11 - 15
days
Embryo
16 - 20
days
Death
6 - 10
days
Stage of
11 - 15
days
Embryo
16 - 20
days
Death
ou
0.0
0-5
days
21 days pipped
6 - 10
days
Stage of
11 - 15
days
Embryo
16 - 20
days
Death
o  Hatchlings transferred to brooder battery
Figure 2. Effect of TCDD (left), PeCDF (middle), TCDF (right) on incidence of embryo mortality (number of embryos in stage / number of fertile eggs).
o  14-day-old chicks
TCDD Deformities
Tissue weighing
Beak
4
Cranial
Deformities
3
4%
2
2%
1
0%
Vehicle
Control
Egg Mortality
6%
0.0745
0.0994
0.224
0.311
Dose (pmol/g egg)
0.820
3.17
6.68
PeCDF Deformities
Limb
5
8%
Trunk
Beak
4
Cranial
Deformities
6%
3
4%
2
2%
1
0
0%
Vehicle
Control
0.141
0.235
0.388
0.599
Dose (pmol/g egg)
1.07
4.08
6.76
21 days pipped
TCDF Deformities
5
Limb
8%
Trunk
Beak
4
Cranial
6%
Deformities
Incidence
8%
Trunk
Incidence
Limb
5
3
4%
2
2%
1
0
0%
Vehicle
Control
0.131
0.170
0.288
0.654
1.12
4.77
14.2
Dose (pmol/g egg)
100%
Congener
Estimate [95% Confidence Interval]
pmol/g egg
0.22 [0.042 - 0.42] 0.075 [0.014 - 0.14]
0.31 [0.092 - 0.59] 0.095 [0.028 - 0.18]
LD50
pmol/g egg
TCDD
3.60 [2.3 - 6.3]
1.20 [0.74, - 2.0]
60%
PeCDF
0.61 [0.28 -1.2]
0.21 [0.095 - 0.41]
TCDF
1.20 [0.62, - 2.2]
0.37 [0.19 - 0.67]
Mortality
0.57 [0.29 - 0.90]
PeCDF
TCDF
40%
LD80
PeCDF
0.180 [0.093 - 0.29]
TCDF
LD80
pmol/g egg
22 [11 - 77]
1.7 [0.92 - 6.3]
7.10 [3.5 - 25]
0.58 [0.32 -2.1]
4.5 [2.4 - 15]
1.40 [0.73 - 4.6]
Figure 3: Deformities observed in pheasant embryos exposed to TCDD (left), PeCDF (middle) or TCDF (right). Cranial deformities included exencephaly and microphthalmos or
anophthalmos; bill deformities included incomplete or lack of upper or lower beak development and crossbill; trunk deformities included gastroschisis, achondroplasia and edema; limb
deformities included club foot, curled toes and extra limb formation.
o  There were no dose-related changes in body weight or relative organ weights in hatchlings and 14-day-old chicks.
ng/g egg
TCDD
PeCDF
TCDF
TCDD
ng/g egg
TCDD
80%
LD50
ng/g egg
LD20
Vehicle Control
Background Mortality
o  Species sensitivity based on LD50 values generated by probit analysis:
  TCDD: Chicken > Pheasant > Quail
  PeCDF: Chicken ≈ Pheasant > Quail
  TCDF: Chicken > Pheasant > Quail
(Cohen-Barnhouse et al., 2008 [WP210]; 2009 [MP178])
o  To assess the relative potencies (RePs) of TCDF and PeCDF compared to
TCDD in the Common pheasant (Phasianus colchicus torquatus).
Gr
ou
Gr
0-5
days
0
20%
o  To confirm the proposed avian sensitivity classification in ovo using TCDD,
and two TCDD-like compounds, PeCDF and TCDF.
0.1
se
0.0
21 days pipped
14.2
p
p
p
ou
Gr
0.1
se
6 - 10
days
Stage of
4.77
0.2
Do
0-5
days
1.12
0.4
0.3
6.76
0.2
Do
0.0
0.654
0.5
se
Incidence
0.3
6.68
0.1
4.08
0.288
0.6
Do
0.3
1.07
0.4
0.170
0.7
0.599
0.5
Vehicle Control
0.131
0.8
0.388
0.6
Dose
(pmol/g egg)
0.9
% of post 11-day embryos with deformities
Tagged 4-day-old chicks
3.17
0.2
  Weighed
  Tissues removed, weighed and processed
for histology
•  Brain, heart, bursa and spleen
•  Liver
-  CYP1A4 and CYP1A5 mRNA
abundance (Yang et al., 2009 [#537])
-  Analyzed for compound concentration
Brooder battery
0.820
0.4
  Stage of embryo at death
  Incidence and type of deformities
0.7
0.311
0.5
TCDD Embryo Mortality 0.235
% of post 11-day embryos with deformities
Opening of unhatched eggs
Vehicle Control
0.141
0.8
0.224
0.6
o  Unhatched eggs opened to determine:
Sealing injection site with
melted paraffin
Dose
(pmol/g egg)
0.9
0.0994
0.7
Incidence
Injection with positive
displacement pipettor
PeCDF Embryo Mortality Incidence
0.8
o  Eggs incubated until hatch or d 24
(37.6ºC, 52% humidity)
o  A molecular basis for differences in sensitivity to TCDD-like compounds
among avian species has been suggested.
o  Proposed TCDD species-sensitivity ranking: (Kennedy et al., 2009 [#247])
  White Leghorn chicken > Common pheasant > Japanese quail
•  Triolein (Vehicle Control)
•  TCDD: 7 doses (0.0745 - 6.68 pmol/g egg)
•  PeCDF: 7 doses (0.141 - 6.76 pmol/g egg)
•  TCDF: 7 doses (0.131 - 14.5 pmol/g egg)
Drilling shell using Dremel® tool
Vehicle Control
0.0745
Incidence
Candled egg with marked
injection side
Dose
(pmol/g egg)
0.9
o  Injection of 0.1 µL/g egg into the air cell
of Common pheasant eggs (30g) with:
LD20
o  Differences have been attributed to amino acid substitutions in the ligandbinding domain (LBD) of the aryl hydrocarbon receptor (AhR).
TCDD Embryo Mortality o  1,760 eggs candled, weighed and
allocated into treatment groups
% of post 11-day embryos with deformities
Differences in 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) sensitivity between
avian species is due, in part, to variations in amino acid substitutions of the
ligand-binding domain (LBD) found in the aryl hydrocarbon receptor (AhR).
Existing dose-response data for cohorts with known AhR constructs suggest
that avian species containing the AhR LBD as found in the White Leghorn
chicken will be most sensitive to dioxin-like compounds, those with constructs
characteristic of species including the Common pheasant will be moderately
sensitive and those with AhR LBD constructs characteristic of species
including the Japanese quail will be least sensitive. To confirm this hypothesis
and to test for applicability across TCDD-like compounds, a series of egg
injection studies was conducted with the aforementioned species. Data
associated with the effects of TCDD, 2,3,7,8-tetrachlorodibenzofuran (TCDF)
and 2,3,4,7,8-pentachlorodibenzofuran (PeCDF) on Common pheasant
hatchability, stage of embryo mortality and occurrence of deformities in
hatchlings are presented. Doses ranging from 0.0745 to 14.5 pmol/g egg (0.024
to 4.33 ng/g egg) were injected into the air cell prior to incubation. Hatchlings
were raised for 14 days post-hatch to assess growth and survivability. Samples
of chicks at day-of-hatch and 14 days-of-age were weighed, euthanized,
necropsied and processed for histological assessment. Subsamples of the
liver were analyzed for chemical concentration and cytochrome P4501A
induction. LD50 values (95% confidence intervals) based on hatchability for
TCDD, PeCDF and TCDF were 3.6 (2.3 – 6.3), 0.61 (0.28 – 1.2) and 1.2 (0.62 – 2.2)
pmol/g egg. The relative potencies (RePs) of PeCDF and TCDF compared to
TCDD were 5.8 and 3.0, respectively. The ReP for PeCDF from this study was
similar to the ReP value in a complimentary study based on induction of CYP1A
activity in cultured pheasant hepatocytes, while the ReP for TCDF was greater
in the present study compared to the ReP based on CYP1A activity (3.0
compared to 0.90). It would be of interest to determine if dietary exposure to
TCDD-like compounds in the Common pheasant results in similar ReP values
as determined by egg injection and enzyme induction studies.
0%
0.001
0.01
0.1
1
10
Log Dose (pmol/g egg)
Figure 1. Mortality of pheasant eggs injected with TCDD, PeCDF or TCDF. LD values
corrected for background mortality (18%).
o  TCDD was not the most potent TCDD-like compound for Common pheasants
  TCDD < TCDF < PeCDF
o  RePs (95% confidence intervals) were:
  PeCDF: 5.8 (5.4 – 8.4)
  TCDF: 3.0 (2.8 – 3.8)
•  ReP values greater than 1.0 are an unexpected finding based
on the current toxicity equivalency factor (TEF) system.
o  These results are similar to those from a complimentary in vitro
study in which the ReP for PeCDF in Common pheasants was also
greater than 1.0 based on CYP1A activity in cultured hepatocytes.
Funding for this project was provided through an un-restricted grant from The Dow Chemical Co., Midland, MI.