Arch. Toxikol.

3t, 99--108 (1973)

9 by Springer-Verlag t973

Originalarbeiten/ Original Investigations

Formation of Cyanide from o-Chlorobenzylidene

Malononitrile and Its Toxicological Significance
L a r s F r a n k e n b e r g a n d B e SSrbo
Division of Experimental Defence Medicine, Rssearch Institute of National Defence,
Department l , Sundbyberg, Sweden
Received July 23, 1973

Abstra~. Mice received o-chlorobenzylidene malononitrile (CS) by i.p. injection

(0.5 LDs0) or by aerosol exposure (20,000 mg rain-1 m-a). Increased excretion of
thioeyanate in the urine was observed, indicating a transformation of CS to cyanide
in rive. Determinations of cyanide in whole blood after i.p. administration of CS
verified a rapid transformation of the agent to cyanide. A correlation between the
time course of cyanide levels and symptoms was observed. Toxicity of injected
CS was significantly reduced by pretreatment with thiosulfate, slightly reduced
by nitrite and not affected by C%EDTA.
Thiocyanate excretion, blood cyanide levels and protective effect of antidotes
were also evaluated after administration of 0.5 LD~0 of malononitrile and potassium
cyanide. The importance of cyanide formation for the toxicity of CS is discussed.
Key words: o-Chlorobenzylidene Malononitrile - - Malononitrile - - Potassium
Cyanide - - Cyanide Formation - - Thiocyanate Excretion - - Sodium Thiosulfate - -
Sodium Nitrite - - Cobalt-EDTA.

Zusammen/~sung. M~use wurden mit o-Chlorbenzylidenmalononitril (CS)

durch i.p. Injektion (0,5 LDs0) oder Aerosol (20000 mg min-1 m -8) behandelt. Eine
gesteigerte Ausscheidung yon Rhodanid im H a m wurde beobachtet, was eine
Umwandiung yon CS in Cyanid in rive andcutet.
Bestimmungen yon Cyanid in Vollblut best~itigten eine schnelle Bildung yon
Cyanid nach der Injektion yon CS.
Eine Korrelation zwischen dem Verlauf der Cyanidkonzentrationen und den
Vergiftungssymptomen der Tiere wurde beobaehtet. Die Toxicitiit yon injiziertem
CS wurde bei prophylaktischer Behandlung mit Thiosnifat deutlich reduziert,
w~hrend Nitrit eine geringere und Co2EDTA keine Wirkung hatte. Die Aus-
scheidung yon Rhodanid, die Cyanidkonzentrationen des Blutes und die Wirkung
der Antidote wurde auch bei Behandlung mit 0,5 LDs0 yon Malononitril und
Kaliumcyanid untersucht.
Die Bedeutung der Cyanidbildung f'dr die Toxicit~t yon CS wurde diskutiert.
Schliisselw~rter: o-Chlorbenzylidenmalononitril - - Malononitril - - Kalium-
cyanid - - Cyanidbildung - - Rhodanidausscheidung - - Natriumthiosulfat - -
Natriumnitrit - - Kobalt-EDTA.

L o w c o n c e n t r a t i o n s of o - c h l o r o b e n z y l i d e n e m a l o n o n i t r i l e (CS) in air
p r o d u c e a n i n t e n s e i r r i t a t i o n in t h e eyes a n d u p p e r a i r w a y s o f h u m a n
8 Arch.Toxikol.,Bd. 81
iO0 L. Frankenberg and B. S6rbo

beings (Punte et a/., i963) and the compound has therefore found ex-
tensive use as a tear gas. Although the risks involved in the employment
of CS for this purpose are often considered to be slight, animal experi-
ments have demonstrated that high air concentrations of the compound
can be lethal (Ptmte eta/., t962; Ballantyne and Callaway, 1972), and
alleged toxic reactions in human beings exposed to the agent have also
been reported (Rose and Smith, t969). It is known that CS hydrolyzes
spontaneously to malononitrile (Patai and Rappaport, i962) and the
latter is converted to cyanide in animal tissues (Nash et al., t950; Stern
et al., i952). This led Jones and Israel (1970) to suggest that toxic effects
of CS may arise from a gradual conversion of the compound in vivo to
cyanide, with malononitrile as an intermediate. In support of their
proposal, these authors noted that symptoms of poisoning in mice after
injections of CS and malononitrile were very similar and the LDs0 values
for the compounds were nearly the same expressed on a mole-to-mole
basis. Furthermore, they reported that thiosulfate, a well-known antidote
to cyanide and malononitrile (Heymans and Masoin, t897; Nash et al.,
i952) also acted as an antidote to CS. This antidotal action of thiosulfate
to CS was confirmed by Cuccinell et al. (t971), who also reported that
cyanide was detectable in the plasma of dogs injected with CS, but very
few experimental data were presented. Although these studies indicated
that cyanide may arise from CS in vlvo, they did not explain the im-
portance of this mechanism in the toxicity of CS.
The aim of the present investigation was to provide more quantitative
information about the cyanogenic properties of CS in relation to the
toxicity of the compound. One approach was based on the fact that
cyanide is rapidly converted in the mammalian body to thiocyauate,
which is excreted with the urine (Young and Maw, 1958). Determination
of the thiocyanate excretion following the administration of a compound
may thus allow estimation of its cyanogenic ability. Such experiments
were performed on mice which received CS either by injection or by
aerosol exposure. The appearance of cyanide in blood following the
injection of CS was also studied. Finally, assuming that CS exerts its
toxic action by release of cyanide, the protective effect of certain well-
established cyanide antidotes against the lethality of injected CS was
ewluated. Comparative studies were performed with malononitrile
and potassium cyanide.

Experimental

Mate~al8
o-Chlorobenzylidene malononitrile (CS) prepared according to Corson and
Stoughton (1928) was obtained from the Division of Applied Organic Chemistry
 Formation of Cyanide from o-Chlorobenzylidene Malononitrile lOl

of our institute. Dicobalt ethylene-diaminetetraacetate (Co~ EDTA) was used in

the form of the commercially available antidote preparation Kelocyanor (Laroche
Navarron, Paris). Other compounds were commercial products. The experiments
were carried out on male C 57 Black mice weighing i8 to 39. g.

Methods
The excretion of thioeyanate after administration of cyanogenic eompounds
or cyanide was determined in the following way. The animals were placed in pairs
in metabolic cages and kept on a standardized diet. Urine from each pair of animals
was collected under toluene for 24-h periods. After two control periods, the animals
were injected i.p. with the compounds studied or exposed to CS aerosol, and the
urine was then collected for the next two 24-h periods. All urine samples were
diluted to 20 ml with water and deproteinized by the addition of an equal volume
of 5 % triehloroacetie acid followed by centrifugation. The thioeyanate content
of the supernatant was then determined by a modification of the method of Boxer
and Rickards (1952), as described elsewhere (Nystr5m and S6rbo, 1957); thio-
eyanate is oxidized by permanganate in acid solution to hydrogen cyanide, which
is then separated by aeration into sodium hydroxide and determined by a specific
colorimetric reaction. Control experiments revealed that CS, but not malononitrile,
interfered in this assay by conversion to hydrogen cyanide in the oxidation step.
However, CS was easily separated from thioeyanate by extraction with n-heptanone.
As preliminary experiments demonstrated that the thiocyanate values obtained
in urine samples from animals injected with CS or exposed to CS aerosol did not
change after extraction of the samples with n-heptanone, this treatment was
omitted in further experiments. These results indicate that CS was not excreted
in the urine or decomposed during the collection period.
Cyanide determination was carried out in whole blood, collected in a heparinized
syringe by heart puncture. A measured volume (about 0.5 ml) was rapidly intro-
duced into the aeration tube of the aeration apparatus described by Boxer and
Richards (1952). The aeration tube contained l0 ml 0.05 M sulfuric acid together
with 3 drops of n-oetanol (to prevent foaming) and the receiving tube 4 ml 0A M
sodium hydroxide. The hydrogen cyanide liberated in the aeration tube was
transferred to the receiving tube by 45 min aeration and then determined by the
colorimetrie method of Asmus and Garschagen (19531. CS (but not malononitrile)
interfered in the colorimetric assay, but was not transferred by aeration from the
sample to the receiving tube. Thus the entire procedure could safely be used to
determine cyanide in the presence of CS.
CS, dissolved in polyethylene glycol 200, was given by i.p. injection in a volume
of 2 ml/kg in the case of sublethal doses, while higher doses were given in volumes
of 4 ml/kg due to solubility requirements. Other compounds were injected i.p.
as aqueous solutions in volumes of 8 ml/kg. LD60 was determined according to
Weii (1952) from results obtained with 4 dose levels of the compound studied and
4 animals in each case. Mortality was determined I week after administration of
the compounds, although death generally occurred within 24 h.
The aerosol exposure was carried out in an aerosol chamber with a volume of
2601. CS aerosol was generated with a spray device containing 1% CS in methylene
chloride and with dichlorodifluoromethane as the propellent gas. The concentra-
tion of CS was calculated from the amount of CS dispersed into the chamber and
the chamber volume. Immediately after exposure the animals were thoroughly
vacuum cleaned to remove CS from the fur.
8*
102 L. l~rankenberg and B. S6rbo

Results
Thiocyanate Excretion Following Injection o/CS,
Malononitrile and Cyanide
Evaluation of CS, malononitrile and cyanide for their ability to
induce thioeyanate excretion in mice following i.p. injection gave the
results in Table i. The compounds were studied at an equitoxic dose
level corresponding to 0.5 LDs0 (c.f. Table i), the highest possible dose
which would allow complete survival of the animals during the ex-
perimental period. The results show that there was extensive formation
of thioeyanate from CS and malononitrile, of a similar magnitude to
that obtained from cyanide. They suggest that cyanide formation
in vivo m a y accmmt for the toxicity of CS, as well as for malononitrile.

Table 1. Thiocyanate excretion following i.p. administration of nitriles

or cyanide to mices
Thiocyanate excretion (~moles]kg body weight/24 h)
Before treatment After treatment Yieldb
Compound Day 1 :Day 2 Day I Day 2 (%)

CS 18.7 2 2.4 25.0 2 3.6 86.8 + 12.1 29.7 + 3.6 49.9

Malononitrile 25.4 + 2.0 22.8 + 2.7 102 + 5.1 33.0 2 t.9 78.9
KCN 24.5 2 1.5 22.9 + 1.4 50.6 2 4.4 27.7_+2.6 78.4

s The excretion values are the mean 2 S.E.M. from six cages, each containing
two animals.
b The yield of thiocyanate relative to the compound administered is the dif-
ference between the sum of the excretion values after treatment and the sum of
the values before treatment, divided by the dose of compound administered. The
latter (corresponding to 0.5 LDso) was 146 ~moles/kg body weight in case of CS,
t10 ~moles/kg in case of malononitrile and 39.4 ~moles/kg in case of KCN.

Thiocyanate Excretion Following Exposure to GS Aerosol

These experiments were performed in order to assess whether cyanide
formation also occurs after inhalation of CS, which m a y be of practical
importance when the agent is used as a tear gas.
Mice were exposed to a CS aerosol with an exposure level of 20000 mg
rain -1 m -3, corresponding to about 0.5 LDa0 (Punte et al., i962) and
thioeyanate was subsequently determined. The results (Table 2) demon-
strate a conspicuous increase in thioeyanate excretion, suggesting that
significant amounts of cyanide are also formed after administration by
this route. I t should be noted that the animals showed only slight
symptoms during and after exposure to the aerosol.
 Formation of Cyanide from o-Chlorobenzylidene Malononitrile t03

Table 2. Thiocyanate excretion following exposure of mice to CS aerosol=

Thiocyanate excretion (~moles/kg body weight/24 h)
Before treatment After treatment
Day t_ Day 2 Day i Day 2
18.8 + 1.6 25.8 +_1.5 t40.4 + 5.8 36.9 _+ 2.6

The animals were exposed to a CS aerosol of about 3500 mg/ma for 6 rain.
The excretion values are mean + S.E.M. from six cages, each containing two mice.

I I I I

o, oe

E Oos

o
E
"; qo4

0,o2
/ ',

I I ! I
9/. 8 16 30
T i m e (rain)

Fig. 1. Blood levels of cyanide following i.p. injections of 0.5 LDso of nitrfles or
KCN. [3 • CS (30 mg/kg); 9 . . . . . 9 malononitrile (7.3 mg/kg); A-.-.-A,
KCN 2.7 mg/kg

Cyanide Concentration in Whole Blood in Relation to Symptoms

Following Injections o/CS, Mcdononitrile and Cyanide
After an i.p. injection of 0.5 LDs0 of CS or malononitrile the mice
developed toxic symptoms within 3 to 4 rain; these were paralysis and
respiratory depression. The animals recovered slowly and respiration
was still moderately to severely depressed 30 rain after injection. De-
termination of cyanide in blood showed t h a t high concentrations were
reached rapidly, with peak levels 4 to i6 rain after injection in case of
CS and after 8 rain in case of malononitrile (Fig. i). The blood cyanide
concentrations fell rather slowly and more t h a n haft of the peak values
were still present 30 rain after injection. When an equitoxie dose of
104 L. Frankenberg and B. SSrbo

cyanide was given symptoms appeared within 3 rain but vanished

rapidly and the animals recovered completely within 30 rain. The peak
cyanide concentration in blood following injection of cyanide (Fig. l)
was reached within 2 min; it was somewhat lower than that obtained
after administration of CS or malononitrile, and declined rapidly. There
is thus a correlation between the time course of toxic symptoms and the
blood cyanide levels after the administration of the nitriles and cyanide,
which is consistent with the hypothesis that cyanide is the toxic agent
formed from CS and malononitrile.

EUect of Cyanide Antidotes on Toxicity o/ CS in Comparison with Their

Antidotal Action against Malononitrile and Cyanide
Antidotes with different action mechanisms (Friedberg, t968) were
chosen for this study, and their effects on CS toxicity were compared
with those obtained in experiments with malononitrile and cyanide. The
first antidote studied was thiosulfate, which is involved in the enzymatic
detoxifieation of cyanide to thiocyanate. It was found (Table 3) that
the acute toxicity of CS injected i.p. was significantly reduced by pre-
treatment with thiosuffate, in confirmation of previous reports in the
literature. However, thiosulfate was less effective against CS than
against cyanide or malononitrile. The second antidote, inorganic nitrite,
induces the formation of methemoglobin, which inactivates cyanide by
formation of cyanometheinoglobin. Nitrite had a weak antidotal effect
against CS and was only moderately active against cyanide and malono-
nitrile (Table 2). The third cyanide antidote studied was Co~ EDTA,
which forms a strong complex with cyanide. It was found to be inactive
against CS (Table 2), and to have a weak antidotal effect against malono-
nitrile although it was a very potent antidote to cyanide.

Discussion
Results presented here demonstrate that administration of CS to
mice leads to an increased excretion of thiocyanate, indicating in vivo
formation of cyanide from CS. Gal and Greenberg (i952) demonstrated
a similar excretion of thiocyanate in rats receiving certain substituted
malononitriles, although CS was not included in their study. It should
be noted that CS and malononitrile contain two nitrile residues and may
thus in theory give rise to two cyanide ions per molecule of the parent
compound. However, the yields of thiocyanate (when corrected for the
incomplete yield of thioeyanate obtained from injected cyanide, e.f.
Table i) correspond in our experiments where CS was injecteded to only
one mole of thiocyanate per mole of CS. This indicates that only one
of the two nitrile residues in the malononitrile is converted to cyanide
 o
Table 3. Protective effect of cyanide antidotes against nitriles and cyanlde~
Antidote CS Malononitrile KCN
LD~o DRF LDs0 DRF LDs0 DRF
(mg/kg) (mg/kg) (mg/kg)

-- 60.1 ( 47.5--- 75.9) i.0 14.5 ( 12.8-- 16.5) 1.0 5.29 ( 4.32-- 6.48) i.0 ?
Thiosulfate 147 (120--t80) 2.5 133 ( 1 0 8 - - 1 6 3 ) 9.2 29.7 (24.3--36.4) 5.6
Nitrite 92.8 (75.8---114) t.5 43.5 ( 32.7-- 58.0) 3.0 18.3 (14.9 --22.4) 3.5
Co2 EDTA 61.1 ( 40.1-- 93.3) 1.0 29.0 ( 23.7-- 35.5) 2.0 32.9 (24.7 --43.8) 6.2

The antidotes were given i 0 rain before the cyanogenic compounds at the following dose levels: NasS20s. 5 l~s0 2.0 g/kg. NAN02
75 mg/kg and Cos EDTA 50 mg/kg. All injections were i.p. The LDso values are given with 95 % confidence intervals within brackets.
DRF (Dose Reduction Factor) is the ratio between LDs0 with and without anr
106 L. Frankenberg ~nd B. S6rbo

in vivo. A similar proposal was made by Heymans and Masoin (i897)

when they found malononitrile to be isotoxie with cyanide on a mole-to-
mole basis.
The transformation of CS to cyanide in vivo was also directly demon-
strated in the present investigation by the significant cyanide levels
found in blood taken from CS-injected animals. The time course of toxic
symptoms was also related to that of the blood cyanide concentration,
suggesting that cyanide formation is a major factor of CS toxicity.
On the other hand, our results from studies on the protective effect
of cyanide antidotes against CS were less clear-cut. Although thiosulfate
afforded some protection against CS, it was more effective against
cyanide and even more effective against malononitrile. The superiority
of the antidotal effect of thiosulfate against malononitrile in comparison
with that against cyanide, which was also observed by Nash et al. (1950),
may be explained as follows. Administration of cyanide by i.p. injection
results in an initially high blood cyanide concentration, which rapidly
decreases, whereas malononitrile liberates cyanide slowly for a long
time. As thiosulfate is a cyanide antidote with a slow onset but a long
duration of action (Friedberg, i968) it ought to be more effective against
malononitrile than against cyanide. However, although the cyanide
concentration in blood after injection of CS followed a time course
practically identical with that after malononitrile (Fig. l), the protection
given by thiosulfate against CS was markedly inferior to that against
malononitrile. This may indicate that other mechanisms than cyanide
formation are involved in the toxicity of CS. This interpretation is also
on line with our findings that nitrite and Co~ EDTA were less effective
against CS than against malononitrile. It should be noted in this connec-
tion that Co~ EDTA was a much better antidote to cyanide than to
malononitrile, which may be due to its short duration of action (Fried-
berg, 1968).
The results of the present investigation thus suggest that cyanide
formation is an important mechanism in the toxicity of injected CS,
although other mechanisms may also be involved. This is probably also
the case after inhalation of CS, as our results demonstrate a high excre-
tion of thiocyanate in the urine following exposure by this route. Al-
though a quantitative evaluation of the yield of thiocyanate from
inhaled CS is not possible as the amount of CS retained in the airways
is unknown, our data clearly indicate significant resorption of CS from
the respiratory tract. This is substantiated by the recent findings of
Leadbeater (1973), who determined CS and two of its metabolites
(o-chlorobenzylmalononitrile and o.chlorobenzaldehyde) in the blood
and was thus able to demonstrate significant absorption of CS from the
respiratory tract of animals exposed to the agent. However, other
 Formation of Cyanide from o-Chlorobenzylidene Malononitrile i07

mechanisms t h a n cyanide formation m a y be of u t m o s t importance for

the toxicity of inhaled CS. Thus the symptoms and histopathological
findings in animals which received lethal exposures to CS aerosols,
indicated t h a t lung damage was the cause of death (Punte et aL, i962;
Ballantyne and Callaway, i972). Although the mechanism of the irritant
action of CS is still unknown it m a y be related to the reactivity of CS
with certain thiol compounds of biological importance, especially di-
hydrolipoic acid (Cuccinell et al., i97i).

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Veterin~ir Lars Frankenberg

F6rsvarets forskningsanstalt
S-172 04 Sundbyberg 4
Sweden