Page 1 of 4 NDMRB-RA-119 V1 November 2017 (OL)

NDM Research Building

NDMRB-RA-119

Risk Assessment: Use of Cholera toxin for cell culture

Scope Cholera toxin is the virulent factor from Vibrio cholerae that leads to severe diarrhoea followed by dehydration in humans. It is an adenylate cyclase activator and is used as a supplement in tissue culture medium to stimulate the growth of certain cell types in vitro, such as the breast epithelial cell line MCF10a. Biochemically, it is involved in the calcium transport pathway. When handling the powdered form and any solutions you must wear PPE – safety glasses, gloves and lab coat. It is provided as a lyophilised powder, which should ideally be ordered in vials with septum so that liquid can be injected into the vial, thus avoiding exposure to the powdered form. The toxin should be dissolved in water to a concentration of 1mg/ml before filtered and aliquoted for use in cell growth medium (working concentration ~0.1ug/ml). In liquid form (e.g. in cell growth medium), it should be deactivated by adding 10-12% sodium hypochlorite 1:1 (v/v) for one hour before being disposed to drain with copious amounts of water. Contaminated plastics should be put in a container and soaked in 10-12% sodium hypochlorite before being rinsed and disposed of in the black bin waste. It is stable for 1 or 3 years in liquid or powder form (respectively) and should be stored at 2-8°C in suitable secondary containers clearly labelled. It will lose biological activity upon freezing or prolonged exposure to pH <6 or >8. No staff member may work with cholera without notifying the lab manager and going through the safety documentation. Name of assessor: Olivia Lombardi & Andrea KK Date of

Assessment: Jan 2018 Review

Date: Every three years

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Risk Matrix: Risk Matrix Likelihood

High Medium Low Negligible Consequence Severe High High Medium Effectively Zero

Moderate High Medium Medium/low Effectively Zero Insignificant Medium/Low Low Low Effectively Zero Negligible Effectively Zero Effectively Zero Effectively Zero Effectively Zero

Risk Assessment: Hazard

(Cause and consequence) Affected Groups Existing controls Risk Further Action

Exposure to Cholera toxin (via skin absorption, ingestion) can cause acute toxicity - severe diarrhoea and dehydration – and may be fatal. Exposure to Cholera toxin and sodium azide via inhalation or contact with skin/eyes can cause respiratory, skin and eye irritation.

Staff/students/visitors Although protocols report that optimal MCF10a cell growth requires cholera toxin, cell viability will be tested with and without this medium supplement in order to check whether it is indeed essential.

Restrict use whenever possible to the liquid aliquots when available. When preparing liquid stocks from powder stock, inject liquid via septum, ensuring to only apply gentle pressure (minimising splash risk). Sterilise and use in medium within a tissue culture hood. Store liquid stocks in a separate, clearly labelled box within fridge.

User must wear gloves, safety specs and lab coat at all times.

See specific COSHH risk assessment.

Medium None

Splash risk due to sterilisation using a syringe and syringe filter

Staff/students/visitors This must be done in a biological hood and safety glasses, gloves and lab coat must be worn.

Never force any liquid through the syringe filter, only gentle pressure should be applied

Medium None

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Exposure to other tissue culture suite users

Staff/students/visitors Any hood that has been used in tissue culture that involves cholera must be deep cleaned after use to avoid exposing others.

All spills/splashes must be cleared away immediately

All plastic ware that has been used in tissue culture involving cholera must be treated with 1-%-12% sodium hypochlorite for 1 hour before disposal. Bins must be emptied upon completion of work and the biohazard bag taken immediately to the autoclave room

Medium None

Spills during storage – exposure to other users

Staff/students/visitors All solutions and powders must be stored in secondary leak proof containers and must be clearly labelled.

Medium None

Exposure to powdered form Staff/students/visitors Preferably this pathogen should be ordered in vials with septum so that liquid can be injected into the vial and thus avoiding exposure to the powdered form.

It must be handled in a biological safety cabinet at all times

Medium None

Signed By Author: Approved by (sign and print): Reviewed by: Review date:

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