Chemical Safety

 and Pollution Prevention

 (7101)

EPA No. tbd

Draft of December 17, 2014

Endocrine Disruptor 

Screening Program Test 

Guidelines

OCSPP 890.2100:

Avian Two-generation 

Toxicity Test in the 

Japanese Quail

PUBLIC REVIEW DRAFT
Public Review Draft of December 17, 2014

Page 2 of 30

NOTICE

This guideline is one of a series of test guidelines established by the United States Environmental 

Protection Agency’s Office of Chemical Safety and Pollution Prevention (OCSPP) for use in testing 

pesticides and chemical substances to develop data for submission to the Agency under the Toxic 

Substances Control Act (TSCA) (15 U.S.C. 2601, et seq.), the Federal Insecticide, Fungicide and 

Rodenticide Act (FIFRA) (7 U.S.C. 136, et seq.), and section 408 of the Federal Food, Drug and 

Cosmetic Act (FFDCA) (21 U.S.C. 346a). Prior to April 22, 2010, OCSPP was known as the Office of 

Prevention, Pesticides and Toxic Substances (OPPTS). To distinguish these guidelines from guidelines 

issued by other organizations, the numbering convention adopted in 1994 specifically included OPPTS 

as part of the guideline’s number. Any test guidelines developed after April 22, 2010 will use the new 

acronym (OCSPP) in their title.

The OCSPP test guidelines serve as a compendium of accepted scientific methodologies and 

protocols that are intended to provide data to inform regulatory decisions under TSCA, FIFRA and/or 

FFDCA. This document provides guidance for conducting the test, and is also used by EPA, the public 

and the companies that are subject to data submission requirements under TSCA, FIFRA and/or the 

FFDCA. As a guidance document, these guidelines are not binding on either EPA or any outside 

parties, and the EPA may depart from the guidelines where circumstances warrant and without prior 

notice. At places in this guidance, the Agency uses the word “should.” In this guidance, the use of 

“should” with regard to an action means that the action is recommended rather than mandatory. The 

procedures contained in this guideline are strongly recommended for generating the data that are the 

subject of the guideline, but EPA recognizes that departures may be appropriate in specific situations. 

You may propose alternatives to the recommendations described in these guidelines, and the Agency 

will assess them for appropriateness on a case-by-case basis. 

For additional information about these test guidelines and to access these guidelines 

electronically, please go to http://www.epa.gov/ocspp and select “Test Methods & Guidelines” on the 

left side navigation menu. You may also access the guidelines in http://www.regulations.gov grouped 

by Series under Docket ID #s: EPA-HQ-OPPT-2009-0150 through EPA-HQ-OPPT-2009-0159, and 

EPA-HQ-OPPT-2009-0576.

DRAFT DOCUMENT DISCLAIMER: This draft document is distributed solely for the purpose of 

external review. It has not been formally disseminated by the EPA and should not be construed to 

represent any Agency determination or policy. The information correction process under the Agency’s 

Information Quality Guidelines does not apply until this document is formally disseminated by the EPA 

in its final form. This draft document should only be cited or quoted in the context of providing 

comments. 
Public Review Draft of December 17, 2014

Page 3 of 30

OPPTS 890.2100: Avian Two-generation Toxicity Test in the Japanese Quail.

(a) Scope.

(1) Applicability. This guideline is intended to meet testing requirements of the Toxic 

Substances Control Act (TSCA) (15 U.S.C. 2601, et seq.), the Federal Insecticide, Fungicide, and 

Rodenticide Act (FIFRA) (7 U.S.C. 136, et seq.), and the Federal Food, Drug, and Cosmetic Act 

(FFDCA) (21 U.S.C. 346a).

(2) Background. The Endocrine Disruptor Screening Program (EDSP) reflects a twotiered approach to implement the statutory testing requirements of FFDCA section 408(p) (21 

U.S.C. 346a). In general, EPA intends to use the data collected under the EDSP, along with other 

information, to determine if a pesticide chemical, or other substances, may pose a risk to human 

health or the environment due to disruption of the endocrine system.

This test guideline is intended to be used for a definitive test in characterizing the potential 

adverse apical effects posed to avian fauna from a putative vertebrate hormone active substance. 

This test, if triggered, would follow other guidelines in the OCSPP 890 series that make up the 

full screening battery under the EDSP to assess the possible adverse consequences that occur 

from the potential to interact with the estrogen, androgen, or thyroid hormone systems identified 

in Tier 1 screening. The determination whether to invoke a Japanese Quail Two-Generation Test 

will be made on a weight-of-evidence basis taking into account data from the Tier 1 assays and 

other scientifically relevant information available. The fact that a substance may interact with a 

hormone system, however, does not mean that when the substance is used, it will cause adverse 

effects in humans or ecological systems.

Chemicals that go through Tier 1 screening and are found to have the potential to interact with the 

estrogen, androgen, or thyroid hormone systems will proceed to the next stage of the EDSP where 

EPA will determine which, if any, of the Tier 2 tests are necessary based on the available data. 

Tier 2 testing, such as with this test, is designed to identify any adverse endocrine-related effects 

caused by the substance in the relevant taxonomic group, and to establish a quantitative 

relationship between the dose and that endocrine effect.

(3) Source. OCSPP developed this through a process of harmonization with the 

Organization for Economic Cooperation and Development (OECD) (Ref. 1-2).

(b) Purpose. 

The purpose of this guideline is to describe a laboratory testing procedure for evaluating 

the impact of pesticides and other chemicals upon avian development, growth, and reproduction 

through two generations. Data collected from such tests may be used in the assessment of risks to 

birds exposed to chemicals. These data from a two-generation test are principally intended to 

evaluate the adverse consequence of exposure to a “possible” endocrine disruptor to the 

reproductive viability of F1 birds, but may also provide information to confirm a chemical as an 

endocrine disruptor. The test is designed for dietary exposure, but also may include exposure 

through drinking water if more appropriate for the chemical.
Public Review Draft of December 17, 2014

Page 4 of 30

(c) Introduction. 

This guideline describes a two-generation test. The test monitors health and reproductive 

parameters in Japanese quail (Coturnix japonica) adults and their progeny, including viability of 

second generation offspring. A range of fitness, biochemical, behavioral, and histological 

endpoints are included to identify and characterize potential adverse effects which may be 

incident to endocrine perturbations.

(d) General Experimental Design.

Principle of the Test. Birds in the parent (F0) generation are hatched from eggs obtained 

from an in-house culture or a recognized lineage from a supplier and are paired at 4 weeks of age, 

and acclimated for 2 weeks prior to beginning chemically treated diet. Dosing begins in the F1 

generation at hatch. Parameters for adult (F0 and F1), egg production (F0 and F1), and offspring 

health (F1 and F2) are evaluated by making statistical comparisons between treated groups and 

the control group. The no observed effect concentration (NOEC) and the lowest observed effect 

concentration (LOEC) are determined for health, fitness and endocrine parameters.

(e) Description of the Method.

(1) Test animals. The test species is the Japanese quail (Coturnix japonica). Birds 

should be drawn from a strain that has an adult body weight in the range of 200-250g and that 

generally meet the performance criteria for the test (see Table 5).

Japanese quail generally start laying eggs at approximately six weeks of age. Once egg laying has 

begun it will take about two to three weeks for birds to reach full egg production. Eggs are 

generally fertile when birds are eight weeks of age.

Birds used in the test should appear healthy and be free of abnormalities or injuries that may 

affect test results. Birds should not receive any dietary medications beginning one week prior to 

the start of the test and continuing until the test is terminated. All F0 birds used in a test should 

be from the same hatch.

(2) Husbandry. Temperature, ventilation, and light controlled facilities are needed 

throughout the test. Recommended ventilation is about 8 to 15 air changes per hour. Sound and 

vibrations or other disturbances in the rooms should be kept to a minimum to minimize stress.

Artificial lighting should approximate the daylight visual spectrum and be automatically 

controlled. The photoperiod for adult birds, from the start of acclimation onwards, and the chicks 

is 16 or 17 hours light and 7 or 8 hours of darkness. Birds should be exposed to light intensity of 

at least 10 lux, measured at the level of the feeder. The light intensity is dictated by the amount 

required for daily observations. Light intensity that is too high will encourage aggressive 

behavior.
Public Review Draft of December 17, 2014

Page 5 of 30

Incubators and hatchers, preferably with automatic temperature and humidity controls and an eggturning device, are recommended. In addition, suitable equipment is required to maintain stored 

eggs within the temperature and humidity ranges specified.

Suitable pens of appropriate size for adults and for chicks are required. Wire pens with slanting 

floors and egg-catchers or other measures to prevent breakage of eggs are recommended for 

adults. Pens for both adults and chicks should preferably be of stainless or galvanized steel or 

other inert materials. When using wire meshing for the floors, the wire mesh should be of a size 

sufficient to prevent foot injury (trauma or pododermatitis) but large enough to allow excreta to 

drop through. Measures should be taken to minimize spillage of diet (e.g., covering the food 

troughs with a wire grid).

Birds should be placed in breeder pens at four weeks of age, and then housed in opposite sex pairs 

(one male/one female). The parental cages should be distributed in such a way that crosscontamination and positional effects are avoided. Pen mate aggression can be a problem and 

measures must be taken to minimize injuries, stress and mortality resulting from such aggression. 

If separation becomes necessary, pairs should be reintroduced daily to maintain fertility.

For rearing chicks, the use of rearing pens with thermostatically controlled warm compartments 

or cages, which are free of drafts and have a radiator (e.g., ceramics), is recommended. Sufficient 

space for feeding and drinking must be provided, especially during the first week after hatching, 

to promote the weaker animals accessing food and water resources. Chicks should be identified 

individually or by pen of origin. They may be housed in groups of approximately equal number, 

by treatment group. The optimal number of chicks per pen depends on pen size. Birds must be 

separated into pairs (one male/one female) when 4 weeks old after sex has been determined.

Guidance for housing conditions is provided in Table 1.

Table 1. Housing Conditions

Age

(week)

Temperature

(

oC)

Relative humidity

(%)

Minimum floor area

(cm2/bird)

1 35-38 40-80 50

2 30-35 40-80 75

3-4 23-27 40-80 100

>4 16-27 40-80 625

Note: The acceptability of housing conditions of adults will be evaluated on the basis of results 

of reproductive performance.

(3) Feeding. Diet and drinking water are provided ad libitum. The diet should be 

described in the study report and should meet the specific nutrient requirements of Japanese quail. 

The caloric contents of the diet must be reported. If the same diet is used for chicks and adults, 

extra calcium should be added to the adult diet to accommodate egg production. During the 

treatment period, birds are fed basal diet mixed with the test substance at specified concentrations.

Guidance for recommended nutritional values is provided in Table 2.
Public Review Draft of December 17, 2014

Page 6 of 30

Table 2. Recommended Nutritional Values

Nutrient

Adults (> 4weeks)

Recommended range (%)

Chicks

Recommended range (%)

Crude protein 27 to 29 27 to 30

Crude fibre 3.5 to 5.0 3.0 to 6.0

Crude fat 2.5 to 7.0 5.5 to 7.5

Calcium 2.6 to 3.6 0.75 to 1.2

Phosphorous 0.9 to 1.1 0.6 to 1.0

(f) Procedure.

(1) Test Animals. Care should be taken to ensure that birds are drinking water as they 

transition from group pens to the breeder pens. An acclimation period of at least two weeks is 

required for the F0 generation prior to the start of treatment. The test begins with the

administration of treated diet (i.e. when birds are 6 weeks old). Breeding pairs should be 

maintained in a 16:8 or 17:7 light:dark photoperiod for the duration of the study. Onset of egg 

laying can take place during the acclimation period.

Prior to the start of treatment, individual F0 generation birds are weighed, sexed (preferably, 

using genetic techniques), and impartially allocated to pens and treatment groups. Birds are 

paired by known lineage such that males from one line are paired with females from another line. 

Pairs should be observed for signs of excessive aggression, and if present the sexes separated 

except for periodic conjugal visits. A translucent separator wall may be used for this purpose in 

each pen. Otherwise, the pairs will be housed together in the same pen. When together, signs of 

excessive aggression should be monitored and the males and females separated if injury is 

imminent. The number of pairs allocated to each group (for example, 16) should be sufficient to 

ensure that there are 12 breeding pairs in the F0 control group that have successfully produced 

eggs at the end of the treatment period.

Eggs are incubated from F0 post-treatment birds beginning week 5 post-treatment (11 weeks posthatch) to examine fertility. Broods from the post-treatment 6th and 7th week are used to establish 

the first generation offspring (F1). Up to 6 eggs are randomly selected from the 6th and 7th week 

production (12 and 13 weeks post-hatch) and incubated to hatch for each parental pair, but a 

minimum of 40 eggs must be set for hatching for each test level. If during the 6th and 7th posttreatment weeks, a treatment level does not produce at least 40 eggs, that level is dropped from 

further analysis in the test. At approximately two weeks of age F1 control and treatment chicks 

are genetically sexed. At four weeks of age, F1 control and treatment group birds are weighed 

and randomly allocated to pens (optimally 12) by pairs based on genetic sex, with male offspring 

from odd numbered parental pens being paired with females from even numbered parental pens 

and even numbered pen males being paired with odd numbered females. The F1 generation 

breeding birds are maintained until 12 to 13 weeks post-hatch. Eggs collected from F1 pairs at 

onset of egg production through 11 weeks post-hatch will be used to evaluate fertility. Eggs 

collected from weeks 12 to 13 post-hatch will be used to establish the F2 generation, optimally 6 

eggs will be collected from each F1 breeding pair again as done for the F0 breeding pairs. F2 
Public Review Draft of December 17, 2014

Page 7 of 30

hatchling survival will be observed at least through day 14. Observations can be extended to 

examine timing of sexual maturity in the F2 generation if warranted.

(2) Dietary Concentrations. The concentration of the test substance in the diet is 

expressed as weight of the test substance per unit weight of diet with known water content: 

mg/kg of diet (or weight of test substance per volume of water when birds are exposed through 

drinking water).

Dietary concentrations of the test substance should be chosen on the basis of toxicological data 

from a range-finding study or other preceding avian tests (e.g., OCSPP 850.2300). Information 

may also be gained from tests with rodents or other mammals. The highest concentration tested 

should be a level that is expected to reveal significant effects on adult health or reproductive 

parameters. If no significant effects are expected, then the highest concentration should exceed 

the expected concentration of the chemical in the environment or approach the currently 

recommended limit for avian reproduction studies of 5,000 ppm. However, the highest dose 

administered should not cause mortality or other severe signs of parental toxicity that will 

preclude the evaluation of reproductive parameters. The lowest concentration tested should be 

chosen so that no effects are seen on adult health or reproductive parameters. Intermediate 

concentrations should be geometrically spaced between the highest and lowest doses. A 

minimum of four concentrations and a control are required for the definitive test.

A range-finding test is recommended to determine the concentrations to be used in the definitive 

test. If a range-finding test is performed, a minimum six week dietary exposure period may 

provide information helpful in determining appropriate test concentrations for the definitive test 

and to evaluate any potential avoidance effects.

(3) Preparation and monitoring of the test diet. To prepare the test diets, the 

appropriate amounts of test substance are mixed into the diet. The mixing method should be 

developed so as to obtain a homogeneous distribution of test substance in the diet. The use of a 

premix is advisable.

If necessary, a vehicle of negligible toxicity (e.g., food grade corn oil, water, etc.) may be used to 

ensure a uniform distribution. Acetone may be used to dissolve a test substance, provided it is 

allowed to evaporate before feeding the diet to the birds. The amount of vehicle used should be 

as low as possible and should not exceed 2% by weight of the diet. When used, a constant 

amount of vehicle should be added to each test group and the control group diet, in order to keep 

the caloric value of the diet equal between dosage groups.

The frequency of diet preparation should be chosen so that degradation or volatilization of the test 

substance in the diet does not allow the actual concentration to fall below 80% relative to initial 

concentration. The frequency of diet renewal should not be more than once a day and not less 

than once a week. Residual feed should be visibly inspected for mold and if acceptable, weighed 

and mixed with fresh feed supplied to the birds. It may be desirable to keep all feed frozen until 

use.
Public Review Draft of December 17, 2014

Page 8 of 30

Stability of the chemical in feed must be analyzed under typical test conditions prior to the start of 

the test or in parallel with the range-finding study. Stability data from previous (mammalian) 

feeding studies may be used as guidance, if available. Stability should be verified during the 

definitive reproduction test. In order to check stability of the test substance under test conditions, 

feed should be sampled and analyzed from the feed hoppers at the end of the first feeding period, 

i.e., before diet in the feeding bowls is renewed, and again at the end of the last feeding period. A 

sufficient number of samples should be taken into account for variability.

Homogeneity of the test substance in the diet may be evaluated prior to the test or samples for 

homogeneity measurements may be taken at the first mix prepared for the study.

To verify test concentrations, samples of diets fed to the birds should be taken every time new 

diet is mixed during the treatment period to allow measurement of the actual concentration of the 

test substance.

Both F0 and F1 generations are maintained on treated diet until test termination. All F2 chicks 

are maintained on untreated diet.

(4) Measurements and Observations.

(i) Adults. Prior to start of the treatment period, egg production of all bird pairs from the 

F0 generation (including potential replacements) should be recorded. Pairs that do not lay eggs 

by the 2nd week post-treatment (8 weeks post-hatch) should be excluded from further study.

During the treatment period, the parental birds are observed daily to detect any overt signs of 

toxicity or other clinical signs. The following observations are to be performed on adults during 

the study:

Clinical signs of toxicity and health conditions should be evaluated at least once daily, during the 

acclimation and treatment periods. Observations should include mortality and clinical sign of 

toxicity such as lethargy, depression, wing droop, ruffled feathers, lacrimation, feces, etc. Any 

injuries sustained and subsequent treatment should also be recorded. Clearly moribund 

individuals or those otherwise in severe distress should be immediately euthanized.

 Food consumption (per pair) should be recorded at least weekly as often as food is 

replaced in the feeders. Any apparent food spillage should be noted.

 Body weights should be determined at least at pairing and at the end of the 

treatment period. There should be no significant difference in mean body weight 

between test groups at the start of the treatment period. If there is a significant 

difference between test groups, birds should be again randomized.

 Observations of secondary sex characteristics (e.g., male/rusty or female/mottled 

plumage, production of the first egg or first foam), cloacal length, should be 

recorded beginning on the 4th week and every week after until distinctly 
Public Review Draft of December 17, 2014

Page 9 of 30

dimorphic traits are expressed (F0 and F1 generations). If plumage is not 

distinctly “male” or “female”, note the plumage as “ambiguous”.

(ii) Offspring – eggs. Eggs are collected from all pens at least once daily, numbered 

according to pen of origin and stored; broken eggs are numbered, recorded and then discarded. 

The total number of eggs laid per pen should be accounted for at the end of the study.

The eggs are stored in a cold storage facility, for a maximum of 2 weeks, prior to setting in the 

incubator (see Table 3). Cool storage prevents embryo development and aids in synchronizing 

development. Before placing the eggs in the incubator, they are candled to check for 

abnormalities and fine cracks that can be identified only by candling. After removing all cracked 

and abnormal eggs, the remaining eggs as appropriate are equilibrated to room temperature and 

set in the incubator. All cracked and abnormal eggs should be recorded.

Table 3. Conditions for egg storage, incubation and hatching.

Temperature (oC) Relative humidity (%) Turning

Storage 13-16 55-75 Optional

Incubation 37.5-37.8 50-70 Yes

Hatching 37-37.5 70-75 No

Eggs produced by F0 and F1 parents will be collected weekly to test egg shell thickness. Eggs 

collected from F0 pairs during the 5th, 6th, and 7th post-treatment week will be incubated to 

assess embryo viability (embryonic day (ED) 8 and 15) and establish the next generation (6th and 

7th post-treatment weeks). Eggs collected from the F1 generation during post-pairing weeks 1 

through 4 will also be incubated through embryonic day 15 to evaluate fertility at ED8 and ED15. 

Incubation is best performed when eggs are set in an incubator with temperature and humidity 

control and an automatic turning device. From the second day of incubation onwards, eggs 

should be turned at least three times per day. If turned by hand, eggs should be turned an odd 

number of times a day.

(iii) Egg shell thickness/strength. During the study, one egg per pen is collected from 

odd number pens in odd numbered weeks and from even numbered pens in even numbered weeks 

to evaluate eggshell strength and thickness. If F0 egg production is limited during the 5th (<5 

eggs), 6th, and 7th weeks post-treatment (<7 eggs total for both weeks), forego egg collection for 

shell measurements during those weeks.

Eggshell strength is measured in Newtons using a strength tester. The egg is placed on its side on 

the test stand so the compression head will contact the egg at the equator. Then to determine 

eggshell thickness, each egg will be cut open around the equator and washed out; subsequently 

the shells are left to dry with the membrane intact for at least 48 hours at room temperature. Shell 

thickness is measured at a minimum of four points around the girth using a calibrated micrometer. 

Eggshells are measured to at least 0.01 mm and the mean value calculated per egg. Eggs other 

than those collected for production of the next generation (post-hatch weeks 12 and 13), and those 
Public Review Draft of December 17, 2014

Page 10 of 30

collected for eggshell thickness and strength (one egg/pen biweekly), are incubated to examine 

fertility and embryo viability and then discarded prior to hatch. Egg yolks from eggs broken 

during shell measurements can be used for biochemical analyses.

(iv) Incubated eggs. Eggs from F0 will be collected during the 11th, 12th, and 13th posthatching weeks. Eggs collected during the 11th week will be candled to estimate fertility and 

embryo viability on ED 8 and 15, respectively. On ED 15, eggs will be opened and embryo 

weight will be recorded. Tissues will be collected as described below from two ED 15 chicks per 

pen. All eggs collected from F1 pairs will be collected and those not removed because of being 

cracked/abnormal or removed for eggshell measurements will be incubated as previously 

indicated (section 4.iii).

Only eggs (up to 6 per pair) collected from weeks 12 and 13 will be allowed to hatch to establish 

the F1 generations. F1 birds are maintained on treated diet from hatch until test termination. All 

F2 offspring will be maintained on untreated diet and survival through day 14 is recorded.

The onset of sexual maturity (egg laying or foam production) or absence thereof will be recorded 

for F1 and may be recorded for F2 hatchlings if observations are extended. The timing of sexual 

maturity will be considered when egg or foam production and appropriate behavioral responses 

occurs in 90% of the control (i.e., birds maintained on an untreated diet) and appropriate 

behavioral responses among paired birds. If time to sexual maturation is monitored in the F2 

generation, F2 observations will extend to the period required for sexual maturation of the control 

group. This should suffice to adequately assess potential pubertal delays among treated birds.

Fertility and embryo viability are checked by removing the eggs from the incubator and candling 

them. All incubated eggs are candled for fertility after 8 days of incubation (ED 8) and again for 

viability of embryos approximately 2 days before hatching (ED 15). During candling, eggs 

should not be allowed to cool to room temperature, since this may delay embryonic development. 

After hatching, chicks should be dry before they are taken out of the hatcher. Those chicks that 

have not hatched within approximately 24 hours of the majority of chicks hatching, should be 

considered unhatched. No assistance should be given to chicks during hatching.

(v) Egg Parameters. During the treatment periods, the following egg information is 

recorded:

 Eggs laid

 Eggs cracked

 Eggs broken

 Description of all egg abnormalities

 Eggs set

 Eggshell strength

 Eggshell thickness

 Fertile eggs 

 Infertile eggs

 Fertility (ED8)

 Embryo viability (ED15)
Public Review Draft of December 17, 2014

Page 11 of 30

 Eggs that hatch

 Eggs that do not hatch 

Typical values for reproductive parameters in Japanese quail are shown in Table 4.

Table 4. Typical values for reproductive parameters in Japanese quail.

Parameter Units Normal JQ values

Egg laid/hen #/day 0.66 to 0.89

Eggs cracked/broken* % of eggs laid 0 to 10%

Eggs set #/pair

Eggshell strength Per egg (Newtons)

Eggshell thickness Mean/egg (0.01 mm) 0.19 to 0.22

Fertile eggs (ED8) %eggs set

Viable embryos (ED15) % of eggs set 85 to 96%

Hatchability % hatching of viable embryos 70 to 98%

Hatchling survival to day 14 % of hatched eggs that survive to 14 days 85 to 97%

* Describe any egg abnormalities.

If reproductive parameters of control birds do not meet these values, test procedures and 

husbandry conditions should be checked for potential problems.

If a pair is removed during the study due to death, injury, etc., data from all preceding weeks are 

evaluated. If data from a pen in which mortality occurred or a bird was injured are used in study 

results, this should be justified.

(vi) Chicks. The following observations on F1 and F2 chicks are recorded during the 

treatment periods:

 Day of hatch

 Hatching mortality

 Hatchling morphology and/or abnormalities

 Chick body weight at hatching

 Clinical signs of toxicity/disease

 Number of 14-day-old surviving chicks

 Chick body weight at 14 days after hatching

 Time to reproductive maturation (F2*)

 Phenotypic sex

 Genetic sex (F0*, F1, F2)

 Mating behavior (F0,F1,F2*)

*optional
Public Review Draft of December 17, 2014

Page 12 of 30

(5) Performance Criteria. The minimum performance criteria that should be achieved 

are shown in Table 5.

Table 5. Performance Criteria.

Parameter Acceptable level

Test Substance concentrations in diet or 

water Losses < initial concentrations

Morbidity/Mortality during test duration <10%

Parental mortality during acclimation <3%

Eggs laid/pair >5/wk

Viable embryos >80% of eggs set

Normal hatchlings >85% of viable embryos

Cracked eggs < 10%

Successful egg production > 12 breeding pairs in the F0 control 

group 

(6) Pathology.

(i) Adults. All adults that survive to the end of the treatment period are killed as 

humanely as possible. All adult birds are to undergo necropsy and gross examination. Adult birds 

that die, or are killed during the course of the treatment period, will be subjected to the same 

procedures. A more thorough examination of histopathology and biochemical endpoints will be 

examined in at least 8 males and 8 females from each treatment group (including control 

animals). Trunk blood will be collected for biochemical analyses. The left thyroid gland is to be 

stored for glandular thyroxine (T4) content. Organs including kidneys, liver, spleen, adrenal 

glands, reproductive organs and associated structures, cloacal gland, bursa, and right thyroid 

gland should be excised and preserved in Davidson's fixative or a suitable alternative for 

histopathological examination. A detailed description of excision, fixation, and tissue preparation 

is provided in Appendix I (pages 3-41) of this document. Any morphologic abnormality should 

be recorded at the time of necropsy. Fixed organ (or group of organs) weight will be collected 

when organs are removed for histopathology.

(ii) Chicks (14 days). The F2 generation will be terminated 14 days post-hatch, unless 

timing of reproductive maturity or reproductive behaviors were observed to have been altered in 

the F1 generation (at which point, F2 offspring will be maintained until the onset of reproductive 

maturity). Histopathological and biochemical samples will be collected from at least 8 male and 

8 female birds from each treatment group (including controls). Organs collected for 

histopathology will be identical to those collected from adult animals. Reproductive organs from 
Public Review Draft of December 17, 2014

Page 13 of 30

14 day old chicks will be fixed in situ. A detailed description of excision, fixation, and tissue 

preparation is provided in Appendix I (pages 93- 114) of this document. 

(iii) Embryos. For each mating pair, two late-stage embryos (ED 15, 2 days prior to 

hatch) that are not allowed to hatch (i.e., from 5

th week post-treatment eggs) are to undergo 

necropsy and tissues preserved for histopathology as described above for 14 day old chicks. 

Thyroid glands will be collected from two late-stage embryos per pair for biochemical analyses. 

Birds (adults or chicks) which are in severe distress will be killed in extremis. If one member of a 

pair dies or is killed in extremis during the treatment period, the other member of the pair is killed 

as well. Tissue samples collected from the control and the high dose groups in the F1 generation 

will be examined to assure that endocrine effects are neither masked nor imitated by systemic 

toxicity. Tissues examined will include liver, kidney, adrenal, reproductive tissues (gonads, 

accessory sex tissues, reproductive glands and ducts) and thyroid gland (Table 6). If no signs of 

overt general toxicity are observed, future histopathological examination will be limited to 

endocrine organs and tissues (Tables 7 and 8). 

Table 6: Samples examined general toxicity from control and high dose of F1 generation.

Treatment Number Tissues Purpose

Control 8M / 8F

Liver, kidney, adrenal, 

thyroid, reproductive 

tissues

Examine tissues for 

potential systemic (nonendocrine related) 

toxicity

High dose 8M / 8F

Table 7. Samples examined for endocrine-specific effects from all generations in control, low, 

and high dose

Generation Treatment Sample size Tissues Purpose

F0 Control 8M / 8F

Ovary, oviduct, shell 

gland, cloacal glands, 

bursa (F)

Testis, cloacal glands, 

bursa epididymis/vas 

(when present; M)

To examine treatmentrelated effects on the 

endocrine system of 

Japanese quail exposed to 

a putative endocrine 

disrupting compound.

Low 8M / 8F

low mid 8M / 8F

high mid 8M / 8F

high 8M / 8F

F1 Control* 8M / 8F

Low 8M / 8F

low mid 8M / 8F

high mid 8M / 8F

high 8M / 8F

F2† Control 8M / 8F

Low 8M / 8F

low mid 8M / 8F

high mid 8M / 8F

high 8M / 8F

* May be 14 day old chicks or 6 week old juvenile/adults
Public Review Draft of December 17, 2014

Page 14 of 30

Table 8. Samples examined for endocrine and developmental effects on embryonic day (ED) 15 

in ovo quail

Generation Treatment Sample size Tissues Purpose

F1 Control 5

Liver, kidney, adrenal, 

thyroid, reproductive 

structures, gonad

Examine potential 

developmental effects of 

exposure to putative 

endocrine disrupting 

compound in ovo.

Low 5

low mid 5

high mid 5

high 5

F2 Control 5

Low 5

low mid 5

high mid 5

high 5

(7) Biochemical/Endocrine Measurements and Observations. Plasma treated with 

heparin or EDTA and is preferred over serum for hormone measurements, due to greater 

methodological recoveries. A minimum volume of 600 μl is required for thyroid (250 μl of 

unextracted plasma) and steroid hormone analyses (350 μl of diethyl ether extracted plasma).

(i) Thyroid hormone measurements. Measurements of thyroid function include thyroid 

gland weight, plasma thyroxine (T4), glandular T4 content, and thyroid histology/histopathology. 

Samples will be collected from embryonic and adult life stages. Thyroxine content in eggs 

collected from post-treatment week 5 incubated to ED 15, can be used in lieu of embryo glandular 

T4 measurements. After assessing late embryo viability two days prior to hatch, the embryo is 

removed from the shell and necropsied. Thyroid glands are dissected. Fixed thyroid glands for 

histology will be collected from two embryos and unfixed thyroid glands for biochemistry will be 

collected from two embryos for each male-female pair. In addition, during necropsy of the adult 

Japanese quail, thyroid glands are dissected and the left thyroid gland will be used for glandular 

T4 analysis and right gland (preserved in situ) used for histological assessment as above. A 

detailed description of blood, thyroid gland, and yolk preparation for thyroid hormone analysis 

and immunoassay results are provided in Appendix 7 of this document. If alternative sample 

preparation or analytical methods are used, validation must be adequately demonstrated.

(ii) Steroid hormone measurements. Steroid hormones shall be analyzed in at least 8 

male and 8 female quail blood samples collected from each treatment group (including controls) 

at necropsy, and from egg yolks resulting from eggshell thickness/strength measures. A detailed 

description of blood, thyroid gland, and yolk preparation for steroid hormone analysis and 

immunoassay results are provided in Appendix 7 of this document. If alternative sample 

preparation or analytical methods are used, validation must be adequately demonstrated.

(iii) Vitellogenin measurements. Vitellogenin is a protein that is generally controlled by 

estrogen. It is present in females of oviparous species during egg development. It is not 

expressed in normal males due to lack of estrogen, however in males that have been exposed to 

estrogenic contaminants, it is expected that vitellogenesis will be stimulated. Vitellogenin may be 

measured from adults plasma samples collected at necropsy, when appropriate, to evaluate 

estrogenic potential of a test chemical.
Public Review Draft of December 17, 2014

Page 15 of 30

(8) Behavioral Measurements. Initiation of normal male mating behavior is important 

for successful reproduction. Mating behavioral observations will be conducted, preferably 

beginning at the onset of maturation, on males of the F1 (F2, optional) generation. Trials shall be 

performed to assess mating behaviors including time to mount attempts, number of mount 

attempts, and number of successful cloacal contacts. Behavioral observations shall be conducted 

on naive birds raised singly or in same-sex pairs. At sexual maturity (i.e., egg laying in females 

and foam production in males), 7 naive male-female pairs will be introduced and behavior will be 

observed during three minute trials for three consecutive days. Trials may be truncated after 

successful mating or if bird aggression becomes an issue. At the conclusion of the trial, birds will 

be returned to their single or same-sex housing. The same individuals shall be re-introduced for 

three consecutive days or until successful mating is observed. If behavioral observations of the 

F1 generation suggest a treatment-related effect, observations may be extended to the F2 

generation.

Handling-related disturbance during introduction and separation of birds involved in behavior 

observation trials may perturb egg production, and therefore, observational data should be 

collected from distinct pairs not used in collection of other data endpoints (e.g., egg production, 

fertility, etc.).

(9) Data Collection and Reporting

(i) Treatment of Results. Data are collected in a spreadsheet form and transfered to and 

analyzed with a statistical software package. Statistical analyses of the data should preferably 

follow procedures described in the document Current Approaches in the Statistical Analysis of 

Ecotoxicity Data: A Guidance to Application (OECD 2006).

All adult health data should be recorded per individual bird (food consumption per pair). Since 

the F0 parental pair (or pen) is the primary statistical unit and the F1 parental pair (or pen) is the 

secondary unit, all reproductive data should be related by lineage. The raw data values should be 

reported by pen to provide sufficient detail for an independent statistical analysis.

Measurements of endpoints will be evaluated by comparing values obtained from birds in treated 

groups with values obtained from control birds.

Moreover, mortality, clinical observations or pathological findings may be evaluated statistically 

to verify any apparent relationship between adult and treatment or when effects may be borderline 

(e.g.; pathological findings).

(ii) Statistical Treatment. Prior to using parametric statistical analysis, variables should 

be checked for normality. If the assumptions of parametric statistics are violated, data should be 

transformed and retested. Transformations should follow commonly accepted, scientific 

approaches. For example, proportions should be arcsine transformed, skewed data should be 

retested after applying log or negative log transformation. If data are still not normally 

distributed, EPA recommends examining data for possible alternate (e.g., binomial) distributions. 

Monotonicity and homogeneity of variance can be tested within the statistical model. Only after 

other statistical approaches have been exhausted does EPA suggested an analysis of ranked data.
Public Review Draft of December 17, 2014

Page 16 of 30

Statistical comparisons of endpoints between generations, beyond noting the NOEC/LOEC for a 

particular endpoint for each generation, are not desired as the experimental design and timing of 

sample collections vary by generation.

 Response variable calculation. For all equations in this guideline, the index j = 1 

to the total number of pens per treatment group (typically 16).

 Adult body weight gain. The change in adult body weight (males and females are 

tracked separately) between test initiation and test termination,

j bw , for pen j is 

the measure used in this test guideline to evaluate the inhibitory effects of the test

substance on adult growth. The change in adult body weight (male or female), 

assuming one bird of each sex per pen, is calculated using Equation 1. 

j jt2 jt1

bw  bw bw Equation 1

where:

bwjt = male (or female) body weight in pen j at time t, where t1 is test initiation; and t2 is test termination.

 Average hatchling weight. The response measure for hatchling weight is the 

average hatchling weight per pen which is calculated using Equation 2.

j

NH

k

k j

j NH

HATWT

HATWT

j



 1

Equation 2

where:

k = index number of a hatchling in pen j from 1 to NHj;

NHj = total number of hatchlings in pen j; and

HATWTkj = body weight of hatchling k in pen j.

 Average 14-d survivor weight. The response measure for 14-d old survivor 

weight is the average survivor weight per pen which is calculated using Equation 

3.

j

HS

k

k j

j HS

SURVWT

SURVWT

j



 1

 Equation 3

where:

k = index number of a 14-d old hatchling in pen j from 1 to HSj;

HSj = total number of 14-d old surviving hatchlings in pen j; and

SURVWTkj = body weight of 14-d old surviving hatchling k in pen j.
Public Review Draft of December 17, 2014

Page 17 of 30

 Average egg shell thickness. The response variable for egg shell thickness is the 

average thickness per pen which is calculated using Equation 4.

j

m

k

k j

j m

THICK

THICK

j



 1 Equation 4

where:

k = index number of egg shell thickness measurement in pen j from 1 to mj;

mj = total number of eggs with shell thickness measured in pen j; and

THICKkj = average shell thickness of egg k in pen j., measured as described in Section (f) 

4(ii) of this guideline.

 Total food consumption per adult per pen. Total food consumption per adult 

bird between test initiation and test termination, TFOODj, for pen j is the measure 

used in this test guideline to evaluate adversion or inhibitory effects of the test 

substance on consumption of food by adults. The total food consumption per adult 

bird per pen is calculated using Equation 5. Additionally, the weekly (or biweekly 

after the onset of laying) food consumption rate per adult (e.g., t1, t2, etc.) during 

the course of the test is calculated and plotted to assess effects on the pattern of 

food consumption.





















term

t jt

jt

j m

FOOD

TFOOD

1

 Equation 5

where:

t = index of weekly and biweekly measurements of food consumption, with t=1 being week 1 of the study 

and t=term being the test or exposure termination week;

FOODjt = total food consumption in pen j at time t;

mjt = number of adult birds in pen j at time t.

 Proportion of uncracked eggs. The proportion of uncracked eggs is calculated 

using Equation 6.

 

j

j j

j EL

EL EC UE 

 Equation 6

where:

ELj = total number of eggs laid in pen j; and

ECj = number of eggs cracked in pen j.

 Proportion of normal eggs. The proportion of normal eggs is calculated using 
Public Review Draft of December 17, 2014

Page 18 of 30

Equation 7.

 

j

j j j

j EL

EL EC EA NE  

 Equation 7

where:

ELj = total number of eggs laid in pen j;

ECj = number of eggs cracked in pen j; and

EAj = number of irregular or abnormal eggs in pen j.

(iii) Descriptive Statistics.

 Environmental conditions.

o Calculate descriptive statistics (mean, standard deviation, minimum, 

maximum) for temperature, relative humidity, and light intensity during the 

three exposure phases (initial, photostimulation and laying) for adults, and for 

chicks or ducklings in the brooder pens.

o Calculate descriptive statistics (mean, standard deviation, minimum, 

maximum) for temperature, and relative humidity during egg storage and 

incubation.

 Dietary test substance concentrations. Calculate descriptive statistics (mean, 

standard deviation, coefficient of variation, minimum, maximum) by treatment 

level of the test substance concentration in the diet.

 Basal diet. Calculate descriptive statistics (mean, standard deviation) of the 

percentages by weight of protein, fat, fiber, ash, calcium, and phosphorus.

 Reproductive response variables. For each treatment group including the 

control, calculate and plot summary statistics (mean, median, minimum, 

maximum, first quartile, and third quartile) for each reproductive response variable 

in Table 5. Additionally, calculate the standard deviation, coefficient of variation, 

standard error of mean, and 95% confidence interval of mean for each treatment 

group including the controls.

 Inhibitory effects (Percent inhibition). Except for the two response variables, 

number of cracked eggs and number of irregular and abnormal eggs, all other 

response variables are expected to exhibit increasing inhibition or reduction in the 

measured response with increasing test substance concentration in the diet. For all 

response variables in Appendix 2 the percent inhibition (%I) as compared to the 

control at each test substance concentration is calculated using Equation 8.
Public Review Draft of December 17, 2014

Page 19 of 30   

C

C X

I

100 %

 Equation 8

where:

C = the control mean treatment response value (e.g., number of eggs laid); and

X = the test substance treatment mean response value (e.g., number of eggs laid). Stimulation or a greater 

response in the test substance treatment than the control is reported as negative %I.

 Stimulatory effects. For the response variables number of cracked eggs and 

number of irregular and abnormal eggs, the interest is in the increase or stimulation 

of these events with increasing test substance concentrations rather than in their 

reduction or inhibition. The percent stimulation or increase is also calculated using 

Equation 8 except stimulation is reported as negative values of %I. Negative %I 

values indicate an increased or stimulatory effect over the control response. If 

working with negative numbers is confusing, the analyst may find multiplying the 

%I value by -1 reduces confusion when discussing the increase in cracked eggs 

and irregular or abnormal eggs with increased test substance concentration.

 No observed effect concentration (NOEC) and lowest observed effect 

concentration (LOEC). A NOEC and LOEC are determined for each of the 

reproductive response variables in Appendix 2 using appropriate statistical 

methods. All methods used for statistical analysis should be described completely. 

Experimental units (replicates) are the individual pens within each treatment level. 

The overall study NOEC and LOEC are the lowest values (most sensitive) of all 

response variables considered.

(iv) Statistical Analyses By Variable Class

 Continuous. Continuous data should be analyzed using a simple ANOVA by 

generation with treatment as the effect. For most continuous variables, a normal 

distribution can be assumed and tested within the model (normality preferably 

using the Shapiro-Wilk or Anderson-Darling test and variance homogeneity tested 

using the Levene test; both tests are performed on the residuals from an ANOVA). 

The exception may be endpoints, where the range values within a treatment group 

is greater than one order of magnitude (e.g., blood, yolk, and glandular endocrine 

data). In this case, a log transformation can be applied to normalize data prior to 

the one factor ANOVA.

Where non-normality or variance heterogeneity is found, a normalizing, variance 

stabilizing transformation should be sought. If the data (perhaps after a 

transformation) are normally distributed with homogeneous variance, a significant 

treatment effect is determined from Dunnett’s test. If the data (perhaps after a 

transformation) are normally distributed with heterogeneous variance, a significant 

treatment effect is determined from the Tamhane-Dunnett or T3 test or from the 

Mann-Whitney-Wilcoxon U test. Where no normalizing transformation can be 

found, a significant treatment effect is determined from the Mann-Whitney-
Public Review Draft of December 17, 2014

Page 20 of 30

Wilcoxon U test using a Bonferroni-Holm adjustment to the p-values. The 

Dunnett test is applied independently of any ANOVA F-test and the MannWhitney test is applied independently of any overall Kruskall-Wallis test. In 

addition, regardless of the results of the previous tests, a step-down JonckheereTerpstra trend (JT) test should also be applied to the data as long as a monotonic 

response was observed.

 Proportional. A binomial model is recommend for anlysis of proportional 

variables. For monotonic data, the Rao Scott Cochran-Armitage test is preferred. 

When the denominators of the proportions representing responses of replicate pairs 

of birds are very large, generally approaching 50 for example, it was recommended 

that analysis of the response be performed on the means of the arcsine-square root 

transformed replicate proportions using Dunnett's test. Where the data differ 

significantly from monotonicity, the Rao-Scott adjustment can be applied to the 

Fisher-Exact test. A Dunnett test on arcsine-square root transformed proportions is 

also acceptable, but should be weighted by replicate size if there are marked 

variations in replicate sizes.

 Time-to-event. Time to event endpoints are often right censored (i.e., the event 

does not occur within the observation period). The recommended statistical 

analysis for these types of endpoints is the Cox Proportional Hazard mixed effects 

model, so long as the event occurs over a range of at least six days.

 Ordered categorical. Histopathology severity scores can be analyzed using a 

relatively new approach, Rao Scott Cochran-Armitage by slices (RSCABS; Green 

et al. 2014). Both SAS and R-based software versions are available free of charge 

from the publication's authors. This approach accounts for both severity and 

frequency of specific pathologies.

Phenotypic sex based on secondary sex characteristics (i.e., plumage) may be 

recorded as "male", "ambiguous", or "female". Analyses of severity of phenotypic 

deviation from genetic sex should be assigned on an ordinal scale of 0 to 3 

indicating divergence of each birds secondary sex characteristics expectation based 

on its known genotypic sex. For a male, for example, the scale would be 0=normal 

male phenotype, 1=mostly male phenotype, 2=indeterminate phenotype, 3=female 

phenotype. Thus, statistical analysis of each genetic sex will be performed 

independently.

(v) Test Report. In addition to data indicated in the sections above and described in 

Appendices 1 – 4, the final report should include the following information:

 Test substance:

o Identification, including chemical name

o Batch or lot number
Public Review Draft of December 17, 2014

Page 21 of 30

o Degree of purity

o Chemical stability under the conditions of the test

o Volatility

 Test species: Name of species tested (scientific name), strain or origin, source and 

age of birds at start of test (in weeks)

 Test conditions:

o Housing conditions (type, size and material of pens for adults and for chicks, 

additional floor covering, adjustments made to pen floors to facilitate egg 

collection and prevent breakage, temperature, humidity, ventilation, 

illumination intensity and photoperiod, water supply); any changes to these 

during the test; measures taken to minimize food spillage;

o Description of the untreated diet, manufacturer, composition, caloric content of 

diet and carrier, results of periodically performed contaminant and nutrient 

analysis of diet and drinking water contaminant analysis;

o Test groups (number of concentrations used, nominal concentrations); details 

of type of carrier used and concentration as percentage of diet; test substance 

concentrations must be reported as mg/kg diet and as mg/kg body weight;

o Specific analytical method used to determine the concentrations of test 

substance in the diet as well as actual values of homogeneity, stability and 

accuracy of preparation in diet under test conditions, as recorded in the study;

o Description of the type and frequency of the procedure used to prepare the test 

diets; description of the manner of administrating the test diets; storage 

conditions;

o Description of acclimation, stabilization and pre-treatment procedures, and 

method of assigning pairs to test groups (F0 and F1); arrangement of pens; 

number of pairs per dose group; measures taken, if any, to reduce pen-mate 

aggression;

o Frequency, duration and methods of observation;

o Information on the period and conditions of storage of eggs and on the 

incubation method;

o Method of marking all birds and eggs;

o Adherence to performance criteria;

o Description of genotyping methods;
Public Review Draft of December 17, 2014

Page 22 of 30

o Description of all biochemical analysis methods;

o Description of incidence of mortality, indicating the number of dead animals 

and the time of death during the test;

o Description of all clinical signs of toxicity and other abnormal behavior, 

including time of onset, duration and severity, and number of affected birds 

per test group and in the control group; any injuries sustained and subsequent 

treatment;

o Individual weights of the birds that died or were killed in extremis during the 

test along with macroscopic pathological findings and adult fixed organ 

weights and results of histopathological investigations;

o Weekly food consumption per test group during treatment periods and 

extrapolated mean food consumption per pair; and expressed as mg/kg body 

weight per bird per day; an indication of any apparent food spillage;

o Results of range-finding test (when performed);

o LOEC;

o NOEC.
Public Review Draft of December 17, 2014

Page 23 of 30

(g) Literature.

(1) Report of the SETAC/OECD Workshop on Avian Toxicity testing. (1996). 

Environment Health and Safety Publication - Series on Testing and Assessment: No. 

5. OECD/GD (96)166, Paris.

(2) OECD Guidelines for the Testing of Chemicals, Section 2 Effects on Biotic Systems 

(1993). ISSN : 2074-5761 (online) DOI : 10.1787/20745761

(3) Endocrine Disruptor Screening and Testing Advisory Committee: Final Report. 

(1998) http://www.epa.gov/scipoly/oscpendo/pubs/edspoverview/finalrpt.htm.

(4) Green J.W., Springer T.A., Saulnier A.N., Swintek J. (2014). Statistical analysis of 

histopathology endpoints. Environ Toxicol Chem. 33(5):1108-1116.

(h) Glossary.

 14-day-old survivors are birds that survive for 2 weeks following hatch.

 Cracked eggs are eggs determined to have cracked shells when inspected with a 

candling lamp. Fine cracks cannot be detected without using a candling lamp and if 

undetected will bias data by adversely affecting measures of embryo development.

 Eggs set refers to all eggs placed under incubation, i.e. total eggs produced minus 

cracked eggs and those selected for analysis of eggshell thickness. The number of 

eggs set, itself, is an artificial number, but it is essential for the statistical analysis of 

other development parameters.

 Eggshell thickness refers to the thickness of the shell and the membrane of an egg at

several points around the girth after the egg has been opened, washed out, and the 

shell and membrane dried for at least 48 hours at room temperature. Values are 

expressed as the average thickness of these several measured points in millimeters 

(mm).

 Hatchlings, normal refers to embryos that mature, pip the shell, and liberate 

themselves from the eggs on day 17-22 of incubation.

 Live 17-day embryos refers to embryos that are developing normally after 17 days of 

incubation. This is determined by candling the eggs.

 Viable embryos (or fertile eggs) refers to eggs in which fertilization has occurred and 

embryonic development has begun. This is determined by candling the eggs 8 days 

after incubation has begun. It is difficult to distinguish between the absence of 

fertilization and early embryonic death. The distinction can be made by breaking 

open eggs that appear infertile and examining further. This distinction is especially

important when a test substance induces early embryo mortality.
Public Review Draft of December 17, 2014

Page 24 of 30

(i) List of Appendices. Page

(1) Sampling Design for the Avian 2-Generation Toxicity Test in Japanese 

Quail................................................................................. 25

(2) Sample Collection and Apical Endpoints from the Avian 2-Generation Toxicity 

Test in the Japanese Quail..................................................................... 26

(3) Weekly Schedule of Actions by Generation for the Avian 2-Generation Toxicity 

Study............................................................................................ 27

(4) Avian 2-Generation Toxicity Test Variables by Type............................... 28

(5) Recommendations for Preparation of Sample.................................... 29

(6) Avian 2-Generation Toxicity Test Histopathology Guidance................ 31
Public Review Draft of December 17, 2014

Page 25 of 30

APPENDIX 1: Sampling Design for the Avian 2-Generation Toxicity Test in Japanese 

Quail. 

Generation

Timing F0 F1 F2

ED# 8 Fertility/embryo viability

ED 15 Fertility/embryo viability

Sample collection (PT wk 5 

eggs)

Trunk blood

Thyroid gland

Histopathology

HATCH Begin treated diet Untreated diet

Week 1 Observations of secondary sex characteristics

Clinical observations (survival)

Behavioral observations

Week 2

Week 3

Week 4 Pairs established

2 week acclimation period*

Daily clinical observations

Pairs established Observations of sexual 

maturity in F2 birds 

(optional)

Sexual maturity

 Egg production

 Foam production

Sexual maturity

 Egg production

 Foam production

 Mating behavior in 

satellite group

Week 6 Begin treated diet*

All wks 

posttreatment

Daily clinical observations

Daily egg collections

Biweekly egg collection (shell 

thickness/strength)

Weekly food consumption

Daily clinical observations

Daily egg collections

Biweekly egg collection 

(shell thickness/strength)

Weekly food consumption

Set eggs from wks 1-4 to 

ED8

5 wk posttreatmentEgg collection

 Fertility/embryo data 

to ED15

Egg collection

Fertility/embryo data to 

ED15

6-7 wk posttreatmentEgg collection

 Set for F1

 Termination (unless 

extended)

 Trunk blood

 Thyroid gland

 Preserve organs

Egg collection

 Set for F2

 Termination

 Trunk blood

 Thyroid gland

 Preserve organs

Sexual maturity 

(delayed?)‡

Termination

 Trunk blood

 Thyroid gland

 Preserve organs

8-9 wk post 

treatment†

Possible egg collection 

Termination

# ED = embryonic day.

*F0 birds are given two weeks to acclimate after sexual maturity is reached, as indicated by the initiation of foam 

production, egg laying, and mating behavior among pairs. The earliest treatment would occur when parental birds are 

6 weeks old.

‡If egg production during the 5th post-treatment week is not sufficient for evaluating fertility and embryonic sample 

collection, Parental pairs (F0) can be held for an 8th and 9th treatment week for additional egg collection prior to 

termination.
Public Review Draft of December 17, 2014

Page 26 of 30

APPENDIX 2: Sample Collection and Apical Endpoints from the Avian 2-Generation 

Toxicity Test in the Japanese Quail. 

Generation Levels Treatment Sample sizes Endpoints

F0 5 (control + 4 

treated (tx))

> 6 wks 12 to 16 pairs(prs)/level

(controls must end with 12 prs)

60 to 80 prs total

Survival

Body weight (wt)

Histopathology

Egg parameters 

Fertility 

Biochemistry (Biochem)

F1 5 (control + 4 tx) eggs

embryos

> hatch

Sexual 

maturity

3/pr (biweekly)

(180 to 240 total)

4/pair (2 for histo & 2 for 

biochem)

(240-320 total)

Ideally 6 eggs/pr

(360 to 480 eggs)

Must include 40 /level

7 prs/level

(35 prs total)

Shell thickness

Shell strength

Yolk steroids

Tissue histology

Hormone biochem

Survival

Body wt

Sexual development

Histopath

Egg parameters 

Fertility 

Biochemistry

Mating behavior

F2 5 (control + 4 tx) If extended:

eggs

eggs

embryos

> hatch

Sexual 

maturity

3/pr (biweekly)

(600+ total)

All from wks 1-4

4/pair (2 for histo & 2 for 

biochem)

Ideally 6 eggs/pr

(360 to 480 eggs)

Must include 40/level

(200 total)

7 prs/level*

Shell thickness

Shell strength

Yolk steroids

Fertility (Embryonic Day 

(ED) 8)

Tissue histology 

Hormone biochem

Survival

Body wt

Sex ratio

Sexual development

Histopathology

Egg parameters 

Fertility 

Biochemistry

Mating behavior

*Mating behaviors are observed in F2 generation only if F1 generation displays altered mating behaviors relative to controls.
Public Review Draft of December 17, 2014

Page 27 of 30

APPENDIX 3: Weekly Schedule of Actions by Generation for the Avian 2-Generation 

Toxicity Study.

Week F0 F1 F2

Age

egg 

lot Action Age

egg 

lot Action Age Action

 set eggs

2

3 0 hatch

4 1

5 2 genetic sex

6 3

7 4 pair

8 5

9 6 treated diet/eggs?

10 7 1 not set

11 8 2 not set

12 9 3 not set

13 10 4 not set

14 11 5 set eggs (to ED15)

15 12 6 set eggs (F1)

16 13 7 set eggs (F1) hatch ED15 (wk 5)

17 14 terminate F0 adults 0 hatch (wk 6)/treated diet

18 1

19 2 genetic sex

20 3

21 4 pair

22 5

23 6 eggs?

24 7 1 set eggs (to ED8)

25 8 2 set eggs (to ED8)

26 9 3 set eggs (to ED8)

27 10 4 set eggs (to ED8)

28 11 5 set eggs (10 ED15)

29 12 6 set eggs (F2)

30 13 7 set eggs (F2) hatch ED15 (wk 5)

31 14 8 terminate F1 adults 0 hatch (wk 6)/untreated diet

32 1

33 2 genetic sex

34 3

35 4

36 5

37 6 pair for mating behavior

38 7 terminate F2 
Public Review Draft of December 17, 2014

Page 28 of 30

APPENDIX 4: Avian 2-Generation Toxicity Test Variables by Type.
Public Review Draft of December 17, 2014

Page 29 of 30

APPENDIX 5: Recommendations for Preparation of Samples.

(a) Blood Collection.

(1) The tube for blood collection may be a hard plastic centrifuge tube (no plasticizers) or 

borosilicate glass, depending on the size needed for volume and centrifuge. 

(2) The volume of whole blood collected should be ~2.5X the volume of serum or plasma 

needed. 

(3) For serum, the collection tubes contain no additives. 

(4) For plasma, an anticoagulant (0.5 M EDTA, 10 μl per ml blood) is added to each tube 

prior to collection and the syringe (if applicable) is rinsed with the EDTA solution by 

drawing it up through the needle into the syringe and expelling it, leaving a small 

amount in the needle. 

(5) Samples may be collected at the time of euthanasia; if the neck is severed, then trunk 

blood can be collected into the tube. 

(6) If the sample is taken by venipuncture from older animals, the needle size is 16-20 

gauge; smaller gauge results in hemolysis if the blood sample is squeezed back through 

the barrel of the needle (better to remove the needle and then expel the blood sample 

into the tube). 

(7) For serum, allow samples to stand for 30-45 min at room temperature to clot and then 

centrifuge at 1,500xg for 10 min at 4oC. 

(8) For plasma, invert the tubes immediately after collection to allow the anticoagulant to 

mix with the blood. 

(9) If a number of samples are to be collected prior to centrifuging in order to fill the 

centrifuge rotors, the tubes should be kept chilled by putting them into a rack on ice. 

(10) Samples should be centrifuged at 1,500xg for 10 min at 4oC. Serum and plasma should 

be clear and light tan in color. 

(11) A Pasteur pipet with a bulb may then be used to siphon the serum or plasma sample and 

transfer it to a labeled polypropylene microcentrifuge tube for storage. 

(12) The sample should be kept frozen at -20oC or below. Labeling, using indelible pen 

should include code for the sample, date, and other identifiers specific for the sample. 

(13) Samples should be stored at – 20oC and shipped on dry ice. 

(14) A sample inventory in the form a spreadsheet, clearly indicating bird identification and 

approximate plasma volume, must be provided in both an electronic and a hard copy.

(15) Assuming the samples cannot be diluted to run in the assays, the approximate total 

amount of plasma required to conduct all assays is 600μl.

(b) Thyroid gland samples.

(1) Thyroid gland samples from adults, hatchling, and embryos should be provided frozen in 

2-ml round bottom centrifuge tubes clearly and legibly labeled with the bird 

identification. 

(2) The thyroid glands need to be weighed at collection. If the weight of the gland is more 

than 10 mg, a piece between 5-10 mg needs to be accurately weighed and placed in the 

sample tube. 

(3) The glands should be stored at -20oC and shipped on dry ice. 
Public Review Draft of December 17, 2014

Page 30 of 30

(4) A sample inventory in the form of a spreadsheet, clearly indicating bird identification, 

total thyroid gland weight, and weight of the sample collected into the tube needs to be 

provided in both an electronic and hard copy.

(c) Yolk samples.

(1) Yolk samples should be collected with clean separation from albumin because albumin 

may interfere with the assay. 

(2) The entire yolk should be weighed and homogenized with an equal volume of ultrapure 

deionized water. 

(3) A 1-ml aliquot of the homogenized, diluted yolk should be put into a 1.7-ml centrifuge 

tube that is clearly and legibly labeled with the bird identification. 

(4) Samples should be stored at – 20oC and shipped on dry ice. 

(5) A sample inventory in the form of a spreadsheet, clearly indicating bird identification, 

total yolk weight, amount of water used for dilution, and volume (if other than 1-ml) 

needs to be provided in both an electronic and a hard copy.