In vitro toxicity of several dithiocarbamates and structure-activity relationships.

Article abstract of DOI:10.1002/jat.868

Dithiocarbamates (DTCs) are chemicals featuring a great chelating capacity. The toxicological study of DTCs is very important in view of their relatively simple synthesis and wide array of sanitary and industrial applications. In this study, the toxicity of some of the more recently synthesized DTCs is determined using an extremely simple bioassay, described in previous studies, based on the inhibition of growth of Escherichia coli (IGEC). The lowest-observed-effect concentration (LOEC), the median effective concentration (EC(50)) and no-observed-effect concentration (NOEC) of the following sodium dithiocarbamates was determined: N-benzyl-N-methyldithiocarbamate x 2H(2)O, N-benzyl-N-isopropyldithiocarbamate x 3H(2)O, N-benzyl-N-ethyldithiocarbamate x 2H(2)O, N-butyl-N-methyldithiocarbamate x 2H(2)O, N,N-dibenzyldithiocarbamate x 2H(2)O and N-benzyl-2-phenethyldithiocarbamate x 4H(2)O. Our results showed N,N-dibenzyl-DTC to be the least toxic of the tested substances, with an EC(50) value of 1,269.9 micro g ml(-1), whereas N-butyl-N-methyl-DTC and N-benzyl-N-methyl-DTC, with respective EC(50) values of 14.9 micro g ml(-1) and 23.5 micro g ml(-1), were the most toxic. Regression analysis showed, through exponential models, that the degree of toxicity of this group of substances correlated with the molecular weight of the compound, the molecular weight of the smallest chemical radical linked to the dithiocarbamate group and the number of benzene rings present in the molecule. The consideration of these models allows us to establish that in general terms the toxicity of DTCs decreases exponentially with a greater molecular weight and the number of benzene rings. Copyright 2002 John Wiley & Sons, Ltd.

Full text of DOI:10.1002/jat.868

JOURNAL OF APPLIED TOXICOLOGY
J. Appl. Toxicol. 22, 353–357 (2002)
Published online in Wiley InterScience (www.interscience.wiley.com). DOI:10.1002/jat.868
In vitro Toxicity of Several Dithiocarbamates
and Structure–Activity Relationships
N. Segovia,1 G. Crovetto,2 P. Lardelli1 and M. Espigares1*
1
Department of Preventive Medicine and Public Health, University of Granada. Campus Universitario de Cartuja, 18071
Granada, Spain. Fax: 958 249958. E-mail: mespigar@ugr.es
2
Department of Physical Chemistry, University of Granada. Campus Universitario de Cartuja, 18071 Granada, Spain. E-mail:
crovetto@ugr.es
Key words: dithiocarbamates; toxicity; bioassay; Escherichia coli; amines; LOEC; EC50; NOEC.
Dithiocarbamates (DTCs) are chemicals featuring a great chelating capacity. The toxicological study of DTCs
is very important in view of their relatively simple synthesis and wide array of sanitary and industrial appli-
cations. In this study, the toxicity of some of the more recently synthesized DTCs is determined using an
extremely simple bioassay, described in previous studies, based on the inhibition of growth of Escherichia
coli (IGEC). The lowest-observed-effect concentration (LOEC), the median effective concentration (EC50)
and no-observed-effect concentration (NOEC) of the following sodium dithiocarbamates was deter-
mined: N -benzyl-N -methyldithiocarbamate·2H2O, N -benzyl-N -isopropyldithiocarbamate·3H2O, N -benzyl-
N -ethyldithiocarbamate·2H2O, N -butyl-N -methyldithiocarbamate·2H2O, N ,N -dibenzyldithiocarbamate·-
2H2O and N -benzyl-2-phenethyldithiocarbamate·4H2O. Our results showed N ,N -dibenzyl-DTC to be the
least toxic of the tested substances, with an EC50 value of 1269.9 µg ml−1, whereas N -butyl-N -methyl-DTC
and N -benzyl-N -methyl-DTC, with respective EC50 values of 14.9 µg ml−1 and 23.5 µg ml−1, were the most
toxic. Regression analysis showed, through exponential models, that the degree of toxicity of this group of
substances correlated with the molecular weight of the compound, the molecular weight of the smallest chem-
ical radical linked to the dithiocarbamate group and the number of benzene rings present in the molecule.
The consideration of these models allows us to establish that in general terms the toxicity of DTCs decreases
exponentially with a greater molecular weight and the number of benzene rings. Copyright  2002 John
Wiley & Sons, Ltd.
mechanisms.21–25 Iron DTCs have been used for treat-
INTRODUCTION
ing AIDS and neurodegenerative diseases.26,27 In addi-
tion, these chemicals have important applications in the
production of petroleum derivatives, lubricants and poly-
mers, where they are used as accelerators of vulcanization,
antioxidants and antihumidity agents.28–30 Some DTCs are
excellent reagents for the analysis of trace metals, by
means of enhancement techniques.31,32
Dithiocarbamates (DTCs) are chemicals with a great
chelating capacity and their synthesis is relatively sim-
ple. The most commonly used method involves the reac-
tion of carbon disulphide with a large range of primary
and secondary amines in the presence of an alkali metal
hydroxide.1 Because of the great variety of synthesized
compounds, DTCs have a wide variety of applications.
Many are used as pesticides (e.g. propineb, zineb, maneb,
mancozeb, ziram, thiram), leading to analytical methods
designed to detect the presence of these potentially haz-
ardous chemicals and their degradation products in differ-
ent media.2–5 Other current uses of DTCs are as antiviral
agents,6 antidotes for preventing the effects of phytotoxic
agents,7 bactericides and antimicrobial agents,8 antitumour
drugs9 and prophylactic or therapeutic agents for metal
toxicity.10–19 Although DTCs are suitable for the removal
of metals from organisms, they also are able to alter redis-
tribution, resulting in increased toxicity.20 Their capacity
for inhibiting apoptosis has been shown through different
Generally, DTCs compounds have low levels of toxic-
ity, although some used as pesticides have been found
to produce adverse health effects. Pesticide DTCs can
produce disturbances of the peripheral and the cen-
tral nervous systems. Thiram and ziram have a pow-
erful mutagenic capacity,33,34 and thiram also has been
shown to induce stress protein overproduction, even
at sub-inhibiting concentrations,35 and to suppress pro-
oestrogens while increasing luteinizing hormone and dis-
rupting ovulation.36 Genotoxic effects have been described
for the DTCs having considerable commercial use.37,38
Bearing in mind the widespread use of these com-
pounds, the study of their possible effects on health is very
important. The toxicity of some of the more recently syn-
thesized DTCs has been determined using an extremely
simple bioassay based on the inhibition of growth of
Escherichia coli (IGEC) described previously. Toxic sub-
stances produce an alteration of bacterial metabolism lead-
ing to inhibition of growth. The predictive power of
* Correspondence to: M. Espigares, Department of Preventive Medicine
and Public Health, University of Granada, Campus Universitario de
Cartuja, 18071 Granada, Spain. E-mail: mespigar@ugr.es
Received 5 November 2001
Revised 7 June 2002
Copyright  2002 John Wiley & Sons, Ltd.
Accepted 13 June 2002

354
N. SEGOVIA ET AL.
toxicity test data is limited by interspecies differences.
However, the data for metabolic inhibition on E. coli
can be extrapolated to human cells. Some data regarding
the predictive power of this biotest have been published
previously.39,40
The objective of the present study was to assess the
toxicity of a group of newly synthesized DTCs using the
IGEC bioassay, and to evaluate statistical models based on
quantitative structure–activity relationships for estimating
the possible relationship between some chemical charac-
teristics of these compounds and their degree of toxicity.
without toxic was used together with a tube for each sep-
arate concentration of the tested toxic, and all were inoc-
ulated with an 18-h pre-culture until obtaining an initial
absorbance (650 nm) of 0.05. Incubation was for 5 h of
°
the exponential phase in a thermostated bath at 37 C with
agitation, with absorbance measured every hour. From the
absorbance rates of each tube we calculated the values for
instantaneous growth rates (IGR)
IGR = ln(At /At−1
)
where At is the absorbance at time t hours, At−1 is the
absorbance at the previous hour and t is the time in hours.
On the basis of the IGR, the lowest-observed-effect con-
centration (LOEC) is calculated, along with the median
effective concentration (EC50) and no-observed-effect con-
centration (NOEC) for each toxic substance tested.42
MATERIALS AND METHODS
Chemicals
The sodium DTCs were produced by the interaction of
amines, S2C and NaOH, in agreement with previously
described methodology.40,41 The amines were treated with
equimolar aqueous solutions of NaOH. Additional S2C
was introduced in small fractions to maintain the reac-
tion medium at room temperature. It was agitated for 8 h
and then filtered and evaporated to dryness in a vacuum
Statistical analysis
The LOEC is the minimal effective concentration tested
that presents statistically significant differences with
respect to the control, using Student’s t-test for two
independent samples. In order to obtain EC50 and NOEC,
the percentage inhibition (PI ) with respect to the control
is calculated for each of the IGR values using the
following expression
°
at a temperature no higher than 60 C. The solid por-
tion was treated with acetone, filtered, evaporated and
treated with ethylic ether to remove impurities. Finally,
the product obtained was recrystallized in acetone with a
few drops of benzene. The sodium DTCs were identified
and characterized on the basis of elemental analysis (C, N
and H), thermogravimetric and differential thermal anal-
ysis curves, fusion range, thin-layer chromatography and
P I = (IGRc − IGRtox) × 100/IGRc
where IGRc is the value of the negative control and
IGRtox is the value of the culture with a specific con-
centration of toxicant in the same experiment and at
the same time.
1
UV–visible, IR, H-NMR and mass spectra.
From the interval of concentrations determining a linear
reduction of IGR, the regression curve is obtained with
the concentrations as an independent variable and PI as
a dependent variable. The EC50 and NOEC values are
calculated from this linear model, respectively assigning
to PI the values of 50 and zero. The NOEC is considered
equal to zero when calculations using the model give a
negative value.
For synthesis of the DTCs, the following amines were
used: N,N-dibenzylamine (Boheringer), N-benzyl-N-
ethylamine (Aldrich), N-benzyl-N-isopropylamine (Boh-
eringer), N-benzyl-N-methylamine (Aldrich), N-ethyl-N-
methylamine (Aldrich) and N-benzyl-2-phenethylamine
(Boheringer). From these amines, the following sodium
DTCs were then synthesized: N-benzyl-N-methyldi-
thiocarbamate·2H2O, N-benzyl-N-isopropyldithiocarba-
mate·3H2O, N-benzyl-N-ethyldithiocarbamate·2H2O, N-
butyl-N-methyldithiocarbamate·2H2O, N,N-dibenzyldi-
thiocarbamate·2H2O and N-benzyl-2-phenethyl-dithio-
carbamate·4H2O.
All statistical analyses were performed using the Statis-
tical Package for the Social Sciences (SPSS for Windows,
v. 10.0).
Structure–activity relationship modelling
IGEC bioassay
The statistical models based on quantitative struc-
ture–activity relationships (QSARs) for estimating toxi-
city were obtained with the data on EC50 from the DTCs
studied in the present work, in addition to our previously
published data40 obtained applying the same methodology
to another group of DTCs. The independent variables con-
sidered were the molecular weight of each compound, the
molecular weight of the two chemical radicals constitut-
ing the molecule of each DTC and the number of benzene
rings in each compound.
The toxicity of the synthesized DTCs was determined by
means of the IGEC bioassay, which has been described
in previous work.39,40 The toxic effect is assessed by test-
ing growth inhibition of the bacterial strain Escherichia
coli K12 W3110 thy− (CECT 416, Spanish Type Cul-
ture Collection). The assay was carried out in tubes
containing 10 ml of Vogel–Bonner culture medium sup-
plemented with 50 mg 1−1 of thymine and 20 g 1−1 of
glucose. The DTCs were prepared in concentrated solu-
tions allowing the desired concentration to be attained for
each assay by addition. In accordance with their solu-
bility, N-benzyl-N-methyl-DTC, N,N-dibenzyl-DTC and
N-butyl-N-methyl-DTC were prepared in aqueous solu-
tions, whereas N-benzyl-N-isopropyl-DTC, N-benzyl-N-
ethyl-DTC and N-benzyl-N phenethyl-DTC were dis-
solved in DMSO. For each experiment, a control tube
RESULTS
The inhibiting effect of the new sodium DTCs has been
assayed in different concentrations to a maximum of
Copyright  2002 John Wiley & Sons, Ltd.
J. Appl. Toxicol. 22, 353–357 (2002)

IN VITRO TOXICITY OF DITHIOCARBAMATES
355
Benzyl-methyl-DTC
Benzyl-ethyl-DTC
Benzyl-isopropyl-DTC
Butyl-methyl-DTC
Dibenzyl-DTC
0.5
0.4
0.3
0.2
0.1
0
Benzyl-phenethyl-DTC
0
0.1
1
5
10
25
50
100
500
1000
Concentration (µg/ml)
Figure 1. Mean value of the instantaneous growth rate (IGR) for the different concentrations of dithiocarbamates (DTCs).
1000 µg ml−1; this limit was not surpassed in order to
avoid precipitation of the compounds, which would alter
the toxic effect and interfere with absorbance determi-
nations. For N-benzyl-N-phenethyl-DTC, the maximum
concentration tested was 100 µg ml−1, in view of the lim-
ited solubility of the compound. For N,N-dibenzyl-DTC,
complete inhibition was not attained at the concentration
of 1000 µg ml−1 but a significant inhibition was obtained
with respect to the control, which allowed the param-
eters of toxicity to be calculated. The action of each
DTC was represented graphically using the IGR as the
measurement of the effect of each concentration assayed
(Fig. 1).
The toxic potential power expressed as EC50 was related
to some simple chemical characteristics of the molecules
(Table 2), giving the regression models shown in Table 3.
The statistically significant models best fitted to the data
are all exponentials.
DISCUSSION
Because the synthesis of sodium DTCs and their areas of
application are ever-increasing, the toxicological knowl-
edge about them is crucial. The IGEC biotest has demon-
strated its utility in the evaluation of DTC toxicity and
although it is a bacterial model its simplicity and low cost
are strong advantages.
The degree of toxicity of the tested DTCs is highly
variable, as can be seen in the results shown in Table 1.
All three parameters—LOEC, EC50 and NOEC—show
that N,N-dibenzyl-DTC is the least toxic and N-butyl-
N-methyl-DTC the most toxic of this group of DTCs.
The toxic effects of the DTCs assayed can be observed
in the graphic representations of IGR in conjunction with
Table 1—Lowest-observed-effect concentration (LOEC), median effective concentration (EC50) and no-observed-effect
concentration (NOEC)
Compound
LOEC (µg ml−1
)
Statistical
EC50 (µg ml−1
)
NOEC (µg ml−1
)
significance
N-Benzyl-N-ethyl-DTC Na·2H2O
N-Benzyl-2-phenethyl-DTC Na·4H2O
N-Benzyl-N-isopropyl-DTC Na·3H2O
N-Benzyl-N-methyl-DTC Na·2H2O
N-Butyl-N-methyl-DTC Na·2H2O
N,N-Dibenzyl-DTC Na·2H2O
25
100
25
10
5
0.000
0.001
0.020
0.000
0.000
0.004
28.9
104.9
190.1
23.5
14.9
1276.9
6.3
22.1
0
0.7
0
1000
60.4
Copyright  2002 John Wiley & Sons, Ltd.
J. Appl. Toxicol. 22, 353–357 (2002)

356
N. SEGOVIA ET AL.
Table 2—Characteristics of the sodium dithiocarbamates tested using the IGEC bioassay, and parameters related
to the molecular structure
EC50 (µg ml−1
)
Mol. wt.
R1
R2
Benzene
ringsc
a
b
Compound
N-Benzyl-N-ethyl-DTC Na·2H2O
N-Benzyl-N-isopropyl-DTC Na·3H2O
N-Benzyl-N-methyl-DTC Na·2H2O
N-Benzyl-2-phenethyl-DTC Na·4H2O
N-Butyl-N-methyl-DTC Na·2H2O
N,N-Dibenzyl-DTC Na·2H2O
28.9
190.1
23.5
104.9
14.9
1276.9
65.0
43.0
269.4
301.4
255.4
381.5
221.3
331.5
225.3
179.3
299.4
299.4
193.3
91.1
91.1
91.1
105.1
57.1
91.1
29.0
15.0
136.1
136.1
29.0
29.0
43.1
15.0
91.1
15.0
91.1
29.0
15.0
15.0
15.0
15.0
1
1
1
2
0
2
0
0
1
1
0
N,N-Diethyl-DTC Na·3H2Od
N,N-Dimethyl-DTC Na·2H2Od
(+)-ψ-Ephedrine-DTC Na·2H2Od
(−)-Ephedrine-DTC Na·2H2Od
N-Ethyl-N-methyl-DTC Na·2H2O
301.0
55.0
31.8
a R1 = molecular weight of largest radical.
b R2 = molecular weight of smallest radical.
c Number of benzene rings in the molecule.
d Previously published data.40
Table 3—Regression of different structural characteristics and
EC50
its concentrations (Fig. 1). For all the DTCs, inhibition
presenting statistically significant differences was seen
compared with the control. In the case of N,N-dibenzyl-
DTC, the maximum concentration assayed (limited by its
solubility) did not produce total inhibition, although the
differences with the controls were statistically significant.
The same was found to occur with N-benzyl-2-phenethyl-
DTC, although in this case the maximum concentration
allowing testing was 100 µg ml−1. Taking into account the
assayed concentrations, the LOEC was obtained (Table 1).
It showed great variability, with values ranging from 5 to
1000 µg ml−1, despite the common chemical structure of
the substances.
Independent
variable
Model
R2
Statistical
significance
M
Molecular
weight (M)
Largest
EC50 = e2.214+0.013
—
0.378
—
0.044
NS
radical (R1)
Smallest
radical (R2)
Number of
benzene rings (N)
R
EC50 = e27.939+0.029
0.431
0.418
0.028
0.032
2
N
EC50 = e29.656+1.122
The EC50 has a greater value from a toxicological
standpoint and it points to N,N-dibenzyl-DTC (Table 1),
with a value of 1276.9 µg ml−1, as being the least toxic
of the DTCs studied overall with the IGEC bioassay
(Table 2), including the DTCs tested in our previous
work.40 On the other hand, we found N-butyl-N-methyl-
DTC and N-benzyl-N-methyl-DTC, with respective EC50
values of 14.9 and 23.5 µg ml−1, to be the most toxic
DTCs, again including those of previous studies.
All these substances present a similar chemical struc-
ture: a common dithiocarbamate group joined by two
chemical radicals. This leads us to infer that the degree
of toxicity may be related to specific structural differences
within this family of substances. In the regression analysis,
the molecular weight of the compound was included, as
well as the molecular weight of each one of the chemical
radicals linked to the dithiocarbamate group and the num-
ber of benzene rings present in the molecule (Table 2).
With the exception of the molecular weight of the largest
radical, the variables fit an exponential model (Table 3).
The consideration of these models leads us to certain
general proposals. First, a greater molecular weight might
imply a lesser toxicity. The toxicity of this group of com-
pounds decreases exponentially as the molecular weight
increases. This occurs even with the smallest chemical
radical: the greater its molecular weight, the lesser its tox-
icity. In fact, the EC50 values (Table 2) indicate that the
compounds containing a methyl radical are the most toxic.
Furthermore, when the number of benzene rings is con-
sidered as an independent variable, identical results are
obtained because the greater number of rings coincides
with a lesser toxicity (Table 3).
We may conclude that this study of several DTCs,
using the IGEC bioassay, manifests a large variability
in their toxicity. N,N-Dibenzyl-DTC is the least toxic
and N-butyl-N-methyl-DTC is the most toxic of the sub-
stances analysed, and a statistically significant regression
was found between the degree of toxicity, expressed as
EC50, and the different parameters related to the molecu-
lar structure.
Acknowledgements
The authors thank Ce´sar Criado Sa´nchez for his capable assistance in the
laboratory and Jean Sanders for her helpful suggestions while translating
and editing the manuscript.
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