The views expressed and conclusions reached in this document, together with the recommendations for a standard, are those of NIOSH, after review of the evidence and consideration of the comments of reviewers. These views and conclusions are not necessarily those of the consultants, other federal agencies, and professional societies that reviewed the document, or of the contractor. recommends that employee exposure to ethylene dibromide in the workplace be controlled by adherence to the following sections.

# CRITERIA DOCUMENT: RECOMMENDATIONS FOR AN OCCUPATIONAL EXPOSURE STANDARD FOR ETHYLENE DIBROMIDE
The standard is designed to protect the health and provide for the safety of employees for up to a 10-hour workday, 40-hour workweek, over a working lifetime.
Compliance with all sections of the standard should prevent adverse effects of ethylene dibromide on the health of employees and provide for their safety. Techniques recommended in the standard are valid, reproducible, and available to industry and government agencies. Sufficient technology exists to permit compliance with the recommended standard. Although NIOSH considers the workplace environmental limit to be a safe level based on current information, the employer should regard it as the upper boundary of exposure and make every effort to maintain the exposure as low as is technically feasible. The criteria and standard will be subject to review and revision as necessary.
The possible health effects of employees chronically exposed to ethylene dibromide may include the induction of cancers, malformations and heritable changes in offspring, and sterility. Ethylene dibromide may also cause adverse effects to the liver, kidneys, heart, and other internal organs and systems. Direct contact of the skin with ethylene dibromide may induce chemical burns as well as systemic effects, and ingestion of ethylene dibromide may damage internal organ systems, or even be fatal.
The alkylation of cellular constituents, including the genetic material, deoxyribonucleic acid (DNA), is the most plausible molecular basis for the induction of adverse effects after exposure to ethylene dibromide.
These criteria and the recommended standard apply to occupational exposure of workers to the brominated hydrocarbon BrCH2CH2Br, hereinafter referred to as "ethylene dibromide." Synonyms for ethylene dibromide include ethylene bromide, dibromoethane, sym-dibromoethane, 1,2dibromoethane, glycol dibromide, and EDB. The major uses of ethylene dibromide are as an additive to leaded gasoline and as a component of fumigants.
"Occupational exposure to ethylene dibromide" is defined as work in any establishment where ethylene dibromide is manufactured, blended, stored, used, handled, or otherwise present. The "action level" is defined as one-half of the recommended workplace exposure concentration designated as a ceiling limit for ethylene dibromide. Exposure to airborne ethylene dibromide at concentrations less than the action level, as determined in accordance with Section 8, will not require adherence to Sections 2, 3, 4(a), or 8(c,d). If exposure to other chemicals also occurs, the employer shall comply with any applicable standard for the other chemicals.

# Section 1 -Environmental (Workplace Air) (a) Concentration
The employer shall control workplace concentrations of ethylene dibromide so that no employee is exposed in his workplace to concentrations greater than 1.0 mg/cu m (0.13 ppm) as a ceiling limit, as determined by a sampling period of 15 minutes.
(b)

# Sampling and Analysis
Procedures for the collection and analysis of workroom air samples for compliance with the standard shall be as provided in Appendices I and II, or by any methods shown to be at least equivalent in precision, sensitivity, and accuracy to the methods specified.

# Section 2 -Medical
Medical surveillance shall be made available to employees as outlined below:
(a) Comprehensive preplacement and annual medical examinations unless a more frequent schedule is indicated by professional medical judgment based on such factors as emergencies, variations in work periods, and the preexisting health status of the individual worker.
(b) These examinations shall include at least:
(1) Comprehensive or interim medical and work histories, with special emphasis directed to disorders of the heart, liver, kidneys, and nervous system.
(2) A comprehensive physical examination, with particular emphasis given to cardiovascular, pulmonary, neurologic, hepatic, and renal systems, and to the skin.
(3) An evaluation of the employee's physical ability to safely wear a negative or positive pressure respirator.
(c) Employees shall be counseled by the physician to ensure that each employee is aware that ethylene dibromide has been shown to induce in experimental animals adverse effects in reproductive processes, including abnormalities in offspring, mutations, and stomach cancer following direct administration.
The relevancy of these findings in animals to male or female employees has not yet been established. They do indicate, however, that both employers and employees should do everything possible to minimize exposures to ethylene dibromide. If a physician becomes aware of any adverse effects on the reproductive system, or cancers in individuals who have been exposed to ethylene dibromide, or any abnormal babies born to parents either or both of whom have been exposed to ethylene dibromide, such information should be forwarded to the Director, National Institute for Occupational Safety and Health, as promptly as possible.
(d) Medical attention shall be provided promptly to all employees suspected of being exposed to ethylene dibromide vapor at or above the action level or to liquid ethylene dibromide.
A 48-hour medical observation period for delayed systemic or dermal effects is recommended.
(e) Examinations of current employees shall be performed as soon as practicable after the promulgation of a standard based on these recommendations.
(f) The employer shall maintain medical records for all persons exposed to ethylene dibromide at or above the action level.
All medical records, including information on required medical examinations and supporting documents, shall be kept for at least 30 years after the termination of the individual's employment.
(g) The medical representatives of the Secretary of Health, Education, and Welfare, of the Secretary of Labor, of the employee or former employee, and of the employer shall have access to these medical records.

# Section 3 -Labeling and Posting
All labels and warning signs shall be printed both in English and in the predominant language of non-English-reading workers. Illiterate workers and workers reading languages other than those used on labels and posted signs shall receive information regarding hazardous areas and shall be informed of the instructions printed on labels and signs.
(a) Labeling
The following warning label shall be affixed in a readily visible location on ethylene dibromide processing or other equipment and on ethylene dibromide storage tanks or containers:  (C) During emergencies when air concentrations of ethylene dibromide may exceed the recommended occupational exposure limit.
ETHYLENE

# C2)
When a respirator is permitted by paragraph (a)(1) of this section, it shall be selected and used pursuant to the following requirements:
(A)
The employer shall ensure that no employee is exposed to ethylene dibromide because of improper respirator selection, fit, use, or maintenance.
(B) The employer shall establish and enforce a respirator program meeting the requirements of 29 CFR 1910.134 as amended.
(C) The employer shall provide respirators in accordance with Table 1-1 and shall ensure that the employee uses the respirator provided when necessary.
(D) Respiratory protective devices described in   (1) Supplied-air respirator with full facepiece, helmet, or hood (2) Self-contained breathing appara tus with full facepiece Type C supplied-air respirator with full facepiece operated in pressuredemand or other positive pressure mode or with full facepiece, hood, or hel met operated in continuous-flow mode
(1) Self-contained breathing appara tus with full facepiece operated in pressure-demand or other positive pressure mode (2) Combination respirator that in cludes Type C supplied-air respirator with full facepiece operated in pressure-demand or other positive pressure or continuous-flow mode and auxiliary self-contained breathing apparatus op erated in pressure-demand or positive pressure mode (3) Supplied-air suits may be neces sary (b)
Eye Protection
Eye protective devices shall be provided by the employer and used by each employee where contact of ethylene dibromide with the eyes is likely.
Chemical safety goggles or plastic face shields (8-inch minimum) with goggles made completely of ethylene dibromide-resistant materials shall be used. Selection, use, and maintenance of eye protective devices shall be in accordance with 29 CFR 1910.133.
(c) Protective Clothing
Protective clothing shall be resistant to the penetration and to the chemical action of ethylene dibromide. Additional protection, including gloves, bib-type aprons, boots, and overshoes, shall be provided for, and worn by, each employee while in any operation that may cause direct contact with liquid ethylene dibromide. Supplied-air hoods or suits resistant to penetration by ethylene dibromide shall be worn when entering confined spaces, such as pits or storage tanks. In situations where heat stress is likely to occur, supplied-air suits, preferably cooled, are recommended.
The employer shall ensure that all personal protective clothing is inspected regularly for defects and is maintained in a clean and satisfactory condition by the employee. Records of such training should be kept for inspection by authorized personnel as required. This program shall be held for all employees with occupational exposure to ethylene dibromide at intervals not greater than quarterly, or whenever there is a process change. For all work areas where emergencies may occur, the employer shall take all necessary steps to ensure that employees are instructed in and follow the procedures specified below and any others appropriate to the specific operation or process.
Procedures shall include at least prearranged plans for :
( (2) Evacuation alarm systems shall be provided by the employer.
(3) Personal protective equipment and clothing as specified in Section 4 shall be used by trained personnel essential to emergency operations.
(4) Nonessential employees shall be evacuated from hazardous areas during emergencies. Perimeters of these areas shall be delineated, posted, and secured. The employees in adjacent areas shall be trained in evacuation procedures if these work areas become involved.

# C5)
Only personnel trained in the emergency procedures and protected against the attendant hazards shall shut off sources of ethylene dibromide, clean up spills, control and repair leaks, and fight fires in ethylene dibromide areas.
(6) Firefighting procedures shall be established for areas where flammable materials are used with ethylene dibromide. Chemical foam, carbon dioxide, or dry chemicals shall be used for fighting fires in areas where ethylene dibromide is present.
Proper protective respirators and clothing shall be worn by all personnel in the hazard area until concentrations of airborne ethylene dibromide have been demonstrated by monitoring to be below the recommended occupational exposure limit.
Showers, eyewash fountains, and washroom facilities shall be provided and so located as to be readily accessible to workers in all areas where skin or eye contact with liquid ethylene dibromide is likely. If liquid ethylene dibromide is splashed on the clothing or skin, contaminated clothing shall be promptly removed and the skin washed thoroughly with soap and water for at least 15 minutes. If liquid ethylene dibromide gets into the eyes, they shall be irrigated immediately with copious quantities of running water for at least 15 minutes. (1) Suitable engineering controls designed to limit exposure to ethylene dibromide to that prescribed in Section 1(a) shall be used. The use of completely enclosed processes is the recommended method of control for ethylene dibromide. Local exhaust ventilation may also be effective, used alone or in combination with process enclosure. When a local exhaust ventilation system is used, it shall be designed to prevent the accumulation or recirculation of ventilation control or process air in the workroom, to maintain ethylene dibromide concentrations below the limit of the recommended standard, and to remove ethylene dibromide from the breathing zones of employees. Exhaust systems discharging into outside air must conform with applicable local, state, and federal air pollution regulations. Ventilation systems shall be subjected to regular preventive maintenance and cleaning to ensure effectiveness, which shall be verified by periodic airflow measurements at least every 3 months. Measurements of system efficiency shall also be made immediately by personnel properly attired in specified protective equipment when any change in production, process, or control might result in increased concentrations of airborne ethylene dibromide. Tempered makeup air shall be provided to work areas in which exhaust ventilation is operating.
(2) Forced-draft ventilation systems shall be equipped with remote manual controls and shall be designed to turn off automatically in the event of a fire in the work area.

# C2)
Prompt medical attention shall be provided if there is known or suspected exposure to ethylene dibromide, whether or not symptoms are present.

# C3)
The employer shall ensure that safety showers, eyewash fountains, and other emergency equipment is in proper working order through regularly scheduled inspections performed by qualified maintenance personnel.
(4) Ethylene dibromide operating systems shall be inspected daily for signs of leaks by personnel attired in specified protective equipment. All equipment including valves, fittings, and connections shall be checked for tightness and good working order. All newly made connections shall be checked for leaks immediately after ethylene dibromide is introduced by trained personnel attired in prescribed personal protective equipment.
If there is a leak, the leak shall be corrected immediately. Work shall resume normally only after necessary repair or replacement has been completed, the area has been ventilated, and the concentration of ethylene dibromide has been determined by monitoring to be below the recommended occupational exposure limit.
(6) Transportation and use of ethylene dibromide shall comply with all applicable local, state, and federal regulations. Where ethylene dibromide is used as a fumigant, strict adherence to the pesticide container label requirements for application and personal protection shall be followed. Additional standards for pesticide use by agricultural workers can be found in 40 CFR 170. (7) When ethylene dibromide containers are being moved, or when they are not in use and are disconnected, valve protection covers shall be in place.
Containers shall be moved only with the proper equipment and shall be secured to prevent dropping or loss of control while moving.
(8) Process valves and pumps shall be readily accessible and should not be located in pits and congested areas.
Containers and systems shall be handled and opened with care. Approved protective equipment as specified in Section 4 shall be worn while opening, connecting, and disconnecting ethylene dibromide containers and systems. Adequate ventilation shall be available to prevent exposure to ethylene dibromide when opening containers and systems.
(10) Personnel shall work in teams when ethylene dibromide is first admitted to a system, while repairing leaks, or when entering a confined or enclosed space.
(11) Any odor of ethylene dibromide shall be reported to a responsible authority and an alarm sounded immediately.

# (d) Work Areas (1) Ethylene Dibromide Hazard Areas
A hazard area shall be considered as any space workers may enter that has physical characteristics and sources of ethylene dibromide that could result in air concentrations exceeding the recommended limit.
Exits shall be plainly marked and shall open outward. Emergency exit doors shall be conveniently located and shall open into areas which will remain free of contamination in an emergency. At least two separate means of exit shall be provided from each room or building in which ethylene dibromide is stored, handled, or used in quantities that could create a hazard.

# Confined or Enclosed Spaces
Entry into confined spaces, such as tanks, pits, process vessels, tank cars, sewers, or tunnels, where there may be limited egress shall be controlled by a permit system. Permits shall be signed by an authorized employer representative certifying that preventive and protective measures have been followed.
Confined spaces which have contained ethylene dibromide shall be thoroughly ventilated to ensure an adequate supply of oxygen, tested for ethylene dibromide and other contaminants, and inspected for compliance with these requirements prior to each entry. Adequate ventilation shall be maintained while workers are in the space. Leakage of ethylene dibromide into the confined space while work is in progress shall be prevented by disconnecting and blanking the ethylene dibromide supply lines. An individual entering confined spaces shall be furnished with appropriate personal protective equipment and protected by a lifeline harness tended by another worker outside the space, who shall also be equipped for entry with approved personal protective equipment and who has contact with a third party. Communication (visual, voice, signal line, telephone, radio, or other suitable means) shall be maintained by the standby person with the employee inside the confined or enclosed space. A third employee, equipped to proceed to the aid of the other two if necessary, shall have general surveillance of their activities.
(e) Storage
(1) Storage facilities shall be designed to contain spills completely within a surrounding dike and to prevent contamination of workroom air.
Storage of ethylene dibromlde in the same area as reactive metals, such as aluminum or magnesium, or as liquid ammonia shall be prohibited.
(3) Ethylene dibromide shall be stored in tightly closed containers in a well-ventilated area away from excessive heat and sunlight.

# C4)
Storage containers shall be periodically inspected for leakage.
(5) Ventilation switches and emergency respiratory equipment shall be located outside storage areas in readily accessible locations which will be free of ethylene dibromide in an emergency.
(f) Spills, Leaks, and Waste Disposal
(1) If ethylene dibromide leaks or is spilled, the following steps shall be taken:
(A) Evacuate all nonessential personnel from the area.

# (B)
Adequately ventilate the area of the spill or leak to prevent accumulation of the vapor.
(C) If in liquid form, collect spilled material for reclamation or absorb in vermiculite, dry sand, earth, or similar nonreactive material.
(D) If in solid form, collect spilled material in the most convenient and safe manner for reclamation or for disposal.
(2) Personnel entering the spill or leak area shall be furnished with appropriate personal protective equipment. All other personnel shall be excluded from the area.
All wastes and residues containing ethylene dibromide shall be collected in ethylene dibromide-resistant containers and incinerated or buried in such a manner that no ethylene dibromide or toxic decomposition products are released to the environment. Clothing contaminated with liquid ethylene dibromide shall be immediately removed and placed in a closed container in a well-ventilated area for later disposal or decontamination. Employers shall require personnel who work with ethylene dibromide to shower before leaving the workplace at the end of a workday.
(c) Employers shall ensure that employees who handle ethylene dibromide wash their hands thoroughly with soap and water before eating, smoking, or using toilet facilities.

# (d)
The storage, dispensing, preparation, and consumption of food, beverages, or tobacco shall be prohibited in ethylene dibromide work areas.
(e)
The employer shall ensure that personnel who launder and clean clothing or equipment contaminated with ethylene dibromide are provided adequate personal protective equipment to prevent exposure and shall ensure that these employees are aware of the potential hazards of exposure to ethylene dibromide. where ethylene dibromide is handled, processed, or stored. Records of these surveys, including the basis for concluding that environmental concentrations are below the recommended limit or below the action level, shall be maintained. Surveys shall be repeated every month or whenever a process change is made.
(b) Where exposure concentrations have not been determined, they shall be determined as soon as practicable after the promulgation of a standard based on these recommendations.

# (c)
Requirements set forth below apply to work areas in which the concentration of ethylene dibromide has been determined to be at or above the action level.
(1) An adequate number of samples shall be collected monthly in accordance with Appendix I for the evaluation of the work environment with respect to the occupational exposure of the employees.
(2) Environmental samples shall be taken when a new process is installed or process changes are made which may cause an increase in environmental concentrations. Significantly increased production, relocation of existing operations, interruption of normal maintenance schedules, or other functions which may increase ethylene dibromide concentrations shall require resampling and analysis.
(3) In all monitoring, samples shall be collected in accordance with the provisions prescribed in Section 1(b).
The minimum number of representative exposure determinations for an operation or process shall be based on variations in exposures and production schedules and in accordance with the provisions prescribed in Section 1(b).
(5) If initial, periodic, or special evaluations indicate that the recommended limit is exceeded, corrective engineering or other control measures shall be immediately instituted to ensure the safety of employees until a concentration below the recommended occupational exposure limit is achieved.
In such cases, sampling of each operation and work location shall be conducted at least weekly until two consecutive employee exposure measurements, taken at least 1 week apart, reveal that the employee is not exposed to ethylene dibromide above the recommended occupational exposure limit. Employers shall notify in writing, within 5 days, every employee who is found to be exposed to ethylene dibromide above the recommended environmental limit. Adherence to the recommended standard for occupational exposure to ethylene dibromide is intended to minimize the potentials for the induction of sterility, carcinogenesis, mutagenesis, or teratogenesis in employees and their offspring from exposure to ethylene dibromide in the workplace.
Adherence to the recommended standard is also intended to minimize the hazards from skin penetration and irritation associated with exposure to liquid ethylene dibromide and the delayed and insidious long-term systemic effects resulting from exposure to bath liquid and vaporous ethylene dibromide.
III.

# BIOLOGIC EFFECTS OF EXPOSURE
Ethylene dlbromide, also known as 1,2-dibromoethane and ethylene bromide, is a clear, colorless, heavy liquid at room temperature with a distinctive odor described as "characteristic, mildly sweet" . The minimum concentrations of ethylene dibromide in air reported to be detectable by odor range from 10 ppm (77 mg/cu m)  to 25 ppm (192.5 mg/cu m) . Selected chemical and physical properties of ethylene dibromide are listed in Table XII
Ethylene dibromide is a reactive molecule which may form covalent bonds under biologic conditions . Because of the two replaceable bromine atoms, ethylene dibromide is considered to be a bifunctional alkylating agent that is capable of introducing cross-links into biologic materials .
It tends to react with nucleophilic organic compounds  more readily with those that are relatively easily polarized, such as those containing sulfhydryl or amino groups, than with those that are less readily polarized, such as acids and ketones.
The half-life of ethylene dibromide in water at 20 C and at a pH of 7 is about 14 years .
Although the biologic half-life of ethylene dibromide in humans is unknown, the presence of large numbers of nucleophiles in biologic tissue suggests that the half-life would be considerably shorter than that in water. The approximate biologic halflife of ethylene dibromide after intravenous (iv) injection in rats was less than 2 hours, and in chicks, it was less than 12 hours . An approximate biologic half-life of 14C-labeled ethylene dibromide in mice and in guinea pigs can be estimated from the data presented by Edwards et al  and Plotnick and Conner  as less than 48 hours. This indicates that either spontaneous reactions with nucleophiles, enzymatically catalyzed degradative reactions, or efficient excretory mechanisms predominate in biologic systems.
Alkyl bromides, such as ethylene dibromide, readily react with thiols, amines, alcohols, and other nucleophilic biochemical constituents  . The initial monoalkylation product between ethylene dibromide and substrate heteroatoms, such as nitrogen, oxygen, or sulfur, is a "half mustard" (a thiane) reagent which may spontaneously cyclize under biologic conditions to form a strained three-membered ring. This highly reactive intermediate product may then undergo a second alkylation reaction with cellular constituents. When both alkylation reactions occur with cellular DNA, the covalent cross-links between the DNA strands may prevent the normal separation of the strands during DNA synthesis and subsequent cell division . Thus, such bifunctional alkylating agents as ethylene dibromide tend to possess a considerably greater biologic activity than monofunctional agents of the same primary reactivities .
The covalent reaction of ethylene dibromide with biologic materials may alter the chemical behavior and physical characteristics of the cellular constituents so as to prevent the altered molecules from functioning normally in physiologic processes. The formation of stable reaction products may account, in part, for the subsequent deleterious effects observed in biologic systems exposed to ethylene dibromide. The alkylation by foreign chemicals of the biologic materials controlling cellular metabolism is the most plausible basis for the induction of genetic and neoplastic alterations after the exposure of biologic populations to alkylating agents. When the risk of induction of adverse biologic changes, such as mutation and neoplasia, increase as a cumulative function of the total dosage, then the observation of measurable effects may not be possible until after long periods of exposure to low concentrations of the inducing agent .

# Extent of Exposure
Ethylene dibromide-manufacturing processes are based on the bromination of ethylene . Natural bromide-containing brines are treated with chlorine to release elemental bromine through anionic replacement.
The reaction between gaseous ethylene and liquid bromine (which may contain traces of chlorine) yields a mixture of ethylene dibromide and reaction side-products, including very small amounts of vinyl bromide, ethyl bromide, and ethyl chlorobromide . If the reaction temperatures and pressures are carefully controlled, the purity of the ethylene dibromide can approach 99.95% . Ethylene dibromide can also be produced by the hydrobromination of acetylene , although this process is of little commercial value today.
In 1921, the antiknock properties of tetraalkyl lead compounds in the internal combustion engine were discovered . To prevent the deposition of lead on the cylinder walls, a substance capable of reacting with the lead to aid its removal from the engine was needed. Ethylene dibromide was found to be such a substance and has been used since then in leaded gasoline as a lead scavenger.
The US production of ethylene dibromide increased from an estimated 64 million pounds in 1940 to over 331 million pounds in 1973 . This fivefold increase can be related to the increased consumption of gasoline containing ethylene dibromide as an additive, which has always been its largest use . About 85% of the ethylene dibromide produced during the The production of ethylene dibromide is the largest single use of bromine, and, as such, the locations of manufacturing plants have usually been near the major sources of bromine . Until 1961, the major source of bromine for ethylene dibromide manufacture was seawater; since 1961, the major source of bromine has been underground brine deposits, and the manufacturing of ethylene dibromide has been redistributed to be near these natural bromide brine wells in Michigan and Arkansas . A number of other occupations in which a potential for ethylene dibromide exposure exists are listed in Table XII
last
NIOSH estimates that approximately 9,000 employees (manufacturing, formulating, fumigating) are potentially exposed to ethylene dibromide in the workplace.
If one includes gasoline station attendants, this figure becomes much larger (about 660,000).

# Historical Reports
One of the first instances of ethylene dibromide intoxication was described by Marmetschke  in 1910. This case history described the accidental use of ethylene dibromide instead of ethyl bromide as an anesthetic or narcotic agent. Although this report is historical rather than clinical, it is one of the few relating to human exposure which give the amount of ethylene dibromide administered to each patient and the clinical symptoms resulting from the exposures. Thus, this report will be discussed in detail in Effects on Humans.
Another early case of occupational exposure to ethylene dibromide, which will also be discussed in Effects on Humans, was described in 1928 by Kochmann .

# Effects on Humans
In 1910, Marmetschke  wrote about a case of ethylene dibromide poisoning that occurred as the result of the mistaken administration of ethylene dibromide to a patient instead of ethyl bromide. A woman was administered the contents of a 70-g bottle of ethylene dibromide in aliquots of about 10 ml (22.0 g) through a gauze mask without producing the expected anesthesia. She coughed constantly during the administration and was dizzy at the completion of the application. She began to vomit and continued vomiting throughout the night.
The patient complained of a burning sensation in her chest and of inability to sleep. During the night, she had diarrhea, and the next day she became dizzy again after exertion. She became very restless and nervous and complained of having trouble in breathing. She continued to complain of thirst, abdominal pain, and a burning sensation in her chest.
The next day, she had uterine hemorrhaging, and died approximately 44 hours after she received the ethylene dibromide.
The results of the autopsy showed that the skin was a very pale blue and that the vessels of the skin were filled with blood Pflesser  subjected eight human volunteers and himself to the gauge fluid and to each of the components separately to determine their effects on the skin. One milliliter of the gauge fluid was placed on the hands of the volunteers and rubbed between the hands for about 1 minute.
Two hours later, the hands were washed with soap and water. None of the subjects developed any symptoms of adverse effects on the day of the test or later. Pflesser observed that, after rubbing the fluid between the hands for 1 minute, the liquid could no longer be seen on the skin, and he concluded that it had been absorbed or had evaporated.
In an additional experiment , the same volunteers rubbed 1 ml of the gauge fluid into the skin of the right forearm for 1 minute. Again, washing with soap and water was done after 2 hours. No symptoms of irritation were seen immediately after the exposure or later.
In a third experiment , the same volunteers were subjected to 1 ml of the gauge fluid on a cotton swab that was placed on the left forearm and covered; exposure time was 2 hours. After a few minutes, all eight subjects noted a sensation of heat at the application site, sometimes complaining of a pronounced burning of the skin. At the end of the 2-hour exposure, a strong reddening of the skin had appeared at the application site in all cases, and, in one case, pinhead-sized blisters had developed.
During the next 12 hours, a severe burning pain developed in addition to small blisters which merged into large ones.
The blisters initially contained a clear, amber-yellow, thick liquid, but the liquid had a pronounced tendency to coagulate because of its high protein content. The area immediately surrounding the application site was edematous and swollen, and, in some cases, the swelling extended from the fingertips to the upper arm. In one case, the axillary lymph nodes were moderately swollen and painful. Even with topical cod liver oil treatment, the skin did not grow back until 13-17 days after exposure, and surface scars persisted for at least 4 months. Pflesser  stated that all subjects felt severely ill during the first days following exposure. In two subjects, dermatitis, consisting of severe swelling and reddening of the skin accompanied by intensive itching, persisted in the area of the application for several weeks.
To determine which component of the gauge fluid was responsible for the adverse effects on the skin, Pflesser  subjected the same eight subjects and himself to each of the individual components. The results of the tests with the paraffin oil, Sudan Red B, and tetrachlorethane were negative.
During the 1-hour exposure to 0.5 ml of tetrachlorethane, several individuals noted a slight sensation of heat at the application site, but no symptoms similar to those for the gauge fluid were subsequently noted. All volunteers rubbed 0.5 ml (1.1 g) of ethylene dibromide into the skin of the forearm for 1 minute and washed with soap and water 30 minutes later. All subjects developed swelling and reddening of the exposed area, as well as itching, during the 24 hours immediately after exposure. The symptoms subsided without supportive treatment within 2-3 days.
In an additional experiment , the subjects received 0.5 ml (1.1 g) of ethylene dibromide applied to the skin on a swab, the swab and the application site being covered for 10 minutes. The swab was then removed and the area was washed with soap and water. A sensation of heat or slight burning was noticed during the exposure; and, during the next 24 hours a painful reddening and swelling of the skin developed. However, no blistering was noticed in any of the subjects. The injuries disappeared in 3-5 days without leaving visible traces.
In a subsequent experiment , this procedure was repeated except that the application site was covered for 30 minutes. After this exposure period, the swab was removed and the area was washed with soap and water.
During the 30-minute exposure, the subjects reported the customary heat or burning sensation at the application site. After exposure, a very painful inflammation of the skin occurred, including reddening, swelling, and blistering, within 15-20 hours. The damaged skin grew back after 7-13 days of supportive treatment. The author experienced such a strong burning pain in his skin immediately after application that increased so rapidly that the swab containing the ethylene dibromide had to be removed after 3 minutes.
The pain and reddening of the skin subsided, but then increased again after a few hours until a blister the size of a crab apple had formed at the application site after 18 hours. Very strong pain was associated with the blister. When the blister was opened, Pflesser collected 21 ml of a clear exudate, which coagulated soon afterward, and over 245 ml of fluid within the first 3 days after application. In addition, the author stated that when the blister appeared on the right arm, all places that had previously been exposed to ethylene dibromide or to the gauge fluid on both the left and right arms began swelling, reddening, and itching. A moderately strong, painful glandular swelling occurred in the left armpit (opposite the arm of the application site). Pflesser also reported that only a slight edema had occurred surrounding the application site after the 1st day, but that, on the 2nd day, an extensive edema of the entire right forearm and right hand suddenly developed. During these first few days after exposure, there was a pronounced feeling of illness and a slight temperature increase in addition to the physical manifestations at the exposure site. Healing of the damaged skin was complete after 17 days of zinc-sulfur ointment supportive treatment, leaving only a surface scar.
From this series of experiments, Pflesser  concluded that ethylene dibromide was the component of the gauge fluid that was responsible for the swelling, reddening, and blistering of the skin of the seaman on the destroyer. He also concluded that the effects and their intensities depended on the duration of exposure to the skin, and that covering the application site to prevent evaporation greatly increased the extent of damage from exposure. Pflesser concluded that ethylene dibromide was absorbed through the skin, causing tissue death, general inflammation, and plasma exudation. He further postulated that ethylene dibromide possessed a sensitization potential, citing the symptoms that appeared at previous application sites after his own exposure for 3 minutes to 0.5 ml of ethylene dibromide as proof of the assumption.
In 1960, Olmstead   Macroscopic and microscopic examinations of the internal organs showed a pronounced granular degeneration of the parenchymal tissue of the kidneys and smaller amounts of damage in the pancreas, spleen, heart, liver, and adrenals. In addition, the authors observed that ethylene dibromide exposure produced swelling and a generalized interstitial edematous degeneration of the endothelial lining of the abdominal vascular system that was not described further.
Thomas and Yant  noted that commercial and purified ethylene dibromide applied to the shaved abdomens of three groups of two rats each over a 2-cm square area killed all of the animals within 6-18 hours at doses of 0.25, 0.50, or 1.00 ml (0.55, 1.1, or 2.2 g)/animal. No attempt was made to determine the minimum lethal dose. The application site showed marked hyperemia of the small cutaneous blood vessels, and the abdominal muscles became contracted and remained tense. By 20 minutes, the reflexes became weak and the animals were scarcely able to stand. A slight, temporary increase in activity was noted during the next 10 minutes, but the general appearance remained that of great weakness. Macroscopic and microscopic examinations of tissues were similar to those in guinea pigs after vapor inhalation. The only difference observed between the two routes of exposure concerned the spleen: gross examination of the guinea pigs exposed to the vapor of airborne ethylene dibromide revealed that spleens were pale and edematous, whereas spleens of the dermally exposed rats were highly congested and edematous. No microscopic characterization of these differences was given.
In 1928, Lucas  published the results of an experiment in which two adult rabbits inhaled ethylene dibromide vapor in quantities sufficient to produce light or deep anesthesia (concentrations not reported). Very light anesthesia was maintained in one rabbit by inhalation of ethylene dibromide for about 10 minutes. The rabbit vocalized during exposure, presumably responding to the irritating effects of ethylene dibromide.
Respiration became extremely rapid during exposure and there was considerable phonation. The mucous membranes of the mouth were a peculiar "old rose" color after recovery from the anesthesia. This may have been due to a combination of vascular congestion and cyanosis. Death occurred within 18 hours, and, at autopsy, the liver was enlarged and mottled.
Microscopic examination of the liver showed slight-to-moderate diffuse fatty changes, which tended to be more marked in the portal regions.
Another rabbit deeply anesthetized by inhalation of ethylene dibromide for about 12 minutes exhibited signs of marked irritation from the gas, considerable phonation, rapid breathing progressing as the anesthesia continued, and snuffling in its breathing after recovery from the anesthesia . Death occurred within 15 hours. Autopsy showed the lungs to be enlarged and filled with a frothy exudate. About 10 ml of fluid was found in the pleural cavity. The liver was swollen and markedly congested. Lucas postulated that these results may have been caused by the decomposition of ethylene dibromide to hydrogen bromide and that local high concentrations of this material would be sufficient to bring about the observed changes. The next day, the animals began to tremble while in the cage, tears began forming, and the nasal mucosa was strongly reddened. After 11 exposures, the survivors were very weak and maintained a reclining posture, and strong trembling was observed, especially in the extremities. These conditions persisted until death occurred. There was a general weight loss during the exposures. Autopsy showed that the body cavities contained a clear, yellowish liquid. The lungs contained dark red discolorations and were judged by the author to be partially nonfunctional. The spleen was slightly enlarged and the kidneys were swollen and yellow colored.
Kochmann diagnosed the condition of the cats after exposure to ethylene dibromide as follows: rhinitis, conjunctivitis, ascites, pleural effusion, pneumonia in the left superior lobe of the lung, and incipient fatty degeneration of the liver and possibly degeneration of the tubules of the kidneys. Similar adverse signs of intoxication, including rhinitis, conjunctivitis, anorexia, and loss of weight, appeared in rabbits.
Autopsies of the rabbits showed that the small intestine contained an excessive amount of liquid, that the colon contained blood, and that the liver and kidneys were hyperemic.
In an additional experiment, the animals inhaled ethylene dibromide at a concentration of 0.01% (770 mg/cu m) once for 30 minutes, and the author  noted that there was a reduction in appetite, a corresponding loss of weight, a distinctly reduced hemoglobin content in the blood, and a slow recovery to the pretreatment conditions of the test animals. disappeared immediately after the guinea pigs were removed from the exposure chamber. Paralysis of the rear extremities occurred 24 hours after the sixth exposure, but it had partially disappeared after 5 additional days and was completely gone after another 8 days. The authors concluded that the adverse effects, such as paralysis of the extremities and spasms of the diaphragm, appeared at a lower concentration and in a much shorter time than did the adverse effects in a concurrent experiment with methyl bromide. They also concluded that the signs of toxicity remained longer than those observed from methyl bromide.
In 1929, Kistler and Luckhardt  reported the effects on respiration, muscle reflexes, and blood pressure in dogs exposed to ethylene dibromide by inhalation, ingestion, and injection. Dogs, anesthetized with sodium barbital, were sequentially injected iv with 0.2-1.0 ml (0.44-2.2 g) of ethylene dibromide for a total of 5.5 ml (12 g).
Marked decreases in respiratory rate, blood pressure, and muscle reflexes resulted.
Ethylene dibromide at 0.3 ml (0.66 g) injected iv caused cessation of respiration which was "momentary" or lasted as long as 40 seconds in the dogs tested. The cessation was followed by panting and a gradual return to almost normal breathing rate. The same dose produced a 75% decrease in blood pressure, the fall being immediate and sharp. The blood pressure gradually returned toward normal in a period of 2-12 minutes; but, in some cases, it remained 30% below normal. Ethylene dibromide at doses of 0.1-0.3 ml (0.22-0.66 g) injected iv caused a profound and rapid depression of the knee jerk reflex, which never returned to normal after the exposure.
The effects on dogs exposed to 1.0-10.0 ml (2.2-22.0 g) of ethylene dibromide vaporized from an ether bottle were essentially the same as those caused by the iv injections, differing only slightly in the extent of the decreases . The recovery times were described as being prolonged because of the longer exposure times, but complete experimental details were not given.
The investigators   The autopsy showed bronchopneumonic foci and severe hyperemia in both lungs.
A spherical thrombus was found in the heart, and the liver showed pronounced fatty degeneration.
The effects noted above in the hearts of the dogs exposed to ethylene dibromide were also found in isolated, perfused hearts from male frogs (Rana temporaria) subjected to 400, 800, or 1,600 ppm of ethylene dibromide in Ringer's solution . Immediate diastolic arrest occurred at 800 and 1,600 ppm with no recovery of function. At 400 ppm, there was diastolic arrest after 1 minute, but washing produced a gradual recovery to normal function.
No cardiac arrest or change in the heart rate was found at a concentration of 200 ppm of ethylene dibromide.
In 1946, Aman et al  studied the effects of repeated oral administration of ethylene dibromide on rats and guinea pigs. Nineteen animals were administered ethylene dibromide at average daily doses of 0, Total accumulated dosages of ethylene dibromide ranged from 0 to 1,420 mg. One rat, given 20 mg of ethylene dibromide daily, died after 3 months. All other rats and guinea pigs appeared to have gained weight normally during the experimental periods, and no untoward signs of toxicity were noted. Autopsy and microscopic examinations were not mentioned. The authors concluded that daily administration of ethylene dibromide for periods up to 4 months did not adversely affect the growth and outward physical appearance of rats and guinea pigs. The lack of experimental detail and data, the ambiguity of the data given, the inadequate number of control and experimental animals, and the absence of autopsies make this study difficult to evaluate and its importance questionable.
In 1952, Rowe et al  investigated the effects of ethylene dibromide administered to rats, guinea pigs, rabbits, mice, chickens, and monkeys by single oral intubations, eye contact, dermal contact, dermal absorption, single exposure inhalation, or repeated exposure inhalations.
The ethylene dibromide used in these studies was the 99% pure, commercial quality product, except that the inhalation studies were conducted with a repurified commercial product of essentially 100% purity. Ethylene dibromide was administered in undiluted form for eye and dermal contact, dermal absorption, and inhalation studies; in olive oil and acacia emulsion or olive oil solution for oral intubation studies; in propylene glycol solution for eye contact studies; or in butyl carbitol acetate solution for dermal contact studies.
Fifty-five female rabbits, 28 chicks, 40 male and female guinea pigs, 40 female rats, 60 male rats, and 20 female mice were administered ethylene dibromide at sufficient doses to enable calculation of single oral-dose LD50's of 55 mg/kg, 79 mg/kg, 110 mg/kg, 117 mg/kg, 146 mg/kg, and 420 mg/kg, respectively . The LD50's for male and female rats were significantly different (P<0.05). Of the five species tested, rabbits were the most sensitive to ethylene dibromide and mice were the least sensitive.
Rowe et al  found that undiluted ethylene dibromide promptly caused obvious pain and conlunctival irritation when introduced into the eyes of rabbits. The undiluted material was in contact with the eyes for 30 seconds before one eye was thoroughly flushed for 3 minutes with running water; the other eye was not washed. Both eyes of each rabbit were then observed for injury. The conjunctival irritation cleared within 48 hours, and a very slight amount of superficial necrosis of the cornea healed promptly and completely. When a 10% solution of ethylene dibromide in propylene glycol was tested in rabbits bv the same procedure used with the undiluted material, it produced a more severe response than did the undiluted material. Moderate conjunctival irritation developed within 2 hours and persisted for 48 hours before remission began to occur.
Moderate-to-severe corneal injury also persisted for about 48 hours before tissue repair became evident. Healing was complete 12 days after exposure without corneal scarring being evident. No injury to the iris or the lens of the eye was noted. A 1% solution in propylene glycol elicited a response in the rabbit eye very similar to that of the undiluted material.
The authors noted that prompt washing of the treated eyes had a beneficial effect in all cases, slightly reducing the intensity of the response and shortening the healing time.
Application of undiluted ethylene dibromide or 10% solutions in butyl carbitol acetate to the shaved skins of rabbits killed the animals within 24 hours . It was apparent that a few hours of confined contact of the material with the skin caused marked erythema and edema. When evaporation was not inhibited by a covering, only slight erythema was noted. A 1.0% solution of ethylene dibromide in butyl carbitol acetate applied to rabbit ears 10 times in 14 days caused only slight erythema and exfoliation. When the solution was applied repeatedly for 10 times in 14 days onto the shaved abdomen of the rabbit and then bandaged, marked erythema and edema, which progressed to necrosis and exfoliation, were observed. Healing was complete without scarring within 7 days after termination of both exposures.
Rowe et al  found that absorption of undiluted ethylene dibromide at doses of 210, 300, 650, or 1,100 mg/kg through the intact skin killed 1 of 15 rabbits at 210 mg/kg and all 5 rabbits at 1,100 mg/kg when the exposures lasted for 24 hours. In all of the exposed animals, the material produced a moderate-to-severe erythema, edema, and necrosis of the skin and caused scar formation in the survivors. Marked central nervous system (CNS) depression was seen in rabbits at all of the doses. At the 650 and 1,100 mg/kg doses, the animals felt cold to the touch. Deaths occurred within 4 days or not at all. The authors did not calculate an LD50 for dermal absorption in the rabbits; however, an LD50 of approximately 400 mg/kg for ethylene dibromide can be estimated from the dose-response data.
These investigators  also studied the effects of single exposures of ethylene dibromide vapor to groups of 4-30 rats of both sexes at concentrations of 100, 200, 400, 800, 1,600, 3,000, 5,000, or 10,000 ppm (770, 1,540, 3,080, 6,160, 12,300, 23,100, 38,500, or 77,000 mg/cu m, respectively) and to guinea pigs of both sexes at 200 or 400 ppm (1,540 or 3,080 mg/cu m ) . At each concentration, the rats were exposed to the vapor for durations ranging from 0.02 to 16.0 hours to allow for the determination of concentration versus period of exposure values that represent death in essentially all of the rats, death in 50% of the rats, and death in essentially none of the rats. The findings of this study are presented in Table III-l. These data suggest that at concentrations above about 5,500 mg/cu m (715 ppm), the Haber product  becomes actually almost a constant but that, at lower concentrations of ethylene dibromide, the Haber product increases rapidly as the concentration is lowered further. This suggests, in turn, that exposure of the rat to a concentration of about 5,500 mg/cu m (715 ppm, 29.2 /jmoles/liter) of ethylene dibromide saturates some detoxification mechanism. Whether this is metabolic or excretory in nature cannot be judged from these data. Adapted from Rowe et al  Ethylene dibromlde produced only slight anesthetic effects at the concentrations used . Depression of the CNS was observed in rats exposed at the higher concentrations (unspecified). Deaths usually occurred within 24 hours at the higher concentrations and were caused by respiratory or cardiac failure. Deaths occurring from exposures at lower concentrations (unspecified) were generally delayed, sometimes for as long as 12 days after exposure. The majority of these deaths were caused by pneumonia. These animals usually lost weight, appeared rough and unkempt, became quite irritable, discharged what appeared to be a blood-tinged fluid from the nose, and finally died. Animals surviving the exposure at the lower concentrations exhibited a similar progression of toxic signs for several days before recovery was apparent. Rats exposed at concentrations producing mortality and killed for autopsy 16-24 hours after exposure showed an increase in the weight of the lungs, liver, and kidneys. The lungs were congested, edematous, hemorrhagic, and inflamed; the liver had cloudy swelling, centrilobular fatty degeneration, and necrosis; and the kidneys exhibited slight interstitial congestion and edema, with slight cloudy swelling of the tubular epithelium in some cases. Guinea pigs appeared to be slightly less susceptible than rats to the effects of ethylene dibromide vapor when exposed to the same concentrations. All guinea pigs exposed at 400 ppm (3,080 mg/cu m) for 7 hours died, whereas all those exposed at 400 ppm (3,080 mg/cu m) for 7 hours and at 200 ppm (1,540 mg/cu m) for 7 hours lived.
The authors made no attempt to determine concentrations and corresponding exposure durations that would produce an LD99.99, LD50, or LD0.01 in guinea pigs, but they did postulate that 30 ppm (231 mg/cu m) for 5 hours was the most severe repeated exposure without detectable adverse effects.
Rowe et al  subjected rabbits, monkeys, guinea pigs, and rats to Two groups of controls, well-matched with the experimental animals with respect to number, age, sex, and body weight, were used in the experiment.
One group was exposed to the same experimental regimen as the group exposed to ethylene dibromide, but in a chamber ventilated with clean air. The second control group was simply maintained in the animal quarters.
During the test period, 10 of 20 ethylene dibromide-exposed male rats died, primarily from pneumonia and upper respiratory tract infections, and 3 of 20 ethylene dibromide-exposed female rats died from unspecified causes . Twenty-three additional female rats subjected to 13 7-hour exposures at 25 ppm (192.5 mg/cu m) in 17 days showed no ill effects and were found to have total liver lipid contents similar to those of control rats.
Groups of 8 male and 8 female guinea pigs tolerated 145 7-hour exposures at 25 ppm (192.5 mg/cu m) in 205 days without evidence of adverse effects as judged by the same criteria used to evaluate the rat data, except that liver lipid determinations were not conducted. Mortality during the experiment, caused by pulmonary infections, was 50% in male and 25% in female guinea pigs exposed to ethylene dibromide. Eighteen of the 20 animals in both the male and female control groups exposed to clean air survived the experimental regimen, but only 7 and 8 of the 20 females and males, respectively, in the unexposed control groups survived. An additional group of 8 female guinea pigs received 13 7-hour exposures at 25 ppm (192.5 mg/cu m) in 17 days without exhibiting adverse effects.
Rabbits, 3 male and 1 female, and monkeys, 1 male and 1 female, were subjected to 152 7-hour exposures in 214 days and 156 7-hour exposures in 220 days, respectively, at 25 ppm (192.5 mg/cu m ) . There were no signs of adverse effects in the rabbits and monkeys when judged by the above criteria for the guinea pig data. Since an unusually high mortality rate was observed in the nonexposed control rats but not in the air-exposed control animals, no decisive conclusions can be drawn from the published data with respect to the hazard resulting from exposure to ethylene dibromide at 25 ppm (192.5 mg/cu m ) .
Rowe and his colleagues  found that rats, guinea pigs, monkeys, and rabbits did not tolerate ethylene dibromide well at a concentration of 50 ppm (385 mg/cu m) administered 7 hours/day, 5 days/week, for 70-90 days.
The group of 20 male and 20 female rats receiving 63 exposures in 91 days exhibited increased liver and kidney weights in both sexes, increased lung weights in males, decreased spleen weights in females, and decreased testis weights in males at autopsy. The group of 8 male and 8 female guinea pigs receiving 57 exposures in 80 days showed decreased rates of growth and final body weights, increased organ weights, slight central fatty degeneration of the livers, and slight interstitial congestion and edema with degeneration of the tubular epithelium in the kidneys. The 1 male and 3 female rabbits receiving 59 exposures in 84 days showed only small increases in liver and kidney weights; no other adverse effects were noted.
The male and female monkeys receiving 49 exposures in 70 days appeared ill, nervous, and unkempt throughout the experimental period. Liver weights were increased and very slight central fatty degeneration of the liver was noted; no other adverse effects were noted from microscopic examination of the body organs.
At concentrations of 100 ppm (770 mg/cu m) of ethylene dibromide vapor, 10 female rats steadily lost weight and 3 died after 1, 5, and 7 exposures, respectively, for 7 hours/day . The remaining rats appeared thin and unkempt at the time of autopsy after seven exposures in 9 days.
The stomachs were full of food which appeared blood tinged, and lung, liver, and kidney weights were increased markedly. Microscopic examination showed some thickening of the alveolar walls with slight leukocytic infiltration of the lungs, widespread cloudy swelling of the liver, and slight congestion and hemosiderin deposition in the spleen. Rabbits subjected to the same concentration suffered severe intoxication to the extent that two of four died after the second 7-hour exposure and the third died while receiving the third exposure.
The fourth animal was killed after receiving the fourth exposure in 4 days. Microscopic examination of tissues from the last two rabbits showed widespread central fatty degeneration of the liver with some necrosis.
The authors  concluded from the above series of experiments that ethylene dibromide was a fairly toxic, markedly irritating material. They postulated that ingestion of 1.5-2.0 ml (3.3-4.4 g) of undiluted ethylene dibromide could jeopardize the life of the average human and that this amount of material could easily be swallowed by accident. Rowe et al pointed out that, although ethylene dibromide was not likely to cause permanent injury to the eye, it probably would cause appreciable pain and suffering; thus, eye protection was strongly recommended for those handling ethylene dibromide. Although the hazard of contact with uncovered skin was not thought to be particularly serious unless repeated or prolonged, marked irritation and rapid absorption of ethylene dibromide occurred when it was confined to the skin.
Ethylene dibromide vapor at 50 ppm (385 mg/cu m) repeated daily was not well tolerated by the species tested, whereas adverse effects were not believed to have resulted from exposure at 25 ppm Ethylene dibromide administration was discontinued in two of the four bulls to ascertain the reversibility of the exposure, and was later readministered to one of these two to determine the time needed for ethylene dibromide's action to be effective .
In the first animal, almost normal spermatozoa (the acrosomes were not entirely restored) were obtained after exposure had been discontinued for 1 month; semen of normal density, spermatozoic motility, and sperm cell forms was obtained 3.5 months after discontinuation.
In the second bull, normal semen was obtained 10 days after administration was stopped. After 2 further weeks of renewed exposure, semen from the second bull began exhibiting the same abnormalities as described above for bulls receiving ethylene dibromide on the continuous schedule. A previously unexposed 16-month-old bull of the same breed was fed ethylene dibromide at 2 mg/kg/day for 3 weeks. After 2 weeks, his semen exhibited the abnormalities described above, which persisted for a month after exposure ended. The semen quality improved, but normal semen was not obtained until 2-3 months after administration stopped.
From these experiments, the authors  concluded that quantities of ethylene dibromide, equivalent to two or three times that expected in insufficiently aired grains, caused definite reproductive impairment when fed daily or every other day to bulls. Semen density and spermatozoic motility decreased sharply and the spermatozoa were of abnormal shape after only 2 weeks of exposure. Recovery occurred in 10 days-3 months in the animals tested. Resumption of exposure caused the abnormalities to return.
Studies to determine whether the reproductive impairment was prolonged after repeated removal and reinstatement of ethylene dibromide diets were not conducted. However, in the one bull that was reexposed after a period of discontinuation, the time necessary for recovery of normal spermatozoic production increased from 10 days after the first discontinuation to 2-3 months after the second.
In Eleven bulls (D Amir, written communication, August 1976) given ethylene dibromide in olive oil were 15-24 months of age and weighed 400-500 kg.
The two remaining bulls given ethylene dibromide in olive oil were 4.5 and 5 years of age and weighed 950 and 1,050 kg, respectively. Six young bulls, 15-24 months of age, were used as controls; three were given olive oil and three remained as negative controls. Six of the 11 young bulls given ethylene dibromide and the 6 control bulls were castrated or killed (number killed or castrated not specified) 1 day after receiving the last oral dose. Semen was collected from the seven remaining bulls two or three times a week for 2 months after the first dose and once or twice a week for the next 2 months. Spermatozoic concentration, motility, and malformation were determined microscopically from at least 400 randomly chosen spermatozoa from each semen sample. In the control bulls, 2-4% of the spermatozoa taken from various places along the genital tract had misshapen heads, and distribution of the malformed spermatozoa was uniform throughout the tract.
In the animals that were killed or castrated, the number of spermatozoa exhibiting abnormal, mainly pear-shaped heads was markedly increased in the ethylene dibromide bulls over that of the controls and varied in distribution in the genital tracts of the different bulls. Amir
 attributed the distributional pattern of abnormal spermatozoa in the genital tract to the variation in the time of release of the spermatozoa through the ductus deferens caused by individual variations in the doseresponse threshold of the bulls.
In the semen samples collected from the five young bulls, maximum numbers of spermatozoa with misshapen heads appeared in the ejaculates 2-10 days after the cessation of ethylene dibromide administration . Again, these differences were thought to be caused by differences in the sperm transit time through the epididymis as well as to the variation in release time of the affected spermatozoa from the testes. The effect of ethylene dibromide was more acute in the adult than in the young bulls. The concentrations of spermatozoa in the younger bulls were only slightly affected by the doses, but were greatly reduced in the older bulls. Normal spermatozoic concentrations were regained after 1 and 4 months in the two older bulls.
During the period of low spermatozoic concentration, the ejaculates of the older bulls contained much spermatozoic debris and many spermatids and spermatocytes; the debris presumably came from further disintegration of the abnormal spermatozoa. The number of abnormal spermatozoa in the young bulls decreased to about normal within 3 weeks after the last dose, but remained elevated in the two adult bulls for about 15 weeks. The author concluded that ethylene dibromide affected spermiogenesis in the young bulls since the abnormalities in the spermatozoa observed in the ejaculates persisted for a period that coincided with the duration of spermiogenesis in the bull, which is about 3 weeks. Amir also concluded that either ethylene dibromide affected earlier stages of the spermatogenic process in adult bulls or its elimination from the circulation of young bulls was more rapid.
In 3 weeks. They also observed that hens that had ceased egg production could not be induced to lay again by feeding them a bromide-free diet, even after feeding continued for several months.
To determine the effects of small amounts of ethylene dibromide in grain on egg laying, the authors  subjected 4 groups of 16 6-month-old hens each at concentrations of ethylene dibromide in grain ranging from 0 to 30 ppm (total ration concentrations of 0-15 ppm). In addition to the ethylene dibromide, small amounts of "residual bromide," defined as the bromine-containing material remaining in sorghum grain that had been fumigated with ethylene dibromide and then aerated to remove free ethylene Sixty 1-day-old female chicks were divided equally into two groups, the first was fed twice daily a commercial mash containing 40 ppm of ethylene dibromide and the second served as controls in a paired-feeding design.
Weights and feed consumption were recorded weekly up to 10 weeks of age.
Feed intake, weight gain, onset of egg production at 4.5-5 months, and incidence of double-yolked eggs were the same for the experimental and control groups.
The weight of eggs from the group receiving ethylene dibromide was significantly lower (P<0.01) than from the controls and the number of eggs laid by the hens receiving ethylene dibromide approached statistical significance, lowering below that of the control hens.
In a second experiment , two groups of 12 1-year-old hens in full egg production were fed for 4 weeks a commercial mash with or without 100 ppm of ethylene dibromide. At the end of 4 weeks, egg weight in the hens fed ethylene dibromide had significantly decreased from an average of 63 to 43 g. Both groups were then artificially inseminated twice at 7-day intervals with 0.1 ml of semen/hen. Eggs collected between the 2nd day after the first insemination and the 7th day after the second insemination were examined for embryos. A striking reduction in the fertilization rate, with no live embryos at all, was noted in the ethylene dibromide-fed group; only 2 of 16 eggs were fertilized as compared with 48 of 56 eggs from the control group, and both fertilized eggs from the ethylene dibromide group contained dead embryos.
Similarly, a third experiment  was conducted to determine the effects of ethylene dibromide on male chicken growth rate and sexual development.
Three groups of 20 3-day-old cockerels were fed mash containing 0, 80, or 180 ppm of ethylene dibromide in which the amount of feed intake by the 180-ppm group determined the amounts of food given to the control and 80-ppm groups. Three additional groups of 25 cockerels were each fed mash containing 0, 150, or 300 ppm of ethylene dibromide without restrictions on the intake. At 6 weeks, the cockerels fed 150 ppm of ethylene dibromide showed a reduced weight gain when compared with the controls. Only cockerels fed the 300-ppm ethylene dibromide diet without restrictions on food intake showed significant growth retardation and an apparent feed intake depression at 12 weeks of age; however, the weight gain to feed intake ratio was not significantly different. The cockerels receiving the regulated diets were killed at 3 months of age, and the bromide content of the testes, spermatozoic count and activity, and testes weight were determined. Significant amounts of bromide were found in the testes of the ethylene dibromide-fed groups, but differences were not observed in spermiogenic activity, spermatozoic count, or testes weight between the control and ethylene dibromide-fed groups. The remaining unrestricted-intake cockerel groups were examined at the age of 9 months by collecting semen samples three times at weekly intervals and were killed at 12 months of age for testicular examination and measurement of body, testes, and comb weights. Semen collected and examined from ethylene dibromide-fed cockerels did not differ significantly from that of the controls. Comb weights at death declined sharply with increasing concentrations of ethylene dibromide in the diet; however, body and testes weights were not affected by the ethylene dibromide.
Another experiment  was conducted to determine if ethylene dibromide administration to adult cockerels would affect fertility rates and hatchability of eggs. Eleven mature cockerels each were fed for 105 days a control mash or mash containing 300 ppm of ethylene dibromide.
Semen was collected about 2, 4, 8, and 10 weeks after the beginning of the experiment. No significant differences were seen between the semen of the controls and that of the cockerels fed ethylene dibromide with respect to ejaculated volume and spermatozoic motility and concentration. Subsequent fertilization tests in hens conducted with the pooled semen collected from the ethylene dibromide-fed and control groups gave no significant differences in fertilization rate or hatchability of eggs at 60 or 105 days.
The authors  concluded that prolonged feeding of mash containing ethylene dibromide significantly depressed growth of male chickens when fed without restrictions, but that the depression seemed to result from reduced food intake and not from the direct action of ethylene dibromide. They also concluded that ethylene dibromide had no effect on the onset of egg production in hens fed from birth, on sexual development in males and females, and on sperm characteristics or fertility in mature males.
However, statistically significant reductions in egg size and egg fertility were noted in hens fed ethylene dibromide. previously given ethylene dibromide as described above.
The weights of whole eggs and yolks were compared for each hen prior to, during, and for 10 days after the injections. Injection of the purified hormone did not change the total egg weight or the weight of the yolks, which both remained lower than comparable control values. The authors concluded from the above four experiments that ethylene dibromide did not affect pituitary FSH concentrations in treated hens. The injection of FSH preparations did not reverse the adverse effects of ethylene dibromide; therefore, the effects of ethylene dibromide on egg laying did not seem to be connected with impaired formation or release of FSH.
In an attempt to clarify the causes of smaller eggs in laying hens fed ethylene dibromide-fumigated mash In 1970, Edwards et al  investigated the antifertility effects of ethylene dibromide in an unspecified number of adult male Wistar rats.
Doses of 10 mg/kg were injected ip into 250-g rats for 5 consecutive days.
The average live litter size from six female rats serially mated on a weekly schedule with the males receiving ethylene dibromide decreased substantially during the 3rd week and drastically (to zero) during the 4th
week after administration. From the authors' data, the average litter size appears normal during the 5th week and for every week thereafter. The authors concluded that the antifertility effects resulted from ethylene dibromide selectively damaging the spermatid cells, since the short course of exposure produced only transient sterility in rats and corresponded in time to changes that would result from spermatid damage. In an additional study , the major metabolite of ethylene dibromide in urine, S-(2hydroxyethyl)-cysteine, was reported to have produced no effect on male mouse fertility at a dose of 1,000 mg/kg when administered daily for 5 days by oral intubation, although experimental details and data were not presented.
The data indicate that oral exposure to ethylene dibromide induces temporary sterility in male rats. However, further evaluation of the data is not possible because of the lack of experimental details, such as number of test animals or any data about control animals, that were not given by the authors in the publication. Differences have been reported in the abilities of rats and chickens to detoxify ethylene dibromide. Nachtomi et al  and Nachtomi and Alumot  observed that rats possessed a much greater ability to conjugate glutathione with ethylene dibromide than chicks . In addition, the rat liver formed significant amounts of lipids containing conjugated double bonds as a result of the peroxidation of lipids induced by ethylene dibromide in the liver microsomal supernatant fraction, whereas chicken liver was relatively inactive . Also, the concentration of triglycerides increased significantly in the rat liver, probably as a direct result of the formation of conjugated double bonds discussed above.
In 1970, Edwards and coworkers  reported the tissue distribution of radioisotope at various time periods after an ip injection of 40 mg/kg of carbon-labeled (14 C) ethylene dibromide in mice.
The data are presented in Table XII-3. One hour after injection, most of the 14Cradioactivity was found in the small intestine, with smaller amounts being found, in descending order, in the kidneys, liver, blood plasma, whole blood, large intestine, fat, and spleen. After 3 hours, the majority of the radioisotope was present in the large intestine, kidneys, and blood plasma, with smaller amounts being present in the whole blood, liver, small intestine, and spleen.
After 24 hours, all tissues were below 1.0% retention of 14C-radioisotope except for the whole blood, blood plasma, stomach, and kidneys. After 1 hour, essentially all of the administered radioisotope was present in the various mouse tissues, whereas only 89% was present after 3 hours and 16% after 24 hours. It is of interest to note that 3.1, 4.4, and 0.66% of the administered radioisotope was detected in the tail of the epididymis 1, 3, and 24 hours after injection, respectively. Smaller amounts of 14C-radioisotope, up to 1.5% at 3 hours, were also present in the testes. The data presented by the authors suggest that ethylene dibromide is rapidly distributed to a wide variety of tissues. Major concentrations of radioisotope accumulated in the liver, kidneys, and digestive tract.
There also seems to be a rapid depletion of radioisotope from the tissues, but the residual radioactive material is still widely distributed throughout the body tissues.
In 1976, Plotnick and Conner  conducted a similar study of the tissue distribution of carbon-labeled (14 C) ethylene dibromide in guinea pigs.
The ethylene dibromide was administered in a single ip injection of 30 mg/kg and tissue samples were collected 4-72 hours after the injection.
The data are presented in Tables XII-4 and XII-5. At all intervals studied, the highest concentration of radioactivity derived from ethylene dibromide as lig/g of tissue was found in the kidneys, liver, and adrenal glands. At all time periods studied, the organs containing the highest percentage of the administered dose were the liver and kidneys.
Approximately 66% of the injected dose was excreted in the urine over the 72-hour study, 15% of which was in the first 4 hours, and approximately 3% was excreted in the feces. Plotnick and Conner mentioned that unpublished preliminary studies by their laboratory "strongly suggest that excretion of unchanged ethylene dibromide in the expired air also represents a significant (10-12% of the dose) route of excretion."
From the data of Edwards et al  and Plotnick and Conner , it seems that a relationship may exist between the tissue distribution and the destructive tissue changes, particularly with respect to the liver and kidney damage, in other animal  and human  studies reported previously in this chapter.

# Carcinogenic, Mutagenic, and Teratogenic Studies (a) Carcinogenesis
In 1973, Olson et al  reported the results of a preliminary investigation to determine the potential carcinogenic effects of ethylene dibromide in rats and mice of both sexes by oral administration. Further statements by Powers et al  and Ward and Habermann   Initially the only deleterious effect of ethylene dibromide on the rats was a depression in weight gain . As early as 10 weeks after the initiation of the experiment (dose not stated), one squamous cell carcinoma of the stomach was noted in one male and in one female rat. Squamous cell carcinomas of the stomach became more prevalent as the experiment progressed, developing in 83 of 100 male rats and in 70 of 100 female rats intubated with ethylene dibromide.
The tumors originated in the forestomach, invaded locally, and eventually metastasized throughout the abdominal cavity. Only one mammary tumor in a female rat was noted in the concurrent corn oil controls; however, in the untreated control group, 6 of 20 male rats and 14 of 20 female rats developed tumors, none of which were squamous cell carcinomas of the stomach. Tumor incidence was 68% in the male rats receiving ethylene dibromide at 80 mg/kg/day versus 98% in those receiving 40 mg/kg/day, and it was 58% in the female rats at 80 mg/kg/day versus 82% at 40 mg/kg/day. Olson et al  postulated that this decreased incidence of tumorigenesis may have resulted from the earlier death of the animals receiving the higher dose or because the animals did not receive ethylene dibromide for a 14-week period between weeks 16 and 30.
The induction sequence and results of microscopic examinations were discussed in detail only for the male rats receiving the 40 mg/kg dose, since the authors considered the induction and progression of the tumors to be similar in all experimental groups . All 50 male rats had stomach lesions, although one did not contain a neoplasm. The forestomachs had diffuse squamous cell hyperplasia with many papillomatous projections.
Focal areas of invasion were reported from the origin of the carcinomas.
Invasion occurred through the stomach wall to the peritoneal cavity, where nodules were reported in 70% of the rats. The metastatic tumors were less differentiated or formed keratin pearls, often accompanied by abcesses or peritonitis.
The authors  stated that other types of tumors and lesions were also present in a few rats, including mesotheliomas, intestinal tumors, nodular hyperplasias of the liver, and poorly differentiated stomach tumors.
Each group of mice received ethylene dibromide in doses of 60 or 120 mg/kg/day for 13 weeks . After 13 weeks, the doses were increased to 100 and 200 mg/kg/day but were reduced to the original doses after 2 weeks because of toxicity. At 42 weeks, the daily dose of ethylene dibromide for all mice was changed to 60 mg/kg/day. At 42 weeks, one male and one female mouse developed squamous cell carcinomas of the stomach at the higher dose regimen, and three males and two females at the lower regimen. High early mortality was encountered in mice receiving the higher dose (40%). No It seems that ethylene dibromide caused an increased incidence of gastric carcinoma in rats treated by intubation with either 40 mg/kg/day or an ordered combination of 80, 0, and 40 mg/kg/day. Carcinomas were found to originate at the site of administration (the stomach) in both species, to invade locally, and eventually to metastasize throughout the abdomen . Also, 72 of 145 mice that developed squamous cell carcinomas were diagnosed after week 59, whereas the vehicle controls were killed at week 59.

# (b) Mutagenesis
In 1972, Buselmaier et al  reported the results of an investigation to determine the potential for ethylene dibromide to induce mutations in the host-mediated assay with Salmonella typhimurium G46 and Serratia marcescens a21 Leu-in NMRI mice and in the in vitro plate test with Salmonella typhimurium G46. Immediately after ip inlection of each bacterial strain, 500 mg/kg of ethylene dibromide emulsified in edible oil was injected subcutaneously into one hind leg of six 10-to 12-week-old mice. Three hours after the injections, they were killed and the fluid from the peritoneal cavity was collected for plating to determine the number of mutant colonies induced. In the comparative in vitro plate test with Salmonella typhimurium, a concentration equivalent to the 500 mg/kg dose was applied to a filter paper disc in the center of the plate, and after a 12-hour incubation, the number of mutant colonies were counted.
The investigators  found that ethylene dibromide was definitely mutagenic in the host-mediated assay with Salmonella typhimurium G46 and in vitro with the same organism.
The mutation frequencies for Salmonella typhimurium G46 in the host-mediated assay were 0.77 x 10"8 in the control test and 6.23 x 10-8 in the experimental test. The ethylene dibromideexposed mutation frequency was significantly different (P<0.01) from the control.
The in vitro plate test results were also positive. Ethylene dibromide did not induce a significant number of mutants in the hostmediated assay with Serratia marcescens a21 (6.93 x 10"7 for control, 2.43
x 10" 7 for ethylene dibromide-exposed). The authors  concluded that ethylene dibromide did not require activation through in vivo metabolism to exert its mutagenic effects on Salmonella typhimurium, nor did metabolism of ethylene dibromide sufficiently deactivate its mutagenic potential, since both the host-mediated assay and the in vitro plate tests were positive.
In 1972, Epstein et al  published the results of a screening study on the detection of chemical mutagens by a dominant lethal assay in ICR/Ha Swiss mice. Ethylene dibromide was tested along with 173 other industrially important chemicals or chemical mixtures. Ten male mice, 8to 10-weeks-old, were given 0, 50, or 100 mg/kg of ethylene dibromide for 5 consecutive days by oral administration and were then mated weekly with three different virgin 8-to 10-week-old female mice for 8 consecutive weeks. One male mouse died at the 50 mg/kg dose and two males died at the 100 mg/kg dose during the experiment. Two different groups of seven or nine male mice received single ip injections of 18 or 90 mg/kg of ethylene dibromide, respectively, and were then mated as above. The mated females were killed about the 13th day after presumptive mating and were scored for the number of total implants, live implants, early fetal deaths, and pregnancies. The experimental animal scores were contrasted to concurrent control scores.
The authors included ethylene dibromide in a class of agents which did not meet "any screening criteria for mutagenic effects," although specific data for ethylene dibromide were not presented. No effect of ethylene dibromide on fertility was reported in this paper, which differs from the finding of Edwards et al .
In 1973, Clive  tested the mutagenic potential of ethylene dibromide on the back mutation frequency of the thymidine kinase locus in a mammalian somatic cell tissue culture derived from mouse lymphoma cells.
The L5178Y mouse lymphoma cells were exposed to ethylene dibromide at concentrations of 0.0-3.0 mM in culture media for 2 hours. The exposed cells displayed an induced mutational frequency that was dose related and typical of other alkylating agents, such as methyl methanesulfonate and ethyl methanesulfonate.
Thirteen new mutations for each 10,000 surviving cells were found under these experimental conditions .
The frequency of induced mutations generally increased monotonically after exposure of the cells to increasing concentrations of ethylene dibromide. The induced mutation frequency indicated that ethylene dibromide was mutagenic over the entire range of experimental concentrations when plotted against the growth inhibition, although less potent than ethyl methanesulfonate (an induced mutational frequency of about 2/10,000 cells versus one of 7/10,000 cells at 60% growth inhibition, respectively). The induced mutational frequency in L5178Y mouse lymphoma cells corresponded with that of a mutational frequency of over 600 R of X-irradiation at the highest concentration.
Clive concluded that ethylene dibromide was mutagenic to the L5178Y mouse lymphoma cells although not as potent as ethyl methanesulfonate.
In 1974, Meneghini  reported the results of an investigation to determine the potential for ethylene dibromide to induce DNA repair synthesis in cultured opossum lymphocytes. Two-year-old opossums, Didelphis virginiana, were used to obtain lymphocytes for culture. Blood was removed from their tails and lymphocyte suspensions were prepared and maintained at 37 C. Samples containing about 5 x 10^ cells/ml were incubated with various concentrations of ethylene dibromide for 1 hour at 37 C. At the end of this period, the cells were incubated in culture media with 3H-thymidine for 4 hours to allow for DNA radiolabeling to occur. Control cells were processed as above, but without exposure with ethylene dibromide.
At concentrations between 0.001 and 1 mM, ethylene dibromide was very effective in inducing DNA repair in opossum lymphocytes . Repair induction decreased at a concentration of 10 mM of ethylene dibromide, which was presumed to be the result of repair-inhibition phenomena such as those observed for ultraviolet-induced repair. The author indicated that ethylene dibromide was very effective in inducing DNA repair in a manner similar to other alkylating agents, such as methyl methanesulfonate and ethyl methanesulfonate, which were also studied. He stated that only compounds that interact with DNA through covalent bonds induce repair synthesis. Therefore, these data suggest that ethylene dibromide, or its metabolites, may interact via covalent bonding with DNA.
In 1974, Vogel and Chandler  measured the possible genetic effects induced by ethylene dibromide in sex-linked lethal tests with fruit flies, Drosophila melanogaster. The adult Berlin K male flies were allowed to feed on a 0.3-mM ethylene dibromide solution for 3 days before being allowed to mate with two females for 3 days. A sequence of two 3-day brood periods was initiated (broods 1 and 2), followed by one 4-day brood period (brood 3).
At the end of each 3-day breeding period, the treated males were transferred to a new vial and were allowed to mate with two new females.
The frequencies of recessive lethal mutations in meiotic and postmeiotic germ cells were determined in the offspring of treated and control males. Ethylene dibromide induced significant numbers of recessive lethal mutations in the offspring of all three broods. These were 0.50, 1.49, and 1.30% in broods 1, 2, and 3, respectively, and an average lethal frequency for all broods of 1.10%. The authors concluded that the brood pattern resulting from ethylene dibromide exposure was consistent with the action of chemicals that affect spermatids and spermatocytes because of the higher incidence of recessive lethal mutations in broods 2 and 3. The authors considered ethylene dibromide to be a bifunctional alkylating agent capable of introducing cross-links into large biologic molecules, and to be definitely mutagenic in Drosophila melanogaster.
In 1971, Ames  published the results of an experiment to determine the mutagenic potential of ethylene dibromide to the his-G46 and TA 1530 strains of Salmonella typhimurium. Ethylene dibromide inhibited the growth of the E coli pol Al-strain preferentially to isogenic pol A+ strain as indicated by zone of inhibition diameters of 20 versus 15 mm, respectively, at concentrations of 10 /I (22.0 mg)/plate .   for the induction of back mutations at the his-G46 locus in Salmonella typhimurium.
In 1975, Alper and Ames  published the results of a study to determine whether ethylene dibromide or 39 other agents could cause mutants by chromosomal deletions of various lengths in Salmonella typhimurium LT2
and Salmonella typhimurium galE503. Bacteria were poured onto agar plates as suspensions in top agar and were incubated for 8 hours. A few drops of the test compound, 1-5 il (2.2-11.0 mg), were placed near the edge of the plate immediately after the top agar had solidified.
The number of colonies clustered within a 3.5-cm radius of the spot where the compound was applied was used as an index of the extent of mutagenesis. The zone of inhibition for ethylene dibromide was reported as less than 1.5 cm from the application spot, but specific data were not presented. Although no experimental data were given, ethylene dibromide was included in a list of compounds which failed to increase the frequency of deletion mutants by as much as fourfold over that of the control.
In 1974, Sparrow et al 

# Ethylene dibromide has been reported to induce mutations in
Neurospora crassa , but complete experimental details were not given in these abstracts.
(c)

# Teratogenesis
In 1976, Short et al  examined the effects of ethylene dibromide vapor on rats and mice during organogenesis. The production of congenital defects was used as a measure of inherent toxicity. Female Charles River CD rats or female CD-I mice were caged overnight with proven male breeders; successfully bred females were identified the next morning by the presence of sperm in vaginal smears from rats or of copulation plugs in mice.
Pregnant animals were divided into a control group of 18 rats and 17 mice which would receive a normal diet without inhalation exposure to ethylene dibromide, a group of 18 rats and 13 mice to receive 31.6 ppm (243.32 mg/cu m) of ethylene dibromide and a normal diet, and a group of 17 rats and 9 mice to receive a restricted diet without exposure to ethylene dibromide.
All groups of animals were housed in the inhalation chambers for 10 consecutive days beginning on day 6 of gestation. During this time, the groups to be given ethylene dibromide were exposed to the vapor at an average concentration of 31.6 1.9 ppm (243.32  14.63 mg/cu m) for 23 hours a day. Rats and mice were killed on gestational day 20 or 18, respectively. Fetuses were surgically removed from the dams, weighed, examined for external anomalies, and fixed for either soft-tissue or skeletal examinations. Ethylene dibromide exposure did not produce mortality in either pregnant rats or mice during the 10-day inhalation period; however, two of nine pregnant mice from the restricted diet group died. Both rats and mice exposed to ethylene dibromide consumed less feed and gained significantly (P<0.05) less weight than the controls during the exposure period, although feed consumption and weight gain returned to normal after cessation of the exposure on the 15th day of pregnancy.
Ethylene dibromide-exposed rats consumed about twice as much food daily as did the feed-restricted rats (10.5  0.6 versus 5.0  0.1 g/day, respectively), but only about one-half of that consumed by the controls (10.5  0.6 versus 21.1  0.4 g/day, respectively).
Rats exposed to ethylene dibromide at a concentration of 31.6 ppm  The authors  concluded that the above-mentioned effects of ethylene dibromide exposure in mice were most likely attributable to malnourishment rather than to ethylene dibromide exposure. It is apparent from the authors' data that some of the anomalies may be compounded by the presence of ethylene dibromide. In addition, if the malnourished controls and ethylene dibromide-exposed animals had been compared with the nonexposed controls at the same statistical probability levels, only the ethylene dibromide-exposed animals would have been significantly different from the controls.
Skeletal anomalies, in particular the variations in ossification of the incus and supraoccipital bones, were much more prevalent in the ethylene dibromide-exposed mice than in the feedrestricted mice (80-90% of the litters versus 33-50%, respectively). The authors also attributed many of the observed anomalies in the rat dams and fetuses exposed to ethylene dibromide to malnutrition rather than to direct effects of ethylene dibromide exposure. However, they concluded that the increase in the fourth ventricular "hydrocephaly," the reduction in the occurrence of the fourteenth rib, and the increase in the frequency of wavy ribs could be correlated to ethylene dibromide exposure in the rat.
The significance of the effects of malnutrition in rats is not clear, and even though ethylene dibromide-exposed rats consumed less food than did controls, they consumed more food than did the rats in the feed-restricted group. It is apparent, however, that an increase in the incidence of anomalies in fetal rats was induced by exposure to the one concentration of ethylene dibromide, but no dose-response relationship for this action is available.

# Correlation of Exposure and Effect
Neither studies that correlate workplace concentrations of ethylene dibromide with observed toxic effects nor any epidemiologic studies have been found. Few reports of human exposure to ethylene dibromide exist. Adverse effects resulting from exposure of experimental animals to ethylene dibromide are similar to those described for human exposures and include ocular, dermal, and respiratory irritation and systemic effects on the liver, kidneys, spleen, circulatory system, and nervous system.
The effects of ethylene dibromide exposure on the eyes have been noted in several animal species. Merzbach  discovered that a dog exposed for 1 hour to 1 ml (2.2 g) of ethylene dibromide vaporized into a 100-liter chamber showed signs of ocular irritation during the exposure.
Five hours after the exposure ended, a milky-blue corneal opacity developed which became more pronounced and developed into purulent conjunctivitis in both eyes and an ulcer in one eye. Kochmann  showed that 100 ppm (770 mg/cu m) of ethylene dibromide for 30 minutes/day produced conjunctivitis in cats exposed to the vapor repeatedly until death occurred at approximately 10 days. Rowe et al  reported that instillation of undiluted ezhylene dibromide into the eyes of rabbits caused conjunctival izritation that cleared within 48 hours and left only very slight superficial corneal necrosis which healed completely. Rowe et al  also found that a 10% solution of ethylene dibromide in propylene glycol produced a more severe reaction in the eye than did the undiluted material.
Moderate conjunctival and corneal irritation developed and persisted for 48 hours; healing was complete within 12 days without corneal scarring. A 1% solution produced an effect similar to that caused by the undiluted material. These reports indicate that ethylene dibromide can cause adverse ocular effects after exposure to 1 and 10% solutions, as well as to the undiluted material.
The skin of experimental animals is susceptible to penetration and local surface effects resulting from exposure to ethylene dibromide.
Thomas and Yant  noted that liquid ethylene dibromide at doses of 0.25, 0.50, and 1.0 ml (0.55, 1.1 and 2.2 g)/animal produced marked hyperemia of the small cutaneous blood vessels of the shaved abdomens of rats. All animals died within 6-18 hours after application. Rowe et al  found that undiluted ethylene dibromide or a 10% solution in butyl carbitol acetate killed within 24 hours all rabbits to which it was applied dermally; subsequent experiments showed that the dermal LD50 was about 400 mg/kg. Marked erythema and edema were noted when the material was prevented from evaporating from the skin, whereas only slight erythema was noted when evaporation was not inhibited. Undiluted doses of about 210 mg/kg produced a moderate-to-severe erythema, edema, and necrosis of the skin. Similar results were described by Pflesser  in human volunteers exposed to 0.5 ml (1.1 g) of liquid ethylene dibromide for 1-30 minutes by placing small quantities on their arms or hands. Confinement produced more extensive erythema and edema surrounding the application site, and, in one case, blistering occurred as a result of continued contact between ethylene dibromide and the skin. These investigations indicate that localized surface effects on the skin, such as erythema, edema, blistering, or necrosis, may occur after contact with either undiluted or 10% solutions of ethylene dibromide. Percutaneous absorption of 10% solutions has also caused death in experimental animals.
Respiratory irritation after single exposures to ethylene dibromide vapor has been reported in guinea pigs at concentrations of 8,000 ppm (61,600 mg/cu m) for 30 minutes, 4,000 ppm (30,800 mg/cu m) for 60 minutes, and 2,000 ppm (15,400 mg/cu m) for 150 minutes , and in cats exposed at 100 ppm (770 mg/cu m) for 30 minutes . In cats, three 30-minute exposures at 100 ppm (770 mg/cu tn) produced signs of nasal irritation that consisted of a strong reddening of the nasal mucosa . Lucas  demonstrated that a 12-minute exposure of a rabbit to ethylene dibromide vapor caused the lungs to become enlarged and filled with a frothy exudate.
Merzbach  found that 5 ml (11.0 g) of ethylene dibromide allowed to vaporize in a 100-liter chamber produced severe bleeding in the right lung of a dog exposed for 1 hour. Another dog exposed to the vapor of 1 ml (2.2 g) for 1 hour developed severe hyperemia and bronchopneumonic foci in both lungs. Rowe et al  showed that death occurring in rats exposed to ethylene dibromide at concentrations of 100-10,000 ppm (770-77,000 mg/cu m) for 0.02-16.0 hours was caused by respiratory or cardiac failure at the higher exposures and by pneumonia at the lower ones. The lungs of animals exposed at these concentrations were congested, edematous, hemorrhagic, and inflamed.
Respiratory irritation has also been noted after the repeated inhalation exposure of cats at 100 ppm (770 mg/cu m) to ethylene dibromide for 30 minutes daily for an average of 10 days. This concentration produced dark red discolorations in the lungs, and the lungs were partially nonfunctional . Rowe et al  reported on lethal dose rates in rats exposed to ethylene dibromide for 0.02-16.0 hours at concentrations of 100-10,000 ppm (770-77,000 mg/cu m). Death was caused by respiratory or cardiac failure at the higher concentrations and by pneumonia at the lower ones.
Half of the male rats exposed at 25 ppm (192.5 mg/cu m) for 7 hours/day for 151 exposures in 213 days died during the experiment, primarily from pneumonia and upper respiratory tract infection. Pulmonary infections were also responsible for a 50% mortality in male guinea pigs and a 25% mortality in females exposed at 25 ppm (192.5 mg/cu m) for 7 hours/day for 145 exposures in 205 days .
Systemic parenchymal cell damage, particularly in the liver, kidneys, and spleen, develops after single and repeated exposure of experimental animals to ethylene dibromide vapor. Thomas and Yant  observed that guinea pigs receiving single exposures of ethylene dibromide vapor at concentrations of 8,000 ppm (61,600 mg/cu m) for 30 minutes, 4,000 ppm (30,800 mg/cu m) for 60 minutes, or 2,000 ppm (15,400 mg/cu m) for 150 minutes had a slight granular degeneration of the parenchymal tissue of the liver. Rabbits exposed to an unknown concentration of ethylene dibromide vapor for 10 or 12 minutes had enlarged livers, with slight-to-moderate diffuse fatty changes evident in the rabbit exposed for 10 minutes .
Merzbach  published a study detailing similar results in a dog exposed to 1 ml (2.2 g) of vaporized ethylene dibromide in a 100-liter chamber for 1 hour; autopsy showed that a pronounced fatty degeneration of the liver had occurred. Rowe et al  observed that rats exposed at 100-10,000 ppm (770-77,000 mg/cu m) of ethylene dibromide for 0.02-16.0 hours developed cloudy swelling, centrilobular fatty degeneration, and necrosis of the liver. Repeated exposure at approximately 100 ppm (770 mg/cu m) for 30 minutes/day for up to 10 days caused incipient fatty degeneration in the liver of cats . According to Rowe et al , rats exposed to 100 ppm (770 mg/cu m) of ethylene dibromide for 7 hours/day for seven exposures in 9 days had a cloudy swelling of the liver, whereas rabbits exposed to the same concentration for three or four exposures had widespread central fatty degeneration and some necrosis of the other cells of the liver. Rowe et al  reported that slight central fatty degeneration of the liver developed in guinea pigs repeatedly exposed to 50 ppm (385 mg/cu m) of ethylene dibromide for 7 hours/day for 57 exposures in 80 days and in monkeys repeatedly exposed to the same concentration for 49 times in 70 days.
Renal damage, in the form of pronounced granular degenerative changes in the parenchymal tissues, occurred in guinea pigs exposed to ethylene dibromide at concentrations of 8,000 ppm (61,600 mg/cu m) for 30 minutes, 4,000 ppm (30,800 mg/cu m) for 60 minutes, or 2,000 ppm (15,400 mg/cu m) for 150 minutes . Rowe et al  demonstrated that the kidneys of rats exposed at 100-10,000 ppm (770-77,000 mg/cu m) of ethylene dibromide vapor for 0.02-16.0 hours showed slight interstitial congestion and edema, with slight cloudy swelling of the tubular epithelium in some cases.
Repeated exposures at approximately 100 ppm (770 mg/cu m) for 30 minutes/day for an average of 10 days in cats produced a swelling and discoloration of the kidneys and possibly a slight degeneration of the tubules . Rowe et al  reported that repeated exposure of guinea pigs at 50 ppm (385 mg/cu m) of ethylene dibromide for 7 hours/day for 57 times in 80 days produced a slight interstitial congestion and edematous condition in the kidneys which was accompanied by degeneration of the tubular epithelium. From these data, it can be concluded that renal damage can occur in experimental animals after exposure to single or repeated exposures of ethylene dibromide.
Adverse splenic effects have been seen in experimental animals after exposure to ethylene dibromide. Single exposures of ethylene dibromide at 8,000 ppm (61,600 mg/cu m) for 30 minutes, 4,000 ppm (30,800 mg/cu m) for 60 minutes, or 2,000 ppm (15,400 mg/cu m) for 150 minutes produced a slight granular degeneration of the parenchymal tissue in the spleen of guinea pigs . Rats exposed to 0.25, 0.50, or 1.0 ml (0.55, 1.1, or 2.2 g) of ethylene dibromide applied on the abdomen died within 6-18 hours; autopsy showed that the spleens of these animals were highly congested and edematous . Kochmann  noted a slight enlargement of the spleens of cats exposed at 100 ppm (770 mg/cu m) of ethylene dibromide for 30 minutes/day for an average of 10 days. Rowe et al  observed that rats receiving seven exposures to ethylene dibromide at 100 ppm (770 mg/cu m), 7 hours/day, for 9 days developed a slight congestion of the spleen and some hemosiderin deposition. The results from these experiments indicate that adverse effects to the spleen can occur after exposure to ethylene dibromide by inhalation or by percutaneous absorption.
Cardiovascular system effects have been noted in experimental animals subjected to single or multiple exposures of ethylene dibromide.
Concentrations of 8,000, 4,000, and 2,000 ppm (61,600, 30,800, and 15,400 mg/cu m) of ethylene dibromide for 30, 60, and 150 minutes, respectively, produced a slight granular degeneration of the muscular tissue of the heart and a generalized interstitial edematous degeneration of the endothelial lining in the abdominal vascular system in guinea pigs . Merzbach  exposed a dog to the vapor of 5 ml (11.0 g) of ethylene dibromide in a 100- postulated that death resulting from exposure of cats and rabbits at 50-100 ppm (385-770 mg/cu m) of ethylene dibromide for up to 22 days was caused by injury to the circulatory system, especially to the heart and blood vessels. Rowe et al  also concluded that deaths occurring at the higher concentrations after exposure of rats at 100-10,000 ppm (770-77,000 mg/cu m) of ethylene dibromide for 0.02-16.0 hours were caused by cardiac or respiratory failure.
Few studies have specifically addressed the potential of ethylene dibromide to adversely affect the CNS. Rowe et al  reported that CNS depression was observed at the higher concentrations in rats exposed to 100-10,000 ppm (770-77,000 mg/cu m) for 0.02-16.0 hours, but did not further elucidate the nature of the effects. Rowe et al   Experiments with bulls receiving 4 mg/kg on alternate days for 7 or 10 doses showed that the abnormal spermatozoa were present in both the epididymis and testes after 7 doses. After 10 doses, abnormal spermatozoa constituted 67% of the population in the ductus deferens and 77% of the ejaculate, with almost all the abnormal spermatozoa exhibiting tail and acrosomal defects  . These spermatozoic abnormalities may lead to a decrease in the fertility of the bulls and might induce sterility if they continued long enough, although reports have not been found that experimentally test this hypothesis. In rats, ip injections of ethylene dibromide at a dose of 10 mg/kg for 5 days produced a decrease in the average live litter size in female rats mated with experimental males . This decrease in fertility occurred during the 3rd and 4th weeks following administration, which corresponds to the maturation cycle of spermatids in the genital tract of rats. This finding, which corresponds with the findings of the reports of spermiogenic impairment in bulls, supports the postulated sterilizing effect discussed above. It is important to point out that reports have not been found delineating a no-effect level for the reproductive system abnormalities caused by the repeated administration of ethylene dibromide to bulls.
In chickens, the function of the female reproductive system is impaired by ethylene dibromide exposure , but the male system appears to be unaffected . Cockerels fed 300 ppm of ethylene dibromide for 12 months showed significant growth retardation at 12 weeks of age; however, semen collected between 9 and 12 months did not differ from control semen, and body and testicular weights were comparable with those of controls . Semen collected between 2 and 10 weeks from cockerels fed 300 ppm for 105 days showed no significant differences when compared with control semen with respect to ejaculated volume and spermatozoic motility and concentrations. Subsequent fertility tests at 60 or 105 days in hens gave no significant differences in fertilization rates or hatchability of eggs. These results indicate that the reproductive system in male chickens was not detectably affected by ethylene dibromide under these experimental conditions. This is in contrast to the effects reported in two mammalian species, cattle and rats, in which definite impairments of the male reproductive system have been noted. A striking reduction (12 versus 86% in the control) in the fertilization rate was seen in hens fed 100 ppm of ethylene dibromide for 4 weeks; all embryos in the eggs from the experimental group were dead  . These results indicate that the female reproductive system in chickens is affected by ethylene dibromide. Although no reports have been found that explore the potential of ethylene dibromide to affect fertility in females of mammalian species, the avian data suggest the potential of ethylene dibromide to impair female fertility. However, until experiments are conducted with mammalian species to assess this potential, extrapolation of the effects seen in chickens to mammals and humans must be uncertain.
There has been only one study concerning the potential of ethylene dibromide to induce cancer in rats and mice . In this study, rats and mice were given the maximum tolerated dose (80 mg/kg for rats, 120 mg/kg for mice) and one-half the maximum tolerated dose by daily intubation for 54 or 62 weeks, except when toxicity forced the total discontinuation of administration or reduction of the maximum tolerated dose to that of one-half the maximum tolerated dose during the experiment. The tumors (squamous cell carcinomas), which were first noted during the 10th week of ethylene dibromide administration in rats, originated in the forestomach, invaded locally, and metastasized throughout the abdominal cavity. The fraction of animals with tumors was greater in the male rats than in the females (an average of 83% of the males developed tumors versus 70% of the females), and was greater at the lower dose than at the higher dose in rats at the termination of the experiment at 54 weeks (98 versus 68% in males and 82 versus 58% in females, respectively). The concurrent control populations did not develop squamous cell carcinomas of the stomach. The fraction of mice that developed squamous cell carcinomas was 74% in males and 72% in females by the termination of the experiment at weeks .
The irregularities in the dose regimens of both species, the use of the suggested maximum tolerated dose, and the route of administration do not negate the importance of the fact that ethylene dibromide has induced carcinomas in two mammalian species.
The data from this single study indicate that ethylene dibromide is a carcinogen after daily introduction of about one-half the maximum tolerated dose into the stomach of rats and mice for up to 62 weeks.
The mutagenic potential of ethylene dibromide has been established in a wide spectrum of procaryotic and eucaryotic mutational test systems. It has induced mutations in vertebrate cell cultures , insects , bacteria , plants , and fungi . It has induced mutations or mutagenic events in a number of experimental test systems, including the recessive lethal test in Drosophila melanogaster , the host mediated assay in mice with Salmonella typhimurium , and a backward mutation system with mouse lymphoma cells .
In addition, positive mutagenic induction has been reported in tests with back mutational systems in certain strains of Salmonella typhimurium .
Ethylene dibromide has also been reported to have given negative results in the dominant lethal test in mice  and in the back mutational test system with Serratia marcescens in the host mediated assay .
The dominant lethal test conducted in male mice given 50 or 100 mg/kg of ethylene dibromide for 5 consecutive days by oral intubation or given 18 or 90 mg/kg by ip injection did not induce a significant increase in the number of dead implants when compared with controls , although specific data were not presented for evaluation. Buselmaier et al  reported that ethylene dibromide was mutagenic in the host mediated assay with Salmonella typhimurium G46 at a dose of 500 mg/kg injected subcutaneously in mice and in vitro with the same organism at an equivalent dose. The mutational frequency for the experimental group was significantly (P<0.01) different from the control mutational frequency. The positive results in the in vivo and in vitro tests indicate that metabolic activation is not necessary for ethylene dibromide to exert its mutagenic effects and that metabolic deactivation does not reduce the mutagenic effect. Buselmaier et al  reported that a similar in vivo test with Serratia marcescens, also a back mutational test system, was negative; this finding may only indicate that species differences exist in reactions with ethylene dibromide.
In a recessive lethal test with Drosophila melanogaster, Vogel and Chandler  reported that 0.3 mM of ethylene dibromide fed to adult males for 3 days induced significant numbers of recessive lethal mutations in the offspring of three successive broods. The brood patterns resulting from ethylene dibromide exposure indicated that ethylene dibromide affected spermatozoic maturation more than spermatozoic formation. The authors considered ethylene dibromide typical of a bifunctional alkylating agent and capable of introducing cross-links into biologic molecules.
A mammalian somatic cell tissue culture of L5178Y mouse lymphoma cells was exposed to 0.0-3.0 mM of ethylene dibromide in culture media . The induced mutagenic frequency was dose related, typical of other alkylating agents tested, and approximately equivalent to a dose of 600 R of X-irradiation at the highest concentration. Meneghini  noted that ethylene dibromide very effectively induced DNA repair synthesis in opossum lymphocyte cell cultures at concentrations between 0.001 and 1 iriM but decreased at 10 mM. Only compounds that interact with DNA through covalent bonds induce repair synthesis; therefore, ethylene dibromide is typical of other alkylating agents in forming covalent bonds with DNA.
Bacterial and other plant systems have also been used to show the mutagenic potential of ethylene dibromide. Ames   hours.
The ability of ethylene dibromide to induce mutations in a wide variety of test systems is suggestive of its potential to induce mutations in human populations, but there is no evidence available to enable an adequate assessment of the quantitative aspects of the relative risks for human populations. Since ethylene dibromide is a bifunctional alkylating agent, the most plausible basis for the induction of mutations in these systems is the covalent bonding of ethylene dibromide to the genetic material, DNA. The linear relation between the number of induced mutations and the concentration of ethylene dibromide in Tradescantia  is consistent with the idea of covalent bonding between ethylene dibromide and DNA.
Only one study pertaining to the potential of ethylene dibromide to induce fetal anomalies has been found . Fetuses from pregnant mice and rats exposed to approximately 31.6 ppm (243.32 mg/cu m) of ethylene dibromide for 23 hours/day during days 6-15 of gestation were significantly different from fetuses of pregnant control mice and rats. These differences include costal anomalies and hydrocephaly in rat fetuses, and additional anomalies in other ossification processes in mice fetuses.
Fetuses from control mice fed a feed-restricted diet during the experiment to simulate malnourishment exhibited some of the effects found in ethylene dibromide-exposed mice fetuses; however, the frequency of the type and number of anomalies formed in the malnourished group was reduced, but the degree of significance was not as great as that found in the ethylene dibromide-exposed mice fetuses. Although it can be argued that the effects produced by ethylene dibromide exposure are similar in part to those produced by malnourishment, the anomalies in both rat and mouse fetuses from ethylene dibromide-exposed dams were significantly different from those produced by malnourishment alone when both were compared with those found in fetuses from control dams. From these data, it is evident that ethylene dibromide caused fetal abnormalities in mice and rats that were not caused by malnourishment alone.
In summary, it is concluded from the data from human and experimental studies that the adverse systemic effects resulting from exposure to ethylene dibromide may include ocular, dermal, and respiratory irritation,      The concentration of airborne ethylene dibromide on the premises of an oil refinery ranged from 0. In this report, both the most likely and worst-case emission concentrations were calculated, using the average value of 0.9-1.3 g of ethylene dibromide/gal of automotive fuel (one equivalent of organic bromine is added to gasoline for each two equivalents of lead added). The estimated emission rate for ethylene dibromide by measurement was reported to be 0.000063 g/g lead/gal. The reported losses for entrainment and spillage each were 0.000047 g/g lead/gal. Thus, the total loss because of refueling was reported to be 0.000157 g/g lead/gal. Evaporative losses were calculated to be 0.00014 g/g lead/gal from the automobile fuel tanks.
in
The most likely estimate of loss from the carburetor was 0.0016 g/g lead/gal, whereas the worst case was 0.0043 g/g lead/gal. Exhaust emissions were also calculated, the most likely being 0.0065 g/g lead/gal and the worst case being 0.3 g/g lead/gal. Summarizing the three types of emissions, the total emissions calculated for refueling, evaporation, and exhaust was most likely 0.008397 g/g lead/gal and the worst case, 0.304597 g/g lead/gal. The total emissions can also be estimated directly in terms      Liquid absorption media, such as a 1:1 mixture of monoethanolamine and dioxane, have been used as trapping solutions for ethylene dibromide . This medium decomposes ethylene dibromide and many other brominated organic contaminants to inorganic bromide and, as such, is not specific for ethylene dibromide.
Porous polymer beads have been used as a collection medium for chlorinated and brominated hydrocarbons . This procedure has not been tested specifically for ethylene dibromide but has been used successfully for closely related halogenated hydrocarbons; thus, it seems feasible that ethylene dibromide could also be collected by this method. The same column is used for sample collection and gas-liquid chromatographic analysis, but only one analysis can be made on each sample.
Silica gel has been used as a collection medium for ethylene dibromide . One advantage of using a solid adsorbent is that sample loss cannot occur from spillage during sampling or in transit for analysis.
However, silica gel is a polar adsorbent and shows pronounced selectivity in adsorbing polar molecules, particularly water . Studies with silica gel tubes indicated that water vapor could displace organic molecules during a normal sampling operation . Although similar studies have not been conducted for ethylene dibromide, it is reasonable to assume that similar displacement of significant quantities of ethylene dibromide by water vapor could occur.
Activated charcoal has been used as an adsorbent in conjunction with gas-liquid chromatography . Charcoal is an excellent collecting medium because of its nonpolarity and its affinity for organic vapors and gases. As such, water vapor does not readily displace organic molecules as is the case with silica gel. However, adsorption and desorption efficiencies may vary with different batches of charcoal; therefore, it is necessary to determine the desorption efficiency for each new batch of charcoal.
Charcoal tubes containing as much as 600 mg of activated charcoal are commercially available ,
In the past several years, direct-reading instruments and devices have been developed which make continuous or "on-the-spot" monitoring of halogenated hydrocarbons, including ethylene dibromide, feasible. These devices, when properly calibrated and used within their performance characteristics and limitations, can be helpful in monitoring airborne halogenated hydrocarbons . Colorimetric indicator tubes are available from at least three sources  which provide semiquantitative measurement in the range of 1-200 ppm, although no detector tubes have been certified as yet by NIOSH for response to ethylene dibromide. The use of infrared light absorption at a wavelength of 8.4 jum  is claimed to detect airborne ethylene dibromide at a concentration of 0.1 ppm. These direct-reading instruments are portable and also may be used as part of a multipoint sampling system for continuous, unattended monitors.
Other sampling devices used for the collection of organic solvents and halogenated hydrocarbons may be adaptable to ethylene dibromide collection. These include sampling bottles , bubblers , and plastic bags . Chemical analysis has been used to estimate ethylene dibromide concentrations in the collection media. Aeration has been used by Peterson et al  to desorb ethylene dibromide from the silica gel; pyrolysis of the free ethylene dibromide produced bromine and hydrobromic acid. These products were collected in a second absorption solution of 1% sodium carbonate and 1% sodium formate in deionized water and quantitated by titrimetric analysis of the inorganic bromide present. Aman et al  132 estimated the airborne ethylene dibromide concentration by absorbing the free ethylene dibromide with 1% sodium hydroxide and by quantitating the hypobromite formed iodometrically. Dumas  determined airborne ethylene dibromide concentrations by coulometric titration of sodium bromide after absorption of ethylene dibromide in methanolic sodium hydroxide and digestion by boiling under reflux for 15 minutes on a steam bath. These methods, like most chemical methods, require somewhat bulky apparatus and are not specific for ethylene dibromide since any aliphatic brominated compound will interfere and be included in the quantitative estimate.
Physicochemical methods have been developed or adapted to identify and quantitate concentrations of airborne ethylene dibromide. Christie et al  modified a refrigerant leak-detector lamp to detect organic halogen compounds, including ethylene dibromide, in the atmosphere at a minimum concentration of 5 ppm. The procedure is dependent on visual estimation of the intensity of a flame color in response to the vapor concentration and is not specific for ethylene dibromide.
Flame chemiluminescence has been reported by Crider  to detect ethylene dibromide concentrations in air of 0.03 ppm. This method has not been tested on mixtures of halogenated hydrocarbons, and it is too early in its development to enable prediction of the practicality of the method for area or personal monitoring in an occupational environment.
Other physicochemical methods have been developed for halogen compounds, but they have not been tested for identification and quantitation of ethylene dibromide. One of these methods, based on nitrogen enhancement of an AC spark, has been used for continuous measurement of halogenated compounds in field situations . The instrument is lightweight, portable, fast-responding, and reportedly capable of detecting halogenated hydrocarbons in the ppm range. However, until testing with ethylene dibromide has been conducted, the feasibility and reliability of this instrument is not known.
In recent years, gas-liquid chromatography has become the most prevalent method for the detection and analysis of organic materials . Direct analysis of airborne ethylene dibromide has been reported to result in measurement of amounts as small as 0. (1) the delayed and insidious onset of symptoms, (2) an odor threshold which is not adequate to provide warning of dangerous concentrations, (3) its irritating and penetrating effects on the skin, and (4) its potential for causing cancer, mutations, sterility, and fetal anomalies.
The principal method for manufacturing ethylene dibromide is the bromination of ethylene . Small quantities of vinyl bromide, ethyl bromide, and ethyl chlorobromide may be formed as impurities in the production of ethylene dibromide , and caution must be taken to avoid exposure to these substances as well.
Ethylene dibromide is nonflammable and nonexplosive at ordinary temperatures, but since it can be decomposed to toxic and corrosive compounds, such as hydrobromic acid, by contact with open flames or red-hot surfaces , it should be appropriately stored and handled to prevent such contacts. Special precautions are necessary for maintenance and emergency repair work, such as welding, cutting, or any spark-and flame-generating operations. Furthermore, it is recommended that smoking be prohibited in workplace areas where ethylene dibromide is manufactured, handled, blended, or stored. Since decomposition may occur  and highly toxic aldehyde vapors and acid gases may be emitted , ethylene dibromide must be protected from direct light and excessive temperature. Ethylene dibromide reacts rapidly with certain metals, such as aluminum and magnesium, to form combustible and explosive organometallic compounds  and with liquid ammonia , Therefore, it is recommended that reasonable precautions be taken to keep ethylene dibromide separated from these materials.
Engineering controls should be used to keep the concentration of airborne ethylene dibromide below the recommended occupational exposure All containers used to transport, hold, or process ethylene dibromide should be made of ethylene dibromide-resistant materials, such as lined steel or stainless steel, and should be periodically inspected for signs of wear, corrosion, or leaks by manual and instrumental means. All valves, pipes, and seals used in pumping ethylene dibromide from processing areas to storage areas or transportation loading sites should be made of ethylene dibromide-resistant materials and should be periodically inspected for possible leaks, weak points, or signs of wear.
Ethylene dibromide is a severe eye and skin irritant in humans (G Ter Haar, written communication, January 1977) and can be absorbed through the intact skin of animals in quantities sufficient to present an imminent hazard . In view of this, the use of personal protective equipment, including nylon-impregnated neoprene gloves , ethylene dibromideresistant and fire-retardant clothing, rubber boots or overshoes, bib-type aprons, and chemical safety goggles is recommended when contact by liquid ethylene dibromide with the skin and eyes is possible.
In addition, it is recommended that proper respiratory protection be worn when entering an area where the concentration of the vapor of ethylene dibromide may be greater than the recommended occupational exposure limit. Clothing should be immediately removed if it becomes contaminated, and the skin of the exposed area should be thoroughly washed with water.
Personnel working with ethylene dibromide must be instructed in emergency procedures and participate in periodic, simulated emergency drills.
All personnel not involved in the specified emergency operations must be immediately evacuated from the area. Special training sessions must be held and written emergency procedures must be updated periodically.
These should include the location, use, and maintenance of first-aid, firefighting, and decontamination equipment.
To prevent the adverse effects caused by ethylene dibromide, it is recommended that exposure to ethylene dibromide vapor or liquid be kept at a minimum. Routine visual and functional inspections must be made by trained personnel to ensure that processes in which ethylene dibromide is used are completely closed. If leaks or spills occur in the processing, handling, or storage of ethylene dibromide, these must be promptly corrected, regardless of the ethylene dibromide concentration in the environment. It is recommended that nonessential personnel be evacuated from the immediate area where a leak or spill has occurred until decontamination of the area is complete. If employees must withdraw samples from a process involving the use of ethylene dibromide, an impervious suit, including gloves, boots, and air-supplied hood, should be worn. An effective exhaust system for trapping any ethylene dibromide vapor may be used in place of the air-supplied hood. Any waste or residues containing ethylene dibromide should be incinerated, buried, or otherwise disposed of so that no ethylene dibromide is released into the environment.
Applicable local, state, and federal regulations should be followed. Air exhausted from ethylene dibromide workplace areas must be decontaminated by incineration, chemical treatment, or other effective means so that the release of ethylene dibromide into the environment will be minimized.
Safety showers and eyewash fountains should be located in or near areas where ethylene dibromide exposures are likely to occur and should be properly maintained.
It is recommended that employees immediately remove contaminated clothing, flush all affected skin surfaces with water for at least 15 minutes, and then obtain medical attention.
Since ethylene dibromide is toxic when ingested and has caused death in a woman after ingestion , it is recommended that handwashing facilities, soap, and water be made available to the employees. As a good hygiene practice, it is recommended that employees wash their hands before consuming beverages or food, using tobacco, or using toilet facilities.
The employer should provide lunchroom facilities physically separated from the ethylene dibromide work areas.
It is recommended that any contaminated article of personal protective equipment be discarded or, if feasible, decontaminated with soap and water. It is also recommended that employer-supplied clothing be worn while working with ethylene dibromide and that the employer arrange to have this clothing laundered daily and properly maintained. The employer should inform the launderers of the possible hazard of coming into contact with contaminated clothing and advise him on safe methods of handling such material.
The employer should provide separate locker and change facilities for work and street clothes. As a good hygiene practice, it is recommended that shower facilities be provided for the employees and that they be required to shower before leaving the workplace at the end of the work shift.
Since ethylene dibromide is a component of some insecticidal fumigants and conventional work practice guidelines are inappropriate to protect agricultural workers from the hazards of exposure, it is recommended that the label precautions on such pesticides be followed and stringently adhered to. These label requirements usually specify allowable time limits before a fumigated space or area may be reentered and safe practices for the application of the particular fumigant mixture. Specific requirements of worker protection standards for agricultural pesticides can be found in 40 CFR 170.
In summary, precautions should be exercised with ethylene dibromide to prevent serious consequences which may result from ingestion, inhalation, and skin or eye contact. Processes in which ethylene dibromide is used in large quantities should be carried out in closed systems.
Well-designed hoods, ventilation systems, and exhaust systems should be used to maintain concentrations below those specified by this standard.
Personal protective equipment and clothing should be worn by employees engaged in the manufacture, handling, or blending of ethylene dibromide.
It is important that employees be informed of the hazards associated with ethylene dibromide before job placement and whenever changes are made in any process that may alter their exposure.

# VI. DEVELOPMENT OF STANDARD Basis for Previous Standards
In 1953, the American Conference of Governmental Industrial Hygienists (ACGIH) adopted a threshold limit value (TLV) of 25 ppm as an 8hour TWA concentration for 1,2-dibromoethane (ethylene dibromide) .
No specific basis for this TLV has been found. In 1954, the ACGIH  changed the name to ethylene dibromide in the official TLV list, and, in 1956, they added the value of 190 mg/cu m to the official entry . In al  reported that ethylene dibromide was readily absorbed through the intact skin of rabbits and from the gastrointestinal tract of rats, mice, guinea pigs, chickens, and rabbits. Ethylene dibromide vapor caused CNS depression, pulmonary irritation, and liver and kidney damage in rats and guinea pigs after single exposures. Rats, guinea pigs, monkeys, and rabbits tolerated repeated exposures of ethylene dibromide vapor at 25 ppm for 7 hours/day, 5 days/week, for about 6 months without adverse effects, but these species did not tolerate well a similar exposure at 50 ppm.
In 1965, the ACGIH  recommended that special emphasis be given to the potential for skin absorption of 1,2-dibromoethane by adding the designation "skin" after the name in the TLV list. Such a notation refers to the potential contribution to overall exposure by the dermal route, including mucous membranes and eyes, either by airborne, or more particularly, by direct contact with ethylene dibromide. This designation was intended to indicate that measures for the prevention of absorption from skin and mucous membranes were necessary if the TLV was to be successful in limiting occupational exposure to a safe level. In 1965, the ACGIH  also recommended that the TLV of 25 ppm as a TWA concentration for ethylene dibromide be tentatively changed to a ceiling limit of 25 ppm.
The basis  for this limit was primarily the report by Rowe et al , discussed previously, and the paper by Lucas . Lucas  observed that single 10-to 12-minute exposures of rabbits to a concentration of ethylene dibromide vapor sufficient to produce anesthesia resulted in rapid breathing, phonation, and death within 15-18 hours. The shift of the TLV from a TWA value to a ceiling limit was made final in 1967.
In 1971, the ACGIH  recommended changing the ceiling limit of 25 ppm to an 8-hour TWA concentration of 20 ppm (145 mg/cu m ) . The 1971 Documentation of the Threshold Limit Values for Substances in Workroom Air  cited several studies with no particular emphasis on how the limit was set. These reports included Rowe et al , Lucas , Kochmann
, Olmstead , Rowe et al , and McCollister et al , which are discussed in Chapter III. Presumably, the study by Rowe et al  was the principal basis on which the new limit was set. The proposed value of 20 ppm as a TWA concentration was adopted in 1973 . The basis for this change as stated in the 1974 supplement of the 1971 Documentation , did not differ from that stated in 1971.
In 1976, the ACGIH  added to the TWA value a tentative short term exposure limit (STEL) of 30 ppm (220 mg/cu m) , which is defined as a maximal concentration to which workers can be exposed for a period up to 15 minutes continuously. No more than 4 such excursions are permitted each day, with at least 60 minutes between successive exposure periods. Also, the daily TLV-TWA is not to be exceeded.
Florida, Mississippi, Pennsylvania, and South Carolina have adopted a TWA concentration of 25 ppm (190 mg/cu m) as their environmental limit for ethylene dibromide .
In Finland, the German Democratic Republic, and Yugoslavia, the maximum allowable concentration (MAC) for the workplace environment is 190 mg/cu m (25 ppm) . In the Rumanian Socialist Republic, 200 mg/cu m (approximately 26 ppm) of ethylene dibromide is the maximum concentration allowed in the occupational environment, whereas in Poland, the MAC allowed for ethylene dibromide is 100 mg/cu m (13 ppm) . Although the USSR does not currently recommend a standard for ethylene dibromide, the US recommendation of 145 mg/cu m (20 ppm) is stated to be inadmissibly high.
The 1976 edition of the Handbook for Chemists, Engineers and Physicians  states that, in all likelihood, the permissible concentration should be on the same order as the Russian MAC for dichloroethane (12.5 ppm) or lower . No bases for these standards have been found.
The current federal standard (29 CFR 1910(29 CFR .1000) for occupational exposure to ethylene dibromide is 20 ppm as an 8-hour TWA limit, with an acceptable ceiling concentration of 30 ppm. A maximum peak above the acceptable ceiling concentration for an 8-hour work shift of 50 ppm not to exceed 5 minutes (Federal Register 40:103, May 28,1975) is also permitted.
This standard was adopted from the American National Standards Institute (ANSI) recommendation Z37.31-1970 , which was based on the reports of Rowe et al  and Olmstead , and the summary presentation by Irish , which includes synopses of several other reports  discussed in Chapter III. , and conjunctival irritation was noted in rabbits after instillation of undiluted, 10%, or 1% solutions of ethylene dibromide . The irritation subsided within 2-12 days in the rabbits without causing corneal scarring. Marked hyperemia of the cutaneous blood vessels surrounding the application site was found after 0.25, 0.50, or 1.0 ml (0.55, 1.1, or 2.2 g) of ethylene dibromide was applied to the abdomen of rats . Rabbits responded similarly when undiluted or 10% solutions of ethylene dibromide were applied to the abdomen . Application of 210 mg/kg of the undiluted chemical caused marked erythema, edema, and necrosis of the skin.
Respiratory tract irritation caused by ethylene dibromide vapor has been seen in guinea pigs after single exposures at 2,000 ppm (15,400 mg/cu m) for 150 minutes , and for cats after exposures as low as 100 Other systemic damage also occurred in animals after single and repeated exposures to ethylene dibromide vapor.
Guinea pigs exposed at concentrations of ethylene dibromide of 2,000 ppm (15,400 mg/cu m) for 150 minutes developed a pronounced granular degeneration of the parenchymal tissue of the kidneys and a slight degeneration of the parenchymal tissue of the liver, spleen, and heart . Rats exposed at concentrations between 100 and 10,000 ppm (770 and 77,000 mg/cu m) of ethylene dibromide for 0.02-16.0 hours developed cloudy swelling, centrilobular fatty degeneration, and necrosis of the liver, cloudy swelling and a slight interstitial congestion and edema of the kidneys, and CNS depression at the higher concentrations .
Repeated inhalation exposures of rats for 7 days and rabbits for 3-4 days at 100 ppm (770 mg/cu m) of ethylene dibromide for 7 hours/day produced slight congestion of the spleens of rats, cloudy swelling and a slight leukocytic infiltration in the livers of rats, and widespread central fatty degeneration and some necrosis in the livers of rabbits .
Exposure of guinea pigs and monkeys at 50 ppm (285 mg/cu m) of ethylene dibromide for 7 hours/day, 5 days/week, for 70-80 days caused only slight central fatty degeneration of the liver . Cats exposed at 100 ppm (770 mg/cu m) of ethylene dibromide for 30 minutes/day for about 10 days had enlarged spleens. Death resulted from circulatory system damage to the heart and vessels .
CNS effects, such as agitation, restlessness, body tremors, or unconsciousness, are caused by exposure to ethylene dibromide ], but they have not been sufficiently described to permit an adequate evaluation or quantitation of their relevance or validity.
The information provided by the few reports on humans  and such experimental animal data as those given in the following references  indicates that many effects produced by ethylene dibromide on humans and animals are similar and differ only in magnitude. Respiratory tract irritation, damage to the liver, kidneys, spleen, and lungs, irritation of the skin and eyes, and gastrointestinal disturbances are the predominant effects of ethylene dibromide exposure. Since systemic effects may occur from ingestion, inhalation, or dermal contact with ethylene dibromide, NIOSH recommends that work practices be used to minimize employee exposure to ethylene dibromide liquid or vapor through inhalation, body or eye contact, or ingestion.
Mammalian studies indicate that reproductive abnormalities, including antifertility  and spermatozoic anomalies , occur from exposure to ethylene dibromide. One report  indicated that five ip injections of 10 mg/kg given to male rats produced a decrease in fertility only during the 3rd and 4th weeks after injection. Since spermatids require about 3-4 weeks to mature into spermatozoa in the rat, the evidence indicates that ethylene dibromide affects the development of the spermatids that were present at the time of injection. This is further supported by the return of normal fertility 5 weeks after the injection. Three studies with bulls In another experiment , production of abnormal spermatozoa occurred with a dose as small as 4 mg/kg given on alternate days for seven doses. These spermatozoic abnormalities would greatly reduce the fertility, even if they did not cause total sterility.
Although these effects were from the ingestion of ethylene dibromide and not from inhalation or dermal contact, they indicate that a hazard, including decreased fertility and even temporary sterility, may result from inhalation or percutaneous absorption of ethylene dibromide.
One study, reported by several authors , was conducted to determine the carcinogenic properties of ethylene dibromide. Rats and mice given daily oral doses by gavage of ethylene dibromide at 40 and 60 mg/kg, respectively, for 52-64 weeks developed squamous cell carcinomas in the stomach. These carcinomas invaded locally and metastasized throughout the abdominal cavity.
Male and female rats developed stomach carcinomas as early as 10 weeks after the start of administration of ethylene dibromide.
The carcinomas became more prevalent as the daily doses of ethylene dibromide were continued, and the final percentage of male rats with tumors after administration of 40 mg/kg/day was 98% after termination of the experiment at 54 weeks. Male rats were more susceptible to tumorigenesis than female rats; 80% of all the males in the study developed tumors versus 38% of all the females. The concurrent control populations did not develop squamous cell carcinomas. More than 70% of all the mice were reported to The mutagenic potential of ethylene dibromide is well established in both animal and plant systems. It induces mutations in vertebrate cell cultures , insects , bacteria , plants , and fungi  .
One study with Drosophila melanogaster  showed that ethylene dibromide induced a significant number of recessive lethal mutations in three successive broods of offspring. The adult males fed 0.3 mM of ethylene dibromide for 3 days produced subsequent brood patterns indicative of impaired spermatozoic maturation rather than of impaired formation.
Studies with mouse lymphoma cells  indicated that a dose-related effect, typical of those of other alkylating agents tested, existed over the range of 0.0-3.0 mM ethylene dibromide. The effect of the highest concentration was approximately equal to that of a dose of 600 R of Xirradiation.
A study in a host-mediated assay system  with Salmonella typhimurium G46 in mice suggested that ethylene dibromide was mutagenic at a dose of 500 mg/kg.
A second part of this study  showed that an equivalent dose produced a positive mutagenic effect on Salmonella typhimurium G46 in vitro, indicating that ethylene dibromide did not require metabolic activation and was not deactivated by metabolism.
Similar positive mutagenic results occurred in Salmonella typhimurium TA Several studies  suggest that the mechanism of mutagenic activity of ethylene dibromide is based on its ability to alkylate, or covalently bond to, DNA in the exposed cells. Ethylene dibromide is a bifunctional alkylating agent capable of introducing cross-links into biologic materials  by displacement of the two reactive bromine atoms by reacting with amine, sulfhydryl, carboxy or other electron-donating groups.
Ethylene dibromide, or its metabolites, has interacted with DNA through covalent bonds to induce DNA repair synthesis in opossum lymphocyte cells . Another indication of ethylene dibromide's ability to alkylate DNA is that ethylene dibromide is mutagenic in Salmonella typhimurium TA 1530 and TA 1535, both transitional mutational systems . These data  suggest strongly that the most plausible chemical basis for the mutagenic activity of ethylene dibromide in procaryotic and eucaryotic organisms is its alkylation of cellular constituents such as DNA. This broad biologic reactivity suggests that ethylene dibromide may be capable of increasing spontaneous mutation rates in humans. However, the quantitative aspects of this potential have not been determined. Since the process of induction of mutations is a stochastic, virtually irreversible process, any increased frequency of mutation in exposed populations would accumulate as a function of the total absorbed dose of ethylene dibromide. Therefore, an adequate assessment of the importance of the plant and submammalian animal data in extrapolating to the concentrations of airborne ethylene dibromide present in the workplace environment is difficult, but the widespread mutagenic activity of ethylene dibromide does give cause for concern about damage to the genetic mechanisms in employees working with it.
The teratogenic effects found in a rat and mouse study  involved brain and costal anomalies in the offspring of dams exposed to ethylene dibromide. Pregnant rats and mice were exposed at a concentration of about 32 ppm of ethylene dibromide for 23 hours/day on days 6-16 of gestation. Some of the effects were attributed to malnourishment, but the abnormalities in the ethylene dibromide-exposed rats and mice were significantly different, both qualitatively and quantitatively, from those in the nonexposed controls; some of these abnormalities did not appear in mice fed a restricted amount of the normal diet whereas others appeared in both the restricted and the control mice with about the same incidences.
These data suggest that ethylene dibromide causes fetal anomalies in mice and rats that are not caused by malnourishment alone. Since inhalation is one of the major routes of exposure for the employee, these data suggest also that the babies of female employees may be subject to increased risks of developmental defects if their mothers are exposed to ethylene dibromide in the workplace during the critical phases of pregnancy.
The total risk to the health of employees exposed to ethylene dibromide is the result of the compounded risks from carcinogenicity, mutagenicity, teratogenicity, sterility, and damage to the kidneys, liver, spleen, respiratory tract, central nervous system, circulatory system, skin, and eyes. Although no comprehensive epidemiologic studies have been conducted to assess adequately these risks in the industrial environment, evidence of their existence in experimental animal systems or in isolated human exposures to ethylene dibromide has been discussed above and in Chapter III. Experimentation conducted with animal models, as outlined above, generally supports the findings observed in the limited number of human exposures.
Concern for employee health requires that the probability of the occurrence of long-term effects of ethylene dibromide be minimized. The preliminary report available to NIOSH from a review of the mortality experience of 161 employees of one manufacturer   induced stomach cancer in the rat. In addition, the extensive experimental evidence on the induction of adverse effects in lower species 9,33,62,66,69] and the formation of stable covalent bonds between ethylene dibromide and cellular constituents  indicate that the occupational exposure limit should be lowered to decrease the potential hazard to employees.
Because of the intrinsic, stochastic, and virtually irreversible character of the chemical reactions that initiate the carcinogenic and mutagenic processes, the risk of adverse effects is a function of the rate of absorption and the total absorbed dose. Consequently, this risk can be reduced to any value necessary to protect the employees by decreasing both the absorption rate (to prevent saturation of the enzymatic detoxification mechanisms) and the total lifetime dose (to reduce the probability of deleterious stochastic processes, such as carcinogenesis and mutagenesis, from occurring).
The unusual complexity of the dynamics of the cellular response mechanisms to ethylene dibromide intoxication is emphasized by the doserate effect relationship observed for induction of gastric neoplasms.
Daily intragastric doses of 40 and 60 mg/kg induced tumors in rats and mice within 10 weeks , whereas daily 7-hour inhalation exposures of about 49 mg/kg did not result in the observation of tumors even after 30 weeks .
The data suggest that one of the major factors in the development of gastic carcinomas is direct contact between the mucosal cells and ethylene dibromide, and that a major difference in tumor induction may exist between the two routes of exposure. However, the animals used in the inhalation study were not maintained until the end of their normal lifespan; therefore, a direct comparison between the final incidences of tumors initiated by the two routes of exposure cannot be made.
A plausible pharmacokinetic explanation for the observed differences exists. Since the rat's capacity to metabolize ethylene dibromide  exceeds the rate of absorption (at 25 ppm) by a factor of at least 100, the concentration of ethylene dibromide in tissues should be considerably less than that in the air, which is about 1.02 /umoles/liter at 25 ppm . The concentration of ethylene dibromide in corn oil used in the intubation study ranged between 0.066 and 0.132 moles/liter. Consequently, the mucosal cells of the stomach may have been exposed for short periods to concentrations of ethylene dibromide up to 100,000 times that to which lung tissue would be exposed during inhalation exposures at 25 ppm (192.5 mg/cu m ) . The saturation of enzymatically catalyzed detoxification and repair mechanisms may occur in the stomach tissues, resulting in an amplification of the tissue damage, which otherwise may be minimal. These pharmacokinetic considerations are consistent with the experimental results of Rowe et al , where the product of the concentration and duration of exposure to produce 50% mortality among the exposed rats was found to be approximately constant for higher concentrations of ethylene dibromide but not for lower ones. The evidence indicates that tissue detoxication and repair mechanisms exist, but that they cannot negate completely the intrinsic capacity of ethylene dibromide to alkylate cellular constituents.
Because of this, exposure to ethylene dibromide has potentials for the A major factor in the development of gastric carcinoma in the rats and mice is considered to be the direct contact between mucosal cells and concentrated ethylene dibromide in a quantity which exceeded the ability of the tissues to handle the chemical. Mutagenic effects from ethylene dibromide have been demonstrated in microbes, plants, insects, and mammalian cells in vitro, presumably due to its ability to alkylate, or covalently bond, to DNA in a cross-linking mechanism by virtue of its bifunctional structure. Although this potential has not been demonstrated in humans, the broad biologic activity of ethylene dibromide warrants concern about possible damage to genetic mechanisms in employees exposed to it. Teratogenic effects which seem to be due to maternal exposure to ethylene dibromide have been confined to brain and costal anomalies in fetal mice and rats, but malnourishment as a contributing factor cannot be discredited unequivocally.
Although it is not possible at this time to state categorically an exposure concentration at which ethylene dibromide may be regarded to be completely without risk, NIOSH considers that the recommended occupational exposure limit should be substantially lower than the current federal standard of 20 ppm as an 8-hour TWA limit, 30 ppm ceiling. The recommended occupational exposure limit for ethylene dibromide, at the least, should be reduced sufficiently to keep the total lifetime dose well below the cumulative doses shown to be hazardous in animal experiments. In considering the alkylating capability of ethylene dibromide and the potential adverse effects on the organism which may result, especially at the subcellular level, NIOSH recommends that the occupational exposure limit for ethylene dibromide be reduced to a ceiling concentration of 1.0 mg/cu m (0.13 ppm) for any 15-minute sampling period. This represents a reduction to one-two-hundred and thirtieth of the current federal ceiling limit for ethylene dibromide and is a level at which an employee would inhale a maximum of about 686 mg/kg of ethylene dibromide during a 40-year working lifetime, which is substantially below that total dose known to induce adverse effects in experimental animals. It is believed that so long as care is taken to prevent entrance of any appreciable amount of ethylene dibromide into the digestive tract, adherence to this exposure limit will protect against acute adverse effects and will reduce the potential long-term effects to a negligible level. NIOSH concludes that reduction to a ceiling concentration of 1.0 mg/cu m (0.13 ppm) will protect employees from acute illness resulting from exposure to ethylene dibromide and will reduce markedly, and perhaps remove entirely, any hazard of adverse effects on health from long-term exposure to this chemical. Studies should be conducted to determine the feasibility of using body fluids, such as blood or urine, as the basis of a method for biologic monitoring of workers that are occupationally exposed to ethylene dibromide.
(h)

# Long-term Animal Exposure Studies
Long-term exposure of several animal species at a variety of concentrations of ethylene dibromide vapor approaching the recommended environmental limit is needed. These studies should simulate occupational exposure conditions of 8-10 hours/day, 4-5 days/week, for at least 18-24 months and the animals maintained until the end of their natural life.
These studies should be properly designed and performed to allow for assessment of general body parameters, biochemical/physiologic parameters, and gross or microscopic examinations of involved organs including at least the liver, lungs, spleen, kidneys, CNS, and circulatory system.
In addition, repeated long-term experiments should be performed to determine the effects of ethylene dibromide absorption through the skin.
Similar schedules and experimental designs as those for inhalation studies should be followed.
The National Cancer Institute has informed NIOSH that a long-term experiment to study the possible carcinogenic effects from the inhalation of ethylene dibromide is presently being conducted. Several studies  have indicated that ethylene dibromide, or its metabolites, is widely circulated throughout the body and remains widely distributed in the body tissues for a considerable time. These studies also indicated that ethylene dibromide, or its metabolites, is excreted in the urine and eliminated in the feces.  The sampling train consists of a charcoal tube and a vacuum pump.
(1) Charcoal tubes: Glass tubes, with both ends flamesealed, 7-cm long, with a 6-mm OD and a 4-mm ID, containing two sections of 20/40 mesh activated charcoal separated by a 2-mm portion of polyurethane foam. The activated charcoal is prepared from coconut shells and is fired at 600 C prior to packing. The primary section contains 100 mg of charcoal, the backup section, 50 mg. A 3-mm portion of polyurethane foam is placed between the outlet end of the tube and the backup section. A plug of silylated glass wool is placed in front of the primary section.

# METHOD FOR SAMPLING ETHYLENE DIBROMIDE IN AIR
The pressure drop across the tube when in use must be less than 1 inch of mercury at a flowrate of 1 liter/minute. Tubes with the above specifications are commercially available.
(2) Pump:
A battery-operated pump, complete with clip for attachment to the employee's belt, capable of operation at 200 ml/minute or less with a controlled accuracy of + 5%.

# (b) Calibration
The accurate calibration of a sampling pump is essential for the correct interpretation of the volume sampled. The frequency of calibration is dependent on the use, care, and handling to which the pump is subjected.
Pumps should also be recalibrated if they have been misused or if they have just been repaired or received from a manufacturer. If the pump receives hard usage, more frequent calibration may be necessary. Maintenance and calibration should be performed on a regular schedule and records of these should be kept.
Ordinarily, pumps should be calibrated in the laboratory both before they are used in the field and after they have been used to collect a large number of field samples. The accuracy of calibration is dependent on the type of instrument used as a reference.
The choice of calibration instrument will depend largely on where the calibration is to be performed. (2) Break the tips of a charcoal tube to produce openings of at least 2 mm in diameter.
Repeat the procedure in (7) above at least three times, average the results, and calculate the flowrate by dividing the volume between the preselected marks by the time required for the soapbubble to traverse the distance. If, for the pump being calibrated, the volume of air sampled is calculated as the product of the number of strokes times a stroke factor (given in units of volume/stroke), the stroke factor is the quotient of the volume between the two preselected marks divided by the number of strokes. Therefore, if the analysis cannot be performed within 16-24 hours after sampling has been completed, the samples must be stored at -25 C or below. Refrigerated samples may be stored for two weeks. (25 liters), the probable range of this method is 40-800 nanograms/sample solution at a detector sensitivity that gives nearly full deflection on the strip chart recorder for a 1-mg sample. The method may be capable of measuring much smaller amounts if the desorption efficiency is adequate and if a photon-ionization detector is used on the chromatograph. Desorption efficiency must be determined over the range used.
The upper limit of the range of the method is dependent on the adsorptive capacity of the charcoal tube. This capacity varies with the concentrations of ethylene dibromide and other substances in the air. The first section of the charcoal tube held at least 21.4 mg of ethylene dibromide when a test atmosphere containing 446 mg/cu m of ethylene dibromide in dry air was sampled at 200 ml/minute for 240 minutes; at that time, the concentration of ethylene dibromide in the effluent was less than 2% of that in the influent. If a particular atmosphere is suspected of containing a large amount of contaminant, a smaller sampling volume should be taken.

# Interferences
Compounds which have about the same retention time as ethylene dibromide and which are detected by the electron capture detector will interfere with the analysis. This type of interference can be overcome by changing the operating conditions of the instrument, usually by modifying the column, the column temperature, or both.
Ethylene dibromide will not be efficiently trapped when the amount of water vapor in the air is so great that condensation occurs in the trapping media.
Lesser amounts of water vapor in the air may severely decrease the breakthrough volume. When interfering compounds are known or suspected to be present in the air, such information including their suspected identities could be transmitted with the sample.

# Precision and Accuracy
The relative standard deviation for the combined analytical and sampling method in the prescribed range of 203-2,370 nanograms/charcoal tube was 0.070.

# Advantages and Disadvantages of the Method
This method uses a sampling device that is small, portable, and involves no liquids.
Interferences are minimal and can usually be eliminated by altering chromatographic conditions. Analysis of the charcoal tubes can be accomplished rapidly. Simultaneous analysis of two or more compounds suspected of being present in the same sample can usually be accomplished by simply changing chromatographic conditions.
One disadvantage of the method is that the amount of sample which can be collected by this method is limited by the weight of ethylene dibromide which the tube will hold before breakthrough. When the sample value obtained for the backup section of charcoal exceeds 25% of that found in the front section, the possibility of appreciable sample loss exists.
Recoveries of ethylene dibromide from charcoal are decreased upon storage of the charcoal tube samples at room temperature, particularly with lesser amounts of analyte. The use of an internal standard is required in order to attain good precision. Other organic compounds in high concentrations may displace ethylene dibromide from the charcoal. High humidity may decrease the absorptive efficiency and capacity of the charcoal. The precision of the method is limited by the reproducibility of the pressure drop across the charcoal tube. This drop will affect the flowrate and the volume of air sampled, since the pump is usually calibrated for one tube only.
(e)

# Measurement of area:
The areas of the sample peaks are measured by electronic integration or some other suitable method of area measurement.
Preliminary sample results are read from a standard curve prepared as outlined below.
(f)
The approximate retention times of ethylene dibromide and two potential internal standards under the GC conditions described in this method are: In the case of the internal standard method, prepare standard curves by plotting concentration in mg/ml versus the ratio of the peak areas of ethylene dibromide to n-pentadecane.
Compound

# Calculations
Read the weight in milligrams of ethylene dibromide corresponding to the total peak area from the standard curve.
No volume corrections are needed because the standard curve is based on mg ethylene dibromide/ml of benzene-methanol and the volume of sample injected is identical to the volume of the standards injected. Chemical substances should be listed according to their complete name derived from a recognized system of nomenclature.
Where possible, avoid using common names and general class names such as "aromatic amine,"
"safety solvent," or "aliphatic hydrocarbon" when the specific name is known.
The "%" may be the approximate percentage by weight or volume (indicate basis) which each hazardous ingredient of the mixture bears to the whole mixture. This may be indicated as a range or maximum amount, ie, "10-40% vol" or "10% max wt" to avoid disclosure of trade secrets.
Toxic hazard data shall be stated in terms of concentration, mode of exposure or test, and animal used, eg, "100 ppm LC50-rat," "25 mg/kg LD50-  The "Health Hazard Data" should be a combined estimate of the hazard of the total product. This can be expressed as a TWA concentration, as a permissible exposure, or by some other indication of an acceptable standard. Other data are acceptable, such as lowest LD50 if multiple components are involved.
Under "Routes of Exposure," comments in each category should reflect the potential hazard from absorption by the route in question. Comments should indicate the severity of the effect and the basis for the statement if possible. The basis might be animal studies, analogy with similar products, or human experiences. Comments such as "yes" or "possible" are not helpful. Typical comments might be:
Skin Contact-single short contact, no adverse effects likely; prolonged or repeated contact, possibly mild irritation.
Eye Contact-some pain and mild transient irritation; no corneal scarring.
"Emergency and First Aid Procedures" should be written in lay language and should primarily represent first-aid treatment that could be provided by paramedical personnel or individuals trained in first aid.
Information in the "Notes to Physician" section should include any special medical information which would be of assistance to an attending physician including required or recommended preplacement and periodic medical examinations, diagnostic procedures, and medical management of overexposed employees. Respirators shall be specified as to type and NIOSH or US Bureau of Mines approval class, ie, "Supplied air," "Organic vapor canister," etc.
Protective equipment must be specified as to type and materials of construction.    Adapted from reference 9

# PUBLIC HEALTH SERVICE C E N T E R F O R D I S E A S E C O N T R O L N A T I O N A L I N S T I T U T E F O R O C C U P A T I O N A L S A F E T Y A N D H E A L T H R O B E R T A . T A F T L A B O R
(f) 99:1 Benzene-methanol (v/v).

# Analysis of Samples
All glassware used for the laboratory analysis should be washed in detergent and rinsed with tap and distilled water.