m f e t y in the chemkal laboratory
edited by MALCOLM M. RENFREW University of ldaho Moscow, ldaho 83843
Carcinogens and Mutagens in the Undergraduate Laboratory Joy Melnikow, James R. Keeffe, and R. L. Bernstein San Francisco State University, San Francisco, CA 94132 Ames svstem (7). and a literature search of exposure to a carcinogen and the onset of cancer may be 20 years or more. Thus carcinogens are often not handled as carefully as chemicals with immediate damaging effects. According to one current estimate, 90% of cancers result from exposure to environmental factors, including exposure to chemicals, cigarette smoke, air-borne particulate matter such as asbestos, as well as dietary and alcohol intake (I). Three epidemiological studies (in the United States, Sweden, and Britain) have indicated that chemists, particularly those working in laboratories, have an unusually high death rate from cancer (2-4). This tragedy may be due in part to the fact that many veteran chemists handle potentially dangerous materials with the "contempt bred by familiarity." However, although those who work with carcinogens for long periods undergo the greatest risk, a brief exposure may be sufficient to induce cancer. N-nitrosodithylamine and N-nitroso-di-nbutylamine have been shown to cause cancer in test animals following a single dose 15). Occupational exposures to benzene as brief as two years have been associated with leukemia (6). Hence, all students and instructors participating in chemistry courses need to be aware of the hazards of handling carcinogens. Concern a t San Francisco State University over such health hazards motivated our investigation. The project was a cooperative effort of faculty and students in the Departments of Chemistry and Biology. It involved two stages: to identify carcinogens and suspected carcinogens used in the labs, and to make recommendations for their elimination or control. These recommendations are based on toxicological properties, volatility, how the chemicnls were used in the labs. and in what
mals are presumed to cause cancer in humans. This assumption is now commonly accepted in the scientific community and by some government regulatory agencies l1,8). (2) Chemicals shown to be mutagenic in the Ames test should be treated as sus~eeted
tories to target potentially carcinogenic substances (9,10). Ninety percent of known carcinogens are detected as mutagens in this test, and 87% of "nanearcinogens" are negative in the assay (21). About 13% of "noncarcinogens" are detected as mutagens in the test and constitute apparent false positives. However, many of these compounds may have been inadequately tested in animals. Thus the actual rate of false positives may be lower (10). It is clear that mutagenicity and carcinogenicity are only parts of the problem of chemical hazards, and the focus in this paper derives in part from the availability of a convenient testing procedure for mutagenicity (7). But it is also true that these two hazards are among the most insidious of laboratorydangers. Moreover, it is likely that a t least one other hazard, teratogenieity, correlates well with mutagenicity (12). See discussion under General Recornmendotions below. In analyzing the data we collected and in making recommendations for changes in the labs, we preferred to err on the side of caution, rather than continue to risk potentially dangerous exposures to suspected eareinogens.
Methods
Identifying Carcinogens and Suspected Carcinogens The identification process consisted of mutaeenicitv testing with bacteria using the Presented in part a t the 4th Annual Biological Sciences Undergraduate Research Conference, University of Santa Clara, May 5, 1979.
We tested chemicals for mutagenicity according to procedures described by Ames, et al. (7). The Ames system uses specially selected mutant strains of the bacterium Salmonella typhimurium which are unable to synthesize histidine, an amino acid required for growth. Mutagens can reverse the orianal mutation in the histidine genes, enabling the bacteria to synthesize histidine. Bacteria then grow and reproduce in histidine-free media, forming visible colonies on a Petri plate. Chemicals were tested for mutagenicity on
the two most sensitive tester strains (91, TA98 (sensitive to frameshift mutations) and TAlOO (sensitive to base-pair suhstitution mutations), both with and without the S9 ' fraction of rat liver. The extract S9 incorporates elements of mammalian metabolism into the test, allowing the detection of mutagens which require metabolic activation by mammalian enzymes not present in Salmonella. We selected chemicals for testing by their structural resemblance to known carcinogens or because of their widespread use. Many had not been previously tested for mutagenicity or carcinogenicity. All chemicals except pdinitrobenzene were obtained from the chemistry stockroom a t San Francisco State University. Compounds were classified as mutagenic if they produced at least twice the spontaneous background number of mutant (histidine independent) colonies or generated a reproducible dose-related increase in the number of mutant colonies (13). Such a dose-response relationship far m-dinitrobenzene is shown in Figure 1. Carcinogens were identified hy a literature search of animal carcinogenicity studies, epidemiological studies of human exposures, and reports by the National Institute for Occupational Safety and Health (NIOSH). The International Agency for Research on Cancer (IARC) has prepared a series of monographs reviewing all available data on the carcinogenicity of 368 chemicals (14). These monographs often provided the basis for distinguishing a compound as a definite carcinogen rather than as a suspected one. Routes bv which humans absorb various chemicals were obtained or estimated from published toxicological data (15-19).
Results and Specific Recommendations Many common laboratory chemicals are mutaeens and carcinoeens. Of 31 chemicals " tested in our laboratory for mutagenicity, 11 (35%)were mutagenic. These compounds and their possible routes of absorption are shown in Table 1.Themajority are nitroaromaties or hydrazines; hoth classes are easily absorbed through the skin (20, 21). Although further testing will be required to identify these mutagens definitively as carcinogens,
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(Continued on page A121 Volume 58, Number 1. January 1981 / A l l
we feel it is prudent to handle these eompounds as suspected carcinogens until additional data from animal tests become avail^ able. Table 1. Chemicals U s e d in Chemistry L a b Courses a t S a n Francisco S t a t e University W h i c h were Found t o b e Mutagenic in Our St~dy.~
Chemical
S9
Hydrarines 2.4-Dinitrophenylhydrazine 2.4-DNP Derivativeof acetophenone Hydrarine
.Through Skin
+
Phenylhydrazine
+ + +
NitroaromatiCs mDinitrobenzene
i
pDinitrobenzene
-
mNitroaniiine
+
pNitrotoluene
-
pNitrophenylhydrarine
Major Route of Exposure
? Through skin Through skin Through skin Through skin Through skin Through skin Through skin
Miscellaneous compounds Benzylamine PhenyliSOthioCyanate a he following compounds were not mutagenic under the conditions of our experiments: o-dinitrobenzene, o-
ninhydrin. picric acid. and tr8ethanolsmine. Compounds which require funher testing for definitive results are: Z,&dniIrophenol, pnitroaniline, phenylisocyanate, and o-phenanfhroline
Thirty-two known or suspected carcinogens were identified among the laboratory chemicals surveyed. These compounds and .their routes of absorption are listed in Table 2. Of these,pnly benzene (5, 14) and benzyl chloride (23) are implicated as human carcinogens by epidemiological studies, and are known to cause cancer in one or more species of rodents. N-Nitrosodiethvlsminp has All other compoundsare known or suspected to cause cancer in one or more species of rodent. In making recommendations for specific compounds, we t r i d to take intoaccount how they were used in the labs and in what quantity, as well as how they might be absorbed by humans. T h e following specific recommendations were made for those eompounds which seemed to present the most serious hazards. Nitrosamines
Nitrosamines are extremely hazardous substances which should be entirelv elimi-
A12 / Journal of Chemical Education
alkyl or alkylaryl amines during a qualitative test designed to characterize unknown amines. N-Nitrosodiethylamine and N-nitrosodi-n-hutylamine have been shown to cause cancer in test animals following a single dose. They are extremely volatile and are absorbed through the skin (24,251. Because of this risk the nitrous acid test orohahlv
available for testing. Nitroaromatics and Aromatic Amines
Compounds in these classes are highly toxic and are readily absorbed through the skin. A serious short-term toniceffect of these compounds is methemaglubinemia, a condition in which the ability of hemoglobin to transfer oxygen to the tissues is diminished. This condition can result from continuing skin absorption due to a spill onto shoes or clothing, or when a large area of skin is en^ posed for a short period. Clinical signs of methemoglohinemia include: cyanosis, a blue coloration noticeable first amund the lips, ears, and fingertips; headache; weakness; dizziness; and shortness of hreath (26). Most of the nitraaromatics we tested are mutagenic (see Table 1).Ames, et al., have shown that many aromatic amines found in hair dyes are highly mutagenic. Several of these hair dye mutagens have nitro suhstituents and are similar in structure to the nitroaromatic mutagens shown in Tahle 2, for example, 4-nitm-o-phenlenediamine (27). Several mutagenic hair dye components are heing tested by the National Cancer Institute, and appear to be carcinogens (121.
T h e use of nitraaromatics and aromatic amines should be avoided whenever possible because of their acute toxic effects and because many, though nut all, are likely to he careinogenic. Among aromatic amines, some compounds may be carcinogenic while structurally similar eompounds are not. In the case of nitroaromatics, slight variations in structure probably d o not correlate with variations in carcinogenic properties (28). All nitroaromaties and aromatic amines should be handled only with impervious gloves. Organic Solvents
Many organic solvents are known or suspected carcinogens (see Tahle 2), including all chlorinated hydrocarbons. These should always be used under a fume hood, since they are absorbed by inhalation. Carbon tetrachloride is particularly dangerous because in addition to being carcinogenic, it has immediate toxic effects on the liver and kidneys, and may be absorbed through the skin in toxic quantities (291. If i t is absolutely necessary to work with this solvent, suitable gloves should be worn. Such gloves should also be worn when working with N,N-dimethylformamide or dimethyl sulfoxide, as these may be dangerous transmitters of solutes. Benzene is also a dangerous solvent which has been related to an increased incidence of leukemia in exposed workers and is known to be a ~ a w e r f u hone-marrow l toxin
may have effects on bone marrow similar to those of benzene, and it is not a recom-
Table 2. Carcinogens U s e d in Undergraduate Chemistry L a b s a t S a n Francisco S t a t e University.
Chemical Acetamide Ambenzene Benzene Benzoyi chioride Benzoyl peroxide Benzyl chloride 2.2'-bipyridine Carbon tetrachloride Chloroform Chromium (VI) o-Dianisidine Diethyl~ulfate Dioxane Hydrarine isobutyl alcohol Maleic anhydride Methyl iodide Napthalene KNitrosodiethylamine KNitrosodi-+b~tylamine KNitrosodi-~propylamine Phenylhydrazine Phenol Semicarbazide Succinic anhydride Tetrachloroethylene Thioacetamide Thiourea o-Toluidine pTOluidine Zinc sulfate Zinc chloride
Reference
(5).v. 7,197 (5).v. 8.75 IS, 141 suspect (23,40) suspect (40) (14. 191 Suspect (40)
15). v. 1.53 (401 ( 14, 2Zl (5).v. 4.44 ( 5 ) , v. 6.55 (17) (5).v. 4. 127 suspect (401 suspect (40) (741 ~ ~ ~ p e ~ t ~ 4 0 1 ( 5 ) ,v. 1. 107 ( 5 ) , v. 4. 197
-
suspect (40) suspect (40) (14) (14) suspect (40) 15). v. 7.77 (5). v. 7,95 suspect (40) suspect (40) suspect (401 SUSO~C 1401 ~
-
Major Route of Exposure
ingestion ingestion inhalation, through skin inhalation inhalation, through skin inhalation ? inhalation, through skin inhaiation inhaiation. through skin inhalation, through skin ? inhalation. through skin inhalation. through skin inhalation ? ? inhalation inhalation, through skin inhalation, through skin inhalation, through skin ingestion inhalation. through skin ? ? inhalation ingestion inhalation, through skin inhalation. through skin ? ?
Table 3. Hazardous Solvents and Possible Substitutes Approx. Cost
Hazardous Solvent
Substitute
bp ('C)
P (20°)
hb
Sol. (H20)*
(per 5 gal.)=
CCI, Tetrachlomethylene
1.4-Dioxane Tetrahydrofuran 1.2-Dirnethoxyethane Benzene Cyclahexane Teluene Xylene(s) Toluene lsabutyl alcohol ~ B u t yalcohol l tor near roomtemp, me symbols i = immiscible;p = lessthan 10-15 giloo g n20:m = miscible (in the absence of elecfrolyfes). An emoirical solvent .nolsriw ~.garameter. See K . Dimrofh. efel.,Ann.. 661. 1 (1963); E. M. Kosower. ''An lntrduction to Physical Organic Chemistry." Wiley, New York. 1968, pp. 304-305. ~ondiscounted,commercial grade prices are quoted where available.
mended substitute (31). The substitutions shown in Table 3, while not entirely eliminating the hazards of working with organic solvents, should reduce the hazard somewhat. Some chlorinated hydrocarbons are recommended a s substitutes for others because, although likely t o be carcinogenic, they are less toxic and are not absorbed through the skin. Not all recommended substitutes have been exhaustively tested, and we suggest that solvents always be handled with caution, particularly with regard to inhalation hazards. Toluene, far example, while probably not carcinogenic, is known to have tonic effects on the centralnervous system and may contain benzene, a known carcinogen, as a contaminant ( 3 2 ) .
by the skin and lungs (35).Students analyzing phenyl esters should he warned about these hazards.
2.4-Dinitrophenylhydrazine, Phenylhydrazine, and Semicarbazide These compounds, commonly used to make derivatives of aldehydes and ketones, should be used only in small amounts as prepared solutions from dropper bottles. These reagents and their derivatives should be handled with gloves. Probably most, if not all hydrazines are carcinogenic.
Azobenzene This compound has been used a t San Francisco State University to introduce thin-layer chromatography. It could easily be replaced by spinach homogenate or some other natural products mixture, which would safely demonstrate the same technique.
Toiuidine and Thiourea These chemicals are both present in a reagent used to assay glucuse-l-phosphate.
(Continued a n page A14)
Unknowns and Practical Exams Mutagens and carcinogens should not be given out as unknowns for qualitative or quantitative analysis. Students should he warned t o avoid skin contact with all unknowns given out for analysis. This precaution is oarticularlv imoortant in the case of nitroaromatics and aromatic amines, which are easily absorbed through the skin.
" .
Chromium ( VO Chromic acid should he used only in small amounts from dropper bottles. Its use for cleaning glassware should be discourage6 non-toxic substitutes are available ( 3 3 ) .Experiments involving synthesis or analysis of chromium coordination compounds could be replaced with experiments using coordination eomolexes of a nonearcinoeenic metal such
respect ( 3 4 ) .
Phenol When possible phenol should be eliminated. In addition to being a suspected carcinogen, 1%solutions can cause skin necrosis. Phenol demyelinates and otherwise destroys nerve fibers. Solutions are readily absorbed by the skin, and its vapors are abiorbed well Volume 58, Number 1, January 1981 / A13
quiz (39). All mutaeens. carcinoeens. and susoected
This assay could he replaced by Nelson's test or another test using noncarcinogenic reagents.
General Recommendations for Reducing Carcinogenic Hazards Since it is impossible t o remove all hazardous chemicals from chemistry lab courses, the general recommendations discussed helaw were developed to minimize hazards. T h e specific recommendations do not deal with all the carcimmens and mutaeens idencompounds used in chemistry lab courses at San Francisco State University. Those c o m ~ pounds tested for mutagenicity were tested only under standard conditions. Special testing conditions are required to detect highly volatile mutagens and thofie which diffuse slowly through agar (7). I t is important to realize that failure to produce twice the background number of mutant colonies does not label a chemical as safe. I t means only that it is not sufficiently mutagenic to double the number of mutations produced spontaneously. In a large population less than twice the natural number of mutations can itself be a large number. These considerations suggest that more carcinogens are used in university chemistry labs than are listed in this paper. Some have not yet been identified, and it is sometimes difficult to omit or find a safe substitute for those mutagens and carcinogens which have been identified. However, recognition of carcinogenic hazards and a flexible, innovative attitude toward chemistry lab curricula can help to alleviate the problem. Organic laboratory texts are increasingly attentive to tonic and carcinogenic hazards (36.37). Yip and Dalton (37) have written a n organic chemistry lab text which attempts to avoid the use of all known mutagens, carcinogens, and teratoeens. handling any mutagen or carcinogen. These recommendations are based on long experience teaching chemistry lab courses and an a review of publications by NIOSH and 0 t h ~ ers which suaaest methods for handlina toxic chemicals ahh known or suspected
