B. Decontamination Methods 
Physical and chemical means of decontam- 
ination fall Into four main categories: Heat; 
Liquid Decontaminants; Vapors and Gases; 
and UV Radiation. 
1. Heat. The application or heat, either 
moist or dry, is recommended as the most 
effective method of sterilization. Steam at 
121 C under pressure in the autoclave is the 
most convenient method of rapidly achiev- 
ing sterility. Dry heat at 160 to 170 C for 
periods to 2 to 4 hours is suitable for 
destruction of viable agents on impermeable 
nonorganic material such as glass, but is not 
reliable in even shallow layers of organic or 
inorganic material that can act as insulation. 
Incineration is another use of heat in the 
decontamination of microorganisms and also 
serves as an efficient means for disposal. 
2. Liquid Decontaminants. In general, the 
liquid decotaminants find their most prac- 
tical use in surface decontamination and, at 
sufficient concentration, as decontaminants 
of liquid wastes for final disposal in sanitary 
sewer systems. 
There are many misconceptions concerning 
the use of liquid decontaminants. This is 
due largely to a characteristic capacity of 
such liquids to perform dramatically in the 
test tube and to fail miserably in a practical 
situation. Such failures often occur because 
proper consideration was not given to such 
factors as temperature, time of contact, pH. 
concentration, and the presence and state of 
dispersion, penetrability and reactivity of 
organic material at the site of application. 
Small variations in the above factors may 
make large differences in effectiveness of de- 
contamination. For this reason, even when 
used under highly favorable conditions, com- 
plete reliance should not be placed on liquid 
decontanimants when the end result must 
be sterility. ' 
There are many liquid decontaminants 
available under a wide variety of trade names. 
In general, these can be categorized as halo- 
gens, acids or alkalies, heavy metal salts, 
quaternary ammonium compounds, phenolic 
compounds, aldehydes, ketons, alcohols and 
amines. Unfortunately, the more active the 
decontammant the more likely it is that the 
decontaminant will possess undesirable char- 
acteristics, such as the possession of corrosive 
propertie. None is equally useful or effective 
under all conditions. 
3. Vapors and Gases. A variety of vapors 
and gases possess decontamination properties. 
The most useful of these are formaldehyde 
and ethylene oxide. When these can be em- 
ployed in closed systems and under controlled 
conditions of temperature and humidity, ex- 
cellent decontamination can result. Vapor 
and gas decontaminants are primarily useful 
in decontaminating: (i) Biological Safety 
Cabinets and associated effluent air-handling 
systems and air filters; (ii) bulky or station- 
ary equipment that resists penetration by 
liquid surface decontaminants; (iii) instru- 
ments and optics that might be damaged 
by other decontamination methods; and (iv) 
rooms and buildings and associated airhan- 
dling systems. 
4. Radiation. The usefulness of ultraviolet 
(UV) irradiation as a decontaminant is lim- 
ited by its low penetrating power. No infor- 
mation is available regarding the effective- 
ness of UV irradiation for decontaminating 
microorganisms containing recombinant DNA 
molecules. Dependence on UV must be based 
on the results of experiments imitating par- 
ticular anticipated environmental conditions 
and applications. Ultraviolet light is gen- 
erally of limited application and is primarily 
useful in air locks and animal holding areas 
for controlling low levels of airborne con- 
taminants. 
No one procedure or material wm solve 
all decontamination problems. The only 
NOTICES 
method of assuring the efficacy of selected 
methodologies is to critically examine the 
results obtained in practical tests with the 
microorganism(s) of Interest. 
C. Laboratory Spills 
A troublesome problem that may occur 
in the laboratory is the decontamination of 
an overt biological spill. The occurence of a 
spill poses less of a problem if it occurs 
in a Biological Safety Cabinet provided splat- 
tering to the outside of the cabinet does 
not occur. Direct application of concentrated 
liquid decontaminant and a thorough wipe 
down of the internal surfaces of such cab- 
inetry will usually be effective for decon- 
taminating the work ozne but ' gaseous de- 
contaminants would be required to rid the 
interior sections of the cabinet of contam- 
inants. Each researcher must realize that 
in the event of an overt accident, research 
materials such as tissue cultures, media, and 
animals within such cabinets may well be 
lost to the experiment. 
The greater problem arises if the incident 
occurs in the open laboratory. All laboratory 
protocols should be designed to prevent such 
'occurrences. The first action in the event 
of an overt laboratory spill is evacuation of 
tho affected area to minimize the exposure 
of personnel involved. Next, the spill area 
must be isolated to prevent exposure of per- 
sonnel and experimental materials beyond 
those involved in the immediate area of the 
spill. The procedures adopted must be rap- 
idly effective and must not create additional 
aerosol or foster mechanical transfer of ma- 
terials to unaffected areas. Personnel carry- 
ing out the procedures must be provided 
with protective clothing and equipment, in- 
cluding respiratory protection. Consideration 
must be given to the safe disposal of all ma- 
terials and liquids resulting from cleanup 
procedures. Reentry of personnel to the area 
should be avoided until it can be reasonably 
established that the area has been effectively 
idecontaminated. Further specific details are 
provided in Section VIII. 
D. Disposal 
Decontamination and disposal in infec- 
tious disease laboratories are closely interre- 
lated acts in which decontamination con- 
stitutes the introductory phase of disposal. 
All materials and equipment used in re- 
search on recombinant DNA molecules will 
ultimately be disposed of; however, in the 
sense of daily use, only a portion of these 
will require actual removal from the lab- 
oratory complex or on-site destruction. The 
remainder will be recycled for use either 
within the same laboratory or in other lab- 
oratories that may or may not engage in DNA 
recombinant research. Examples of the latter 
that immediately come to mind are: re- 
usable laboratory glassware, instruments 
used in necropsy of infected animals, and 
laboratory clothing. Disposal should there- 
fore be interpreted in the broadest sense of 
the word, rather than in the restrictive sense 
of dealing solely with a destructive process. 
The principal questions to be answered 
prior to disposal of any objects or materials 
from laboratories dealing with potentially 
infectious microorganisms or animal tissues 
are: 
1. Have the objects or materials been ef- 
fectively decontaminated by an approved 
procedure? 
2. If not, have the objects or materials 
been packaged in an approved manner for 
immediate on-site incineration or transfer 
to another laboratory? 
3. Does disposal of the decontaminated 
objects or materials involve any additional 
potential hazards, biological or otherwise, to 
personnel either: 
(1) Those carrying out the Immediate dis- 
posal procedures or 
27931 
(11) Those who might come into contact 
with the objects or materials outside the 
laboratory complex? 
Laboratory materials requiring disposal 
will normally occur as liquid, solid, and ani- 
mal room wastes. The volume of these can 
become a major problem when there is the 
requirement that all wastes be decontami- 
nated prior to disposal. It is mast evident 
that a significant portion of this problem 
can be eliminated if the kinds of materials 
initially entering the laboratory are reduced 
In any case, and wherever possible, materlali 
not essential to the research should be re* 
tained in the nonresearch areas for dis- 
posal by conventional methods. Examples are 
the packaging materials in which goods are 
delivered, disposable carton-cages for trans- 
port of animals, and large carboys or tanks 
of fluids which can be left outside and drawn 
from as required. Reduction of this bulk will 
free autoclaves and other decontamination 
and disposal processes within the laboratory 
for the more rapid and efficient handling of 
materials known to be contaminated. 
Inevitably, disposal of materials raises the 
question, “How can we be sure that the 
materials have been treated adequately to 
assure that their disposal does not constitute 
a hazard?” In the small laboratory, the prob- 
lem is often solved by requiring that each 
investigator decontaminate all contaminated 
materials not of immediate use at the end 
of each day and place them in suitable con- 
tainers for routine disposal. In larger lab- 
oratories where the mass of materials for 
disposal becomes much greater and sterili- 
zation and decontamination bottlenecks 
occur, materials handling and disposal will 
likely be the chore of personnel not engaged 
in the actual research. In either situation, 
a case can be made for establishing- a posi- 
tive method of designating the state of mate- 
rials to be disposed of. This may consist of a 
tagging system stating that the materials 
are either sterile or contaminated. 
Disposal of materials from the laboratory 
and animal holding areas will be required for 
research projects ranging in size from an In- 
dividual researcher to those involving large 
numbers of researchers of many disciplines. 
Procedures and facilities to accomplish this 
will range from the simplest to the most 
elaborate. The primary consideration in any 
of these is to dispel the notion that labora- 
tory wastes can be disposed of in the same 
manner and with as little thought as house- 
hold wastes. Selection and enforcement of 
safe procedures for disposal of laboratory 
materials are of no less importance than the 
consideration given to any other methodol- 
ogy for the accomplishment of research ob- 
jectives. 
Materials of dissimilar nature will be 
common in laboratories studying recombi- 
nant DNA molecules. Examples are combi- 
nations of common flammable solvents, 
chemical carcinogens, radioactive isotopes, 
and concentrated viruses or nucleic acids. 
These may require input from a number of 
disciplines in arriving at the most practical 
approach for their decontamination. 
E. Characteristics of Chemical Decontami- 
nants in Common Use in Laboratory 
Operations 
Every person actively working with viable 
microorganisms, no matter how remote the 
field of specialization, will, from time to 
time, find it necessary to decontaminate by 
chemical methods work areas and materials, 
equipment, and specialized instruments. 
Chemical decontamination is necessary be- 
cause the use of pressurized steam, the most 
rapid and reliable method of sterilization, 
is not normally feasible for decontaminating 
large spaces, surfaces, and stationary equip- 
ment. Moreover, high temperatures and 
moisture often damage delicate instruments. 
FEDERAL REGISTER, VOL. 41, NO. 1 31— WEDNESDAY, JULY 7, 1976 
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