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UNITED STATES ENVIRONMENTAL PROTECTION AGENCY

WASHINGTON, D.C. 20460

OFFICE OF CHEMICAL SAFETY AND

 POLLUTION PREVENTION

MEMORANDUM

Date: February 2, 2016

SUBJECT: Response to Public Comments: Process for Establishing and Implementing 

Alternative Approaches to Traditional In Vivo Acute Toxicity Studies

PC Code: NA DP Barcode: xxx

Decision No.: NA Registration No.: NA

Petition No.: NA Regulatory Action: NA

Risk Assessment Type: NA Case No.: NA

TXR No.: NA CAS No.: NA

MRID No.: NA 40 CFR: 40 CFR§180.342

FROM: Evisabel Craig

Chris Schlosser

Health Effects Division (7509P)

THROUGH: Anna Lowit, Senior Scientist

Health Effects Division (7509P)

TO: Jennifer McLain, Deputy Director

Antimicrobials Division (xxx)

In early 2015, the EPA published a draft document delineating the proposed process for establishing and 

implementing alternative approaches to traditional in vivo acute toxicity studies. Members of the private 

sector, non-governmental organizations and the general public had the opportunity to provide comments 

to further strengthen and improve the draft document. The EPA has taken into consideration all 

comments received and made appropriate changes to the revised document. The following 

memorandum constitutes a formal response to the aforementioned comments.
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Overall Positive Comments

During the public comment period, EPA received an array of comments from the private sector, nongovernmental organizations and private individuals. Overall, the majority of comments received by EPA 

were positive and supportive of the proposed guidance along with the direction that the agency is headed 

with respect to expanding the implementation of alternative approaches. A few comments asked for 

clarifications or revisions of the document, and responses to these are summarized below. Comments 

have been summarized and responses organized based on the section of the document they pertain to.

Collaboration

ACC, CLA and NC3R – suggest that EPA work with other federal agencies (FDA, CPSC, etc.) and 

OECD in the development of test guidelines for any specific alternative method being proposed to 

ensure consistency and harmonization. 

EPA is already working with other federal, state and international agencies and will continue to do so to 

ensure consistency and harmonization of test guidelines, and has included language to that effect on 

page 7 of the Process guidance.

Syngenta – state that the proposed approach needs to be part of the NAFTA regulatory cooperation 

process to be successful. 

The Agency is aware of the importance of international cooperation. OPP has already been collaborating 

with PMRA on the use of alternative approaches for the acute 6-pack battery. However, this guidance is 

drafted in the context of FIFRA section 6(a)(2), which is a US-specific statute. Interested parties are 

encouraged to contact Canada PMRA regarding their efforts on alternative methods to in vivo toxicity

testing.

Other Alternative Methods

Biocides Panel - Acknowledge that there are existing alternative approaches to the six studies 

requirement including waiving and bridging, as discussed in EPA’s Guidance for Waiving or 

Bridging of Mammalian Acute Toxicity Tests for Pesticides and Pesticide Products (Acute Oral, 

Acute Dermal, Acute Inhalation, Primary Eye, Primary Dermal, and Dermal Sensitization) (dated 

March 1, 2012).1 

A citation of the aforementioned bridging guidance document has been added.

CLA, PETA/PCRM – would like to have in silico methods included, not just in vitro. 

A reference to in silico methods has been included.

 

1 Available at http://www2.epa.gov/sites/production/files/documents/acute-data-waiver-guidance.pdf.
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Evaluation Phase

ARDF and Syngenta – Text places too much emphasis on comparing new alternative methods to 

current “in vivo” standards. The draft guidance should do more to: 1. move away from apical

endpoints and towards pathways-based approaches; 2. incorporate more guidance on integrated 

testing strategies-how they could be developed and applied to the chemicals under consideration.

As guidelines for in vivo studies are referenced in the current data regulations at 40 CFR part 158, any

developed alternative approaches will most often be compared to these standards already in place to 

determine the suitability of the alternative approach in replacing the required studies, except in cases 

where the alternative approach has been shown to be more predictive of human effects than the in vivo

laboratory animal study. These comparisons do not preclude use of adverse outcome pathways as 

scientific foundation for developing or evaluating alternative approaches for use as stand-alone 

replacements or as part of an integrated approach on testing and assessment (IATA) process or 

integrated testing strategy (ITS). 

With respect to developing integrated testing strategies, the agency expects IATAs/ITS to be developed 

on a case-by-case basis for various endpoints or effects; this level of detail is beyond the scope of the 

“Process For Evaluating & Implementing Alternative Approaches to Traditional In Vivo Acute Toxicity

Studies for FIFRA Regulatory Use” document.

Syngenta – Provide clarification on who is responsible for data generation, is it voluntary? Also, it is 

not clear in the current document how something is determined to be useful for regulatory purposes. 

In the near future, the Agency’s expectation is that data to be used will likely come from existing 

sources of data (e.g. industry or in-house research and development). Data may come from any source 

and participation in this initiative is voluntary. However, the Agency notes that limited participation by

industry may lead to slower adoption of alternatives methods and/or narrow adoption with respect to 

chemical space of applicability. Considerations to determine whether data are useful for regulatory

purposes are discussed in section IIA (e.g. the degree to which a method has been validated by an 

international validation process).

CLA - suggest EPA consider test methods which have been vetted through an international validation 

process (i.e., OECD) in order to ensure consistency in test methods and data interpretation, 

particularly when conducting any evaluation correlating in vitro to in vivo results. 

The Agency notes that OECD does not validate methods, per se. The role of the OECD is develop 

guidelines from well-established methods. There are validation organizations around the world who

work collaboratively to achieve validation 

(https://ntp.niehs.nih.gov/pubhealth/evalatm/iccvam/international-partnerships/index.html). The issue

identified in this comment is addressed in point 1 under “considerations during the evaluation phase” in 

the draft guidance. The Agency concurs that well-established methods will be the focus during the 

evaluation phase.
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PETA/PCRM - suggest the EPA add language stating that the agency should be consulted prior to the 

data being generated to ensure the study is conducted in a manner that meets any applicable 

standards for evaluating the method. Add language clarifying suitability of methods for all classes vs. 

class-specific methods, and if class-specific methods are acceptable. 

The Agency expects to use data that has already been generated. For new data generation, EPA 

encourages and welcome open dialogue. The Agency often reviews study protocols and invites the 

submission of protocols. The suitability of a method, whether class-specific or across many classes, will 

be assessed during the evaluation phase as stipulated in point 3 of the evaluation phase section.

Transition Phase

ARDF – Do not extend public comments into the transition phase, work with state and international 

regulators early in the process. Discourage use of “in vivo” studies as early as possible. 

The previously titled transition phase has been renamed “the proposal and public comment phase”. As 

discussed in IIA, throughout the course of an evaluation, the Agency will engage diverse stakeholders 

including states, NGOs, international partners and other federal agencies.

Syngenta and PETA/PCRM - No length of time or volume of data required was identified for the 

transition phase. Concerns were raised regarding cost for potential registrants who must utilize more 

than one test method for one endpoint for an indeterminate period of time. Suggestions include that 

methods that already have been substantially evaluated (for example at OECD) and/or undergone 

peer review, be subject to an abbreviated transition phase. Also, it is suggested that the EPA set up 

some form of outreach (ideally including incentives such as fast-track review) that would encourage 

companies to submit data so that, with a greater volume of submittals, the transition period could 

hopefully be shortened.

The previously titled transition phase has been renamed “the proposal and public comment phase”. The 

Agency will work diligently after the public comment period closes to issue a revised policy in a timely

manner to ensure rapid application. Typical comment periods are 30-90 days but extensions may be 

granted whenever appropriate. Upon finalization of the policy, it will be the choice of the submitter to 

use the alternative approach or the traditional in vivo test. The statement encouraging data submission 

during the comment period seemed to confuse readers so it has been removed. However, consistent with 

EPA’s regulations, companies may submit data generated with an alternative method at any time. 

EPA appreciates the suggestion of incentives and encourages the commenters to provide specific ideas 

to the agency.

Implementation Phase

PETA/PCRM – Provide discussion on the phase-out of in vivo methods in the event that one or more 

acute in vitro methods are deemed acceptable by the agency. We would hope that a “test on animals 

only as a last resort” policy could be developed.
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Our 2013 document, Guiding Principles for Data Requirements, states that “The goal is to ensure there 

is sufficient information to reliably support registration decisions that are protective of public health and 

the environment while avoiding the generation and evaluation of data that do not materially influence 

the scientific certainty of a regulatory decision. It is important to only require data that adequately 

inform regulatory decision making and thereby avoid unnecessary use of time and resources, data 

generation costs, and animal testing.”

6a2

CLA - Clarity is also sought regarding the timing of if and when FIFRA §6(a)(2) reporting will be 

required for a new method, and whether the requirement for reporting would apply to specific 

individual methods or to multiple approaches combined in an integrated fashion.

As stated in section C (implementation phase) and section III (page 7), data generated with an 

alternative method for which the Agency has issued a final policy detailing how the data generated with 

the method (or a set of methods) should be interpreted with respect to EPA labeling requirements will be 

reportable under section 6(a)(2). In the evaluation phase 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.

2

 Through its evaluation process, the Agency will 

determine whether the alternative method(s) may meet OPP’s regulatory needs. Whether the 

requirement for reporting would apply to specific individual methods or to multiple approaches 

combined in an integrated fashion will depend on the particular policy at hand. The critical factor is 

whether the Agency has determined how to interpret the data with respect to EPA’s regulatory criteria. 

If the interpretation is dependent on using results from a set of methods combined in an integrated 

fashion, only such data are reportable. However, if the Agency issues criteria for interpreting data from 

individual methods with respect to EPA’s regulatory criteria, whether or not those individual methods 

may also be combined in an integrated fashion, data from the individual methods would be reportable. 

Syngenta - Should an agreement of not changing already approved label/registration, like EDSP 

program used, be included for participants evaluating alternative assays? We’re interested in the 

comments on PRF of effects. Currently, our criteria state that, when used as a screen, in vitro results 

are not referable. In this case referability is entirely dependent on the in vivo data. When used as a 

stand-alone assay an in vitro test would be referable. We need to understand exactly how the EPA’s 

comments would affect this. 

FIFRA section 6(a)(2) provides that “if 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 is required irrespective of 

whether the study is in vitro, in vivo, used as a screen or as a stand-alone assay. The 2009 “Endocrine 

Disruptor Screening Program; Policies and Procedures for Initial Screening” document, section K states 

 

2 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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that “Under FIFRA section 6(a)(2), pesticide product registrants are required to submit adverse effects 

information about their products to the EPA”, which corresponds to the discussion in “Process for 

Evaluating & Implementing Alternative Approaches to Traditional In Vivo Acute Toxicity Studies for 

FIFRA Regulatory Use.”

ARDF – Establish a framework for “systematic review” instead of a “weight-of-evidence” approach 

in order to provide a scientific method for data integration. 

In recent years, the National Academy of Sciences has encouraged the Agency to move towards 

systematic review processes to enhance the transparency of scientific literature reviews that support 

chemical-specific risk assessments to inform regulatory decision making3. According to the National 

Research Council, systematic reviews “have several common elements: transparent and explicitly

documented methods, consistent and critical evaluation of all relevant literature, application of a 

standardized approach for grading the strength of evidence, and clear and consistent summative 

language. ” EPA’s Office of Chemical Safety and Pollution Prevention is currently developing 

systematic review policies and procedures. Weight of evidence analysis is the Agency’s process for 

evaluating the strength of the scientific evidence along with integrating such evidence as a component of 

systematic review. 

ARDF and NC3R – Is a safe harbor from 6A2 possible? Is it possible to elaborate on how this will be 

done - are there plans to establish a framework? (In considering data for 6a2).

EPA believes that it would be inconsistent with its FIFRA mandate to create a “safe harbor” that would 

allow registrants to withhold information “if the registrant knows, or reasonably should know, that if the 

information should prove to be correct, EPA might regard the information alone or in conjunction with 

other information about the pesticide as raising concerns about the continued registration of a product or 

about the appropriate terms and conditions of registration of a product prevent unreasonable adverse 

effects of pesticides.” (§ 159.195(a)). However, EPA emphasizes that information submitted pursuant to 

section 6(a)(2) will not necessarily dictate regulatory decisions or label amendments. 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. 

General Comments

PETA/PCRM – On page 3, footnote 5 indicates that the document does not apply to in vitro or 

alternative methods used to develop adverse outcome pathways. We are not sure what this refers to as 

the assays involved in evaluating skin sensitization, for example, i.e., DPRA, KeratinoSens, and 

hCLAT, all measure events along the AOP for this endpoint. We would ask that the OPP provide 

more clarification on this footnote.

Footnote 5 has been removed.

 

3 NRC 2011. “Review of the Environmental Protection Agency’s Draft IRIS Assessment of Formaldehyde” ; 

NRC 2014. “Review of EPA's Integrated Risk Information System (IRIS) Process”