0.195
0.5
0.416
0.4
0.767
0.3
1.57
0.2
3.07
ou
10 - 14
days
Embryo
15 -18
days
Death
0.6
0.142
0.5
0.335
0.4
0.693
0.3
1.38
0.2
2.49
TCDD
0.27 [0.14 - 0.39]
0.087 [0.045 - 0.13]
0.8
PeCDF
TCDF
0.36 [0.27 - 0.44]
0.16 [0.090 - 0.23]
0.120 [0.092 - 0.15]
0.049 [0.028 - 0.070]
TCDD
0.66 [0.47 - 0.90]
0.21 [0.15 - 0.29]
0.6
PeCDF
TCDF
LD80
0.75 [0.64 - 0.87]
0.33 [0.23 - 0.45]
pmol/g egg
0.26 [0.22 - 0.30]
0.10 [0.070 - 0.14]
ng/g egg
TCDD
1.70 [1.2 - 2.8]
0.55 [0.39 - 0.90]
PeCDF
1.60 [1.3 - 2.0]
0.54 [0.44 - 0.68]
TCDF
0.69 [0.51 - 1.1]
0.21 [0.16 - 0.34]
Mortality
Gr
se
5-9
days
TCDD
PeCDF
LD80
TCDF
ng/g egg
LD50
15 -18
days
Death
19 days
- pipped
Dose
(pmol/g egg)
0.9
Vehicle Control
0.8
0.0742
0.7
0.148
0.6
0.245
0.5
0.516
0.4
1.05
0.3
1.83
0.2
4.02
o  To assess the relative potencies (RePs) of TCDF and PeCDF compared to
TCDD in the White Leghorn chicken (Gallus gallus domesticus).
0-4
days
0.0
0.001
0.01
0.1
Log Dose (pmol/g egg)
1
10
Figure 1. Mortality of chicken eggs injected with TCDD, PeCDF or TCDF. LD values corrected
for background mortality (13%).
se
0.0
Background Mortality
o  To confirm proposed avian sensitivity classification using TCDD, and two
TCDD-like compounds, PeCDF and TCDF.
Gr
0.1
Vehicle Control
5-9
days
Stage of
Do
0.2
ou
p
LD20
10 - 14
days
Embryo
TCDF Embryo Mortality Incidence
pmol/g egg
LD50
0.4
Do
0-4
days
Stage of
ng/g egg
0.0963
0.195
0.416
0.767
1.57
10 - 14
days
Embryo
15 -18
days
Death
3.07
Trunk
25%
Beak
3
Cranial
20%
Deformities
15%
2
10%
1
5%
0
0%
Vehicle
Control
0.0438
0.0867
19 days
- pipped
Figure 2. Effect of TCDD (top), PeCDF (middle), and
TCDF (bottom) on incidence of embryo mortality.
0.142
0.335
0.693
1.38
2.49
Dose (pmol/g egg)
TCDF Deformities
Trunk
100%
Beak
B
3
Incidence
0.0867
p
0.0438
0.7
0.0
pmol/g egg
0%
0.0494
Vehicle Control
1.0
Estimate [95% Confidence Interval]
5%
4
4
0.1
Congener
10%
1
Dose
(pmol/g egg)
0.8
Egg Mortality
15%
2
A
0.9
Tagged 2-day-old chicks
20%
Deformities
Vehicle
Control
19 days
- pipped
PeCDF Embryo Incidence
Brooder battery
Cranial
PeCDF Deformities
Dose (pmol/g egg)
Mortality   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
3
0
Gr
Stage of
25%
Beak
C
80%
Cranial
Deformities
60%
2
40%
1
20%
0
0%
Vehicle
Control
0.0742
0.148
0.245
0.516
1.05
1.83
4.02
% of post day-10 embryos with deformities
  Stage of embryo at death
  Presence and type of deformities
5-9
days
Trunk
Do
0-4
days
se
0.0
4
Incidence
0.6
TCDD Deformities
Incidence
0.0963
0.1
o  14-day-old chicks
Chick necropsy
0.0494
0.7
o  Unhatched eggs opened to determine:
o  Hatchlings transferred to brooder battery
Sealed egg
0.8
p
o  Eggs incubated until hatch or d 23
(37.6ºC, 52% humidity)
Sealing injection site with
melted paraffin
Injection with positive
displacement pipettor
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 (0.0494 – 3.07 pmol/g egg)
  PeCDF: 7 doses (0.0438 – 2.49 pmol/g egg)
  TCDF: 7 doses (0.0724 – 4.02 pmol/g egg)
Vehicle Control
ou
Hole drilled using Dremel® tool
Incidence
Candled egg with marked
injection side
Dose
(pmol/g egg)
0.9
% of post day-10 embryos with deformities
o  Injection of 0.1 µL/g egg into the air cell of
White Leghorn chicken eggs (58g) 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  2,200 eggs candled, weighed and
allocated into treatment groups
% of post day-10 embryos with deformities
Sensitivity to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-like compounds varies
between avian species. This variation is, in part, due to differences in amino
acid substitutions of the ligand-binding domain (LBD) found in the aryl
hydrocarbon receptor (AhR). A proposed avian sensitivity model for TCDD-like
compounds places species in one of three categories, where; those highly
sensitive to TCDD share an AhR LBD construct similar to the White Leghorn
chicken, species moderately sensitive share a construct similar to the Common
pheasant, and those least sensitive share constructs similar to the Japanese
quail. A series of egg injection studies was conducted to confirm the proposed
avian sensitivity classification. The effects of TCDD, 2,3,7,8-tetrachlorodibenzofuran (TCDF) and 2,3,4,7,8-pentachlorodibenzofuran (PeCDF) on White
Leghorn chicken hatchability, stage of embryo mortality and occurrence of
deformities are reported here. Doses ranging from 0.0438 to 4.02 pmol/g egg
(0.0149 to 1.23 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, and necropsied. Selected tissues from 14-day-old chicks
were removed, weighed and processed for histological assessment.
Subsamples of the liver were analyzed for chemical concentration and
cytochrome P4501A induction. Based on hatchability data, LD50 values (95%
confidence intervals) for TCDD, PeCDF and TCDF were 0.66 (0.47 – 0.90), 0.75
(0.64 – 0.87) and 0.33 (0.23 – 0.45) pmol/g egg, respectively. The relative
potencies (RePs) of PeCDF and TCDF compared to TCDD were 0.88 and 2.0,
respectively. The ReP for PeCDF from this study (0.88) is similar to the REP
from a complimentary study where the value based on CYP1A activity in
cultured chicken hepatocytes was 1.0. However, the ReP for TCDF from the
present study was greater compared to the REP based on CYP1A activity (2.0
vs. 0.9). It would be of interest to determine if dietary exposure to TCDD-like
compounds in the White Leghorn chicken results in similar REP values as
determined in the egg injection and enzyme induction studies.
Dose (pmol/g egg)
Figure 3: Deformities observed in chicken embryos exposed to TCDD (top-left), PeCDF (top-right), or TCDF (bottom-right).
Cranial deformities included exencephaly (A) or anencephaly (B) and microphthalmos or anophthalmos; beak deformities
included incomplete or lack of upper or lower beak development (C) and crossbill; trunk deformities included gastroschisis,
achondroplasia and edema.
o  There were no dose-related changes in body weight or relative organ weights in hatchlings and 14-day-old
chicks.
o  Species sensitivity based on LD50 values generated by probit analysis was:
  TCDD: Chicken > Pheasant > Quail
  PeCDF: Chicken ≈ Pheasant > Quail
  TCDF: Chicken > Pheasant > Quail
(Cohen-Barnhouse et al., 2008 [WP210]; 2009 [MP178])
o  TCDD was not the most potent TCDD-like compound for White Leghorn chickens
  TCDD ≈ PeCDF < TCDF
  RePs (95% confidence intervals) were:
•  PeCDF: 0.88 (0.74 – 1.0)
•  TCDF: 2.0 (2.01 – 2.02)
o  These results differ from a complimentary in vitro study where no significant differences between RePs
were found for these compounds based on CYP1A activity in cultured chicken hepatocytes.
Funding for this project was provided through an un-restricted grant from The Dow Chemical Co., Midland, MI.