1

DRAFT September 2011



### OECD GUIDELINE FOR TESTING OF CHEMICALS

Avian Two

- generation Toxicity Test in the Japanese Quail

INTRODUCTION

1. The purpose of this guideline is to describe a laboratory testing procedure for evaluating the impact of pesticides and other chemicals upon avian health and reproduction. Data collected from such tests may be used in the assessment of risks to birds exposed to pesticides and other chemicals under fie ld conditions.

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

chem ical as an endocrine disruptor.

2. The test is designed for dietary exposure, but also may include exposure through drinking water if more appropriate for the chemical. This guideline describes a

two

- generation test. The test monitors health and reproduct ive parameters in Japanese quail ( Coturnix japonica ) adults and their progeny, including viability of second generation offspring.

3. The number of second generation (F2) 14

- day old survivors per F1 generation hen is the primary biological endpoint of this test. Additional hormone and morphological endpoints are included to assess and confirm potential endocrine disruption.

PRINCIPLE OF THE TEST

4. Birds in the parent (F0) generation are paired at 4 weeks of age, and acclimated for 2 weeks prior beginnin g 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.

VALIDITY CRITERIA

5. The test substance concentration in the diet to which birds are exposed should be satisfactorily maintained and reported. Losses of 20% or less relative to initial concentrations are generally considered acceptable. Higher rates of loss must be investigated and explained.2

6. Parental mortality during the two week acclimation s hould not exceed 3%. When possible, all mortalities in the control group should be explained. At least eight breeding F1 pairs that have produced eggs must be available in the control group at the end of the test period.

DESCRIPTION OF THE METHOD

Test animals

7. The test species is the Japanese quail ( Coturnix japonica ).

8. Japanese quail generally start laying eggs at or later than six weeks of age. Once egglaying has begun it will take about two to three weeks for birds to reach full egg production. De pending on the strain, eggs will be fertile when birds are about eight to ten weeks of age.

9. 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

medicati ons 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.

H usbandry

10. 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.

11. Artificial lighting should approximate the daylight visual spectrum and be

automa tically 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 f eeder. The light intensity is dictated by the amount required for daily observations. Light intensity that is too high will encourage aggressive

behavior.

12. Incubators and hatchers, preferably with automatic temperature and humidity controls and an egg

- tur ning device, are necessary. In addition, suitable equipment is required to maintain stored eggs within the temperature and humidity ranges specified.

13. 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 m esh 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).3

14. 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 aggress ion can be a problem and measures must be taken to minimize injuries, stress and mortality resulting from such aggression. Pairs should be reintroduced daily to maintain fertility.

15. For rearing chicks, the use of rearing pens with thermostatically contr olled 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 acc essing 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 separ ated into pairs (one male/one female)

when 4 weeks old after sex has been determined.

16. 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.

Feeding

17. Diet and drinking water are provided ad libitum . The diet should be described 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 for the adult di et. During the treatment period, birds are fed basal diet mixed with the test substance at specified

concentrations.

18. Guidance for recommended nutritional values is provided in Table 2.

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 6.04

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

TEST DESIGN

19. 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 the test. The test begins with

the ad ministration of treated diet ( i.e. when birds are 6 weeks old, at the earliest ). Breeding pairs should be maintained in a 16:8 or 17:7 light:dark photoperiod for the duration of the study. Onset of egglaying can take place during the acclimation period.

P rior 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 wi th 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 pair s 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 en sure 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 collected from F0 post

- treatment week 5 (and possibly, week 8 and/or 9) to examine fertility. Brood s from the post

- treatment

6 th and 7 th week are used to establish the first generation offspring (F1). Optimally, 6 eggs are set for each parental pair, but a minimum of 40 eggs must

be set for each test level. If during the 6 th and 7 th post

- treatment weeks, a

treat ment 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 c ontrol and

treatment group birds are weighed and randomly allocated to pens by pairs 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 6 to 7 weeks post

- fertility. Eggs collected from F1 pairs during weeks 1 through 5 will be used to evaluate fertility. Eggs collected from weeks 6 and 7 post

- pairing

will be used to establish the F2 generation , optimally 6 eggs will be collected from each F1 breeding pair. F2 offspring survival will be observed though day 14. Observations can be extended to examine timing of sexual maturity in the F2 generation.5

Dietary concentrations

20. 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 weight of water when birds are exposed through drinking water). The concentration should also be expressed as mg/kg body weight per bird per day.

21. 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. Information may a lso be gained from tests with rodents or other mammals. The highest concentration 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 concen tration to be tested should be the expected concentration of the chemical in the environment with the addition of a safety factor (e.g., 5X). However, the highest dose administered should not cause mortality or other severe signs of parental toxicity tha t 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 hi ghest and lowest doses. A minimum of four concentrations and a control are required

for the definitive test.

22. A range finding test can be performed before initiating the main study to aid in establishing test concentrations and to evaluate any potential av oidance effects.

Preparation and monitoring of the test diet

23. 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

24. 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 di et, in order to keep the caloric value of the diet equal between dosage groups.

25. 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 t he birds. It may be desirable to keep all feed

frozen until use.6

26. 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 (ma mmalian) 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 fe ed 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.

27. Homogeneity of the test sub stance 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.

28. To verify test concentrations, samples of diets fed to the birds should be taken every time new diet is mix ed during the treatment period to allow measurement of

the actual concentration of the test substance.

29. Both F0 and F1 generations are maintained on treated diet until test termination.

All F2 chicks are maintained on untreated diet.

Measurements and obser vations

Adults

30. 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, et c. 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

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

• Body weights should be determined at least at the start of treatment 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 4 th week and every week after until distinctly dimorphic traits are expressed (F0 and F1 generations). If7

plumage is not distinctly “male” or “female”, note the plumage as “ambiguou s”.

Offspring

– Eggs

31. Eggs are collected 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.

32. 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 of embryos of eggs. Before placing the eggs in the i ncubator, 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 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 ( 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

33. Eggs from F0 and F1 pairs will be collected during the 5 th , 6 th , and 7 th post

- pairing weeks. Eggs collected during the 5 th week will be candled to estimate fertility and embryo viability on embryonic days (ED) 8 and 15, respectively. On ED 15, eggs will be opened and embryo weight and sex will be recorded. Tissues will be collected (see 43 and 44 below) from two ED 15 chick s per pen. In addition, trunk blood and thyroid glands will be collected for biochemical analyses from two ED 15 chicks per pen. If egg production from the 5 th week is limited (i.e. < 4 eggs with viable ED15 embryos/pen are produced), the priority is as f ollows: 1 st
embryo for histology, 2 nd embryo for biochemistry, 3 rd embryo for histology, 4 th
embryo for biochemistry. If the eggs produced during week 5 are not sufficient (e.g. < 3/pair) to estimate fertility, additional eggs can be collected from breedi ng pairs during weeks 8 and 9. F1 birds are maintained on treated diet until test termination. Eggs collected from weeks 6 and 7 will be set, artificially incubated, and allowed to hatch to establish the F1 generation. All F2 offspring will be

maintaine d on untreated diet and survival through day 14 is recorded.

The onset of sexual maturity (egglaying or foam production) or absence thereof can be recorded for F2 hatchlings. The timing of sexual maturity will be considered egg or foam production in 90% of the control (i.e. birds maintained on an untreated diet) and appropriate behavioral responses among paired birds. Observations will extend for an additional week beyond sexual maturity of the control groups to estimate potential pubertal delays among treated birds.8

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 egg production is limited during the

5 th (<5 eggs) , 6 th , and 7 th weeks (<7 eggs total for both weeks), forego egg collection for shell measurements. 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. 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

- treatment weeks 6 and 7)

, embryonic survival and fertility (post

- treatment week 5, and if necessary post

treatments weeks 8 and 9), and those collected for eggshell thickness and strength (one egg/pen/2 weeks), can be discarded after enumeration and examination for

cracks or abno rmalities. Egg yolks from eggs broken during shell measurements can be used for biochemical analyses (see 46 and 47 below).

35. Eggs collected from the F0 and F1 generation during the 5 th , 6 th , and 7 th post

pairing week will be incubated to assess embryo viability (5 th post

- pairing week) and establish the next generation (6 th and 7 th post

- pairing weeks). Eggs collected from the F1 generation during post

- pairing weeks 1

- 4 will also be incubated through embryonic day 8 to evaluate fertility. 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

turne d an odd number of times a day.

36. 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, for only eggs collected during p ost

- pairing weeks

5

- 7, again for viability of embryos approximately 2 days before hatching (ED 15). Eggs from post

- pairing weeks 6 and 7 will be transferred to a hatcher. 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 assis tance should be given to chicks during hatching.

Egg parameters

37. Prior to start of the treatment period, egg production of all F0 generation pairs of birds available for the study (including potential replacements) should be

recorded. These data wi ll be used to exclude pairs that do not lay eggs from9

further study. Eggs will be discarded after candling unless otherwise indicated (see appendix).

38. During the treatment periods, the following information is recorded:

• Eggs laid

• Eggs cracked

• Eggs broken

• Description of all egg abnormalities

• Eggs set

• Eggshell strength

• Eggshell thickness

• Fertile eggs

• Infertile eggs

• Early viability (ED8)

• Late viability (ED15)

• Eggs that hatch

• Eggs that do not hatch

39. 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.

40. Typical control values for reproductive parameters in Japanese quail are provided in Table 4. If reproductive parameters of control birds do not meet these values, the test procedure and husbandry conditions should be checked for potential problems.

Table 4: Typical values for reproductive parameters in Japanese quail

Number of eggs laid per hen per day 0.66

– 0.89 Percentage of cracked/broken eggs 0 to 10% Viability (percentage fertile, live embryos of eggs set) 85 to 96% Hatchability (percentage hatching of fertile eggs) 70 to 98% Percentage hatchlings that survive 14 days 85 to 97%

Mean number of 14

- day old survivors per hen per day 0.34

– 0.71 Eggshell thickness (mm) 0.19 to 0.22

Chicks

41. The following observations on F1 and F2 chicks are recorded during the treatment periods:

• Day of hatch

• Hatching mortality10

• 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 optional)

• Ph enotypic sex

• Genetic sex (F0, F1, F2)

• Mating behavior (F2 optional)

Pathology

4 2

. 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 pathology assessme nt. Adult birds that die, or are killed during the course of the treatment period, will be subjected to the same procedures. Trunk blood will be collected for biochemical analyses. The left thyroid gland is to be stored for glandular T4 content. Organs including brain (POM, pituitary, pineal gland), kidneys, liver, adrenal glands, reproductive organs and associated structures, cloacal gland, bursa, and right thyroid gland should preserved in situ using

Davidson’s fixative for further h istopathology. 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.

4 3

. 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

adults. Trunk blood and thyroid glands will be collected from two late

- st age embryos per pair for biochemical analyses. If a mating pair produces fewer than 4 eggs with viable late

- stage embryos, see section 34 above for sample prioritization.

44. 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.

Biochemical/endocrine measurements and observations

4 5

. Thyroid hormone measurements

– Measurements of thyroid function include thyroid gland weight, glandular thyroxine ( T4 ) content, and thyroid histology/histopathology. Samples will be collected from embryonic and adult life stages. Eggs collected from post

- treatment week 5 incubated to ED 15

, can be used for e mbryonic 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 collected11

from two embryos and unfixed th yroid 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 used for histological assessment

as above.

4 6

. Steroid hormone measurements

- Non

- invasive procedures are preferable for periodic measurements of estradiol and testosterone in developing birds, adults, and eggs. One approach for measuring steroid hormones in the developing birds and adults is through fecal/urate material. Steroids are to be measured from fecal/urate material using radioimmunoassay (RIA) starting Day 1 of adult pairing , and weekly thereafter. A dults are housed in pairs, and the f ecal/urate material shall be linked to the pen. Steroid hormones shall be analyzed in t runk

blood collected from two late

- stage embryos per male

- female pair, from blood samples collected from all animals at necropsy, and from egg yolks resulting from

eggs hell thickness/strength measures

.

4 7

. 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 o f estrogen, however in males that have been exposed to estrogenic contaminants, it is expected that vitellogenesis will be stimulated. Vitellogenin is to be measured from trunk blood of the late embryos used for steroid and thyroid hormone analys e s and from adults at necropsy , when appropriate, to evaluate estrogenic potential of a test chemical

.

Behavioral measurements

4 8

. Males

– 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

. T rials shall be performed to assess mating behavior s 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 behavio r will be observ ed 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 indivi duals shall be re

- introduced for three consecutive day s 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 distur bance during introduction and separation of birds involved in behavior observation trials may perturb egg production, and therefore,12

observational data should be collected from distinct pairs not used in collection of other data endpoints (e.g. egg product ion, fertility, etc.).

DATA COLLECTION AND REPORTING

Treatment of results

49. Numerical data should be presented in tabular form. 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 an alysis.

50. Measurements of endpoints made on adult birds will be evaluated by comparing values obtained from birds in treated groups with values obtained from control birds. The following adult health endpoints should be analyzed for both F0 and

F1 bird s, using appropriate statistical procedures:

• Food consumption

• Body weight

• Number of eggs per hen per day

51. Moreover, mortality, clinical observations or pathological findings may be evaluated statistically to verify any apparent relationship between adult and

treatment.

52. The following reproductive data (both continuous and discrete variables) are to be evaluated statistically by comparison with the control group using appropriate

methods:

• Number of fertile eggs as a percentage of eggs set

• Number of early viable embryos as a percentage of eggs fertile

• Number of late viable embryos as a percentage of early viable embryos

• Number of eggs hatched as a percentage of late viable embryos

• Number of 14

- day old chicks as a percentage of eggs hatched

• Number o f 14

- day old chicks as a percentage of eggs set

• Number of 14

- day old chicks per hen per day

• Number of cracked eggs as a percentage of eggs laid

• Number of abnormal eggs as a percentage of eggs laid

• Mean egg shell thickness

• Mean eggshell strength

• Mean hatc hling body weight

• Mean 14

- day old chick body weight

• Sex ratio of chicks13

53. The following parameters may be useful to evaluate statistically, depending on the findings:

• Number of hatchlings per hen per day

• Number of normal hatchlings as a percentage of total hatchlings

• Number of infertile eggs as a percentage of eggs set

• Number of early embryonic deaths as a percentage of fertile eggs

• Number of late embryonic deaths as a percentage of viable eggs

54. The following endocrine and physiological endpoints ( both continuous and discrete variables) are to be evaluated statistically by comparison with the control group using appropriate methods:

• Fixed weight of adult testes, ovaries, thyroid, adrenals, oviduct, cloacal gland,

liver

• Histology of thyroid, adrenal s, gonads, brain

• Testicular spermatid counts and morphology (optional)

• Gross anomalies of the genital tract

• Feather dimorphism

• Cloacal gland size, 1 st appearance of foam

• 1 st egg laid

• Mating behavior (time to mount, number of mount attempts, successful cloacal contact)

• Fecal/urate steroid hormones (estradiol, testosterone)

• Egg steroid content (estradiol, testosterone)

• Serum steroid hormones (estradiol, testosterone, vitellogenin when appropriate)

• Glandular thyroid hormone

55. A “no observed effect concentration” (NOEC) expressed in mg/kg diet and mg/kg body weight per day should be determined for all health and reproductive parameters evaluated.

Test report

56. The final report should include the following information:

Test substance:

• identific ation, including chemical name

• batch or lot number

• degree of purity

• chemical stability under the conditions of the test

• volatility14

Test species:

• name of species tested (scientific name), strain or origin, source and age of birds at start of test (in week s)

Test conditions:

• 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,

illuminati on intensity and photoperiod, water supply); any changes to these during the test; measures taken to minimize food spillage

• 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

• test groups (number of concentrations used, nominal concentrations); details of type of carrier used and concentration as percentage of diet; test substance

concentrations m ust be reported as mg/kg diet and as mg/kg body weight

• 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 condition s, as recorded in the study

• 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

• 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

• frequency, duration and methods of observation

• information on the period and c onditions of storage of eggs and on the

incubation method

• method of marking all birds and eggs

Results:

• description of incidence of mortality, indicating the number of dead animals and the time of death during the test

• description of all clinical signs o f 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

• macroscopic pathological findings and adult fixed organ weights and results of histopathological investigations

• mean body weights of F0 males and F0 females per pen at start of treatment and at the end of the F0 treatment period (end of week 6 of treatment); mean15

body weights of F1 males and F1 females per pen at pairing and at the end of the test; individual weights of the birds that died or were killed during the test

• 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

• results of range

- finding test (when performed)

• lowest observed effect concentration (LOEC)

• no observed effect concentration (NOEC)

Reproductive Data (tabulated separately for each generation):

• number of eggs laid per hen and per day

• all eggs should be traceable to their pen of origin

• all egg abnormalities should be described and reported

• numbers of eggs cracked and the percentage of eggs cracked of eggs laid

• eggshell thickness, eg gshell strength measurements

• number of fertile eggs and the percentage fertility related to eggs set (F0

collection weeks 5

- 7; F1 collection weeks 1

- 7)

• number of infertile eggs and the percentage infertility related to eggs set (F0

collection weeks 5

- 7; F1 collection weeks 1

- 7)

• number of early viable embryos and the percentage viability related to fertile

eggs

• number of early embryonic dead and the percentage related to fertile eggs

• number of late embryonic dead and the percentage related to 8

- day viable embryos (collection week 5)

• number of normal hatchlings per hen per day and the percentage related to 15

- day viable embryos (collection week 5)

• number of normal hatchlings as a percentage of fertile eggs (collection weeks 6 and 7)

• number of normal hatchlin gs as a percentage of eggs set (collection weeks 6 and 7)

• number of normal hatchlings and the percentage related to total number of hatchlings (collection weeks 6 and 7)

• number of 14

- day old chicks per hen and per day (collection weeks 6 and 7)

• number of 1 4

- day old chicks as a percentage of (normal) hatchlings (collection weeks 6 and 7)

• number of 14

- day old chicks as a percentage of eggs set (collection weeks 6 and 7)

• mean body weights of chicks on the day of hatching and after 14 days per pen,

per test gro up

• description of abnormal behavior of chicks, of severe birth defects and their general state of health, during the first 14 days after hatching, clinical signs of toxicity

• observations of hatchling mortality

• NOEC (and LOEC) per parameter that was evaluat ed statistically16

A description of statistical methods used in the analysis of data.

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. (1993). Section 2

- Effect on Biotic Systems: Test Guideline 206: Avian Reproduction Test (adopted April 1984).

(3) OECD Guidelines for Testing of Chemicals. Proposal f or a new test guideline: Avian reproduction toxicity test in the Japanese quail or Northern bobwhite. (September 1999 draft)

(4) Endocrine Disruptor Screening and Testing Advisory Committee: Final Report. (1998)17

APPENDIX

Table 1. Sampling design for the Avian Two

- Generation Toxicity Test in the Japanese Quail. (ED = embryonic day).

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

* 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 week s old

. †If egg produ ction 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 prior to termination

.18

Table 2. Sample C ollection and Apical Endpoints from the Avian Two

- Generation Toxicity Test in the Japanese Quail.

Generation Levels Treatment Sample sizes Endpoints

F0 5 (control + 4 tx) > 6 wks 12 to 16 prs/level (controls must end with 12 prs) 60 to 80 prs total Survival Body wt Histopathology Egg parameters Fertility Biochemistry

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 (ED 8)

Tissue histology

Hormone biochem

Survival Body wt

Sex ratio Sexual development Histopath Egg parameters Fertility Biochemistry

Mating behavior

* Mating behaviors are observed in F2 generation only if F1 generation displays altered mating behaviors relative to

controls.19

Table 3. Weekly schedule of actions by generation for avian 2

- generation toxicity study.

week F0 F1 F2

age egg lot action age egg lot action age action

1 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