Guidance for Reviewing OCSPP 850.2100 Avian Oral Toxicity Studies Conducted with Passerine Birds
Memorandum
August 20, 2014
SUBJECT: Guidance for Reviewing OCSPP 850.2100 Avian Oral Toxicity Studies Conducted with Passerine Birds
FROM: /s/ Donald J. Brady, Director, Environmental Fate and Effects Division (7507P), Office of Pesticide Programs
TO: Environmental Fate and Effects Division (7507P), Office of Pesticide Programs
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Purpose
The purpose of this document is to provide guidance to Environmental Fate and Effects Division (EFED) scientists for reviewing Avian Acute Oral Toxicity Studies conducted with passerine birds under the OCSPP 850.2100 guideline. The information provided in this document may alleviate the need to request a protocol prior to requesting a registrant to conduct a definitive test. EFED scientists may still consider requesting protocols depending on the properties of the pesticide being tested.
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Introduction
As a result of the revised 40 CFR Part 158 pesticide data requirements (July 1, 2008; 72 FR 60957, October 26, 2007), avian acute oral toxicity data (OCSPP Guideline 850.2100) are now required for one passerine species in addition to either a waterfowl or upland game species for all new federal actions involving outdoor end-use pesticides (i.e., Section 3 New Use or New Chemical Registrations, and Registration Reviews). An updated version of the OCSPP 850.2100 guideline, published in January 2012, addresses some issues related to conducting acute oral toxicity tests with passerines. Given the recentness of the passerine data requirement, the Office of Pesticide Programs (OPP) has requested the submission of protocols by registrants/laboratories prior to conducting an avian acute oral test with a passerine species. Several dozen passerine avian acute oral toxicity protocols have been received and reviewed by EFED scientists and discussed within EFED's Terrestrial Biology Technical Team (TBTT). Through this process, additional insights have been gained into passerine-specific issues encountered while conducting the test. This guidance compiles these insights and is intended to facilitate the review and classification of studies with passerines conducted under the OCSPP 850.2100 guideline.
The information in this document may alleviate the need to request a protocol prior to conducting a definitive test. However, EFED scientists may still consider requesting protocols depending on the properties of the pesticide being tested. In addition, much of the information in this guidance document relates to only a few passerine test species. For species that have not commonly been tested under the OCSPP 850.2100 guideline, prior submission of a protocol addressing the husbandry or test conditions of the test species may be useful. Appendix A of this document provides an optional footnote that can be used in the data call-in table to indicate that a protocol does not need to be submitted to EPA for review prior to test initiation if it reflects the recommendations in this and other Agency guidance documents.
This guidance is relevant only to the conduct of the OCSPP 850.2100 avian acute oral toxicity test with a passerine species, and does not address test design or review issues related to the OECD Test Guideline (TG) 223. There is a separate guidance memo (USEPA, 2011) on how to classify studies conducted under the TG 223 guideline. EFED generally recommends against reliance on TG 223 for passerine species since this guideline was not tested or validated for passerines.
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General considerations
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General test guidance
In reference to the general conduct of an acute oral toxicity study with passerine birds, the current OCSPP 850.2100 guideline (January 2012) paragraph (d)(2)(ii) states the following:
"Successful adherence to elements in paragraph (h) of this guideline when testing passerine species may depend upon special provisions regarding husbandry, diet, acclimation of wild-caught birds, handling and age at dosing. For tests conducted with passerine species it is recommended, prior to test initiation, to submit protocols to the regulatory Agency for approval that identify husbandry, dietary, holding and acclimation methods, age at dosing and any other special provisions planned (see in particular references in paragraphs (j)(2) and (j)(4) through (j)(9) of this guideline). To be complete, the protocol should also contain information on all of the items discussed in this guideline."
The information compiled in this document may alleviate the need to request a protocol prior to conducting a definitive test. Please see Section 2 of this document for more information.
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Range-finding, definitive, and limit tests
Regurgitation has been reported by numerous laboratories in limit, range-finding, and definitive toxicity tests conducted with passerines. If regurgitation is observed during these tests, refer to Section 5 of this document as well as the separate guidance memo entitled, Guidance for Use When Regurgitation is Observed in Avian Acute Toxicity Studies with Passerine Species (USEPA, 2012).
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Test standards
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Test organism
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Species
In reference to species choice for an acute oral toxicity study with passerine birds, the current OCSPP 850.2100 guideline (January 2012) paragraph (e)(3)(i) states the following:
"At this time, there is no identified preferred passerine species; examples of potential passerine species that have been used in acute oral testing include the house sparrow (Passer domesticus), zebra finch (Taeniopygia guttata), and red-wing blackbird (Agelaius phoeniceus). Regardless of the passerine species chosen, a protocol should be submitted to the Agency prior to test initiation for review."
There is not yet sufficient information to designate one or more preferred species for passerine acute oral toxicity testing. Considerations concerning the appropriateness of a given species for conduct under the OCSPP 850.2100 guideline include the following:
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the degree of phylogenetic or taxonomic relatedness, and therefore potential biological relevance, between the test species and native passerines;
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the ability to successfully maintain the species in the laboratory;
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possible genetic distinctions among lab colonies due to lack of outbreeding (the OCSPP 850.2100 guideline suggests that test birds come from breeding stocks that are periodically outbred);
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social behavior impacts of care and husbandry (e.g., anti-social behavior such as pecking); and
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sensitivity of the species to chemical exposure as compared to other passerine species, when such information is available.
In addition to this information, any data on the background mortality rate for each passerine species being tested from the testing lab would provide a stronger statistical basis for interpreting effects observed in pesticide-treated birds relative to control birds. EFED is currently analyzing data for passerine sensitivity to determine suitable test species. Submitted background mortality information is being reviewed to determine if the mortality rate for a species in question is less than 1% under laboratory conditions (a standard set by data from bobwhite quail).
Please see Section 2 of this document regarding the need to submit protocols prior to submission of a definitive test to EPA.
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Source
In reference to the use of wild-caught birds for an acute oral toxicity study with passerines, the current OCSPP 850.2100 guideline (January 2012) paragraph (e)(3)(i) states the following:
"The Agency recommends against the use of wild-caught birds. However, where suitable cultured supplies of passerine species may not be available, protocols submitted in advance should detail why wild stock must be used and have the necessary elements identified in this guideline addressed (see paragraph (d)(2)(ii) of this guideline)."(Please see Section 3 of this document for paragraph (d)(2)(ii) of OCSPP 850.2100)
If passerines from a wild stock are used in a study, the reviewer should determine that use of wild-caught birds did not interfere with the quality or outcome of the test.
Since testing with passerines is a relatively recent data requirement, there is little information available on the establishment and performance of breeding stocks of potential test species. Therefore, information on husbandry conditions of the laboratory stock birds (source colony) provided in the study report, including details of history/origin of the colony, genetic composition (the OCSPP 850.2100 guideline suggests periodic outbreeding is needed), and presence of mortality and general health of the colony, can be used by the reviewer to evaluate the appropriateness of the source of birds used for testing. Historical information on the source colony helps ensure reliability of the results associated with any studies conducted with these birds.
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Age, sex, and size
In reference to the age and weight of birds used for acute oral toxicity testing, the current OCSPP 850.2100 guideline (January 2012) paragraph (e)(3)(iii)(B) states the following:
"For a satisfactory test, all upland and waterfowl birds used in a test should be the same age, plus or minus (±) 1 week. Currently, due to culturing and/or husbandry practices of Passeriformes, passerine birds used in a test may not be ± 1 week; however this may change if husbandry practices evolve. More consistent responses may be attained if the range of body weights is no greater than ±10% of the mean body weight for the test population."
In altricial species, it may be difficult to use birds of the same age for testing, especially if breeding colonies do not track individual birds. Based on the information provided in the study report, the reviewer should determine that every attempt was made to use birds of similar age in submitted studies. However, a study with a passerine species should not necessarily be rejected solely based on not meeting the age criterion if the reviewer does not believe it affected the quality or outcome of the study.
The OCSPP 850.2100 guideline recommends that the birds used in the test should be assigned randomly to treatment and control pens, and randomization should be done at the initiation of the acclimation period. The reviewer should pay special attention to instances in which test birds are grouped by body weight, which may compromise the randomization process.
Regarding the sexing of individual birds for testing, the reviewer should assess whether blood samples used to determine the sex of the birds were taken far enough in advance of the test to ensure that the individuals had ample time to recover from the handling stress.
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Health status
Zebra finches or other colonial passerine species may exhibit antisocial behavior, such as pecking, which may be a sign of poor health. In general, unhealthy birds should not be used as test specimens. The reviewer should determine if such behaviors may affect species performance in a manner that would impact the quality or outcome of the study.
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Care and handling
Test birds should be handled only as much as necessary to conform to test procedures. In addition, many passerines are small and could potentially be more sensitive to handling stress than other commonly tested bird species. Reviewers should determine if handling during sexing, weighing, and dosing caused undue stress to the animals or affected the quality or outcome of the study.
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Diet and feeding
In reference to the diet and feeding of passerine birds during an acute oral toxicity test, the current OCSPP 850.2100 guideline (January 2012) paragraph (e)(3)(viii) states the following:
"Passerine species may require more complex diets. Feed should be withheld from all test groups for a minimum of 15 hours prior to administration of the test substance but feed should be available ad libitum during the study. This fasting period should be decreased, as appropriate, for the selected passerine species."
For passerine species, the reviewer should determine whether the duration of fasting was long enough to ensure crop clearance but not so long as to cause undue stress or starvation of the birds. Available information indicates that feed withholding times of approximately 2-3 hours for zebra fiches and 3-4 hours for canaries may be sufficient to allow gut clearance (Twigg et al., 2009; Malone, 1965). If zebra finches or canaries are subjected to longer fasting times than the ones stated above, or if the test is conducted with another passerine species, the reviewer should look for any information provided in the study report on why a specific feed withholding period was chosen as well as assess whether the fasting duration affected the health, behavior, and performance of control birds during the test.
Measurement of food consumption following dosing may be difficult due to the large amount of chaff and spillage associated with consuming seeds in certain types of commercial zebra finch diets. The use of pelleted feed during the acclimation period and test may allow for more accurate food consumption measurements and can aid in the clearing of feed from the digestive tract during fasting periods. The reviewer should assess whether feed spillage affected the evaluation of food consumption of test birds.
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Administration of test substance
In reference to the dosing of passerine species, the current OCSPP 850.2100 guideline (January 2012) paragraphs (e)(4) and (e)(4)(i)(B) state the following, respectively:
"After acclimation under paragraph (e)(3)(iv) of this guideline, feed should be withheld from all test groups for a minimum of 15 hours prior, except potentially for Passeriformes, which may require less time to clear the digestive system of confounding substances, to administration of the test substance."
"Materials with known toxic or emetic properties should not be used. The absence of the crop in many passerine species requires using smaller, softer tubing than with waterfowl."
Please see Section 4(a)(vi) of this document for discussion of feed withholding times prior to dosing of passerine species.
During administration of the test substance, the addition of a nontoxic, colored dye to the test material and use of clean white paper to line the cages can facilitate the observation of regurgitation if it occurs.
Split-dosing with capsules has been used in some studies. If split-dosing is employed, the reviewer should assess whether the timing between doses compromised the intended design of the test, i.e., administration of the test substance as a single oral dose. Considerations for assessing whether the use of split-dosing affected the outcome of the test include:
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the physico-chemical properties of the test substance;
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the gut transit time of the test species; and
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the absorption and metabolism of the test substance based on poultry data from OPP's Health Effects Division (OCSPP Guideline 860.1480).
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Pens
In reference to pen size for passerine species, OCSPP 850.2100 paragraph (e)(7)(ii)(A) states the following:
"Appropriate sized pens for passeriformes potentially may be different to accommodate bird size and social behaviors."
The reviewer should ensure that the size, shape, and design of pens used prior to or during the test did not adversely impact bird health or behavior, while also allowing for natural behaviors of the particular species. Small pens and a lack of perches are associated with disease as well as behavioral issues including dominance (pecking of other birds), anxiety, and depression (Bateson and Feenders, 2010). Furthermore, the placement of perches near the food and/or water supply may lead to contamination from bird droppings (Harding 1999). Longer cages typically offer greater potential for flight as opposed to taller, shorter cages with the same intrinsic volume (Bateson and Feenders, 2010).
If conditions warrant use of pen dividers, the reviewer should determine that the dividers were used consistently among all pens. However, while this may be sufficient for more solitary passerine species, other species may avoid food consumption to be in closer proximity to individuals in an adjacent cage. The reviewer should determine whether the species being used was adversely affected by being placed in a solitary environment (Bateson and Feenders, 2010).
The reviewer should determine whether the cage material contributed to injury of the test species. Small birds, including many passerines, may be injured in cages built with metal wire mesh, whereas plastic cages or those made from plastic mesh, may offer a safer environment (Harding, 1999).
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Environmental conditions
In reference to environmental conditions for passerine birds during an acute oral toxicity test, the current OCSPP 850.2100 guideline (January 2012) paragraph (e)(8) states the following:
"Environmental conditions should be appropriate to the study species. Conditions should be modified as appropriate for passerine species"
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Temperature
The OCSPP 850.2100 guideline (January 2012) paragraph (e)(8)(i) specifies a temperature range of 15-27°C (70-80°F) for mallard duck and northern bobwhite quail. The reviewer should assess whether the temperature range in a study using passerines was appropriate for the species. Fluctuations in temperature can affect metabolism and induce breeding behavior (Bateson and Feenders, 2010).
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Humidity
The OCSPP 850.2100 guideline (January 2012) paragraph (e)(8)(ii) recommends a relative humidity of between 45 and 70% for mallard duck and northern bobwhite quail, but notes that relative humidity is not as critical as some other variables. For passerine studies, the reviewer should assess whether the relative humidity was maintained within a range sufficient to provide effective disease prevention and to minimize changes in behavior and reproductive status. For canaries, breeding conditions are typically associated with high humidity (i.e., 60 to 80%) (Couteel, 2003). For zebra finch, increases in humidity can induce reproductive behaviors (Bateson and Feenders, 2010). Lower humidity can minimize the development of pathogens (Couteel, 2003).
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Lighting and photoperiod
In reference to photoperiod for passerine birds during an acute oral toxicity test, the current OCSPP 850.2100 guideline (January 2012) paragraph (e)(8)(iii) states the following:
"A photoperiod of 10 hours light and 14 hours dark is recommended in order to prevent birds from coming into reproductive condition for upland and waterfowl species. However, this may potentially be different for passerine species and should be adjusted as appropriate to ensure birds are not entering reproductive condition."
The reviewer should note whether the photoperiod used in an acute oral toxicity test with a passerine species was appropriate to avoid inducing reproductive condition in the test species. For canaries, 8 to 10 hours of light is typically used for non-breeding periods (Couteel, 2003). For zebra finches, 8 hours of light and 16 hours of darkness help to reduce breeding behavior (Hayne, 1995). The reviewer should also take note of whether a transition period (approximately 15-20 minutes), which minimizes stress on birds, was used between light and dark cycles.
The reviewer should also assess whether the type of lighting used in the study caused stress to the birds. Low frequency fluorescent light (i.e., below 150 Hz) may aggravate birds due to their ability to perceive the flicker of fluorescent lighting in this range (Bateson and Feenders, 2010).
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Regurgitation
In reference to regurgitation during an acute oral toxicity test, the current OCSPP 850.2100 guideline (January 2012) paragraph (e)(8)(iii) states the following:
"Birds are monitored closely for the first 60 to 120 minutes after dosing. Any regurgitation should be noted. Additional observations of test birds are made a minimum of three times on the day of dosing and at least daily (where feasible, twice daily observations are recommended) throughout the remainder of the test period."
The OCSPP 850.2100 guideline recommends that birds be monitored closely for the first 60 to 120 minutes after dosing. Monitoring for regurgitation of the administered dose is particularly important since regurgitation has been reported by numerous laboratories in limit, range-finding, and definitive toxicity tests conducted with passerines. The addition of a nontoxic, colored dye to the test material and use of clean white paper to line the cages can facilitate the observation of regurgitation.
If regurgitation is observed in a range-finding and/or definitive test, then the EFED regurgitation guidance memo, Guidance for Use When Regurgitation is Observed in Avian Acute Toxicity Studies with Passerine Species, should be consulted (USEPA, 2012). According to this memo, replacement of regurgitating birds with new birds is not recommended under any circumstance as this strategy may eliminate individuals that are more sensitive to the test substance. Eliciting a regurgitation response can be interpreted as a potentially adverse effect of the test material. If birds cannot eat, they are unlikely to survive or reproduce.
As described in the EFED regurgitation guidance memo, options for proceeding after regurgitation is observed in a range-finding and/or definitive test include using a different dosing method (e.g., using gelatin capsules, reducing dosage volume) and repeating the study with a different passerine species.
If regurgitation remains a potentially confounding factor, a further option described in the memo is to conduct a 5-day subacute dietary toxicity test following the OCSPP 850.2200 guideline and using the species that presented the strongest response (e.g., regurgitation) in the attempted acute oral toxicity test(s). The EFED regurgitation guidance memo recommends that a study protocol be submitted to EPA prior to initiating a subacute dietary test with a passerine species. Food consumption should be carefully measured so that an ingested dose can be calculated.
The reviewer should assess whether a dietary study can be considered acceptable in lieu of an acute oral study based on the registrant's ability to demonstrate that the steps presented in the EFED regurgitation guidance memo and summarized here were followed. In making this decision, the reviewer should pay close attention to the rationale for the testing approach, including data detailing regurgitation and other outcomes observed in all attempted acute oral studies, and outcomes of any alternatives that were explored.
Alternatively, if regurgitation is observed in an acute oral study in a dose-dependent pattern with no regurgitation at the lowest dose tested, the NOAEC for regurgitation can be used as the regulatory endpoint.
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References
The following references should be consulted for additional background material on this test guideline:
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Bateson, M., Feenders, G. 2010. The use of passerine bird species in laboratory research: Implications of basic biology for husbandry and welfare. ILAR Journal 51(4): 394-408
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Couteel, P.T. 2003. Veterinary aspects of breeding management in captive passerines. Seminars in Avian and Exotic Pet Medicine 12(1):3-10.
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Harding, C.F. 1999. Husbandry and care of passerine birds. Poultry and Avian Biology Reviews 10(2): 79-83.
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Hayne, M.D. 1995. Guidelines for housing excruciatingly happy zebra finches. Lab Animal June 1995.
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Malone, C.R. 1965. Dispersal of plankton: rate of food passage in mallard ducks. Journal of Wildlife Management 29(2): 529-533.
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Twigg, L.E., Lowe, T.J., Taylor, C.M., Calver, M.C., Martin, G.R., Stevenson, C., How, R. 2009. The potential of seed-eating birds to spread viable seeds of weeds and other undesirable plants. Austral Ecology 34: 805-820.
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USEPA. 2012. Guidance for use when regurgitation is observed in avian acute toxicity studies with passerine species. Memorandum to all managers and staff of the Environmental Fate and Effects Division, Office of Pesticide Programs, United States Environmental Protection Agency. Dated April 9, 2012.
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USEPA. 2011. Guidance for Classifying Studies Conducted Using the OECD Test Guideline 223 (TG223) (Acute Avian Oral Sequential Dose Study). Memorandum to all managers and staff of the Environmental Fate and Effects Division, Office of Pesticide Programs, United States Environmental Protection Agency. Dated November 9, 2011.
EFED Passerine Guidance Team
Elyssa Arnold
Scott Glaberman
Andrew Sayer
Robin SternbergTBTT Co-Chairs
Catherine Aubee
Hannah Yingling -
Appendix A. Optional Data Call-In Footnote.
The following language can be used in the data call-in table to indicate that a protocol does not need to be submitted to EPA for review prior to test initiation if it reflects the recommendations in this and other Agency guidance documents:
"The OCSPP 850.2100 guideline currently recommends the submission of a protocol for EPA review prior to initiation of tests conducted with passerine species. Data submitters are encouraged to consider the recommendations contained in relevant EPA reference documents (i.e., OCSPP 850.2100, EFED Guidance for Reviewing OCSPP 850.2100 Avian Oral Toxicity Studies Conducted with Passerine Birds, EFED Guidance for Use when Regurgitation is Observed in Avian Acute Toxicity Studies with Passerine Species) when preparing test protocols. A protocol does not need to be submitted to EPA for review prior to test initiation if it reflects these recommendations. If a data submitter elects to submit a protocol to EPA, in order to facilitate the review process, any aspects of a proposed study design that differ from this guidance should be noted and accompanied by a descriptive rationale which addresses why they are not expected to adversely impact the quality of the resulting study."