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Mutagenic action of methyl 2-cyanoacrylate vapor
Mutation Research, 102 (1982) 373-381
373
Elsevier Biomedical Press
Mutagenic action of methyl 2-cyanoacrylate vapor Margrethe Andersen, Mona-Lise Binderup, P. Kiel, H. Larsen and Jette Maxild Department of Microbiology, The Royal Danish School of Pharmacy, 2 Universitetsparken, DK.2100 Copenhagen ~ (Denmark)
S.H. Hansen Department of Chemistry BC, The Royal Danish School of Pharmacy, 2 Universitetsparken, DK-2100 Copenhagen ~ (Denmark) (Received 29 March 1981) (Revision received 23 April 1982) (Accepted 3 May 1982)
Summaff Alkyl 2-cyanoacrylate adhesives were tested for mutagenicity in Salmonella typhimurium strains TA98, TA100, TA1535 and TA1538. Both a normal spot test and a spot test specially designed to test volatile compounds were used. The adhesives were also tested in the plate incorporation assay. These investigations showed that methyl 2-cyanoacrylate adhesives are mutagenic in strain TA100. The spot test for volatile compounds showed that it is the vapors from the methyl 2-cyanoacrylate monomer that are responsible for the mutagenic effect. One can conclude that working with methyl 2-cyanoacrylate adhesives entails exposure to vapors with a mutagenic effect and may therefore pose a carcinogenic hazard. Because the adhesives are used in industry, their mutagenic effect has a special importance in work environment.
The production of alkyl 2-cyanoacrylate (Fig. 1) started in the United States in 1958. Since then, a number of different cyanoacrylate adhesives have been produced. Because of their properties, cyanoacrylate adhesives are now used all over the world. They are advantageous because they adhere to many different'materials, e.g. glass, steel, plastic and rubber, and can be used where adhesion has to take place rapidly [10]. Monomeric alkyl 2-cyanoacrylate is a liquid. The commercialized alkyl 2Reprint requests to: Pauli Kiel, The Royal Danish School of Pharmacy, Department of Microbiology, 2 Universitetsparken, DK-2100 Copenhagen ~b, Denmark. 0165-1218/82/0000-0000/$02.75 © Elsevier Biomedical Press
374 C_=N
CH2=(~-c~O_R Fig. 1. Structure of alky] 2-cyanoacrylate.
cyanoacrylate adhesive may contain various additives, e.g. thickening agents, colors and preservatives. Polymerization takes place within a couple of seconds upon contact with even very weak bases, as for example water and alcohol, without requiring heat or the addition of a catalyst [9]. The manufacturers recommend methyl 2-cyanoacrylate adhesives both in household and industrial applications. The adhesives are much used in the electronics industry. Butyl and isobutyl 2-cyanoacrylate adhesives are used especially in the fields of medical science. They are used to close wounds [1,2], to stop bleeding [5], and in a number of special ways in surgery, e.g. brain surgery [21]. Alkyl 2-cyanoacrylate adhesives are used by dentists in the sealing of teeth, to stop bleeding [5], and in the prevention of caries [20]. Our attention has been drawn to methyl 2-cyanoacrylate adhesive with the trade mark TIXO K100 by a woman who has worked with the product. She suspects that her work with the adhesive could be the cause of a miscarriage, and the birth of an anencephaloid child. However, no epidemiological work has been done on the possibility of a teratogenic effect of alkyl 2-cyanoacrylates; therefore, there is no evidence for the woman's suspicions. As a large number of industrial workers, in a number of industrial fields all over the world, are exposed daily to vapor from adhesives, we found it valuable to examine various alkyl 2-cyanoacrylate adhesives in the Salmonella/microsome mutagenicity test.
Materials and methods
Alkyl 2-cyanoacrylate adhesives tested The methyl 2-cyanoacrylate adhesives, TIXO K100 (Hapas A / S ) and Cyanolite 102 (3M A/S), the ethyl 2-cyanoacrylate adhesive, Cyanolite 201 (3M A/S), the allyl 2-cyanoacrylate adhesive, Cyanolite 303 (3M A/S), and the butyl 2-cyanoacrylate adhesive, Histoacryl (m (Braun, Melsungen AG) were all tested in a spot test without modifications, and in a spot test specially designed to test volatile substances, and finally in the plate incorporation assay. Acetone (Merck t.a.) and dimethylformamide (Merck, Uvasol) were used as the solvents in the plate incorporation assay. Tetrachloromethane (Merck t.a.) was used as solvent in the determination of ]H-NMR spectra.
Mutagenicity testing The alkyl 2-cyanoacrylate adhesives were tested in the Salmonella/microsome mutagenicity test, with the histidine-requiring mutants of Salmonella typhimurium.
375 The test was carried out on strains TA100 and TA1535 (reverting by base-pair substitution) and TA98 and TA1538 (reverting by frame-shift mutation), both with and without the addition of the rat-liver preparation $9 mix. Rat-liver enzymes were induced by adding sodium phenobarbital (0.1%) to the drinking water in the last week before the rats were killed. The rat-liver microsome fraction $9 and the N A D P H *-generating system were prepared according to Ames et al. [3]. To each plate, 150/xl S9 in 500 #1 $9 mix was added. The spot test was carried out in two different ways.
The spot test (without modifications) The spot test was carried out as described by Ames at al. [3], without modification. A drop of the adhesive was placed directly on the agar surface. Three different sizes of drop (about 5, 10 and 20 mg) were investigated for each adhesive. Three plates were made f o r each drop size. The plates were incubated at 37°C for 2 days. The spot test for volatile compounds To test whether the mutagenic activity of the adhesives was due to volatile compounds, the spot test was also carried out as follows. As soon as the agar overlay with bacteria and $9 mix had hardened, a sterile microscope glass cover slip (22 mm in diameter) was placed in the middle of the plate. A drop of adhesive was then placed on the cover slip. To stop vapors from escaping, the petri dishes were then sealed with tape (Samson, Plastic electrical tape 0.2 m m / 1 5 mm) (Fig. 2). All the adhesives were examined by using three different sizes of drop, about 5, 10 and 20 mg, with three plates per size of drop. Plate incorporation assay The plate incorporation assay was carried out with the test strain TA100 and without $9 mix. The test was carried out as described by Ames et al. [3], without modification. The adhesives were dissolved in 100 #1 acetone and incorporated into the agar overlay. Amounts from 0.1 to 5.0 mg of adhesive per plate were tested. Proton magnetic resonance spectra (1H-NMR) Proton magnetic resonance spectra were recorded on a Jeol C-60 H L 60 MH z proton magnetic resonance spectrometer with tetramethylsilane as internal standard. The test solutions were made by dripping the adhesives directly into the tetrachloromethane.
Results
When the drop of adhesive was placed directly on the agar surface, polymerization took place immediately, activated by the humidity in the agar overlay. As the * NADPH, reduced form of nicotinamideadenine dinucleotidephosphate.
376
revertant colony ~/' :.:.::!~..
: ;i".~;.-'ig. inhibition zone
~ i
- - m -'~ ,i adhesive ~.., c o v e r s l i p
" .....................
drop
~:a~-~:. "' ..... :;:: "."
background
growlh
Fig. 2. Spot test method for testing the mutagenic effect of volatile compounds. drop of adhesive polymerized, a white ring-shaped film formed on the surface of the agar around the drop of adhesive. The same phenomenon was observed in the spot test for volatile compounds. Polymerization took place in the same way both with and without $9 mix in the plates. In both spot test methods the methyl 2-cyanoacrylate adhesives formed the largest white rings and the butyl 2-cyanoacrylate adhesive the smallest ones. After two days of incubation at 37°C, the spot test plates were examined for inhibition zones in the background lawn.of bacterial growth, and for accumulation of revertant colonies near the zone of inhibition. An accumulation of revertant colonies near the inhibition zone was regarded as a mutagenic effect. The inhibition zone and the white ring had the same diameter in the case of each adhesive. In both spot test methods the two methyl 2-cyanoacrylate adhesives, TIXO K100 and Cyanolite 102, had a mutagenic effect in the test strain TA100 (Fig. 3). There was a mutagenic effect both with and without the $9 mix. No mutagenic effect was observed with test strain TA1538, TA1535 or TA98. N o mutagenic effect was observed with the ethyl, allyl and butyl 2-cyanoacrylate adhesives in the spot tests. In the plate incorporation assay, precipitation of polymerized adhesive could be clearly seen in the agar overlay, when a dose greater tl~an 1.0 mg per plate (2 ml agar overlay) was used. Inhibition of the background growth could be detected in the case of TIXO K100 and Cyanolite 102 as from concentrations of 0.5 mg (4.5/~moles) per plate, whilst with ethyl, allyl and butyl 2-cyanoacrylate adhesives, it could be detected as from concentrations of between 2.0 and 5.0 mg per plate. The results of the plate incorporation assay with the two methyl 2-cyanoacrylate adhesives and the butyl 2-cyanoacrylate adhesive tested in strain TA100, are presented in Fig. 4. Corresponding results were obtained in three independent experiments. The figure shows that the mutagenic effect of methyl 2-cyanoacrylate adhesives increased as the amount of adhesive increased, between 0.1 and 0.5 mg (0.9-4.5 /~moles) of adhesive per plate. In the assays with ethyl, allyl and butyl 2-cyanoacrylate adhesives, no increase in the number of revertants with increase in dose was observed. To find out whether the mutagenic effect was due to additives, the following adhesives were examined with ] H - N M R spectroscopy: Cyanolite 201, Cyanolite 102, TIXO K100 and Histoacryl.
377
Fig. 3. Mutagenic action of the methyl 2-cyanoacrylate, TIXO K100, on Salmonella typhimurium TAI00. The photograph shows the spot test method for testing volatile compounds. An accumulation of revertant colonies near the inhibition zone is seen.
378 600 500
a /*00
Z 300 .~ 200
IO0
0123&56.7891.0 Dose per ptafe (rag)
Fig. 4. The mutagenic effect on Salmonella typhimurium TAI00 of the two methyl 2-cyanoacrylate adhesives and the butyl 2-cyanoacrylate adhesive in the plate incorporation assay without the addition of $9 mix. Each point is an average of numbers scored on three to five plates. The limits of the vertical lines indicate the lowest and highest values obtained with each dose. A, methyl 2-cyanoacrylate, Cyanolite 102; O, methyl 2-cyanoacrylate, TIXO KI00; f-q, butyl 2-cyanoacrylate, Histoacryl R.
In the spectra of the two methyl 2-cyanoacrylate adhesives some very broad signals could be seen, situated in the zones of 0.8-1.0 ppm, 1.3-2.0 p p m and 3.5-3.7 ppm. These signals could not be attributed to the monomer, and further identifications have not been attempted. Shaking the tetrachloromethane part together with water resulted in polymerization of the adhesive; but the broad signals described above could still be observed unchanged in the organic phase. That, or the substances that were the cause of the irregularity in the N M R spectrum, were therefore isolated in the followingway. One gram of adhesive was dissolved in 2 0 m l tetrachloromethane and then shaken together with 200 ml distilled water. This caused the adhesives to polymerize. The tetrachloromethane part, containing the additives, was separated from the rest and evaporated. The residue from the evaporation, 0.1 g (0.9/~moles) from T I X O K 1 0 0 and 0.005-0.01 g (0.045-0.09 gmoles) from Cyanolite 102, was dissolved in dimethylformamide, and was tested for mutagenicity in t h e same way as were the adhesives, both in the spot test and in the plate incorporation assay. In the plate incorporation assay an amount corresponding to between 0.1 mg (0.9 #moles) and 10 mg (90/tmoles) of adhesive per plate was tested. The evaporation residue did not reveal any mutagenicity. To find out whether the mutagenic effect observed could be due to a product formed by a possible depolymerization of the polymerized adhesive, e.g. formaldehyde, we also examined an already polymerized adhesive. A drop of adhesive was placed on a microscope glass cover slip, which was then placed on the surface of a Vogel-Bonner plate. The petri dish was sealed with tape and maintained at 37°C. After 24 h the cover slip with the polymerized adhesive was transferred to a plate with agar overlay, containing TA100. After 48 h at 37°C no inhibition zone and no ring of revertants could be seen around the cover slip.
379
Discussion
The two methyl 2-cyanoacrylate adhesives tested were mutagenic in the Salmonella/microsome mutagenicity test in strain TA100 both with and without $9 mix. Mutagenicity testing of the additives isolated from the two methyl 2-cyanoacrylate adhesives did not reveal any mutagenic effect. This means that it must be the methyl 2-cyanoacrylate monomer itself that is the cause of the mutagenic effect. The polymer of methyl 2-cyanoacrylate degrades in vitro to formaldehyde and ultimately to cyanoacetate [15]. In their study of the bacteriotoxic effect of cyanoacrylate adhesives [17], Lehman et al. suggest that this effect might be due to products formed as a result of aqueous degradation of the polymers. It has also been suggested that the toxic effect of alkyl 2-cyanoacrylates in animal experiments might be due to products of degradation. In the experiments with the polymerized cyanoacrylate adhesives we found that, under these test conditions, no toxic or mutagenic substances were released. This tends to indicate that both the bacteriotoxic and mutagenic effects are due to the methyl 2-cyanoacrylate monomer. Bonichi et al. [7] and Blum et al. [6] come to the same conclusion according to the toxicity in their studies of the antimicrobial effect. Blum et al. show, moreover, that it is the vapors from the adhesive that are the cause of the growth-inhibiting effect of alkyl 2-cyanoacrylate. Our experiments in the spot test for volatile compounds show, in agreement with the findings of Blum et al., that it is the vapors from the methyl 2-cyanoacrylate adhesives that are responsible for the growth-inhibiting and mutagenic effects. The shape of the dose-response curve (Fig. 4) from the plate incorporation assay also tends to indicate that it is the monomer that is mutagenic. Firstly, the effect did not increase with concentrations higher than 0.5 mg (4.5 ttmoles) per plate, the concentration at which precipitation of polymers can clearly be seen in the agar overlay, and secondly, the greatest increase in revertants can be seen between 0 and 0.1 mg (0.9/~moles) per plate. The observation of a far greater number of revertants per plate in the spot test than even with the highest concentrations in the plate incorporation assay also indicates that it is the vapors from the monomer that are responsible for the mutagenic effect. Alkyl 2-cyanoacrylate monomers have also been tested in implantation studies in rats, dogs and chimpanzees [12,15,16,22]. These studies showed that the methyl 2-cyanoacrylate monomer is the most histotoxic of all the alkyl 2-cyanoacrylates and causes necrosis, edema and cell death. The toxic effect has in many cases been ascribed to degradation products of the polymer [15,22]. Only few investigations have been made to determine the possible carcinogenicity of alkyl 2-cyanoacrylate monomers. In one non-positive investigation [18] the animals were only treated once with a butyl 2-cyanoacrylate spray. In another experiment, designed to provoke epidermoid carcinomas in the oral tissues [11], isobutyl 2-cyanoacrylate was used as a control, bonding DMBA powder (dimethylbenzanthracene, a well-known carcinogen) to the tongues of rats. After 6 months of applications there were no epidermoid carcinomas, either with the isobutyl 2-
380 cyanoacrylate adhesive alone, or with both the D M B A and the adhesive together. F r o m these studies it is therefore impossible to conclude anything about a possible carcinogenic effect of alkyl 2-cyanoacrylate monomers. We do not know of any studies where vapors of cyanoacrylate monomers have been tested for mutagenic or carcinogenic effect. In the field of occupational health and safety research, unlike fields of toxicological research, attention has been paid especially to the vapors of cyanoacrylate adhesives and to their removal. Because the vapor is irritating, a maximal concentration of 2 to 3 p p m is recommended for work spaces [4,19]. This recommendation is based upon the findings of a study [19], where volunteer subjects were exposed to concentrations of methyl 2-cyanoacrylate monomer ranging from 1 p p m to 60 ppm. In this study nose irritation was found from 3 p p m and eye irritation from 5 ppm. An outbreak of irritant dermatitis, caused by cyanoacrylate adhesive, among electronic assembly workers, has been reported [8]. The concentration of cyanoacrylate vapors in the air, and the outbreaks of dermatitis, fell when the relative humidity of the work environment was raised above 55% [8]. The basis for setting the threshold limit value at 2 p p m is thus solely the acute toxic effect. In no way has account b e e n taken of any possible long-term effect. N o relevant studies of the mutagenic or carcinogenic effect of cyanoacrylate adhesives have been made, and these substances are not to be found in I A R C ' s monographs. However, substances such as acrylonitrile (I) and methylmethacrylate (II) (Fig. 5), which are chemically closely related to cyanoacrylate, in a number of studies are referred to as mutagens or carcinogens [ 13,14]. In the case of acrylonitrile the first results of an epidemiological investigation show that occupational exposure to acrylonitrile increases the risk of cancer of the lungs and of the large intestine. It is concluded [13] that acrylonitrile should be regarded as if it were carcinogenic to humans. The harmful effects of cyanoacrylate adhesives on humans will especially appear in work environment where humans are exposed every day to the adhesives. The adhesives give rise to a number of acute complaints in humans, such as irritation of the nose and eyes and dermatitis. Since vapors of the methyl 2-cyanoacrylate monomer~ studied in this investigation, change the genetic information of Salmonella typhimurium TA100, it is reasonable to conclude that a large number of workers, amongst others workers in the electronics industry, in their work with cyanoacrylate adhesives are exposed to vapors that could pose a carcinogenic risk to humans. There is no reason to suppose that this effect can be prevented merely by ensuring that the threshold limit value of 2 p p m is observed.
CH2:CH-C-=N CH2:C-c~,O_cH3 I II Fig. 5. The chemical formulas of the two substances acrylonitrile (I) and methylmethacrylate (II), which are chemically closely related to alkyl 2-cyanoacrylate.
381
Acknowledgement This study was supported by the Danish Medical Research Council.
References 1 Alhopuro, S., A. Rintala, H. Salo and V. Ritsil~i, Tissue adhesives versus sutures in closure of incision wounds, A comparative study in human skin, Ann. Chir. Gynaecol., 65 (1976) 308-312. 2 Antonsen, H., G. Lose and B.K. Olsen, Histoacryl(R)-lim anvendt til lukning af traumatiske shr (Employment of Histoacryl (R) glue for closure of traumatic wounds), Ugeskr. Laeger, 141 (1979) 369-371. 3 Ames, B.N., J. McCann and E. Yamasaki, Methods for detecting carcinogens and mutagens with the Salmonella/mammalian microsome test, Mutation Res., 31 (1975) 347-364. 4 Benson, A.L., G.C. Coletta and P.L. Levins, Removal of cyanoacrylate vapor from work spaces by activated carbon, Am. Ind. Hyg. Assoc. J., 36 (1975) 741-744. 5 Bhaskar, S.N., and J. Frisch, Use of cyanoacrylate adhesive in dentistry, J. Am. Dent. Assoc., 77 (1968) 831-837. 6 Blum, G.N., W.A. Nolte and P.B. Robertson, In vitro determination of the antimicrobiai properties of two cyanoacrylate preparations, J. Dent. Res., 54 (1975) 501-503. 7 Bonichi, O.F., V. Cattaneo, S. DeRysky and A.G. Siccardi, Antibacterial substances diffusible from isobutylcyanoacrylate polymers formed from solutions of monomer in chloroform and ether, IRCS Medical Science: Biomedical Technology; Dentistry and Oral Biology; Microbiology, Parasitology and Infectious Diseases; Pharmacology, 3 (1975) 553. 8 Calnan, C.D., Cyanoacrylate dermatitis, Contact Dermatitis, 5 (1979) 165-167. 9 Coover, H.W. Jr., F.B. Joyner, N.H. Shearer Jr. and T.H. Wicker Jr., Chemistry and performance of cyanoacrylate adhesives, Soc. Plastics Eng. J., 15 (1959) 413-417. 10 Coover, H.W. Jr., and J.M. Mclntire, Cyanoacrylate adhesives, in: I. Skeist (Ed.), Handbook of Adhesives, 2nd edn., Van Nostrand-Reinhold, New York, 1977, pp. 569-580. 11 Giunta, J., and G. Shklar, Studies on tongue carcinogenesis in rats using DMBA with and without cyanoacrylate adhesive, Arch. Oral Biol., 17 (1972) 617- 622. 12 Houston, S., D.K. Ousterhout, K.H. Sleeman and F. Leonard, The effect of n-butyl 2-cyanoacrylate on liver function, J. Biomed. Mater. Res., 4 (1970) 25-28. 13 International Agency for Research on Cancer, Monographs on the evaluation of the carcinogenic risk of chemicals to humans, Vol. 19, Lyon, 1979, pp. 73-113. 14 International Agency for Research on Cancer, Monographs on the evaluation of the carcinogenic risk of chemicals to humans, Vol. 19, Lyon, 1979, pp. 187-211. 15 Kulkarni, R.K., G.A. Hanks, K.C. Pani and F. Leonard, The in vivo metabolic degradation of poly(methyl cyanoacrylate) via thiocyanate, J. Biomed. Mater. Res., 1 (1976) 11-16. 16 Lehman, R.A.W., G.J. Hayes and F. Leonard, Toxicity of alkyl 2-cyanoacrylates, I. Peripheral nerve, Arch. Surg., 93 (1966) 441-446. 17 Lehman, R.A.W., R.L. West and F. Leonard, Toxicity of alkyl 2-cyanoacrylates, II. Bacterial growth, Arch. Surg., 93 (1966) 447-450. 18 Matsumoto, T., and C.A. Heisterkamp, Long-term study of aerosol cyanoacrylate tissue spray: Carcinogenicity and other untoward effects, Am. Surg., 35 (1969) 825-827. 19 McGee, W.A., F.L. Oglesby, R.L. Raleigh and D.W. Fasset, The determination of a sensory response to aikyl 2-cyanoacrylate vapor in air, Am. Ind. Hyg. Ass. J., 29 (1968) 558-561. 20 Pugnier, V.A., Cyanoacrylate resins in caries prevention: A two-year study, J. Am. Dent. Assoc., 84 (1972) 829-831. 21 St6wsand, D., and K. Buhl, Friih- und Spatbefunde nach Biobond-Beschickung von intrakraniellen Aneurysmen (Early and late results after coating intracranial aneurysms with biobond), Acta Neuroclair., 32 (1975) 73-82. 22 Woodward, S.C., J.B. Herrmann, J.L. Cameron, G. Brandes, E.J. Pulaski and F. Leonard, Histotoxicity of cyanoacrylate adhesive in the rat, Ann. Surg., 162 (1965) 113-122.
373
Elsevier Biomedical Press
Mutagenic action of methyl 2-cyanoacrylate vapor Margrethe Andersen, Mona-Lise Binderup, P. Kiel, H. Larsen and Jette Maxild Department of Microbiology, The Royal Danish School of Pharmacy, 2 Universitetsparken, DK.2100 Copenhagen ~ (Denmark)
S.H. Hansen Department of Chemistry BC, The Royal Danish School of Pharmacy, 2 Universitetsparken, DK-2100 Copenhagen ~ (Denmark) (Received 29 March 1981) (Revision received 23 April 1982) (Accepted 3 May 1982)
Summaff Alkyl 2-cyanoacrylate adhesives were tested for mutagenicity in Salmonella typhimurium strains TA98, TA100, TA1535 and TA1538. Both a normal spot test and a spot test specially designed to test volatile compounds were used. The adhesives were also tested in the plate incorporation assay. These investigations showed that methyl 2-cyanoacrylate adhesives are mutagenic in strain TA100. The spot test for volatile compounds showed that it is the vapors from the methyl 2-cyanoacrylate monomer that are responsible for the mutagenic effect. One can conclude that working with methyl 2-cyanoacrylate adhesives entails exposure to vapors with a mutagenic effect and may therefore pose a carcinogenic hazard. Because the adhesives are used in industry, their mutagenic effect has a special importance in work environment.
The production of alkyl 2-cyanoacrylate (Fig. 1) started in the United States in 1958. Since then, a number of different cyanoacrylate adhesives have been produced. Because of their properties, cyanoacrylate adhesives are now used all over the world. They are advantageous because they adhere to many different'materials, e.g. glass, steel, plastic and rubber, and can be used where adhesion has to take place rapidly [10]. Monomeric alkyl 2-cyanoacrylate is a liquid. The commercialized alkyl 2Reprint requests to: Pauli Kiel, The Royal Danish School of Pharmacy, Department of Microbiology, 2 Universitetsparken, DK-2100 Copenhagen ~b, Denmark. 0165-1218/82/0000-0000/$02.75 © Elsevier Biomedical Press
374 C_=N
CH2=(~-c~O_R Fig. 1. Structure of alky] 2-cyanoacrylate.
cyanoacrylate adhesive may contain various additives, e.g. thickening agents, colors and preservatives. Polymerization takes place within a couple of seconds upon contact with even very weak bases, as for example water and alcohol, without requiring heat or the addition of a catalyst [9]. The manufacturers recommend methyl 2-cyanoacrylate adhesives both in household and industrial applications. The adhesives are much used in the electronics industry. Butyl and isobutyl 2-cyanoacrylate adhesives are used especially in the fields of medical science. They are used to close wounds [1,2], to stop bleeding [5], and in a number of special ways in surgery, e.g. brain surgery [21]. Alkyl 2-cyanoacrylate adhesives are used by dentists in the sealing of teeth, to stop bleeding [5], and in the prevention of caries [20]. Our attention has been drawn to methyl 2-cyanoacrylate adhesive with the trade mark TIXO K100 by a woman who has worked with the product. She suspects that her work with the adhesive could be the cause of a miscarriage, and the birth of an anencephaloid child. However, no epidemiological work has been done on the possibility of a teratogenic effect of alkyl 2-cyanoacrylates; therefore, there is no evidence for the woman's suspicions. As a large number of industrial workers, in a number of industrial fields all over the world, are exposed daily to vapor from adhesives, we found it valuable to examine various alkyl 2-cyanoacrylate adhesives in the Salmonella/microsome mutagenicity test.
Materials and methods
Alkyl 2-cyanoacrylate adhesives tested The methyl 2-cyanoacrylate adhesives, TIXO K100 (Hapas A / S ) and Cyanolite 102 (3M A/S), the ethyl 2-cyanoacrylate adhesive, Cyanolite 201 (3M A/S), the allyl 2-cyanoacrylate adhesive, Cyanolite 303 (3M A/S), and the butyl 2-cyanoacrylate adhesive, Histoacryl (m (Braun, Melsungen AG) were all tested in a spot test without modifications, and in a spot test specially designed to test volatile substances, and finally in the plate incorporation assay. Acetone (Merck t.a.) and dimethylformamide (Merck, Uvasol) were used as the solvents in the plate incorporation assay. Tetrachloromethane (Merck t.a.) was used as solvent in the determination of ]H-NMR spectra.
Mutagenicity testing The alkyl 2-cyanoacrylate adhesives were tested in the Salmonella/microsome mutagenicity test, with the histidine-requiring mutants of Salmonella typhimurium.
375 The test was carried out on strains TA100 and TA1535 (reverting by base-pair substitution) and TA98 and TA1538 (reverting by frame-shift mutation), both with and without the addition of the rat-liver preparation $9 mix. Rat-liver enzymes were induced by adding sodium phenobarbital (0.1%) to the drinking water in the last week before the rats were killed. The rat-liver microsome fraction $9 and the N A D P H *-generating system were prepared according to Ames et al. [3]. To each plate, 150/xl S9 in 500 #1 $9 mix was added. The spot test was carried out in two different ways.
The spot test (without modifications) The spot test was carried out as described by Ames at al. [3], without modification. A drop of the adhesive was placed directly on the agar surface. Three different sizes of drop (about 5, 10 and 20 mg) were investigated for each adhesive. Three plates were made f o r each drop size. The plates were incubated at 37°C for 2 days. The spot test for volatile compounds To test whether the mutagenic activity of the adhesives was due to volatile compounds, the spot test was also carried out as follows. As soon as the agar overlay with bacteria and $9 mix had hardened, a sterile microscope glass cover slip (22 mm in diameter) was placed in the middle of the plate. A drop of adhesive was then placed on the cover slip. To stop vapors from escaping, the petri dishes were then sealed with tape (Samson, Plastic electrical tape 0.2 m m / 1 5 mm) (Fig. 2). All the adhesives were examined by using three different sizes of drop, about 5, 10 and 20 mg, with three plates per size of drop. Plate incorporation assay The plate incorporation assay was carried out with the test strain TA100 and without $9 mix. The test was carried out as described by Ames et al. [3], without modification. The adhesives were dissolved in 100 #1 acetone and incorporated into the agar overlay. Amounts from 0.1 to 5.0 mg of adhesive per plate were tested. Proton magnetic resonance spectra (1H-NMR) Proton magnetic resonance spectra were recorded on a Jeol C-60 H L 60 MH z proton magnetic resonance spectrometer with tetramethylsilane as internal standard. The test solutions were made by dripping the adhesives directly into the tetrachloromethane.
Results
When the drop of adhesive was placed directly on the agar surface, polymerization took place immediately, activated by the humidity in the agar overlay. As the * NADPH, reduced form of nicotinamideadenine dinucleotidephosphate.
376
revertant colony ~/' :.:.::!~..
: ;i".~;.-'ig. inhibition zone
~ i
- - m -'~ ,i adhesive ~.., c o v e r s l i p
" .....................
drop
~:a~-~:. "' ..... :;:: "."
background
growlh
Fig. 2. Spot test method for testing the mutagenic effect of volatile compounds. drop of adhesive polymerized, a white ring-shaped film formed on the surface of the agar around the drop of adhesive. The same phenomenon was observed in the spot test for volatile compounds. Polymerization took place in the same way both with and without $9 mix in the plates. In both spot test methods the methyl 2-cyanoacrylate adhesives formed the largest white rings and the butyl 2-cyanoacrylate adhesive the smallest ones. After two days of incubation at 37°C, the spot test plates were examined for inhibition zones in the background lawn.of bacterial growth, and for accumulation of revertant colonies near the zone of inhibition. An accumulation of revertant colonies near the inhibition zone was regarded as a mutagenic effect. The inhibition zone and the white ring had the same diameter in the case of each adhesive. In both spot test methods the two methyl 2-cyanoacrylate adhesives, TIXO K100 and Cyanolite 102, had a mutagenic effect in the test strain TA100 (Fig. 3). There was a mutagenic effect both with and without the $9 mix. No mutagenic effect was observed with test strain TA1538, TA1535 or TA98. N o mutagenic effect was observed with the ethyl, allyl and butyl 2-cyanoacrylate adhesives in the spot tests. In the plate incorporation assay, precipitation of polymerized adhesive could be clearly seen in the agar overlay, when a dose greater tl~an 1.0 mg per plate (2 ml agar overlay) was used. Inhibition of the background growth could be detected in the case of TIXO K100 and Cyanolite 102 as from concentrations of 0.5 mg (4.5/~moles) per plate, whilst with ethyl, allyl and butyl 2-cyanoacrylate adhesives, it could be detected as from concentrations of between 2.0 and 5.0 mg per plate. The results of the plate incorporation assay with the two methyl 2-cyanoacrylate adhesives and the butyl 2-cyanoacrylate adhesive tested in strain TA100, are presented in Fig. 4. Corresponding results were obtained in three independent experiments. The figure shows that the mutagenic effect of methyl 2-cyanoacrylate adhesives increased as the amount of adhesive increased, between 0.1 and 0.5 mg (0.9-4.5 /~moles) of adhesive per plate. In the assays with ethyl, allyl and butyl 2-cyanoacrylate adhesives, no increase in the number of revertants with increase in dose was observed. To find out whether the mutagenic effect was due to additives, the following adhesives were examined with ] H - N M R spectroscopy: Cyanolite 201, Cyanolite 102, TIXO K100 and Histoacryl.
377
Fig. 3. Mutagenic action of the methyl 2-cyanoacrylate, TIXO K100, on Salmonella typhimurium TAI00. The photograph shows the spot test method for testing volatile compounds. An accumulation of revertant colonies near the inhibition zone is seen.
378 600 500
a /*00
Z 300 .~ 200
IO0
0123&56.7891.0 Dose per ptafe (rag)
Fig. 4. The mutagenic effect on Salmonella typhimurium TAI00 of the two methyl 2-cyanoacrylate adhesives and the butyl 2-cyanoacrylate adhesive in the plate incorporation assay without the addition of $9 mix. Each point is an average of numbers scored on three to five plates. The limits of the vertical lines indicate the lowest and highest values obtained with each dose. A, methyl 2-cyanoacrylate, Cyanolite 102; O, methyl 2-cyanoacrylate, TIXO KI00; f-q, butyl 2-cyanoacrylate, Histoacryl R.
In the spectra of the two methyl 2-cyanoacrylate adhesives some very broad signals could be seen, situated in the zones of 0.8-1.0 ppm, 1.3-2.0 p p m and 3.5-3.7 ppm. These signals could not be attributed to the monomer, and further identifications have not been attempted. Shaking the tetrachloromethane part together with water resulted in polymerization of the adhesive; but the broad signals described above could still be observed unchanged in the organic phase. That, or the substances that were the cause of the irregularity in the N M R spectrum, were therefore isolated in the followingway. One gram of adhesive was dissolved in 2 0 m l tetrachloromethane and then shaken together with 200 ml distilled water. This caused the adhesives to polymerize. The tetrachloromethane part, containing the additives, was separated from the rest and evaporated. The residue from the evaporation, 0.1 g (0.9/~moles) from T I X O K 1 0 0 and 0.005-0.01 g (0.045-0.09 gmoles) from Cyanolite 102, was dissolved in dimethylformamide, and was tested for mutagenicity in t h e same way as were the adhesives, both in the spot test and in the plate incorporation assay. In the plate incorporation assay an amount corresponding to between 0.1 mg (0.9 #moles) and 10 mg (90/tmoles) of adhesive per plate was tested. The evaporation residue did not reveal any mutagenicity. To find out whether the mutagenic effect observed could be due to a product formed by a possible depolymerization of the polymerized adhesive, e.g. formaldehyde, we also examined an already polymerized adhesive. A drop of adhesive was placed on a microscope glass cover slip, which was then placed on the surface of a Vogel-Bonner plate. The petri dish was sealed with tape and maintained at 37°C. After 24 h the cover slip with the polymerized adhesive was transferred to a plate with agar overlay, containing TA100. After 48 h at 37°C no inhibition zone and no ring of revertants could be seen around the cover slip.
379
Discussion
The two methyl 2-cyanoacrylate adhesives tested were mutagenic in the Salmonella/microsome mutagenicity test in strain TA100 both with and without $9 mix. Mutagenicity testing of the additives isolated from the two methyl 2-cyanoacrylate adhesives did not reveal any mutagenic effect. This means that it must be the methyl 2-cyanoacrylate monomer itself that is the cause of the mutagenic effect. The polymer of methyl 2-cyanoacrylate degrades in vitro to formaldehyde and ultimately to cyanoacetate [15]. In their study of the bacteriotoxic effect of cyanoacrylate adhesives [17], Lehman et al. suggest that this effect might be due to products formed as a result of aqueous degradation of the polymers. It has also been suggested that the toxic effect of alkyl 2-cyanoacrylates in animal experiments might be due to products of degradation. In the experiments with the polymerized cyanoacrylate adhesives we found that, under these test conditions, no toxic or mutagenic substances were released. This tends to indicate that both the bacteriotoxic and mutagenic effects are due to the methyl 2-cyanoacrylate monomer. Bonichi et al. [7] and Blum et al. [6] come to the same conclusion according to the toxicity in their studies of the antimicrobial effect. Blum et al. show, moreover, that it is the vapors from the adhesive that are the cause of the growth-inhibiting effect of alkyl 2-cyanoacrylate. Our experiments in the spot test for volatile compounds show, in agreement with the findings of Blum et al., that it is the vapors from the methyl 2-cyanoacrylate adhesives that are responsible for the growth-inhibiting and mutagenic effects. The shape of the dose-response curve (Fig. 4) from the plate incorporation assay also tends to indicate that it is the monomer that is mutagenic. Firstly, the effect did not increase with concentrations higher than 0.5 mg (4.5 ttmoles) per plate, the concentration at which precipitation of polymers can clearly be seen in the agar overlay, and secondly, the greatest increase in revertants can be seen between 0 and 0.1 mg (0.9/~moles) per plate. The observation of a far greater number of revertants per plate in the spot test than even with the highest concentrations in the plate incorporation assay also indicates that it is the vapors from the monomer that are responsible for the mutagenic effect. Alkyl 2-cyanoacrylate monomers have also been tested in implantation studies in rats, dogs and chimpanzees [12,15,16,22]. These studies showed that the methyl 2-cyanoacrylate monomer is the most histotoxic of all the alkyl 2-cyanoacrylates and causes necrosis, edema and cell death. The toxic effect has in many cases been ascribed to degradation products of the polymer [15,22]. Only few investigations have been made to determine the possible carcinogenicity of alkyl 2-cyanoacrylate monomers. In one non-positive investigation [18] the animals were only treated once with a butyl 2-cyanoacrylate spray. In another experiment, designed to provoke epidermoid carcinomas in the oral tissues [11], isobutyl 2-cyanoacrylate was used as a control, bonding DMBA powder (dimethylbenzanthracene, a well-known carcinogen) to the tongues of rats. After 6 months of applications there were no epidermoid carcinomas, either with the isobutyl 2-
380 cyanoacrylate adhesive alone, or with both the D M B A and the adhesive together. F r o m these studies it is therefore impossible to conclude anything about a possible carcinogenic effect of alkyl 2-cyanoacrylate monomers. We do not know of any studies where vapors of cyanoacrylate monomers have been tested for mutagenic or carcinogenic effect. In the field of occupational health and safety research, unlike fields of toxicological research, attention has been paid especially to the vapors of cyanoacrylate adhesives and to their removal. Because the vapor is irritating, a maximal concentration of 2 to 3 p p m is recommended for work spaces [4,19]. This recommendation is based upon the findings of a study [19], where volunteer subjects were exposed to concentrations of methyl 2-cyanoacrylate monomer ranging from 1 p p m to 60 ppm. In this study nose irritation was found from 3 p p m and eye irritation from 5 ppm. An outbreak of irritant dermatitis, caused by cyanoacrylate adhesive, among electronic assembly workers, has been reported [8]. The concentration of cyanoacrylate vapors in the air, and the outbreaks of dermatitis, fell when the relative humidity of the work environment was raised above 55% [8]. The basis for setting the threshold limit value at 2 p p m is thus solely the acute toxic effect. In no way has account b e e n taken of any possible long-term effect. N o relevant studies of the mutagenic or carcinogenic effect of cyanoacrylate adhesives have been made, and these substances are not to be found in I A R C ' s monographs. However, substances such as acrylonitrile (I) and methylmethacrylate (II) (Fig. 5), which are chemically closely related to cyanoacrylate, in a number of studies are referred to as mutagens or carcinogens [ 13,14]. In the case of acrylonitrile the first results of an epidemiological investigation show that occupational exposure to acrylonitrile increases the risk of cancer of the lungs and of the large intestine. It is concluded [13] that acrylonitrile should be regarded as if it were carcinogenic to humans. The harmful effects of cyanoacrylate adhesives on humans will especially appear in work environment where humans are exposed every day to the adhesives. The adhesives give rise to a number of acute complaints in humans, such as irritation of the nose and eyes and dermatitis. Since vapors of the methyl 2-cyanoacrylate monomer~ studied in this investigation, change the genetic information of Salmonella typhimurium TA100, it is reasonable to conclude that a large number of workers, amongst others workers in the electronics industry, in their work with cyanoacrylate adhesives are exposed to vapors that could pose a carcinogenic risk to humans. There is no reason to suppose that this effect can be prevented merely by ensuring that the threshold limit value of 2 p p m is observed.
CH2:CH-C-=N CH2:C-c~,O_cH3 I II Fig. 5. The chemical formulas of the two substances acrylonitrile (I) and methylmethacrylate (II), which are chemically closely related to alkyl 2-cyanoacrylate.
381
Acknowledgement This study was supported by the Danish Medical Research Council.
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