Biopolymer facilities, like any other laboratory, are centers of research creativity. They act as resources to the biomedical community to provide highly specialized services within the culture of a basic research setting, collaborate with scientific colleagues, and develop and refine technology for analytical and synthetic work.
Biopolymer facilities can also be a valuable resource to science of another kind. Within the biomedical research community, the highly trained chemists and staff who operate these facilities are perhaps best prepared to recognize and control the hazards associated with handling flammable, corrosive, toxic, and reactive laboratory chemicals. The organized and systematic nature of their work has fostered a respect for the value of managing chemicals carefully, from the time of purchase through their life cycle in the laboratory. The constituents of hazardous waste streams are well characterized, making it easier to achieve pollution prevention objectives and compliance with environmental regulations. Other research laboratories would be well advised to model the safety practices that are common in biopolymer facilities, and bioploymer facilities should share their experience with the scientists they support.
Historical Perspective
For nearly three centuries, societies have trusted laboratory chemists to manage their occupational health risks. Bernardino Ramazzini, who is regarded as the father of occupational medicine, included in his classic De morbis artificum diatriba (1) a discussion of maladies of chemists who work in laboratories synthesizing medically important compounds. In addition to drawing attention to health risks of working chemists, several cultural observations were made that have persisted since his treatise was published in 1700. Ramazzini wrote:
Chemists boast that they have mastered the art of subduing every kind of mineral, yet they themselves do not come off scot-free from their pernicious influence. They very often bring on themselves the same ailments as do other workers who deal with minerals, and in spite of their persistent denials the color of their faces reveals the fact.
He punctuated this conclusion with a personal observation and conveyed his great admiration for chemists and their profession:
I used to know Carlo Lancillotti, my compatriot, a well-known chemist; he was palsied, blear-eyed, toothless, short of breath, and disgusting; the mere sight of him was enough to ruin the reputation of the medicaments, the cosmetics especially, that he used to sell. But far be it from me to condemn such researches as mischievous. Chemists certainly deserve praise, for so devoted are they to the investigation of abstruse matters and to the enrichment of natural science that they do not hesitate to risk their lives for the good of the public. It is not their fault that when they are trying to temper the virulence of minerals they are unable to take sufficient precautions.
A major contribution of Ramazzini's study of occupational illnesses was his emphasis on prevention and the elucidation of practical methods for controlling hazards of the workplace, but he chose not to address the work practices of the laboratory chemist:
I should be insulting the chemists if I suggested any remedy, preventive or curative, for every occasion when they derive more loss than profit from the practice of their profession. For there is hardly any disease for which the chemists cannot produce from their narthecium, as they call it, a ready and effectual remedy.
Government involvement in regulating occupational health risks for chemists is fairly recent. In 1974 the U.S. Occupational Safety and Health Administration (OSHA) issued carcinogen standards that brought into conflict the long-held trust that chemists knew best how to protect themselves from occupational health risks. Specific standards appropriate for industry were prescribed for handling carcinogens in laboratories, even though the circumstances of laboratory and industrial carcinogen use differed significantly. It became apparent that laboratory chemists needed to become major participants in the formulation of OSHA regulatory policy and standards.
The expertise and experience of working chemists were used in a project of the National Research Council that resulted in the 1981 publication of "Prudent Practices for Handling Hazardous Chemicals in Laboratories" by the National Academy Press (2). This publication has become an authoritative reference, and it also contributed to a policy decision by OSHA to develop performance standards for the regulation of hazardous chemicals in laboratories rather than to pursue the conventional strategy of establishing prescriptive standards. This represented a fundamental shift in OSHA regulatory policy.
The success of performance standards, however, depends on the genuine commitment of chemists to establish and follow practices for controlling hazards and reducing occupational health risks to an acceptable minimum. The OSHA laboratory performance standard was issued in 1990 (3). Its cornerstone is the requirement that an employer prepare a written chemical hygiene plan, which includes the necessary work practices, procedures, and policies to protect laboratory workers from hazardous chemicals. All laboratory staff should be familiar with the provisions of the plan and proficient in carrying out the practices necessary for their protection.
Hazard Recognition and Risk Assessment
People who work in biopolymer facilities are exposed to a great variety of hazards. They range from hazards common to most workplaces, like those of ordinary fire and tripping, to hazards that require extensive precautions to adequately control, like the use of hydrogen fluoride (HF) for cleaving synthetic peptides from resins. There are numerous sources of information that can aid in the recognition of hazards. Most institutions are required to maintain an OSHA Log 200 of work-related injuries and illnesses. This source may have useful information regarding the injuries and illnesses of laboratory workers doing similar tasks.
A knowledgeable person from an institution's environmental health and safety office who is familiar with laboratory practice can be a helpful resource. Such a person can best help the laboratory recognize hazards by accompanying staff in a systematic walk through the laboratory area. Information obtained from this collaborative approach is better received than inspection reports that may be sent to the laboratory several weeks after an on-site review. Hazard identification should never be a punitive exercise but should be performed in the spirit of reinforcing good work habits.
There are several important authoritative references for researching the hazardous properties of chemicals and assessing health risks associated with occupational exposures (4-6). Although Material Safety Data Sheets (MSDS) provide information on the hazardous properties of chemicals, they do not provide relevant precautionary information. A valuable, new reference is the National Research Council's comprehensive revision of its 1981 report, "Prudent Practices in the Laboratory: Handling and Disposal of Chemicals" (7). The report includes concise technical summaries of important safety information for 88 chemicals that are commonly used in laboratories, including acetonitrile, cyanogen bromide, dichloromethane, dimethyl-formamide, ethyl acetate, HF, methanol, tetrahydrofuran, and trifluoroacetic acid (TFA). These summaries, called Laboratory Chemical Safety Summaries, are similar in format to MSDS but are written to provide information relevant to the laboratory use of chemicals.
Injury and Illness Experience
The injury and illness experience of the Howard Hughes Medical Institute (HHMI) for the four previous calendar years is summarized in Tables I and II. These data represent all injuries regardless of their severity; fewer than one-fourth of the injuries meet the criterion for being recordable under the OSHA Log 200 system. HHMI encourages employees to report all injuries, because the composite data help identify and rank the importance of existing hazards and allow a more thorough assessment of occupational risk. The Institute's incidence rate per 100 full-time employees for recordable occupational injuries and illnesses was 1.3 in 1993. This compares favorably to rates reported for 1993 by the Bureau of Labor Statistics of the U.S. Department of Labor for the following groups: hospitals, 11.8; medical and dental laboratories, 5.8; and colleges and universities, 4.6.
The incidence data in Table I suggest that research technicians and laboratory support personnel have more exposure to laboratory hazards than other employees. A review of the injury reports indicated that falls, tripping, improper lifting techniques, and bumping against walls, counter tops, and carts contributed to most of the increase in injuries among staff in 1993. Many of these accidents occurred in unfamiliar settings and were associated with moves to new laboratories or to the occupancy of the new HHMI headquarters facility in Chevy Chase, MD.
However, the increased injury rate for laboratory support staff could not be explained by new and unfamiliar work settings. The predominant work activities of this position category are washing, sterilizing, and stocking laboratory glassware. After review, it was concluded that glassware washing facility managers should emphasize high safety standards and the staff should increase their awareness of common workplace hazards and guard against any relaxation in safety performance. An effort to enhance safety leadership at the management level and safety training of staff was undertaken to reverse the injury trend, and the decreased injury rate for 1994 indicated this effort was worthwhile.
The types and numbers of occupational injuries experienced by HHMI laboratory employees are presented in Table II. Lacerations are the most common injury and are caused principally by exposure to broken glassware, razor blades, and sharp implements used to open packaging materials. Eye injuries are potentially the most serious. Chemical exposures to the eye accounted for 24 of the 32 reported eye injuries during this four- year period. Although employees who work in biopolymer facilities account for less than two percent of the total HHMI workforce, they experienced 6 of the 48 chemical exposures reported during this four-year period. These injuries resulted from accidents with syringes, release of chemicals from pressurized lines, and skin exposures from chemically contaminated equipment and from HF. In 1995 there were two reports of chemical exposures to the eye among HHMI biopolymer facility employees.
Burns and repetitive stress injuries account for about 75% of injuries included in the "Other" category in Table II. Nineteen ergonomic work-related injuries were reported in HHMI laboratories from 1991 through 1994 and of these, two resulted in lost work days. No burn or repetitive stress injuries were reported to have occurred within the Institute's biopolymer facilities.
Commonly observed hazards
Site visits by safety professionals can help identify previously unrecognized or common hazards that a complacent staff has allowed to go uncorrected, and such visits occur within HHMI laboratories. Commonly observed hazards include cluttered corridors, Bunsen burners left on and unattended, eye hazards and inadequate eye protection, razor blades and other sharps on benches and sinks, water on floors, bottles of chemicals on floors, dust accumulation near balances and scales, unsecured gas cylinders, cluttered workspace in chemical fume hoods, gel boxes without protective covers, excessive accumulation of wastes, and careless disposal of pipettes and pipette tips. Such hazards are less frequently observed in biopolymer facilities.
Assessing chemical hazards
Flammability, corrosiveness, reactivity, and explosivity are hazardous properties of chemicals that are usually well understood, but toxicity is the least predictable chemical property. Data from animal studies are often the best available information to assess toxicity in humans and are usually reported as a lethal dose 50 (LD50) value, expressed in milligrams of chemical per kilogram of test animal body weight. These values help rank the intrinsic toxicity of chemicals in humans. One ranking system correlates these values (single oral dose in rats) with descriptions of severity (8): extremely toxic, highly toxic, moderately toxic, slightly toxic, and practically non-toxic describe ranges of LD50 values of 1 or less, 1-50, 50-100, 500-5000, or more than 5000 mg chemical/kg body weight, respectively.
Dose, duration, and the route and frequency of exposure are all important factors in determining the seriousness of an exposure hazard. In the biopolymer laboratory two routes of exposure are of primary concern: inhalation and contact with the skin or eyes. The opportunity for ingestion should never be present in the laboratory, and the use of syringes presents risk of accidental self-injection. Chemicals that are highly corrosive, like TFA, represent the greatest risk of contact harm. A few chemicals like cyanogen bromide, which can cause acute toxic effects through inhalation, require strict control to avoid exposure. For most chemicals used in the biopolymer laboratory, it is unlikely that under standard operating procedures an inhalation exposure could approach the permissible exposure limits (PELs) for hazardous chemicals regulated by OSHA; PELs are concentration levels that are not to be exceeded by the average exposure over an 8-hour working day. Routine protocols primarily involve chemicals in closed systems, and open vessel operations with volatile chemicals are usually confined to chemical fume hoods. A potential inhalation exposure to a concentration above a PEL might possibly occur during certain accident situations, such as immediately following the spillage of a large quantity of a highly volatile chemical or when a vent line from equipment becomes disconnected and goes unrecognized.
Equipment hazards
Automation has significantly increased the demand for electrically powered laboratory equipment. For example, the typical reversed-phase HPLC unit may require multiple outlet receptacles, and automated DNA sequence analysis equipment incorporates high voltage electrophoresis. Some equipment may run unattended for hours or days. Electric shock can be life-threatening even at relatively low current (e.g., 25 mA), and the processing of large volumes of flammable chemicals with automated equipment that occurs in the biopolymer facility presents a fire hazard. A recent incident occurred in an HHMI laboratory where the top buffer chamber of an automated DNA sequencer caught fire during unattended use; the fire was started by arcing caused at a loose electrode connection. Leaking buffer chambers can also cause arcing.
Chemicals can leak from connecting points in pressurized delivery tubing. This is a potential hazard often unrecognized by individuals who are merely observing the operation of automated equipment. The principal hazard to staff from pressurized chemical delivery tubing is the potential for eye exposure.
Chemical fume hoods are designed to capture contaminants that are accidentally released within the hood enclosure and move them away from the breathing zone of the laboratory worker. If there is insufficient airflow to capture and transport contaminants, the worker is placed at risk of an inhalation exposure. Performance tests to demonstrate proper operating conditions should be conducted annually and anytime a change has been made to the ventilation system. A potentially hazardous situation arose in one HHMI biopolymer facility when maintenance workers stopped the exhaust fan for a hood that was being used to contain an HF cleavage reaction. This placed both the technician and maintenance workers at unnecessary risk. Close coordination and effective communication between staff and building engineers is essential whenever maintenance is performed on chemical fume hoods.
Precautionary Measures
The best safety habits to acquire in the biopolymer laboratory are those that enable workers to avoid exposures to chemicals. These habits are formed by mastering scientific protocols and maintaining standard operating procedures for automated equipment. Other effective habits involve a safety discipline--conscientious and routine use of appropriate safety procedures to protect staff from chemical exposures. A comprehensive review of safe practices for protecting the eyes, skin, and respiratory tract from exposures to chemicals is available (7) and is briefly described below.
Avoiding eye exposures
Eye protection should be worn when handling hazardous chemicals and when operating automated equipment where chemicals are transported in closed systems under positive pressure. Safety glasses with side shields provide minimum protection and should be limited to activities involving splash hazards like opening or closing a bottle or monitoring the operation of automated equipment. Goggles should be worn when handling liquids that are highly corrosive such as TFA and HF. It is prudent to supplement the protection provided by glasses or goggles by wearing a face shield when either large volumes of hazardous chemicals are handled or a high risk of a splash hazard is present.
Avoiding skin exposures
The hands, wrists, forearms, and face are the most likely areas of the body to come into direct contact with chemicals and other hazardous agents. Wearing gloves is a simple and effective way to protect hands from chemical contact and avoid transferring contaminants to other parts of the body. An effective practice is to select gloves that are both comfortable to wear and offer some chemical resistance and to change them frequently and whenever they are contaminated. An important step in good glove use is to always wash hands thoroughly with soap and water when changing to fresh gloves and after working with any hazardous substance. Wearing laboratory coats can reduce the potential for accidental exposure to skin and personal clothing. The hazards associated with open sleeves can be avoided by selecting coats with an elastic band at the end of the sleeves, by taping the open sleeve ends of conventional lab coats, or by using disposable sleeves with elastic ends.
Avoiding inhalation exposures
Any procedure that may create an inhalation exposure should be carried out in a functional chemical fume hood. The vertical or horizontal sash should be positioned so that the work opening is within the safe operating range and allows convenient use. The designed containment efficiency of the hood can best be achieved by working four to six inches inside the face of the hood. The hood work surface should be clear of clutter to ensure that the airflow is unobstructed. In case of malfunction, stop work, close the sash, and immediately inform the group that has responsibility to correct the problem.
Guidelines for the safe handling of hydrogen fluoride in the synthesis of peptides
HHMI assembled a workgroup of experienced scientists, technicians, and environmental health and safety advisors in 1989 to evaluate practices used in HF cleavage reactions in peptide synthesis (9). The group conducted a risk assessment, recommended practices for safely carrying out the reaction, and prepared a guide for physicians who may be called upon to respond to accidental exposures to HF.
The workgroup concluded that the potential dangers inherent in the use of HF required consideration of less hazardous substitute peptide synthesis methods such as the use of Fmoc chemistry. Some use of HF will still continue because substitute methods are not available for all syntheses that can be performed with Boc chemistry. Infrequent use of HF, however, raises several concerns. There will be greater difficulty in sustaining proficiency when the HF procedure is no longer performed on a routine basis. An even greater concern is the hazard associated with the storage of HF cylinders. A representative from Mattheson Gas Products has suggested that storage of HF cylinders beyond six to twelve months is inadvisable, because lecture cylinders have been known to explode during undisturbed storage. This precautionary note should persuade users of HF to dispose of unempty cylinders within a precisely defined time period. The complete report is available from the HHMI Office of Laboratory Safety (Fax: (301) 215-8828).
Reducing electric shock and fire hazards
Laboratory staff responsible for operating electrically powered equipment must have a clear understanding of its proper use and demonstrated proficiency in carrying out standard operating procedures. Each electrical receptacle should accept three-prong plugs and provide a ground connection. The receptacle type should be appropriate for the equipment connected to it. The use of strip outlets and extension cords should be avoided. Equipment that may be left on and unattended should include a fuse or other overload protection device that will disconnect the circuit in the event of equipment failure or overload. It is desirable to have a main circuit breaker box located near the exit door of the laboratory for easy access in the event of an emergency.
Adequate provisions for safe flammable storage is a necessity. A typical biopolymer facility may process more than 60 liters of flammable solvents weekly and accumulate an equal volume of liquid flammable wastes. Approved flammable material storage cabinets should be used to store stock purchases of flammable solvents. The flammable storage cabinet beneath a chemical fume hood should be used as a temporary storage area for accumulated wastes. The transfer of waste from an equipment reservoir to a waste container can be performed safely in the hood. Wastes should be removed from the laboratory on a regularly scheduled basis to avoid excess accumulation. If flammable solvents or wastes are transferred from metal containers or through metal lines, the containers and lines should be bonded and grounded properly to discharge static electricity.
Safety training
The OSHA laboratory standard mandates periodic training for laboratory workers. Most institutions have adopted an annual or biennial training cycle. This frequency may be adequate to introduce new institutional policy, review progress toward achieving safety goals, or to conduct formal training programs, but safety instruction and guidance needs to become a continuing process in the laboratory to maintain an appropriate level of safety awareness and performance. HHMI has produced a series of laboratory safety training videos that were designed to provide a technical review in safety for experienced staff and to introduce new staff to good laboratory practices. The videos are available from HHMI as a public service to the academic and research communities (see inset).
Emergency Response
Most institutions in which biopolymer facilities operate have generic emergency response plans. Emergency situations that could arise in the biopolymer facility, however, may vary in intensity because of their high fuel load of flammable solvents, array of potential ignition sources, and the quantity of hazardous chemicals. Additional guidance for staff may be needed to assure the most appropriate response to emergency situations. Plans should be periodically reviewed to ensure they remain up-to-date, and all laboratory workers in the facility should know the procedures for responding to emergencies. Procedures for responding to fire and chemical spills should be practiced because these are the most likely emergencies to occur or if they were to occur could cause serious consequences. General guidance for these types of emergencies are provided below.
Responding to fire
Small fires can be extinguished without evacuation. However, an immediate readiness to evacuate is essential in the event the fire cannot be controlled. The first step is to alert people in the laboratory and activate the fire alarm. Turn off any electrical equipment near the fire. Then smother the fire or use an appropriate fire extinguisher. Fire extinguishers, however, should be used only by trained personnel. Aim the extinguisher at the base of the fire. The following guidelines should be observed in the event of a major fire: alert people in the area to evacuate, activate nearest fire alarm, disconnect room circuit breaker if it is safe to do so and close doors to confine fire, evacuate to a safe area or exit building through a stairwell, and a person knowledgeable of the incident and the laboratory should be available to assist emergency crews.
Responding to chemical spills
The biopolymer facility should have spill kits with instructions, absorbents, reactants, and protective equipment available to clean up minor spills. Laboratory staff should know how to clean up minor spills. The following procedures are appropriate for responding to major spills: attend to injured or contaminated persons and remove them from exposure, alert people in the laboratory to evacuate, turn off ignition and heat sources, call the institution's emergency response number, close doors to affected area, and have a person knowledgeable of the spill and the laboratory assist emergency personnel.
Responding to chemical exposures to eyes and skin
Skin that has been splashed with a chemical should be flooded with running water from a faucet or safety shower for at least 5 minutes. Contaminated clothing should be removed at once. Splashes in the eye require immediate rinsing of the eyeball and inner surface of the eyelid with water continuously for 15 minutes. The eye needs to be forcibly held open to ensure effective washing behind the eyelids. In the case of an eye exposure, the person should seek medical attention. This may also be advisable in the case of an exposure to the skin.
Pollution Prevention
Laboratory workers today have an increased sensitivity to the value of protecting the quality of the natural environment. This consciousness has led to new initiatives for reducing the quantity of hazardous wastes that need to be disposed in chemical landfills or incinerated. Much progress has been made in this regard as a consequence of miniaturizing chemical laboratory operations. Waste minimization can also occur by recycling and developing synthesis procedures that generate less hazardous waste.
References
1. B. Ramazzini, De Morbis Artificum, translation by W. Wright, New York Academy of Medicine, Hafner Publishing Company, New York, 1964.
2. NRC Committee on Hazardous Substances in the Laboratory, Prudent Practices for Handling Hazardous Chemicals in Laboratories, National Academy Press, Washington, D.C., 1981.
3. Occupational Safety and Health Administration (OSHA), 29CFR 1910.1450, Occupational Exposure to Hazardous Chemicals in Laboratories (Laboratory Standard), U.S. Government, Washington, D.C., 1990.
4. P. Patnaik, A Comprehensive Guide to the Hazardous Properties of Chemical Substances, Van Nostrand, New York, 1992.
5. American Conference of Governmental and Industrial Hygienists (ACGIH), Threshold Limit Values for Chemical Substances and Physical Agents and Biological Exposure Indices, ACGIH, Cincinnati, OH, 1994.
6. R.J. Lewis, Sax's Dangerous Properties of Industrial Materials, 8th Edition, Van Nostrand Reinhold, New York, 1992.
7. NRC Committee on Prudent Practices for Handling, Storage, and Disposal of Chemicals in Laboratories, Prudent Practices in the Laboratory: Handling and Disposal of Chemicals, National Academy Press, Washington, D.C., 1995.
8. H. Hodge and J. Sterner, 1949, Tabulation of toxicity classes, Am. Ind. Hyg. Quart., 10:39.
9. E. Barkley, J. Elliott, J. Gorka, R. Randall, C. Riley, and J. Stewart, 1990. Research hazard review on use of anhydrous hydrogen fluoride in the synthesis of peptides, Office of Laboratory Safety, HHMI, Chevy Chase, MD 20815.
Click here to view a list of safety training videos offered by Howard Hughes Medical Institute
W. Emmett Barkley is the Director of Laboratory Safety at Howard Hughes Medical Institute and may be contacted at HHMI, 4000 Jones Bridge Rd., Chevy Chase, MD 20815-6789. Clark T. Riley is the Biopolymers Facilities Director at HHMI/The Johns Hopkins University and may be reached at HHMI/Johns Hopkins University, 725 N. Wolfe St., 807 PCTB, Baltimore, MD 21205-2105.
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