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Introduction Background to COM consideration of Formaldehyde
1. Formaldehyde is produced worldwide on a large scale and is used in the production of phenolic, urea, melamine and polyacetal resins. Formaldehyde is also used as an intermediate in the manufacture of industrial chemicals and as an aqueous solution (formalin) as a disinfectant and preservative in many situations. Formaldehyde also occurs as a natural product in most living systems and in the environment. There are also a number of non occupational sources of exposure including vehicle emissions, from building and household materials, from food and cooking and from tobacco smoke. Most formaldehyde released to the environment is rapidly degraded and human exposure from environmental sources is most likely to occur when there is a continuous source present.1,2
2. Formaldehyde is a normal intermediary metabolite in humans. It is estimated that endogenous blood concentrations of formaldehyde are approximately 0.1 mM. Absorbed formaldehyde can be metabolised to formate and enter the one carbon pool for incorporation in DNA, RNA and proteins. Other pathways of metabolism include oxidation to carbon dioxide. Absorbed formaldehyde is rapidly excreted. A small proportion of material may be metabolised by a number of non saturable pathways at the site of contact to form cross links with proteins and a small amount to DNA. The Committee agreed that a consideration of the toxicokinetics of formaldehyde would be important with regard to consideration of potential for systemic mutagenicity.
3. Formaldehyde has recently been considered by IARC and placed into group 1 (carcinogenic to humans).1 There is sufficient evidence that formaldehyde causes nasopharyngeal cancer in humans. There was limited evidence that formaldehyde causes sinonasal cancer in humans. The working group concluded that '..there is strong but not sufficient evidence for a causal association between leukaemia and occupational exposure to formaldehyde. Increased risk for leukaemia has consistently been observed in studies of professional workers and in two of three of the most relevant studies of industrial workers These findings fall slightly short of being fully persuasive because of some limitations in the findings from cohort and garment workers in the USA and because they conflict with the non-positive findings from the British cohort of workers'. This conclusion stimulated some discussion in the published literature regarding the possible mechanism of formaldehyde induced increased risk of leukaemia.3 The IARC working group commented on the possible mechanism and '..noted evidence for clastogenic damage to circulatory cells, but overall since there was no good animal models for acute myeloid leukaemia, did not reach a conclusion with regard to the mechanism of acute myeloid leukaemia reported in epidemiological studies'.1 Introduction to COM discussion 4. The COM was asked to consider the evidence for systemic mutagenicity from animal experiments and biomonitoring studies of workers exposed to formaldehyde. The objective was to consider the potential for systemic mutagenicity following inhalation exposure, the predominant route of exposure in the occupational groups for which evidence of leukaemia had been reported. 5. The COM were aware of a number of recent internationally recognised reviews which had reported on both carcinogenicity and mutagenicity and agreed there was no need to specifically review all of the mutagenicity data on formaldehyde.1,2,4 This included a recently published evaluation of the mode of action (MOA) for nasopharyngeal cancer.4 The COM acknowledged that formaldehyde is a direct acting in vitro mutagen in bacterial and mammalian cells (including rodent and human cell lines). Mutagenic effects reported included point mutations, chromosome aberrations, sister chromatid exchanges, DNA strand breaks and UDS in rat nasal turbinate cells.1,24 With regard to the MOA for rat nasopharyngeal tumours, most reviewers had considered the formation of formaldehyde DNA-protein cross links (DPX) with a similar dose-response to the formation of nasal tumours in rats, with consequent marked local effects on cytotoxicity, cell proliferation and local site of contact mutagenic effects as key elements in the proposed MOA. The magnitude of the formaldehyde induced local site of contact cell proliferation had been emphasised in the available reviews.2,4 6. The Committee agreed to focus its initial discussion on the available peer reviewed scientific literature regarding toxicokinetics of absorbed formaldehyde and the evidence in the published literature for systemic in-vivo mutagenicity.1-30 The kinetics of absorbed formaldehyde
7. The COM considered information from published inhalation studies in rats and Rhesus monkeys (using either single exposure to 6 ppm for 6 h or repeated exposure to the same concentrations 5 days/week for 4 weeks with sampling after the last exposure) and agreed the majority of absorbed formaldehyde was incorporated into intermediary metabolism (³91%) with small amounts bound as DPX (ca £9%).3,14,15 Importantly there was no evidence for an increase in blood formaldehyde concentrations in these studies. In some relatively old published experiments the metabolic incorporation and covalent binding of formaldehyde was investigated in nasal tissue and bone marrow following inhalation exposure of rats to formaldehyde (dual labelled 3H- and 14C-) at a number of concentrations up to 15 ppm for 6 h. There was evidence for covalent binding of formaldehyde to rat nasal DNA but no evidence for binding to bone marrow.15 In a further study using a similar inhalation exposure rats were pretreated with an intraperitoneal injection of phorone designed to deplete non protein sulphydryl levels and hence increase the potential for DNA binding of absorbed formaldehyde. Metabolic incorporation into rat nasal tissue and bone marrow were significantly decreased, but there was no evidence for covalent binding to bone marrow DNA.16
8. The committee noted that a number of research groups had attempted to model the systemic uptake and distribution of formaldehyde in humans exposed to formaldehyde at exposure levels close to or at the UK occupational exposure standard of 2 ppm for 8 hours.2,3,5,29 These studies had concluded that the systemic blood levels of formaldehyde resulting for such exposures would be £0.1% of the endogenously formed blood concentrations of formaldehyde (ie approximately 0.0001mM compared to an endogenous level of approximately 0.1 mM). 9. The Committee concluded that systemic exposure to formaldehyde at potential cancer target organs resulting from inhalation exposure would be a negligible amount compared to endogenously formed formaldehyde. Members acknowledged that there was limited information on the proportion of free and bound formaldehyde and the potential for release of adsorbed formaldehyde bound to macromolecules but overall considered that the potential for redistribution of formaldehyde was very small compared to endogenously formed formaldehyde. Potential for in vivo systemic mutagenicity
10. The COM noted that a number of test materials had been used in in vivo studies in experimental animals which included aqueous solutions (30-50%) stabilised with 10% methanol. Alternatively paraformaldehdye hydrolysed using sodium hydroxide might be used as a source of formaldehyde generation. Formaldehyde is intrinsically reactive and thus unstabilised solutions may be oxidised to formic acid and at low temperatures a precipitate of trioxymethylene may be formed.2 The precise composition of the test material used was not clear in many of the studies reviewed but was presumed to be predominantly formaldehyde. Members agreed the presence of methanol would complicate the evaluation of in vivo genotoxicity studies. 11. The COM agreed that the available in vivo tests for micronucleus induction and chromosome aberrations in bone marrow in rodents using inhalation exposure or intraperitoneal administration were predominantly negative.7-11 A slight and apparently dose related increase in micronucleated cells/1000 PCEs was noted in mice given two intraperitoeneal doses of formaldehyde (derived from paraformaldehdye at 6.25, 12.5 or 25 mg/kg bw) separated by 24 hours at two samplings (16 h and 40 h post final dose).8 It was noted that the study authors had not considered that a statistically significant increase had been documented in this study. The Committee considered a recently published in vivo comet assay in rats exposed by inhalation to up to 10 ppm 6 h/day for 5 days/week. An apparent dose-related increase in comet tail moment had been reported following examination of 50-100 cells/animal in peripheral blood lymphocytes and in the liver. It was noted that in principle a cross linking agent such as formaldehyde might be expected to reduce comet tail moment and it was possible that the effects might have been due to oxidative damage and possibly apoptosis. The COM noted that formaldehyde induced disturbances of protein and lipid oxidation in this study.13 12. The Committee considered the evidence for a dominant lethal effects and noted two positive results had been reported. Thus a single intraperitoneal dose of formaldehyde (50 mg/kg bw) administered to Q strain mice with separate matings each week for seven weeks resulted in increased embryonic death in the 1st and 3rd weeks.6 There was no evidence for a clastogenic effect on spermatocytes in this study. The COM concluded it was unlikely that the effects reported resulted from a systemic mutagenic effect of formaldehyde. 13. In a separate investigation an increase in the number of abnormal spermatozoa and evidence for a dominant lethal effect was reported in a study using isogenic University of Lagos rats given intraperitoneal doses of 0.125-0.5 mg/kg bw of formaldehyde.12 These doses were reported to be between 1/16 and ¼ of the intraperitoeneal LD50 in this strain of rat and are very much lower than the dose levels used in other in-vivo mutagenicity studies with formaldehyde. The dominant lethal study examined matings 1-7 days, 8-14 days and 15-21 days post dosing. A significant increase in the number of dead implants was reported in the period 1-7days post dose which was accompanied by a reduction in sperm counts and abnormal spermatozoa.12 Members noted that the use of methanol to stabilise formaldehyde could have contributed to the observed effects on spermatozoa morphology reported in rats. 14. The COM concluded that the mechanism by which formaldehyde could have induced the observed effects in the dominant lethal studies was unclear but did not involve a direct systemic mutagenic response.
15. The Committee noted clear evidence for an increase in micronucleated cells of the basal epithelium of the stomach in a study in rats where a single oral dose of 200 mg/kg formaldehyde was administered.11 In a separate study using repeated inhalation exposure of rats to formaldehyde (up to 15 ppm 6 h/day for 1 or 8 weeks), lung lavage samples in addition to bone marrow samples were examined for chromosome aberrations. There was no evidence for a clastogenic effect in bone marrow samples but a statistically significant increase in chromosome aberrations was noted in lung lavage samples.10 16. The COM agreed that formaldehyde was a site of contact in-vivo mutagen. Members noted that the site of contact effects in the gastrointestinal tract might not all be due to formaldehyde reacting directly within the target cells as the effects extended down the gastrointestinal tract further than would be expected for a highly reactive chemical. Biomonitoring studies of formaldehyde exposure17-28 17. Biomonitoring studies of genotoxicity in workers exposed during a variety of activities including manufacture of formaldehyde and use of formaldehyde in mortuary and anatomy departments and in paper impregnation were evaluated. A further group used in biomonitoring studies were dialysis patients where the dialysis equipment was sterilised with formaldehyde. A recent study of volunteers exposed to formaldehyde at levels below the occupational exposure standard was also retrieved.31 A number of these studies had reported evidence for an increase in MN or DPXs in PBLs.17-28 18. The COM considered these data with regard to the evaluation of data from toxicokinetic studies and in-vivo genotoxicity studies in experimental animals which suggested there would be no biological rationale for a direct systemic mutagenic effect of formaldehyde in biomonitoring studies. None of the studies collected the appropriate information previously identified by COM to assess background variation in results. Thus members noted that the quality of the biomonitoring studies was poor with limited account for confounding factors, including age, and also considered that the method for determination of DNA-protein cross links (SDS separation of protein-linked DNA) in PBLs had not been adequately validated. The COM suggested that a secondary mechanism could be responsible for the positive findings in peripheral blood lymphocytes. 19. The COM considered that no definite conclusions could be reached with regard to the small increases in DNA-protein cross links reported in the studies published by Saham et al.22,26 20. The Committee considered if any of the available studies was of sufficient quality to draw conclusions with regard to systemic mutagenicity of formaldehyde. Members noted the study by Ye and colleagues27 clearly showed an increase in micronuclei in nasal mucosal cells in workers exposed to formaldehyde during manufacture whilst no concurrent increase in micronuclei in peripheral blood lymphocytes was noted.27 The increase in SCE formation in peripheral blood lymphocytes in this study may have resulted from a secondary mechanism following oxidative DNA damage. 21. Members commented on the publication by Orsiere T et al (2006).28 The apparent increase in micronuclei with centromeres was not consistent with the proposed mechanism of formaldehyde effects cross linking DNA and proteins. It was noted the protocol was not optimal for identification of aneuploidy, and that individual data were not available. Members considered that no definite conclusions could be reached on the data presented in this publication. 22. There was no evidence for an increase in micronuclei in buccal smears from volunteers exposed for up to 4 h/day for 10 working days to levels of formaldehyde below the UK occupational exposure limit (ie < 2 ppm).31 23. The Committee concluded that there was no convincing evidence regarding direct systemic mutagenic effects of formaldehyde from the available biomonitoring studies. The COM agreed a secondary mechanism might be involved with regard to the genotoxic effects documented in peripheral blood lymphocytes in the biomonitoring studies reviewed. Additional in vitro study of formaldehyde induced DPX 24. The Committee noted that recent in vitro studies had demonstrated that DPXs formed from formaldehyde were effectively removed at concentrations up to 100 µM.30 COM conclusions 25. The COM concluded that the amount of formaldehyde systemically available following inhalation exposure at the occupational exposure standard would be negligible. 26. The COM was aware that formaldehyde was a direct acting in vitro mutagen. The COM concluded that there was no convincing evidence from in vivo mutagenicity studies in experimental animals and from biomonitoring studies of genotoxicity in workers exposed to formaldehyde for a direct in vivo systemic mutagenic effect of inhaled formaldehyde. A secondary mechanism might be involved in the genotoxic effects documented in peripheral blood lymphocytes in the biomonitoring studies reviewed. 27. The COM concluded that there was no reason to consider that direct systemic mutagenicity would be involved in the mechanism of formaldehyde induced systemic tumourigenicity. 28. The Committee concluded that for occupational and environmental exposure to formaldehyde, the pattern of metabolism and distribution of formaldehyde indicate that a threshold for in vivo systemic mutagenicity is likely. November 2007 References 1. IARC (2004). Summary of Data Reported and Evaluation. (http://monographs.iarc.fr/ENG/Meetings/88-formaldehyde.pdf). IARC monograph vol 88 in preparation. 2. IPCS (2002). Formaldehyde. Concise International Chemical Assessment Document 40. 3. Heck H d'A and Casanova M (2004). The implausibility of leukaemia induction by formaldehyde: a critical review of the biological evidence on distant-site toxicity, Regulatory Toxicology and Pharmacology, 40, 92-106. 4. McGregor D et al (2006). Formaldehyde and Gluteraldehyde and nasal cytotoxicity: case study within the context of the 2006 IPCS human framework for the analysis of cancer mode of action in humans. (Draft in confidence document provided to the secretariat). 5. Overton JH et al (2001). Dosimetry modelling of inhaled Formaldehyde: The human respiratory tract. Toxicological Sciences, 64, 122-134. 6. Fontignie-Houbrechts N (1981). Genetic effects of formaldehyde in the mouse. Mutation Research, 88, 109-114. 7. Gocke E et al. (1981). Mutagenicity of cosmetics ingredients licensed by the European Communities. Mutation Research, 90, 91-109. 8. Natarajan AT et al (1983). Evaluation of the mutagenicity of formaldehyde in mammalian cytogenetics assays in-vivo and in-vitro. Mutation Research, 122, 355-360. 9. Kligerman AD et al (1984). Cytogenetic analysis of lymphocytes from rats following formaldehyde inhalation. Toxicology Letters, 21, 241-246. 10. Dallas CE et al (1992). Cytogenetic analysis of pulmonary lavage and bone marrow cells of rats after repeated inhalation. J of Applied Toxicology, 12, 199-203. 11. Migliore L et al (1989). Micronuclei and nuclear anomalies induced in the gastrointestinal epithelium of rats treated with formaldehyde. Mutagenesis, 4, 327-334, 1989. 12. Odeigah PGC (1997). Sperm head abnormalities and dominant lethal effects of formaldehyde in albino rats. Mutation research, 389, 141-148. 13. Im H et al(2006). Evaluation of toxicological monitoring markers using proteomic analysis in rats exposed to formaldehyde. J of Proteome Research, 5, 1354-1366. 14. Casanova M et al (1988). Formaldehyde concentrations in the blood of Rhesus monkeys after inhalation exposure. Fd Chem Tox, 26, 715-716. 15. Casanova-Schmitz M et al (1984). Differentiation between metabolic incorporation and covalent binding in the labelling of macromolecules in the rat nasal mucosa and bone marrow by inhaled 14C- and 3H- formaldehyde. Toxicology and Applied Pharmacology, 76, 26-44. 16. Casanova M and Heck H D'A. Further studies of the metabolic incorporation and covalent binding of inhaled 3H and 14C formaldehyde in F344 rats: Effects of glutathione depletion. Toxicology and Applied Pharmacology, 89, 105-121. 17. Goh K and Cestero RVM (1979). Chromosomal abnormalities in haemodialysis patients. J of Medicine, 10, 167-174. 18. Fleig I et al (1982). Cytogenetic analysis of blood lymphocytes of workers exposed to formaldehyde in formaldehyde manufacturing and processing. Journal of Occupational Medicine, 24, 1009-1012. 19. Thomson EJ et al (1984). Chromosome aberrations and sister chromatid exchange frequencies in pathology staff occupationally exposed to formaldehyde. Mutation research, 141, 89-93. 20. Bauchinger M and Schmid E (1985). Cytogenetic effects in lymphocytes of formaldehyde workers of a paper factory. Mutation Research, 158, 195-199. 21. Suruda A et al (1993). Cytogenetic effects of formaldehyde exposure in students of mortuary science. Cancer Epidemiology, Biomarkers & Prevention, 2, 453-460. 22. Shaham J et al (1996). DNA-protein crosslinks, a biomarker of exposure to formaldehyde in vitro and in vivo studies. Carcinogenesis, 17, 121-125. 23. Vasudeva N and Anand C (1996). Cytogenetic evaluation of medical students exposed to formaldehyde vapour in the gross anatomy dissection laboratory. Clinical and Program Notes, 177-179. 24. Ying CJ et al (1997). Micronuclei in nasal mucosa, oral mucosa and lymphocytes in students exposed to formaldehyde vapour in anatomy class. Biomedical and Environmental Sciences, 10, 451-455. 25. Ying CJ et al (1999). Lymphocyte subsets and sister chromatid exchanges in the students exposed to formaldehyde vapour. Biomedical and Environmental Sciences, 12, 88-94. 26. Shaham J et al (2003). DNA-protein cross links and p53 protein expression in relation to occupational exposure to formaldehyde. Occupational and Environmental Medicine, 60, 403-409. 27. Ye X, et al (2005). Cytogenetic analysis of nasal mucosa cells and lymphocytes from high level long-term formaldehyde exposed workers and low-level short-term exposed waiters. Mutation Research, 588, 22-27. 28. Orsiere T et al, (2006). Genotoxic risk assessment of pathology and anatomy laboratory workers exposed to formaldehyde by use of personal air sampling and analysis of DNA damage in peripheral lymphocytes. Mutation Research, 605, 30-41. 29. Franks SJ (2005). A mathematical model for the absorption and metabolism of formaldehyde vapour by humans Toxicology Applied Pharmacology, 206, 309-20. 30. Schmid O and Speit G (2007). Genotoxic effects induced by formaldehyde in human blood and implications for the interpretation of biomonitoring studies. Mutagenesis, 22, 69-74. 31. Speit G et al (2007). Assessment of local genotoxic effects of formaldehyde in humans measured by the micronucleus tests with exfoliated buccal mucosal cells. Mutation Research, 627, 129-135.
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