/ . Embryol. exp. Morph. Vol. 16, 1, pp. 29-39, August 1966
Printed in Great Britain
29
The relationship of chemical structure to teratogenic
activity among bisazo dyes: a re-evaluation
By J. B. LLOYD 1 & F. BECK2
From the Departments of Biochemistry and Anatomy,
University College of South Wales and Monmouthshire
INTRODUCTION
Although the potency of trypan blue as a teratogenic agent in rodents has
been known for almost twenty years the mechanism of its action remains
obscure. The dye produces a variety of biological effects (see review by Beck &
Lloyd, 1966) and most of these have at some time been suggested as possibly
relevant to its teratogenicity. The investigation of such correlations would
clearly be assisted by determining whether the biological effects of the dye are
individually modified by small changes in its chemical structure, particularly
if these changes also result in differences in its teratogenicity.
The first teratological study of dyes related to trypan blue was made by
Gillman, Gilbert, Spence & Gillman (1951), who obtained negative results in
rats with six dyes (methylene blue, trypan red, Sudan IV, Bismarck brown,
Niagara blue and sky blue), but gave no experimental details of their investigation. Later Wilson (1955) studied the effects of fourteen dyes on rat gestation
by injecting 10 mg of each subcutaneously on each of days 7, 8 and 9 of
pregnancy. He obtained large numbers of malformed offspring at term only from
trypan blue, while Evans blue, Niagara blue 4B and Niagara sky blue 6B, the
four dyes most closely related to trypan blue in structure, were found to be
teratogenic but to a far lesser degree. In the same year Tuchmann-Duplessis &
Mercier-Parot (1955) reported that azo blue was not teratogenic. Beaudoin &
Pickering (1960) tested Niagara blue 2B (see Table 1 for formula) and sixteen
specially synthesized compounds resembling all or part of the trypan blue
molecule. Each dye was administered by a single injection of 140 mg/kg intraperitoneally into rats on the 8th day of gestation. Only two of these substances
produced malformed offspring at term and then in very small numbers. Beaudoin
(1961) also tested several dyes against the developing chick, and found that only
trypan blue, Evans blue and Niagara blue 4B were more teratogenic than saline.
1
Author's address: Department of Biochemistry, University College of South Wales and
Monmouthshire, St Andrews Place, Cardiff, U.K.
2
Author's address: Department of Anatomy, University College of South Wales and
Monmouthshire, Newport Road, Cardiff, U.K.
30
J. B. LLOYD &F. BECK
A search of the literature indicates therefore that trypan blue is the only dye
able to cause a high incidence of foetal malformation, that even slight modification of its formula reduces its teratogenic activity very greatly and that the
majority of related dyes are completely inactive. Certain features of this earlier
work, however, suggest the need for a re-assessment of the situation.
Authenticity and purity of dye samples used
Commercially obtained dyestuffs frequently contain large quantities of both
inorganic and organic contaminants. The principal inorganic impurity is
sodium chloride, which is used in the salting-out stage of manufacture, but
which is usually not removed from the final product as it improves the dyeing
qualities. Sodium chloride though not, of course, likely to modify the teratogenic properties of a dye, is present in some samples in such large quantities that
it displaces dyestuff as the principal component. A recent investigation (Lloyd
& Beck, 1963), has shown that among a random sample of bisazo dyes marketed
for biological staining the salt content ranged from nil to almost 80 %. It hardly
needs to be emphasized that if such dyes were used without purification for
teratogenesis experiments, the dose required to elicit a given response would
vary within wide limits; the different teratogenic dose-levels reported by various
workers for trypan blue may well be a reflexion of this. In addition, paper
chromatography has shown that bisazo dyes often contain substantial quantities
of coloured impurities (Lloyd & Beck, 1963) and variation between samples in
the nature and amount of these impurities has led to much confusion. Indeed it
has only recently become apparent (Beck & Lloyd, 1963 a) that the active
teratogen in commercial trypan blue is trypan blue itself and that the coloured
contaminants do not modify the activity except in so far as they act as diluents.
Again paper chromatography frequently reveals a qualitative difiFerence between
the principal coloured components of the various manufacturers' samples of
dye (Lloyd & Beck, 1963). This calls in question the authenticity of some of the
samples available and suggests the need for a chemical test to verify the identity
of each sample. Such a test has been devised (Lloyd & Beck, 1964) and its
application has confirmed the impression that mislabelling of dyes is not
infrequent. One example will suffice to illustrate this point: of six samples of
Evans blue obtained for the present investigation only four were authentic, one
being trypan blue and another Niagara blue 6B. Clearly, had no check been
made on them before determining their teratogenic activity, the results would
have been misleading and contradictory.
Dosage
Most previous workers have confined their investigations to administration
of the dyes at a single dose level. Our experience with trypan blue (Beck & Lloyd,
1964) has shown that adequate assessment of teratogenic potency cannot be
made in this way because an ' optimum teratogenic dose' exists, below which
Chemical structure and teratogenic activity
31
response falls off rapidly and above which embryonic death masks any abnormalities of embryonic development. A strong possibility exists, therefore, that
the low incidence of malformed foetuses observed after administration of certain
dyes is not the result of a low intrinsic potency but of the administration of a
suboptimal dose. Similarly, a high maternal or foetal mortality might indicate
a potent teratogen given in too large a dose.
The interpretation of intrauterine death
Beck & Lloyd (1963 b) have shown that if trypan blue is given at 8-5 days at
50 mg/kg (the optimum teratogenic dose) the primary response is embryonic malformation but that, during the remainder of gestation, many of the more severely
affected foetuses die, leading at term to the presence of a relatively small number
of malformed young accompanied by evidence of extensive foetal resorption.
Consequently if a dye (e.g. Niagara blue 2B; Beaudoin, 1962) induces foetal
resorptions but few if any malformed survivors at term, the implication may be
that the dye is a potent teratogen inducing malformations which prove lethal
before term, a possibility which can be verified by examining treated embryos
early in gestation.
The above considerations, as well as differences among earlier reports regarding the route and time of administration, the use of single or multiple doses, the
definition of abnormality and the presentation of results have led us to investigate the teratogenic activity of four dyes closely related to trypan blue. Compounds of known composition and purity were used, and were applied at several
different dose-levels. Time and route of injection were standardized as were the
time of killing of the mother and the assessment of malformation among the
offspring. In addition, experiments were carried out to determine serum levels
of each of the dyes in adult rats at various times after injection.
MATERIALS AND METHODS
Dyes
Table 1 shows the structural formula of trypan blue and the four dyes used
in the present investigation. Apart from Evans blue they differ only in the
substituents on the biphenyl nucleus; Evans blue and trypan blue are isomers,
differing in the positions of the sulphonate groups on the naphthalene rings.
Except in the case of Afridol blue, which was especially synthesized by Ciba
Clayton Ltd., several commercial samples of each dye were obtained. One of
each was selected, having been confirmed as authentic by paper electrophoresis
of its reduction products (Lloyd & Beck, 1964), and shown to be reasonably
free of coloured impurities by ascending paper chromatography in «-butanol:
pyridine:water, 1:5:4 (Lloyd & Beck, 1963). A solution in water was then
dialysed against running tap water for several days to remove salt and other
diffusible impurities, filtered through sintered glass, and passed through a column
32
J. B. LLOYD & F. BECK
of Dowex 50 W (x 4,20-50 mesh, H+ form) to remove final traces of cations and
to convert the dye into the free acid form. The solution was evaporated to dryness in vacuo, the dye dried overnight in vacuo at 50 °C over P2O5 and subsequently a 1 % solution in distilled water prepared. The purity of each dye was
estimated by titration with 0-05 N titanous sulphate solution and was found to
be 90% for Niagara blue 2B, 91 % for Niagara blue 4B, 91 % for Afridol blue
and 100 % for Evans blue.
Table 1. Structural formulae of dyes used
NH
OH
OH
•N=N—( v
,)—(K
Name
Colour index no.
(Colour Index,
1956)
Trypan blue
Niagara blue 2 B
Niagara blue 4 B
Afridol blue
Evans blue
23850
22610
24400
—
23860
NH,
/V-N=N
Substituents
A
f
X
Q
R
CH 3
H
OCH 3
Cl
CH 3
SO 3 H
SO3H
SO3H
SO3H
H
H
H
H
H
SO3H
Teratogenesis experiments
Wistar rats from an inbred laboratory strain propagated by brother-sister
mating were used, and pregnancy diagnosed by vaginal smearing. Taking
midnight of the night of mating as the time of conception, animals were injected
subcutaneously at 8-5 days with a 1 % solution of dye and killed with chloroform
at 20-5 days. The uteri were removed, foetal resorption sites counted and live
embryos examined for evidence of external malformation. In the case of Niagara
blue 4B two further groups were killed at 11-5 and 14-5 days following dye
treatment at 8-5 days, and in the case of Evans blue additional animals were
killed at 20-5 days following 7-5-day injection.
Serum level determinations
Male rats of the same strain as those in the teratogenesis experiments were
used for dye serum level determinations after a preliminary experiment had
shown that there was no sex-dependent difference in response. Animals were
injected subcutaneously with a 1 % solution of dye and individuals killed at
varying intervals after injection by withdrawal of blood from the aorta under
light ether anaesthesia. The blood was allowed to clot and the serum (isolated
after centrifugation) was suitably diluted with pH 8-6 sodium barbitone-hydro-
Chemical structure and teratogenic activity
33
chloric acid buffer. The dye concentration was estimated by measuring light
extinction at 600 m/i with a Unicam SP600 spectrophotometer.
RESULTS
The results of the teratogenesis experiments are presented in Table 2 and Fig. 1,
the data for trypan blue having been presented in a previous publication (Beck &
Lloyd, 1964). It may be seen that in addition to trypan blue, Niagara blue 2B
and Afridol blue are potent teratogens, the three dyes producing a very similar
Table 2. Teratogenic response to four bisazo dyes injected
subcutaneously at 8-5 days of pregnancy
No. of mothers
Dye
Niagara blue 2B
Niagara blue 4B
Afridol blue
Surviving Total Resorbed Abnormal Normal
,
to
\ /—
>
t—
Dose Inimplanta- r— A
(mg/kg) jected term
tions No. %* No • 0//o * No. 0//o *
A
- *
150
200
300
11
13
14
7
13
12
13
11
10
14
8
11
10
9
11
12
12
5
13
11
11
5
10
14
8
11
9
8
86
7
33
46
53
31
39
64
50
22
32
16
57
45
71
50
7
7
70
13
100
150
200
8
8
10
8
7
7
72
70
74
31
66
58
50
100
150
200
50
75
100
150
25
50
100
Evans blue
97
114
128
53
122
109
114
50
123
134
77
113
80
1 0-9 89
90
28-7 4
3-5 77
40-7 32 22-2 50
100
0
0
0
33-5
2-4 88
3
36-4 0
0
70
56-6 0
0
50
100
0
0
0
17-2
9 7-6 92
260 11 7-7 91
21-8 13 16-3 48
50-3 21 17-2 35
58-7 10 13-2 25
81-7
7 10-6
8
1
16-3
1 1 56
2-5 39
48-4 2
95-2 4
4-8
0
79-8
2
2-4 14
N
901
67-8
37-1
0
641
63-6
43-4
0
75-2
66-3
61-9
32-5
28-1
7-7
82-6
491
0
17-9
* Percentages represent the arithmetic means of the percentage within each individual
litter; this enables the standard error of the mean resorptions and malformations for each
dose to be calculated and shown in Fig. 1.
pattern of response, the chief difference being that whereas trypan blue has an
optimum teratogenic dose in the region of 50 mg/kg Niagara blue 2B and
Afridol blue are maximally active as teratogens at several times this dose. The
teratogenic potencies (the percentage of external malformation observed at
term at the optimum teratogenic dose) of the three dyes are strikingly similar.
In contrast Niagara blue 4B and Evans blue, although producing much intrauterine mortality, do not at any dose lead to the appearance of more than a very
few malformed foetuses at term.
Fig. 2 presents graphically the marked variation observed in toxicity of the
3
JEEM l6
34
J. B. LLOYD & F. BECK
various dyes to the mother; Niagara blue 4B is seen to be the most potent of
the dyes in this respect, killing over 50 % of mothers before term when administered at 150 mg/kg. Direct measurement of the toxicity of Niagara blue 4B and
of the less toxic dye Afridol blue were also performed on male rats. The results
confirmed those seen in Fig. 2 in that the Ld50 (Karber, 1931) over a 12-day
period was 179 mg/kg for Niagara blue 4B and over 400 mg/kg for Afridol blue.
100 - i
50
100
Niagara blue 2B
80604020 -
100-
250
300
250
300
250
300
250
300
80 - Niagara blue 4B
6040 20 -
10080-
50
100
60-
A
4020-
100-i
150
200
Afridol blue
.Ai
50
Evans blue
100
150
X
40-
200
-I
8060-
*-
-?''''
2050
100
150
200
Dose (mg/kg)
Fig. 1. Dosage-response curves for the teratogenic activity of five
bisazo dyes.
, Resorptions;
, abnormalities.
35
Chemical structure and teratogenic activity
60 -i
Niagara blue 4B
50-
40-
Evans blue
£ 30
\
Niagara blue 2B
£ 20
I
v*- 1 " 1 " 1
10-
/
50
—|
100
1
150
Afridol blue
Trypan blue
1
200
Dose (mg/kg)
1—
250
—r~
300
Fig. 2. Maternal mortality within 12 days of administration of various bisazo dyes.
Trypan blue (174)
Hours
Fig. 3. Serum levels following injection of 50 mg/kg of various dyes
(figures in parenthesis are the average levels over the first 24 h).
3-2
36
J. B. LLOYD & F. BECK
Fig. 3 shows the serum levels of the five dyes at intervals after subcutaneous
injection of 50 mg/kg. By calculation of the areas under the curves it is possible
to obtain the average concentration over the first 24 h, and these values are
given in parenthesis.
The results suggest an explanation of the different optimum teratogenic doses
of the three active dyes; they imply that, in order to reach serum levels comparable with those reached by trypan blue when administered at 50 mg/kg,
Niagara blue 2B and Afridol blue would have to be given at significantly higher
doses. It may therefore be supposed that differences between these dyes as
teratogens are in part reflexions of differences in their handling by the mother,
particularly in rate of release from the subcutaneous injection site.
Table 3. Teratogenic response to Evans blue injected
subcutaneously at 7-5 days of pregnancy
No. of mothers
Dose
(mg/kg)
Injected
100
150
10
6
Surviving Total
to
implanterm tations
10
6
76
71
Resorbed
A
,
>
0/ *
No.
/o
27
65
301
90-9
Abnormal
A
,
,
0/ *
No.
/o
9
0
12-7
0
Normal
,
*
0/
No.
/o *
40
6
57-2
9- 1
*See footnote to Table 2.
Evans blue differs markedly from the other dyes in its very slow release into
the bloodstream, the maximum concentration being reached only after 36 h
(Fig. 3). Since the average serum level during the first 24 h is only 27 /*g/ml, it
might be estimated that doses of 300 mg/kg or higher would be needed to give
teratogenic effects. At this dose the dye would be highly toxic to the mother
(Fig. 2) leading almost invariably to maternal death before term. An alternative
method of achieving adequate serum levels utilizes the fact that average serum
levels in the period 24-48 h after injection is 60 /tg/ml. Consequently two series
of rats were injected with Evans blue at 7-5 days of pregnancy at doses of 100
and 150 mg/kg. The results (Table 3) show that considerable teratogenic
potency is observed at the lower of the two doses. With Evans blue, therefore,
a teratogenic effect similar to that shown by the other dyes can be demonstrated
if adequate serum levels can be maintained over the susceptible period. Wilson's
(1955) demonstration of the teratogenic potency of Evans blue is therefore
explicable, for he administered the dye in divided doses, beginning at day 7
(7-5 days of pregnancy according to the present authors' convention), and his
results, when allowance is made for a different method of presentation, are not
unlike those in Table 3.
From Fig. 3 one would predict that Niagara blue 4B might have a high teratogenic potency at doses of about 150 mg/kg because at this level of dosage the
Chemical structure and teratogenic activity
37
average serum levels attained in the first 24 h might be expected to be comparable with those following the optimum teratogenic doses of the other dyes.
However, in this case the complicating factor of a high toxicity alters the
picture. At 150 mg/kg Niagara blue 4B causes a 55 % maternal mortality and
the total resorption of litters in the survivors, suggesting that the foetal mortality
observed at term following the administration of Niagara blue 4B may well
represent not malformed embryos dying before 20-5 days but a direct toxic
effect comparable with that observed among treated mothers. In order to test
this hypothesis two further series of animals were injected at 8-5 days of pregnancy with Niagara blue 4B at 100 mg/kg and killed at 11-5 or 14-5 days. The
results (Table 4) show that the vast majority of resorption sites seen at term are
due to death of conceptuses within 3 days of injection. This suggests that a direct
toxic effect rather than the secondary consequences of induced malformation is
responsible for the embryonic loss.
Table 4. The effect of 100 mg/kg Niagara blue 4B injected
subcutaneously at 8-5 days of pregnancy
No. of mothers
A
,
> Total Resorbed
A
Day
In- Sur- implan- ,
>
killed jected viving tations No.
%*
11-5
14-5
20-5
8
9
13
8
9
11
73
86
114
34
49
73
47-8
61-7
566
Abnormal
A
,
*
No.
%*
4|
1
0
7-3
10
0
Normal
,
* *
No.
%*
35
36
41
44-9
37-3
43-4
* See footnote to Table 2.
t Including two embryos showing doubtful abnormalities of the body axis.
DISCUSSION
The evidence presented is consistent with a hypothesis that the dyes investigated will demonstrate high teratogenic potency if present in the blood at an
average concentration of 150-300 /*g/ml for a period of 24 h between 8-5 and
9-5 days of gestation. In the case of four dyes, trypan blue, Niagara blue 2B,
Afridol blue and Evans blue, this has been shown to be the case. The remaining
dye Niagara blue 4B is highly toxic to the maternal organism when present in
these concentrations, and the ensuing maternal and embryonic death masks any
teratogenic effects.
The four examples investigated are probably not the only teratogenic bisazo
dyes. Beaudoin (1% 4 ) has found that Congo Red (C.I. 22120), previously
thought to be inactive (Wilson, 1955), is capable of producing malformations
if given at higher dose. The malformations observed, however, were all of the
viscera, so that a direct comparison with the present results is not possible.
38
J. B. LLOYD & F. BECK
Although alterations in chemical structure are certain to modify embryopathic
activity to some extent, the increasing probability that most, if not all, bisazo
dyes are potential teratogens should direct attention from the differences
between 'teratogenic' and 'non-teratogenic' dyes towards a study of the means
whereby they elicit their response.
SUMMARY
1. Samples of five bisazo dyes, trypan blue, Niagara blue 2B, Niagara blue
4B, Afridol blue and Evans blue, of confirmed identity and known purity, have
been tested as teratogens by sucbutaneous injection into the Wistar rat. Each
dye except Niagara blue 4B, if administered at an appropriate dose and stage
of pregnancy, shows high activity. Niagara blue 4B at low dose produces a few
malformed survivors at term, but in higher doses leads to considerable maternal
and embryonic mortality.
2. Serum dye concentrations at intervals after subcutaneous injection were
calculated and it was found that when injected at doses which gave maximum
teratogenic response serum levels of the same order over the susceptible period
were maintained for each dye. The hypothesis is advanced that the observed
differences in teratogenic behaviour of the dyes to some extent reflect differences
in the handling of the dyes by the mother rather than absolute differences in
activity.
RESUME
Relation entre la structure chimique et Vactivite teratogene
de colorants azoiques: une re-evaluation
1. Des echantillons de cinq colorants azoiques, le bleu trypan, le bleu Niagara
2B, le bleu Niagara 4B, le bleu Afridol et le bleu Evans, de nature certaine et de
purete connue, ont ete testes en tant que substances teratogenes par des injections sous-cutanees chez le rat Wistar. Chaque colorant, a l'exception du bleu
Niagara 4B, administre a une dose et a un stade de la gestation adequats, montre
une haute activite. Le bleu Niagara 4B, a faible dose, produit quelques survivants malformes qui vont jusqu'a terme, tandis qu'a hautes doses il cause une
mortalite maternelle et foetale considerable.
2. Les concentrations des colorants dans le serum ont ete calculees a differents intervalles de temps apres les injections sous-cutanees et les resultats
montrent que, lorsque les injections sont faites a des doses qui ont l'effet
teratogene maximum, les teneurs du serum, pour chaque colorant, sont maintenues du meme ordere au cours de la periode sensible. Une hypothese est
avancee: les differences observees dans les effets teratogenes des colorants
refleteraient, dans une certaine mesure, plutot des differences dans le sort de ces
colorants dans l'organisme maternel que des differences absolues d'activite.
Chemical structure and teratogenic activity
39
We thank Mr A. Griffiths for carrying out some of the spectrophotometric analyses, Ciba
Clayton Ltd. for preparing and supplying a sample of Afridol blue, the Medical Research
Council and the Spastics Society for financial support, and Professors K. S. Dodgson and
J. D. Lever for their encouragement and criticism. We should also like to acknowledge the
excellent technical assistance of Miss G. M. Perry and Miss B. Humphreys.
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(Manuscript received 29 November 1965)