Fd Cosmet. ToxicoL Vol. 9, pp. 405-412. Pergamon Press 1971.

Printed in Great Britain

Study of the Teratogenicity of 2,3,7,8-Tetrachlorodibenzo-p-dioxin

in the Rat
G. L. SPARSCHU,F. L. DUNN and V. K. ROWE
Biochemical Research Laboratory, The Dow Chemical Company,
Midland, Michigan 48640, USA

(Received 9 November 1970)

Abstraet--2,3,7,8-Tetrachlorodibcnzo-p-dioxin was given orally to pregnant rats on days 6-15

of gestation, at levels of 0 (control), 0"03,0"125,0"5, 2"0 and 8"0/~g/kg/day.No adverse effect on
the foetuses was noted at the 0.03-/~g/kg level, but in the groups given doses of 0.125-2.0
/~g/kg, foetal mortality, early and late resorptions and foetal intestinal haemorrhagc were
observed, with the incidence increasing as the dose increased. There was no evidence of
maternal toxicityat 0"03and 0"125/~g/kg/day,but at 0.5 and 2"0/~g/kg/daythem was a decrease
in maternal weight gain. Severe maternal toxicity and embryotoxicity were evident at the
8.0-t~g/kg/day dose, maternal weight loss and early resorptions occurring in all cases. The
results suggest that teratogenic effects attributed earlier to 2,4,5-tdchlorophenoxyaceticacid
may have been due to 2,3,7,8-tetrachlorodibcnzo-p-dioxin,which was present as a contaminant
at a level of about 30 ppm in the sample of the herbicide tested.

INTRODUCTION
In the latter part of 1969, it was revealed by the National Cancer Institute that a study
conducted at the Bionetics Research Laboratories, Division of Litton Industries, had shown
2,4,5-trichlorophenoxyacetic acid (2,4,5-T) to be teratogenic (Courtney, Gaylor, Hogan,
Falk, Bates & Mitchell, 1970).
During the process of 2,4,5-T manufacture, it is possible for varying amounts of 2,3,7,8-
tetrachlorodibenzo-p-dioxin to be formed. A small sample of the 2,4,5-T used in the Bio-
netics study was obtained and, when analysed by The Dow Chemical Co., Midland, Mich.,
was found to contain approximately 30 ppm 2,3,7,8-tetrachlorodibenzo-p-dioxin. This
quantity of impurity represents appreciable contamination because the content of the
2,3,7,8-tetrachlorodibenzo-p-dioxin in most commercial samples of 2,4,5-T is less than
1 ppm. Therefore a study of 2,3,7,8-tetrachlorodibenzo-p-dioxin was initiated to evaluate
its foetotoxic and teratogenic properties.
Past knowledge and experience have shown 2,3,7,8-tetrachlorodibenzo-p-dioxin to be
highly toxic. The LDso for male and female rats is 23 and 45/~g/kg respectively, while that
for male guinea-pigs is 0.6 ~g/kg (The Dow Chemical Co., unpublished data. Kimmig
& Schulz (1957) concluded that 2,3,7,8-tetrachlorodibenzo-p-dio×in (alternatively numbered
and designated 2,3,6,7-tetrachlorodibenzodioxin) was responsible for human cases of
chloracne, and showed that it had strong chloracne-inducing activity in the rabbit-ear test.
Furthermore, 2,3,7,8-tetrachlorodibenzo-p-dioxin is extremely toxic in the chick-embryo
assay, producing 100~o mortality at a dosage level of 0.05 ~g/egg (Higginbotham, Huang,
Firestone, Verrett, Ress & Campbell, 1968).
405
406 G. L. SPARSCHU, F. L. DUNN and V. K. ROWE

EXPERIMENTAL
Material. The 2,3,7,8-tetrachlorodibenzo-p-dioxin used in this study was a white solid of
m.p. 303-305°C (elemental analysis: C, 45, H, 1.34 and CI, 42 .7/o, o/. theoretical values C,
44.7, H, 1.25 and CI, 44-0~). Using mass spectrometric analysis, the composition was
found to be: <0"02~o dichloro-, 7~o trichloro-, 9 1 ~ tetrachloro- and 2~o pentachloro-
dibenzo-p-dioxin.
Animals and husbandry. Sprague-Dawley (Spartan strain) rats were used in this study.
Virgin female rats approximately 130 days old (body weight 290-300 g) were mated with
110-day-old male rats. After breeding, the females were housed individually and maintained
on Laboratory Chow (Ralston Purina Co., St. Louis, Missouri) and tap water ad lib.
Experimental design and conduct. Untreated females were mated with untreated males and
the day on which sperm was observed in the vaginal smears was designated day 0 of gesta-
tion. The females were divided into five treatment groups comprising 10-14 animals and a
control group of 31 animals. Oral administration of 2,3,7,8-tetrachlorodibenzo-p-dioxin, by
gavage, was begun on day 6 and concluded on day 15 of gestation.
Preparation of the solutions for oral administration entailed dissolving the 2,3,7,8-
tetrachlorodibenzo-p-dioxin in acetone and adding an appropriate aliquot of the acetone
solution to corn oil (USP), making a 9 : 1 corn oil-acetone solution. The concentrations of
the 2,3,7,8-tetrachlorodibenzo-p-dioxin corn oil-acetone solutions used for dosing at levels
of 0.03, 0-125, 0.5, 2-0 and 8.0/zg/kg/day were 1.25, 5.0, 20.0, 80.0 and 320.0 tzg/100 ml
respectively. The total volume of test solution given to each dam was between 0.6 and
0.8 ml/day, and the control rats were given an equivalent volume of the corn oil-acetone
vehicle. All doses were based on body weights at day 0.
The female rats were observed daily throughout the experimental period for indications
of toxicity. Maternal body weights were recorded on days 0, 6, 13 and 20 of gestation.
Pregnancy was terminated on day 20 by decapitation of the females and Caesarean section.
The corpora lutea were counted and implantation sites were inspected in situ. Resorptions
were classified as early or late. Only resorptions with foetal tissue readily apparent were
classified as late. Non-viable foetuses that did not show maceration or liquefaction were
classified as dead foetuses and not as resorptions. All live and dead foetuses were removed,
weighed, sexed and examined for external malformations. They were also examined for the
presence of haemorrhage in the gastro-intestinal tract by incision of the abdominal wall and
exposure of the stomach and intestine. One-third of the foetuses from each litter were fixed
in 95 ~ ethanol prior to clearing and alizarin-red staining for skeletal examination. The
remaining two-thirds were placed in Bouin's fixative for subsequent Wilson sectioning
(Wilson & Warkany, 1965) of the head, neck and thorax, and dissection of the abdominal
viscera. In addition, representative sections of foetal thoracic and abdominal viscera were
sectioned at 6 # and stained with haematoxylin and eosin for histological evaluation.

RESULTS
No adverse clinical signs were seen in any of the dams in the control group or in the
experimental groups at dosage levels of 0.5/Lg/kg/day or less. Of 11 dams given 2.0/zg/kg/
day, three had vaginal haemorrhage from day 15-20 of gestation. Six of the eight dams
given doses of 8.0 t~g/kg had vaginal haemorrhage at various intervals from days 13 to 20
of gestation. One dam died several hours before Caesarean section. The signs prior to
death in this animal consisted of vaginal haemorrhage of 5 days duration and extreme
 TERATOLOGY OF 2,3,7,8-TETRACHLORODIBENZO-p-DIOXIN 407

p a l l o r o f the m u c o u s m e m b r a n e s a n d eyes. In general, the females on the 8-0-t~g/kg dose

level were thin and showed signs o f debilitation.
M a t e r n a l body-weight gains were depressed in d a m s given doses o f 0.5, 2.0 and 8.0
t~g/kg/day but not at the lower dosage levels (Table 1). D e p r e s s i o n o f weight gain was slight
b u t statistically significant at 0.5 t~g/kg, essentially no weight gain occurred at 2-0 t~g/kg a n d
a p p r e c i a b l e weight loss occurred at 8-0/zg/kg.

Table 1. Body weights of pregnant rats treated orally with 2,3,7,8-tetrachlorodibenzo-p-dioxin on

days 6-15 of gestation

Body weight (g) on day

Dose No. of
(v~g/kg/day) rats 0 6 13 20

0 (control) 24 298 318 347 423

0"03 10 290 325 340 416
0"125 10 303 328 354 435
0"5 12 290 311 323* 388*
2-0 11 296 320 322* 327**
8-0 8 295 317 302* 284**

Values are means from the numbers of animals stated and those marked with asterisks differ
significantly (Student's t test) from control values: *P< 0"01 ; **P< 0.001.

The n u m b e r o f pregnancies in each g r o u p a n d the total a n d mean n u m b e r o f i m p l a n t a t i o n

sites and c o r p o r a lutea are shown in Table 2. N o significant difference was a p p a r e n t in the
mean n u m b e r o f i m p l a n t a t i o n sites or c o r p o r a lutea. The n u m b e r o f viable foetuses,
r e s o r p t i o n sites and d e a d foetuses at d a y 20 o f gestation are given in Table 3. A t 0.03
/zg/kg/day, no deviation f r o m n o r m a l was seen, whereas at 0.125 t~g/kg/day there were
three dead foetuses. A t b o t h levels all resorptions occurred early, as they did in the controls.
In the g r o u p given doses o f 0-5 t~g/kg the n u m b e r o f viable foetuses was reduced slightly,
resorptions (early and late) were increased, a n d there were six d e a d foetuses. A t 2.0/zg/kg
only seven live foetuses were f o u n d a n d all o f these occurred in f o u r o f the 11 litters; b o t h
early a n d late resorptions were found. A t 8.0 tLg/kg there were no live foetuses a n d all
resorptions had occurred early in pregnancy.

Table 2. Numbers of pregnancies, implantations and corpora lutea in female rats treated orally with
2,3,7,8- tetrachlorodibenzo-p-dioxin on days 6-15 of gestation

Implantation sites Corpora lutea

Dose
(tzg/kg/day) Fertility* Total Mean/dam 4- SD Total Mean/dam 4- SD

0 (control) 24/31 309 13 4- 3.7 419 17 + 3"4

0-03 10/13 147 15 4- 2.5 184 18 4- 3.5
0'125 10/13 146 15 4- 3-6 202 20 4- 3"3
0.5 12/14 157 13 4- 3-6 205 17 4- 3.4
2-0 11/14 136 12 4- 3.8 198 18 -4- 7-0
8"0 8/10 110 14 4- 1"9 168 21 4- 4-6

*No. of pregnancies/no, of rats bred.

FOOD 9 / 3 - - F
 QO

P
Table 3. Numbers of viable foetuses, resorption sites and dead foetuses from female rats treated orally with
.r-
2,3,7,8-tetrachlorodibenzo-p-dioxin on days 6-15 of gestation

Viable foetuses Resorption sites

Dose No. of Total dead
~g/kg/day) litters examined Total Mean/litter 4- SD Early Late Total Mean/litter 4- SD foetuses .m
.r-
0 (control) 24 246 10 4- 4.3 63 0 63 3 4- 2"8 ~7
0"03 10 115 12 4- 3"8 31 0 31 3 4- 2"0
0"125 I0 124 12 4- 2"9 19 0 19 2 4- 1.1 ;Z
0.5 12 93 8 + 4"1" 32 26 58 5 4- 2.3* Z
2"0 11 7t -- 87 42 129 12 4- 3"9**
8"0 8 0 -- 110 0 110 14 4- 1"9'*
<

tThe viable foetuses were from four litters.

Values marked with asterisks differ significantly (Student's t test) from the control values : *P< 0"05 ; **P< 0.001. o
t~
 TERATOLOGY OF 2,3,7,8-TETRACHLORODIBENZO--p-DIOXIN 409

Table 4 shows the sex distribution and mean b o d y weights o f the viable foetuses at all
dosage levels. The sex ratios at 0.03 and 0.125 /zg/kg" were c o m p a r a b l e to those in the
controls, but at 0-5 and 2.0/~g/kg there was a t r e n d suggesting that the males were m o r e
susceptible to the foetotoxic action o f 2,3,7,8-tetrachlorodibenzo-p-dioxin. There was not,
however, a statistically significant difference in the sex ratios at these two dosage levels.
At 0.03/zg/kg the foetal weights were similar to those in the control group. A t b o t h 0.125
and 0.5/~g/kg the weights o f the foetuses were very slightly depressed, a n d at 2.0/zg/kg the
foetuses were distinctly smaller, being only a b o u t two-thirds o f the weight o f the c o n t r o l
foetuses.

Table 4. Body weights and sex ratios of foetuses from female rats treated orally with
2,3,7,8-tetrachlorodibenzo-p-dioxin on days 6-15 of gestation

Males Females

Dose No. of Mean foetal No. of Mean foetal Sex ratio

(/~g/kg/day) live foetuses weight (g) q- SD live foetuses weight (g) 4- SD M:F

0 (control) 117 4"03 5:0"37 129 3"89 ± 0"39 48:52

0.03 55 4"14 q- 0"26 60 3.98 ± 0"35 48:52
0.125 66 3.85 4- 0"35** 58 3.71 ± 0"37** 53:47
0"5 39 3"86 4- 0"61" 54 3"78 ± 0"54 42:58
2-0 3 2"72 4- 0'25*** 4 2"69 4- 0"19"** 43:57
8"0 0 - - 0 -- - -

Values marked with asterisks differ significantly (Student's t test) from the control values:
*P< 0"05; **P< 0"01 ; ***P< 0-001.

Table 5. Gross abnormalities in foetuses from female rats treated orally with
2,3,7,8-tetrachlorodibedzo-p-dioxin on days 6-15 of gestation

Intestinal haemorrhage

Dose Foetuses affected/ Litters affected/ Subcutaneous Tail and

~g/kg/day) foetuses examined litters examined oedema limb malformations

0 (control) 0/246 0/24 0 0

0.03 0/115 0/10 0 0
0.125 18/127 7/10 0 1
0.5 36/99 10/12 0 0
2.0 4/7 2/4 3 1
8.0 . . . .

The abnormalities observed during e x a m i n a t i o n o f individual foetuses at the time o f

Caesarean section are s u m m a r i z e d in Table 5. N o a b n o r m a l i t i e s were observed in t h e
control foetuses o r in the foetuses from the g r o u p receiving 2,3,7,8-tetrachlorodibenzo-p-
dioxin in doses o f 0.03/~g/kg. F o e t a l intestinal h a e m o r r h a g e was observed with increasing
frequency at dosage levels o f 0.125, 0-5 a n d 2.0/~g/kg. Occasionally gastric h a e m o r r h a g e
was observed, b u t only in conjunction with severe intestinal h a e m o r r h a g e . There was a
combined t o t a l o f 58 foetuses with intestinal h a e m o r r h a g e , 27 male a n d 31 female. Sub-
cutaneous o e d e m a was seen macroscopic.ally in three o f the seven foetuses from the g r o u p
 0
r,
Table 6. Soft tissue abnormalities in foetuses from female rats treated orally with 2,3,7,8-tetrachlorodibenzo-p-dioxin
on days 6-15 of gestation

Dilated renal pelvis Hydroureter

Dose No. of Subcutaneous Dilated lateral
/.tg/kg/day foetuses examined Right Left Bilateral Right Left Bilateral oedema ventricles
.w

0 (control) 154 11 7 8 6 4 5 0 0
0"03 73 I0 I 3 3 0 2 0 0
0"125 80 I0 I 1 1 0 0 22 0
0'5 65 l 5 lO 0 0 2 31 I
2"0 4 1 0 0 0 0 0 4 l .<
8"0 0 . . . . . . .

r~
 TERATOLOGYOF 2,3,7,8-TETRACHLORODIBENZO-p-DIOX1N 411

on 2.0/zg/kg/day. Oedematous foetuses were not observed macroscopically at lower doses.

The malformations observed consisted of a rudimentary tail in one foetus from the group
given 0.125 /~g/kg/day, and two misshapen limbs and a kinked tail in a foetus from the
2-0-/zg/kg group. Examination of the skeletal preparation of the latter foetus revealed that
the visible limb malformations were not of skeletal origin.
The results of the examination for soft tissue abnormalities are given in Table 6. The
frequency of dilated renal pelvis and hydroureter among the various groups did not indicate
a relationship to compound or dose. Subcutaneous oedema, however, while not observed
in the controls or in the 0.03-tzg/kg dosage group, was apparent, after Wilson sectioning,
in a considerable number of the foetuses from the higher dosage levels.
Skeletal examinations (Table 7) revealed delayed ossification of some sternebrae and skull
bones and wavy thirteenth ribs, but these findings occurred throughout the various groups,
including controls.

Table 7. Skeletal abnormalities in foetuses from female rats treated orally with
2,3,7,8-tetrachlorodibenzo-p-dioxin on days 6-15 of gestation

Dose ~g/kg/day)

Skeletal abnormalities 0 0-03 0.125 0"5 2"0 8"0

Number of foetuses examined 92 42 44 28 3 0

Incompletely ossified sternebrae
Fifth 51 28 15 19 0 --
Second and fifth 23 10 21 9 0 --
Multiple 14 1 4 8 3 --
Malaligned sternebrae 1 0 0 0 0 --
Wavy ribs (thirteenth) 7 4 7 20 0 --
Incomplete ossification
Interparietal 8 3 10 9 3 --
Parietals 5 2 5 12 2 --
Frontals 0 0 2 1 2 --
Occipital 2 0 0 0 2 --
Nasal 0 0 0 5 2 --

Microscopic findings in the foetuses confirmed gross observations. The only renal lesions
observed were varying degrees of dilated renal pelves in the control and test foetuses.
Gastro-intestinal lesions were confined to the foetuses from dams given 2,3,7,8-tetra-
chlorodibenzo-p-dioxin doses of 2.0, 0.5 and 0.125 /zg/kg and consisted of luminal
haemorrhage. Occasionally in the intestine there was haemorrhage in the lamina propria
and submucosa with focal necrosis and sloughing of the mucosa.

DISCUSSION
Courtney et aL (1970) reported an increase in mortality, gastro-intestinal haemorrhage
and cystic kidneys in rat foetuses from dams treated with 2,4,5-T containing approximately
30 ppm 2,3,7,8-tetrachlorodibenzo-p-dioxin. The doses of 2,4,5-T administered were 4.6,
10.0 and 46.4 mg/kg/day, which included approximately 0.15, 0-3 and 1-5 /zg 2,3,7,8-
tetrachlorodibenzo-p-dioxin/kg/day.
On the basis of the dosage levels of 2,3,7,8-tetrachlorodibenzo-p-dioxin, the observations
of intestinal haemorrhage and foetal mortality in the present study are similar to the
412 G . L . SPARSCHU, F. L. DUNN and V. K. ROWE

findings o f C o u r t n e y et al. (1970). H o w e v e r , cystic kidneys were n o t o b s e r v e d in the present

study after gross or histological e x a m i n a t i o n o f the foetuses. N o m o r p h o l o g i c a l d e s c r i p t i o n
was given by C o u r t n e y et al. (1970) to s u b s t a n t i a t e t h e use o f the t e r m 'cystic k i d n e y ' .
I n t h e p r e s e n t study, a high i n c i d e n c e o f m a t e r n a l a n d foetal toxicity was a s s o c i a t e d with
t h e oral a d m i n i s t r a t i o n o f 2 , 3 , 7 , 8 - t e t r a c h l o r o d i b e n z o - p - d i o x i n to p r e g n a n t rats on days 6 - 1 5
o f gestation. T h e p r e s e n c e o f 2 , 3 , 7 , 8 - t e t r a c h l o r o d i b e n z o - p - d i o x i n in the s a m p l e o f 2,4,5-T
tested in rats in the Bionetics L a b o r a t o r i e s c o u l d well h a v e a c c o u n t e d f o r the r e p o r t e d
o b s e r v a t i o n s which were a t t r i b u t e d to 2,4,5-T.

Acknowledgements--The authors thank Mrs. Roselle Lisowe and Mrs. Susan McCollister for their expert
technical assistance. They also express their gratitude to Dr. N. Skelly for the synthesis of the 2,3,7,8-
tetrachlorodibenzo-p-dioxin sample and to Dr. R. LaVier for his helpful advice.

REFERENCES
Courtney, K. Diane, Gaylor, D. W., Hogan, M. D., Falk, H. L., Bates, R. R. & Mitchell, I. (1970). Tera-
togenic evaluation of 2,4,5-T. Science, N. Y. 168, 864.
Higginbotham, G. R., Huang, Anita, Firestone, D., Verrett, Jacqueline, Ress, J. & Campbell, A. D. (1968).
Chemical and toxicological evaluations of isolated and synthetic chloro derivatives of dibenzo-p-dioxin.
Nature, Lond. 220, 702.
Kimmig, J. & Schulz, K. H. (1957). Occupational chloracne caused by aromatic cyclic ethers. Dermatologica
115, .540.
Wilson, J. G. & Warkany, J. (1965). Teratology: Principles and Techniques. Ist ed. p. 263. The University of
Chicago Press, Chicago.

Etude des effets t~ratog~nes de la 2,3,7,8-t~trachioro-dibenzo-p-dioxine chez

le rat
R&~um6---De la 2,3,7,8-t6trachloro-dibenzo-p-dioxine a 6t6 administr6e par vole orale ~. des
rats femelles gravides, du 6e au 15e jour de gestation, aux doses de 0 (t6moin), 0,03, 0,125,
0,5, 2,0 et 8,0 ag/kg/jour. La dose de 0,03/Lg/kg n'a eu aucun effet nocif sur les foetus. De la
mortalit6 foetale, des r6sorptions pr6coces et tardives et des h6morragies intestinales foetales
ont 6t6 observ6es dans les groupes qui recevaient de 0,125 h 2,0 tzg/kg. La fr6quence de ces
accidents augmentait avec la dose. Aucun indice de toxicit6 pour la m6re n'a 6t6 relev6 aux doses
de 0,03 et de 0,125 ~g/kg/jour, mais le gain de poids des m~res ralentissait aux r6gimes 5. 0,5 et
de 2,0 t~g/kg/jour. La dose de 8,0/~g/kg/jour a provoqu6 dans tousles cas des pertes de poids
chez les m~res et des r6sorptions foetales pr6coces, ce qui d6montre sa nette toxicit6 chez la
m/~re et son embryotoxicit6. Les r6sultats sugg~rent que les effets t6ratog~nes attribu6s aupara-
vant b. racide 2,4,5-trichloro-ph6noxy-ac6tique pourraient 6tre le fair de la 2,3,7,8-t6trachloro-
dibenzo-p-dioxine: l'6chantillon d'herbicide utilis6 pour l'essai en 6tait pollu6 :~ 30 ppm
environ.

Untersuchung der Teratogenitfit von 2,3,7,8-Tetrachlordibenzo-p-dioxin an der

Ratte
Zusammenfassung--2,3,7,8-Tetrachlordibenzo-p-dioxin wurde oral an trfichtige Ratten am 6.
bis 15. Tag der T#ichtigkeit in Konzentrationen von 0 (Kontrolle), 0,03, 0,125, 0,5, 2,0 und 8,0
vg/kg/Tag verabreicht. Bei der Konzentration 0,03/*g/kg wurde keine nachteilige Wirkung auf
die Foeten bemerkt, aber in den Gruppen, die Dosen yon 0,125-2,0 t-*g/kg erhielten, wurden
totale Mortalitiit, friJhe und spiite Resorptionen und foetale Eingeweideblutungen beobachtet,
wobei die Hiiufigkeit mit der Erh/Shung der Dosis zunahm. Es gab kein Anzeichen von Toxizitiit
des Muttertieres bei 0,03 und 0,125 t~g/kg/Tag, aber bei 0,5 und 2,0 ~g/kg/Tag kam es zu einer
Verminderungder Gewichtszunahme der Muttertiere. Starke Muttertiertoxizitiit und Embryo-
toxizit~.t war bei derDosis 8,0tzg/kg/Tag zu beobachten, wo in allen Fiillen Gewichtsverluste der
Muttertiere und Friihresorptionen eintraten. Die Ergebnisse lassen annehmen, dass die friiher
der 2,4,5-Trichlorphenoxyessigsiiure zugeschriebenen teratogenen Wirkungen dem 2,3,7,8-
Tetrachlordibenzo-p-dioxin angelastet werden miissen, das als Verunreinigung in einer Kon-
zentration yon etwa 30 ppm in der Probe des gepriaften Herbicids vorhanden war.