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Ecotoxicological information

Toxicity to birds

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Reference
Endpoint:
long-term toxicity to birds: reproduction test
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2010-07-22 till 2010-09-01
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 206 (Avian Reproduction Test)
Deviations:
not specified
GLP compliance:
yes
Dose method:
other: dietary exposure
Analytical monitoring:
yes
Vehicle:
yes
Details on preparation and analysis of diet:
Diet Preparation
Test diets were prepared by mixing bisphenol A into a premix that was used for weekly preparation of the final diet. Control diet and each of the four treated diets were prepared weekly beginning on July 28, 2010 and presented to the birds on Wednesday of each week. Dietary concentrations were adjusted for purity of the test substance and are presented as parts per million active ingredient (ppm a.i.).

Diet Sampling
Homogeneity of the test substance in the diet was evaluated by collecting six samples from each of the 1 ppm a.i. and 1000 ppm a.i. treated diets on Day 0 of Week 1. Samples were collected from the top, middle and bottom, of the left and right sections of the mixing vessel. One sample from the control diet and two samples from the 30 and 300 ppm a.i. diets were also collected on Day 0 of Week 1. Control and treatment group diet samples were
also collected from the feed troughs on Day 7 of Week 1 to assess stability of the test substance under actual test conditions. Additionally, a sample was collected from the control and two from each of the treatment group diets on Day 0 of Weeks 2 through 9 of the test to measure/verify test concentrations. The diet samples were transferred to the Wildlife International, Ltd. chemistry facility and stored frozen prior analysis.

Analytical Method
The method used for the analysis Bisphenol A in avian diet was based upon methodologyn developed by Wildlife International Ltd.
Samples were extracted with acetonitrile: water (90:10). Concentrations of Bisphenol A in extracts of the samples were determined by high performance liquid chromatography using an Agilent 1200 High Performance Liquid Chromatograph (HPLC) equipped with a Jasco FP-1520 fluorescence detector. High performance liquid chromatograph separations were achieved using an Inertsil C18 analytical column (250 mm x 4.6 mm I.D., 5 µm particle
size).
Calibration standards of Bisphenol A, ranging in concentration from 0.035 to 2.00 µg a.i./mL, were analyzed with each sample set. Linear regression equations were generated using the peak area responses versus the respective concentrations of the calibration standards. The concentration of test substance in the samples was determined by substituting the peak area responses of the samples into the applicable linear regression equation.
The Limit of Detection (LOD) was set at the lowest analytical standard analyzed 0.035 µg a.i./mL Bisphenol A (0.875 ng on column). The method limit of quantitation (LOQ) for these analyses was set at 0.35 ppm a.i. based upon product of the lowest analytical standard 0.035 µg a.i./mL and the dilution
factor (10) of the matrix blank extract. Measured values greater than or equal to the LOQ were reported.
Along with the sample analyses, nine matrix blanks were analyzed to determine possible interferences.
No interferences were observed at or above the LOQ standard during the sample analyses.
Avian diet samples were fortified at 0.600, 120 and 1200 ppm a.i. and analyzed concurrently with the samples to determine the mean procedural recovery. The method yielded mean procedural recoveries of 100 %, 102 %, 101 %, 100 %, 101 %, 101 %, 100 %, 98 % and 101 %. These values correspond to each sample set analyzed during the definitive study. Sample measured concentrations were not corrected for the mean procedural recoveries from each sample set.
sample set analyzed during the definitive study
Test organisms (species):
Coturnix coturnix japonica
Details on test organisms:
TEST ANIMALS: Japanese quail (Coturnix japonica)
AGE TEST ANIMALS: 4 weeks of age at the initiation of the test
SOURCE TEST ANIMALS: Wildlife International, Ltd.; 8598 Commerce Dr.; Easton, MD 21601; U.S.A.

Test Organisms
Pen-reared Japanese quail were obtained from Wildlife International, Ltd., Easton, MD 21601, U.S.A. At the start of acclimation, the Japanese quail were apparently healthy and phenotypically indistinguishable from wild type. The birds were from the same hatch, approaching their first breeding season and had not been used in any previous testing. At the start of acclimation, a random number generating function in a spreadsheet program was used to randomize pen assignment for each bird. Immediately prior to test initiation, all potential study birds were examined for physical injuries and
general health. Birds that did not appear healthy, either due to injury or inability to acclimate to laboratory conditions, or were outside the weight range for the test, were excluded from the study. All birds were four weeks of age at test initiation (first day of exposure to test diet) and ranged in weight from 80 to 118 grams at test initiation. Sex of the birds was determined by a genetic (DNA) sexing.
Total exposure duration (if not single dose):
9 wk
No. of animals per sex per dose and/or stage:
Numbers of birds
Male-Treatment group and Females-Treatment group (ppm a.i.):
Control: 15
1: 15
30: 16
300: 16
1000: 15

Control animals:
yes
Nominal and measured doses / concentrations:
NOMINAL TEST CONCENTRATIONS: 0, 1, 30, 300 and 1000 ppm a.i.
Details on test conditions:
Housing and Environmental Conditions
Housing and husbandry practices were conducted so as to adhere to the guidelines established by the National Research Council (6). The adult birds were housed indoors in batteries of pens manufactured by Georgia Quail Farm Manufacturing (GQF Model No. 0330), measuring approximately 25 X 51 cm. The pens had sloping floors that resulted in ceiling height ranging from 20 to 26 cm. The pens were constructed of galvanized wire mesh and galvanized sheeting. Sisal ropes were added to each pen for animal enrichment.
Each pen was equipped with feed and water troughs. Weekly, sufficient feed for the feeding period was placed in the trough for each pen and presented to the birds. During the feeding period additional feed was weighed and added to the troughs as needed. Water troughs were changed and water
added as necessary to provide potable water (generally every 2-3 days).
Only birds associated with this study were maintained in the study room in order to avoid excessive disturbances. The average temperature in the adult Japanese quail study room during the course of the test was 24.8 ± 0.7 °C (SD) with an average relative humidity of 64 ± 10 % (SD). The air handling system in the study room was designed to vent up to 15 room air volumes every hour and replace them with fresh air.
The photoperiod in the adult Japanese quail room was maintained by a time clock. The photoperiod during the test was 17 hours of light per day, to induce egg laying, and was maintained at thatlength until the adult birds were euthanized. Throughout the test, the birds received a mean of
approximately 68 lux (~ 6 ft. candles) of illumination provided by fluorescent lights that closely approximated the color spectrum of noonday
sunlight.
Details on examinations and observations:
Observations
The test birds were acclimated to the facilities and study pens for two weeks prior to initiation of the test. During acclimation, all birds were observed daily. Birds exhibiting abnormal behavior or debilitating physical injuries were not used for the test. During the study, all adult birds were observed
daily for signs of toxicity or abnormal behavior. Additionally, all offspring were observed daily from hatching until 14 days of age. A record was maintained of all mortalities and clinical observations.

Adult Body Weight and Feed Consumption
Adult body weights were measured at test initiation, at the end of Week 2, and at adult termination. Body weights were not measured during egg laying because of the possible adverse effects handling may have on egg production.
Feed consumption for each pen was measured weekly throughout the test. Feed consumption was determined by weighing the freshly filled feeder on Day 0, recording the amount of any additional diet added during the week and weighing the feeder and remaining feed at the end of the feeding period (Day 7). An attempt was made to minimize feed wastage by the birds by using externally mounted feeders designed with a “feed-saver” lip. Feed wastage was further reduced by placement of a piece of wire grid on the top of the feed. The wire grid allowed birds to feed unencumbered, but prevented the birds from “scooping” feed out of the feeder. The amount of feed wasted by the birds was not quantified, since the wasted feed was normally scattered and mixed with water and excreta. Therefore, feed consumption is presented as an estimate of total feed consumption.

Egg Collection and Storage
Eggs were collected daily from all pens, when available. The eggs were stored in a cold room until incubation. The cold room was maintained at a mean temperature of 14.1 ± 0.1 °C (SD) with a mean relative humidity of approximately 89 ± 0 % (SD). Groups of eggs were identified by an alphabetic lot
code. All eggs laid in a weekly interval were considered as one lot.

Candling and Incubation
At the end of the weekly interval, all eggs were removed from the cold room, counted and eggs were selected by indiscriminate draw for egg shell thickness measurement. The remaining eggs were candled with a Speed King (Model No. 32) egg-candling lamp to detect egg shell cracks or abnormal
eggs. Cracked or abnormal eggs were recorded and discarded. All eggs to be incubated were fumigated with formaldehyde gas in an airtight cabinet with a circulating fan for approximately two hours, to reduce the possibility of pathogen contamination prior to incubation. Formaldehyde gas was generated by combining 20 g of potassium permanganate and 19 mL of 37 % commercial grade formalin in a porcelain bowl at the base of the airtight cabinet.
All eggs not discarded or used for egg shell thickness measurements were placed in a NatureForm Incubator (Model No. NMC 4000). In the incubator the temperature was maintained at an average 37.4 ± 0.0 °C (SD) with an average relative humidity of 55 ± 0 %(SD). The incubator was equipped with a pulsator fan and blades that produced a mild breathing air movement designed to eliminate intracabinet temperature and humidity variation during incubation. In order to prevent adhesion of the embryo to the shell membrane, the incubator was also equipped with an automatic egg rotation device, designed to rotate the eggs from 45° off of vertical in one direction to 45° off of vertical in the opposite direction (total arc of rotation was 90°) every hour through Day 15 of incubation. Eggs were candled on Day 8 of incubation to determine embryo viability and on Day 15 to determine embryo survival.

Hatching and Brooding
On Day 15 of incubation, the eggs were placed in a NatureForm Incubator (Model No. NMC-4000 or 2340) configured for hatching the eggs. Pedigree baskets constructed of galvanized steel wire mesh were used to keep hatchlings separated by parental pen of origin. Eggs were not rotated in the incubator during the hatching period. The average temperature in the hatching compartment was 37.3 ± 0.0 °C (SD) with an average relative humidity of 58 ± 1 %(SD).
All hatchlings, unhatched eggs and egg shells were removed from the hatcher on Day 18 or 19 of incubation. The body weights of surviving hatchlings were recorded and the group body weight by pen was determined. Hatchlings were leg banded for identification by pen of origin and then routinely housed according to the appropriate parental concentration grouping in brooding pens until 14 days of age. The hatchlings were fed untreated diet without the addition of 5 % supplemental limestone. At 14 days of age, the body weights of surviving hatchlings were recorded and the average body weight by parental pen of all surviving chicks was determined. The chicks were euthanized with carbon dioxide and disposed of by incineration.
Hatchlings were housed in batteries of brooding pens manufactured by Beacon Steel Company (Model B735Q). Each pen measured approximately 72 X 90 X 23 cm high. The external walls and ceilings of each pen were constructed of galvanized wire mesh and galvanized sheeting. Floors were of
galvanized wire mesh. Thermostats in the brooding compartment of each pen were set to maintain a temperature of approximately 38 °C from the time of hatching until the birds were 14 days of age. The average ambient room temperature was 26.6 ± 1.7 °C (SD) with an average relative humidity of 44 ± 12 % (SD). The photoperiod for the hatchlings was maintained by a time clock at 16 hours of light per day.

Egg Shell Thickness Measurements
Weekly throughout the egg laying period, one egg was collected, when available, from each of the odd numbered pens during odd numbered weeks (1, 3, 5 & 7) and from each of the even numbered pens during the even numbered weeks (2, 4, 6 & 8). The eggs were opened at the waist, the contents removed, and the shells thoroughly rinsed with water. The shells were then allowed to air dry for at least one week at room temperature. The average
thickness of the dried shell plus the membrane was determined by measuring five points around the waist of the egg using a micrometer. Measurements were made to the nearest 0.002 mm.

Necropsy and Tissue Sampling
Adult birds that died or were euthanized during the course of the study were subjected to a gross necropsy. At the conclusion of the exposure period, all surviving adult birds were euthanized with carbon dioxide gas and necropsied. Twelve adult birds (six males and six females) per group were necropsied for tissue/gland excision, collection and preservation. For offspring, typically and if available, one male and one female from each pen for each lot were indiscriminately chosen for tissue collection at 14 days of age. Sex of offspring was determined by a gross visual examination of the gonads.
Birds for potential tissue collection were first anesthetized with carbon dioxide gas. Trunk blood was then collected via cardiac puncture. Collected blood was placed in a micro tube with EDTA, to prevent coagulation, and then stored frozen. Additional carbon dioxide gas was then used for euthanasia.
The following tissues were collected: head, heart, liver, spleen, kidneys, thyroid glands, bursa of Fabricius, adrenal glands, gonads and associated structures (oviduct, shell gland, epididymis, ductus deferens) and cloacal area. The entire head was collected. The caudal cranium (occipital region) was opened the to allow for fixative to penetrate the brain, pituitary gland and the pineal gland. The area around the thyroid glands was collected without the glands being dissected away from surrounding tissue. The heart, liver and spleen were each individually collected. Viscera not being collected, skin and limbs were dissected away. The lumbar and pelvic regions of the birds were collected leaving the adrenal glands, kidneys, gonads and
associated structures, bursa of Fabricius and cloacal area in place.
Initially, the collected tissues were placed in Davidson’s fixative. After two days, the tissues were removed from Davidson’s solution and placed in 10 % neutral buffered formalin for storage. Tissues for each bird were grouped together and placed in individual, labeled containers. All bird carcasses and tissues remaining after collection were disposed of by incineration.
Reference substance (positive control):
no
Duration (if not single dose):
9 wk
Dose descriptor:
NOEC
Effect level:
1 000 mg/kg bw
Mortality and sub-lethal effects:
Mortalities
One incidental mortality occurred and one bird escaped its pen and was considered to be a mortality. A third bird was injured due to penwear and penmate aggression and housed in an individual pen. The extensive injuries precluded timely conjugal visits, therefore the bird was euthanized and reproductive data from this pen were excluded.
The single mortality in the control group was the female in Pen 105. The bird was found dead on Day 6 of Week 3. Prior to death, the bird was observed with a severe neck laceration, and feather loss on the head and neck. The neck laceration was sutured closed and the male was moved to a separate pen. At necropsy the bird was noted as thin and weighed 79 g. Externally, there was an extensive lesion on the head, neck and upper back. Internally, there was a loss of muscle mass with the keel being prominent. On the neck there was a subcutaneous hematoma and cellulitis. In the abdominal cavity there were extensive lesions consistent with egg-yolk peritonitis. Necropsy of the female’s penmate was not remarkable.
On Day 3 of Week 5, the male from Pen 117 of the 1 ppm a.i. treatment group was noted as missing. A subsequent search of the test room and laboratory areas failed to located the missing bird. Reproductive data from this pen were excluded as if a mortality had occurred. Necropsy of the male’s penmate showed a slightly mottled liver, but was otherwise unremarkable.
The single mortality in the 1000 ppm a.i. treatment group was the male in Pen 179, euthanized due to his injuries on Day 6 of Week 9. Prior to euthanasia, the bird was observed with a head lesion, an ocular injury and a thin appearance. At necropsy the bird weighed 102 g. Externally, there was scar
tissue formed over the head lesion and the right eye was slightly swollen. Internally, there was old intracranial bleeding in the occipital region of the skull. Necropsy of the male’s penmate showed a right persistent oviduct filled with approximately 10 mL of a clear fluid, but was otherwise unremarkabl
e.
No other mortalities occurred during the course of the study. Due to the nature of the lesions observed at necropsy, none of the mortalities that occurred were considered to be related to treatment.

RESULTS:

There were no treatment-related mortalities, overt signs of toxicity or treatment-related effects upon body weight or feed consumption at any of the concentrations tested. Additionally, there were no treatment-related effects upon any of the reproductive parameters measured at the 1, 30, 300 or 1000 ppm a.i. test concentrations. The no-observed-effect concentration for Japanese quail exposed to bisphenol A in the diet during the study was 1000 ppm a.i. (168 mg a.i./kg/day), the highest concentration tested.

Summary of Reproductive Results from a Japanes Quail Reproduction Study with Bisphenol A

Reproductive Parameter

Experimental Group (ppm a.i.)

Control

1

30

300

1000

Number Surviving Replicates

15

15

16

16

15

Total Eggs Laid

611

658

746

631

651

Eggs Cracked

3

4

0

1

3

Eggs Set

553

591

680

572

587

Fertile Embryos

534

583

664

567

564

Viable Embryos

504

537

623

539

515

Live 2-Week Embryos

498

^534

622

353

511

Hatchlings

475

498

595

509

499

14-Day Old Survivors

427

463

556

455

475

Eggs/Hen

41

44

47

39

43

Eggs Laid/Hen/Day1

0.64

0.69

0.73

0.62

0.68

14-Day Old Survivors/Hen

28

31

35

28

32

1 Based on 64 days of egg production

Summary of Reproductive Performance from a Japanese Qail Reproduction Study with Bisphenol A

Reproductive Parameter

Experimental Group (ppm a.i.)

Control

1

30

300

1000

Number Surviving Replicates

15

15

16

16

15

Eggs Laid

611

658

746

631

651

Eggs Laid/Maximum Laid (%)

68

73

78

66

72

Eggs Cracked/Eggs Laid (%)

0

1

0

0

1

Fertile Embryos/Eggs Set (%)

98

99

98

99

96

Viable Embryos/Eggs Set (%)

93

91

91

94

88

Live 2-Week Embryos/Viable Embryos (%)

99

99

100

99

99

Hatchlings/Live 2-Week Embryos (%)

96

94

95

96

97

14-Day Old Survivors/Hatchlings (%)

90

93

93

90

95

Hatchlings/Eggs Set (%)

88

85

86

89

86

Hatchlings/Fertile Eggs (%)

90

86

88

90

89

14-Day Old Survivors/Eggs Set (%)

79

79

81

80

81

Hatchlings/Maximum Set (%)

59

61

69

59

62

14-Day Old Survivors/Maximum Set (%)

53

57

64

53

59

Validity criteria fulfilled:
not specified
Conclusions:
There were no treatment-related mortalities, overt signs of toxicity or treatment-related effects upon body weight or feed consumption at any of the concentrations tested. Additionally, there were no treatment-related effects upon any of the reproductive parameters measured at the 1, 30, 300 or 1000 ppm a.i. test concentrations. The no-observed-effect concentration for Japanese quail exposed to bisphenol A in the diet during the study was 1000 ppm a.i. (168 mg a.i./kg/day), the highest concentration tested.
Executive summary:

The study was conducted according at the method OECD guideline 206 "Avian Reproduction Test". The objective of this study was to evaluate the effects upon the adult Japanese quail (Coturnix japonica) of dietary exposure to Bisphenol A over a period of nine weeks.

Effects on adult health, body weight, and feed consumption were evaluated. In addition, the effects of adult exposure to Bisphenol A on the number of eggs laid, fertility, embryo viability, hatchability, offspring survival, and egg shell thickness were evaluated.

The primary phases of the study and their approximate durations were:

1. Acclimation - 4 weeks.

2. Pre-egg laying (with photostimulation) - 1 weeks.

3. Egg laying - Approximately 8 weeks.

4. Post-adult termination (final incubation, hatching, and 14-day offspring rearing period) – 5 weeks.

The total duration of the study were 14 weeks.

There were no treatment-related mortalities, overt signs of toxicity or treatment-related effects upon body weight or feed consumption at any of the concentrations tested. Additionally, there were no treatment-related effects upon any of the reproductive parameters measured at the 1, 30, 300 or 1000 ppm a.i. test concentrations. The no-observed-effect concentration for Japanese quail exposed to Bisphenol A in the diet during the study was 1000 ppm a.i. (168 mg a.i./kg/day), the highest concentration tested.

Description of key information

There were no treatment-related mortalities, overt signs of toxicity or treatment-related effects upon body weight or feed consumption at any of the concentrations tested. Additionally, there were no treatment-related effects upon any of the reproductive parameters measured at the 1, 30, 300 or 1000 ppm a.i. test concentrations. The no-observed-effect concentration for Japanese quail exposed to bisphenol A in the diet during the study was 1000 ppm a.i. (168 mg a.i./kg/day), the highest concentration tested.

Key value for chemical safety assessment

Long-term EC10, LC10 or NOEC for birds:
1 000 mg/kg food

Additional information

The key study from Frey et al., 2010, was conducted according at the method OECD guideline 206 "Avian Reproduction Test". The objective of this study was to evaluate the effects upon the adult Japanese quail (Coturnix japonica) of dietary exposure to Bisphenol A over a period of nine weeks. Effects on adult health, body weight, and feed consumption were evaluated. In addition, the effects of adult exposure to Bisphenol A on the number of eggs laid, fertility, embryo viability, hatchability, offspring survival, and egg shell thickness were evaluated.

The primary phases of the study and their approximate durations were:

1. Acclimation - 4 weeks.

2. Pre-egg laying (with photostimulation) - 1 weeks.

3. Egg laying - Approximately 8 weeks.

4. Post-adult termination (final incubation, hatching, and 14-day offspring rearing period) – 5 weeks.

The total duration of the study were14 weeks.

There were no treatment-related mortalities, overt signs of toxicity or treatment-related effects upon body weight or feed consumption at any of the concentrations tested. Additionally, there were no treatment-related effects upon any of the reproductive parameters measured at the 1, 30, 300 or 1000 ppm a.i. test concentrations. The no-observed-effect concentration for Japanese quail exposed to Bisphenol A in the diet during the study was 1000 ppm a.i. (168 mg a.i./kg/day), the highest concentration tested.

Halldin et al., 2001 performed three different studies with quail embryos or adults after in ovo exposure to 67 µg/egg (uptake study), 67 µg/egg and 200 µg/g egg (male and female behavioural and reproduction study), or 105 µg/bird (female distribution study). The most relevant studies for the assessment of reproductive effects are the male and female reproductive variable assessment studies. For the male reproductive variable assessment, 7-week old males were individually placed in metal cages. Sexual behaviour tests were performed in the 8th week after hatching. Neck grab and mount attempt were assessed upon placement with a female. Blood for testosterone analysis was collected and testis weight and gonadosomatic index were assessed. Female reproductive variables assessed were egg laying and oviduct pathology. No significant oestrogen-like effects were observed in males or females treated with Bisphenol A. There was a tendency for females exposed to the 200 µg/g egg to retain the right oviduct. Bisphenol A was readily excreted by the laying female as well as the growing embryo. In view of the data on distribution, maternal transfer, embryonic uptake and oestrogenic potency, it was concluded that the risk for adverse reproductive toxicity in avian wildlife resulting from embryonic exposure is probably low.

Sashihara et al., 2003 reported that hatchability was not significantly affected by Bisphenol A exposure to embryos after egg injection. There was no indication that Bisphenol A exposure significantly damaged chick embryos as determined by the stage of development at death. The phenotypic ratio of male chicks in the 10 and 100 ng/µL treatment groups was slightly higher than the 50% ratio found in controls. When compared, the genotype and phenotype ratios completely matched. The results of this study indicate that low doses of Bisphenol A do not affect the hatchability or embryonic development of chickens.

Considering a supporting study to the two weight of evidence studies described above, Berg et al., 2001 assessed embryo development just prior to hatch in eggs that were treated by injection on day 3 of incubation. There was a statistically significant increase in mortality in chickens at both doses but no impact on quail. In quail embryos treated with 200 µg/g Bisphenol A, malformations of the mullerian ducts occurred in 6 of the 14 females. No impact was seen in males or in either sex in chickens. In quail embryos, Bisphenol A did not cause an increased ovotestis frequency compared to controls. In chicken embryos, the ovotestis frequency was 55% following exposure to 200 µg/g Bisphenol A, but none were found in the 67 µg/g dose group. The risk for reproductive impacts in avian wildlife, as predicted by this study, are probably low as the doses required for effects in this study were fairly high.