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- Elimination and excretion
- Effects on Laboratory Mammals and Other Test Systems
- Short-term and sub-chronic exposure studies
- Long-term exposure studies
Kinetics and Metabolism
Inhaled formaldehyde is mostly deposited and readily absorbed in the regions of the upper respiratory tract with which it comes into initial contact, owing to its high water solubility and reactivity with biological macromolecules (Heck et al., 1983; Swenberg et al., 1983). A complex relationship between nasal anatomy, ventilation and breathing patterns (nasal or oronasal) determines where in the upper respiratory tract formaldehyde absorption occurs in species. In rodents, which are obligate nasal breathers, deposition and absorption occurs primarily in the nasal passage. In contrast, primates are oronasal breathers, and although absorption and deposition is likely to occur primarily in the oral mucosa and nasal passages it can also occur in the trachea and bronchus (Monticello et al., 1991). At the site of contact, formaldehyde has been shown to produce intra and intermolecular crosslinks with proteins and nucleic acids (Casanova et al., 1989; 1991). There are no direct toxicokinetic studies on formaldehyde following oral or dermal administration. However, the use of physiochemical and toxicological data allows a qualitative assessment of the toxicokinetic behaviour of formaldehyde to be made for these routes of exposure. On the basis of its low molecular weight, high water solubility and moderate octanol/water partition coefficient (Log P) value, it is likely that significant absorption via the oral route would occur. These physiochemical characteristics of formaldehyde would also favour dermal absorption. The observation of skin sensitisation in animal studies (Section 10.3) indicates that such absorption can occur.
No increase in formaldehyde concentration was seen in blood in humans, rats, and monkeys following exposure to concentrations of 1.9 ppm (2.3 mg/m3), 14.4 ppm (17.3 mg/m3) and 6 ppm (7.2 mg/m3) gaseous formaldehyde, respectively (IPCS, 2002). This has been attributed to the deposition of formaldehyde principally in the respiratory tract and its rapid metabolism (Heck et al., 1985; Casanova et al., 1988). The half-life in circulation has been shown to range from 1 to 1.5 minutes between animal species following intravenous administration (Rietbrock, 1969; McMartin et al., 1979). Such rapid metabolism would inhibit systemic distribution of formaldehyde.
Formaldehyde can be metabolised by a variety of pathways: (1) incorporation into the one-carbon pool pathway, (2) conjugation to glutathione then oxidation by formaldehyde dehydrogenase, and (3) oxidation by the peroxisomal enzyme catalase (Kallen & Jencks, 1966; Uotila & Koivusalo, 1974a; Waydhas et al., 1978). Formaldehyde is rapidly metabolised to formate by a number of widely distributed cellular enzymes, the most important of which is formaldehyde dehydrogenase that metabolises the formaldehyde-glutathione conjugate to formate. Formaldehyde dehydrogenase has been detected in human liver and red blood cells and a number of tissues in the rat including respiratory and olfactory epithelium, kidney and brain (Uotila & Koivusalo, 1974b; Casanova-Schmitz et al., 1984). Both formaldehyde and formate are incorporated into the one-carbon pathways involved in the biosynthesis of protein and nucleic acid via direct reaction with tetrahydrofolate. Formaldehyde can also be oxidised to formic acid by catalase, though this reaction probably represents a minor pathway for formaldehyde metabolism. Additionally, it should be noted that formaldehyde is itself formed endogenously during the metabolism of amino acids and xenobiotics (Johansson & Tjalve, 1978; Upreti et al., 1987). Due to the rapid metabolism of formaldehyde, much of the material is eliminated as carbon dioxide in expired air shortly after exposure, and as formate in urine (Keefer et al., 1987; Heck et al., 1983). Elimination of total radioactivity following exposure of rats to [14C]-formaldehyde indicated that 40% of the inhaled [14C] was excreted in expired air, 17% in urine and 5% in faeces. The rest of the radioactive label (35% to 39%) remained in the tissues and carcass, presumably as products of metabolic incorporation (Heck et al., 1983).
This chapter is a summary of the health effects of formaldehyde. It is mainly based on the Concise International Chemical Assessment Document (IPCS, 2002), the Toxicological Profile (ATSDR, 1999) and the SIDS Initial Assessment Report (OECD, 2002). Articles published post 1998 are summarised in this chapter.
Formaldehyde has been found to be moderately toxic in laboratory animals exposed via inhalation, dermal and oral routes. The acute toxicity of formaldehyde has been studied in several animal species and is summarised in Table 10.1. Table 10.1:Summary of LD50 and LC50 values for formaldehyde
Clinical signs of toxicity, observed following single exposure of formaldehyde vapour at concentrations > 100 ppm (> 120 mg/m3) were hypersalivation, acute dyspnoea, vomiting, muscular spasms, and death (Skog, 1950; Horton et al., 1963; Bitron & Aharonson, 1978).
With the exception of a recently conducted eye irritation study by Maurer et al. (2001) summarised below, the limited data available for skin and eye irritation are from old briefly reported studies. These studies state that aqueous solutions of 0.1% to 20% formaldehyde were irritating to rabbit skin (NRC, 1981), and aqueous solutions of 5% and 15% formaldehyde were irritating to rabbit eyes (Carpenter and Smyth, 1946). In a mouse repeated dermal study (see Section 10.4.3), skin irritation was observed with 0.5% formaldehyde solution and above. No skin irritation was seen at 0.1% (Krivanek et al., 1983). The SIAR (OECD, 2002) and IPCS report (1989) concluded that although formaldehyde solution is known to be a primary skin and eye irritant in animals this is based on anecdotal evidence rather than robust animal studies. Skin irritation studies in animals using gaseous formaldehyde were not found. In a recent well-reported study, Maurer et al. (2001) investigated the ocular irritation of formaldehyde solution in a series of experiments. In a low-volume eye test (LVET), 10 l of 37% formaldehyde solution was applied directly to the cornea of 12 rabbits. Eyes were macroscopically examined to determine the degree and extent of irritation to the cornea, iris and conjunctiva at 3 hours post instillation and 1-4, 7, 14, 21, 28 and 35 days after treatment. The maximum score obtainable was 110 (cornea = 80, iris = 10, conjunctiva = 20). Additionally, from this group of 12 rabbits, 3 animals were sacrificed at 3 hours, 1, 3 and 35 days post-instillation, and the eyes were removed, sectioned, stained and examined by light microscopy to determine the extent of corneal and conjunctiva changes (< 5% slight, 6% to 30% mild, 61% to 90% marked and 91% to 100% severe). Macroscopic observations showed that formaldehyde solution produced irritation of the cornea, conjunctiva and iris 3 hours after application. An irritation score of 53.5/110 was determined. This value increased to a maximum of 80.0/110 (time of scoring not reported). Microscopic examination indicated that severe irritation had occurred to the cornea and conjunctiva. Observations included erosion, denudation and oedema to the corneal and conjunctival epithelium. “Necrosis/loss” of corneal keratocytes was also observed 1 day after instillation in all 3 rabbits. At study termination on day 35, both macro- and microscopic examination revealed corneal irritation in all animals. In a further experiment, Maurer et al. (2001) determined the initial corneal injury 3 hours and 1-day post-instillation of 10 l of 37% formaldehyde solution by post-mortem quantitation of dead corneal epithelium and keratocytes, using a scanning laser confocal microscopy. Post mortem quantitation indicated that corneal injury extended deeply into the stroma, at times to 93.2% of the corneal thickness on day 1. Dead corneal epithelial cells and keratocytes were also observed on day 1.
No internationally validated animal tests are currently available for this endpoint. Data are available from a study investigating effects on the mucociliary clearance and histopathological changes in Fischer 344 (F344) rats using light microscopy after a single 6-hour exposure to 0, 2, 6 or 15 ppm (0, 2.4, 7.2 or 18 mg/m3) gaseous formaldehyde (Morgan et al., 1986). At 15 ppm, slowing or cessation of mucous flow was detected in the nasal tract along with separation of epithelial cells and intravascular margination and local tissue infiltration by neutrophils and monocytes. No effects were seen at 2 or 6 ppm formaldehyde. However, in a study using electron microscopy to investigate histopathological changes in the nasal tract of F344 rats following a single exposure to formaldehyde (Monteiro- Riviere & Popp, 1986), loss of microvilli in ciliated cells, autophagic vacuoles in basal cells and cytoplasmic vacuoles in most cell types were seen at > 6 ppm. Although altered cilia were seen at 0.5 ppm and 2.2 ppm (0.6 mg/m3 and 2.6 mg/m3), such changes were also occasionally reported in control animals. Consequently, it cannot be determined whether these findings at 0.5 ppm and 2.2 ppm are attributable to formaldehyde exposure or inter-animal variations. In an Alarie assay in Swiss mice (Kane & Alarie, 1997), a 10-minute exposure to 3.1 ppm (3.7 mg/m3) formaldehyde was calculated to depress the respiratory rate by 50% (RD50 value). Additionally, tracheal cannulation of mice was seen to produce a minimal decrease in respiratory rate; 4.2% compared to 54% in un- cannulated controls. In a recent modified Alarie assay (Nielsen et al., 1999), respiratory patterns and parameters were continuously measured in BALB/c mice exposed (head only) to formaldehyde at concentrations ranging from 0.2 to 13 ppm (0.24 to 15.6 mg/m3) for 30 minutes. A 10-minute RD50 of 4 ppm (4.8 mg/m3) was calculated, which was reported to be due to irritation of the upper respiratory tract. At concentrations above the RD50 value both upper respiratory tract irritation and bronchoconstriction were involved in the decrease in respiratory rate.
The skin sensitisation potential of formaldehyde solutions has been investigated in numerous studies in the guinea-pig and mouse. A positive response to formaldehyde solution was seen in a large number of these studies. For example, strong positive responses to formaldehyde solution were observed in well- conducted guinea-pig maximisation tests, a Buehler occluded patch test and murine local lymph node assays (Kimber et al., 1991; Hoechst, 1994; Hilton et al., 1996). The details of the studies were summarised in ATDSR (1999). Furthermore, the cytokine secretion profile of formaldehyde was recently determined in mice and compared with that produced by a reference skin and respiratory sensitiser. Previous studies by the authors had shown that skin sensitisers stimulated a cytokine profile associated with the activation of T helper type 1 cells, compared to T helper type 2 cells for respiratory sensitisers. Topical exposure of mice to a 50% formaldehyde solution resulted in a cytokine secretion profile identical to that induced by the reference contact allergen (Dearman et al., 1999). There is no evidence in inhalation studies with rats, mice, hamsters or monkeys that formaldehyde gas induces skin sensitisation.
No internationally validated animal test is currently available that allows prediction of the ability of a chemical to induce respiratory sensitisation. However, data are available from non-validated studies investigating this endpoint in mice and guinea pigs. Formaldehyde was negative in immunoglobulin-E (IgE) tests in the mouse (Potter & Wederbrand, 1995; Hilton et al., 1996;) and guinea-pig (Lee et al., 1984). This predictive test method for assessment of respiratory sensitisation potential measures induced changes in serum concentration of IgE following topical exposure of mice to the test chemical. Furthermore, in a study investigating the cytokine secretion profile in mice (Dearman et al., 1999), topical exposure to formaldehyde did not induce a profile comparable to that of the reference respiratory sensitiser (i.e. secretion of cytokines associated with selective activation of T helper type 2 cells). Data is also available from studies that investigated whether pre-exposure to formaldehyde may enhance allergenic responses to ovalbumin. Compared to controls, a statistically significant increase in specific anti-ovalbumin antibody levels were seen in mice exposed to 1.67 ppm (2.00 mg/m3) formaldehyde daily for 10 days (Tarkowski & Gorski, 1995), and guinea-pigs to 0.25 ppm (0.3 mg/m3) daily for 5 days (Riedel et al., 1996), prior to induction then bronchial challenge with ovalbumin.
Repeated dose studies are available via the inhalation, oral, and dermal routes of exposure.
For repeated inhalation exposure the database is extensive. Studies have generally been conducted in rats, though data are also available in mice, hamsters and monkeys. These studies clearly show that the target organ following formaldehyde exposure is the nasal tract, where effects observed have included alterations in mucociliary clearance, cell proliferation and histopathological changes to the nasal epithelium. In the only study that investigated effects on the nasal mucociliary apparatus (Morgan et al., 1986), male F344 rats were exposed to 0, 0.5, 2, 6, or 15 ppm (0, 0.6, 2.4, 7.2 or 18 mg/m3) formaldehyde 6 hours/day, 5 days/week for up to 2 weeks. Inhibition of mucociliary clearance (i.e. reduced mucous flow rate) was observed at 6 ppm and above in the 9-day exposure group. The inhibitory effect of formaldehyde was mostly observed in the lateral aspect of the nasoturbinate and dorsal or medial aspects of the maxilloturbinate. No evidence of reduced mucous flow rate was seen at 2 ppm.
In the rat, studies with exposure durations from 2 days to a lifetime are available. An overview of the results seen in short-term to sub-chronic exposure studies is presented below [see CICAD (IPCS, 2002) for a more detailed summary of the data]. In short-term to sub-chronic exposure studies with exposure periods of 6-8 hours/day, 5 days/week, conclusive evidence of squamous metaplasia and/or cell proliferation of the nasal epithelium were seen with light microscopy at > 3.2 ppm (3.8 mg/m3) formaldehyde for 2-3 days exposure (Swenberg et al., 1983; Monteiro-Riviere and Popp, 1986; Morgan et al., 1986; Cassee et al., 1996); > 5 ppm (6 mg/m3) formaldehyde in a 4-week study (Wilmer et al., 1987); > 6.2 ppm (7.4 mg/m3) formaldehyde in a 6-week study (Monticello et al., 1991); and > 3 ppm (3.6 mg/m3) formaldehyde in studies with exposure durations of approximately 13-weeks (Feron et al., 1988; Woutersen et al., 1987; Zwart et al., 1988; Wilmer et al., 1989; Casanova et al., 1994).
5 days/week for 4 and 13 weeks compared to controls (Ozen et al., 2002; exposure concentrations confirmed by personnel communication). The effects seen on these testicular trace elements are considered a secondary non-specific consequence of marked general toxicity, seen as growth retardation. Data are also available from short-term to sub-chronic studies in other species. Hyperplasia of the nasal epithelium was seen in mice exposed to 15 ppm (18 mg/m3) gaseous formaldehyde 6 hours/day for 3 consecutive days (Swenberg et al., 1986). In a 13-week mouse study (Maronpot et al., 1986), minimal squamous metaplasia was observed in the nasal tract of 1/10 males, but absent in females, exposed to 4 ppm (4.8 mg/m3) formaldehyde 6 hours/day 5 days/week. Data are also available in the monkey. Histopathological changes in the nasal cavity and upper portion of the respiratory tract (trachea and bronchial biforcation) were seen in male rhesus monkeys exposed to 6 ppm (7.2 mg/m3) formaldehyde 6 hours/day 5 days/week for 1 or 6 weeks (Monticello et al., 1989). A comparative study of the effects of near continuous exposure to formaldehyde (i.e. 22 hours/day 7 days/week) for 26 weeks is available in cynomologus monkeys, F344 rats and Syrian hamsters (Rusch et al., 1983). Comparable effects were seen between F344 rats and cynomologus monkeys at 3 ppm (3.6 mg/m3) formaldehyde. In contrast, no conclusive evidence of histopathological changes in the respiratory tract was observed in hamsters at 3 ppm. Together, the data from these two studies suggests that rats and monkeys may be equally susceptible to epithelial damage from formaldehyde exposure, but a wider regional distribution of formaldehyde occurs in the upper respiratory tract of (rhesus) monkeys than in rats. Although no obvious clinical signs of neurotoxicity or histopathological changes in the brain have been observed in rodent inhalation studies, a recent sub-chronic inhalation study is available investigating the effect of formaldehyde on behaviour in male and female Wistar rats (Pitten et al., 2000). Compared to controls, exposure to 2.6 or 4.6 ppm (3.1 or 5.5 mg/m3) formaldehyde 10 min/day, 7 days/week for 13 weeks was seen to produce a statistically significant increase in the time to find the food, and number of mistakes made in a maze. However, the small group sizes (13-14/dose), assessment of a single neurobehavioral trait and absence of dose-response relationship for observed effects prevent any reliable conclusions being drawn from the data on the neurotoxic potential of formaldehyde. Long-term exposure studiesData are available from seven chronic inhalation studies in rodents. All these studies, which employed an exposure period of 6 hours/day 5 days/week, are presented below. In a study by Kerns et al. (1983), F344 rats and B6C3F1 mice (approximately 120 per species per sex per concentration) were exposed to 0, 2, 5.6 or 14.3 ppm (0, 2.4, 6.7 or 17.2 mg/m3) formaldehyde for up to 24 months. In rats, rhinitis, epithelial dysplasia and squamous metaplasia of the nasal tract was observed at 2 ppm and above. In mice, histological changes were seen at 5.6 ppm and above, along with rhinitis in a “few” animals at 2 ppm (no further details available). In a study by Appelman et al. (1988), male Wistar rats (40 per concentration) were exposed to 0, 0.1, 1 or 9.4 ppm (0, 0.12, 1.2 or 11.8 mg/m3) formaldehyde for 12 months. Rhinitis, hyperplasia and squamous metaplasia were observed in animals at 9.4 ppm only. In a study by Woutersen et al. (1989), male Wistar rats (30 per concentration) were exposed to 0, 0.1, 1 or 9.8 ppm (0, 0.12, 1.2 or 11.8 mg/m3) formaldehyde for up to 28 months. At 9.8 ppm rhinitis, disarrangement of the olfactory epithelium, hyperplasia and squamous cell metaplasia were observed in the nasal tract. No histopathological changes were observed at 0.1 or 1.0 ppm. In a study by Monticello et al. (1996), male F344 rats (90-150 per concentration) were exposed to 0, 0.7, 2, 6, 10 or 15 ppm (0, 0.84, 2.4, 7.2, 12 or 18 mg/m3) formaldehyde for up to 24 months, and effects determined at seven sites within the nasal tract: anterior lateral meatus, posterior lateral meatus, anterior mid- septum, posterior mid-septum, anterior dorsal septum, medial maxilloturbinate and maxillary sinus. At > 6 ppm hyperplasia and squamous metaplasia were observed in the nasal tract, mainly at the anterior lateral meatus. No histopathological changes were observed in the nasal tract at 0.7 or 2 ppm. In a study by Kamata et al. (1997), male F344 rats (36 per concentration) were exposed to 0, 0.3, 2.17 or 14.85 ppm (0, 0.36, 2.6 or 17.8 mg/m3) formaldehyde for up to 28 months. At > 2.17 ppm a statistically significant increase in squamous metaplasia in the nasal tract was observed both in the presence and absence of epithelial hyperplasia. At 0.3 ppm, although not statistically significant, squamous metaplasia was seen in the absence (1/5 animals at 18 months) and presence of hyperplasia (1/5 animals at 24 months and 3/11 animals at 28 months). However, the small group sizes and number of animals at interim sacrifice limits the significance that can be attached to the results of this study. Hyperplasia and squamous metaplasia were observed in the nasal tract of rats in studies by Sellakumar et al. (1985) and Holmstrom et al. (1989) that are of limited value as they only employed a single (high) exposure level; 14 and 12 ppm (16.8 and 14.4 mg/m3) formaldehyde, respectively. In these studies no conclusive evidence of systemic toxicity following inhalation exposure to formaldehyde was seen. The principal non-neoplastic effect observed in animals after repeated inhalation exposure was histological changes at the site of contact (i.e. in the nasal tract) due to irritation. The available data provide a dose-response range for histopathological changes in the nasal tract of rats, with effects being seen at 2 ppm (2.4 mg/m3) and above. Overall, the data also indicate similar effects are observed irrespective of exposure period. Although histopathological changes to the nasal tract were observed in rats at 0.3 ppm following 28 months exposure (Kamata et al., 1997), study limitations reduce the significance that can be attached to the data. Furthermore, no histopathological changes were seen at 0.7 and 1 ppm in studies of 24 and 28 months duration, respectively (Monticello et al., 1996; Woutersen et al., 1989). Consequently, a LOAEC of 2 ppm (2.4 mg/m3) is identified for histopathological changes to the nasal tract from an 18- and 24-month rat studies (Swenberg et al., 1980 and Kerns et al., 1983, respectively) with a NOAEC of 1 ppm (1.2 mg/m3) identified from a rat 28-month study (Woutersen et al., 1989).
Data are available from studies in rats and a dietary study in dogs. In short-term drinking water studies in rats, histopathological changes to the fore- stomach were seen at 125 mg/kg bw/day in a 28-day study following administration of formaldehyde solution (95% paraformaldehyde prill and 5% water) at dose levels of 5, 25, 125 mg/kg bw/d (Til et al., 1988). In contrast, a reduction in body weight gain was seen in a 13-week study [administering formaldehyde solution (95% paraformaldehyde prill and 5% water) in drinking water at 0, 50, 100, 150 mg/kg bw/d] at 100 mg/kg bw/day but no treatment- related histopathological changes were reported up to 150 mg/kg bw/day (Johannsen et al., 1986). A 28-day study is also available investigating the immunotoxicity of formaldehyde solution (28.44%) in male rats (Vargova et al., 1993). Animals were administered 0, 20, 40 or 80 mg/kg bw/day formaldehyde by gavage. Compared to controls, the only effects seen were a statistically significant increase in haematocrit concentration and decrease in body weight gain at > 40 and 80 mg/kg bw/day, respectively. However, the magnitude of changes were < 10% and are not considered biologically significant. Additionally, although lymph node weight was significantly increased at 80 mg/kg bw/day no histopathological changes were seen in the lymph node organs. Consequently, this study is not considered to provide conclusive evidence that formaldehyde possesses an immunosuppressive potential. Data are also available from long-term drinking water studies in the rat. In a study by Tobe et al. (1989), male and female Wistar rats (20 per sex per concentration) were administered formaldehyde solution in drinking water at concentrations of 0, 0.02, 0.1, 0.5% (approximately 0, 10, 50 or 300 mg/kg bw/day formaldehyde solution) for up to two years. However, the small group sizes employed and significant increase in mortality rate at the top dose (45% females and 55% males had died at 12 months) limits the value of this study for identification of a robust no-effect level. In contrast, a 2-year study by Til et al. (1989) was both well conducted and reported. In this study, groups of male and female Wistar rats (70 per sex per concentration) were administered formaldehyde solution (95% paraformaldehyde prill and 5% water) at dose levels of approximately 0, 1.2, 15 or 82 mg/kg bw/day in males and 0, 1.8, 21 or 109 mg/kg bw/day females for up to 2 years. At the top dose, histopathological changes including hyperplasia, hyperkeratosis, and focal ulceration of the forestomach epithelium, as well as focal atrophic gastritis, glandular hyperplasia and ulceration in the glandular stomach, were observed in both sexes. A reduction in body weight gain, liquid intake and an increased incidence in renal papillary necrosis were also seen in both sexes at the top dose. As these findings were not seen in other studies they are considered likely to be a secondary consequence of the severe effects seen in the stomach. No treatment-related effects were seen in either sex in the mid and low dose groups. In a 90-day oral study in dogs administering formaldehyde solution (95% paraformaldehyde prill and 5% water) in drinking water at 0, 50, 100 mg/kg bw/d (Johannsen et al., 1986), no treatment-related effects were reported up to 100 mg/kg bw/day. The absence of toxicity in both the dogs and rats in this study suggests that the target intakes may not have been achieved. Furthermore, it is not reported whether histopathological examination of the stomach was conducted in this study. Therefore, from the available data there is no conclusive evidence of systemic toxicity following oral administration of formaldehyde. The principal non- neoplastic effect observed in animals after repeated oral dosing was irritation at the site of contact (i.e. fore- and glandular-stomach). From the available data, a NOAEL of 15 mg/kg bw/day and a LOAEL of 82 mg/kg bw/day were identified for histopathological changes to the stomach from a well-conducted 2-year oral study in the rat (Til et al., 1989).
The limited data available on the repeat dermal toxicity of formaldehyde solution are from briefly reported mouse initiation/promotion studies (Krivanek et al., 1983; Iversen, 1986). None of these studies showed evidence of systemic toxicity. The study by Krivanek et al. (1983) contained a briefly reported dose ranging test. Groups of female CD-1 mice (number/dose not reported) received 100 l of a 10%, 2% or 1% formaldehyde solution in acetone (equivalent to 10, 2 and 1 mg) 5 days/week for 2 weeks or, 0.5% or 0.1% (equivalent to 0.5 or 0.1 mg) 5 days/week for 3 weeks. Skin irritation was observed at 0.5% and above, whose severity increased with concentration. Systemic toxicity was not seen at any dose level. However, the limited details provided prevent identification of a reliable NOAEL or LOAEL from this study.
A large number of studies have been conducted in vitro with either gaseous or aqueous formaldehyde and a wide variety of endpoints assessed. An overview of these results is presented below [see IARC (1995) for a comprehensive summary of the available data]. The majority of Ames tests with Salmonella typhimurium produced a positive result in the absence of metabolic activation, as seen in more recent studies by Marnett et al. (1985) and Takahashi et al. (1985). Positive results, generally weaker, have also occasionally been reported in the presence of metabolic activation (Connor et al., 1983; Donovan et al., 1983; Pool et al., 1984; Schmid et al., 1986; Temcharoen & Thilly, 1983). Positive results have also been reported in the reverse mutation assay with Escherichia coli in the absence of metabolic activation (Takahashi et al., 1985; O’Donovan & Mee, 1993). In mammalian cells, positive results have been reported in gene mutation assays in the absence of metabolic activation (Goldmacher & Thilly, 1983; Crosby et al., 1988; Liber et al., 1989). Furthermore, loss of heterozygosity analysis following a positive gene mutation assay in the absence of metabolic activation suggested that small-scale chromosomal deletion or recombination is the mechanism of mutation formation in mammalian cells in vitro (Speit and Merk, 2002). Additionally, increased incidences of chromosomal aberrations and SCE have been observed in the presence and absence of metabolic activation (Basler et al., 1985; Galloway et al., 1985; Natarajan et al., 1983; Schmid et al., 1986). Formaldehyde has also been reported to produce DNA damage (single strand breaks), and DNA-protein cross-links (DPX) in the absence of metabolic activation (Ross et al., 1981; Grafström et al., 1984; Grafström, 1990).
In somatic cells Data are available from a number of in vivo studies that are presented below. Some of these studies did not follow validated test methods with regard to the tissues examined or the exposure duration employed (i.e. prolonged). In a bone marrow cytogenetic assay (Natarajan et al., 1983), no increased incidence in chromosome aberrations or micronuclei were seen in male and female CBA mice that received two intraperitoneal injections of formaldehyde solution (concentrations not stated) over 24 hours for total doses up to 25 mg/kg bw. Additionally, no increased incidence in chromosome aberrations was seen in spleen cells. In a further ip bone marrow study (Gocke et al., 1981), no significant increase was seen in micronuclei in male and female Sprague-Dawley following a single injection of formaldehyde solution (concentration not reported) up to 30 mg/kg bw. No information on cytotoxicity was reported for either of these studies. In an inhalation bone marrow cytogenetic study by Kitaeva et al., 1990 (reported in Russian, summary from IPCS, 2002), a statistically significant increase in the proportion of cells with chromosomal aberrations (chromatid or chromosome breaks) were seen in female Wistar rats exposed to 0, 0.42, or 1.3 ppm (0, 0.50 or 1.56 mg/m3) gaseous formaldehyde for 4 hours/day for 4 months (0.7%, 2.4% and 4%, respectively). No further details are reported in the CICAD (IPCS, 2002). Whereas, no significant increase in chromosome aberrations was seen in the bone marrow of male Sprague-Dawley rats exposed up to 15 ppm (18 mg/m3) formaldehyde 6 hour/day, 5 days/week for 1 or 8 weeks (Dallas et al., 1992). A marginal but statistically significant increase in chromosome aberrations (predominantly chromatid breaks) was seen in pulmonary lavage macrophages in the same study at 15 ppm only following 1 and 8 weeks exposure (7.6% and 9.2%, respectively, compared to 3.5% and 4.8% in controls). No information on cytotoxicity was reported. In a further inhalation study (Kligerman et al., 1984), no significant increase in SCE or chromosome aberrations were seen in peripheral lymphocytes from male and female F344 rats exposed up to 15 ppm (18 mg/m3) formaldehyde 6 hour/day for 5 days. No information on cytotoxicity was reported.
In germ cellsData are also available from studies determining the genotoxicity of formaldehyde in germ cells. None of the following studies reported information on cytotoxicity. In an ip study (Fontignie-Houbrechts, 1981), no chromosome aberrations were seen in spermatocytes from male Q mice 8-15 days after a single injection of 50 mg/kg bw formaldehyde solution. A dominant lethal assay was also conducted in this study, in which male Q mice were mated for 7 weeks following a single ip injection of 50 mg/kg bw formaldehyde solution. Compared to controls, a statistically significant increase in post-and pre-implantation loss was seen at week 1 and pre-implantation loss at week 3. However no significant effects was seen on the number of pregnant females or live embryos per dam. Therefore, this study is not considered to have demonstrated a genotoxic effect. Additionally, no significant increase in potential dominant lethal findings were seen after single ip injection of up to 40 mg/kg bw formaldehyde solution (reported to be the ip LD50) to male ICR/Ha mice which were mated for 3 or 8 weeks (Epstein et al., 1972), and following ip injection of 20 mg/kg bw formaldehyde solution (reported to be the ip LD50) to male CD-1 mice which were mated for 8 weeks (Epstein et al., 1968). In contrast, daily ip injection of rats with 0.125, 0.25 or 0.5 mg/kg bw/day formaldehyde solution (1/4 to 1/16 of the determined ip LD50) for 5 days resulted in a dose related statistically significant increase in epididymal sperm head abnormalities (> 106%) and decrease in epididymal sperm count (> 41%) at 0.125 mg/kg and above compared to controls (Odeigah, 1997). This study also included a dominant lethal assay in which male rats received daily ip injections of 0, 0.125, 0.25 or 0.6 mg/kg bw/day for 5 days prior to mating for 3 weeks. A significant and dose related decrease was seen in the number of pregnant females mated 1-7 and 8-14 days after treatment of males with > 0.125 mg/kg bw/day (6-19/24 pregnancies compared to 29/30 in the control group), together with a significant dose related increase in the number of dead implants per dam in females mated 1- 7 days after treatment of males with > 0.125 mg/kg bw/day (> 1.23 compared to 0.43 in controls) and was associated with a corresponding decrease in the number of live foetuses per dam (< 5.95 compared to 7.43 in controls).
The carcinogenic potential of formaldehyde has been investigated in a number of animal studies, predominantly by the inhalation route of exposure.
In the only study conducted in both sexes, groups of F344 rats (approximately 120 per sex per concentration) were exposed to 0, 2.0, 5.6 or 14.3 ppm (0, 2.4, 6.7 or 17.2 mg/m3) formaldehyde 6 hours/day, 5 days week for up to 24 months (Kerns et al., 1983). All animals were subject to a complete and thorough gross and microscopic examination. A significant increased incidence in nasal squamous cell carcinomas was observed in both sexes at 14.3 ppm in the presence of irritation to the nasal tract. The overall incidence in this tumour type at 0, 2.0, 5.6 and 14.3 ppm was 0/118, 0/118, 1/119 and 51/117 in males, and 0/118, 0/118, 1/116 and 52/119 in females, respectively. There were no significant tumour findings in any other tissue. In a further study, groups of male F344 rats (90-150 per concentration) were exposed to 0, 0.7, 2, 6, 10 or 15 ppm (0, 0.84, 2.4, 7.2, 12 or 18 mg/m3) formaldehyde 6 hours/day, 5 days/week for up to 24 months (Monticello et al., 1996). This study is considered the most extensive bioassay conducted to date as proliferative responses were determined at the anterior lateral meatus, posterior lateral meatus, anterior mid-septum, posterior mid- septum, anterior dorsal septum, medial maxilloturbinate and maxillary sinus sites within the nasal tract after 3, 6, 12 and 18 months exposure, as well as at the end of the study. The overall incidence of nasal squamous cell carcinoma in animals was 0/90, 0/90, 0/90, 1/90, 20/90 and 69/147 exposed to 0, 0.7, 2, 6, 10 and 15 ppm, respectively. These tumours were mainly located in the anterior lateral meatus, the posterior lateral meatus and the mid-septum. Nasal polypoid adenomas, located in or adjacent to the lateral meatus, were also observed at 10 ppm (5/90 rats) and 15 ppm (14/147 rats) only. Both tumour types were observed in the presence of irritation to the nasal tract. Additional bioassays are available in male F344 rats [Tobe et al., 1985 (cited in IPCS, 2002); Kamata et al., 1997]. Exposure-responses in these studies were similar to those seen in the studies by Monticello et al. (1996) and Kerns et al. (1983), that is, an increased incidence in nasal tumours at concentrations > 5.6 ppm (> 6.7 mg/m3) formaldehyde in the presence of irritation (i.e. tumours observed at approximately 14 ppm [16.8 mg/m3] in the studies by Tobe et al., 1985 and Kamata et al., 1997). Data are available in other strains of rat. In a study in male Sprague-Dawley rats employing a single exposure concentration to formaldehyde (Sellakumar et al., 1985), a significant increase in the incidence of nasal squamous cell carcinoma was observed in animals exposed to 14 ppm (16.6 mg/m3) formaldehyde 6 hours/day, 5 days/week for approximately 24 months compared to controls (0/99 and 38/100, respectively). These tumours were mainly located at the naso- maxillary turbinates and nasal septum and observed in the presence of irritation to the nasal tract. There were no significant tumour findings in any other tissue. In a study in male Wistar rats (26-28/concentration) no significant increase in nasal tumours was observed in animals exposed to 0, 0.1, 1, or 9.8 ppm (0, 0.12, 1.2 or 11.8 mg/m3) formaldehyde 6hours/day, 5days/week for 28 months (Woutersen et al., 1989).
Data are available from drinking water studies in the rat. In the study summarised below by Soffritti et al. (1989) the dose administered were reported in mg/L only. Therefore, the default values in Table 10.2 have been applied to convert mg/L to mg/kg bw. These values are taken from Gold et al. (1984). Table 10.2: Default values for dose calculations
In the most comprehensive study available (Til et al., 1989), male and female Wistar rats (70 per sex per dose) were administered formaldehyde solution in drinking water for up to 24 months at dose levels that equated to approximately 0, 1.2, 15 or 82 mg/kg bw/day in males and 0, 1.8, 21 or 109 mg/kg bw/day in females. Selected organs of animals in the low and mid dose groups were examined at necropsy (including the stomach), while a complete and thorough gross and microscopic examination was conducted on control and top dose group animals. There were no significant tumour findings in any tissue. Similarly, no significant tumour findings were seen in selected organs (including the stomach) from male and female Wistar rats (20 per sex per dose) administered formaldehyde solution in drinking water for up to 24 months at dose levels that equated to approximately 0, 10, 50 or 300 mg/kg bw/day (Tobe et al., 1989). In contrast, Soffritti et al. (1989) reported a marked increased incidence in tumours in Sprague-Dawley rats (50 per sex per group) administered 1500 mg/L (the top dose level) for life. These tumours were leukaemias (all ‘haemolymphoreticular neoplasias’) in males and females (22% and 14%, respectively, compared to 4% and 3% in controls), along with adenomas of the stomach (4%), intestinal adenocarcinomas (2%) and leiomyosarcomas (4%) in males, and intestinal leiomyomas in females (6%). No gastrointestinal tumours were seen in control animals. Using the default values given in Table 10.2, the daily intake of aqueous formaldehyde at the top dose was estimated to have been 75 and 100 mg/kg bw/day in males and females, respectively. However, the pooling of tumour types reported as leukaemias and lymphomas, together with the final report of this study by Soffritti et al. (2002) that reports an increased incidence of these tumours compared to the original summary (with no explanation provided by the authors), means no reliable conclusions can be drawn from the data for these tumours. The later report by Soffritti et al. (2002) provides information on tumour incidences in additional tissues to those reported earlier. Although an increase in testicular interstitial cell adenomas was seen in males, it was not dose related or statistically significant at the top dose. Similarly, although a statistically significant increase was seen for all mammary tumours in females at the top dose (24% compared to 11% in controls), the increase was not dose related, while no dose related or statistically significant increase was seen for specific histologic tumours of the mammary gland. In an initiation/promotion study in male Wistar rats (Takahashi et al., 1986), papillomas of the forestomach were reported in the presence of irritation in 8/10 animals administered approximately 0.5% formaldehyde solution in drinking water for 32 weeks. No forestomach tumours were seen in control animals.
No standard studies are available. Data are available from mouse initiation/promotion studies. No skin tumours were seen in mice (16-20 per sex per dose) topically administered 1% or 10% formaldehyde solution only 3 times/week for 26 weeks (Krivanek et al., 1983,) or 10% formaldehyde solution only 2 times/week for 60 weeks (Iversen, 1986). However, the small group sizes and short duration of exposure to formaldehyde used in these studies prevent any reliable conclusions on the carcinogenic potential of formaldehyde by the dermal route.
In the only reproductive study available, a 1-generation study in minks (Li et al., 1999), groups of 12 females were fed 0, 550 or 1100 ppm formaldehyde solution in the diet from 1 month prior to mating (with untreated males) until weaning of kits. However, dose levels of formaldehyde in the feed were determined to be 17, 291 and 662 ppm. No toxicity was observed in parental females. No effect was observed on fertility index or litter size. A statistically significant decrease in kit survival was reported at birth at the top dose (87% compared to 96% in controls). Kit survival was unaffected 3 and 6 weeks post partum. The decrease in kit survival at birth was observed in the absence of a significant increase in mean number dead kits/dam or decrease in live kits/dam. These mean values are considered more reliable markers of adverse effects on fertility. Consequently, it is concluded that no adverse effects on fertility were observed in this study. However, the absence of parental toxicity means there are concerns that formaldehyde was not robustly tested in this study. Data are also available from a study by Ward et al. (1984) that investigated the reproductive effect of formaldehyde in both mice and humans. In this study, administration of 100 mg/kg bw/day formaldehyde solution (the only dose level tested) to mice via gavage for 5 consecutive days had no effect on epididymal sperm morphology. Furthermore in a rat 2-year repeated oral study, no histological changes were observed in the testes or ovaries up to and including the top dose, 82 mg/kg bw/day (Til et al., 1989). Similarly, in repeated inhalation studies of 18 months duration and longer, no histological changes were observed in reproductive organs at the maximum exposure concentration: 14.3 ppm (17.2 mg/m3) in rats and mice (Kerns et al., 1983). Although changes were seen in testicular trace element concentrations (zinc and copper) at 10.2 ppm (12.2 mg/m3) and 20.3 ppm (24.4 mg/m3) gaseous formaldehyde (see details in section 10.4.1), they were considered to be a secondary non-specific consequence of severe general toxicity; reductions in body weight gain of 38% to 87% (Ozen et al., 2002). In contrast, effects on male reproductive organs were observed in rodent intraperitoneal (ip) studies. In rats, ip administration of formaldehyde solution for 30 consecutive days resulted in a statistically significant decrease in testicular weight at > 5 mg/kg bw/day (magnitude not reported), a statistically significant decrease in epididymal sperm count (44%), mobility (4%) and viability (17%) at 10 mg/kg bw/day, and histological changes in Leydig cells at > 10 mg/kg bw/day (Chowdhury et al., 1992; Majumder & Kumar, 1995). In further studies, ip administration of formaldehyde for 5 consecutive days resulted in a statistically significant increase in epididymal sperm head abnormalities (> 106%) in rats at > 0.125 mg/kg bw/day (Odeigah, 1997), and in mice a statistically significant decrease in sperm mobility (5%) and viability (53%) at > 4 mg/kg bw/day, and sperm count (54%) at > 10 mg/kg bw/day (Yi et al., 2000). However the relevance of these studies are questionable, as ip administration is not a relevant route of human exposure.
Data are available from studies via inhalation, oral and dermal routes of exposure. In an inhalation study (Saillenfait et al., 1989), groups of 25 mated female Sprague-Dawley rats were exposed (whole-body) up to 0, 5.2, 9.9, 20 or 39 ppm (0, 6.2, 11.9, 24.0 or 46.8 mg/m3) gaseous formaldehyde for 6 hours/day from day 6 to 20 of gestation. At 39 ppm only, a statistically significant decrease in dam body weight gain (51%) and male (21%) and female (19%) foetal body weight was observed compared to controls. A slight (5%) but statistically significant decrease in male foetal body weight was also seen at 20 ppm. No other treatment- related effects were observed on development. The slight decrease in foetal body weight in males only at 20 ppm is not considered sufficient magnitude to be biologically significant. While the statistically significant decrease in foetal body weight gain at 39 ppm was seen in the presence of a substantial decrease in dam body weight gain, and is therefore considered to be a secondary non-specific consequence of severe maternal toxicity.
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