effects Test Guidelines: OPPTS 870.1300: Acute Inhalation Toxicity. 

1998. 

36. OECD, OECD Guidelines for the Testing of Chemicals: Test No. 420: Acute Oral Toxicity 

- Fixed Dose Procedure. 2001. 

37. OECD, OECD Guidelines for the Testing of Chemicals: Test No. 423: Acute Oral toxicity - 

Acute Toxic Class Method. 2001. 

38. OECD, OECD Guidelines for the Testing of Chemicals: Test No. 425: Acute Oral Toxicity: 

Up-and-Down Procedure. 2006. 

39. OECD, OECD Guidelines for the Testing of Chemicals: Test No. 402: Acute Dermal 

Toxicity. 1987. 

40. OECD, OECD Guidelines for the Testing of Chemicals: Test No. 403: Acute Inhalation 

Toxicity. 1981. 

41. OECD, OECD Guidelines for the Testing of Chemicals: Test No. 451: Carcinogenicity 

Studies. 1981. 

42. OECD, OECD Guidelines for the Testing of Chemicals: Test No. 453: Combined Chronic 

Toxicity/Carcinogenicity Studies. 1981. 

43. USEPA, Health Effects Test Guidelines: OPPTS 870.4200: Carcinogenicity. 1998. 

44. USEPA, Health Effects Test Guidelines: OPPTS 870.4300: Combined Chronic 

Toxicity/Carcinogenicity. 1998. 

45. NTP, Specifications for the conduct of studies to evaluate the toxic and carcinogenic 

potential of chemical, biological and physical agents in laboratory animals for the National 

Toxicology Program. 2006. 

46. OECD, OECD Guidelines for the Testing of Chemicals: Test No. 471: Bacterial Reverse 

Mutation Test. 1997. 

47. USEPA, Health Effects Test Guidelines: OPPTS 870.5100: Bacterial Reverse Mutation 

Test. 1998. 

48. OECD, OECD Guidelines for the Testing of Chemicals: Test No. 473: In Vitro Mammalian 

Chromosome Aberration Test. 1997. 

49. USEPA, Health Effects Test Guidelines: OPPTS 870.5375: In Vitro Mammalian 

Chromosome Aberration Test. 1998. 

50. OECD, OECD Guidelines for the Testing of Chemicals: Test No. 474: Mammalian 

Erythrocyte Micronucleus Test. 1997. 

51. USEPA, Health Effects Test Guidelines: OPPTS 870.5395: Mammalian Erythrocyte 

Micronucleus Test. 1998. 

52. OECD, OECD Guidelines for the Testing of Chemicals: Test No. 475: Mammalian Bone 

Marrow Chromosome Aberration Test. 1997. 

53. USEPA, Health Effects Test Guidelines: OPPTS 870.5385: Mammalian Bone Marrow 

Chromosome Aberration Test. 1998. 

54. OECD, OECD Guidelines for the Testing of Chemicals: Test No. 476: In Vitro Mammalian 

Cell Gene Mutation Test. 1997. 

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U.S. EPA Design for the Environment Program Version 2.0 

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55. USEPA, Health Effects Test Guidelines: OPPTS 870.5300: In Vitro Mammalian Cell 

Gene Mutation Test. 1998. 

56. OECD, OECD Guidelines for the Testing of Chemicals: Test No. 483: Mammalian 

Spermatogonial Chromosome Aberration Test. 1997. 

57. USEPA, Health Effects Test Guidelines: OPPTS 870.5380: Mammalian Spermatogonial 

Chromosome Aberration Test. 1998. 

58. OECD, OECD Guidelines for the Testing of Chemicals: Test No. 486: Unscheduled DNA 

Synthesis (UDS) Test with Mammalian Liver Cells In Vivo. 1997. 

59. OECD, OECD Guidelines for the Testing of Chemicals: Test No. 424: Neurotoxicity Study 

in Rodents. 1997. 

60. OECD, OECD Guidelines for the Testing of Chemicals: Test No. 426: Developmental 

Neurotoxicity Study. 2007. 

61. USEPA, Health Effects Test Guidelines: OPPTS 870.6300: Developmental Neurotoxicity 

Study. 1998. 

62. OECD, OECD Guidelines for the Testing of Chemicals: Test No. 408: Repeated Dose 90-

Day Oral Toxicity Study in Rodents. 1998. 

63. OECD, OECD Guidelines for the Testing of Chemicals: Test No. 409: Repeated Dose 90-

Day Oral Toxicity Study in Non-Rodents. 1998. 

64. OECD, OECD Guidelines for the Testing of Chemicals: Test No. 411: Subchronic Dermal 

Toxicity: 90-day Study. 1981. 

65. OECD, OECD Guidelines for the Testing of Chemicals: Test No. 413: Subchronic 

Inhalation Toxicity: 90-day Study. 1981. 

66. USEPA, Health Effects Test Guidelines: OPPTS 870.3100: 90-Day Oral Toxicity in 

Rodents. 1998. 

67. USEPA, Health Effects Test Guidelines: OPPTS 870.3150: 90-Day Oral Toxicity in 

Nonrodents. 1998. 

68. USEPA, Health Effects Test Guidelines: OPPTS 870.3250: 90-Day Dermal Toxicity. 

1998. 

69. USEPA, Health Effects Test Guidelines: OPPTS 870.3465: 90-Day Inhalation Toxicity. 

1998. 

70. OECD, OECD Guidelines for the Testing of Chemicals: Test No. 407: Repeated Dose 28-

day Oral Toxicity Study in Rodents. 1995. 

71. OECD, OECD Guidelines for the Testing of Chemicals: Test No. 410: Repeated Dose 

Dermal Toxicity: 21/28-day Study. 1981. 

72. OECD, OECD Guidelines for the Testing of Chemicals: Test No. 412: Repeated Dose 

Inhalation Toxicity: 28-day or 14-day Study. 1981. 

73. OECD, OECD Guidelines for the Testing of Chemicals: Test No. 422: Combined 

Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening 

Test. 1996. 

74. USEPA, Health Effects Test Guidelines: OPPTS 870.3050: Repeated Dose 28-day Oral 

Toxicity Study in Rodents. . 2000. 

75. USEPA, Health Effects Test Guidelines: OPPTS 870.3200: 28-Day Dermal Toxicity. 

1998. 

76. OECD, OECD Guidelines for the Testing of Chemicals: Test No. 415: One-Generation 

Reproduction Toxicity Study. 1983. 

77. OECD, OECD Guidelines for the Testing of Chemicals: Test No. 416: Two-Generation 

Reproduction Toxicity. 2001. 

78. USEPA, Health Effects Test Guidelines: OPPTS 870.3800: Reproduction and Fertility 

Effects. 1998. 

79. OECD, OECD Guidelines for the Testing of Chemicals: Test No. 421: 

Reproduction/Developmental Toxicity Screening Test. 1995. 

80. USEPA, Health Effects Test Guidelines: OPPTS 870.3550: Reproduction/Developmental 

Toxicity Screening Test. 2000. 

81. USEPA, Health Effects Test Guidelines: OPPTS 870.3650: Combined Repeated Dose 

Toxicity Study with the Reproduction/Developmental Toxicity Screening Test. 2000. 

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82. OECD, OECD Guidelines for the Testing of Chemicals: Test No. 414: Prenatal 

Development Toxicity Study. 2001. 

83. OECD, OECD Guidelines for the Testing of Chemicals: Test No. 406: Skin Sensitization. 

1992. 

84. OECD, OECD Guidelines for the Testing of Chemicals: Test No. 429: Skin Sensitization: 

Local Lymph Node Assay. 2002. 

85. USEPA, Health Effects Test Guidelines: OPPTS 870.2600: Skin Sensitization. 1998. 

86. OECD, OECD Guidelines for the Testing of Chemicals: Test No. 203: Fish, Acute Toxicity 

Test. 1992. 

87. USEPA, Ecological Effects Test Guidelines: OPPTS 850.1075: Fish acute toxicity test, 

freshwater and marine . . 1996. 

88. OECD, OECD Guidelines for the Testing of Chemicals: Test No. 202: Daphnia sp. Acute 

Immobilisation Test. 2004. 

89. USEPA, Ecological Effects Test Guidelines: OPPTS 850.1010: Aquatic Invertebrate 

Acute Toxicity Test, freshwater daphnids. . 1996. 

90. USEPA, Ecological Effects Test Guidelines: OPPTS 850.1035: Mysid acute toxicity test. 

. 1996. 

91. OECD, OECD Guidelines for the Testing of Chemicals: Test No. 201: Alga, Growth 

Inhibition Test. 2006. 

92. USEPA, Ecological Effects Test Guidelines: OPPTS 850.5400: Algal toxicity, Tiers I and 

II. . 1996. 

93. USEPA, Ecological Effects Test Guidelines: OPPTS 850.1085: Fish Acute Toxicity 

Mitigated by Humic Acid. . 1996. 

94. USEPA, Ecological Effects Test Guidelines: OPPTS 850.1025: Oyster acute toxicity test 

(shell deposition). . 1996. 

95. USEPA, Ecological Effects Test Guidelines: OPPTS 850.1045: Penaeid acute toxicity 

test. . 1996. 

96. USEPA, Ecological Effects Test Guidelines: OPPTS 850.1055: Bivalve Acute Toxicity 

Test (embryo larval). . 1996. 

97. USEPA, Ecological Effects Test Guidelines: OPPTS 850.4400: Aquatic plant toxicity test 

using Lemna spp. Tiers I and II. . 1996. 

98. OECD, OECD Guidelines for the Testing of Chemicals: Test No. 301: Ready 

Biodegradability. 1992. 

99. OECD, OECD Guidelines for the Testing of Chemicals: Test No. 310: Ready 

Biodegradability - CO2 in sealed vessels (Headspace Test) 2006. 

100. USEPA, Fate, Transport and Transformation Test Guidelines: OPPTS 835.3110: Ready 

Biodegradability. 1998. 

101.	 Aronson, D., et al., Estimating biodegradation half-lives for use in chemical screening. 

Chemosphere, 2006. 63(11): p. 1953-1960. 

102. OECD, OECD Guidelines for the Testing of Chemicals 303A: Aerobic Sewage 

Treatment: Activated Sludge Units. 2001. 

103. USEPA, Fate, Transport and Transformation Test Guidelines: OPPTS 835.3240 

Simulation Test—Aerobic Sewage Treatment: A. Activated Sludge Units. 2008. 

104. OECD, OECD Guidelines for the Testing of Chemicals: Test No. 309: Aerobic Mineralisation in 

Surface Water - Simulation Biodegradation Test 

2004. 

105. USEPA, Fate, Transport and Transformation Test Guidelines: OPPTS 835.3190 - 

Aerobic Mineralization in Surface Water – Simulation Biodegradation Test. 2008. 

106. OECD, OECD Guidelines for the Testing of Chemicals: Test No. 314: Simulation Tests to Assess 

the Biodegradability of Chemicals Discharged in Wastewater. 2008. 

107. USEPA, Fate, Transport and Transformation Test Guidelines: OPPTS 835.3280 

Simulation Tests to Assess the Primary and Ultimate Biodegradability of Chemicals 

Discharged to Wastewater. 2008. 

108. USEPA, Fate, Transport and Transformation Test Guidelines: OPPTS Harmonized 

Guideline 835.3170 - Shake Flask Die-Away Test 1998. 

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109. USEPA, Fate, Transport and Transformation Test Guidelines: OPPTS Harmonized 

Guideline 835.3180 - Sediment/Water Microcosm Biodegradation Test 1998. 

110. OECD, OECD Guidelines for the Testing of Chemicals: Test No. 305: Bioconcentration: 

Flow-through Fish Test. 1996. 

111. USEPA, Ecological Effects Test Guidelines: OPPTS 850.1710: Oyster BCF. . 1996. 

112. USEPA, Ecological Effects Test Guidelines: OPPTS 850.1730: Fish BCF. . 1996. 

30