Toxicity of inhalational anaesthesia: long-term exposure of anaesthetic personnel—environmental pollution

5 Toxicity of inhalational anaesthesia: long-term exposure of anaesthetic personnel environmental pollution

P. J. A R M S T R O N G A. A. SPENCE

Environmental pollution from waste anaesthetic gases continues. This problem has been extensively investigated and special attention has been paid to assessing its effects on exposed personnel. Recently, it has been realized that it may have wider consequences on the environment with possible deleterious changes in both the ozone layer and the 'greenhouse' effect. Leaks of waste gases occur from breathing systems, masks, exhaust outlets, spillage from the filling of vaporizers and exhalation from recovering patients. Prolonged exposure to high concentrations of anaesthetic gases causes abnormal effects and it was postulated that in developing embryos chronic exposure to trace concentrations could induce a similar outcome. Much time and effort was spent in the 1970s and early 1980s on both animal and human studies. Epidemiological surveys were performed on operating room workers to see if adverse effects did occur. Scavenging of anaesthetic gases became the norm for operating rooms and different techniques (e.g. total intravenous anaesthesia) were attempted. Recommended maximum levels of pollution were suggested in some countries. Most of the evidence indicated that no problem existed and the importance of the matter to the anaesthetic world and, consequently, the number of studies investigating it, declined. Further impetus to exploring the dangers of pollution by waste gases came from the discovery that nitrous oxide (N20) has a specific toxic effect, Not only did this indicate a direct link between exposure and toxicity, but it also gave new clinical avenues to investigate, as knowledge of how it caused this toxicity allowed different methods of examining the dangers to be developed. Another stimulus to further investigation of anaesthetic gas pollution was an increased awareness that environmental damage was caused by industrial pollutants and that anaesthetic gases were implicated. In this chapter we discuss the evidence relating to possible toxicity from waste anaesthetic gases, first on exposed operating room workers and then on global environmental pollution and its effects on the Earth. Bailli&e's ClinicaIAnaesthesiology-Vol. 7, No. 4, December 1993 ISBN 0-7020-1750-7

915 Copyright 9 1993, by Bailli6re Tindall All rights of reproduction in any form reserved

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Toxic mechanisms Some anaesthetic gases have specific toxic effects besides their more general metabolic actions. The pathogenesis is understood in some cases whilst in others it is still vague (Brown and Gandolfi, 1987). To classify a drug as having a specific toxic effect needs a known mechanism, specific lesions and a clear indication that they cease or decrease after withdrawal of the suspected substance (Spence, 1987). For example, methoxyflurane causes high output renal failure in a dose dependent manner because it is metabolized to produce fluoride ions that are toxic to the kidney (Mazze et al, 1971). N 2 0 is known to inhibit methionine synthase by its reaction with vitamin B12, the consequences of which include alterations in folate metabolism with decreases in D N A synthesis. The clinical sequelae of this includes the development of megaloblastic bone marrow with reduction in resistance to infection. Potential teratogenic effects may also occur (Armstrong and Spence, 1991). In contrast, halothane, whilst considered to have a clinically significant effect on the liver, cannot be shown to have a dose-dependent effect and its pathogenesis is still unknown. The effects of enflurane and isoflurane are even more unclear. Measurement of anaesthetic gas pollution The concentrations of waste anaesthetic gases depend on many factors and vary greatly within any working area. All operating room staff are exposed to some pollution, although the actual amounts will depend on these factors. Other occupational groups also exposed to these waste gases include midwives (Munley et al, 1986) and ambulance men (Ilsley et al, 1989). Pollution measurement methodology must include standardization of sampling sites and the timing of samples. Comparison between different operating rooms or anaesthetic methods is therefore difficult and the actual methods used to measure pollution greatly affect the results. The method of choice for individual assessment is to measure individual blood concentrations of gases (Krapez et al, 1980), although the standard approach has been to assess the operating room air concentrations. Many methods have been used to measure them using a variety of techniques, although all have two basic stages. Firstly, the environmental gas is sampled and then its air and pollutant concentrations measured. Sampling can be active or passive. Active methods involve sucking air into a storage or analytical device in contrast to passive systems in which gas diffuses into small samplers packed with a molecular sieve (Gray et al, 1987). This passive system allows pollution levels to be measured over longer times. Air collection is either continuous, giving a time-averaged value, or intermittent ('grab'). As concentrations vary over time, an individual grab sample will not reflect standard pollution levels unless multiple samples are taken and averaged. Sampling may be from the individual's breathing space (personal sampling) or from the work area (area sampling). The former measures inhaled gas levels and is thought to assess individual pollution more accurately, whilst area sampling gives a more general estimate. Operating room gas concen-

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trations vary greatly. For example, when an infra-red camera was used, localized N20 clouds could be seen in some parts of the operating room in contrast to other areas where there was little or no pollution (Allander et al, 1981). Halothane concentrations differed up to 16-fold, depending on the position of the sampler (Langley and Steward 1974). Area sampling is therefore too inaccurate for assessment of individual gas inhalation. Specific gases can be measured using techniques which include gas chromatography, mass spectroscopy and infra-red methods. Many factors can affect overall levels. Scavenging (Davenport et al, 1980, Gray, 1989) and intravenous techniques obviously reduce levels whilst paediatric anaesthesia increases them (Wood et al, 1992). Low flow anaesthesia and the use of tracheal tubes or laryngeal masks decreases pollution (Sarma and Leman 1990). Generally, operating room N20 levels average from 0 to 1000ppm and halothane levels are 0-15 ppm. It is likely that both enflurane and isoflurane have similar levels when used in preference to halothane. The levels of these drugs tend to be greater in dental surgeries as increased leakage from the breathing system occurs (Table 1). A variety of recommended maximum levels has been suggested. Those from the National Institute of Occupational Safety and Health (NIOSH) are 25 ppm N20 and 0.5 ppm halothane (NIOSH 1977). In Sweden, the recommended 8 h time-averaged exposure is 100 ppm (Sik et al, 1990). In contrast, in the UK, the Department of Health and Social Security (DHSS) recommendation is that the limit should be as low as possible, allowing local Health and Safety Executive inspectors to make their own interpretations (DHSS, 1976). However, anaesthetic gases and vapours are now considered to be 'hazardous to health' and a full Control of Substances Hazardous tc, Health (COSHH) assessment is being made. Discussions are underway to find acceptable recommended levels (Halsey, 1991). ASSESSMENT OF THE DANGERS OF ANAESTHETIC GAS POLLUTION ON OPERATING ROOM WORKERS

A variety of methods has been used to assess the dangers of occupational exposure to waste anaesthetic gases. Animals have been exposed to trace concentrations of anaesthetic gases and examined both biochemically and histologically for evidence of toxicity. Many clinical epidemiological trials on exposed operating room workers have been undertaken in an attempt to find if their patterns of illness deviate from the expected norm. All methods have advantages and problems and none alone is sufficient to obtain a precise picture of the problem, if one exists. Epidemiological studies of morbidity and mortality

Most of the epidemiological studies that have examined the possible dangers from exposure to anaesthetic gas pollution have an intrinsic problem in their design. Inevitably, they study personnel working in operating rooms. In this environment there are many factors that might influence health, so are these

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studies a reflection solely of the effect of anaesthetic gas exposure alone or are other agents involved? Working in operating rooms can be stressful and this could be a cause of any possible increase in spontaneous abortions (Fink and Cullen, 1976). Harmful biological agents can be encountered in operating rooms and these include patient's blood which may be infected with potentially virulent organisms (e.g. hepatitis B and HIV). Chemical agents may be the cause of increases in morbidity and mortality (e.g. methylmethacrylate). In some operating rooms there is an increase in exposure to X-ray radiation. Studies need to be tightly designed utilizing close parallel control groups who have otherwise identical work conditions. Most of the published studies have examined the effect of working in an operating room rather than that of exposure to trace concentrations of anaesthetic gases. Other problems exist in the design of these studies. In the exposed groups there has been little or no attempt to quantify the amounts and types of anaesthetic gas exposure. Different workers vary in the extent of their exposure, and in individuals this may alter from day to day. With these differences, it is difficult to define a fixed level of exposure of the subject group and it is difficult to show any dose-dependent toxicity. In an attempt to overcome this problem, Cohen et al (1980) stratified dentists who used only N 2 0 for their work into three groups: none, light (1-2999 h/10 years) and heavy ( > 3000 h/10 years) usage. However, even this permitted only a broad banding of N20 exposure and wide variations exist in the 'light' and 'heavy' groups. There are problems in data collection as most studies have been retrospective using postal surveys with individuals asked to report their past medical history. This form of enquiry has many problems (Spence, 1987). Recall of past medical illness may be inaccurate and some individuals are more motivated than others to respond. This bias in recall may be especially true when people have a vested interest in the outcome. For example, 70% of the exposed group compared with only 41% of the control group responded in the 1974 American Society of Anesthesiologists survey. Different motivations may cause variations in the accuracy of recall of problems and these differences may bias the results. Verification of data is difficult in postal surveys because it is often impossible to check the accuracy of the data. It is well known that surveys and interviews are inaccurate compared with information obtained from the medical records for finding the incidence of diseases (Lilienfeld and Lilienfeld, 1980). One way of overcoming this is the use of registers of health and occupations because the data they contain is more accurate and complete. However, there are a limited number of registers in existence and so only a few studies have used them (Ericson and K~illen 1979, 1985, Hemmininki et al, 1985). With all these problems, are these epidemiological studies useful and their results meaningful? In assessing the effects of operating room pollution, it is important that that no detrimental effects, however minor or uncommon, are overlooked. Therefore the power of the study must be high and one method of achieving this is to increase the size of the study sample. As clinical studies tend to involve only a few people, potential abnormalities

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may be overlooked. Epidemiological studies are able to assess large numbers and the chances of detecting any minor increases in the incidence of any problem are improved. If several studies are analysed together, the overall strength of the method of study is improved (Vessey, 1978). All the published studies have tended to concentrate on a few major potential abnormalities, including the examination of the incidence of carcinogenesis and mortality in all exposed workers and of teratogenesis, fetotoxicity and congenital abnormalities in pregnant women.

Mortality and carcinogenesis Four studies have looked at cancer morbidity (Corbett et al, 1973a; American Society of Anesthesiologists (ASA), 1974; Tomlin, 1979; Cohen et al, 1980) and five have examined cancer mortality (Bruce et al, 1968, 1974, Doll and Peto, 1977; Lew, 1979; Neil et al, 1987). Corbett et al (1973a) surveyed 621 female nurse anaesthetists with an 84.5% response rate (525 responses). They found an incidence of 33 malignancies in 31 nurses. The control group was based on statistics from the Connecticut Tumor Registry and there was an expected tumour rate of 402.8/100 000 compared with 1333/100 000 found in the nurse group. The ASA (1974) published a national study of occupational disease in operating-room personnel using a postal survey involving 49 585 exposed individuals and 23 911 non-exposed controls. The incidence of tumour was increased only in women (by 130-200%) and, statistically, only the incidences of leukaemias and lymphomas were significant (p = 0.05). Cohen et al (1980) looked at the incidence of tumours in 30 650 dentists and 30 547 female assistants by dividing them into anaesthetic and non-anaesthetic gas users. They were further subdivided into dentists who only gave N20 (81.3%) and those who used N20 in combination with other anaesthetics (19%). Therefore, their control groups were carefully selected to ensure that only the effects of anaesthetic gases or N20 were assessed, with all other confounding factors being excluded. Male dentists had no increase in turnout rates, whilst female assistants showed only a slight, although not statistically significant, increase. All five anaesthetic mortality surveys have shown no abnormal pattern. Bruce et al (1968) found 17 anaesthetists had died from tumours of the lymphoid and reticuloendothelial systems as compared with an expected 8.9 (p = 0.05), although their second survey failed to confirm this (Bruce et al, 1974). Doll and Peto (1977) examined 20 540 male British doctors aged over 35 years in 1951 for 20 years. Their occupations were found from the Medical Directory in 1952, and 547 and 704 were full- and part-time anaesthetists respectively. No increase in mortality was seen, although there was a slight increase in death from pancreatic cancer. Lew (1979) examined the mortality rates of male and female members of the ASA from 1954 to 1976. The death certificates of 610 deceased male anaesthetists (out of 637 deaths) were examined and no increase in cancer rates as compared with controls was found. Female anaesthetists showed similar negative findings (66 deaths). Neil et al (1987) followed up 3769 male anaesthetists in the UK between 1957 and 1983, all Fellows of the Faculty of Anaesthetists. During

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the period of study 221 died. The control group was composed of males from social class 1 and the total standardized mortality ratio (observed/expected deaths expressed as a percentage) was 68% (95% CI, 59-77%). For cancer alone it was 50% (95% CI, 36-67%) and no increase in the incidence of leukaemias or lymphomas was seen. Overall, it appears from these studies that the evidence for any increase in the incidence of carcinogenicity or mortality after exposure to anaesthetic gases is slight. In an attempt to improve the validity of these findings, expert analysis of these studies was performed in 1982 by Professor Colton (Buring et al, 1985) who used the relative risk (RR) measurement to find the any possible association between exposure and disease: RR-

Rate of disease among exposed individuals Rate of disease among unexposed individuals

Each separate study was analysed and, if its methodology was sound, its results were pooled and the R R for each disease calculated. When the R R is greater than 1.0 an association becomes possible and if less than 1.0 it is unlikely. The higher the RR, the greater the probability of an association. An overall R R for malignancies for men was calculated at 1.07 and for women 1.4, implying that there is no connection between exposure and mortality and morbidity of cancer in men, whilst in women it is possible although the evidence is tenuous.

Fetotoxicity and teratogenicity Anxiety over the possibility of fetotoxicity and teratogenicity occurring in exposed female workers has prompted several studies (Askrog and Harvald, 1970; Knill-Jones et al, 1972; ASA, 1974; Corbett et al, 1974; Tomlin, 1979; Cohen et al, 1980; Ericson and K~illen, 1979, 1985). Initial work suggested that an increase in spontaneous abortions could occur, but the studies were flawed as their controls were inadequate. Buring et al (1985) pooled the results from six studies to calculate several different RRs. For spontaneous abortion in exposed women, it was 1.30 (95% CI, 1.2-1.4) and for congenital abnormalities it was 1.20 (95% CI, 1.0-1.4). Nurses were less affected than doctors. However, it is possible that this small increase in risk may have been a result of bias in the studies, although the possibility of an association cannot be excluded. Cohen et al (1980) examined pregnant dental workers exposed to N20 and demonstrated what they regarded as a dose dependent increase in spontaneous abortions, which reached a maximum of 105% above normal in chair-side assistants. A small increase (50%) in congenital abnormalities among children of the same chair-side assistants was also found, although this was not dose dependent. In Sweden, a Medical Birth Registry, started in 1973, contains information on prenatal care, delivery and postnatal examinations for 99% of all pregnancies. Similarly, there is a register for nurses and one for abortions (spontaneous and induced). Using these registers, Ericson and K~illen (1979) scrutinized the cases of 494 women from 1973-1975 who had worked

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for at least 50% of their pregnancy in operating rooms. No increase in abnormalities was seen, except for the possibility of a shorter gestation period. Similar work on the same registers over a longer period (1973-1978) included 1323 operating-room nurses with 1382 medical-ward nurses as controls and showed similar results (Ericson and Kfillen, 1985). The Nurse Register allowed a case-control within-group study to be made. A total of 25 operating-room nurses whose children had died perinatally or had severe congenital malformations were chosen, as were 50 nurses from the same group who had a normal childbirth. Questionnaires were sent to these 75 women (74 replies) and no difference in exposure patterns to operating room gases was found. A recent study investigated the effects of N20 exposure on the fertility of American female dental assistants by assessing the time it took for conception to occur (Rowland et al, 1992). Seven thousand women were randomly selected from a dental register and sent screening questionnaires to which 69% replied. A total of 459 women were suitable for further study (had a planned pregnancy within the last 4 years and worked for at least 30 h/week for 6 months before the start of unprotected intercourse). These individuals had a telephone interview, of which 418 were completed. Exposure was assessed by the interviewee who estimated her average hours of exposure to N 2 0 per week and whether there was scavenging in the room. Fecundity ratios (the ratio of the rate of conception in exposed to unexposed women) were calculated. Eighty-seven per cent of the women became pregnant within 13 menstrual cycles. With increasing N 2 0 exposure, the fecundity ratio decreased (implying decreased fertility), corresponding to a 6% reduction in the probability of conception per cycle for each hour of exposure to unscavenged N20 (Table 2). In 19 women there was an association between decreased fertility and increased exposure to N20. There are design problems with this study because it is retrospective, there may be responder bias, estimations of exposure levels are crude and the study results depend heavily on recall of events that may easily be forgotten. However, this study does demonstrate a dose-dependent decrease in the time taken for conception in N 2 0 exposed women. It is a completely new method of assessing N 2 0 toxicity in exposed operating-room personnel and may be a more sensitive indicator of adverse effects. Further work is required into these findings.

Table 2. Fecundity rates of dental assistants exposed to N 2 0 in scavenged and unscavenged operating rooms.

N 2 0 exposure (h/week) ~< 1 2-4 5-9 /> 10 ~<5 t> 5

No. of women

Adjusted fecundity rate

Scavenged

21 20 9 10 85 36

0.94 1.03 0.45 0.37 1.05 1.15

No No No No Yes Yes

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Prior to this study, the results of the other epidemiological studies were reassuring for pregnant operating-room workers. Whilst most of the studies have inbuilt problems in their design and set-up, pooling of the results of several studies have shown, at most, a low possibility of problems during pregnancy. Further work using better controlled groups and improved methods of data collection indicate that pollution by anaesthetic gases is not as great a hazard as was thought 10-15 years ago. However, there is still sufficient doubt about the safety of trace concentrations of anaesthetic gases for further research to be needed. Clinical studies (in vivo and in vitro) It is possible to perform only a few experiments which directly study the effects of trace concentrations of anaesthetic gases on exposed personnel and hence most of the evidence about their potential toxicity depends upon animal studies. However, extrapolation of results between species is unsound as the relative susceptibilities of different animals to the gases are hard to predict (Cohen et al, 1980). For example, when exposed to N~O, rats have no haematological sequela, unlike humans who become megaloblastic. In addition, most studies have used small numbers of animals which reduces the power of the experiments. However, animal studies are useful in determining how toxicity can occur and whether potential problems may exist.

Mutagenicity Anaesthetic gases may be mutagenic and carcinogenic. N20 disrupts DNA synthesis (Nunn, 1987) and therefore is potentially mutagenic. Some anaesthetics contain a double bond and these classes of compounds are known mutagens (Simmon and Baden, 1980). As carcinogens are always mutagens (Duncan and Brookes, 1973), mutagenicity is an essential test for any potential carcinogenic chemical. It is also simpler and cheaper to test for mutagenicity, as standard carcinogenic bioassays requires life-long exposure of a large number of animals. Anaesthetics, both in short- and long-term exposures, have been investigated extensively for mutagenicity (Baden and Simmon 1980). Sturrock (1977) examined the effects of halothane both with and without N20 on Chinese hamster lung fibroblasts using the 8-azaguanine assay system. There was no colony formation when these cells were exposed to 75% N20 with 1-3% halothane for 24h, suggesting that no significant mutation had occurred. Similar results were found for 1-3% chloroform. White et al (1979) examined the frequency of sister chromatid exchanges (SCE) in Chinese hamster ovarian cells, a rapid in vivo assay of genetic damage. Exposure to 1 minimum alveolar concentration (MAC) N20, diethyl ether, trichloroethylene, halothane, enflurane, isoflurane, methoxyflurane or chloroform had no effect. The test was positive when fluroxene or divinyl ether was used. Baden et al (1979) used the Salmonella microsome assay of Ames et al (1973). Two histidine-dependent strains of Salmonella

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typhimurium which

only survive and grow in histidine-depleted growth media if mutagenic change converts them into independency were exposed to N20 and cyclopropane with negative results. The results for trichloroethylene were equivocal, whilst those for divinyl ether were strongly positive. Combinations of NzO with 1-2% halothane, enflurane or isoflurane had no mutagenic effect on Drosophila flies (Baden and Kundumal, 1986). Overall, it appears that modern anaesthetics have no mutagenic properties at high concentrations and hence it is unlikely that they have effects at lower concentrations, even if exposure is chronic. The urine of operating room personnel was found to contain no mutagenic compounds (Baden et al, 1980). Lymphocytes from operating-room personnel exposed to anaesthetic waste gases for up to 26 years had no increased incidence of chromosomal abnormality or SCE numbers compared with unexposed controls (Husum and Wulf, 1983). Trainee nurse anaesthetists were examined for SCE formation before they were exposed to any operating room pollution, and when the incidence of their formation was compared with that found after exposure (for up to 32 months) no increase was seen (Husum et al, 1985). It is unlikely from this evidence that anaesthetic waste gases are mutagenic.

Carcinogenicity Is it implausible that anaesthetic gases are carcinogens without being mutagens. To confirm this, a study of isoflurane exposure to mice in utero was performed (Corbett, 1976). There was an increase in the incidence of liver tumours in male mice alone, although a second study using isoflurane, halothane, enflurane and NzO for 2 h on alternate days in utero in the second half of pregnancy was negative (Eger et al, 1978). In mice exposed to either 0.1 or 0.4% isoflurane in air 4 h daily, 5 days a week for 78 weeks, there was no increase in carcinogenesis (Baden et al, 1988). Exposure of either male or female rats to either 1 or 10 ppm halothane with 50 or 500 ppm NzO for 7 hours a day, 5 days a week for 104 weeks, caused no increase in malignancy in their reticuloendothelial systems (Coate et al, 1979a). Overall, there is no evidence for any mutagenic or carcinogenic properties of N20.

Teratogenicity There have been many animal studies on the possibility that chronic exposure to anaesthetic gases causes an increased incidence of teratogenicity. There are many teratogenic mechanisms (mutation, interference with cell division, lack of cell energy and precursors for development, enzyme inhibition, and cell membrane changes) and it is possible that anaesthetic agents are teratogenic for a variety of these reasons. NzO has been shown to have fetotoxic and teratogenic effect (Fink et al, 1967). Pregnant rats who had inhaled 50% N20 showed increases in the incidence of fetal resorption, mortality and visceral and skeletal abnormalities. This was a direct NzO effect rather than a generalized effect of anaesthetics because xenon, a gas with similar properties to N20, had no teratogenicity in

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similar doses (Lane et al, 1980). Further work has been carried out to find when in pregnancy the teratogenic effect is most profound and how much exposure is needed. Shephard and Fink (1968) exposed pregnant rats to 24 h continuous 50% NzO from days 5 to 11 of pregnancy. Skeletal abnormalities were greatest on day 11 but were also increased from days 5 to 8. Hence, it appears that N20 has different effects at different times during rat pregnancy. There is a trace concentration below which no teratogenicity occurs. Pregnant rats were exposed to either 100, 1000, or 15 000 ppm N20, either continuously or for 8h a day on either days 8-13, 10-13, 12-19, 14-19 or 10-19 of pregnancy. Intermittent exposure had no effect except during days 10-13 when there was an increase in fetal death rate. In the continuousexposure group, 1000 and 15 000 ppm exposure for 24 h caused increased fetal death rates and decreased implantations, whilst 100 ppm had no effect (Corbett et al, 1973b). Exposure of rats to low N20 concentrations throughout their pregnancy showed that 250 and 500 ppm N20 had no effect, whilst 1000ppm N20 caused a reduction in litter numbers with increased frequency of fetal resorption and decreased fetal weight (Vieira et al, 1980). Hence, in rats, it appears that concentrations of N20 greater than 500 ppm are required with continuous exposure for teratogenesis to occur. This is similar to the lowest concentration (450ppm) that inhibits rat liver methionine syntase (MS) activity (Sharer et al, 1983). In intermittently exposed rats the threshold was above 1000ppm (Vieira et al, 1983). In contrast, some studies have been unable to demonstrate this teratogenicity. Mice who were exposed for 4 h a day from days 6 to 15 of pregnancy to 0.5 %, 5% or 50% N20 with 21% oxygen had normal pregnancies (Mazze et al, 1982). The mechanism of this N20 effect is still unknown. Maternal and fetal MS was inhibited in a dose-dependent manner, although fetal inhibition was slower, probably due to the time required for N20 equilibration between maternal and fetal blood (Baden et al, 1984). In vitro N20 inactivation of fetal MS was similar to that of their mothers (Baden et al, 1987). Is the teratogenicity due to abnormal folate and DNA metabolism from MS inhibition? N20 does decrease cellular proliferation in mice fetuses exposed briefly to N20 (Rodier et al, 1986). However, evidence has accumulated that other mechanisms may be responsible. If isoflurane (0.35%) and N20 (50%) are given to rats the incidence of teratogenicity is reduced (Fujinaga et al, 1987a), although addition of fentanyl to N20 caused no reduction (Mazze et al, 1987). In addition, if this effect is mediated via folate inhibition, folinic acid should protect the animals by 'springing' the folate trap. Unfortunately, it did not (Keeling et al, 1986, Fujinaga et al, 1987b). N20 causes a high adrenergic tone with an increase in plasma noradrenaline levels (Smith et al, 1970) and it is possible that blood flow to the uterus could be reduced enough to compromise the fetus. As both isoflurane and halothane prevent this high adrenergic tone and dilate the arterial supply to the uterus it is possible that they are able to diminish the vasoconstrictor effects of N20. However, when a whole-embryo culture system was used to expose 100 10-day-old rat embryos to either 75% N20 or N2 it was found

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that the DNA content in the N2O exposed rats was much lower and seven rats developed abnormally (Fujinaga et al, 1988). Exposure of rat fetuses to 75% N20 on day 9 in the same system increased abnormalities (Baden and Fujinaga, 1991). This whole-embryo culture system removes any maternal influences that N20 has and therefore allows the direct effects of N20 on the fetus to be assessed. Are the toxic effects of N20 multifactorial? Whilst N20 can clearly have teratogenic effects, it appears unlikely that these will manifest at concentrations below 500 ppm which is above recommended maximum.pollution levels, although there are circumstances when levels can be greater than this. The evidence for trace concentrations of the other anaesthetic gases being teratogenic is much weaker. Isofturane (0.35%) was not teratogenic (Fujinaga et al, 1987a), nor was halothane (1 or 10 ppm in N20) (Coate et al, 1979b). It is unlikely these agents are teratogenic, especially at trace concentrations.

Sperrnatogenesis Men may be susceptible to waste anaesthetic gas pollution, with the development of sperm abnormalities. Exposure of 135 young male rats to 20% N20 resulted in normal-looking testes, although their weight decreased after 28 days exposure. Histologically, there was damage to the spermatogenie cells and suppression of spermatogenesis, whilst the supporting cells remained normal. The earliest exposure causing this was 2 days and all rats were abnormal after 14 days. Recovery took 6 days (Kripke, 1976). Young male rats exposed to trace concentrations of N20 (50 and 500 ppm) and halothane (1 and 10 ppm) for 7 h a day, 5 days a week for up to 52 weeks, showed an increase in chromosomal damage in their spermatogonial cells at both concentrations (Coate et al, 1979b). However, other studies have been negative. Exposure of male mice to 8% or 80% N20 had no effect (Lane et al, 1981). Male mice exposed to 0.5%, 5% or 50% N20 for 4h each day, 5 days a week, showed no effect after 14 weeks (Mazze et al, 1983). Anaesthetists exposed to operating room pollution through their normal working practices for at least 1 year had no abnormalities in concentration or morphology of sperm (Wyrobek et al, 1981). Hence, it is possible that if N20 is present either at a high enough concentration or for a long enough time in trace concentrations, abnormal spermatogenesis may occur. Anaesthetists showed no obvious effect to general exposure, although personnel exposed to high concentrations have not yet been assessed.

N20 and methionine synthase inhibition There are other potential areas of concern about exposure to trace concentrations of anaesthetic gases. N20 pollution of operating rooms has been studied extensively as it has been shown to have a specific toxicity the pathogenesis of which is almost understood. Experiments have been performed both on animals and humans to assess the impact of this toxicity.

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Other anaesthetic gases have not been investigated as thoroughly in vivo. Human methionine synthase (MS) appears more resistant than rat MS to inhibition from acute exposure to high concentrations of N20 with a 50% inhibition half-life of 46 and 5.4 rain, respectively (Royston et al, 1989). Some inhibition does occur with trace concentrations. Rats exposed to 2% N20 for 15 h had a 30% decrease in liver MS activity (Kondo et al, 1981). Rats exposed to 700-1600ppm NzO (average 1100ppm) showed a 27% reduction in liver MS activity, although it was normal after 15 days exposure (Koblin et al, 1981). A dose-dependent increase in inhibition was found in rats, the lowest concentration having an effect after 24h exposure was 1000ppm and for longer exposures was 500ppm. Extrapolation of this indicates that the minimum N20 concentration for MS inhibition 840 ppm for 24 h and 450 ppm for longer exposures (Sharer et al, 1983). The effects of trace concentrations on human MS have not been studied. However, attempts have been made to assess toxicity using indirect measurements by examining the consequences of human MS inhibition which include disturbances of folate metabolism and megablastosis. Salo et al (1984) examined operating room workers' blood. They studied eight male anaesthetists and 118 operating-room nurses who worked in scavenged operating rooms. In addition, ten nurses and doctors who worked in an unscavenged operating room were examined. All had normal haematological profiles, except for two anaesthetists who had raised mean corpuscular volumes (MCV) and one had five segmented neutrophils. However, this study will only detect severe abnormalities as abnormal neutrophil appearance is a late effect of bone marrow depression. Sweeney and et al (1985) examined N20 pollution in dentists using a more sensitive assay. They studied the bone marrow of 21 dentists and measured their N20 exposure using a personal sampler over a 3-11 week period. The average exposure was 159-4000ppm N20. Deoxyuridine suppression tests (a method of analysing megaloblastic marrow) were abnormal for two subjects and that of a third was slightly irregular. In addition, these two subjects also had abnormal blood films and microscopy of their marrow showed megablastosis. All three dentists had a raised N20 exposure (time-averaged exposure of 1900, 2500, and 1800 ppm) and calculation of total exposure doses showed that they had the 1st, 3rd and 5th greatest exposures. Nunn et al (1982) found no change in methionine concentrations in anaesthetists exposed to N20, although this is a crude assay of MS inhibition as many factors influence methionine concentrations in the plasma. Catabolism of histidine requires a functioning folate metabolic pathway which, when inhibited, results in excretion of formiminoglutamic acid (FIGlu). As MS inhibition causes an abnormality in folate metabolism, it will cause an increase in FIGlu excretion. This test can be used to assess N20 toxicity on folate metabolism. Ten anaesthetists had normal excretion, indicating that they had no abnormality of folate metabolism, in spite of administering N20 regularly (Armstrong et al, 1991). This test is a sensitive indicator of N20 toxicity as it occurs before DNA formation is reduced and, if folate metabolism is inhibited, potentially several serious consequences may occur. The overall evidence from these biochemical test, indicate that

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exposure to trace concentrations of N20 lacks effect on metabolism unless high concentrations are inhaled.

Immune system Anaesthetic gases, especially N20, may affect the immune system (Duncan and Cullen, 1976; Hill et al, 1978). A variety of studies have shown conflicting results with reductions seen in both total and T lymphocyte numbers in 14 anaesthetists (Pettingale et al, 1978), in contrast to negative results from other anaesthetists (Bruce, 1972; Salo and Vapaavuori, 1976; Ziv et al, 1988). Recently, immunological changes were studied in anaesthetic personnel who worked in unscavenged operating rooms. They were exposed to 10--60 times the normal concentrations of halothane and N20 (timeaveraged exposure of 85-1500 ppm, using active-area sampling and infrared spectroscopy). Whilst the control group was not fully comparable as they did not work in operating rooms, all subjects were repeat tested after a 3-4 week vacation to see if improvement had occurred. There was little difference between the exposed and control groups, but there was improvement in a variety of immunohaematological profiles after the 4-week break from exposure (Peric et al, 1991). It is most likely that these observations are due primarily to increases in N20 exposure rather than to halothane, although exposure levels were unusually high.

Hepatoxicity Halothane is known to be hepatotoxic. Rats, mice and guinea-pigs all exposed to 15 ppm halothane or isoflurane for 35 days had no liver damage nor was any injury to other organs found (Stevens et al, 1975). In contrast, rats exposed to 50 ppm halothane for 12 weeks had hepatocellular injury (Plummer et al, 1983). However, continuous exposure to 20 ppm halothane, isofturane or enflurane for 30 weeks caused no harm (Plummer et al, 1986). Thus, extrapolating from these animal studies and from known levels of anaesthetic gas pollution, it is unlikely that waste gases have any hepatic effects on operating-room workers.

Behavioural aspects Anaesthetic gases cause profound behavioural disturbances in high doses. However, it appears that moderately high concentrations of anaesthetic agents (5-10% MAC) are needed to produce detrimental effects, although this conclusion was based on laboratory investigations (Smith and Shirley, 1978). It is possible that the behavioural effects could be increased by other factors in operating rooms. Twenty two anaesthetists working in an operating room (average time-weighted exposure to 58ppm N20 and 1.4ppm halothane) were shown to have similar mood and cognitive functions as when working outside the operating room (Stollery et al, 1988). Hence it appears that at the concentrations found in operating rooms, exposed personnel show no obvious behavioural changes.

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THE ENVIRONMENT AND ANAESTHETIC GAS POLLUTION

Concern about the possible deleterious consequences of anaesthetic gas pollution on the environment has grown, especially as it is now realized that these gases may destroy the ozone layer and increase the 'greenhouse' effect. Chloroflurocarbons (CFC) are stable compounds used as aerosol propellants, refrigerants, cleaning solvents and foaming agents. They are carbon molecules containing both fluorine and chlorine atoms, and it was suggested in 1974 that they may destroy the ozone layer. This layer of gas (03) lies high up in the atmosphere in the midstratosphere, its function being to filter out dangerous radiation (Molina and Rowland, 1974). On release into the air, the CFC molecules pass initially into the lower atmosphere (troposphere) where they are totally inert because their main method of degradation is by photolysis from ultraviolet radiation and most of this is screened out by the ozone layer. However, the CFCs gradually diffuse further up the atmosphere to eventually reach the ozone layer where they are destroyed by the unscreened radiation. This causes the formation of free chlorine which catalyses ozone destruction in a vicious circle. Eventually, the free chlorine dissolves in water as hydrochloric acid and returns to earth in rain. The cycle can take up to 2 years to complete with up to 100 000 molecules of ozone being destroyed (Rowland, 1989). C1 + O3----~C10 + O2 C102 + O--~C1 + 02 Bromine may be up to 80 times as effective as chlorine in destroying ozone (Hammitt et al, 1987). All the main anaesthetic gases contain fluorine and carbon and may act in a similar manner as CFCs. Halothane which contains a bromide atom may be more dangerous. However, several factors decrease the importance of this ozone destructive effect by anaesthetic agents. Their atmospheric lifetimes are much less than those of the CFCs as they can react with free hydroxyl radicals in the troposphere. It takes 2 years for the agents to reach the stratosphere and therefore only small amounts will arrive. Secondly, their potential ozone depletion efficiency is small when compared with that of CFCs (Table 3). As fluorine has little effect on ozone, the greater the number of fluorine atoms present, the less the destruction. Sevoflurane contains only fluorine and will Table 3. The effects of anaesthetic gases on the ozone layer and the 'greenhouse' effect.

Compound CFC-11 (CFC13) CFC-12 (CF2C12) Halothane (CF3CHCIBr) Enflurane (CFzHOCF2CFC1H) Isoflurane (CF2HOCHC1CF3) Sevoflurane (CFH2OCH(CF3))

Lifetime in Greenhouse Global atmosphere Potential ozone warming production (years) depletionefficacy effect (t/year) 76 140 2 6 5 1.4

1.0 1.0 0.36 0.02 0.01 0

0.39 1.00 0.004 0.04 0.03 0.005

350000 400000 1000 220 800

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have no effect on ozone. Thirdly, only small amounts are manufactured globally in comparison with CFCs. Overall, it is probable that their contribution to ozone depletion is less than 0.0005 of the total (Brown et al, 1989). The 'greenhouse' effect is produced by an inability of the Earth to radiate excess heat away into space. In order to lose heat, infra-red radiation (8-20 ~m) is radiated away from the Earth. However, this frequency of infra-red radiation is readily absorbed by polyatomic molecules (carbon dioxide, ozone, water, etc.) and then re-emitted in all directions. Accumulations of these compounds in the atmosphere will reduce the amount of radiation passing into space and the Earth will heat up. CFCs are up to 10 000 times more efficient at absorbing this radiation than CO2 and therefore, may make a large contribution to the 'greenhouse' effect (Ramanathan et al, 1985). Because of their relatively small production the halogenated hydrocarbon anaesthetics have only a small 'greenhouse' warming potential in comparison with CFCs (Brown et al, 1989). However, in contrast, N20 has a long atmospheric half-life of 150 years. It is a significant 'greenhouse' gas and it is increasing in concentration. From 1979 to 1982 there was an annual increase in its atmospheric concentration of approximately 0.8 ppb (0.25% yearly) (Khalil and Rasmussen, 1985)). N20 has an equivalent 'greenhouse' effect to that of carbon dioxide and is produced from many sources, including microbial nitrification and denitrification of fertilizers. Anaesthetic N20 production (estimated at i x 109 litre in 1988) is small when compared with these methods (3-10% of microbial nitrification alone) (Logan and Farmer, 1989). N20 has no direct effect on ozone, although it can be oxidized to nitric oxide which then enters the NOx catalytic cycle. This system has similar effects on ozone as the chlorine cycle, and because these oxides of nitrogen are only removed when they react with other radicals they have the potential to inflict great damage on the ozone layer. Overall, the effect of anaesthetic-gas pollution on the environment is not as severe as was initially thought. Volatile anaesthetics appear relatively benign. So 'ozone friendly' are they in relation to CFCs that they are considered as a replacement for industrial CFC use (Logan and Farmer, 1989). N20 may have more deleterious consequences as it increases the 'greenhouse' effect and destroys ozone, but its overall anaesthetic production is small in comparison with other processes. SUMMARY

The effects of pollution from waste anaesthetic gases are still unclear, but they may cause adverse effects in both exposed operating-room personnel or have a more global impact on the environment. A standardization of measuring operating-room pollution has been developed utilizing a time averaged value. The amount that occurs varies greatly, although is drastically reduced by scavenging. There appears to be little evidence that it increases the incidence of tumours in exposed workers. However, there is some evidence suggesting that it may have unfavorable effects on pregnant

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women and, recently, it has been suggested that it may reduce conception rates. There is evidence of a small effect on the immune system, although this is probably of no clinical relevance. Its effects on the environment are mostly benign as the volatile anaesthetics are ozone friendly. However, N 2 0 does increase the 'greenhouse' effect, although anaesthetic use, when compared with other methods of N 2 0 production, is small. Overall, the evidence for a severe problem with pollution from anaesthetic waste gases is small.

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Coate WB, Ulland BM & Lewis TR (1979a) Chronic exposure to low concentrations of halothane-nitrous oxide. Lack of carcinogenicity in the rat. Anesthesiology 50: 306-309. Coate WB, Ulland BM & Lewis TR (1979b) Chronic exposure to low concentrations of halothane-nitrous oxide. Reproductive and cytogenetic effects in the rat. Anesthesiology 50: 310--318. Cohen EN, Brown BW, Wu ML, Whitcher CE, Brodsky LR, Gift HC, Greenfield W, Jones TW & Driscoll EJ (1980) Occupational disease in dentistry and chronic exposure to trace anaesthetic gases. Journal of the American Dental Association 101: 21-31. Corbett TH (1976) Cancer and congenital abnormalities associated with anesthesia. Annals of the New York Academy of Science 271: 58-66. Corbett TH, Cornell RG, Endres JL & Leiding K (1973a) Incidence of cancer among Michigan nurse anesthetists. Anesthesiology 38: 260-263. Corbett TH, Cornell RG, Endres JI & Millard RI (1973b) Effects of low concentrations of nitrous oxide on rat pregnancy. Anesthesiology 39" 299-310. Corbett TH, Cornell RG & Endres JL (1974) Birth defects among children of nurseanesthetists. Anesthesiology 43: 341-348. Davenport HT, Halsey MJ, Wardley-Smith B & Bateman PE (1980) Occupational exposure to anaesthetics in 20 hospitals. Anaesthesia 35: 354-359. DHSS (Department of Health & Social Security) (1976) Health services development. Pollution of operating departments, etc, by anaesthetic gases. Health Circular HC(76)38, London: Department of Health. Doll R & Peto R (1977) Mortality among doctors of different occupations. British Medical Journal 1: 1433-1436. Duncan ME & Brookes P (1973). The induction of azoguanine-resistant mutants in cultured Chinese hamster cells by reactive derivatives of carcinogenic hydrocarbons. Mutation Research 21: 107-111. Duncan PG & Cullen BF (1976) Anaesthesia and immunology. Anesthesiology 45: 271-282. Eger EI, White AE, Brown CL, Biara CG, Corbett TH & Stevens WC (1978) A test of the carcinogenicity of enflurane, isoflurane, halothane, methoxyflurane and nitrous oxide in mice. Anesthesia and Analgesia 57: 328-345. Epistat Associates (1982). Evaluation of the epidemiological evidence for occupational hazards of anesthetic gases. Colton T (ed.) Park Ridge, Illinois. American Society of Anesthesiologists. Ericson A & K~illen AJ (1979) Survey of infants born in 1973 or 1975 to Swedish women working in operating rooms during their pregnancies. Anestheisa and Analgesia 58: 302-305. Ericson A & K/illen B (1985) Hospitalisation for miscarriage and delivery outcome among Swedish nurses working in operating rooms 1973-1978. Anesthesia and Analgesia 64: 981-988. Fink BR & Cullen BF (1976) Anaesthetic pollution: what is happening to us? Anesthesiology 45: 79-83. Fink BR, Shephard TH & Blandau RJ (1967) Teratogenetic activity of nitrous oxide. Nature 214: 146-148. Fujinaga M, Baden JM, Yhap EO & Mazze RI (1987a) Reproductive and teratogenic effects of nitrous oxide, isoflurane and their combination in Sprague-Dawley rats. Anesthesiology 67: 960-964. Fujinaga M, Baden JM, Yhap EO & Mazze RI (1987b) Halothane and isoflurane prevent the teratogenic effects of nitrous oxide in rats. Folinic Acid does Not. Anesthesiology 67: A456. Fujinaga M, Mazze RI, Baden JM, Fantel A G & Shepard TH (1988) Rat whole embryo culture: an in vitro model for testing nitrous oxide teratogenicity. Anesthesiology 69: 410--404. Gray WM (1989) Occupational expoure to nitrous oxide in four hospitals. Anaesthesia 44: 511-514. Gray WM, O'Sullivan J, Houldsworth HB & Musgrove N (1987) The use of diffusive samplers to measure occupational exposure to waste anaesthetic gases. In Berlin A, Brown RH & Saunders KJ (eds) Diffuse Sampling. An Alternative Approach to Workplace Air Monitoring, pp 89-92. London: Royal Society of Chemistry. Halsey MJ (1991) Occupational health and pollution from anaesthetics: a report of a seminar. Anaesthesia 46: 486-488.

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Hammitt JK, Camm F, Connel PS et al (1987) Future emission scenarios for chemicals that may deplete stratospheric ozone. Nature 330: 711-716. Hemmininki K, Kyyr6nen P & Lindbohm ML (1985) Spontaneous abortions and malformations in the offspring of nurses exposed to anaesthetic gases, cytotoxic drugs and other potential hazards in hospital based on registered information of outcome. Journal of Epidemiological and Community Health 39: 141-150. Hill GE, English JB, Stanley TH, Kawamura R, Loeser E A & Hill HR (1978) Nitrous oxide and neutrophil chemotaxis in man. British Journal of Anaesthesia 50: 555-558. Hillman KM, Saloojee Y, Brett II & Cole PV (1981) Nitrous oxide concentrations in the dental surgery. Anaesthesia 36: 257-262. Husum B & Wulf HC (1983) Sister chromatid exchanges in lymphocytes in operating room personnel. Acta Anaesthesiologica Scandinavica 24: 22-24. Husum B, Wulf HC & Niebuhr E (1985) Monitoring of sister chromatid exchanges in lymphocytes of nurse anesthetists. Anesthesiology 62: 475-479. Ilsley AH, Plummer JL, Cousins MJ, Frondsko RL & Oilligan JE (1989) Atmospheric concentrations of nitrous oxide in ambulances during Entonox administration. Anaesthesia and Intensive Care 17: 83-85. Keeling PA, Roche DA, Nunn JF, Monk SJ, Lumb MJ & Halsey MJ (1986) Folinic acid protects against nitrous oxide teratogenicity in the rat. British Journal of Anaesthesia 58: 528-534. Khalil MAK & Rasmussen RA (1985) Causes of increasing atmospheric methane--a depletion of hydroxyl radicals and the rise of emissions. Atmospheric environment 19: 397-407. Knill-Jones RP, Rodrigues LV, Moir DD & Spence A A (1972) Anaesthetic pracitice and pregnancy. A controlled study of women anaesthetists in the United Kingdom. Lancet i: 1326-1328. Koblin DD, Watson JE, Deady JE, Stokstad ELR & Eger EI (1981) Inactivation of methionine synthase by nitrous oxide in mice. Anesthesiology 54: 318-324. Kondo H, Osbourne ML, Kolhouse JF, Binder MJ, Podell ER, Utley CS, Abrams RS & Allen RH (1981) Nitrous oxide has multiple deleterious effects on cobalamin metabolism and causes decreases in activity of both mammalian cobalamin dependent enzymes in rats. Journal of Clinical Investigations 67: 1270-1283. Krapez JR, Saloojee Y, Hinds C J, Hackett GH & Cole PV (1980) Blood levels of nitrous oxide in theatre personnel. British Journal of Anaesthesia 52: 1143-1148. Kripke B J, Kelman AD, Shube NK, Baloyh K & Handler A H (1976) Testicular reaction to prolonged exposure to nitrous oxide. Anesthesiology 44: 104-113. Lane GA, Nahrwold ML, Tait AR, Taylor-Busch M & Cohen PJ (1980) Anesthetics as teratogens. Nitrous oxide is fetotoxic, xenon is not. Science 210: 899-901. Lane GA, Duboulay PM, Tait AR, Taylor-Busch M & Cohen PJ (1981) Nitrous oxide is teratogenic, halothane is not. Anesthesiology 55: A252. Langley DR & Steward A (1974) The effect of ventilation system design on air contamination with halothane in operating theatres, Anaesthesia 46: 736-741. Lew EA (1979) Mortality experience among anaesthetists 1954-1971. Anesthesiology 51: 195-199. Lilienfeld AM & Lilienfeld DE (1980) Foundations of Epidemiology, 2nd edn, p 149. New York: Oxford University Press. Logan M & Farmer JG (1989) Anaesthesia and the ozone layer. Anaesthesia 63: 645-647. Mazze RI, Shue GL & Jackson SH (1971) Renal dysfunction associated with methoxyflurane anaesthesia. Journal of the American Medical Association 216: 278-283. Mazze RI, Wilson AI, Rice SA, & Baden JM (1982) Reproduction and fetal development in mice chronically exposed to nitrous oxide. Teratology 26:11-16. Mazze RI, Rice SA, Wyrobeck WJ, Felton JA, Brodsky JB & Baden JM (1983) Germ cell studies in mice after prolonged exposure to nitrous oxide. Toxicology and Applied Pharmacology 67: 370-375. Mazze RI, Fujinaga M & Baden JM (1987) Reproductive and teratogenic effects of nitrous oxide, fentanyl and their combinations in the rat. British Journal of Anaesthesia 59: 1291-1297. Molina MJ & Rowland FS (1974) Stratospheric sink for chloroflouromethanes: chlorine atom-catalysed destruction of ozone. Nature 249: 810-812. Munley A J, Railton R, Gray WM & Carter KB (1986) Exposure of midwifes to nitrous oxide in four hospitals. British Medical Journal 293: 1063-1064.

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Sturrock J (1977) Lack of mutagenic effect of halothane or chloroform on cultured cells using the 8-azaguanine test system. British Journal of Anaesthesia 49: 207-210. Sweeney B, Bingham RM, Amos RJ, Petty AC & Cole PV (1985) Toxicity of bone marrow in dentists exposed to nitrous oxide. British Medical Journal 291: 567-569. Tomlin PJ (1979) Health problems of anaesthetists and their families in the West Midlands. British Medical Journal 1: 779-784. Vessey MP (1978) Epidemiological studies of the occupational hazards of anaesthesia; a review. Anaesthesia 33: 430-438. Vieira E, Cleaton-Jones P, Austin JC, Moyes DG & Shaw R (1980) Effect of low concentration of nitrous oxide on fetuses. Anesthesia and Analgesia 59: 175-177. Vieira E, Cleaton-Jones P & Moyes D (1983) Effect of low concentrations of nitrous oxide on the developing rat fetus. British Journal of Anaesthesia 55: 67-69. White AE, Takehisa S, Eger EI, Wolff S & Stevens WC (1979) Sister chromatid exchanges induced by inhaled anaesthetics. Anesthesiology 50: 426-430. Wood C, Ewen A & Goresky G (1992) Exposure of operating room personnel to nitrous oxide during paediatric anaesthesia. Canadian Journal of Anaesthesia 32: 682~586. Wyrobeck AJ, Brodsky J, Gordon L, Moore DH, Watchmaker G & Cohen EN (1981) Sperm studies in anesthesiologists. Anesthesiology 55: 527-532. Ziv Y, Shohat B, Baniel J, Ventura E, Levy E & Dintsman M (1988) The immunologic profile of anesthetists. Anesthesia and Analgesia 67: 849-851.