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PROCESS FOR EVALUATING & IMPLEMENTING ALTERNATIVE 

APPROACHES TO TRADITIONAL IN VIVO ACUTE TOXICITY 

STUDIES FOR FIFRA REGULATORY USE

2/4/2016

Office of Pesticide Programs 

U.S. Environmental Protection Agency

Washington DC, 20460
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I. BACKGROUND

EPA’s Office of Pesticide Programs (OPP) has developed a strategic vision1for implementing 

the 2007 National Research Council (NRC) of the National Academy of Science report on 

Toxicity Testing in the 21st Century2. This strategic vision has multiple components involving a 

combination of computational and predictive modeling approaches, in vitro techniques, and 

limited, targeted in vivo testing, to supplement or replace the existing toxicity tests required in 40 

C.F.R. part 1583in support of pesticide registration. In May 2011, the agency sought expert 

advice and input from its Federal Insecticide, Fungicide and Rodenticide Act (FIFRA) Scientific

Advisory Panel (SAP) on OPP's vision and scientific issues associated with adopting integrated 

approaches to testing and assessment (IATA) and strategies for using new computational and 

molecular tools. The Panel concurred that OPP’s strategic vision clearly articulated a sound 

scientific basis for utilizing the NRC’s recommendations regarding “21st Century Toxicity

Testing” in a manner that makes the risk assessment process more efficient and informative. 

And, the Panel commented that utilizing data from rapidly and inexpensively performed in silico

and in vitro technologies appears to be the most logical way to address the need to improve 

efficiency.

As part of the implementation of the strategic vision, OPP developed a document called 

“GUIDING PRINCIPLES for DATA REQUIREMENTS4” which describes some of the key

principles in moving towards smarter testing approaches. In addition to seeking alternative 

testing strategies, in March 2012 the Agency also published a “Guidance for Waiving or 

Bridging of Mammalian Acute Toxicity Tests for Pesticides and Pesticide Products”

5 which 

provides a foundation for reducing redundant or unnecessary testing and thus reducing animal 

use. In 2013, OPP’s policy on the “Use of An Alternative Testing Framework for Classification 

of Eye Irritation Potential of EPA Pesticide Products” was another early step toward this vision. 

As science is rapidly advancing and new technologies are emerging, including some alternatives 

 

1

http://www.epa.gov/pesticides/science/testing-assessment.html#pesticide

2

http://dels.nas.edu/resources/static-assets/materials-based-on-reports/reports-in-brief/Toxicity_Testing_final.pdf

3

http://www.ecfr.gov/cgi-bin/text-idx?tpl=/ecfrbrowse/Title40/40cfr158_main_02.tpl

4

http://www.epa.gov/pesticides/regulating/data-require-guide-principle.pdf

5

http://www2.epa.gov/sites/production/files/documents/acute-data-waiver-guidance.pdf
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assays with Organization for Economic Co-operation and Development (OECD) guidelines,

there is increasing potential for the use of alternative methods in regulatory risk assessment. OPP 

plans to continue to expand its acceptance of alternative methods for acute toxicity testing. The 

agency’s goals for alternative testing approaches include: assessing a broader range and 

potentially more human-relevant adverse effects, faster and less expensive data generation and 

review, and reducing use of laboratory animals in regulatory testing. 

This document describes a transparent, stepwise process for evaluating and implementing 

alternative methods of testing for acute oral, dermal, and inhalation toxicity, along with skin and 

eye irritation and skin sensitization (often collectively referred to as the “six pack studies”).

Included in this document is a discussion of the three major phases of this process, and the 

implications for reporting information under section 6(a)(2) of FIFRA. Having such a process 

and a clear articulation of the related reporting requirements addresses a challenge that has 

previously been associated with adopting alternative methods. Successfully putting this process

into place will require an open dialogue with stakeholders, other regulatory organizations, and 

the scientific community. 

II. STEPWISE PROCESS FOR EVALUATING & IMPLEMENTING 

ALTERNATIVE METHODS

The following text describes the major components of a transparent, stepwise process for

evaluating and implementing in vitro, in silico, or other alternative methods of determining the 

acute toxicity of pesticides and pesticide inerts for use in OPP’s regulatory decision making. 

These components include: 

1. Evaluation 

2. Proposal & Public Comment

3. Implementation

OPP expects the alterative methodologies considered appropriate for this stepwise process to be 

well established by the scientific community with respect to reproducibility and domain of 

applicability; these methods may or may not have OECD guidelines. This process is focused on 

the utility of methods for regulatory application and is thus designed for well-established 
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methods (for example, methods that are documented to be reliable and transferable from one 

laboratory to another). For in vitro methods without a guideline, OPP recommends following 

OECD Guidance Document 211 for describing non-guideline in vitro test methods6. Methods 

still in development or poorly characterized with respect to performance require further research 

and evaluation that is out of the scope of this process. The process does not prescribe a specific 

timeframe for each stage as the analysis required at each stage may differ among various 

methods.

A. Evaluation of An Alternative Method

The purpose of the evaluation phase is to determine whether an alternative method(s) could be 

used for OPP regulatory purposes. The first step in the process is to identify an alternative 

method or system (array) of methods that have the potential to be used in place of a traditional 

acute toxicity test. Organizations or coalitions of stakeholders interested in pursuing an 

evaluation of an alternative method for regulatory applicability are welcome to contact OPP for a 

consultation. Alternatively, EPA may identify promising alternatives. 

The objectives of the evaluation are to determine the reliability of the alternative method,

evaluate the method in the context of regulatory use, and identify uncertainties. Some 

considerations during the evaluation phase are: 

1) the degree to which the method has been vetted by an international validation process 

(e.g., International Cooperation on Alternative Test Methods, ICATM, or its member 

countries; ASTM International7) and/or the availability of an OECD guideline;

2) whether the method is appropriate for single chemicals, mixtures, or both; 

3) the types of chemicals which can be used with the method i.e., the domain of 

applicability;

 

6

http://www.oecd.org/officialdocuments/publicdisplaydocumentpdf/?cote=ENV/JM/MONO(2014)35&doclanguage=

en

7

http://www.astm.org/ABOUT/full_overview.html
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4) whether the method can be used alone as a replacement for an in vivo study or the method 

must be used in combination with others as part of an integrated approach on testing and 

assessment (IATA)8process or integrated testing strategy; 

5) additional strengths and uncertainties associated with the assay such as feasibility, 

sensitivity and specificity, ease of interpretation, costs, etc.; and,

6) whether information derived from the assay(s) is suitable for labeling and regulatory

decisions (including whether the method(s) provide information relevant to risk 

assessment in addition to information for labeling decisions).

Once a proposed alternative method(s) is accepted as a suitable candidate, the evaluation process 

would consist of a number of steps. First, existing data generated using the alternative method(s) 

will be collated and organized (for example in a spreadsheet or database). The data could be 

previously generated or generated explicitly for the purpose of informing an evaluation of 

regulatory applicability. This data compilation could be accomplished in various ways. For 

example, a coalition of interested companies could work with a neutral party to collect and 

aggregate data so that aggregate results could be reported without disclosure of confidential 

business information (CBI), or data could be compiled from the open scientific literature.

Once the data are compiled, OPP anticipates performing an analysis, through which OPP and 

interested stakeholders would cooperatively evaluate the utility of these data in the context of 

regulatory decision making under FIFRA (e.g., approval of uses, application methods, labeling 

requirements for signal words and personal protective equipment). Part of this analysis will 

include comparison with existing data generated through EPA’s approved test methods (i.e., 

consistent with existing test guidance). This analysis would consider how EPA would use data 

 

8

IATA (Integrated Approach to Testing and Assessment): Integrate existing knowledge based on classes of 

chemicals with the results of biochemical and cellular assays, computational predictive methods, exposure studies, 

and other sources of information to identify requirements for targeted testing or develop assessment conclusions. In 

some cases, the application of IATA could lead to the refinement, reduction, and/or replacement of selected 

conventional tests (e.g., animal toxicity tests). IATA also has the potential to further enhance the understanding of 

mode/mechanism of action including the consideration of relevant adverse outcome pathways (AOPs) that provide 

biological linkages between molecular initiating events to adverse outcomes in individual organisms and populations 

that are the bases for human health and ecological risk assessments (NAFTA, 2012). Extracted from 

http://www.oecd.org/officialdocuments/publicdisplaydocumentpdf/?cote=ENV/JM/MONO(2014)19&doclanguage=

en
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from the alternative method to classify products for acute toxicity labeling (Categories I – IV)

9

.

Note that there is not a specific threshold volume of data deemed sufficient for an analysis. 

Whether enough data is available to make a decision regarding suitability for regulatory purpose 

is a factor of multiple considerations such as how well the data set represents the pesticide 

chemical domain and the variability of the data set. Throughout the course of an evaluation, the 

agency will engage diverse stakeholders including states, non-government organizations 

(NGOs), international partners and other federal agencies. The agency will seek peer review of 

the evaluation where appropriate. In general, during the evaluation phase, the Agency does not 

intend to rely on compiled data to make regulatory decisions.

When EPA determines that an alternative method appears suitable for determining a pesticide’s 

acute toxicity category for FIFRA regulatory purposes, EPA intends to publish it for public 

review and comment (e.g., as a draft waiver guidance or draft alternative testing guideline). The 

Agency anticipates that such a determination would come in the form of an EPA publication of a 

draft policy document for public comment on the proposed approach and the appropriateness of 

the alternative method for assessing one or more types of acute toxicity, including the 

establishment of a correlation between the alternative method’s results and an acute toxic effect 

similar to those measured by current guidelines. The draft policy document would also discuss 

any limitations or additional data needs the agency may have. If the Agency identifies in vivo

data needs that cannot be fulfilled with the alternative method, those exceptions will be identified 

in the policy document for that particular method (e.g., in vivo oral acute toxicity data on active 

ingredients may be needed to support ecological risk assessments even if in vitro oral acute 

toxicity alternative methods are accepted for formulations). 

EPA anticipates that the draft policy would also include the Agency’s expectations and 

timeframes for reporting under section 6(a)(2) of FIFRA adverse effects information specific to 

the alternative method.

 

9

http://www2.epa.gov/pesticide-registration/label-review-manual
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In some cases, the method(s) may be found not useful for regulatory purposes. Sometimes, areas 

for additional method development that have the potential to make the method more useful for 

regulatory purposes may be identified. For example, some alternative assays are developed for 

use with a single chemical; because acute toxicity testing is conducted on both single technical 

ingredients and formulations (i.e., mixtures), additional testing or method development may be 

necessary to evaluate the applicability of a particular method for mixtures (i.e., formulations). In 

other cases, the agency may recommend modifications to the test methods or the overall 

alternative approach.

B. Proposal & Public Comment

During this phase, the public will have the opportunity to provide comments on the draft policy

document. The agency will use the public comments to revise the draft policy where appropriate. 

The agency is also aware of the importance of national and international harmonization and will 

continue to work with state, federal and international regulators on use of the alternative method.

C. Implementation

After addressing any potential issues identified through public comments, EPA intends to issue a 

final policy regarding the alternative approach. Subsequently, data generated from the accepted 

alternative approach may be submitted to fulfill the requirement of its standard in vivo

counterpart, as provided in 40 CFR 158.70 and 158.80. Data generated with the method will be 

reportable under section 6(a)(2), if relevant, regardless of when the data were generated (see 

Section III below).

III. APPLICABILITY OF FIFRA 6(a)(2) REQUIREMENTS

FIFRA section 6(a)(2) provides that “[i]f at any time after the registration of a pesticide the 

registrant has additional factual information regarding unreasonable adverse effects on the 

environment of the pesticide, he shall submit such information to the Administrator.” This does 

not mean that the reported information will necessarily lead to regulatory decisions or label 
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amendments: Information is reportable under section 6(a)(2) if it simply “pertain[s] or relate[s]

to unreasonable adverse effects on the environment; it does not have to indicate, establish, or 

prove the existence of such effects.” 43 Fed.Reg. 37611, 37612 (Aug. 28, 1978) (emphasis in the 

original). EPA always considers new information submitted pursuant to section 6(a)(2) in the 

context of the full range of data previously submitted in support of the registration, applying a 

weight of the evidence approach to the multiple lines of available evidence. Likewise, OPP will 

review any information arising from a new test method in relation to other pertinent information 

about the pesticide. 

The process described above for evaluating and implementing alternative methods for in vivo

acute toxicity studies includes comparing existing data previously generated by EPA’s test 

methods with data generated using an alternative test methodology (e.g., OECD Skin Irritation). 

Part of the focus of this comparison is simply to understand both the degree to which there is 

correlation between the standard in vivo and the alternative methods and the degree to which 

determinations regarding adverse effects can be made with the alternative method(s). Pesticide 

registrants cooperating in this evaluation would already have submitted to EPA any information 

required to be reported under section 6(a)(2) arising from previous testing generated using EPA’s 

test guidelines (i.e., in vivo test results) for registered pesticides. As a result, the scope of the 

new information arising from the evaluation that could potentially be reportable under section 

6(a)(2) would be limited to information arising from the alternative methods or from any new 

correlation established between the alternative methodologies and the methods upon which EPA 

currently relies. 

At the beginning of the process, the results of the alternative testing methods are unlikely to be of 

interest to EPA, because the degree to which those results correspond to EPA’s regulatory

criteria is unknown.

10

 Through its evaluation process, the agency will determine whether the 

alternative method(s) may meet OPP’s regulatory needs. If the evaluation is successful in 

 

10 Even in the absence of a reasonably reliable correlation between the OECD methods and EPA’s methods, it is 

possible – though unlikely – that testing under the OECD methods could produce information reportable under 

FIFRA section 6(a)(2). For example, if a product or class of products that routinely show low levels of toxicity in 

[some EPA method] were to consistently show unexpectedly high levels of toxicity under an alternative method, a 

reasonable person might view the divergence in results as raising questions about which method adequately reflects 

the true risk of the product(s). It would be important for EPA to have such information, even if there was little 

short-term likelihood of either a regulatory response or a scientific explanation for the divergence. 
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developing a reasonably reliable correlation between the different test methods, then the results 

of alternative test methods would be more likely to be of regulatory utility to EPA. For example, 

if a reasonably reliable correlation between the alternative assay and EPA’s skin irritation criteria 

were developed, registrants who possess or know of information that in the context of the new 

policy, would raise concerns about the continued registration of a product or about the 

appropriate terms and conditions of the registration of a product, would be required to report that 

information to EPA (§ 159.195(a)). This would apply to data previously developed using 

existing test methods (e.g., the in vivo methods), as well as to data generated during initial 

research of new test methods. It would apply to all registrants i.e., those who are not cooperating 

in the analysis, as well as cooperating registrants.

Information submitted pursuant to section 6(a)(2) will not necessarily dictate regulatory

decisions or label amendments. For the case of acute toxicity studies, an in vitro test result 

corresponding to a higher hazard category than that associated with an acceptable in vivo test 

result would not necessarily require a label change nor would an in vitro result corresponding to 

a lower hazard category necessarily lead to a label change. EPA will consider reported data in 

the context of the full range of data previously submitted in support of the registration, applying 

a weight of the evidence approach.