Download Standard Operating Procedure

Standard Operating Procedure
Colchicine
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Laboratory Safety Manual and Chemical Hygiene Plan.
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☐ Process
☒Hazardous Chemical
☐ Hazardous Class
Purpose
Colchicine has historically been used as a treatment for conditions such as gout. However, despite its
therapeutic properties it is also known as a highly toxic compound causing symptoms similar to arsenic
poisoning. Colchicine inhibits microtubule polymerization by binding to tubulin. One of the primary side
effects is that this prevents the formation of mitotic spindles and thus inhibits effectively blocks mitosis. It
should be treated carefully since it is mutagenic, tumorigenic, and teratogenic. Colchicine is used in
chromosome analysis during karyotyping and in cell synchronization.
Physical & Chemical Properties/Definition of Chemical Group
CAS#: 64-86-8
Class: Highly toxic by inhalation, Highly toxic by ingestion, Irritant, Teratogen
Molecular Formula: C22H25NO6
Form (physical state): powder
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Color: Click here to enter text.
Boiling point: No data
Potential Hazards/Toxicity
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Swallowed: Severely toxic effects may result from the accidental ingestion of the material; animal
experiments indicate that ingestion of less than 5 gram may be fatal or may produce serious
damage to the health of the individual.
Eye: There is evidence that material may produce eye irritation in some persons and produce eye
damage 24 hours or more after exposure. Severe inflammation may be expected with pain.
Skin: The material is not thought to be a skin irritant (as classified using animal models), abrasive
damage however, may result from prolonged exposures. Skin contact with the material may
damage the health of the individual; systemic effects may result following absorption. Open cuts,
abraded or irritated skin should not be exposed to this material. Entry into the blood-stream,
through, cuts, abrasions or lesions, may produce systemic injury with harmful effects. Examine
the skin prior to the use of the material and ensure that any external damage is suitably
protected.
Inhaled: Inhalation of dusts, generated by the material, during the course of normal handling, may
produce severely toxic effects; these may be fatal. The material is not thought to produce
respiratory irritation (as classified using animal models). Nevertheless inhalation of dusts, or
fume, especially for prolonged periods, may produce respiratory discomfort and occasionally,
distress. Persons with impaired respiratory function, airway diseases and conditions such as
emphysema or chronic bronchitis, may incur further disability if excessive concentrations of
particulate are inhaled
Chronic health effects: Ample evidence exists, from results in experimentation,that developmental
disorders are directly caused by human exposure to the material. Long term exposure to high
dust concentrations may cause changes in lung function i.e. pneumoconiosis; caused by particles
less than 0.5 micron penetrating and remaining in the lung.
Colchicine is readily absorbed from the gastro-intestinal tract. Steatorrhoea (fatty stools) and a
histiocytic lymphoma in the jejunum (part of small intestine) developed in a 53 year old man after
taking 0.5 mg daily for 3 years, as a treatment for gout. Fatal agranulocytosis and
thrombocytopenia, with bone marrow hypoplasia, occurred in a patient who had taken colchicine
prophylactically in the recommended doses. Obstructive jaundice could have contributed to the
toxicity of the drug. [Martindale] May cause congenital malformation in the fetus.
In histological studies colchine has been shown to bind specifically with tubulin thus interfering
with microtubule organization and as a result might be an effector in cell division.
Personal Protective Equipment (PPE)
Respirator Protection
Respiratory protection is only required if expected exposures to particulates are high. (AS/NZS 1716 &
1715, EN 143:2000 & 149:2001, ANSI Z88 or national equivalent)
Respirators should be used only under any of the following circumstances:
 As a last line of defense (i.e., after engineering and administrative controls have been
exhausted).
 When Permissible Exposure Limit (PEL) has exceeded or when there is a possibility that PEL will
be exceeded.
 Regulations require the use of a respirator.
 An employer requires the use of a respirator.
 There is potential for harmful exposure due to an atmospheric contaminant (in the absence of
PEL)
 As PPE in the event of a chemical spill clean-up process
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Lab personnel intending to use/wear a respirator mask must be trained and fit-tested by EH&S. This is a
regulatory requirement. (https://www.ehs.ucla.edu/ep/ih/resp)
Hand Protection
Suitability and durability of glove type is dependent on usage. Important factors in the selection of gloves
include: frequency and duration of contact, chemical resistance of glove material, glove thickness and
dexterity. When prolonged or frequently repeated contact may occur, a glove with a protection class of 5
or higher (breakthrough time greater than 240 minutes according to EN 374, AS/NZS 2161.10.1 or
national equivalent) is recommended. When only brief contact is expected, a glove with a protection class
of 3 or higher (breakthrough time greater than 60 minutes according to EN 374, AS/NZS 2161.10.1 or
national equivalent) is recommended. Gloves must only be worn on clean hands. After using gloves,
hands should be washed and dried thoroughly. Application of a non-perfumed moisturizer is
recommended.
NOTE: Consult with your preferred glove manufacturer to ensure that the gloves you plan on using are
compatible with Colchicine.
Refer to glove selection chart from the links below:
http://www.ansellpro.com/download/Ansell_8thEditionChemicalResistanceGuide.pdf
OR
http://www.allsafetyproducts.com/glove-selection-chart-chemical-breakthrough-ratings.html
OR
http://www.showabestglove.com/site/default.aspx
OR
http://www.mapaglove.com/
Eye Protection
Chemical protective goggles with full seal or a shielded mask (gas-type) are recommended. Contact
lenses may pose a special hazard; soft contact lenses may absorb and concentrate irritants. A written
policy document, describing the wearing of lens or restrictions on use, should be created for each
workplace or task. This should include a review of lens absorption and adsorption for the class of
chemicals in use and an account of injury experience. Medical and first-aid personnel should be trained in
their removal and suitable equipment should be readily available. In the event of chemical exposure,
begin eye irrigation immediately and remove contact lens as soon as practicable. Lens should be
removed at the first signs of eye redness or irritation - lens should be removed in a clean environment
only after workers have washed hands thoroughly. [CDC NIOSH Current Intelligence Bulletin 59],
[AS/NZS 1336 or national equivalent].
Skin and Body Protection
For quantities up to 500 grams a laboratory coat may be suitable. For quantities up to 1 kilogram a
disposable laboratory coat or coverall of low permeability is recommended. Coveralls should be buttoned
at collar and cuffs. For quantities over 1 kilogram and manufacturing operations, wear disposable coverall
of low permeability and disposable shoe covers. For manufacturing operations, air-supplied full body suits
may be required for the provision of advanced respiratory protection. Access to an eye wash unit, an
emergency shower and a vinyl suit for emergencies is recommended.
Hygiene Measures
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Engineering Controls
Powders
To prevent contamination and overexposure, no open handling of powder should be allowed.
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Powder handling operations are to be done in a powder weighing hood, a glove box, or other
equivalent ventilated containment system.
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In situations where these ventilated containment hoods have not been installed, a non-ventilated
enclosed containment hood should be used.
Pending changes resulting from additional air monitoring data, up to 300 mg can be handled
outside of an enclosure provided that no grinding, crushing or other dust-generating process
occurs.
An air-purifying respirator should be worn by all personnel in the immediate area in cases where
non-ventilated containment is used, where significant amounts of material (e.g., more than 2
grams) are used, or where the material may become airborne (as through grinding, etc.).
Powder should be put into solution or a closed or covered container after handling.
If using a ventilated enclosure that has not been validated, wear a half-mask respirator equipped
with HEPA cartridges until the enclosure is validated for use.
Solutions Handling
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Solutions can be handled outside a containment system or without local exhaust ventilation
during procedures with no potential for aerosolisation. If the procedures have a potential for
aerosolisation, an air-purifying respirator is to be worn by all personnel in the immediate area.
Solutions used for procedures where aerosolisation may occur (e.g., vortexing, pumping) are to
be handled within a containment system or with local exhaust ventilation.
In situations where this is not feasible (may include animal dosing), an air-purifying respirator is to
be worn by all personnel in the immediate area. If using a ventilated enclosure that has not been
validated, wear a half-mask respirator equipped with HEPA cartridges until the enclosure is
validated for use.
Ensure gloves are protective against solvents in use.
Unless written procedures, specific to the workplace are available, the following is intended as a guide:
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For Laboratory-scale handling of Substances assessed to be toxic by inhalation. Quantities of up
to 25 grams may be handled in Class II biological safety cabinets; Quantities of 25 grams to 1
kilogram may be handled in Class II biological safety cabinets or equivalent containment systems
Quantities exceeding 1 kg may be handled either using specific containment, a hood or Class II
biological safety cabinet.
HEPA terminated local exhaust ventilation should be considered at point of generation of dust,
fumes or vapors.
First Aid Procedures
If inhaled
If fumes or combustion products are inhaled remove from contaminated area. Lay patient down. Keep
warm and rested.
In case of skin contact
Immediately remove all contaminated clothing, including footwear. Flush skin and hair with running water
(and soap if available).
In case of eye contact
Immediately hold eyelids apart and flush the eye continuously with running water. Ensure complete
irrigation of the eye by keeping eyelids apart and away from eye and moving the eyelids by occasionally
lifting the upper and lower lids.
If swallowed
IF SWALLOWED, REFER FOR MEDICAL ATTENTION, WITHOUT DELAY. Where Medical attention is
not immediately available or where the patient is more than 15 minutes from a hospital or unless
instructed otherwise:
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NOTES TO PHYSICIAN
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Basic treatment: Establish a patent airway with suction where necessary. Watch for signs of
respiratory insufficiency and assist ventilation as necessary.
Acute poisoning: In acute poisoning the stomach should be emptied by aspiration and lavage. A
purgative, such as sodium sulfate 30 g in 250 ml water, should be given. Demulcent drinks may
be given freely. Respiration may require assistance. The circulation should be maintained and
fluid and electrolyte imbalance corrected. Morphine sulfate, 10 mg intramuscularly, may be given
to relieve severe abdominal cramps; atropine has also been given [Martindale].
Special Handling and Storage Requirements
Precautions for safe handling: Avoid all personal contact, including inhalation. Wear protective clothing
when risk of exposure occurs. Empty containers may contain residual dust which has the potential to
accumulate following settling. Such dusts may explode in the presence of an appropriate ignition source.
Do NOT cut, drill, grind or weld such containers. In addition, ensure such activity is not performed near
full, partially empty or empty containers without appropriate workplace safety authorization or permit.
Recommended storage methods:
 Glass container: Packaging as recommended by manufacturer. Check that containers are clearly
labeled, and tamper-proof.
 For low viscosity materials: Drums and jerricans must be of the non-removable head type. Where
a can is to be used as an inner package, the can must have a screwed enclosure.
 All inner and sole packaging for substances that have been assigned to Packaging Groups I or II
on the basis of inhalation toxicity criteria, must be hermetically sealed.
Storage requirements: Store in original containers. Keep containers securely sealed. Store in secure
poison area. Refrigerate.
Spill and Accident Procedure
Chemical Spill Dial 911 and x59797
Spill – Assess the extent of danger. Help contaminated or injured persons. Evacuate the spill area.
Avoid breathing vapors. If possible, confine the spill to a small area using a spill kit or absorbent material.
Keep others from entering contaminated area (e.g., use caution tape, barriers, etc.).
Small (<1 L) – If you have training, you may assist in the clean-up effort. Use appropriate personal
protective equipment and clean-up material for chemical spilled. Double bag spill waste in clear plastic
bags, label and take to the next chemical waste pick-up.
Large (>1 L) – Dial 911 (or 310-825-1491 from cell phone) and EH&S at x59797 for assistance.
Chemical Spill on Body or Clothes – Remove clothing and rinse body thoroughly in emergency shower
for at least 15 minutes. Seek medical attention. Notify supervisor and EH&S at x59797 immediately.
Chemical Splash Into Eyes – Immediately rinse eyeball and inner surface of eyelid with water from the
emergency eyewash station for 15 minutes by forcibly holding the eye open. Seek medical attention.
Notify supervisor and EH&S at x59797 immediately.
Medical Emergency Dial 911 or x52111
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Life Threatening Emergency, After Hours, Weekends And Holidays – Dial 911 (or 310-825-1491 from
cell phone) or contact the Ronald Reagan UCLA Medical Center (emergency room) directly at x52111
(located at 757 Westwood Plaza, enter from Gayley Avenue). Note: All serious injuries must be reported
to EH&S at x59797 within 8 hours.
Non-Life Threatening Emergency – Go to the Occupational Health Facility (OHF), x56771, CHS room
67-120 (This is on the 6th floor, 7th corridor, room 120. Enter through the School of Dentistry on Tiverton
Drive and proceed to the “O” elevator to the 6th floor.)Hours: M - F, 7:30 a.m. to 4:30 p.m. At all other
times report to Ronald Regan UCLA Medical Center (emergency room) at x52111. Note: All serious
injuries must be reported to EH&S at x59797 within 8 hours.
Needle stick/puncture exposure (as applicable to chemical handling procedure) – Wash the affected
area with antiseptic soap and warm water for 15 minutes. For mucous membrane exposure, flush the
affected area for 15 minutes using an eyewash station. Page the needle stick nurse by dialing 231 from a
campus phone, enter 93333 when prompted and then enter your extension. Hours: M – F, 8:00 a.m. to
4:00 p.m. At all other times report to Ronald Regan UCLA Medical Center (emergency room) at x52111.
Note: All needle stick/puncture exposures must be reported to EH&S at x59797 within 8 hours.
Decontamination/Waste Disposal Procedure
Label Waste
 Affix an on-line hazardous waste tag on all waste containers using the WASTe Online Tag
Program https://ehs.ucop.edu/waste as soon as the first drop of waste is added to the container
Store Waste
 Store hazardous waste in closed containers, in secondary containment and in a designated
location
 Double-bag dry waste using transparent bags
https://www.ehs.ucla.edu/hazwaste/management/containers
 Waste must be under the control of the person generating & disposing of it
Dispose of Waste
 Dispose of regularly generated chemical waste within 90 days
 Call EH&S at x61887 for questions
 Empty Containers
o Dispose as hazardous waste if it once held extremely hazardous waste (irrespective of
the container size) https://www.ehs.ucla.edu/hazwaste/types/extremely-hazardous
o Consult waste pick-up schedule
https://www.ehs.ucla.edu/hazwaste/management/pick-ups
Prepare for transport to pick-up location
 Check on-line waste tag
 Write date of pick-up on the waste tag
 Use secondary containment
Safety Data Sheet (SDS) Location
Online SDS can be accessed at http://msds.ehs.ucla.edu.
Protocol/Procedure
Quantities covered by this SOP:
0-5 g
0-100 ml at 0.5g colchicine/100ml water
Conditions covered by this SOP:
2°C - 38°C
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General
Colchicine is most commonly used in cells for chromosome analysis. Colchicine is available in both
powdered and dissolved forms. Because of its toxicity all powders and solutions should be prepared and
used in a fume hood. It is recommended that if you are considering using colchicine for chromosome
analysis that you look into potentially using other products such as colcemid which are considered less
toxic.
Chromosome Analysis using Colchicine
Preparation of 0.5 g colchicine/100 ml stock solution from a 5 g stock bottle.
1. In a fume hood weigh out 0.5 g colchicine and dissolve with 100 ml sterile media. Prepared
solution should be labeled appropriately and stored protected from light at 2°C-8°C.
For Monolayer cultures
1. Add 0.1 ml of colchicine (50 µg/ml) per ml of cell culture medium to the proliferating cell culture.
2. Incubate at 37°C for 30 - 60 min. (depending on how rapidly the cells are dividing).
3. Detach the cells from the flask by scraping with a rubber policeman. Do not use trypsin to
dissociate the cells because it can interfere with the chromosomal banding.
For Cell Suspensions
1. Add 0.2 ml colchicine (50 µg/ml) per 2 ml of cell suspension (final cell suspension should be
approximately 2.4 x 106 cells).
2. Incubate at 37°C for 30 - 60 min.
Cell Harvest
1. Transfer cell suspension to a conical centrifuge tube and centrifuge for 5 minutes in a clinical
bench-top centrifuge at 400 x g.
2. Remove supernatant and gently add 2-3 ml of warm (37°C) hypotonic KCI (0.56%). Gently
resuspend cells by pipetting. Incubate at 37°C for 15 - 20 minutes.
3. Centrifuge 5 min. at 400 x g
4. Remove supernatant without disturbing pellet. Gently add 3-4 mls fixative (methanol: glacial
acetic acid, 3:1) down side of tube. Then pipet gently but rapidly to prevent cell clumping.
5. Allow to sit for at least 30 min. Refrigerate if tubes are to be held more than 30 min. The
procedure can be interrupted at this point and resumed later.
6. After 30 min. centrifuge cells at 400 x g, pipet off fixative and resuspend in fresh fixative. Repeat
this step twice more. The last addition of fixative should be just enough to make a thin cell
suspension (solution in tube will look slightly opaque).
Chromosome Slide Preparation
1. Immerse pre-cleaned slides in fixative at least 15 min. prior to use. Wipe slides dry with a
Kimwipe or other lint-free tissue.
2. Drop small drops of cell suspension onto slide surface with a Pasteur pipet and allow it to spread.
If too much suspension is used it will bubble at the edges.
3. As soon as the drop begins to contract and Newton's rings are visible (rainbow colors around the
edge of the drop), blow on the slide surface to accelerate drying. Rapid drying is critical to
obtaining well spread metaphases. Slides may also be dried by using tubing connected to an air
supply or by spraying lightly with Dust-off Plus. The "bomb" method of dropping one drop of
fixative onto the cell suspension once it has started to dry may be used to increase the spreading
of the chromosomes. Use a very small drop of fixative and keep the slide in a flat position while
adding the fixative and until the slide is completely dry. Do not blow on the slide.
4. Repeat steps 2 and 3 until sufficient sample is on the slide. Monitor cell concentration, cell
spreading, and contrast by phase microscopy.
NOTE
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Any deviation from this SOP requires approval from PI.
Documentation of Training (signature of all users is required)
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Prior to conducting any work with Colchicine, designated personnel must provide training to his/her
laboratory personnel specific to the hazards involved in working with this substance, work area
decontamination, and emergency procedures.
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The Principal Investigator must provide his/her laboratory personnel with a copy of this SOP and a
copy of the SDS provided by the manufacturer.
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The Principal Investigator must ensure that his/her laboratory personnel have attended appropriate
laboratory safety training or refresher training within the last one year.
Principal Investigator or Lab Supervisor SOP Approval
Print name_________________________Signature___________________________
Approval Date:
I have read and understand the content of this SOP:
Name
Signature
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Colchicine
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