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Aria pharmacol. et toxicol. 1983, 5 2 , 95-99.

From the Institute of Occupational Health, Department of Industrial Hygieneand Toxicology, Haartmaninkatu 1, SF-
00290 Helsinki 29, Finland

Embryotoxic Effects of Acrolein, Methacrylates, Guanidines and


Resorcinol on Three Day Chicken Embryos
BY
Aila Korhonen, Kari Hemminki and Harri Vainio
(Received May 6, 1982:Accepted August 23, 1982)

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.

Key-words: Embryotoxicity - chicken embryo - acrolein - methacrylate - guanidine - resorcinol.

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.
~

Late deaths All


Dose Non- Mal- affected
(wol Treated Early deaths malformed formed embryos
Treatment Per egg) N N (%I N (%) N (%) N (%)
ACR 0.2 30 30 (100)
0.1 30 23 (77)
0.05 30 14 (47)
0.025 20 0 (0)
MMA 36 30 20 (67)
18 30 8 (27)
9 30 9 (30)
4.5 30 9 (30)
2.3 20 0 (0)
THFMA 11 20 19 (95)
5.5 30 26 (87)
2.8 30 13 (43)
1.4 20 0 (0)
BGDMA 11 20 20 (100)
5.6 30 26 (87)
2.8 30 18 (60)
1.4 30 5 (17)
0.7 20 0 (0)
TPTMA I5 30 28 (93)
7.3 30 9 (30)
3.7 30 5 (17)
1.9 10 0 (0)
DOTG 0.40 20 20 (100)
0.20 30 20 (67)
0.10 30 3 (10)
0.05 30 3 (10)
DPG 0.96 20 20 (100)
0.48 30 28 (93)
0.24 30 17 (57)
0.12 30 4 (13)
0.06 20 2 (10)
RES 7.3 20 20 (100)
3.6 30 27 (90)
1.8 30 4 (13)
0.9 30 1 (3)
AC 5P 1 90 3 (3)
EMBRYOTOXICITY OF RUBBER INDUSTRY CHEMICALS 97

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.

perature was kept at 37.7" and the humidity between 66'31


Results
and 71%) throughout the incubation period. The eggs
were turned 2 to 4 times per day. The results are shown in table 1. The acetone
The affected embryos were classified into the following
background was low, 3 % for the total effect,
categories:
I . Early deaths, embryos which died before day 5 of the including malformations and deaths.
incubation, within two days of the treatment, The most potent of the chemicals was acrolein,
2. Late deaths, non-malformed, externally normal em- with the total effect ED50 0.05 pmol per egg(table
bryos which died between days 5 to 14, 2). The next potent were DOTG, with the ED50
3. Late deaths, malformed, externally malformed em-
0.17 pmol per egg, and DPG 0.20 pmol per egg.
bryos which died between days 5 to 14.
4. Malformed survivors, externally malformed embryos RES and the four methacrylates were much less
alive on day 14 of the incubation. potent, with ED50 values ranging from 2.4 to 22
LD50 and ED50 values were calculated according to pmol per egg. The dose-response curve for MMA
Rosiello ef a/. (1977). using a Wang table computer. was very flat, with the tan a 0.45 (table 2).
The LD50 value for DOTG was approximated on a
probit paper. The curve was so steep, that doubling the LD50 values for total mortality on day 14 of the
dose did not produce enough points for calculation with incubation could be calculated for all the tested
the computer. chemicals excluding MMA (table 2). These values

~ 4 ...*..-. " -....-....- .."...........- ..-".-.-.--

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

’ See text for details.


EMBRYOTOXICITY OF RUBBER INDUSTRY CHEMICALS 99
sidered as the limit for such early deaths in this 4,4'-dithiodimorpholine with the ED50 0.09 pmol
method. Embryos that died during the first two per egg (Korhonen et al. 1982c) and N-pheny1-n'-
days after injection, were difficult to inspect and the isopropyl-p-phenylenediamine with the ED50 value
observation of malformations was very uncertain. 0.11 pmol per egg (Korhonen et al. 1983b) have
The width of the teratogenic dose range in this been more potent than the guanidines. MMA, that
work is considered t o be limited by the LD50 for was much less potent in ourtest than theguanidines,
the early deaths and by the ED50 for the total has been found to be teratogenic in rats at a dose of
effect. The low number of non-malformed late 0.44 ml/kg (Singh et al. 1972).
deaths, that added t o the total effect, has no
practical effect on these numbers. Acknowledgements
Excluding MMA, the tested chemicals caused The project has been supported by the Swedish
complete, regular and steep dose-response curves Work Environment Fund.
for all the measured effects, including the early
deaths. MMA probably diffused poorly from the
air chamber to the embryo. MMA can obviously
cause malformations in the chicken embryo, but
the quantitative relations remain obscure. The References
delay in its penetration through the albumen and
Hamburger, V. & H. L. Hamilton: A series of normal
the vitelline membrane is specific for this method stages in the development of the chick embryo. J.
only. This delay prevents the exact determination Morphol. 195I , 88, 49-92.
of the two median doses, which limit the tcratog- Kankaanpaa, K. J., E. Elovaara & H. Vainio: Embryo-
enic dose range in its both ends. toxicity of acrolein, acrylonitrile and acrylamide in
The effect of the width of the teratogenic dose developing chick embryos. Toxicol. Lett. 1979, 4,
93-96.
range is well illustrated by fig. I . ACR, D P G , Karnofsky, D. A.: Mechanisms of action of certain
T H F M A , and TPTMA, with their largedifferences growthinhibiting drugs. In: Teratology, principles and
between the ED50 and LD50 values, produce the techniques. Eds.: S. G . Wilson and J. Warkany. The
largest amounts of malformed embryos. RES and University of Chicago press, 1965, pp. 185-213.
especially BGDMA had very small differences Korhonen, A,, K. Hemminki & H. Vainio: Application
of the chicken embryo in testing for embryotoxi-
between the LD50 for early deaths. and the ED50. city. Thiurams. Scand. J. Work, Environ. Health 1982a.
They produced small amounts of malformed em- 8, 63-69.
bryos. MMA seems t o have a very long teratogenic Korhonen, A,, K . Hemminki, & H. Vainio: Embryo-
dose range. This may result from its poor penetra- toxicity of industrial chemicals on the chicken embryo.
tion into the embryo. Independent of the dose Thiourea derivatives. Acta pharmacol. et toxicol.
1982b, 51, 3844.
injected into the air chamber, only a teratogenic Korhonen, A,, K. Hemminki & H. Vainio: Embryo-
dose could penetrate t o the embryo during the first toxicity of benzothiazoles, benzenesulfohydrazide and
two days. dithiodimorpholine to the chicken embryo. Arch. En-
Until now, the results from tests with 42 chem- viron. Contam. Toxicol. 1982~.11, 753-759.
Korhonen, A,, K . Hemminki, & H. Vainio: Embryo-
icals are available in our laboratory (Korhonen et
toxicity of industrial chemicals on the chicken embryo.
a/. 1982a,b,c, 1983a & b). In addition, 13chemicals Dithiocarbamates. Teratogenesis, Carcinogenesis and
have been tried, which produced no effects at doses, Mutagenesis 1983a, in press.
that could be injected. The most potent of the tested Korhonen, A,, K. Hemminki & H. Vainio: Embryo-
chemicals, the dithiocarbamates, had ED50 values toxicity of sixteen industrial amines to the chicken
embryo. J. Appl. Toxicol. 1983b. in press.
up from 0.0025 pmol per egg (Korhonen et a / . Rosiello, A. P., J. M. Essigmann & G. N. Wogan:
1983a). Thiurams were the next potent, with ED50 Rapid and accurate determination of the median
values from 0.018 to 0.091 pmol peregg(Korhonen lethal dose (LD50) and its error with a small computer.
er a / . 1982a). Acrolein fits in the middle of this J. Toxicol. Environ. Health 1977, 3, 797-809.
Singh, A. R., W. H. Lawrence & J. Autian: Em-
range, both in this work and in a test made in our
bryonic-fetal toxicity and teratogenic effects of a
laboratory earlier (Kankaanpaa et a / . 1979). group of methacrylate esters in rats. J. Dent. Res.
D O T G and D P G were relatively potent, too. Only 1972, 51, 1632-1638.

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