Mammalian cell lines:C2C12-mouse muscle
Fugene-6 (transfection reagent)
Lipofectamine (transfection reagent)
Hazardous Substances Policy - Assessment
G418
CHEMICAL HAZARD AND RISK ASSESSMENT
School of Biosciences
Name of
supervisor
L.M. Machesky
Assessor
I.M. Olazabal
Assessment Number*
2030
Date of Assessment
24/02/02
Signature
Signature
Notes
A School COSHH form in Word is available on the School Server.
Available from the Health and Safety Unit.
Guidance on making an assessment is given in Making a Chemical Hazard and Risk Assessment.
Guidance is also available from Guidance on Completing the Chemical Hazard and Risk Assessment Form.
Use a continuation sheet to expand any section of this form in hard copy version.
1
LOCATION OF THE WORK ACTIVITY
2
PERSONS WHO MAY BE AT RISK
List names where possible
Laura Machesky, Tom Millard, Juana Magdalena, Sophie Launay, Kam Sethi, Jacqui
Woodings, Celine Costa, Margarita Chibalina, David Knecht, Craig Stovold, Stewart
Sharp, Frank Zimmerman, Jason King, Soha Zouwail, Simone Blagg
3
ACTIVITY ASSESSED
4
MATERIALS INVOLVED
NAME
Stable Transfection of Cells
AMOUNT**
Mammalian cell lines:
C2C12-mouse muscle
Fugene-6 (transfection
reagent)
Lipofectamine
(transfection reagent)
G418
Blastocidin
Puromycin
Dihydrochloride
5
8th Floor labs
Highest
dose: 18 l
Highest
dose: 90 l
1mg/ml
2mg/ml
Highest
dose:
0.1mg/ml
Attach copies of data sheet(s)
HAZARD
RISK PHRASES
Biological
hazard
(Signalling to
Cell Motility)
Harmful
R21/22
Harmful
R21/22
Harmful
Very toxic
Harmful
R20/21/22
R26/27/28
R22
HAZDAT NO***
BIOSCIENCESNO***
R21/22
INTENDED USE
Give brief details and attach protocol/instructions
Fugene-6: is used 18 l per sample to be transfected. The plates will be incubated at 37oC during 24 to 48 hours.
Lipofectamine: 90l per sample to be transfected. The plates will be incubated at 37oC during 24 to 48 hours.
G418: Is used at a concentration varying from 1.2 mg/ml to 0.5 mg/ml on the plates. The plates will be incubated
during two or three weeks at a 37 incubator used exclusively for cell culture.
Blastocidin: Is used at a concentration of 10 g/ml. The plates will be incubated during one or two weeks in a
20oC or 37oC incubator used for Dyctyostelium culture or for mammalian cell culture.
Puromycin Dihydrochloride: Used at a concentration of 0.1 mg/ml. Plates will be incubated during one or two
weeks and reincubated from time to time for a week. Cells will be kept in a 20 oC or 37oC incubator used for
Dyctyostelium culture or for mammalian cell culture.
6
RISKS to HEALTH and SAFETY from INTENDED USE
From personal exposure or hazardous reactions. Refer to OELs, flash points, etc., as appropriate. Are pregnant women, breast-feeding
mothers especially at risk?
G418 and Blastocidin: there is risk of inhalation when weighing powder and of skin contamination if in contact.
Puromycin Dihydrochloride: harmful if ingested
7
CONCLUSIONS ABOUT RISKS
Is level of risk acceptable? Can risk be prevented or reduced by change of substance/procedure? Are control measures necessary?
The level of risk is not acceptable and the following control measures are necessary.
8
CONTROL MEASURES
Additional to Good Chemical Practice
G418, Lipofectamine, Fugene-6: Wear gloves, eye/face protection and suitable clothing. Avoid contact with skin
and eyes. Do not swallow, do not breath dust, use only in fume hood.
Puromycin Dihydrochloride: Wash hands, forearms and face thoroughly after handling the compound and before
eating, smoking, using lavatory, and at the end of the day.
Blastocidin: Store in a closed container in a +4 oC refrigerator. Material is toxic, avoid contact. Avoid prolonged or
repeated exposure. Lab should be equipped with a safety shower and an eye wash station. Wash thoroughly after
handling material.
9
INSTRUCTION/TRAINING
Specify course(s) and/or special arrangements.
Personnel involved in the use of this method must be adequately trained in handling cell culture materials, as well
as toxic reagents.
10
MONITORING
Performance of control measures,
Personal exposure
Health Surveillance
11
WASTE DISPOSAL PROCEDURE
See School Server for Approved Procedure Document on specific Chemical Waste Disposal.
Cells in general and stable transfectants in particular shall be transferred to autoclavable bags and autoclaved for
121oC for 50 minutes. Cells and medium containing, Fugene-6, Lipofectamine, G418 or Blastocidin are killed by
this procedure. Place the waste into a yellow bag and seal for disposal.
Liquid residues are aspirated into a flask containing bleach.
12
REVIEW
Enter the date or circumstances for review of assessment (maximum review interval 5 years)
March 2007 or when more is known about the toxicological properties of Blastocidin.
13
EMERGENCY ACTION
TO CONTROL HAZARDS To stabilize situation eg spread absorbant on liquid spill; eliminate sources of ignition, etc.
Spills and decontamination:
G418: removed spilled material with water. Place into appropriate container for disposal. Avoid dust. Ventilate and
wash spill area. Wear suitable clothing. Use gloves and eye/face protection.
Lipofectamine, Fugene-6: Use an inert absorbent material (vermiculite, dry sand) to contain/pick up the spilled
solution. Place all contaminated material into a suitable container, seal and label for disposal.
Blastocidin: Evacuate area. Wear self-contained breathing apparatus, rubber boots and heavy rubber gloves.
Sweep up and place in a suitable container. Avoid raising dust. Hold for disposal using the special waste
procedure (see Brian Facer in stores). Wash spill site and ventilate area after material pickup is complete.
Puromycin Dihydrochloride: Splash goggles. Full suit. Boots, gloves, protective clothing might not be sufficient,
consult a specialist before handling this product. Use a shovel to put the material into a convenient waste disposal
container. Finish cleaning by spreading water on the contaminated surface and allow to evacuate through the
sanitary system.
TO PROTECT PERSONNEL Evacuation, protection for personnel involved in clean-up, Special First Aid
First aid:
G418, Lipofectamine, Fugene-6, Puromycin Dihydrochloride: Fresh air. If you feel unwell seek medical advice
Skin contact::
G418, Lipofectamine, Fugene-6, Blastocidin, Puromycin Dihydrochloride: Take off contaminated clothes. Wash
with soap and copious amounts of water, cold water in the case of Puromycin Dihydrochloride. Consult a
physician.
Eye contact:
G418, Lipofectamine, Fugene-6, Blastocidin: Rinse eyes with copious amounts of water for at least 15 min, cold
water in the case of Puromycin Dihydrochloride. Assure adequate flushing by separating the eyelids with fingers.
Get medical attention immediately.
Ingestion, inhalation:
G418: If swallowed wash out with water, provided person is conscious. Consult a doctor.
Lipofectamine, Fugene-6: Never give anything by mouth to an unconscious person. Get medical attention
immediately.
Blastocidin, Puromycin Dihydrochloride: Remove to fresh air and monitor breathing. If breathing becomes difficult,
give oxygen and consult a physician. If breathing stops, give artificial respiration and consult a physician. If
ingested, wash mouth out with water and consult the local poison centre and a physician.
Puromycin Dihydrochloride: If ingested do not induce vomiting unless directed to do so by medical advice. Never
give anything by mouth to an unconscious person. Get medical attention immediately. Loosen tight clothing such
as collar, tie, belt or waistband.
Clean-up/decontamination
TO RENDER SITE OF EMERGENCY SAFE
Spills and decontamination:
G418: removed spilled material with water. Place into appropriate container for disposal. Avoid dust. Ventilate and
wash spill area. Wear suitable clothing. Use gloves and eye/face protection.
Lipofectamine, Fugene-6: Use an inert absorbent material (vermiculite, dry sand) to contain/pick up the spilled
solution. Place all contaminated material into a suitable container, seal and label for disposal.
Blastocidin: Evacuate area. Wear self-contained breathing apparatus, rubber boots and heavy rubber gloves.
Sweep up and place in a suitable container. Avoid raising dust. Hold for special waste (see Brian Facer in Stores)
disposal. Wash spill site and ventilate area after material pickup is complete.
Puromycin Dihydrochloride: Splash goggles. Full suit. Boots, gloves, protective clothing might not be sufficient,
consult a specialist before handling this product. Use a shovel to put the material into a convenient waste disposal
container. Finish cleaning by spreading water on the contaminated surface and allow to evacuate through the
sanitary system.
Fire and explosion hazard:
G418: Could cause fire in high temperatures. When heated to decomposition, materials emit toxic fumes (NO 4
and SO4).
Lipofectamine, Fugene-6: use fire-extinguishing media appropriate for site conditions.
Blastocidin: Upon thermal decomposition, it may emit toxic gases.
Puromycin Dihydrochloride: These products are carbon oxides, nitrogen oxides…they emit toxic gases.
For G418, Blastocidin and Puromycin Dihydrochloride , firefighters should wear self-contained breathing
apparatus and full turnout gear.
Emergency action in case of fire:
G418: Fight fire with water, CO2 or powder.
Blastocidin: Extinguishing media: dry chemical powder, CO 2, water, alcohol or polymer foam.
Puromycin Dihydrochloride: Use dry chemical powder, or water spray, fog or foam. Not water jet.
CONTACT
L.M. Machesky
PHONE
42504
10.10.00
*
**
***
Prefix T is used for Teaching Assessment Number.
List the amount by weight of the substance used. (for liquids eg; 100 mls 1M Sodium Hydroxide =
4g).
Hazdat No is the UNICOSHH datasheet report number.
Biosciences No is the Biosciences data sheet number.
UNICOSHH IS A CHEMICAL DATABASE ON THE HEALTH AND SAFETY UNIT SERVER.
BIOSCIENCES DATA SHEETS ARE AVAILABLE IN THE SCHOOL SAFETY OFFICE.