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From the Institute of Occupational Health, Department of Industrial Hygieneand Toxicology, Haartmaninkatu 1, SF-
00290 Helsinki 29, Finland
Absrracr: Acrolein, four methacrylates, two guanidine compounds and resorcinol were tested for embryotoxi-
city on three day chicken embryos. The most potent chemical was acrolein, with the ED500.05 pmol per egg for
the total effect, including deaths and malformations. The substances next in potency were N,N’-di-o-tolyl-
guanidine and N,N-diphenylguanidine, with ED50 values 0. I7 and 0.20pmol per egg, respectively. Resorcinol
and the methacrylates had ED50 values ranging from 2.4to 22.0 Fmol per egg. Acrolein, diphenylguanidine,
tetrahydrofururylmethacrylate and trimethylolpropanetrimethacrylatecaused the largest amounts of mal-
formed embryos.
In mammals, malformations may result from direct acrolein and four methacrylates, as well as two
effects of the chemical on the embryo, from guanidine derivatives and resorcinol, which all are
destruction or modification of some essential phys- used in the rubber industry. Methacrylate esters are
iological function of the pregnant animal, or from used for manufacturing prosthetic devices and
the effect of a metabolite produced by the maternal artificials eyes.
tissues. Direct teratogens could be revealed by the
use of mammalian embryos in vitro. but the method
is laborious and not very suitable for screening Materials and Methods
large amounts of new chemicals. The chicken White Leghorn chicken eggs were obtained from the
embryo method offers a rapid and inexpensive hatchery of Siipikarjanhoitajain Liitto ry, Hameenlinna.
means to check direct teratogenicity of the chem- Finland. The commercial type incubator was made by
icals t o o n e kind of the amniote embryos (Kar- Hameen Insinooritoimisto Oy, Hameenlinna.
nofsky 1965). These results can be compared t o The following chemicals were tested: acrolein (ACR).
methylmethacrylate (MMA), tetrahydrofurfurylmetha-
results with mammalian embryos, in vitro and in crylate (THFMA), 1,3-butyleneglycoldimethacrylate
vivo, t o find out the principal ways of each chemical (BGDMA), trimethylolpropanetrimethacrylate (TPT-
to cause the malformations. MA), N,N’-di-o-tolylguanidine (DOTG), N,N’-diphenyl-
In a series of articles (Korhonen eta/. 1982a, b, c, guanidine (DPG) and resorcinol (RES). Acrolein was of
pract. grade. from Fluka AG, Bucks,Switzerland. All the
1983a & b) we have compared the embryotoxic
other chemicals were technical products from the rubber
effects of several chemicals, used in the manu- factory of Oy Nokia Ab, Nokia, Finland.
facturing of rubber, on three day chicken embryos. Acetone, p.a. grade, from E. Merck, Darmstadt.
In this communication the method is used to test Germany, was used as the solvent for the compounds.
96 AlLA KORHONEN ET AL.
The solution was injected into the egg in a total volume of Eggs containing dead embryos were counted and'dis-
5 pl. Ten control eggs, injected with 5 PI of acetone, were carded. The remaining eggs were then candled every
incubated with each batch of eggs. Results from solvent second or third day. Those containing dead embryos were
controls were not subtracted from experimental values. opened and the embryos checked for external malforma-
Three days old (72-76 hours) embryos were selected tions, and the developmental stage of Hamburger &
by candling. The method of dropping the solution onto Hamilton (1951). The incubation was terminated I I days
the inner shell membrane, on the heart of the embryo is after the injection, after a total incubation of 14 days. The
described in our earlier work (Korhonen et a/. 1982a). remaining eggs were opened and the embryos inspected
Two days after the injection, the eggs were candled again. for survival and for external malformations. The tem-
Table 1.
Embryotoxic effects of acrylates, guanidines and resorcinol o n three day chicken embryos.
~
Table 2.
ED50 and LD5O values for embryotoxic effects of acrylates, guanidines and resorcinol on chicken embryos.
Total effect
95 % Total mortal- Early deaths
confidence ity (on day 14) (days 3-5)
ED50 limits LD50 LD50
(pmoI/egg) (pmoI/egg) (tan a ) (pmoI/egg) (tan a ) (pmol/egg) (tan a )
ACR 0.05 0.03- 0.09 1.4 0.08 1.6 0.10 2.7
MMA 22.0 9.0 -56.0 0.45
THFMA 3.0 2.3 - 3.9 1.5 3.8 1.6 4.1 1.6
BGDMA 2.5 2.1 - 3.1 1.3 2.8 1.3 2.8 1.3
TPTMA 7.8 6.4 - 9.6 1.4 9.3 1.3 13 1.o
DOTG 0.17 0.12- 0.22 1.3 0.2* 8.0* 0.2* 8.0'
DPG 0.20 0.17- 0.25 1.4 0.29 1.4 0.31 1.4
RES 2.4 2.0 - 2.8 2.4 2.7 1.6 2.9 3.9
* Approximated on probit paper.
905 a1 a5 1.0 w 10 50
DOSE (JImol/egg)
Fig. 1. The dose-response curves for malformed embryos produced by acrolein, methacrylates, guanidines and
resorcinol. All malformed embryos surviving the first two days after the treatment are included.
98 AILA KORHONEN ET AL.
followed the order of the ED50 values for the total Umbilical hernias or totally open coelomic cav-
effect. ities were common in series treated with ACR a n d
The LD50 values for early deaths o n day 5 were DPG. Short and twisted backbone occurred fre-
obtained for all the chemicals except MMA. T h e quently in embryos treated with T P T M A .
dose-response curves for the early deaths were very D O T G a n d D P G caused the highest percentages
steep, when D O T G , ACR and RES were used of wing defects. T h e wing could be totally absent,
(table 2). or it could lack the upper arm.
Dose-response curves were plotted for malfor- Absence of legs or short and twisted legs were
med embryos (fig. 1). ACR, and D P G were the observed occasionally, as were oedema and en-
most efficient teratogens, while RES and B G D M A larged lymph sacks o n the rump.
caused few malformed embryos. M M A caused a Single cases of other defects were observed.
constant percentage of malformations in a long These included different degrees of underdevelop-
dose range. T P T M A caused a high percentage of ment of the head and brain, cleft lip, short maxilla,
malformations at the highest tested dose, which underdevelopment of the hip and encephalocoele
was 5 pl of pure TPTMA. or a smaller “pimple” on the head. Several embryos
The malformation types, that are caused in three were firmly attached to the amniotic and inner shell
day chicken embryos by different chemicals, seem membranes a t the point of injection, and died at
t o be very similar (Korhonen e / a/. 1 9 8 2 ~ ) . early stages. Five such embryos were produced by
The malformations caused by the chemicals TPTMA.
tested in this work are given in table 3. T w o types of Late deaths of externally normal embryos oc-
eye defects were observed. One was with a small, curred occasionally. A few cases were produced by
but otherwise normal eye cup, the other with ACR and TPTMA. These chemicals killed also
absence of lids and cornea, or parts of them, but the some malformed embryos after day 5 (table I ) .
eye cup intact. In most embryos with the latter
defect type, the amniotic membrane was attached
Discussion
to the defected area, and often t o the shoulder,
around the wing area, too. When a test chemical can be freely added to the
The small eye cup was relatively uncommon in embryo, malformations can occur only in a limited
all series. The corneal defect was frequently present dose range. Very high doses of any substance will
in embryos treated with ACR, MMA, T H F M A and kill the embryo, before malformations are mani-
DOTG. fested. For practical reasons, two days was con-
Table 3.
Incidence of different malformation types produced by acrylates, guanidines and resorcinol in three day chicken
embryos.
Number of
Corneal Neck Defects Defects Oedema malformed Number
Small & lid Open & back of of or lymph embryos of mal-
eye cup defects coelom defects wings legs blebs Other’ (100%) formations
N (%I) N (‘70) N (%) N (%I) N ( % I ) N ( % I ) N (9‘0) N ( % ) N N
ACR 9 (50) 16 (89) 6 (33) 2 ( 1 1 ) I (6) 3 (17) 18 47
MMA I ( 5 ) 15 (71) 4 (19) 3 (14) 2 (10) 1 (5) 2 (10) 21 28
THFMA 2 (20) 5 (50) I (10) 2 (20) 2 (20) 10 12
BGDMA 2 (67) I (33) 3 3
TPTMA 3 (19) 2 (13) 4 (25) 7 (44) I (6) I (6) 8 (50) 16 26
DOTG 3 (30) 5 (50) 3 (30) 4 (40) 10 15
DPG 2 (12) 7 (41) 10 (59) I (6) 6 (35) 1 (6) 2 (12) 2 (12) 17 31
RES 3 (60) 2 (40) 1 (20) I (20) I (20) 5 8