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 DRAFTPublic 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 sub mission 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 specificall y 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 t hat 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 guidanc e, 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 recommendat ions 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

- H Q

- OPPT

- 2009

- 0159, and

EPA

- HQ

- OPPT

- 2009

- 0576.

DRAFT DOCUMENT DISCLAIMER: T his 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 m eet 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 two

tiered 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 po tential

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 pos sible adverse consequences that occur from the potential to interact with the estrogen, androgen, or thyroid hormone systems identified

in T ier 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 d etermine 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 es tablish 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 p rovide 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

en dpoints 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

fro m 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 prod uction (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 determin ed 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 product ion. 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 pr ior 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 egg

turning 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 st ainless 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 shoul d 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 cross

contamination 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, p airs 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 f or 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 app roximately 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

( o C) Relative humidity

(%) Minimum floor area (cm 2 /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 peri od, 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 ho used 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 t here 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 post

hatch) 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 post

treatment 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 p roduction 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 bre eding pairs. F2Public 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 wat er).

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 mammal s. 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 concentrati on 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 d ietary 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, th e 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 toxi city ( 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

a s 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 frequ ency 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 chemic al 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 veri fied 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 measure ments 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

t est 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 2 nd 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, d uring 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 a lso 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 signific ant 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 distinctlyPublic 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

– e ggs. 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 eg gs should be recorded.

Table 3

. Conditions for egg storage, incubation and hatching

.

Temperature ( o C) 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 a nd 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 incubato r 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 she ll 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 5t h (<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 st and 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 r oom 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 thosePublic 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 colle cted during the 11th, 12th, and 13th post

hatching 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 remove d 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 b irds 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 a n 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 a gain 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 ou t 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 pe riods, 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

 Egg s 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 dea th, 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 toxicit y/disease

 Number of 14

- day

- old surviving chicks

 Chick body weight at 14 days after hatching

 Time to repr oductive maturation (F2* )

 Phenotypic sex

 Genetic sex (F0*, F1, F2)

 Mating behavior (F0,F1,F2*) *optionalPublic 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 biochem ical 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 ex cised 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 abnormal ity 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 repro ductive 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 identica l to those collected from adult animals. Reproductive organs fromPublic 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 c hicks) 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 gla nds 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 (non

endocrine 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 treatment

related effects on the endocrine system of Japanese quail exposed to a putative endocrine disrupting compound. L ow 8M / 8F

low mid 8M / 8F high mid 8M / 8F high 8M / 8F

F1 Control* 8M / 8F

L ow 8M / 8F

low mid 8M / 8F high mid 8M / 8F high 8M / 8F F2† Control 8M / 8F

L ow 8M / 8F

low mid 8M / 8F high mid 8M / 8F high 8M / 8F * May be 14 day old chicks or 6 week old juvenile/adultsPublic 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. L ow 5

low mid 5 high mid 5 high 5

F2 Control 5

L ow 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 μ l is required for thyroid (250 μ l of

unextracted plasma) and steroid hormone analyses (350 μ l 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/histopa thology. 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 tw o 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 ab ove. 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

expresse d 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 appro priate, 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 becom es 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, o bservational 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

E cotoxicity 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 p en) 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 anal ysis.

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 ver ify 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 ass umptions 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 mo del. 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 c alculation

. For all equations in this guideline, the index j = 1 to the total number of pens per treatment group (typically 16).

 Adult b ody w eight g ain. The change in adult body weight (males and females are
tracked separately) between test initiation and test termination,

, 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.

Equation 1

where:

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

 Average h atchling w eight. The response measure for hatchling weight is the average hatchling weight per pen which is calculated using Equation 2.

Equation 2

where:

k = index number of a hatchling in pen j from 1 to NH j ;
NH j = total number of hatchlings in pen j ; and

HATWT

kj = body weight of hatchling k in pen j

.

 Average 14

- d s urvivor w eight. The response measure for 14

- d old survivor weight is the average survivor weight per pen which is calculated using Equation

3.

Equation 3

where:

k = index number of a 14

- d old hatchling in pen j from 1 to HS j ; HS j = total number of 14

- d old surviving hatchlings in pen j ; and

SURVWT

kj = 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.

Equation 4

where:

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

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

THICK

kj = 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, TFOOD j , 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 tota l 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.

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;

FOOD

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

m jt = number of adult birds in pen j at time t

.

 Proportion of u ncracked e ggs. The proportion of uncracked eggs is calculated using Equation 6.

Equation 6

where:

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

EC j = number of eggs cracked in pen j

.

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

Page 18 of 30 Equation 7.

Equation 7

where:

EL j = total number of eggs laid in pen j ; EC j = number of eggs cracked in pen j ; and

EA j = 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 bro oder 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

p ercentages 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 q uartile) 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 i ncreasing 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

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 wit h 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 incre ased 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 inc reased 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

respons e variables considered.

(iv) Statist ical 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 transforma tion 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 Mann

Whitney test is applied in dependently of any overall Kruskall

- Wallis test. In addition, regardless of the results of the previous tests, a step

- down Jonckheere

Terpstra 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, gener ally 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

- Sc ott 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 e vent 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 c ategorical. 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". Anal yses 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 w ould 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 an d described in Appendices 1

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

 Test substance:

o Identification, including chemical name

o Batch or lot numberPublic 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 Des cription 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 tes t 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; descripti on 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; m easures 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 c riteria ;

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 a nd 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).

Env ironment 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) Endocrin e 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

- 11 16.

(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

expresse d 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 T oxicity Test in Japanese Quail

................................................................ ................. 25

(2) Sample Collection and Apical Endpoints from t he Avian 2

- Generation Toxicity Test in the Japanese Quail

....................... .............................................. 26

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

- Generation Toxicity

Stud y...................................................

......................................... 27

(4) Avian 2

- Generation Toxicity Test Variables by Type

................... ............ 28

(5) Recommend ations for Preparation of Sample........................

............ 29

(6) Avian 2

- Generation Toxicity Test Histopathology Guidanc e......

.......... 31Public Review Draft of December 17, 2014

Page 25 of 30 APPENDIX 1 : Sampling D esign 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
post

treatment 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 post

treatment Egg collection

 Fertility/embryo data

to ED15 Egg collection

Fertility/embryo data to

ED15

6

- 7 wk post

treatment Egg 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 pare ntal birds are

6 weeks old.

‡If egg production during the 5 th post

- treatment week is not sufficient for evaluating fertility and embryonic sample collection, Parental pairs (F0) can be held for an 8 th and 9 th treatment week for additional egg collection p rior 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 pair s (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 Histopath ology 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 S tudy.

W eek F0 F1 F2

A ge egg lot A ction A ge egg lot A ction A ge A ction

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 F2Public Review Draft of December 17, 2014

Page 28 of 30 APPENDIX 4 : Avian 2

- Generation Toxicity Test Variables by T ype.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

d rawing 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 s erum, allow samples to stand for 30

- 45 min at room temperature to clot and then

centri fuge at 1,500xg for 10 min at 4 o C. (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 centrif uged at 1,500x g for 10 min at 4 o C. Serum and plasma shou ld 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 samp le should be kept frozen at

- 20 o C or below. Labeli ng, using indelible pen should include code for the sample, date, and other identifiers specific for the sample. (13) Samples should be stored at

– 20 o C and shipped on dry ice. (14) A sample inventory in the form a spreadsheet, clearly indicating bird identificati on 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 600 μ l.

(b) Thyroid gland sampl es.

(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

- 20 o C 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 iden tification, 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

– 20 o C and shipped on dry ice. (5) A sample inventory in the form of a s preadsheet, 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.