CHAPTER XII
PHYSIOLOGICAL ACTION OF
DICHLORETHYLSULPHIDE (MUSTARD GAS) a
When an animal was exposed to the vapors of
dichlorethylsulphide in high concentration it
subsequently showed a complex of symptoms which maybe divided into two
classes: (1) The
systemic effects due to the absorption of the substance into the blood
stream and its distribution
to the various tissues of the body. These effects were not generally
recognized. (2) The local
effects on the eyes, skin, and respiratory tract. These were well
recognized, and consisted mainly
of conjunctivitis and superficial necrosis of the cornea: hyperemia,
edema, and later, necrosis of
the skin, leading to a skin lesion of great chronicity; and congestion
and necrosis of the epithelial
lining of the trachea and bronchi.
The most striking observation regarding the symptoms
of dichlorethylsulphide poisoning was the
latent period, which elapsed after exposure before any serious
objective or subjective effects
were noted. The development of the effects was then quite slow, unless
very high super lethal
doses had been inhaled.
SYSTEMIC EFFECTS
The symptoms observed in dogs when subjected to the
vapors of dichlorethylsulphide which
might suggest absorption into the blood stream through lungs and skin,
and a systemic effect,
were vomiting, diarrhea, hyperexcitability and convulsions, and effects
upon the heart.
Moreover, the condition of the lungs and trachea found at autopsy of
some animals dying from
the inhalation of the gas was not sufficient to account for death.
ABSORPTION THROUGH THE LUNGS
INJECTIONS
In order to become familiar with the effects of the
absorption of dichlorethylsulphide into the
system, dogs were injected with the substance. The simplest method of
introducing the substance
was by subcutaneous or intramuscular injection of olive oil solutions.
The effects observed on an
unanesthetized animal from the injection of a lethal dose in olive oil
were, after a latent period,
salivation, hyperexcitability, and convulsions, diarrhea, slow and
irregular heart, which became
rapid before death, muscular weakness, and finally coma and death.
_
a The data
in this chapter are based on the researches of the pharmacological
research section,
Medical Division, Chemical Warfare Service, conducted in the American
University Experiment
Station Laboratories, Washington, D. C., and published as follows: (1)
Lynch, Vernon, Smith, H.
W., and Marshall, E. K., Jr.: On Dichlorethylsulphide (Mustard Gas). I.
The Systemic Effects
and Mechanism of Action, Journal of
Pharmacology and Experimental Therapeutics. Baltimore,
Md., 1918, xii, No. 5, 265; (2) Marshall, E. K., Jr.,
Lynch, Vernon, and Smith, Homer W.: 11.
Variations in Susceptibility of the Skin to Dichlorethylsulphide. Ibid,
291; (3) Smith, Homer W.,
Clowes, George H .A., and Marshall, E. K. Jr.: IV. The Mechanism of
Absorption by the Skin.
Ibid, 1919, xiii, No. 1, 1.
370
The following protocols illustrate the effects of
subcutaneous and intramuscular injections in
olive oil solution:
EXPERIMENT 1.- Dog V7, male; weight, 12.5
kilos
March 12.
10.15. Pulse
136, respiration 18.
10.28.
Subcutaneous injection of 500 mgm. dichlorethylsulphide in 5 grams
olive oil.
10.45. Behavior
normal. Licks point of injection.
11.00.
Somewhat restless.
12.00. Salivation,
vomiting, diarrhea.
1.15. Pulse 66,
respiration 32.
1.30. Muscular
spasms, especially in hind legs. Convulsions. Animal staggers. Finally
unable to
walk.
1.40.
Intraperitoneal injection of 3 grams chloretone in olive oil.
2.10. Pulse 66
(very strong), respiration 56. Still excitable.
2.45. Pulse 64,
respiration 36.
4.15. Pulse 126,
respiration 26. Given another injection of 500 mgm. sulphide.
5.00. Animal
conscious, but unable to stand.
March 13. Found dead.
Autopsy.-Lungs and trachea
appear normal; stomach contains bile and bloody fluid; intestines
congested with few areas of hemorrhage and contain bloody fluid.
EXPERIMENT 2.-Dog V10, female; weight, 10
kilos.
9.50. Pulse
88, respiration 18.
9.55. Subcutaneous
injection of 1,000 mgm. of dichlorethylsulphide in 10 c.c. of olive
oil, 5 c.c.
on each side.
10.00. Pulse
90, respirations 20. Animal quiet.
10.15. Restless.
10.30. Pulse 118,
respiration 44.
10.50. Pulse
112, respiration very rapid, slight salivation.
11.15.
Salivation, rapid respiration, diarrhea.
12.00. Pulse
44, respiration 102. Animal has previously had very good control of
muscles.
Movements are now stiff, convulsive, and uncontrolled. Tries to
struggle to feet, but topples over
and paws ground convulsively.
1.00. Pulse 36
(very strong, but irregular), respiration 112.
1.45. Pulse
36, respiration 64. Very weak, unable to rise. Has vomited.
2.40. Pulse
54, respiration 82. Vomiting. Now perfectly quiet.
3.00. Pulse
72, respiration 66.
3.20. Pulse
108 (very weak), respiration 54.
3.30. Dies.
Autopsy.-Trachea and lungs appear
slightly congested; stomach filled with bloody fluid;
intestines congested and hemorrhagic; other organs appear normal.
EXPERIMENT 3.-Dog DM203; weight, 17 kilos.
September 23.
10.30. Pulse
88, respiration 18.
10.35.
Intramuscular injection of 240 mgm. (14 mgm. per kilo) of
dichlorethylsulphide in 12 c.
c. of olive oil.
11.00.
Behavior normal. Pulse 114, respiration 30.
11.30. No
toxic symptoms as yet. Pulse 100, respiration 20.
12.00. Pulse
102, respiration panting; is becoming hyperexcitable; appears irritable
and trembles
slightly, but walks without ataxia.
12.45. Pulse
120, respiration 28. Walks uncertainly; salivated; pupils normal.
Injected leg
affected, seems sore, and dog refuses to use it in standing or walking.
1.10. Pulse
120, respiration 30. Injected leg no longer seems sore, hut is wholly
useless.
371
1.45. Pulse
130, respiration 36. Though seemingly conscious, movements are
decidedly
convulsive. No diarrhea, great salivation or hyperexcitability. Pupils
are now greatly dilated.
2.00. Pulse
114, respiration 29. Dog exhibits twitching and slow convulsive
movements. Knee
reflex in injected leg about normal; the leg is inactive during
convulsive movements. Very
irritable and snappy at times; then again is very affectionate.
After drinking heavily,
pulse runs
up to 150, but is very irregular.
2.45. Pulse
144, respiration 60. Shows slight salivation.
4.20. Pulse
174, respiration 66. Still shows slight convulsive tendencies.
September 24.
9.00. Pulse
192, respiration 48. Dog is conscious, but trembles greatly. No
salivation or evidence
of diarrhea. Pulse varies greatly during day.
September 25.
1.00. No
marked change in condition. Pulse running 150 to 180 throughout day,
respiration 24 to
35.
September 26.
10.20. Pulse
156, respiration 32.
4.00. Dog
found dead 77 hours after injection.
Autopsy.-Conjunctivitis; slight cutaneous edema at site of
injection. Tracheal blood vessels
congested; lungs apparently normal, with slight post-mortem changes. No
hemorrhages in
adrenal cortex. Liver, kidneys, and spleen normal; mucous membranes of
gut hemorrhagic and
bloody, chiefly in upper tract; stomach normal.
EXPERIMENT 5.-Dog DM202; weight, 11 kilos.
September 23.
10.30. Pulse
102, respiration 66.
10.40.
Intramuscular injection of 220 mgnm. (20 mgm. per kilo) of dichlorethyl
sulphide in 5.5
c. c. of olive oil.
11.00. Dog
appears normal, except for panting. Pulse 138.
11.30. Pulse
100, respiration still panting.
12.00. Pulse
78, respiration 60. Marked salivation; appears spasmodically ataxic,
with severe
tremors; distinctly hyperexcitable.
12.45. Pulse
78, respiration very irregular. Salivation, but no diarrhea or
vomiting. Exhibits
severe convulsions. Pupils dilated. Injected leg stiff and does not
enter into convulsive
movements, which are of a clonic nature; between convulsive spasms, dog
attempts to rise, but
both rear legs are inactive.
1.10. Pulse
75, respiration 70. Passes small quantity of semiliquid stool without
blood stain.
1.45. Pulse
68, respiration 36. No continued diarrhea.
2.00. Pulse
66, respiration 30. Heart sounds hammer-like, but very irregular in
periodicity and
intensity.
2.15. Pulse
90, respiration 28. Dog attempts to drink with great effort, but cannot
rise or put nose
in water. Continues to lap air. Portrays marked muscular weakness and
incoordination.
2.30. Pulse
68, respiration 39. Eyelids twitching. Reflexes abnormally active.
Heart irregular,
missing every third beat.
2.45. Pulse
150, respiration 40.
3.00. Pulse
150, respiration 48. Heart sounds becoming very faint, with increased
rate.
4.00. Pulse
162, respiration 38. Great muscular weakness. Almost unconscious. Seems
to be
passing into coma.
4.20. Pulse
192, respiration 48. Dog lies in coma.
September 24.
12.00. Found
dead after 13 hours.
Autopsy.- Trachea slightly congested. Lungs show small
circumscribed points of hemorrhage
throughout; considerable post-mortem congestion; adrenals show slight
cortical inflammation.
Kidneys normal, except for slight postmortem congestion. External gut
has too much post-mortem change to describe extent of external
hemorrhage. Stomach is slightly congested. Very
marked hemorrhage in the lumen of jejunum and duodenum; entire gut
blood stained throughout.
Injected leg showed great subcutaneous edema but no muscular changes
beyond a slightly
brighter color than in normal leg.
372
The intravenous injection was much more instructive
but somewhat more difficult. The slight
solubility of the substance in water (about 0.07 percent at 10°
C.), and the rapidity with which an
aqueous solution hydrolyzes were the main difficulties. This could not
be overcome by the
injection of an alcohol or acetone solution, for as soon as these
solutions came in contact with
water the sulphide was precipitated out as fairly large oil droplets.
These difficulties were
overcome by the injection of large amounts of a freshly prepared, cold,
saturated aqueous
solution.
The method of preparing the aqueous solution for
intravenous injection was as follows: Five
hundred cubic centimeters of 0.8 percent saline was cooled to 8 to
10° C. This was placed in a
flask, about 1 c.c. of pure dichlorethylsulphide was added, the flask
tightly stoppered, and
shaken for about one minute. The contents of the flask were transferred
to a separatory funnel
and the oil allowed to settle. An oil film was present on the surface
as well as globules at the
bottom. About 400 c.c. of solution was removed from between the oil and
film, care being taken
to obtain a solution free from oil films or droplets. This was placed
in a bath at 8 to 10° C. and
used as soon as possible.
The solution prepared in this way contained about 0.7 mgm. per cubic
centimeter.
The hydrolysis of the aqueous solution is a
monomolecular reaction. At 10° C. only about 15
percent of the disulphide is hydrolyzed in 10 minutes, while at
37.5° C. over 97 percent is
decomposed in the same time.
The symptoms elicited from an intravenous injection were similar to
those observed from the
subcutaneous injection of olive-oil solutions. While the solution was
being injected, and for
some time after, the animal showed no effects whatever. A record of the
blood pressure, pulse,
and respiration failed to show an appreciable effect at this time.
Within 10 to 20 minutes after injection, however, an
increased salivation was noticed. This soon
developed into a very free flow of rather mucinous saliva. The next
symptom observed was
usually a diarrhea, which might be accompanied ly vomiting. This
diarrhea was present until the
death of the animal, and a few hours after the injection the stools
frequently contained blood.
After injection the respiration became rapid, and if
anesthesia had not been used the animal
showed a distinct hyperexcitability. At this stage he might be
frightened by a slight movement of
the hand or unexpected touch, and the eye reflex might be obtained by
touching almost any part
of the face. The gait soon became unsteady, movements of the muscles
were spasmodic but
uncontrolled, and apparently accompanied by tetanic contractions of the
antagonist. The knee-jerk might be elicited by a touch of the finger.
The animal soon became unable to walk or even
to stand, and the violent spasmodic movements increased to the stage of
convulsions, with
extension of the hind legs and arching of the neck and back. The pulse,
which might have been
somewhat slowed, soon became irregular. Palpation suggested that the
heart was dropping a beat
occasionally. The dropped beat became more and more frequent until
finally the heart was
beating at one-half its former rate. If the chest was opened it was
clearly seen that the ventricles
were beating once to every two beats of the auricles. Later the rhythm
might even become 1 to 3.
Stimulation of the vagi showed some apparent hvperexcitabilitv. The
blood pressure fell very
slowly, and a few hours before death the heart resumed
373
its normal rate. At this stage, or before, it was found that the heart
could not be slowed or in any
way affected by strong stimulation of the vagi, although the
respiration was easily inhibited.
Section of the vagi had no effect upon the heart rate except a slight
slowing. Apparently the
vagus endings were paralyzed. The heart became feeble, there was a
great dilatation in the
splanchnic. area, a high venous pressure, and the arterial blood
pressure was falling slowly. The
convulsions ceased, and the animal lay in a coma. Death came quietly in
less than 24 hours after
the injection and was probably due to the weakening of the heart and
the great dilatation of the
vessels in the splanchnic area. Autopsy revealed a more or less intense
congestion of the
intestinal mucosa which might extend from the pylorus to the anus, and
was frequently
accompanied by hemorrhage into the lumen of the intestine. The
condition suggested the
excretion of the dichlorethylsulphide into the intestine. These effects
upon the heart, the alimentary tract, and the central nervous system
were quite
characteristic and unmistakable.
The following protocols are representative of the
course of an intravenous injection. In all cases
the cold, saturated aqueous solution, prepared as described above, was
used. The solution was
usually injected within 10 to 15 minutes after its preparation. When
injected such a solution
contained about 0.5 to 0.6 mgm. of undecomposed dichlorethylsulphide to
each cubic
centimeter.
EXPERIMENT 6.- Dog, V9, female; weight, 8.2
kilos.
11.40. Pulse
88, respiration 16.
11.50-12.00.
Injection into jugular vein of 120 c. c. fresh aqueous solution (14
c.c. per kilo).
12.05.
Shivering. Pulse 160, respiration 66.
12.20. Slight
salivation, has vomited. Pulse 70, respiration 74.
12.45.
Diarrhea, no blood. Pulse 52 and irregular, respiration 108.
1.20. Highly
excitable, convulsions, readily thrown into spasms-comlparatively quiet
between.
1.45. Pulse
80, respiration 110.
2.15. Pulse
86, respiration 120.
2.45. Pulse
84, respiration 120.
3.45. Pulse
84, respiration 110. Struggling, proftuse salivatioin,has had diarrhea
for some time.
3.50. Killed
with ether.
Autopsy.-Trachea
appears normal. Lungs very small area of congestion in one lobe, a few
emphysematous patches. Esophagus and stomach appear normal. Duodenum,
congested areas
beginning at pylorus. Small and large intestine show distinct congestion.
EXPERIMENT 7.- Dog V17, female; weight, 5.5
kilos.
March 29.
3.10. Pulse
120, respiration 12.
3.12-3.20.
Intravenous injection of 120 c.c. of cold, aqueous solution (22 c.c.
per kilo).
3.25. Pulse
150, respiration 42.
3.35. Pulse
120, respiration 198. Trembling, good control of movements.
3.45.
Vomiting; diarrhea.
4.05. Gait
very unsteady.
4.30.
Vomiting; fluid stools.
4.50.
Struggling, convulsions begin; salivation.
5.00. Still in
convulsions.
March 30.
9.00. Found
dead.
374
Autopsy.-Trachea slightly congested; lungs
appear normal; heart, large, endocardial
hemorrhages; stomach contains bloody fluid; duodenum very deep red,
bloody contents; small
and large intestine deeply congested.
EXPERIMENT 8.- Dog V36,
male; weight, 9.5
kilos.
April 22.
10.45. Pulse
90, respiration 11.
11.02.-11.05.
Injection of 95 c.c. fresh, aqueous solution into jugular vein.
11.16. Slight
salivation, vomiting.
11.25. More
vomiting.
11.40.
Hyperexcitability.
12.45.
Convulsions.
1.00. Pulse
120, respiration 30, convulsions.
In the following experiments the dose given was too
small to produce convulsions, but the other
symptoms were observed.
EXPERIMENT 9.- Dog V20, male; weight, 11 kilos.
April 2.
12.18. Pulse
120, respiration 24.
12.18-12.23.
Injection of 66 c.c. cold, fresh aqueous solution in jugular vein.
12.25. Pulse
116, respiration 21.
12.30.
Perfectly normal.
1.05. Has
vomited.
2.00. Pulse
120, respiration 20; normal movements.
3.00. Pulse
132, respiration 24; salivation; lying quietly, but leg muscle twitched
a good deal.
April 3. Apparently
normal; killed for autopsy.
Autopsy.-Lungs appear normal; duodenum congested and slightly
hemorrhagic; small intestine
contains bloody contents.
EXPERIMENT 10.- Dog V34, male; weight,
7.2
kilos.
9.45. Pulse 96,
respiration 12.
10.05.
Intravenous injection of 36 c.c. cold, fresh aqueous solution (5 c.c.
per kilo).
10.08. Pulse
108, respiration 14.
10.55. Has
vomited.
11.05. Slight
unsteadiness of gait.
2.25. Pulse
132, respiration 32. Gait normal.
EXPERIMENT 11.- Dog V19, female; weight, 17.3
kilos.
April 1.
12.55. Pulse
138, respiration 18.
1-1.05.
Injection intravenously 34 c. C. of cold, fresh aqueous solution.
3.00. Pulse
78, respiration 12, quiet.
April 2.
9.00. Depressed,
very quiet, refuses food. Killed for autopsy.
Autopsy.-Lungs appear normal; duodenum congested, bloody mucous
abundant; small intestine
congested; large intestine normal.
The following protocol is illustrative of the
symptoms of an animal injected very slowly. The
symptoms were the same as when injection was made rapidly, but appeared
to be somewhat
more delayed and miller.
EXPERIMENT 12.- Dog V16, male; weight, 4.5
kilos.
1.30. Pulse
150, respiration 18.
1.55-2.55.
Intravenous injection
of 100 c. c. cold, fresh aqueous solution (22 c.c. per kilo).
2.54. Pulse
125, respiration 48,
slight salivation.
3.00. Pulse 120,
respiration 78,
salivation, seems excitable, defecated, had control of
movements.
3.30. Pulse 120,
respiration 55,
bad control of movements, diarrhea.
3.45. Pulse 60,
respiration 84,
moves around fairly well.
4.00 Movements
Unsteady and
spasmodic, convulsions begin.
4.05. Unable to
stand.
5.00. Respiration
72, lying prone
in convulsions.
March 30. Found dead.
375
Autopsy.-Trachea
normal; lungs, one lobe appears congested; duodenum,
deeply blood-stained
contents; intestines congested with blood-stained contents; heart, few
small subendocardial
hemorrhages.
The injection of saturated aqueous solutions, which
have been allowed
to come to room
temperature or which have been standing for any time, showed much
diminished effects, or, if
several hours old, were entirely without effects.
This was tried by intravenous injection and also on the eye and skin.
Such solutions were known
to be partially or completely hydrolyzed into hydrochloric acid and
dihydroxethylsulphide.
CHART
EXPERIMENT 13.-Dog V11; weight, 4 kilos.
March 22.
3-3.15.
Injection intravenously of 160 c. c. aqueous solution 24 hours
old (40 c. c. per kilo).
4.00. No
effects.
6.00. No
effects.
March 23.
9.00. Dog
perfectly normal. No vomiting or diarrhea during night.
EXPERIMENT 14.-Dog EM970; weight, 8 kilos.
2.40. Pulse
130, respiration 18.
2.50.
Intravenous injection of 10 c.c. per kilo of a saturated aqueous
solution of
dichlorethylsulphide which had stood for three and one-half hours.
3.00. Pulse
160, respiration 18.
3.50. Pulse
160, respiration 22; dog seems normal.
4.15. Pulse
164, respiration 20. Animal appeared perfectly normal while
under observation for
two or three days.
EXPERIMENT 15.-The cold, fresh, aqueous solution was
applied to the
forearms of five men.
After standing at room temperature for two hours itwas again applied.
Twenty-four hours later,
four out of five of the men showed a distinct reaction from the fresh
solution, none exhibited any
effects from the solution two hours old.
EFFECTS OF INHALATION OF LARGE DOSES OF THE VAPOR
Some of the effects which have been observed from
the injection
occurred when a dog was
poisoned by inhalation of the vapor. When a dog was poisoned by
inhalation of a very large dose
of the vapor, practically all the effects obtained by injection
(salivation, vomiting, bloody
diarrhea, hyperexcitability and convulsions, slow, irregular pulse,
becoming rapid before death,
and paralysis of the vagi) were observed. This leaves little room for
doubt that, in high
concentrations, dichlorethylsulphide is absorbed through the lungs and
produced its
characteristic effects upon the body.
EXPERIMENT 16.-Dog V12, male; weight, 11.4
kilos.
March 21.
10.15. Pulse
168, respiration 36. Very active and playful.
10.30-11.30.
In gassing chamber, exposed to 0.3 mgm. per liter of
dichlorethylsulphide-restless,
rapid respiration, excitement, salivation, vomiting
11.40.
Hyperexcitable. Coughing.
11.50. Pulse
108, respiration 56.
1.00.
Defecates. Marked muscular weakness, ataxia.
1.25.
Convulsions.
1.45. Pulse
152, respiration 50.
3.30. Pulse
168, respiration 64; marked muscular weakness; salivation.
4.00. Vomits.
4.30. Pulse
176, respiration 52, pulse becoming feeble. Killed with
ether.
376
Autopsy.-Mouth,
esophagus, and stomach normal; duodenum shows
congestion, contents
bloody; small intestine has congested areas of mucosa; trachea, some
slight membrane; lungs,
one lobe congested and emphysematous; heart, kidneys, liver, and
spleen, normal.
EXPERIMENT 17.-Dog V13, female; weight, 13.5
kilos.
10.00. Pulse 96, respiration 26.
10.05-11.05. In gassing chamber,
exposed to 0.28 mgm. per liter of
dichlorethylsulphide.
Excitement, irritation of upper respiratory tract, salivation, and
vomiting.
11.08. Loose stools.
11.35. Pulse 44, respiration 96;
pulse irregular, expiration spasmodic.
12.35. Pulse 44, respiration 60;
diarrhea, salivation,
hyperexcitability with typical convulsions.
1.20. More violent convulsions.
2.05. Pulse 68, respiration
28. Animal very weak.
2.30. Vomits voluminous, foamy
fluid. Bloody diarrhea.
3.40. Pulse 70, respiration 30.
Very weak, but still struggles some.
4.00. Killed with ether.
Autopsy.-Trachea
slightly congested, contains some fluid; lungs, one
lobe shows slight
hemorrhage, few patches of emphysema; esophagus and stomach normal;
upper intestinal tract
deeply congested, congestion decreases until in ileum, normal; large
intestine congested.
FATE OF DICHLORETHYLSULPHIDE IN THE BODY
Furtherconvincing proof of the absorption of the
sulphide through the
lungs was furnished by the
detection of one of the products of hydrolysis, dihydroxyethylsulphide,
in the urine of animals
poisoned by dichlorethylsulphide by inhalation. This hydrolytic product
could also be found in
the urine after the injection of mustard gas.
Dihydroxyethylsulphide was injected into a dog, the
urine collected and
examined for this
substance. A positive test was obtained. Urine from a normal dog failed
to give a positive
reaction. This was necessary because of the occurrence of ethylsulphide
and its precursors in
normal dog's urine. This proves that the substance was excreted, in
part at least, unchanged. The
injection of dichlorethylsulphide was next tried, and the urine found
to contain the
dihvdroxyethylsulphide. The inhalation experiments were then performed.
EXPERIMENT 18.-Dog V47, female; weight, 13.2
kilos.
May 19. Intravenous
injection of 100 mgm. per kilo of
dihydroxyethylsulphide in 10 c.c. saline.
May 20. No urine passed in cage;
230 c.c. urine by catheter. This was
evaporated under
diminished pressure to small volume, 25 c. c. of concentrated
hydrochloric acid was added, and
distillation carried out under diminished pressure. The distillate was
extracted with ether, and the
extract evaporated. The residue was tested by applying small amounts to
the skin of several
individuals. A typical mustard-gas reaction developed. This was
considered evidence of the
presence of dichlorethylsulphide.
EXPERIMENT 19.-Dog V49, female; weight, 21.4 kilos.
May 20-21. Over a period of 24
hours, injected subcutaneously with 60
mgm. per kilo of
dichlorethylsulphide in olive oil, 10 mgm. per kilo at a time.
Immediately after last injection,
dog was catheterized and 200 c.c. urine obtained. This was concentrated
as above, and tested for
dichlorethylsulphide by applying to the skin of several men. No
reaction obtained. It was then
treated as above, and a slight amount of oily substance obtained on the
distillate. The distillate
was extracted with ether, and the ether evaporated, leaving a globule
of oil. This oil was placed
in a small test tube, and the month of the tube held against the arm
for 3 minutes. After 24 hours
a distinct dichlorethylsulphide effect was obtained. The distillate
contained mustard gas, and the
urine dihydroxyethylsulphide.
377
EXPERIMENT 20.-Dog V54.
May 24.
10.25-10.55.
Gassed in continuous flow chamber for 30 minutes, 0.52
mgm. per liter.
May 25.
9.00. Pulse
102, respiration 34. Great muscular weakness. Unobserved,
but seems to have been
in convulsions.
10.00. Urine
taken with catheter and examined for
dihydroxyethylsulphide.
10.07. Dog
dies in coma.
A severe reaction from the re-chlorinated product
was not obtained, but
a sufficient reaction was
obtained to furnish evidence of dichlorethylsulphide being present.
It was found possible by the application of 0.1 to
0.2 gram per kilo to
the skin of dogs to obtain
the characteristic effects of the absorption of mustard gas;
salivation; vomiting; diarrhea;
hyperexcitability; rapid, feeble pulse and depression, but neither
convulsions nor slowing of the
heart. The product of hydrolysis of mustard gas was also detected in
the urine.
EXPERIMENT 21.-Dog V90; weight, 16 kilos.
11.00. Normal
pulse 108, respiration 36. Chest and abdomen shaved; 1.6
grams of pure
dichlorethylsulphide rubbed into skin with glass rod. Animal placed so
that draft prevented
absorption by inhalation of the vapors.
11.30. Second
application; 1.6 grams rubbed in (total quantity, about
200 mgm. per kilo).
12.00. Pulse
96, respiration 150.
1.40. Pulse
138, respiration panting.
5.00. Pulse
168, respiration panting. Dog placed in clean metabolism
cage. June 4.
9.00. 200 c.
c. of urine collected overnight; examined for
dihydroxyethylsulphide. Positive
findings. Pulse 198, respiration 36. Dog depressed; slight salivation.
June 5.
9.00. Pulse
186, respiration 48. Dog etherized.
MECHANISM OF ACTION
TOXICITY OF PRODUCTS OF HYDROLYSIS
The latent period in the development of the effects
of
dichlorethylsulphide, either local upon the
eyes, skin, or respiratory tract, or systemic upon the heart, nervous
system, and digestive tracts,
suggests that the substance may be altered in the body before
exhibiting its characteristic actions. In fact, the absence of any
immediate effects when the aqueous solution was injected directly
into the blood stream makes this assumption almost imperative. The
simplest chemical change
which this substance undergoes in vitro is hydrolysis into hydrochloric
and
hydroxyethylsulphide. That this change takes place in the animal
organism was shown by the
detection of dihydroxyethylsulphide in the urine. However, an injection
of a hvdrolyzed solution
of dichlorethvlsulphide was without effect. The dihydroxyethylsulphide,
when applied pure to
skin of man and dogs, produced no irritation whatever.c As
much as 0.3
gram per kilo was
injected intravenously into a dog without producing any apparent
effect, immediate or remote.
An injection of 1,400 mgm. per kilo caused only a slight stupor and
loss of coordination, with a
quick return to normal, and none of the symptoms of the
dichlorethylsulphide were present.
c Victor Meyer states that this substance is
nontoxic. Berichte der
deutschen chemischer
Gesellshaft, Berlin, 1886, xix, No. 3, 3259.
378
EXPERIMENT 22.- Dog EM575, male; weight, 7.8
kilos.
November 18.
2.30. Normal
pulse 80, respiration 20.
2.45.
Intravenous injection of 1,400 mgm. per kilo of
dihydroxyethylsulphide in 100 c. c. of
water (total, 10,920 grams). Injection followed by salivation and
slight nausea.
2.50. Dog
shows slight ataxia. Pulse 120, respiration 20.
3.10. Pulse
110, respiration 20.
3.50. Pulse
110, respiration 20. Dog appears stupid, and exhibits as
light ataxia similar to light
alcohol poisoning. No serious symptoms apparent.
5.00. Pulse
105, respiration 20. Dog quiet, perhaps slightly depressed.
Eats and drinks with
indifference.
November 19.
9.00. Dog
normal. Pulse 98, respiration 24.
November 20.
9.00. Dog
normal. Pulse 105, respiration 30.
EXPERIMENT 23.-Dog EM576; weight, 5.9 kilos.
November 19.
10.30. Normal
pulse 100, respiration 20.
10.45.
Intravenous injection of 200 mgm. per kilo (total, 1, 200 mgm.).
Dihydroxyethylsulphide in 50 c. c. of water. Animal
exhibited no
symptoms whatever. Observed
for three days.
EXPERIMENT 24.-The pure dihydroxyethylsulphide was
rubbed into the skin
on four dogs and
five men and produced no irritation, either immediate or remote.
This product of hydrolysis is not responsible for
the effects of
mustard gas.
The other product of hydrolysis is hydrochloric acid and is not a very
toxic substance. Relatively
large amounts can be injected intravenously without producing any
marked effect. This is readily
understood. When injected intravenously it is immediately neutralized
by the "buffer" action of
the blood. The blood does not become acid and the tissues are never
really exposed
to the acid. When
strong solutions are placed on the skin or mucous surfaces, or injected
into the tissues, an
irritating effect is noticed. Hydrochloric acid, however, injected in
very large doses, does
produce very definite effects upon the animal and can cause death. Both
products of hydrolysis
of mustard gas are very readily soluble in water and very sparingly
soluble in organic solvents,
or, in other words, have a low lipoid solubility or partition
coefficient. It would be expected from
this that they would not readily penetrate cells. Harvey has shown this
to be true for hydrochloric
acid.1
THEORY OF ACTION
Dichlorethylsulphide is very slightly soluble in
water and very freely
soluble in organic solvents,
or has a high lipoid solubility or partition coefficient.d It would
therefore be expected to
penetrate cells very readily. Its rapid powers of penetration are
practically proven by its effects
upon the skin. Having penetrated within the living cell, it would
undoubtedly hydrolyze. The
liberation of free hydrochloric acid within the cell would produce
serious effects and might
account for the actions of dichlorethylsulphide. The mechanism of the
action of dichlorethylsulphide may be summarized
as follows: 1. Rapid
penetration of the substance into the cell by virtue of its high lipoid
solubility. 2. Hydrolysis by
the water within the cell, to form hydrochloric acid and
dihydroxyethylsulphide. 3. The
destructive effect of hydrochloric acid upon some part or mechanism of
the cell.
_
d Attempts
to determine accurately the partition coefficient of this
substance are unsuccessful,
due to the rapidity with which it hydrolyzes. It appears to be over
200, using xylene and water at
20 C.
379
Although hydrochloric acid does not penetrate cells
readily and is
easily neutralized by the
buffer action of the fluids of the body, one might expect by flooding
the body with large
quantities of acid to produce some of the characteristic effects of
mustard gas. Stimulation of the
respiratory center is a well-known effect of acid. Convulsions and
salivation may be produced by
injection of hydrochloric acid and it was found possible to produce
slowing of the heart by rapid
injection of this acid.
The delayed action of mustard gas may be explained
by the formation of
some compound with
some constituent of the blood. However, blood taken from dogs which had
been poisoned with
mustard gas and were exhibiting typical symptoms at the time injected
into normal dogs,
produced no effect. Serum treated in vitro with mustard gas and allowed
to stand and then
injected into a dog produced no effect. The fluid which is formed in
the vesicles and blebs
produced by the application of mustard gas to the skin produces no
mustard-gas effects.
EFFECTS OF ADMINISTRATION OF SODIUM BICARBONATE
By the administration of large quantities of sodium
bicarbonate, both
intravenously and by
mouth, the symptoms following an intravenous injection of
dichlorethylsulphide could be
delayed, and convulsions, but not death, were prevented.
Ten cubic centimeters per kilo of the fresh, aqueous
mustard-gas
solution given intravenously
always caused convulsions, violent other symptoms, and death. A series
of 11 dogs injected with
this dose of the solution, were treated with 10 c.c. per kilo of 5 per
cent sodium bicarbonate by
mouth and intravenously every hour for five or six hours. In one of
these animals no benefit was
obtained; in 6, symptoms were delayed and milder; in 4, convulsions
were prevented. Death was
never prevented, although sometimes apparently delayed. The action of
mustard gas on the heart
appeared to be increased, and most of the animals showed a very slow,
failing heart before death.
Since sodium bicarbonate (Harvey) is known to penetrate cells only with
difficulty, much benefit
was not expected. Numerous amines, especially those of high lipoid
solubility, have been tried,
but thus far the effects have not been consistent. A substance
possessing the same physical
properties as mustard gas, but slowly yielding alkali on hydrolysis,
would be ideal to try for
treatment.
EFFECT OF TEMPERATURE ON TOXICITY
The fact that the velocity of hydrolysis of
dichlorethylsulphide is
very much decreased by
lowering the temperature, suggested trying the effects on animals at a
high and low temperature.
The influence of temperature was quite marked on drugs which underwent
a change in the body
before acting. The lethal dose of atoxyl and colchicine was increased
markedly for frogs when
the temperature was lowered, and decreased when it was raised to
37° C.2 Atoxyl is supposed
to be reduced in the body before acting and colchicine is oxidized.
According to the theory
which has been advanced for the mode of action, one might expect that
at a low temperature the
rate of liberation of hydrochloric acid in the cell might he slow
enough to be nontoxic, for a dose
which would prove fatal at a higher temperature.
380
Fish were used for the experiments. Those kept at a
low temperature
survived the same dosage
which proved fatal to those kept at room temperature.
EXPERIMENT 25.-Seventeen healthy catfish were
exposed, in groups of
four to six, to
dichlorethylsulphide by immersing in a quarter saturated solution of
mustard gas in tap water at
100 C. for five minutes. Ten of these were placed in water at room
temperature (24° to 26° C.),
and ten died (100 percent) within 28 hours. The other seven fish were
kept at 8° to 10° C., and
one died in 96 and another in 108 hours, while the other five survived
for 5 days, when
observations were discontinued.
EXPERIMENT 26.-Fifteen goldfish were exposed, as in
experiment 25, to a
half-saturated
solution for 10 minutes. Eight were transferred to a bath at room
temperature (25° C.) and seven
to a bath at 8° to 10°C. Of the first group, four died in 26
hours, two died in 56 hours, and two
died in 6 days. Of the second group, all survived 17 days, when
observations were discontinued.
It is quite noticeable that in the catfish which
were kept at room
temperature hemorrhages
generally developed in the fins and tails and in the ventral surface of
the body after 16 to 20
hours,e while in
those kept cold, no hemorrhage was ever observed.
Symptoms, which were
marked in the first group, were never observed in the second.
Although this evidence fits in perfectly with our
theory, it is well
known that the effect of drugs
is changed by decreasing temperatures. f The following
experiments
indicated that
hydrochloric acid was toxic for fish whether the fish were cooled after
exposure or kept at room
temperature.
EXPERIMENT 27.- Eight catfish were exposed to
0.05 percent hydrochloric
acid for five
minutes. Four were placed at room temperature (25°C.), and four
placed at 8° to 10°C. Of
the first group, all died within 40 minutes; of the second, three died
within 30 minutes, and one
in 150 minutes.
EXPERIMENT 28.-Sixteen goldfish were exposed in the
same manner as in
experiment 27.
Eight were kept at room temperature (20°C.) and eight at 8° to
10°C. Of the first group two
died in 1 hour, and six in 1 hour and 20 minutes. Of the second group
all died within 1 hour.
An experiment on atropine and one on sodium cyanide
indicated that
these substances were just
as toxic for fish whether they were kept at ordinary te npermiture or
cooled, after exposure.
EXPERIMENT 29.-Four goldfish were exposed for
10 minutes to 0.01
percent atropine sulphate
solution. Two were placed in a bath at 8° to 10°C. In the first
group, both died in 6 to 7 hours;
in the second group, both died in 5 to 7 hours.
EXPERIMENT 30.- Six goldfish were exposed for 10
minutes to 0.48
percent solution of
sodium cyanide. Three were kept at 20° C. and three at 8° to
10°C.
They died in 3, 5, and 8
hours, and 4, 6, and 8 hours, respectively .
Catfish survived fifteen times the concentration of
hydrochloric acid
present at the end of 5
minutes in the half-saturated water solution of mustard gas, and three
times the concentration
present at the end of 24 hours. Fish survived 15 minutes' exposure in
this solution after it had
stood 1 hour at room temperature.
It is evident that these experiments on fish tend to
substantiate the
theory of intracellular
liberation of acid.
_
e It is
interesting to note that dichlorethylsulphide appears to act on
the skin of catfish and not
that of goldfish.
f Sollman (A
Manual of Pharmacology. 1917, 94) holds that digitalis,
veratriin, nicotine,
strychnine, tetanus toxin, chloral, and alcohol are rendered more
active, morphine and curare less
active, by raising the temperature for cold-blooded animals.
381
PROPERTIES AND ACTION OF SOME COMPOUNDS RELATED TO
DICHLORETHYLSULPHIDE
Various compounds related to mustard gas were
prepared at one time or
another by the offense
chemical section of the Chemical Warfare Service. A cursory survey of
their lipoid solubility,
rates of hydrolysis, and pharmacological effects was made.
The lipoid solubility was estimated by using xylene
and water. Victor
Meyer3 noted that
ethylsulphide was inactive, while the B-monochlorethylsulphide
was less
active than mustard
gas. Monochlorethylsulphide and the two isomeric B-B-dichlorpropylsulphides
were all highly
(though in different degree) lipoid soluble and all hydrolyzed more or
less rapidly in aqueous
solution. All were active skin irritants, and more or less toxic on
inhalation, producing lesions
and symptoms comparable to those of mustard gas.
The theory of the intracellular liberation of
hydrochloric acid as the
mechanism of action of
dichlorethylsulphide, explains all the experimental facts thus far
observed. The histological
changes in the skin have been stated to resemble hydrochloric acid
burns. According to Warthin
and Weller: 4 "The lesion is a chemical burn unlike that
produced by
heat, electricity, or the
ordinary corrosives such as sulphuric, nitric, and hydrochloric acids,
or strong alkalies. Of all
these agents, the effects are most closely allied to those of
hydrochloric acid, but are much
greater in intensity." Lillie, Clowes, and Chambers,5 in a
study of
the action of mustard gas on
marine organisms, especially starfish eggs, have obtained evidence
which supports our
interpretation of the action of dichlorethylsulphide, e. g., an
intracellular liberation of
hydrochloric acid as the toxic factor.
A theory to explain the edema, fat, infiltration,
multiple hemorrhages,
and necrosis of the central
portion of the liver lobule " by the '"severe tissue effects by the
halogen acids formed in the
tissues" has been advanced by Graham 6 for the delayed
poisoning by
chloroform and other
alkyl halides. Graham's evidence is briefly as follows: Similar
morphological changes can be
produced especially in the liver by injection of hydrochloric acid into
the portal vein. Sections of
the chloroformed liver show an acid reaction with neutral red or Nile
blue immediately, whereas
such changes occur more slowly in the liver of normal animals after
death. Carbon tetrachloride
is more effective than chloroform and methylene chloride less effective
in producing liver
necrosis; this is in the same order as the amount of hydrochloric acid
that can be liberated.
Simultaneous injection of sodium carbonate with the chloroform
anesthesia prevents or
decreases the liver injury and other effects. Other alkyl halides give
a typical morphological
picture of chloroform poisoning. That these substances form halogen
acids is shown by the
excretion of the neutral salts in the urine. Chloral, which does not
yield an appreciable quantity
of hydrochloric acid in metabolism, does not produce these changes.
While Graham did not
discuss the question of how the acid is formed, he suggested Nef's 7 ideas of dissociation,
forming bivalent carbon.
It is evident that the theory suggested in this
chapter is somewhat
different. Graham 6 did not
consider intracellular liberation of acid, and was dealing with
substances which do not readily
yield hydrochloric aci(l on contact with water, but must undergo
relatively slower metabolic
changes by the tissues. The effects of mustard gas are not similar to
those of delayed chloroform
poisoning and, moreover, chloroform is a substance of an entirely
different order of toxicity.
382
It is not impossible to reconcile these two
different types of
poisoning as both being due to
hydrochloric acid. Graham 's suggestion that "the liver is an organ
which most strikingly
manifests the chloroform necrosis, and that this organ is also the site
of a most active
metabolism, harmonize well with this hydrochloric acid theory," may
explain the difference. As
stated above, chloroform must be slowly metabolized to produce
hydrochloric acid. By oxidation
chloroform yields phosgene and hydrochloric acid. Phosgene readily
yields hydrochloric acid on
hydrolysis.
One is inclined to agree with Graham's statement
that the halogen acids
are suggested to be
important factors rather than the only factor involved. Other acids
must play a part, and possibly
other substances than acids are involved.6
With mustard gas and analogous compounds where the
radical, other than
the acid radical, is
nontoxic, the entire responsibility can be laid to the intracellular
production of acid. The question
of cell penetration, as well as ease of hydrolysis and lipoid
solubility, has to be considered in
dealing with a series of compounds.7
It is interesting to note that most of the war gases
can readily yield
a halogen acid by hydrolysis.
Whether the intracellular liberation of acid will explain their
relative toxicity must be decided by
future work. Their partition coefficients, their rate of hydrolysis,
their volatility and other
physical chemical properties may explain the differences in
localization and intensity of action.
The facts which can be gathered from the literature concerning
dimethylsulphate, seem to
indicate that its toxic action may be due to the intracellular
liberation of sulphuric acid. The
symptoms recorded in the literature due to this poison are very
suggestive of mustard-gas
poisoning-local redness and edema, generalized toxic and clonic
convulsions, coma and death.8 The effect on the eyes9 and the
clinical descriptions of several factory cases of poisoning
resemble mustard gas 10. Both Weber 8 and
Michiels,8 who worked
experimentally on
animals with this substance, discuss the possibility of the effects
being due to acid poisoning.
They decide against it because the amount of acid liberated is too
small, the blood gases of the
poisoned animal were found normal, sodium carbonate did not help
poisoned animals, and it is
doubtful whether time elapsed for a toxic dose to hydrolyze. Michiels
found, moreover, that
serum treated with a toxic dose of dimethylsulphide, incubated and
injected into a rabbit, was
nontoxic. So he agreed with Weber that the molecule is toxic and not a
decomposition product.
The evidence appears to be very suggestive for an intracellular
liberation of sulphuric acid as the
toxic factor. It was found that the partition coefficient is about 1.9,
using xylene and water at 20°C., and that this substance hydrolyzes
much more slowly than
dichlorethylsulphide. It would
be expected that the toxicity on intravenous injection would be less
than mustard gas.
Preliminary experiments indicate that 50 mgm. per kilo are necessary to
produce the same
symptoms as are given by 6 mgm. per kilo of dichlorethylsulphide. The
symptoms appeared
much more quickly in the case of the dimethylsulphate. This would
indicate that mustard gas is
prevented for a time from hydrolysis by being held in lipoids, while
dimetlhylsulphate, having a
much lower partition coefficient, is more readily transferred to an
aqueous phase for hydrolysis.
Certain facts noted later as having been observed with respect to the
action of
dichlorethylsulphide on the skin also led to this conclusion.
383
SUMMARY
The results of the experimental work which has been considered may be
summarized as follows:
1. Dichlorethylsulphide is absorbed throughthelungs
and produces
definite, characteristic,
systemic effects.
2. The symptoms of injection of the substance are
salivation, vomiting
and diarrhea, tonic and
clonic convulsions, slow and irregular heart, followed by a rapid
pulse, and stimulation of the
respiration.
3. A dose of six milligrams or less per kilo,
injected intravenously in
aqueous solution, proves
fatal for dogs.
4. Dichlorethylsulphide appears to be excreted in
the urine, in part at
least, as
dihydroxyethylsulphide, which has been shown to be a comparatively
nontoxic body.
5. The lesions in the intestine suggest that
excretion of the substance
may also take place here.
6. The dichlorethylsulphide penetrates the cells,
and in the aqueous
phase of the cell, hydrolyzes
to hydrochloric acid which is responsible for the damage.
7. Sodium bicarbonate in large doses somewhat
alleviates the symptoms,
but does not prevent
death.
8. Fish are much less susceptible to this substance
when kept at a low
temperature after exposure
rather than at room temperature. The hydrolysis of the substance is
much slower at a low
temperature.
9. Monochlorethylsulphide, and
dlichlorpropylsulphide are lipoid
soluble and easily hydrolyzed.
Both act similarly to mustard gas.
10. The evidence leads to the view that
dimethylsulphate acts by
intracellular liberation of
sulphuric acid.
EFFECTS ON THE SKIN
INDIVIDUAL SUSCEPTIBILITY
Every investigator who has worked with
d1ichlorethylsulphidle has
noticed that some
individuals are much more susceptible to the substance than
others.Victor Meyer, its discoverer,
noted that his laboratory assistant was greatly affected by exposure to
this agent, while he
himself was not.11 Similar observations have been made by
many workers
in laboratories.
Despite the great differences in susceptibility, it is very improbable
that any man or beast is
immune to the action of mustard gas. It was possible to demonstrate
beyond a doubt the
individual variation in susceptibility, and methods were evolved by
which determinations of
susceptibility could be made.
METHODS
Two methods were used for determining the cutaneous
sensitivity of
individuals to
dichlorethylsulphide. The first of these methods consisted in exposing
skin to the vapors of the
substance under constant conditions and determining the minimum time of
exposure which was
necessary to produce a visible reaction within 24 hours. The apparatus
which was used in this
method consisted of a small test tube (1 cm. by 10 cm.) containing a
cotton plug saturated with
pure dlichlorethysulphide held by means of a rubber stopper in a larger
test tube which was filled
with water. When not in use the smaller
384
tube was closed with a cork stopper. On being first prepared the tubes
were allowed to stand
unstoppered for about 24 hours in order to remove any volatile
impurities that might be present.
Before use the whole apparatus was placed in a constant temperature
water-bath. In all this work
20° C. was taken as a constant-temperature for making exposures.
The skin was exposed to the
vapors in the smaller tube by holding the mouth of the tube firmly
against the skin. Exposures
were made for different lengths of time in order to determine the
shortest exposure which would
just produce a visible reaction, the minimum burning time. The reaction
was not visible for some
hours, and was noted 24 hours later. The reaction consisted in a
circular area of erythema which
was usually uniform. In certain cases a group of small, red spots might
be all that was noticed.
Longer exposure than the minimal gave rise to edema as well as
erythema, and a still longer one
to subsequent small vesicles or a well-defined blister over the entire
area exposed. In this method
the skin was exposed to supposedly saturated vapor at 20°C.
The second method which was used for studying the
sensitivity of
individuals consisted in
applying to the skin standard solutions of mustard gas in paraffin oil.
For most purposes the best
solutions were a 1 percent (1 gram of pure dichlorethylsulphide in 100
c.c. of oil), a 0. 1 percent
and 0. 01 percent. A small drop of each of these solutions was applied
to the skin of the forearm
and the arm allowed to remain uncovered for about 10 minutes. The
presence or absence of a
positive reaction was indicated by the appearance or absence of
erythema 24 hours later.
Within certain limits the amount of the solution of
a given
concentration of mustard gas which
was applied to the forearm made no difference in determining whether
the reaction was positive
or negative. It made a difference in the intensity of the burn, a large
amount of 1 percent solution
causing a blister, while one-tenth the quantity did not. For this
reason it was advisable not to
employ too large a quantity of the stronger solutions in making the
test. The following
experiments were made, applying at the same time the amount of solution
of mustard gas in
paraffin oil left on a glass rod after dipping it in the solution and
shaking it off, and then
applying about ten times the quantity. The reaction from the larger
quantity is given in brackets.
(Tables 28 and 29.)
TABLE 28.- Irritating effects of
dichlorethylsulphide applied
locally
385
TABLE 29.- Irritating effects
of dichlorethylsulphide applied
locally
The question has been raised as to what length of
exposure was given to
the oil solution on the
arm. Within 5 minutes, where only al small amount of solution was left
on, absorption or
evaporation was complete. When a larger amount of solution was applied,
rubbing off the excess
5 minutes after application made no difference in whether the reaction
was positive or negative.
When the test was applied the subject left the sleeve rolled up for
about 10 minutes and then
continued his usual duty.
Twenty-four hours after making the test was a
convenient time for
reading the results. The
following figures show that any time between 24 to 48 gave the same
result. Less than 24 hours'
readings should not be taken as some individuals were rather slow in
reacting.
Paraffin oil was chosen as the solvent for making
the test, for the
following reasons: It was not
very volatile; the solution of mustard gas in it was fairly stable; it
did not spread greatly when
applied to the skin; and it was probably not readily absorbed.
Mustard-gas vapor was given off,
however, from a 1 percent solution in paraffin oil. A small amount of
this solution placed in a
test-tube and held against the skin of a sensitive individual (for 10
minutes) caused erythema.
Solutions of mustard gas in other solvents than paraffin oil were trieo
on a selective number of
individuals of the resistant, average, and sensitive types. The
solvents used were linseed oil,
cottonseed oil, kerosene, absolute alcohol, and paraffin oil as a
control. The solution in absolute
alcohol was the more reactive, while those in linseed, cottonseed, and
kerosene oils were less
reactive than that in paraffin oil. The greater reactivity in alcohol
was probably due to its
volatility leaving a stronger solution on the skin a few seconds after
it was applied.
The skin of different parts of the body probably
reacted slightly
differently from mustard gas,
although this was not tested fully. The solutions were always placed on
the inner surface of the
forearm. One point which is to be noted is that areas of skin in the
neighborhood of old mustard-gas burns were more sensitive than other
areas. They should be avoided in making the test.
The first method undoubtedly gave the most accurate
results, although
the second was very
much simpler and more rapid in its application. Numerous
386
tests were made on individuals by these methods in order to determine
if each method gave the
same result. Men who were sensitive to one test were sensitive to the
other, and vice versa. Roughly, it may be said that a man who gave a
reaction to the 0.01 percent solution would show
a reaction from a 15-second (or less) vapor exposure, while a man who
showed no reaction to the
1 percent solution would be negative or only faintly positive to a
4-minute vapor exposure.
A method such as the second method described above
might be applicable
in the field or factory,
being used to detect individuals who are especially sensitive to
mustard gas so that they may be
removed from areas which are exposed to this gas. It would be an easy
matter to examine
thousands of men and determine quickly which ones were especially
susceptible to the gas and
which were less susceptible. A sensitive individual living in a region
where mustard gas is
present is practically certain to be a casualty. It would be far easier
to remove him before injury.
This idea may sound very impractical, but it does not seem less
practical than applying
ointments to the body for protection.
RESULTS OF VAPOR METHOD
This method was applied to only a few individuals
working around the
laboratory. The results
are shown in Table 30.
TABLE 30.- Time of exposure to
vapor
tests required to produce a
visible reaction
It is interesting to note that the very extreme
cases had a minimum
burning time of 1 second and
10 minutes, respectively. In other words it may be stated roughly that
one of these individuals
was six hundred times as sensitive as the other. The man who gave a
reaction from a 1-second
exposure blistered from a 5-second exposure. or one to which the vast
majority of individuals
will give no reaction. When first found this subject blistered from the
application of the 0.01
percent solution in paraffin oil. Fortunately, it was possible to
obtain a complete history and
physical examination, but nothing of note was found. This man had never
been exposed to
mustard gas nor had he received even a small experimental burn when
first tested.
RESULTS OF TESTS WITH OIL SOLUTIONS
As has been pointed out, the oil-solution method of
determining
sensitivity was very much easier
and more rapid in its application than the vapor-test
387
method. Results were obtained on rather large groups of individuals,
using this method. Table
31 summarizes the results obtained on a group of men at the American
University Experiment
Station. Practically all these men were indoor workers, and a great
many of them had been more
or less exposed to mustard gas.
TABLE 31.- Skin irritation from
local
application of
dichlorethylsulphide
A similar test on a larger scale was carried out at
the Edgewood
Arsenal, Edgewood, Md. This
was done to determine: (1) In a large group of men, the relative number
of hypersensitive,
average, and resistant individuals. (2) If among factory workers in
mustard, the hypersensitive
individuals were those most frequently burned, and the resistant those
seldom burned.
The method of conducting the test was to treat each
man on the forearm
with a 1 percent and
0.01 percent solution of mustard gas in paraffin oil. Twenty-four hours
later the man was
examined for a positive or negative reaction to these solutions
(indicated by the presence or
absence of visible erythema). In a group of 915 men, the actual time
for 6 workers to apply the
solutions was about 10 minutes, and to read the tests the next day
about 20 to 25 minutes,
including taking the names of the hypersensitive men.
Table 32 indicates the relative number of resistant,
average, and
hypersensitive individuals.
TABLE 32.- Effect in individuals of
local application of
dichlorethylsulphide
The number of men available for testing the second
proposition was too
small to arrive at
definite conclusions (only 31 men from the mustard-gas plant were
present). Tests were also
made on 51 hospital cases suffering from eye or skin effects of mustard
gas. It was evident from
these that average and resistant men were frequently burned. An
examination into conditions at
the plant showed that most of the casualties had occurred in cleaning
pipes and where breakage
occurred, under conditions where any man would be burned. Any
individual will be burned from
the application of the liquid to the skin or from a high enough vapor
concentration. It was true,
however, that where a sensitive and a resistant individual were burned
under the same conditions
the sensitive one had the more severe burn and required longer
treatment.
388
In cases of relatively long exposure to Vapor of
fairly low
concentration, it is certain that
conditions will arise where hypersensitive men will be burned severely
enough to become
casualties, while resistant and average individuals will escape without
burns or only slight ones.
Table 33 is the result of a test made on negroes.
The few of these
previously examined by the
vapor test had been found very resistant. Only the 1 percent and 0.1
percent solutions were used.
TABLE 33.- Sensitivity of negroes
to
dichlorethylsulphide
It is seen from Table 33 that negroes as a race have
a much more
resistant skin than white men.
No negro of the 84 examined reacted to the 0.1 percent solution, and of
course none would react
to a more dilute one. About 10 percent of white men reacted to the 0.1
percent solution, while 2
to 3 per cent reacted to the 0.01 percent solution, or were
hypersensitive. About 78 percent of the
negroes failed to react to the 1 per cent solution, while only 20 to 40
percent of the white men
did not show a reaction.
VARIATIONS IN SUSCEPTIBILITY IN THE SAME INDIVIDUAL
In the above discussion it has been tacitly assumed
that the
sensitiveness of a single individual is
always the same. Such is not the case. The sensitiveness of a single
individual is the same from
day to day provided all conditions are the same, but may vary greatly
under different conditions-the physiological variation in sensitiveness
of the individual.
In order to show that the sensitiveness of the
individual does notary
from day to day under the
same conditions, a number of determinations of susceptibility were made
upon the same
individual on successive weeks. These determinations showed that the
sensitiveness of an
individual did not vary greatly, probably less than 25 percent from day
to day. An individual
who was positive to a 15-second exposure on one day could be negative
to 15 seconds but
positive to 30 seconds a week later. He was never negative to a 1 or
2-minute exposure. Also, an
individual who was negative to a 4-minute burn and positive to a
5-minute burn at one time
could be negative to a 3-minute burn and positive to a 4-minute burn a
week later, but he would
not react to a 1 or 2-minute burn. So constant, in fact, was the
sensitiveness of an individual that
it was possible to plot a curve of the least time required to produce a
burn (erythema) at different
concentrations. If the variations in sensitiveness from day to day had
been great, it would have
been impossible to plot these curves, and there would have been many
burns on the wrong side
of the curve, and many negatives on the other side.
389
There are undoubtedly many conditions which
influence the sensitiveness
of the individual to
mustard gas, but of these only two have been thoroughly investigated;
sweating and moisture. A
number of individuals from the laboratory were selected and given burns
upon the forearm of
different lengths of time, from 1 minute to 5 minutes, according to the
known sensitiveness of
the individual. A very mild burn was desired. The method used for
burning was the "standard
test-tube vapor test.'" The individual was then instructed to go out
and run until the whole body,
including the forearm, was in a profuse sweat. He was then given two
other burns of the same
length exposure, as before, one upon the moist, sweating arm, the other
upon a part of the arm
which was wiped dry with a towel. It was noted that in every case both
of the burns made after
exercising were distinctly more severe than those made before
exercising, and the skin which
was wiped dry usually received a more severe burn than the skin which
was left moist. In
another experiment performed in a similar way the feet of the subject
were kept in a tub of hot
water until the whole body was in a profuse sweat. The results were
essentially the same. This
action may have been due to the effect upon the sweat ducts, as it is
known that the gas enters
most readily these ducts. On the other hand, the action may have been
due to the moisture.
Experiments were carried out on the effect of moisture in the following
way. An area of the
forearm was kept moist for a few minutes with wet cotton. The sponge
was then removed, and
two vapor tests were made, one over the moist area and one over the
normal dry skin. In the first
three cases mild burns were given; in the last three cases, severe
burns. The moist burn was
always the more severe, in one case producing a blister when the other
did not.
SUSCEPTIBILITY OF THE SKIN OF ANIMALS
The paraffin oil test was used on a number of
animals and indicated
that differences in
susceptibility existed in different species and in different
individuals of the same species. Table
34 shows the results.
TABLE 34.- Susceptibility of skin
of
animals to
dichlorethylsulphide
The horse appeared to be the most sensitive and the
monkey and guinea
pig the most resistant
species, while the dog seemed to have a sensitivity as near man as any
of the other species.
However, the number of animals examined was too small for any
far-reaching conclusions, and
it is to l)e noted that no animal exhibited a reaction at all similar
to that in man. No animal has
yet been found which will give a blister from the application of
mustard gas.
390
DISCUSSION
The only factor in the general characteristics of
the skin which would
appear to have a distinct
bearing on the question of sensitivity is the apparent thickness of the
skin. The fact that negroes
as a race are much less susceptible to this substance than the white
race furnishes a clue to the
reason for susceptibility. Any constant differences in the skin of
negroes and white men might
furnish a basis for future work.
The following experiments tend to throw some light
on the mechanism of
absorption of mustard
gas by the skin and, hence, on the question of susceptibility.
Fifteen minutes after pure dichlorethylsulphide was
applied to the skin
it could be almost
entirely removed by long-continued rubbing with kerosene. This
indicated that the substance was
not immediately absorbed into the deeper layers of the skin. A
resistant individual whose normal
minimum burning time was 4 to 5 minutes and who did not react to the 1
percent oil solution,
could be made to give a reaction to a 15 to 30-second exposure or a
0.01 percent oil solution by
covering the area of application with a glass cup immediately after and
leaving it covered for 6
to 8 hours.
It was found that when two vapor tests were made
under identical
conditions on the arm of a
sensitive individual (5 minutes' exposure) and immediately after the
exposure the arms of the
two individuals were impressed on each of the exposed areas for 5
minutes, the burns were
modified in intensity. If the recipients were respectively more or less
resistant than the donor,
that burn on the donor's arm given to the more resistant man was the
mildest. This is very
striking. If a sensitive individual impresses his arm alternately
against burns of the same
concentration and exposure on a resistant and sensitive man, the
recipient receives a more severe
burn from the sensitive than from the resistant man. This would
indicate that the skin of a
resistant individual displays a greater affinity or capacity for
dichlorethylsulphide than that of a
sensitive man. A tentative explanation of this phenomena can be made as
follows: A three-phase
system is involved-the air over the skin surface constitutes the outer
phase; some fatty or
keratinous elements of the skin the central phase; and a cellular
portion of the skin the inner
phase. The central phase is rich in lipoids and poor in water, while
the inner phase is rich in
water and poor in lipoids. After exposure to the vapors of
dichlorethylsulphide the central phase
is the absorbing agent and tends to establish equilibrium with the
other two phases. On account
of the lipoid nature of the central phase no damage is produced here
because the compound is
not hydrolized. On its passage from the central to the inner phase,
hydrolysis takes place within
the cell and damage results when a sufficient concentration of
hydrochloric acid is attained. The
outer phase is constantly being freed from vapor by diffusion and
convection currents, so more
and more can evaporate from the central phase. The susceptibility of an
individual depends on
the relative power of the central phase to hold the poison in an
inactive form (not hydrolyzed)
and prevent its entry into the inner phase at a sufficient velocity to
result in the formation of a
toxic concentration. No attempt was made to localize the central or
inner phase with any definite
structure of the skin. As mustard gas is known to penetrate the
sebaceous ducts, the fat here
might form one phase and the epithelial lining another.
391
PRACTICAL APPLICATION
Anyone who observes the tremendous difference
between individuals is
struck with the practical
use that might be made of this difference. There are individuals who
show distinct burns
(erythema) from a 5-second exposure, while working beside them are men
who require an
exposure of 5 to 10 minutes to produce a burn. The hyperemia produced
by the 5 seconds'
exposure is not serious when limited to a small area, where it is
closely surrounded by normal
tissue, but a hyperemia of no greater intensity when produced over a
large area of the body may
make it imperative that this individual be relieved from duty. The
edema and itching may be
quite distressing, and prevent the patient from sleeping or resting. He
may have to spend most of
his time bathing the injured parts with cold water to get relief. The
man beside him, however,
may be exposed to the same concentration for 10 to 50 times as long and
show absolutely no
effect. The former individual should not be allowed to work in any
region where there is even a
low concentration of mustard gas, for if he does he is certain to be
burned severely, and
practically certain to become a casualty. The latter individual, on the
other hand, would make a
splendid workman in a mustard-gas factory or a splendid soldier in a
territory which has been
shelled with mustard gas, if provided with a mask.
The following suggestions appear warranted from this
investigation in
sensitivity to mustard gas:
1. Sensitivity tests should be made routine in all
plants handling
mustard gas, and only the more
resistant individuals should be stationed there if possible.
Application of a single solution is all
that is necessary. The 1 percent solution will give about 40 percent
of the total men examined to
be used in mustard gas. If a larger percentage is desired, lower
percentage (0.5 percent) solution
can be substituted.
2. The feasibility of withdrawing the hypersensitive
men from the line
from any duty where
mustard gas may be encountered should be considered. This would involve
about 3 percent of
the men. The actual carrying out of sensitivity tests would be very
simple-the application of a
0.01 percent solution of mustard gas in paraffin oil to the forearm of
each man and the
observation of this spot 24 to 36 hours later. The time required would
be much shorter than that
necessary for venereal inspection of a company. The hypersensitive men
are certain to become
casualties if exposed to mustard vapor even in low concentrations, and
in this way be eliminated
afterwards if not before.
MECHANISM OF ABSORPTION BY THE SKIN
CHANNELS OF ABSORPTION
At an early stage in these investigations it was
observed that after
exposure of the skin to either
the liquid or vapor the most efficient method of removal was washing
with some solvent of low
volatility, such as kerosene, followed by thorough rinsing with soap
and water. With such a
treatment it was found possible to apply the oil to the skin and by
washing after 2 or 3 minutes
to either prevent the subsequent delayed reaction entirely or to reduce
it to a slight hyperemia.
The very narrow time limits within which these preventive measures were
effective indicated a
remarkably rapid absorption of at least a
392
part of the gas. It was found, however, that if the sponging with
kerosene was continued
vigorously for 20 to 30 minutes, with frequent renewals of both sponge
and solvent, vesication
could be prevented in most cases after an exposure of 10 minutes or
less and in rare instances
after 15 minutes. (Table 35.)
TABLE 35.- Removal of
dichlorethylsulphide from skin by washing
with kerosene
The following tests were made on the shaved and
dried skin of the
forearm by the "nailhead
method." A nailhead 3 mm. in diameter was pressed firmly against filter
paper saturated with
dichlorethylsulphide and then applied with firm even pressure to the
skin. After a definite period
of time the exposed area was washed or "treated." This period of
undisturbed contact between
the oil and the skin (or, in subsequent experiments, between the vapors
and the skin) will
hereafter be referred to as the length of exposure. Since the reaction
did not appear for several
hours, and since the burns did not develop sufficiently to permit of
accurate comparison in less
than 24 hours, the first readings were made at the expiration of that
period, and final readings
after 48 hours. The reactions as observed at that time are graded as
follows:
+ Mild erythema.
+ + Moderate erythema and swelling.
++ + Mild vesication with slight surrounding
erythema.
+ +.+ + Severe vesication with slight surrounding
erythema.
++ + + + Severe vesication with marked surrounding
erythema and edema.
It should be noted in the table that + + + indicates
the penetration of
sufficient mustard gas to
cause a blistering burn while + and + + indicate that the amount of
mustard gas introduced is
insufficient to cause a blister, but only redness and slight swelling.
Beyond the limits given
above it is unsafe to make quantitative comparisons of two or more
burns.
A 10-minute exposure, if washed with kerosene for 10
minutes or less,
blistered; the actual
blistering could be prevented in most cases by washing for 15 minutes
or more. Little added
benefit could be obtained by treating longer than 25 to 30 minutes.
Where the time of exposure
was increased to 15 minutes or more vesication could barely be
prevented, even after rubbing or
sponging for an hour.
393
That dichlorethvlsulphide was actually being removed
from the skin
after the first few minutes
of treatment was shown by the observation that unless the sponge and
kerosene used were
frequently changed, and the surrounding areas thoroughly washed, the
resulting burn was greatly
increased beyond the size of the original exposure--following in
general the areas wet by
kerosene--and in some cases even the operator's fingers became
contaminated with the gas
and a burn resulted.
Prolonged washing was tried with soap and water
after 10 to 15 minutes
in a similar manner; but
though some benefit was observed, it was not nearly as marked as when
the washing was done
with kerosene or some solvent for the oil (vaseline, acetone, alcohol,
and benzene were tried).
From these observations, it is quite evident that
the
dichlorethylsulphide was at first rapidly
taken up by some element in-or adjacent to-the surface of the skin, and
for 2 to 3 minutes it
might be completely removed, and for 10 to 15 minutes partially
removed, by prolonged
washing with an organic solvent, and to a lesser extent with soap and
water. This conclusion has
been emphasized because it is an important consideration in dealing
with the ultimate absorption
of the substance.
In the absence of accurate information regarding the
physical and
chemical constitution of the
outer layers of the epidermis and the exact location and condition of
the mustard gas when first
taken up, it appears inadvisable to speculate as to whether the gas is
at first absorbed on the skin
surface and then penetrates the sweat glands by capillary or
absorption, or a mixture of both, or
whether it passes to a certain extent directly through the cortical
layers by a process of
absorption or solid solution. Substances like keratin might be expected
to absorb the compound
and fats and lipoids to absorb or dissolve it. It is sufficient for the
moment to consider the skin as
a protective medium taking up the compound on its surface and offering
a definite resistance to
passage of the gas inward. This resistance may be gauged by determining
the amount of gas
which it is necessary to apply externally to insure the delivery to the
inner tissues of a
concentration sufficient to produce recognizable toxic effects. The
nature of this resistance will
become evident after the discussion of the loss of the gas from the
skin by evaporation.
RELATION OF TIME OF EXPOSURE TO CONCENTRATION
It has been shown that individuals vary in their
sensitivity to slight
quantities of
dichlorethylsulphide,11 and a method for demonstrating this
variation
between individuals, by
determining the shortest exposure to the vapors which will produce a
visible reaction, has been
described. Since any individual gives an increasingly severe reaction
with increasing length of
exposures until the full effects of the irritant are obtained (severe
vesication), there should be a
definite relationship between the concentration of the
dichlorethylsulphide and the length of the
exposure required to produce burns of equal intensity. The method used
in working out this
relationship was based upon that used in "gassing chambers." 12
The principle was briefly as follows: Dry air was
bubbled through
dichlorethylsulphide at a
known and constant rate, and mixed with a definite quantity of dry air
also under constant flow
for the purpose of dilution. A suitable glass exposing chamber was
placed in the circuit near the
bubbling tube,
394
using as little rubber tubing as possible for connections. (Rubber
tubing absorbed the gas very
readily.) The concentration could be determined by analysis of the
escaping gas and air mixture
from the open end of the exposing chamber, but could be accurately and
more readily estimated
by the loss-in-weight method, figuring the loss in weight of the
bubbling tube after a certain
period of time in relation to the known quantities of air employed. The
skin of the forearm of the
subject was shaved and sponged with alcohol and the desired exposures
were made. Stress is
again laid on the fact that readings were made after 24 and again after
48 hours, since the burns
did not develop fully until that time. A faint disk of erythema was
considered positive.
While curves drawn were apparently hyperbolic in
form and consequently
asymptotic, there
appeared to be a critical level in concentration at approximately 0.002
to 0.005 mgm. of gas per
liter at which no injurious effects were noted after prolonged
exposures. From this it must be
concluded that the amount of gas finding its way into the interior
tissues after an exposure to the
concentrations in question was no more than can be metabolized by the
cells.This corresponds
with previous observations regarding the threshold concentration of gas
that can be disposed of
without injury in prolonged respiratory tests.
It is essential to mention here that in determining
the toxicity of
dichlorethylsulphide on animals
by inhalation a similar phenomenon was observed; data expressing the
relation of time to
concentration indicated a marked critical condition at this same mean
concentration, 0.005 mgm.
per liter. While the data for different species indicated differences
in sensitivity, all indicated an
ultimate threshold concentration within the limits of 0.01 and 0.001
mgm.per liter. It seems
evident that this concentration represented a border line where the
organism generally can
metabolize the poison without serious effects.
It must be remembered that these experiments were
originally carried
out with a purely practical
military objective: The determination of the relation between the
concentration of gas and the
time of exposure required to produce a burn under field conditions. It
was fully realized that the
loss from the skin by evaporation was considerable, and that exact
scientific data regarding
absorption of the gas by the skin and its penetration into the interior
tissues could be secured
only by covering the exposed area with some impervious substance
immediately after the
completion of treatment. Unfortunately, the suspension of experimental
work on the cessation of
hostilities prevented the carrying out of an elaborate series of
experiments on these lines with
varying concentrations of the compound. But from experiments with one
concentration, to be
reported later in this chapter, it has been demonstrated that the loss
from the skin was extremely
great, far exceeding the amount that penetrated the tissues, and for
this reason it appears
inadvisable to draw any far-reaching conclusions from the preceding
curves.
The fact that a definite proportionality appears to
exist between the
increase in time at a given
concentration and the increase in concentration at a given time
required to produce equivalent
effects in several individuals, suggests that the problem is one of
satisfying the capacity of the
skin to such an extent that the requisite amount of gas may penetrate
to the interior tissues. The
question as to whether this difference is due to the resistant man was
made the subject of further
experiments.
395
EFFECTS OF EVAPORATION
In considering any factor which is known to modify
the final intensity
of a mustard-gas burn,
one must be careful to differentiate between those which modify
absorption only and those
which may have some effect upon the ultimate reaction which follows the
introduction of the
substance into those organized elements of the skin in which
pathological effects are produced.
Where the former may be brought about through simple physical means,
the latter no doubt
involve physiological relations of much more complex nature and are not
dealt with in this
chapter.
As previously explained, no attempt is made to
specify what agencies
are involved in the
preliminary or final absorption of dichlorethylsulphide by the skin.
The detailed localization of
these agencies have not been found necessary to a clear conception of
the general mechanism.
Passing from the external absorbing agent previously indicated, the
dichlorethylsulphide, in all
probability, would pass through the deeper lying elements to the lipoid
constituents of the cells
of the adjacent tissues. If one were dealing with true absorption into
the intermediary binder, the
passage to the deeper tissues would probably be by diffusion from solid
solution; on the other
hand, its retention in this intermediary binder by absorption would
make it highly probable that
capillary and surface tension effects would direct it into the ducts,
from which it would be taken
tip by absorption. On reaching the lipoids of the cell wall or cell
contents proper, the mustard
gas would slowly diffuse into the aqueous constituents of the cell,
where hydrolysis of the
compound would result and its toxic action would begin. Again attention
must be called to the
danger of attempting to localize the site or mechanism of this action
from the evidence on hand.
The nature of the poison may be such as to effect a fatal anesthesia of
the vasomotor nerve
supply, resulting in prolonged vasodilation and subsequent edema and
vessication. On the other
hand, there is abundant evidence from the work done by Lillie, Clowes,
and Chambers on the
penetration of marine eggs to indicate that the intracellular
liberation of acid which would follow
the hydrolysis of the dichlorethylsulphide would cause such profound
changes in the
protoplasmic equilibria of the cells concerned that their normal
metabolism would be
permanently upset and their permeability increased.5 From
the evidence
on hand, one is forced
to believe that equal concentrations of the poison in this last phase
(inside the cells of the tissues
affected) would produce similar and nearly equal reactions in almost
all men. Whereas the
extreme difference in sensitivities of the skin represents an order of
1 to 600, the extreme
difference in toxicity for any one species (dog), by inhalation or
injection, is of a much smaller
order, possibly 1 to 5. Exceptions to this equality of response to
equal intracellular
concentrations have been noted. One case has been reported where the
subject (H. W. S.)
developed an abnormal reaction suggesting an anaphylactic condition.
It has been suggested that after exposure of the
skin to small amounts
of mustard gas,
evaporation from the surface has an important bearing upon subsequent
absorption. To test this
point, exposures to the vapors were made with the test-tube method
previously described. Two
exposures, just sufficient to cause a mild reaction, were made at the
same time and under
identical conditions. One was immediately covered with a shallow glass
thimble of
approximately the same diameter as the exposed area, fastened to the
arm for three
396
hours or more with adhesive tape. The other was left uncovered, and
served as a control. After
24 to 36 hours, when both burns had reached a maximal comparative
development, the covered
burn was seen to be much the worse. A series of experiments performed
in this manner is
tabulated in Table 36.
TABLE 36.- Effects of evaporation
after application of
dichlorethylesulphide
Column I.-Subjects average minimum burning time to
the standard
testtube, 20 0 C.
Column II.-Length of exposures made.
Columns III and IV.-Relative intensity of resulting
burns: + + + severe
erythema; + +mild; +
faint; -negative. (Blistering burns were not made in any case.)
Sensitive men (i. e., men who gave a positive
reaction to short
exposures) generally showed less
increase in reaction than did men who were relatively much more
resistant. This suggested that
the minimal burning time be determined when the exposed skin was
subsequently covered for a
prolonged period and compared with the minimal burning time when
exposures were left
uncovered.
From Table 37 it is apparent that the possible reduction in minimal
burning time in a sensitive
individual by covering the exposure was generally less than in a
resistant individual.
TABLE 37.- Effects of evaporation
after application of
dichlorethylsulphide
This suggests that the skin of a resistant
individual absorbed more of
the dichlorethylsulphide
than the skin of a sensitive one, because the only other apparent wav
in which covering could
influence the final reaction would be by increasing the rate of passage
of the sulphide from the
outer layers of the skin to the inner lavers through an increase in
temperature and thus raise what
would
397
naturally (uncovered) be a subreactive concentration to a reactive one.
On such a basis it would
seem obvious that a sensitive man would show relatively as great an
increase in intensity as a
resistant man. Against the argument that a sensitive man exhibits a
close approach to the
maximum reaction, and therefore can not be made to show a large
increment over his normal
reaction (uncovered), is the fact that individuals were known who
blistered to a 5-second vapor
exposure, and gave severe edema to less than 1 second, and in whose
history there was nothing
to indicate any anaphylactic or other abnormal reaction, beyond their
great sensitivity. This
problem will be considered later.
Another point of interest was the time an exposure
must subsequently be
covered to secure the
maximum reaction on any one individual, thus finding the time during
which the loss by
evaporation would still produce an appreciable decrease in the final
intensity of the burn. An
experiment to determine this is given in Table 38. Exposures of
border-line intensity were made
and covered immediately, and were left covered for varying intervals.
TABLE 38.- Effect of covering
dichlorethylsulphide burns
In another experiment (Table 39) the approximate
rates of evaporation
and absorption were
determined by covering mild exposures after varying intervals of time
had elapsed during which
the skin had been left open to the air.
TABLE 39.- Effect of covering
dichlorethylsulphide burns
From these experiments it is evident that
equilibrium in the skin was
reached in about 45
minutes and that capping had less effect on a sensitive than on a
resistant skin. This rate of
attaining equilibrium must have had a direct bearing upon further
absorption and, ultimately,
upon sensitivity. Since the dichlorethylsulphide was at first absorbed
by the surface of the skin
or some superficial elements adjacent thereto, and could be lost from
this medium by
evaporation, the relation rate at which it passed from this
intermediary binder to some other
phase, in which it was firmly fixed, as against the rate at which it
was lost by evaporation, would
be the determining factor in the question of whether or not a slight
exposure to the vapors would
prove positive or negative.
398
Since the intensity of the burn produced by a given
concentration bore
a direct relation to the
length of the exposure, and since in at least one case (H. W. S.) a
reaction was produced within
10 minutes of exposure, it is probable that the velocity with which the
substance passes from the
surface to the deeper layers was such that the absorption of additional
amounts was not
interfered with. While the determination of the rate at which it passed
from the surface inward
was a problem of considerable complexity, the rate at which it was lost
from the skin by
evaporation was readily determined by a few simple experiments. By
making a series of
exposures of varying periods of time and covering certain of these
exposures to prevent
evaporation, it was possible to determine the time for which a short
exposure must be covered to
give a burn of equal intensity to an exposure of greater length which
was left open to the air.
Two such experiments were made on the skin of the
shoulders of two
subjects (H. E. I and W. B.
Mc.); the burns were covered immediately and left covered for the time
given in the left-hand
columns, Tables 40 and 41. An accurate representation of their relative
severity after they had
reached their maximum comparative development, as determined by the
readings of three
observers, is given. The system of nomenclature is simply an effort to
give the relative severity
of the burns in each set, and comparisons can not be made from one set
to the other.
TABLE 40.- Effect of covering
exposures of varying lengths
In the exposures capped for 2 and 4 minutes it is
probable that the
thimbles were not tight. The
skin used was on a curved surface of the shoulder and the resulting
burns are undoubtedly low in
intensity through this experimental error.
TABLE 41.- Effect of covering
exposures of varying lengths
Where Table 40 is a comparison of more intense
burns. Table 41 is a
comparison of threshold
burns, i. e., where the exposure was just sufficient to produce a faint
reaction.
399
Since a 15-second exposure could be increased by
covering to the
intensity of a 4-minute
exposure, the amount lost by evaporation into the air must have been
far in excess of that which
passed into the deeper tissues. Experimental evidence was insufficient
to determine the relative
distribution between air and the skin. The practical importance of the
fact of evaporation can not
be overlooked, and its bearing on variations in cutaneous sensitivity
will now be considered from
another point of view.
TRANSFER FROM SKIN TO SKIN
An interesting phenomenon was observed when the
untreated normal skin
of one subject was
impressed for 5 minutes upon an area of skin of another subject
previously exposed to the vapors
of dichlorethylsulphide. Under these circumstances, both donor and
recipient might develop
burns (due to the transportation of the poison from one skin to the
other) the intensity of which
would vary according to the circumstances and the respective
sensitivities of the participants.
The degree of transportation was most strikingly observed in the
intensity of the burn on the
donor's arm. If two similar exposures were made on the arm of a
sensitive man, and one of these
burns was treated, so to speak, by contact for 5 minutes with the skin
of a resistant man, the
treated burn would be markedly less severe than the control, in some
cases being entirely
prevented. If, however, the recipient was equally sensitive to or more
sensitive than the donor,
the burns on the latter exhibited far less difference. Both treatments
could be effected at once,
using two recipients, one more resistant, and one less resistant, than
the donor. In such a case the
burn brought in contact with the more resistant skin was the less
severe.
A few experiments of this nature are given in Table
42. The subjects
are divided for clarity into
two classes, (R) resistant and (S) sensitive. It is evident that the
"resistant skin" had reduced the
burn with which it was in contact much more than had the "sensitive
skin." One is forced to the
conclusion that it had actually absorbed more of the gas.
TABLE 42.-Transfer from skin to
skin
of dichlorethylsulphide burns
This same experiment was carried out in the reverse
order. Similar
exposures were made on the
arms of a sensitive and a resistant subject. Immediately after the
exposure, a third subject
received both burns by impressing them against his arm for 5 minutes.
It was seen that a
"sensitive skin" would take more of the poison from a "skin of equal
sensitivity" than from a
more resistant one.
400
Both experiments were, in fact, important evidence
that the skin of a
resistant individual
exhibited a greater affinity or capacity for dichlorethylsulphide than
that of a sensitive one.
Whatever the arrangement of the experiment, the results indicate that
there was an actual
partition of the gas between the two skins, with an evident tendency to
establish an equilibrium
in which the largest portion of the gas would remain in that skin which
possesses the greater
capacity for it. The experiment confirms in a striking manner the
observations noted above, and
explains why equilibrium with the air is attained from a sensitive skin
sooner than from a
resistant one. This is directly contrary to the general belief that a
resistant individual is more
resistant to low concentrations of mustard gas than his fellows because
his skin absorbs less. The
assumption that his skin absorbs more makes it imperative that some
explanation accounting for
the paradox be made.
In order to determine the persistence of the gas on
the surface of the
skin after exposure, an
experiment was made in which a series of 5-minute exposures were capped
for 15 minutes, 30
minutes, 45 minutes, etc., and then impressed on the arm of a subject
of about the same
sensitivity for 5 minutes.
Since the recipient failed to develop a burn after
contact with the
burn on the donor, which had
been capped for 60 minutes, it was apparent the gas had disappeared
from the donor's arm in that
period. This agreed well with the results obtained by capping in Table
39, in which a maximum
absorption was indicated within that time.
TABLE 43.- Sensitivity tests of
dichlorethylsulphide burns
TABLE 44.- Persistence of gas
after
dichlorethylsulphide burns
RELATION OF PHYSICAL PROPERTIES TO PENETRATION
It is generally recognized that substances possessed
of the capacity of
readily penetrating
protoplasm are almost invariably soluble to a certain extent in water
and are also " lipoid
soluble," that is, soluble in fats and organic solvents such as benzene
and xylene, and that the so-called partition coefficient between water
and benzene or xylene is a factor of considerable
importance in conditioning their power to penetrate living cells.
Some 25 compounds were investigated, so-called war
gases, which were
not only highly toxic,
but which irritated the skin. The determination of their partition
coefficients was a very difficult
matter in most cases because
401
of their rapid hydrolysis in water (in such cases a strong acid is one
of the decomposition
products), but it was evident that they were all soluble in both lipoid
solvents and water to some
extent. This fact, with the fact of their hydrolysis, suggested that
the mechanism of their action
could be correlated in a general way with that of dichlorethylsulphide.
Further experimental
work is necessary to establish this fact. This group of compounds in
the category of war gases
vary greatly in their degree of activity and in their specific toxic
effects, and while no far-reaching generalization on this question will
be attempted it is proposed at a subsequent stage in
this chapter to discuss briefly certain cases in which toxic effects
correlate in a measure with
physico-chemical properties.
A series of experiments was performed with a number
of organic bases in
an attempt to
determine their value as a means of counteracting the effects of
mustard gas. It was found that
with the exception of ammonia only those bases which were
lipoid-soluble irritated the skin,
indicating penetration. On the other hand, too high a partition
coefficient (too low solubility in
water) appeared to diminish the irritating activity on the skin. (The
following examples are
representative of the groups tried: Ammonia, propylamine, ethylamine,
amylamine, di-isoamylamine, bornylamine.) Whatever the interpretation
of the details, it is apparent that lipoid
solubility is an important factor in the penetration of the intact skin.
A brief reference should be made to a phenomenon
requiring further
investigation. Field
observers have noted that burns occur more frequently on moist than on
dry polrtions of the
body. This observation was confirmed in the laboratory and,
furthermore, a film of water on the
dry skin was found to facilitate the passage of mustard gas, showing
that the effects observed in
the field were not attributable simply to a possible higher
permeability of freely perspiring areas,
but was in some way attributable to the presence of water on the skin.
To determine whether this was attributable to
surface effects caused by
the presence of a film of
fluid, a series of equal threshold burns were made over the dry skin
and skin wet with various
organic agents, care being taken in the case of each individual to
select the exact time of
exposure required to give a mild burn on the dry skin.
The fact that water is a very poor solvent for
mustard gas and yet
appears to facilitate the
passage of the substance into the tissues as well as the other fluids
which are good solvents for it,
suggests the probability that capillarity rather than solution may play
an important role in
transporting mustard gas from the atmosphere to the point of entry into
the skin, and lends some
support to the view that mustard gas passes down the sweat glands by a
process in which surface
phenomena play an important part.
There is a striking correspondence between the above
results in which a
water film facilitates the
passage of mustard gas into the tissues and the observation of Clowes,
Perrott, and Gordon that
the passage of mustard gas through clothing is facilitated by the
presence of from 3 to 5 per cent
of water.13
DISCUSSION
A variation in sensitivity of the skin of several
hundred to one was
observed in experiments with
saturated vapor or paraffin oil solutions of dichlorethyl-
402
sulphide. In experiments with different concentrations and times of
exposure, using different
species of animals, the variation in the susceptibility of the
individuals of a given species to the
effects of inhalation were of a very much lower order of
magnitude-probably not more than five
to one. Furthermore, on intramuscular or intravenous injection in dogs
variations of no great
order of magnitude were observed. As pointed out in a previous portion
of this chapter, it is
evident that the differences in sensitivity observed were due to
differences in the relative
amounts observed compared with the amounts lost by evaporation. Hence
one is justified in
assuming that in individuals of the same species the amount of mustard
gas required within the
cell to produce pathological effects is roughly the same-that is, the
threshold concentration for
the cell varies very little in different men.
In discussing the mechanism of absorption of mustard
gas by the skin it
is proposed to consider
the problem from the standpoint of a three-phase system in which the
outer phase (A) represents
the external atmosphere containing varying concentrations of mustard
vapors; the middle phase
(B) represents the outer layers of skin through which the mustard must
pass in order to produce a
toxic effect; and the inner phase (C) represents the inner layer of the
skin, particularly the
protoplasmic contents of those cells of the tissues which are directly
affected by the poison.
For purposes of convenience A, B, and C in resistant
individuals have
been designated as Ar, Br,
and Cr, and in sensitive individuals as A8, B8,
and C8.
If, as has been indicated above, the velocity with
which mustard gas
must pass into those
reactive cells which have been designated as C in order to produce a
given pathological effect, is
approximately constant in resistant and sensitive individuals-if the
critical toxic threshold
concentration in Cr is the same as in C8-it necessarily
follows that
variations in concentration of
gas or time of exposure in A required to produce equal effects in Cr
and C. must be attributable
in great part to variation in the resistance offered by Br and B8 to
the passage of the gas.
The explanation for the difference in facility of
passage through Br
and B8 may lie in one or
more of the following causes: (1) Variation in facility of absorption
of mustard gas on interface
between A and B. (2) Variation in the facility with which absorbed
mustard gas passes along
gland surfaces by capillarity or into and through the epidermis by a
process of diffusion of the
absorbed or dissolved gas. (3) Variation in the thickness of Br and B8 or the actual distance to be
traversed from A to C. (4) Variation in the amount of gas required to
saturate constituents of the
skin which may be capable of adsorbing or dissolving large quantities
of the gas (for example,
lipoids, pigment granules, etc., in negro and white skin). (5)
Variation in the facility of passage
of gas from B into C particularly from the lipoid nonaqueous phase into
the aqueous phase of the
protoplasm of those reactive cells in which the pathological effects
are produced.
It is difficult to determine the relative importance
of the r6le played
by each of these factors, but
variation in the resistance of B may be conveniently represented
pictorially either by varying its
thickness, making Br thicker than B8, or by varying the
angle of the
average gradient of
mustard-gas concentration through B required to deliver a given amount
of mustard gas in C; in
which case that of Br would be steeper than B8.
403
It will be seen that in either case, a critical
concentration of
mustard gas would be delivered into
C. with greater facility than into Cr; thus a shorter exposure or a
lower initial concentration
would be required to effect the passage into C, of a reactive quantity
of the poison.
Whatever mode of expression be adopted, the effect
of evaporation after
the exposure is evident.
The concentration at the interface AB would be higher than at the
interface BC, but it would
rapidly decrease as a result of loss into the air.
The delivery of the necessary critical concentration
of gas into C for
a sufficient period of time
to cause pathological changes would obviously depend upon the thickness
of B, upon the angle
of gradient of resistance to passage through B, and upon the facility
with which the
concentration falls off at the interface AB as a result of evaporation.
It appears desirable to
utilize the available experimental data in an attempt to determine,
more specifically the relative
importance of the individual factors enumerated above, viz, surface
adsorption, resistance to
passage of the gas, thickness of the skin, saturation capacity and
threshold concentration required
to produce toxic effects in C.
This last factor- i. e., threshold concentration in
C-must be very low
because (1) concentrations
in the range of 0.002 to 0.005 mg. per liter represent not only
critical points in the curves
determined on the skin but (2) also correspond very closely with the
threshold concentrations
observed in respiratory experiments, and (3) the enormous proportion of
gas lost from the skin
after exposure, as shown by capping experiments, indicates that the
amount which is absorbed is
very small. The extremely small amounts to which some individuals give
a reaction is further
evidence for this fact. This low threshold concentration in C leaves
adsorption on the skin
surface or one or more of the factors involved in the passage through
B, as primarily responsible
for the buffer effect exerted by the skin.
In the case of liquid burns or severe vapor burns
the difference in the
protective mechanism of
resistant and sensitive skins appears to be reduced to a minimum; all
the factors involved are
probably saturated, whatever their capacity.
The observation that the difference between Br and B8 is more and more
accentuated with
reduction in time of exposure or concentration points to surface
adsorption as an important
factor; reasoning in this direction is supported by the following facts:
(1) Ten minutes after exposure of the skin to liquid
mustard gas so
large a proportion of the
mustard gas may be removed by repeated washings with kerosene as to
reduce the amount
passing into the tissues below the critical level required to produce a
blistering burn. (2)
Washing with soap and water produces a corresponding effect, but to a
much less degree. (3)
The presence of mustard gas on the skin surface may be demonstrated
half an hour after
exposure by transmitting a burn to another individual. (4) Since a
15-second exposure may be
raised by capping for an hour to the magnitude of a 3-minute exposure,
the loss from the skin to
the atmosphere must be far in excess of the amount passing into the
interior. This suggests that
the maximum concentration of mustard is on or near the surface. (5) The
curves covering these
capping reactions support the theory that surface adsorption is the
factor of primary importance. (6) The fact that the skin may be left
open to the atmosphere
404
for a prolonged period, in some cases as much as 30 minutes after
exposure, and the intensity of
the burn may still be raised by capping, indicates that loss of gas
from the surface is still taking
place. (7) The affinity of the skin of the resistant individual for
mustard gas is far in excess of
that of a sensitive one. The fact that Br withdraws gas from B8 shows
that Br adsorbs gas more
strongly than B. and that the gas is still on or near the surface. (8)
If exposures on resistant and
sensitive skins have been so adjusted as to give equal burns if
subsequently left uncovered,
capping the exposures gives a worse burn in the resistant than in the
sensitive case. This result
corresponds with the fact that gas may be withdrawn by a resistant skin
from a resistant skin
longer than from a sensitive skin.
From the above considerations it seems probable that
difference in the
adsorptive capacity of the
skin surface is the most important limiting factor in determining the
degree of exposure in A
necessary to effect the delivery of a toxic concentration in C.
In considering the relative importance of factors
other than the
surface adsorption on B and
threshold concentration in C, it is obvious that the greater resistance
of the negro's skin as
compared with the white's skin may reasonably be attributed, in part at
least, to differences in
actual thickness of the skin, to fats and lipoids which appear to be
more plentiful and to pigments
which are known to be more plentiful in the negro skin than in white.
The very short minimum time within which the first
reactions become
visible in certain sensitive
individuals would seem to indicate that the first traces passed through
the skin fairly rapidly.
The experiments with water films seem to support the
view that mustard
gas passes into the
sweat glands from which it is adsorbed after passing by capillarity to
areas adjacent to the cells
of the underlying tissues in which pathological effects are produced.
The capping experiments support this view. The
passage of mustard gas
into the sweat glands
would obviously be facilitated by maintaining the vapor pressure.
These experiments should be considered in the light
of certain
observations concerning the
absorption of dichlorethylsulphide in other parts of the body.
Even though mustard gas is very rapidly hydrolyzed
at body temperature,
inhalation of high
concentrations of the vapor, intravenous injection of large quantities
of the saturated aqueous
solution, and application of the oil to the skin, cause not onlv local
lesions but also marked and
characteristic systemic effects.
It is obvious, therefore, that after its
introduction into the blood
stream a portion at least of the
unhydrolyzed material must be taken up by lipoidal constituents and
thus protected from
immediate hydrolysis. After intradermal, subcutaneous, intraperitoneal
or intramuscular
injections of this substance the severe necrotic lesions which might be
expected from its action
are not observed. The pathological changes consist largely in the
development of intense edema
at the site of injection, with the characteristic effects on the gut
and adrenals. It is possible that,
when it is thus injected directly into the deeper tissues which are
well supplied with blood, and
which lack the nonaqueous binder of the skin, it is rapidly absorbed
without doing much local
damage. Moreover, the bountiful blood supply would go far to counteract
the intracellular
liberation of acid, and would thus circumvent its local toxic action.
This suggestion is supported
by the pathological changes ol)served in the respiratory tract in
animals
405
gassed with mustard gas. Though congestion and necrotic sloughing
appear in the upper tract,
the lower tract is practically undamaged until very high concentrations
are used, when some
edema may appear. Since the lower respiratory tract is primarily an
absorbing tissue, it is in very
close association with the blood stream. The absorbed poison might be
rapidly removed, and
such hydrolysis as would inevitably occur might be counteracted by
neutralization in the buffer
system of the blood.
In considering the toxic action of a series of war
gases it is observed
that there is marked contrast
in the pathological effects exerted upon the upper and lower
respiratory tract and upon the skin.
There appears to be a certain relation between the
areas inhibiting
maximum pathological
changes and certain physical and chemical properties of the gas, for
example, vapor pressure,
lipoid-water distribution coefficient and rate of hydrolysis in water.
Phosgene and superpalite, which have a very high
vapor pressure and
which hydrolyze very
rapidly on contact with water, exert a very destructive effect on the
lower respiratory tract, but
do not markedly affect the upper tract or irritate the skin.
It is obvious that under a given condition of vapor
pressure, lipoid
solubility and other factors, a
given amount of gas finds its way into the cell in a given time and if
the rate of transportation
through the cell is very rapid, the question as to whether or not a
toxic concentration of acid will
be produced within the cell depends upon the rate of hydrolysis, or the
relation between
transportation velocity and rate of hydrolysis.
A low lipoid-water partition coefficient should
facilitate the
decomposition of gas within the cell
by raising the concentration in the water phase and by diminishing the
proportion removed by
the lipoid constituents of the blood.
The failure of such volatile substances as phosgene
and superpalite to
exert any effect on the
skin is readily explained in the light of experiments with mustard gas,
the loss of which from the
skin to the air has been demonstrated to be very great in spite of its
relatively low vapor pressure.
Substances like mustard gas, with a low vapor
pressure and which
hydrolyze comparatively
slowly, would, after their absorption by the lower respiratory tract,
have an opportunity to pass
into the lipoidal phases of the blood before hydrolysis reached such
dimensions within the cell as
to cause pathological effects.
But in the cells of the upper bronchi and the skin,
where
transportation is comparatively
insignificant, an accumulation would result, and ultimately sufficient
acid would he liberated to
produce toxic effects.
Phenyldichlorarsine and ethyldichlorarsine, with a
low vapor pressure
(resembling mustard gas
somewhat in their physical properties) but having a high rate of
hydrolysis, attack the skin and
upper respiratory tract just as mustard gas does, and cause profuse
edema in the lower
respiratory tract just as do phosgene and superpalite.
Unfortunately, these investigations were incomplete
when the suspension
of hostilities brought
an end to experimental work.
Consequently, though a large amount of additional
data is available
suggesting a relation
between the physicochemical and pharmacological properties of
substances which played a rôle
in chemical warfare, it does not appear advisable to attempt further
generalization at the present
stage of our knowledge.
406
SUMMARY
The experimental data indicate -
1. That mustard gas is first absorbed by some
element on or immediately
adjacent to the skin
surface.
2. That while a portion of the mustard gas passes
rapidly inward to a
point from which it can not
subsequently be removed the greater portion remains on or near the
surface for a considerable
period, a proof of which is that it may be removed even after 10 or 15
minutes by persistent
washing with organic solvents.
3. That the amount of mustard gas passing into the
atmosphere from an
exposed surface far
exceeds the amount passing into the inner strata of the skin. This loss
is very great at first and is
still demonstrable after 45 minutes.
4. That the time of exposure necessary to produce a
positive reaction
bears a definite relation to
concentration and varies for different individuals.
5. That a resistant skin absorbs far more gas than a
sensitive skin,
and gas may be withdrawn
from the latter by the former. That difference in sensitivity of
different skins is due principally to
difference in saturation absorptive capacity.
6. That the intracellular threshold concentration of
gas required to
produce pathological changes
in the skin is approximately the same in resistant and sensitive
individuals.
REFERENCES
(1) Harvey, E. N.: The Permeability of Cells for Acids. Publication No.
212. Carnegie Institute
of Washington, D. C., 1915, viii, Part vi, 132.
(2) Sanno, Y.: Ueber den Einfluss der Temperatur auf die
Giftempfindlichkeit des Frosches. Archiv für experimentelle
Pathologie und Pharmakologie. Leipzig, Aug.
7, 1911, lxv, 325.
(3) Meyer, Victor: Physiologische Wirkung der gechlorten
Schwefelathyle. Berichte der
deutschen chemischen Gesellschaft, Berlin, 1887, xx, No. 2,
1729.
(4) Warthin, A. S., and Weller, C. V.: The Pathology of the Skin
Lesions Produced by Mustard Gas. The
Journal of Laboratory and Clinical
Medicine, St. Louis, Mo., 1918, iii, No. 8,
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(5) Lillie, R. S., Clowes, G. H. A., and Chambers, R.: On the
Penetration of Dichlorethylsulphide
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Destructive Action on
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(6) Graham, E. A.: Late Poisoning with Chloroform and Other Alkyl
Halides in Relationship to
the Halogen Acids Formed by Their Chemical Dissociation. Journal
Experimnental Medicine,
New York, 1915, xxii, No. 1, 48.
(7) Nef, J. U.: Dissociationsvorgange bei den Alkylathern der
Salpetersaure der Schwefelsaure
und der Halogenwasserstoffsauren. Annalen
der Chemie, Berlin, 1899,
cccix, 126.
(8) Weber, S.: Ueber die Giftigkeit des Schwefelsauredimethylesters
(Dimethylsulphates) und
einiger Verwandter Ester der Fetreihe. Archiv für Experimentelle
Pathologie und
Pharmakologie, Leipzig, December 19, 1901, xlvii, 113.
Also:
Michiels, J.: Sur la Toxicite du Sulfate Neutre de Methyle. Archives
Internationales de
Pharmacodynamie, Paris, 1911, xii, 467.
(9) Erdmann, P.: Ueher Augenveranderungen durch Dimethylsulfat. Archiv
für
Augenheilkunde, Wiesbaden, 1908-09, lxii, 178.
(10) Weber, S.: Loc. cit.
(11) Meyer, Victor: Ueher Thiodiglycol Verbindungen. Berichte der
deutschen chemischen
Gesellschaft, 1886, Berlin, xix, No. 3, 3259.
(12) Marshall, E. K., jr., and Kolls, A. C.: An Apparatus for the
Administration of Gases and
Vapors to Animals. Journal of
Pharmacology and Experimental
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1919, xii, No. 8, 385.
(13) Personal communication from Dr. G. H. A. Clowes in possessions of
the author of this
chapter.
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