Operating Instructions for - VBG

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L2/S2 Operating Instructions, Committee of Biosavety Officers, DK
FZ Heidelberg

date: 02/12/2007


emergency telephone numbers


Technical Maintenance Squad

2888







Radiation Safety



2457/2699







Safety Engineer



2866







First Aid / DKFZ Doctor


2488



Department of
............................................................



Operating Inst
ructions

for


safe handling of infectious materials and genetically modified organisms




(German abbrev.: GVO)

in laboratories of
biological

safety level 2 (L2/S2)
.


All the rules included in the Federal Law for Protection agai
nst Contagious Diseases (IfSG) and the Genetic
Engineering Act (GenTG), in the corresponding official regulations as well as in other statutory rules and
regulations concerning safety at work and the protection of the environment have priority over these
following rules.



1. Zone of validity

These rules are valid for all L2/S2 laboratories of the department of
..............................................

in the
DKFZ main building
(or other.........)
Im Neuenheimer Feld 280 (or other ), 69120

Heidelber
g.
The
individual laboratory areas carry yellow and black warning labels saying "
biohazard
"
(“
Biogefährdung
”)
and are marked as "gene
-
technological working area of safety level S2" ("gentechnischer Arbeitsbereich der
Sicherheitsstufe S2"). Access to the e
ntire laboratory complex is limited (authorized persons, see points 4
and 5).

Individual instructions as well as such connected to projects or rooms can be added at the end of these rules
(point 11). Separate rules are valid for the L3/ S3 laboratories.



2. Permissions and responsible persons

Permissions according to the Genetic Engineering Act:

Registration as laboratory of safety level S2: Az.:
................

/DKFZ
..................

, date:
......................


Biosafety officer:

..................
.. , deputy: ...................................


Project managers according to the Genetic Engineering Act: ..................................


Federal Law for Contagious Diseases:

Permission for the handling of pathogenic agents:
Az.:
.................
..................

; date:
...........................


responsible person:
...................................


deputy:
...................................



3. Risks

3.1

Working materials:

Organisms / pathogenic agents of risk
-
group II

(see Joint List fo
r the Classification of Biological
Materials, appendix II, Directive 2000/54/EG)
http://www.umwelt
-
online.de/regelwerk/eu/00_54gs.htm



genetically modified organisms (GVO), that require

safety level S2 in the laboratory
, e.g.
______________________
_________________________________

in E
.
coli plasmid or vectors see
http://bvl.bund.de/cln_027/nn_491872/DE/06_Gentechnik/093_ZKBS/03_Organismenliste/organismliste_n
ode.html_nnn=true



nondirected activity performed using


non
-
inactivated materials of human origin

which, corresponding to the Biological Substances E
nactment
(defined in paragraph 4.3.2. of the TRBA 100) are, without exception, classified as potentially infectious,



L2/S2 Operating Instructions, Co
mmittee of Biosavety Officers, DKFZ Heidelberg

date: 02/12/2007


2


e.g. tumor biopsies, blood, serum, primary cell cultures, lymphocyte preparations, and infected established
cell lines.

Definition: Di
rected activity occurs, when the biological material to be used is of a known species, the
activity performed is directed toward the biological material and the exposure of employees is assessable.

The contact of these materials with the human body, e.g. o
ver the mucous membrane (mucosa), eyes, mouth
or skin (especially by skin injury and skin diseases), can lead to infections and diseases. Especially the use
of infectious agents or GVOs (genetically modified organisms)_________________________________ can
under disadventagous circumstances lead to the following specific illnesses_________________________


3.2

The
identity

and
purity

of the
organism

used is to be regularly
reevaluated
, when this is necessary for
the assessment of the organisms danger potenti
al.



4. Regulation for access


The entrance doors to the individual L2/S2 areas are marked with warning labels. Authorized to enter are
people who are permanently employed in these areas as well as employees of the DKFZ or external firms
who are charged b
y the DKFZ with maintenance and repair works and cleaning. Authorized persons are
under medical surveillance and have been instructed about the dangers and rules of conduct in the L2/S2
area (see point 5). Non
-
instructed visitors and those without medical

surveillance (G43) may only have
access to the L2/S2 areas with the permission and presence of a responsible project manager.


If an admitted person knows about an impairment of his/ her physical health situation or is pregnant, he/she
is required to com
municate this to the DKFZ doctor. In agreement with the persons responsible (see point
2) and the head of the department, individual regulations will then be fixed. According to the Law for the
Protection of Expectant and Nursing Mothers, the entire worki
ng place environmenten of the pregnant
woman has to be surveyed for potential hazards. The DKFZ company doctor and the safety engineer must
take part in this survey. Pregnant and stilling mothers underly work restrictions and prohibition of certain
activi
ties. Entrance for children up to the age of 13 is not allowed. Children between the age of 13 and 15
can be allowed to enter in the framework of basic occupational and university preparatory courses which are
under constant supervision, and upon assurance

that to no timepoint can a dangerous situation arize. Parents
must be informed about possible dangers that could occur as well as the methods taken to prevent such
accidents, and give their agreement in writing. Children between 15 and 18 who are particip
ating in a multi
-
year training program can, after extensive work place and and heath safety instructions, and under
supervision of trained personel, work with dangerous chemical and biological reagents, when the substance
concentrations in room air don’t e
xceed standard values and when work with biological agents higher than
security level 2 is excluded. JArbSchG company doctors and safety engineers will accompany all planning,
excecution and supervision.



5. Instructions and medical care


People possess
ing a permission of access will be instructed about the dangers and rules of conduct in the
laboratory area before starting laboratory work. from then on this will occur annually and also in case of the
introduction of new biological agents and procedures;

the contents and the date of instruction are to be
confirmed by signature by the instructed person (see attachment). People carrying out maintenance and
repair works and cleaning are to be previously instructed by an authorized person. Women, in addition,

are
to be instructed about possible risks for expectant mothers.

People possessing a permission of access are examined by the DKFZ doctor before starting their work, from
then on annually and upon ending employment (according to the professional union's

policy G43).
Obligatory examinations also occur before initial work with organisms for which an immunization exists
and for organisms which cause chronic illness, or upon not non
-
directed activity with unfixed blood/ human
samples, when this was not cover
ed during the initial medical examination. Further work relevant medical
examination can be requested by the employee. At the wish of the employee, serum samples can be taken
and stored before starting and after finishing his activities at the DKFZ. Furth
er serum samples can be stored
L2/S2 Operating Instructions, Committee of Biosafety Officers, DKFZ Heidelberg

date: 02/12/2007


3

at the discretion of the company doctor. Blood samples should also be taken immediately after skin injury
by needles, scalpels etc. and after contact of mucosa, eyes or mouth with infectious material (“negative
value”). Free

immunizations against Hepatitis B Virus are offered.


6. Laboratory practice


6.1. Laboratory clothing and personal safety measures

Outside of the S2 area, lockers are at your disposal where you can keep separately lab and street clothing.

Protective clot
hing consisting of a labcoat, trousers and shoes, as well as laboratory goggles are available.
Single
-
use gloves are present in all laboratories.

Labcoats with specific markings are worn in the L2/S2 laboratory area; they must be closed at the front and
mu
st be worn only in the L2/S2 laboratory area. All people entering the laboratory area have to wear these
labcoats. For washing, used protective clothing is rolled (the potentially contaminated outside to the inside)
and collected in coloured (presently vio
let) clothes' sacks in the changing rooms. Obviously contaminated
protective clothes are first autoclaved and then given away for washing (only possible for cotton)

Lab coats must be made available for guest, workmen, or repair specialists.


6.2 General
rules of conduct

To avoid contaminations, eating, drinking, chewing gums and smoking as well as storing food and
application of make
-
up is forbidden in the laboratory area; contamination of long hair is avoided by putting
them up. Eye rubbing, nail chewin
g and hand/ nose contact should be avoided. During work with infectious
materials, the doors and windows of the laboratory are to be kept firmly closed. The laboratories should be
kept tidy and clean. Only the equipment and supplies necessary for immediate

use should be placed on the
lab bench, in order to minimize the effort needed for decontamination. Absorbent material (paper towels,
cardboard boxes) are especially contamination prone and thus are stored in closed cabinets. Supplies for
short term use a
re kept in decontaminatable vessels (plastic
-
boxes or bags). Upon completion of work with
infectious materials, all surfaces of the workplace and equipment are to be decontaminated.

The hygiene plan is to be followed (
http://info/sicherheit/divinfo.htm
). Pipetting is done exclusively with
pipet
-
aids. In the case of pipet
-
aids and membrane pumps, which suck up potentially infectious or GVO
containing liquids, the air eminating from these devices has to
be sterile filtered (for example with a
Vacushield filter or 0.2 µm disposable filter). The tubing used in such devices need to be disinfected after
use (see point 10.7).

Disposable gloves will be used for the handling of infectious materials. These gloves

do not deliver absolute
protection against infection. As such, hands need to be disinfected after glove use. Disposable gloves that
are used in the laboratory can not only carry infectious materials, but also toxic, mutagenic or allergy
causing substances
. Therefore, gloves should never be worn when using common laboratory equipment
such as telephones, refrigerators, water taps or laboratory doors, and should never be worn outside of the
laboratory. To avoid unsettlement of collegues, these rules also ap
ply for newly donned gloves.


The use of object with which a person can injure himself (glass, needles, scalpels) should be kept to a
minimum. Only disposable material (for example Tork Multi, to be found in the DKFZ stockroom) should
be used to clean lab
oratory equipment and surfaces. The multiple use of hand towels, sponges and other
absorbable cleaning materials is not allowed. Plastic brushes with a handle can, however, be used.

Refrigerators or freezers in which potentially infectious materials and
GVOs are stored must be labeled with
the warning sign "biohazard". Due to problems with disinfection, unpainted/ unvarnished wood surfaces are
not allowed.

When leaving the laboratory area, take off your labcoat and hang it up inside the laboratory area. D
isinfect
your hands before washing them. Each standard laboratory contains a hygiene hand
-
basin equipped with a
soap, disinfectant, and towel dispenser. In order to prevent the use of expired disinfection agents and hand
soap, new disinfection agents and s
oap should be changed at least once a year (as a mnemonic device,
perhaps during the yearly security instructions).


6.3 Activities, during which dangerous aerosols are set free,

e.g. pipetting, decanting or mixing liquids, opening vessels that are under e
xcess or negative pressure,
homogenizing or shredding tissue, must be carried out in
Bio
-
Gard
-
hoods (class 2 hoods)
. The operating
instructions fixed at the benches are to be observed. To guarantee
correct ventilation
, assure that only the
L2/S2 Operating Instructions, Co
mmittee of Biosavety Officers, DKFZ Heidelberg

date: 02/12/2007


4

instruments nece
ssary for your actual work are under the hood;
do not cover

the ventilation gaps at the front
and backside of the bench.

Instruments used in the hoods must either be autoclaved after use (point 7) or their surfaces must be
disinfected before taking them ou
t of the bench. Having finished work, switch on the ultra
-
violet light in the
hood.

For
centrifuging
, use exclusively closed centrifuge
-
buckets or rotors with hermetically sealing covers (for
example centrifuge bottles with tightly closing screw caps, swin
gout rotors with hermetically sealed
Makrolon
-
caps, or fixed angle rotors with metal screw caps and gaskets).


6.4 Extractions from human tissues or body liquids,

may be, until
after

the first
denaturalizing treatment

(e.g. phenol), only performed in a B
io
-
Gard
-
Hood
of safety level II.


Packaging of biological substances ADR class 6.2

Risk classification

Packaging instructions
according to
UN
-
number

Container, UN
-
Packaging,
Package

ADR
-
identification

“danger signs”

Risk group 1
: non
-
dangerous transpor
t





B

(undefined risk)

Risk group 3

Risk group 2

(includes GVOs)

UN 3373
: Diagnostic
samples, clinical
samples, etc.

P650

Triple packaging

:

1. Sample tube with
absorbable material

2. Protective receptacle

3. External packaging

External packaging

:
‘B
iological Substance,
Category B







A

(defined risk)

Risk group 4

Risk group 3

(only cultures)

“shipping declaration”

Only over DKFZ
-

Dangerous
GoodsTransport Agent

UN 2814:

Infectious
agents for human and/ or

UN2900:

Infectious
agents for animals.

P62
0
I
1: Certified triple
packaging with water
tight primary container.

P620

I2: ditto

External packaging:
‘Biohazard’

印散p慬⁲敧畬慴i潮猠潦
t桥⁇敲h慮⁐潳a潦fi捥Ⱐ
䅇A

No transportation

of
risk group A samples

Transport of risk group
B upon consultation
wi
th DKFZ
-
Dangerous
Good Transport Agent


Secondary dangers with

Additional labeling

... air transport with dry
ice (solid carbon
dioxide), label class 9
(miscellaneous)

... liquid nitrogen, label
class 2 “gas
-
nonflammable” (green)


6.5 Transport betwee
n laboratories and shipment

Transporte of infectious materials and GVO’s between L2/S2 laboratories occurs only in closed,
unbreakable and hermetically sealed vessels that must not be externally contaminated. For transport of items
for washing and autoclav
ing see point 7. The shipment of samples classified as L2/S2 must be declared as
“potentially infectious substances” of the class 6.2 ADR (European Convention for the International
Transportation of Dangerous Goods on the Highways). Air freight and shipmen
t overseas is regulated by the
International Air Transport Association (IATA). Specific questions should be addressed to the DKFZ’s
Agent Responsible for the Transport of Dangerous Goods, Mr. Buchholz, tel 2746 (pager 551 185).
Packaging for all forms of
transportation are available from Mr. Buchholz. Further information concerning
packaging can be found under
http://www.der
-
gefahrgut
-
beauftragte.de
/gefahrgut
-
verpackung/gefahrgut
-
verpackung_0306.shtml

or
http://www.umwelt
-
online.de/regelwerk/gefahr.gut/adr/adr_rid01/4141g.htm

.




UN 3
373


UN 2814




oder


UN 2900

L2/S2 Operating Instructions, Committee of Biosafety Officers, DKFZ Heidelberg

date: 02/12/2007


5


7. Waste disposal and disinfec
tion of used instruments

Waste that might be contaminated by infectious materials or GVO's, is disposed of only after sterilization
(20 minutes autoclaving at 121°C) in the scullary autoclave (room ______). Whenever possible, potentially
contaminated instr
uments are given away for sterilization after use, otherwise they are disinfected. DNA
-
containg waste is also to be autoclaved. The operating instructions valid for the respective type of autoclave
are to be observed. Autoclaves are only to be operated by
instructed persons.

Closed autoclave drums made of high grade steel are used for interim storage and transport to the scullery
for autoclaving. If an autoclave drum might have been contaminated outside while being loaded, it must be
immediately cleaned wit
h the appropriate desinfectant (see point 10.7).

The autoclaved substances must have been exposed to a temperature of
121
o

C

for
20 minutes
. The
conditions used in autoclaving various types of solid/ porous substances/ waste must be validated for each
ty
pe of autoclave using bio
-
indicators.

Thermal waste sterilization procedures are tested periodically by the
Security Staff Unit (
mailto:Sicherheit@dkfz
-
heidelberg.de
) and the Engineering Department
(
mailto:service
-
technik@dkfz
-
heidelberg.de
) with
bio
-
indicators

(spore packages for solid/ porous
substances, ampulles for liquid substances). At least 10 bio
-
indicators are added to an autoclave run w
ith
waste sacks that correspond to an average run. The 10 bio
-
indicators are positioned in critical areas of these
sacks. If the amount of waste is too tightly packed, spores in the packages will survive the autoclaving since
the hot steam cannot penetra
te into the packing. Autoclaves that use the fractionated vacuum
-
steam
-
procedure (FVDV) or continual temperature
-
pressure
-
recording will be also analyzed at a larger interval.
For further detail see also the
“Working Directives for the Scullary”

section “
Autoclaving of laboratory
waste and washing of article from L2/S2 laboratories (in preparation)”.

After sterilization waste is supposed to be biologically inoffensive and is disposed of according to the
DKFZ
-
directives for the treatment of waste
.


7.1 Wast
e

Contaminated, solid waste

(disposable containers, plastic pipettes, single use gloves etc) are collected in
autoclavable, open, plastic bags (only 2/3 full), place in stainless steel autoclave boxes (ca. 30
-
60x30x30
cm, labeled “Plastik”) and closed for

transport and autoclaving. These autoclave boxes possess a lid valve.
Disposable liquid containing containers must be previously emptied into the liquid waste. Only rest amounts
of liquid are allowed in the solid waste. The lids for the disposable contain
ers should remain open.

Needles

are not to be bent by hand or removed from the syringe. Protective caps must not be put back.
Sharp or pointed single
-
use articles made of metal (i.e. scalpels, lanzets canulae) or glass

are collected
in special plastic cont
ainers, "SHARPS
-
containers", which are secure against perforation. Do not overfill the
container The full container must be autoclaved before disposal as solid waste.

Liquid waste

( e.g. cell culture media) is collected in unclosed plastic/ glas bottles
(maximum amount
1000 ml). These bottles are placed, due to weight considerations, in small stainless steel autoclave boxes
(e.g. 30x30x30 cm, labeled “Flüssigabfall”). No fluids should be able to escape during transport. Bottles
with liquids should remain
open during autoclaving (danger of explosion). The temperature sensor should be
placed in a reference bottle containing the same amount of liquid as the liquid waste (1000 ml).


7.2 Glass pipettes

Glasspipets

are completely submersed in
Sekusept aktiv

for

at least 12 hours in pipet racks possessing a
lifting system (see point 10.7). The handle of the lifting system must be disinfected with disinfection
solution after each possible contamination. The RKI recommends that
Sekusept aktiv

disinfection solution

be changed
daily

in the pipette stands of S2 laboratories to ensure its “complete virusidal effectivity” (use
hand and eye protection!) (see point 10.7). In areas with “limited virusidal dininfection” can the disinfection
solutions change schedule be dep
endent on use. Disinfected glass pipets are carried from the laboratories to
the scullery and cleaned there. When taking the pipets out of the racks, wear thick gloves.


7.3 Reusable, instruments, L2/S2 contaminated washable items

Contaminated auclavable
items containers (e.g. laboratory glassware)
and

laboratory equipment
should be autoclaved together with the L2/S2 solid waste before being sent to the scullary for cleaning.

Non
-
autoclavable instruments

are soaked overnight in
Sekusept
-
aktiv

solution. Eq
uipment that cannot be
submersed in liquid solutions should be intensively washed with a disinfection solution (see point 10.7).


L2/S2 Operating Instructions, Co
mmittee of Biosavety Officers, DKFZ Heidelberg

date: 02/12/2007


6


Large apparatuses can, if necessary, be disinfected by exposing them to formaldehyde vapors under a tent.
This disinfection
procedure may only be performed by the safety engineer. Before instruments are repaired,
they have to be sufficiently disinfected.

Working surfaces
are cleaned with a disinfection solution (see point 10.7) upon finishing the procedure.


7.4 Special waste

Phenol, formaldehyde or other organic solvents must not be autoclaved

in order to avoid danger for the
employee's health in the scullery through their vapors. If these chemicals themselves do not have a
sufficiently disinfecting effect themselves (note the
ir concentration), further disinfection solution (see point
10.7) should be added up to the required concentration.

For
waste containing heat
-
resistant cancerogens
or
radioactively contaminated GVOs
, individual
measures for disinfection must be decided bef
ore starting to work and documented in written form as
described under point 11.

Hazardous waste is to be disposed of after disinfection according to the DKFZ
-
directives for the treatment
of hazardous waste. Further information can be obtained from the saf
ety engineer.



8. Accidents


Keep cool and act prudently!

Whenever someone is injured or contaminated, colleagues are called for help: At the latest, after the first
measures have been taken, a responsible person (see point 2) must be informed immediate
ly, who will then
inform the DKFZ doctor, the safety engineer or rescue personnel and the other responsible persons about the
accident and the biological agents involved. The location of the first aid kit is in building/ room
______________. A doctor sho
uld be consulted after every injury, who will decide whether a serum probe
should be taken. All injuries should be reported to the safety engineer. Objects and biological agents
involved in the accident should be put away for evaluation.


8.1 Emergenci
es


In case of
emergency
, the
safety department

should
immediately

be informed (phone
2888
), which in turn
alarms all the other rescue people.

With the
announcement of an accident
, you must indicate:

-

where

the emergency is taking place,

-

what

has happen
ed,

-

what kind of help

is needed,

-

who

you are.

In case of persisting threat, e.g. fire, the scene of accident must be abandoned according to the official
evacuation plan which is found on each floor of every building. All doors must be shut.


8.2.1 Deco
ntamination of people (after contamination with biological agents)

Contaminated persons

should initially remain at the
scene of accident

to
avoid the spreading of the
contamination
.

Contaminated skin

is to be thoroughly disinfected with Spitacid or Desder
man.

Contaminated eyes

are to be immediately rinsed out under flowing water while opened (10 minutes; the
best thing is to use the eye showers which are fixed to the water
-
taps); Thereafter a specialist must be
immediately consulted.

Contaminated mucous m
embranes

are rinsed thoroughly with water, afterwards a specialist must be
immediately consulted.

In case of
extensive personal contamination
, the contaminated clothes are first to be removed and then the
skin thoroughly disinfected with Spitacid or Desder
man.

In case of
slight skin
-
wounds
(puncture wounds and cuts), let the wound bleed for 1


2 Minutes, then
apply a wound disinfectant, e.g. Neokodan.

In case of
serious wounds
, conventional first aid measures have precedence over disinfection.


L2/S2 Operating Instructions, Committee of Biosafety Officers, DKFZ Heidelberg

date: 02/12/2007


7


Contamina
ted clothes

are kept in an autoclave sack until decontaminated, provisional substitute clothing is
available in the radiation safety department and from the safety engineer.


8.2.2 Decontamination of people (after contamination with toxic agents)

Contamina
ted skin is to be thoroughly cleaned with soap and water or PEG 400
(available from the
safety engineer); do not use organic solvents, since this increases absorption through the skin!


8.3 Decontamination of laboratories

Special caution is necessary when
removing large contaminations (infectious, toxic, carcinogens, mutagens).

To remove extensive contaminations,
special safety clothing
(available in the DKFZ stockroom) to be used
could be:

Tyvek
-
overalls (at tleast type 5, partical density or type 4, liqui
d den
sity according to category III,


DIN EN 468/ EN 469, chemical protection clothing)

Disposable gloves with rubber collar

safety goggles

partical filter P3
-
hals mask made of fleece (FFP3, DIN EN 149:2001)

safety gloves against chemicals and microorganis
ms (DIN EN 374:2003).

Small amounts of the above named articles can be found in the security cabinate building/ room number
____________(see map in the Attachements).

A
contaminated room

is
barred

for people who are not involved in the decontamination and
must not be
entered unless permission was

given

by a responsible person.

Spilled liquids are to be covered with paper hand towels or absorbent cellulose cloth, the covering
subsequently being soaked with disinfection solution (see point 10.7) (never spray
the solution); afterwards,
the liquid is removed by wiping to the centre, and finally the floor is covered again with disinfectant and left
for the appropriate incubation period.

On contaminated surfaces, disinfection solution is applied, coarse dirt is wi
ped off, the surface is then again
disinfected for the appropriate incubation period.



9. Hygiene plan and operating directive


The essential issues of these
operating instructions
are shortly resumed in the L2/Se
-
hygiene plan

(
http://info/sicherheit/divinfo.htm

)and the S2
-
operating directive (
http://info/sicherheit/ba.htm

)
. These
two sheets are hung out in each lab.



10. Additional regulations and liter
ature:


Regulations and literature mentioned in these operating instructions can be read on the following websites:

http://www.umwelt
-
online.de/regelwerk/index.htm


http://frontend.weka
-
intrnet.de/intranet/index.php

(WEKA
-
database, temporary access for 2007)

http://www.gesetze
-
im
-
internet.de/



10.1 Further inernal DKFZ regu
lations (excerpt)

“Regulations for work in the scullary”, chapter “Autoclaving of laboratory waste and the cleaning of
labware from L2/S2
-
laboratories” (
http://infos/biosich
...)
(in preparation)

Fire prevention regulat
ions (
http://info/sicherheit/divinfo.htm

)

waste disposal regulations (
http://info/sicherheit/abfall.htm

)

radiation safety instructions

Users instructi
ons for the central animal facilities

Nitrosamine guidline

material
-

and activity relevant regulations of the DKFZ

Rules for the use of amphotropic retroviruses in the DKFZ



L2/S2 Operating Instructions, Co
mmittee of Biosavety Officers, DKFZ Heidelberg

date: 02/12/2007


8


10.2 Valid legal regulations


Regulations on Containment Levels and Safety Measu
res for Genetic Operations in Genetic Engineering
Installations;

Federal Law for Infectious Diseases; Genetic Engineering Act; Law for Animals' Infectious
Diseases; Federal Regulations for Emissions; Water Conservation Law; Waste Disposal Law;

Law for the
Protection of Expectant and Nursing Mothers; Law for the Protection of Adolescents' at Work;
Law for the Protection of Animals;

Regulations on the Keeping of Records for Genetic Operations; Hygiene Regulations; list of the
disinfectants and disinfection p
rocedures examined and acknowledged by the Federal Health Office;

Regulations for Dangerous Materials; Radiation Safety Regulations; Working Place Regulations; Pressure
Container Regulations.


10.3 Regulations for accident prevention


“Basic rules for prev
ention” (GUV
-
V A1); First Aid (GUV
-
VA5).


10.4 Regulations and instruction sheets

The DIN
-
text and technical Norms can be seen at the Staff Company Safety Unit.

Government regulations and the regulations for the state health services of Baden
-
Württemberg
for
scientific and medical
-
diagnostic laboratories ( IfSG, BioStoffV);

List of disinfection solutions tested and approved by the state health services and their procedures in the
newest version (14
th

edition from 31.05.2002).

Recommendations “Evaluation an
d declaration of the effectivity of disinfection solutions against viruses”,
RKI, State health bullitan 1.2004 (see point 10.7).

List of effective disinfection solutions, regulations of the German Society for Hygiene and Microbiology
(DGHM
-
list, from 21.1
2.2003).

TRGS 525: Technical rules for dangerous substances; Use of dangerous substances in human medical
facilities.

TRG526: Technical rules for dangerous substances; Laboratories (“Laboratory regulations”).

TRBA 100: Protection measures for directived a
ctivity using biological substances in laboratories, with
further standard references.

TRBA120: Care of experimental animals.

TRBA 250: Biological substances used in health care and in care of the needy.

TRBA 500: General Hygienic measures.

DIN
-
paperback 2
22: Medical microbiology.

DIN
-
professional report 7: Reducing the risk of infection in the medical microbiology.

DIN EN 12128 Research development and analysis laboratories
-
Microbiological security levels, with
further standard references.

DIN EN 12469, Pa
rt 1
-
4: Laboratory furnishings: Security benches for microbiological and biotechnological
use.

DN EN 12740, Research, development and analysis laboratories
-
Guideline for the handling, inactivation and
control of waste.

DIN EN 12738
-
Guidline for the enclos
ure of inoculated animals kept in the framework of experiments with
microorganisms.

Bulletin “Safe Biogechnology” from BG Chemie, part 2:

Laboratories (B002); Classification of viruses (B004), of parasites (B005), of bacteria (B006) of fungi
(B007), of r
-
D
NA (recombinant DNA) (B008), of cell cultures (B009.



L2/S2 Operating Instructions, Committee of Biosafety Officers, DKFZ Heidelberg

date: 02/12/2007


9


10.5 Recommendation by the Advisory Committee for Biological Safety (ZKBS)


http://www.bvl.bund.de/nn_491
798/DE/06_Gentechnik/093_ZKBS_node.html_nnn=true


Classification of gene technical work using primary cells from vertebrates (12/97)

Precautions during use of nucleic acids with oncogenic potential

Measures and surface disinfection after gene technical

work with membrane
-
encapsidated
viruses(10/100)

Handling of recombinant vaccinia virus (05/97)

Risk evaluation of the recombinant vaccinia virus MVA (06/02)

Risk evaluation of human adeno
-
associated virus (10/01)

Adenoviral and AAV
-
derived replication de
fective vectors with cell cycle regulating genes (12/04)

Further regulations can be seen in the secretariat of the Commission for Biological Security, Mrs. Ingrid
Reinke, D132a, Tel. 3667
mailto:i.reinke@
dkfz
-
heidelberg.de



10.6 Literature


K.H. Wallhäußer: "Praxis der Sterilisation, Desinfektion, Konservierung", 5. Aufl. 1995, Georg Thieme
Verlag,

HHS Publications No. (NIH)88
-
8395: "Biosafety in Microbiological and Biomedical Laboratories".



11. Speci
al regulations related to individual projects or rooms


This separate page should contain a detailed list of the projects specific danger potential. The regulatory
authority requires submission of point 11 as well as the entire organization plan upon regis
tration of the
facility (point required for approval). After confirmation by the regulatory authority, this single page is
added to the respective DKFZ Operating Instructions and Hygeine Plan, and is placed in every room
registered by the project head. (Er
ase this explanation upon use).


Danger for humans and environment


Gene technically modified organisms are used in the gene technical facility. According to the stand of
scientific knowledge, there appears to be only a minor risk to humans and evironment
. However, all virus
preparations, tissue samples, blood, or other research material from humans, animals and cell cultures are to
be considered as being infectioujs. They can,

-
by human body exposure, cause infections and, in part, serious illnesses.

-
by

inhalation of aerosols, by consumption of pathogen containing materials, through penetration of
pathogens into injured skin, or by splattering of samples into the eyes or mucous membranes, enter into the
body.

-
be present as aerosols by several typical ty
pes of laboratory procedures.

Description of specific danger potentials:


e.g
Lentiviral vectors

Lentiviral vector particles are not replication
-
competent. However, since they can infect human cells and
transfer human genetic information (genes without
a danger potential), they are classified into the risk
group 2. To date, a harmful effect on humans is not known.


e.g.
Epstein
-
Barr
-
Virus (EBV)

Experiments are performed using EBV
-
immortalized human cells. It cannot be excluded that these cells
secrete i
nfectious EBV.

In naïve persons, EBV can cause glandular fever (mononucleosis). The infection rate in persons of 20
-
30
years old is high. The mode of transmission is by droplet infection. There is no prophylactic vaccine
available.


eg.
retroviral vectors


L2/S2 Operating Instructions, Co
mmittee of Biosavety Officers, DKFZ Heidelberg

date: 02/12/2007


10


Retroviral vector particles are not replication competent. However, since they can infect human cells and
transfer human genetic information (genes without a danger potential), they are classified into the risk group
2. To date, a harmful effect on huma
ns is not known.








L2/S2 Operating Instructions, Committee of Biosafety Officers, DKFZ Heidelberg

date: 02/12/2007


11


12. Declaration


I have received and read the safety regulations valid in the department
"
..........................................................
" at the

DKFZ and recognize them as obligatory. I have been instructed
orally and

on a workplace
-
specific basis.


Date: Heidelberg, ........................................



Name in block letters:.......................................




Signature:.......................................