Liquid nitrogen used in the area of medically assisted procreation

Nitrogen, or more specifically molecular nitrogen (N2), is a very common gas on Earth. It is naturally present in the atmosphere and is not toxic. Liquid nitrogen is manufactured from compressed, filtered air. The extracted gases, primarily oxygen and nitrogen, are then liquefied. Liquid nitrogen and nitrogen gas are colourless, odourless and tasteless. At normal atmospheric pressure, liquid nitrogen vaporises at -196°C. Liquid nitrogen is used in an increasingly wide array of applications. One of its uses in the medical field is preservation of human cells and tissues. This application is specifically found in the area of medically assisted procreation.

In the framework of preparation of a ministerial order on good practices in reproductive medicine laboratories, ANSES received a formal request from the French Biomedicine Agency to perform a risk assessment concerning the use of liquid nitrogen in the field of medically assisted procreation (MAP).

In response to this request, a working group was established and included experts from the Agency’s Expert Committees (CES) on “Assessment of the risks related to air environments” and “Assessment of the risks related to chemical substances”, the National Research and Safety Institute (INRS) and recognised experts in reproductive biology or cryogenics.

The expert appraisal focused on three main objectives:

• describe the current regulatory environment concerning the use of liquid nitrogen, excluding industrial manufacturing and distribution of liquid nitrogen;
• assess the risks related to the use of liquid nitrogen in the field of reproductive biology;
• propose recommendations on risk prevention.

Since the formal request received by the Agency concerned only MAP activities, the scope of the risk assessment was restricted to occupational activities in units for medically assisted procreation where liquid nitrogen is present, handling of containers within the facility and the MAP unit, and transport of small-volume containers on public roads.

The Agency’s work

A review of scientific literature was carried out in order to collect general data on liquid nitrogen including physico-chemical properties, stability and reactivity, and to identify and evaluate effects on health.

Occupational risk situations were identified by analysing data collected via:

• the survey of the Biomedicine Agency on conditions in MAP laboratories in France;
• hearings with companies: manufacturers/suppliers of cryogenic equipment, distributors of liquid nitrogen;
• an on-site survery of MAP operations involving the use of liquid nitrogen.

In order to evaluate the hypoxic risk, estimated residual ambient oxygen levels were determined after evaporation of liquid nitrogen in conditions characteristic of those in reproductive biology work areas. Correlations were then established between these estimated levels and the health effects commonly associated with decreased oxygen levels.

These studies, along with the overview of regulations and recommendations applicable to liquid nitrogen, resulted in proposed recommendations for reproductive biology laboratories to guide risk prevention actions concerning liquid nitrogen.

These assessment activities led to publication of a report and an opinion in April 2008.

Results

Nitrogen is one of the main components of air and is not intrinsically toxic. The main hazards related to liquid nitrogen are cold burns and asphyxia as a result of hypoxia. The risk of cryogenic burns is well known given the extremely low temperature of nitrogen in liquid state (-196°C), whereas asphyxia is not nearly as well recognised, even though it can be fatal. The risk of liquid nitrogen-induced hypoxia is related to the ability of liquid nitrogen to rapidly generate a large volume of nitrogen gas via evaporation, thereby reducing ambient oxygen levels by displacement and dilution of oxygen.

The hazards related to the use of liquid nitrogen are difficult to quantify given the wide variety of working conditions and configurations of premises.

To evaluate the hypoxic risk for exposed personnel, the experts estimated the maximum quantity of liquid nitrogen that may evaporate in conditions characteristic of those in reproductive medicine laboratories. This estimate was then used to calculate the maximum ambient oxygen reduction in a work area resulting from this evaporation. Correlations were then established between these residual oxygen levels, calculated using a range of ventilation conditions and work area volumes, and the health effects commonly associated with decreased oxygen levels:

• natural evaporation from the containers: overall results show a low impact of natural evaporation of storage container contents on oxygen levels in the work area, and therefore a low hypoxic risk  ;
• refilling containers: when liquid nitrogen containers are refilled to ensure that the biological products remain immerged, the hypoxic risk appears to be strongly influenced by work area ventilation and volume. This risk is generally averted when work area volumes are high (150 m3), or when the room is mechanically ventilated (20 volwork area.h-1), except in very small-volume spaces of 10 m3 ;
• cooling of a high-volume container initially free from liquid nitrogen: filling a cooling container, a rarely performed operation in the field of MAP, involves relatively high evaporation of liquid nitrogen at the start of filling as a result of the difference in temperature between the sides of the container and the liquid nitrogen. The results of modelling, using experimental measurements specific to the experimental conditions, applied to a large container of 660 litres showed, as expected, a decrease in the oxygen level during the first few minutes of filling. The extent of this decrease was a function of ventilation and work area volume. A risk of serious hypoxia that may even be fatal was found for very low volume 10 m3 rooms, possible effects of variable severity depending on ventilation in rooms of 30 m3, and theoretically no specific risk in a 150 m3 work area, even with an air renewal rate of 0.5 volwork area.h-1 ;
• spilling of the contents of a container: in the scenario concerning accidental spilling of the contents of a container (5, 10 and 60 litres) based on an upper-bound calculation hypothesis for the hypoxic risk, particularly hazardous oxygen level reductions are observed that may lead to decreased physical and intellectual performance ranging from loss of consciousness to death in low volume areas (less than 30 m3). The risk is reduced in large volume work areas (150 m3).

These results clearly confirm that it is beneficial to act on parameters such as work area volumes and ventilation to prevent the hypoxic risk related to use of liquid nitrogen.

Recommendations

Expert appraisal recommendations are presented in their entirety in the risk assessment report. The general principles are outlined below. These recommendations are not intended as a guide for intervention in the case of accidents or large-volume liquid nitrogen spills in a work area.

Implementation of collective protection measures and adaptation of premises

The main recommendation is that a minimum level of 19 % oxygen should be maintained to prevent hypoxia-related effects. This is the value recommended by the National Health Insurance Fund for Salaried Workers (CNAMTS - reference R.276 Tanks and reservoirs). It is also recommended that liquid nitrogen should not be stored or handled in work areas smaller than 20 m3 (i.e. surface area of 8 m2 with a ceiling height of 2.5 m).

Underground premises should not be used for storage or handling of liquid nitrogen. It is important that work areas do not connect to lower-level rooms via stairs or other openings (e.g. service shafts).

In particular, premises must be equipped with oxygen detection and measurement equipment, alarm systems, and suitable two-speed mechanical ventilation. The minimum continual air renewal levels required can be estimated by using the chart included in the collective expert appraisal report.

Premises where liquid nitrogen is handled should be clearly identified. They must also have hazard warnings and be equipped with suitable personal protection equipment, using corresponding pictograms.

Supply and use of personal protection equipment

Handling of liquid nitrogen or materials cooled with liquid nitrogen requires the use of gloves (preferably with long sleeves), protective goggles or visors, closed shoes and an apron made of non-woven material. The working group noted that there are no suitable gloves for handling  straws.

Work procedures

It is recommended that each laboratory define and formalise all safety rules regarding access to facilities (including external persons) and activities involving possible exposure to liquid nitrogen. A staff member should be authorised to work in a laboratory alone only when a suitable safety system is in place.

Storing, filling and equipment for filling

it is recommended that only containers designed to hold liquid nitrogen fitted with integrated closure systems be used. Use of any other unsuitable containers, particularly commercially available insulated bottles with hermetic sealing caps, is prohibited due to the risk of bursting, etc. Preventive measures should be made available to limit evaporation of nitrogen gas in work areas.

Transport

Safety rules are recommended for the movement of liquid nitrogen within the laboratory and elsewhere in the facility. Transport in lifts should only be authorised without personnel present, and only in lifts that can be locked for transport. For small quantities (a few litres), use of transport containers with absorption of liquid nitrogen by a porous material is recommended. Recommendations on the transport of small quantities of liquid nitrogen in light duty vehicles are indicated in the report.

Personnel training

The Agency highlights the importance of personnel training and summarises the main areas of knowledge and competence.

Periodic controls

Attention is drawn to the need to comply with control and maintenance procedures for equipment and facilities.