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PROCESS FOR ESTABLISHING & IMPLEMENTING ALTERNATIVE 

APPROACHES TO TRADITIONAL IN VIVO ACUTE TOXICITY 

STUDIES

12/15/2014

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 NRC 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 1583(40CFR158) in support of pesticide registration. In 

May 2011, the agency sought expert advice and input from its the 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 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 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 

 

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
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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, inhalation toxicity, along with skin and eye 

irritation and skin sensitization (often referred to as the “six pack studies”)5. 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 or other alternative methods in OPP’s regulatory decision 

making with respect to acute toxicity tests. These components include: 

1. Evaluation 

2. Transition

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 

methods (for example, methods that have been ring-tested). Methods still in development or 

poorly characterized with respect to performance require further research and evaluation that is 

 

5 This document does not apply to in vitro or alternative methods used for other purposes such as quantitative 

structure activity relationships, mechanistic or pharmacokinetic data used to support mode of action, adverse 

outcome pathways, or pharmacokinetic models. 
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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 Phase of Alternative Method(s)

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

used for 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., ASTM International6) 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;

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)7process; 

 

6

http://www.astm.org/ABOUT/full_overview.html

7

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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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 confidential business information (CBI) would be concealed. 

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 

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

8

.

Throughout the course of an evaluation, the agency will engage diverse stakeholders including 

states, non-government organizations (NGOs) and other federal agencies. The agency will seek 

peer review of the evaluation when necessary.

OPP’s goal for the evaluation phase is to end with a determination that an alternative method(s)

is suitable for regulatory purposes. Such a determination would come in the form of an EPA 

publication of a draft policy document (e.g., waiver guidance) for public comment on the

proposed approach method and the appropriateness of the alternative method for a stated 

 

8

http://www2.epa.gov/pesticide-registration/label-review-manual
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regulatory purpose, including the establishment of a correlation between method results and a 

toxic effect similar to those measured by current guidelines. The draft policy document would 

also discuss any requirements the agency may have with respect to needing in vivo data. For 

example, depending on the method(s) proposed, a certain amount of in vivo oral acute toxicity

data may be necessary to support ecological risk assessments. At this point, with the publication 

of the draft policy, the agency will provide notice of its expectation and timeframes for reporting 

under section 6(a)(2) of FIFRA.

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 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. Transition Phase

In this phase, OPP would accept data generated from the alternative method(s), which were

conducted following the draft policy document, and submitted along with corresponding data 

from its in vivo method counterpart (or citation to previously submitted in vivo studies). This 

would provide the broader registrant community with the opportunity to practice conducting the 

method(s) and the agency additional experience in assessing the data. During the transition, 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 and international regulators on use of the alternative method.

C. Implementation
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After addressing any potential issues identified through public comments, a final science policy

will be completed and released. Subsequently, data generated from the accepted alternative 

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

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 through the development of 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 
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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.

9

 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 

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. 

 

9 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.