Effect of dimethyl sulfoxide on sporicidal activity of ethylene oxide ga

Spiner, D. R.

English

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Prepared by: 
Protection Branch Report of Test No. 8-69 
The Effect o f  Dimethyl Sulfoxide on the Sporicidal 
Activity of Ethylene Oxide Gas 
9 January 1969 
DAVID R. SPINER 
Decontamination Section 
Protect ion Branch 
Approved by: 
&L+ K &4L- 
ROBERT K. HOFFMAN 
Chief, Decontamination Section 
HERBERT M. DECKER 
Chief, Protection Branch 
Chief, Physical Defense Division 
DEPARTMENT OF THE ARMY 
Fort Detrick, Frederick, Maryland 21701 
https://ntrs.nasa.gov/search.jsp?R=19690005603 2020-03-12T08:25:48+00:00Z
P r o t e c t i o n  Branch Report o f  Test No. 8-69 
The E f f e c t  o f  Dimethyl Su l fox ide  on the  S p o r i c i d a l  
A c t i v i t y  o f  Ethylene Oxide Gas 
A t  the  request o f  the  NASA the  f o l l o w i n g  t e s t s  were conducted t o  
o b t a i n  a qu ick  eva lua t ion  o f  whether there  i s  an enhancement o f  the 
s p o r i c i d a l  a c t i v i t y  o f  e thy lene oxide (EtO) gas by incorpora t ing  
dimethyl  s u l f o x i d e  (DMSO). 
Previous screening tests ,  i n  which a c l o t h  patch contaminated w i t h  
- B. s u b t i l i s  var  n i q e r  (BG) spores was suspended f o r  24 hours i n  a l i t e r  
b o t t l e  over 0.1 m l  o f  DMSO revealed no measurable s p o r i c i d a l  a c t i v i t y ,  
but  a s i m i l a r  t e s t  w i t h  Staphylococcus aureus c e l l s  k i l l e d  100%, showing 
t h a t  DMSO i s  a n t i b a c t e r i a l .  
S i m i l a r  4-hour vapor t e s t s  were conducted us ing  f i v e  chemicals, 
w i t h  and w i thout  0.1 m l  DMSO contained i n  a small  g lass weighing cup. 
A 0.1 m l  amount o f  the  other  chemical was placed on the  bottom o f  the  
b o t t l e  t o  v o l a t i l i z e .  As shown i n  Table I, t h e  a c t i v i t y  o f  f o u r  o f  the 
chemicals was depressed by the presence o f  DMSO. One chemical 's a c t i v i t y  
was not  a f fec ted .  Vapor pressure i s  probably an important f a c t o r  here. 
I t  was noted t h a t  l i t t l e  o f  the  DMSO had v o l a t i l i z e d  and the  l i q u i d  could 
be absorbing some o f  the  other  vapor. The vapor pressure o f  pure DMSO a t  
25' C i s  0.6 mm Hg, which i s  equiva lent  t o  approximately 1.6 m g / l i t e r .  
A t  t h i s  p o i n t  work w i t h  DMSO discontinued, t o  be s t a r t e d  again on ly  when 
t h e  NASA requested more in format ion.  
PROCEDURE 
The present t e s t s  may be grouped i n t o  f o u r  par ts :  (a) exposure o f  
BG spores t o  DMSO vapor (DMSO cont ro ls ) ,  (b) exposure o f  spores t o  Et0 
gas (Et0 cont ro ls ) ,  (c) exposure o f  spores t o  a gaseous mix tu re  o f  E t O -  
DMSO, and (d) exposure o f  spores t o  the  vapor emi t ted from a s o l u t i o n  
of Et0 gas d isso lved i n  l i q u i d  DMSO. 
Tests were conducted a t  2 5 O  C i n  a la rge  des icca tor  f i t t e d  w i t h  two 
stopcocks, except f o r  the  t e s t  (Table 11) i n  which a l i t e r  b o t t l e  was 
used. 
a t  33% RH p r i o r  t o  use. Saturated DMSO vapor was obtained by f l u s h i n g  
ambient a i r  through 40 m l  DMSO i n  a Vigreaux bubbler and i n t o  the des ic -  
ca tor .  Ambient a i r  RH varied, but was no t  under 33%. 
The c l o t h  patches contained about 2 x 105 spores and were s tored 
A 2-hour exposure was chosen f o r  t he  DMSO c o n t r o l  because the t e s t  
The Et0 
samples were exposed f o r  one hour t o  a DMSO vapor f l u s h  fo l lowed by a 1 -  
hour exposure t o  Et0 (100 mg/l) i n  the presence o f  DMSO vapor. 
gas was introduced by reducing the des icca tor  pressure by 42 mm Hg and 
rep lac ing  w i t h  Et0 gas. 
A v a r i a t i o n  o f  the technique involved p l a c i n g  a s o l u t i o n  of 1 p a r t  
Et0 i n  2 p a r t s  l i q u i d  DMSO on the  bottom o f  a des icca tor  t o  v o l a t i l i z e .  
Enough o f  the  s o l u t i o n  was used t o  f u r n i s h  100 mg o f  Et0 gas per  l i t e r  
o f  des icca tor  volume. 
tes ted .  The r e s u l t s  are shown i n  Table 111. 
A week o l d  and a f r e s h l y  mixed s o l u t i o n  were 
A f t e r  exposure t o  the gases, the  c l o t h  patches were placed i n  10 rnl 
d i s t i l l e d  water, shaken v igo rous l y  t o  d is lodge the spores, and s e r i a l  
d i l u t i o n s  p la ted  i n  T'ryptose agar. Colony counts o f  v i a b l e  organisms 
were made a f t e r  48 hours incubat ion a t  37" C. 
The a f f i n i t y  o f  DMSO f o r  Et0 was i l l u s t r a t e d  by p l a c i n g  10 m l  of 
DMSO on the  bottom o f  a la rge  des icca tor  and in t roduc ing  h a l f  an atmo- 
sphere o f  Et0 gas (900 mg/b) by rep lac ing  an equ iva len t  vacuum (380 mm 
Hg). 
2-3 days t o  48 mm Hg (Table I V ) .  
absorbed 732 mg Et0 and i n  2-3 days 1,094 mg EtO.  
W i th in  an hour a vacuum o f  32 mm Hg occurred which increased over 
So i n  on l y  one hour 10 m l  o f  DMSO 
When 25 m l  o f  c h i l l e d  l i q u i d  Et0 i s  placed w i t h i n  a 25 l i t e r  
pressure conta iner  a t  room temperature, i t s  vapor pressure r i s e s  
s t e a d i l y  u n t i l  i t  reaches the t h e o r e t i c a l  380 mrn i n  about two hours 
(Fig.  1 ) .  When the  same experiment i s  repeated however i n  the presence 
of  50 m l  DMSO the r i s e  i s  slower and the  pressure l e v e l s  o f f  a t  a l i t t l e  
over 200 rng Hg, revea l ing  t h a t  almost ha l f  o f  the Et0 i s  adsorbed i n  the 
DMSO, Thus, the  DMSO i s  a c t i n g  as a r e s e r v o i r  (or storage) ho ld ing  the  
Et0 vapor and capable o f  s lowly  re leas ing  i t. 
CONCbUSIONS 
The s p o r i c i d a l  a c t i v i t y  o f  DMSO vapor appears measurable a t  satu-  
r a t i o n  concentrat ion,  but  i t s  low v o l a t i l i t y  makes the r a t e  slow and 
d i f f i c u l t  t o  measure. Exposure t o  E t 0  i n  the  presence o f  DMSO vapor 
2 
seemed t o  cause an increase i n  t h e  number o f  spores k i l l e d  (Test 1115, 
Table 11), as d i d  exposure t o  t h e  vapor f rom a s o l u t i o n  o f  one p a r t  
Et0 i n  two p a r t s  DMSO (Table 111). However, t o o  few data were c o l l e c t e d  
t o  prove s t a t i s t i c a l l y  t h a t  the a d d i t i o n  o f  DMSO vapor s i g n i f i c a n t l y  
enhanced t h e  s p o r i c i d a l  a c t i v i t y  o f  E t O .  I t  i s  d i f f i c u l t  t o  judge 
whether the e f f e c t  i s  a d d i t i v e  o r  synerg is t i c ,  s ince Test IIIA, Table 
11, suggests t h a t  DMSO does not p recond i t ion  the  spores f o r  f a s t e r  Et0 
a c t i v i t y ,  y e t  the vapor pressure t e s t  showed t h a t  Et0 i s  absorbed by 
DMSO, i.e., DMSO on t h e  c l o t h  patches may concentrate Et0 gas. 
3 
Table I. 
Gaseous Fumigant Test with Addition of 0.1 ml Dimethyl Sulfoxide 
Bacillus subtilis var niqer 
1.6 x lob spores/patch Test 0 rgan i sm: 
Exposure : 4 hours 
Chemical 
2,2,4-T r i met h y 1 -3 -h yd roxy - 
pentenoic acid, B-Lactone 
Ethylaziridinyl Formate 
Alpha-bromocyclobutanone 
2-Methyl-2-oxazoline 
Trimethylene oxide 
Per Cent Reduction 
Without DMSO With DMSO 
72 None 
90 None 
56 None 
60 None 
14 12 
4 
Table 11. 
Ef fec t  o f  Dimethyl Su l fox ide  on B a c t e r i c i d a l  A c t i v i t y  
o f  Ethylene Oxide Gas 
Resul ts:  
C l o t h  patch contaminat ion averaged 2 x 10 5 1. s u b t i l i s  var n i q e r  spores, 
Tests conducted a t  25' C, i n s i d e  des iccators .  
Expos u r e  No. o f  
Test I. DMSO Cont r o  1 s (hrs) % Reduction Patches 
A. Saturated DMSO vapor 2 23.3 3 
B. Saturated DMSO vapor 2 14.0 6 
C. 0.1 ml DMSO i n  a 1 l i t e r  b o t t l e  2 0 3 
Test 11. Et0 Contro ls  
A.  100 mg EtO/L 
B. 100 mg EtO/L 
C. 100 mg EtO/L 1 
D. 100 mg EtO/L 2 
Test 111 EtO-DMSO Gaseous Mix 
A. One h r  exposure t o  saturated DMSO vapor, 
remove and expose one hour t o  100 mg 
EtO/L 
B. One hour exposure t o  saturated DMSO vdpor 
fo l lowed by one hour Et0 (100 mg EtO/L) 
s t i l l  i n  presence o f  DMSO vapor. 
C. L i k e  B y  except increase Et0 exposure t o  
two hours. Compare t o  I I D .  
68.0 
42.7 
81.6 
67.0 
71.4 
98.5 
99.6 
3 
4 
3 
4 
3 
3 
4 
5 
Table 111. 
Exposure o f  B a c i l l u s  s u b t i l i s  var  n i q e r  Spores 
t o  Vapor from EtO-DMSO S o l u t i o n  
DMSO-Et0 So lu t ion :  
2 p a r t s  DMSO + 1 p a r t  Et0 
3.4 ml above s o l u t i o n  i n  9.5 l i t e r  des icca tor  (equivalent to  100 mg 
EtO/L volume) 
Expos u r e  No. o f  
(hrs) % Reduct ion$: Patches 
A .  Freshly  mixed 1 95.5 3 
9 .  One week o l d  1 98-05 3 
-I; B a c i l l u s  s u b t i l i s  var n i q e r  spores 
6 
Table I V .  
Absorpt ion o f  Ethylene Oxide Gas by L i q u i d  DMSO 
Time 
(h r s  
0 
1 
2 
60 
Ma nome t e r 
P ressu r e  
(mm Hg) 
0 
-32 
-34 
-48 
Amount o f  
Et0 Absorbed 
by the  DMSO 
(mg) 
0 
732 
775 
1094 
Note: 
Chamber = 9.5 l i t e r  
Amount DMSO = 10 m l  
Amount Et0 = 900 m g / l i t e r  o r  8,550 rng/9.5 l i t e r s  
7 
400 
CI, 
I 
E 300 
E - 
Q) 
3 
a, 
e 
0 
Q 
L 
v) 
v) 
L 
L 
0 200 > 
1 0c 
C 
* THEVAPOR PRESSURE OF ETHYLENE OXIDE WITH 
VD WITHOUT THE PRESENCE OF DIMETHYL SULFOXIDE 
I I I 
5 0  100 150 
Time, minutes 
a 


The effect of dimethyl sulfoxide on the sporicidal activity of ethylene oxide gas

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