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4. Toxicity, animal data

Inhalation

The LC₅₀-value for a 4-hour exposure is reported to 578 mg/m³ for rats and 497 mg/m³ for mice (IPCS 1989).

Formaldehyde is a potent respiratory tract irritant as exposure to 3.8 mg/m³ produces a 50% decrease in respiratory frequency (= RD₅₀) in mice. At this concentration mild histopathological lesions in the anterior nasal cavity was found in the animals following six hours exposure each day for five days (IARC 1995).

Short term, repeated exposures (7-25 mg/m³) of rats produced histological changes in the nasal epithelium, such as degeneration, inflammation, necrosis, squamous metaplasia, and increased cell proliferation (IPCS 1989). In rats exposure to 2.5 mg/m³ formaldehyde 6 hours/day for three days has resulted in increased cell proliferation in the nasal and tracheal mucosa (Roemer et al. 1993).

There is growing evidence that it is the concentration rather than the cumulative dose that determines cytotoxic effects of formaldehyde on the nasal mucosa of rats; concentrations below 1 mg/m³ do not lead to cell damage and hyperplasia (IPCS 1989).

At 0.6 mg/m³ irritation of eyes, nose, and throat were observed in experimental animals (IPCS 1989).

Oral administration

The LD₅₀-value for a single oral administration is reported to 800 mg/kg in rats and 269 mg/kg in guinea pigs (IPCS 1989).

Dermal contact

The LD₅₀-value for a single dermal application is reported to 270 mg/kg for rabbits (IPCS 1989).

Skin irritation

Application of 0.1-20% formaldehyde solutions on the skin of rabbit and guinea pig resulted in mild to moderate irritation (IPCS 1989).

Eye irritation

Experimental application of a drop formalin (approx. 35% formaldehyde solution) to rabbit and guinea pig eyes has caused severe reactions, with oedema of the cornea and conjunctiva, and iritis, graded 8 on a scale to 10 at twenty-four hours but with a tendency to recovery in the course of a month or two. A 0.05% solution applied to rabbit eyes caused complete loss of the top layer or corneal epithelial cells. (Grant 1986).

Skin sensitisation

Formaldehyde has been found to be a skin sensitiser in several animal experiments including the guinea pig maximisation tests in epicutaneous maximisation tests, and in epidermal Draize tests. In the most sensitive guinea pig strain sensitisation has been induced with a 0.01% formaldehyde solution (Nord 1991).

In inhalation experiments guinea pigs exposed to formaldehyde vapours caused skin sensitisation, without causing pulmonary hyperreactivity (IPCS 1989).

A range of subchronic and chronic inhalation experiments have been performed in different animals where especially the early effects in the nasal mucosa have been determined, as these effects may be related to formation of the nasal tumours that occur at longer exposure periods.

From these studies no increases in cell turnover or DNA synthesis have been found in the nasal mucosa after subchronic or chronic exposure to concentrations £ 2 ppm (2.4 mg/m³), while small site-specific increases in the rate of cell turnover and increased DNA synthesis were observed in rats at 3 ppm (3.7 mg/m³) and 6 ppm (7.3 mg/m³), respectively (IARC 1995).

The subchronic and chronic studies have demonstrated that the occurrence and the severity of lesions in the nasal mucosa are more linked to the concentration of formaldehyde than to the total dose or exposure duration (IARC 1995).

Formaldehyde administered in drinking water to rats for a 2 year period has resulted in lesions of the forestomach and the glandular stomach of the animals (raised limiting ridge of the forestomach and gastritis and hyperplasia of the glandular stomach) (IARC 1995).

Til et al. (1989) exposed rats to formaldehyde in drinking water for up to 24 months. The formaldehyde content was continuously adjusted so that the rats would receive daily doses of 5, 25 and 125 mg/kg/day (formaldehyde concentration in drinking water: 0.002, 0.026, and 0.19%). However, the actual daily doses for male/female rats were: 0/0, 1.2/1.8, 15/21 and 82/109 mg/kg per day. At the highest dose level significant lesions in the stomach and forestomach were seen, while no differences compared to control animals were noted at the second highest dose level. Furthermore, significant increased incidences of papillary necrosis of the kidneys were observed at the highest dose level in both male and female animals. A NOAEL of 15 mg/kg per day for male and 21 mg/kg per day for female rats (formaldehyde concentration of 0.026%) was concluded by the authors.

In a study by Tobe et al. (1989) where rats were administered drinking water with a formaldehyde content of 0, 0.02, 0.10, and 0.50% a LOAEL for lesions of the forestomach and glandular stomach was 0.1% (corresponding to 50 mg/kg/day) and NOAEL was found to 0.02% formaldehyde (corresponding to 10 mg/kg/day).

Formaldehyde administered by inhalation, ingestion or by dermal contact to various rodent species did not in a series of reproductive and developmental toxicity studies exert adverse effects on reproductive parameters or foetal development (IPCS 1989, IARC 1995).

Formaldehyde has been reported positive in in vivo tests in rats, where chromosomal anomalies have been found in lung cells after inhalation, while micronuclei formation in the gastrointestinal tract was found after gavage administration. Inhalation of formaldehyde leads to DNA-protein cross-links in the nasal mucosa of rats and monkeys. (IARC 1995).

In in vitro tests formaldehyde induced mutation, gene conversion, DNA strand breaks, and DNA-protein cross links in fungi, and mutation and DNA damage in bacteria. Further, formaldehyde induced DNA-protein cross-links, DNA single-strand breaks, chromosomal aberrations, sister chromatid exchange and gene mutation in human cells in vitro. (IARC 1995).

In general, the available data show that formaldehyde is genotoxic, especially when high concentrations act directly on cells (gene and chromosome mutations). Addition of metabolising systems tends to reduce the activity of formaldehyde. (IPCS 1989).

Inhalation carcinogenicity studies have been performed with mice, various strains of rats, and hamsters.

In two studies with hamsters one with exposure to 10 ppm (12.3 mg/m³) formaldehyde 5 hours/day, 5 days/week for lifetime and one with exposure to 30 ppm (36.9 mg/m³) 5 hours/week for lifetime no tumours of the nasal cavities were found (IARC 1995).

B6C3F1 mice (120 animals of each sex in each exposure group) were exposed to 2, 5.6, and 14.3 ppm formaldehyde (2.5, 6.9, 17.6 mg/m³) 6 hours/day, 5 days/week for up to 24 months. After 24 months of exposure two of 17 remaining animals (reduced number of animals due to interim sacrifices) in the high dose group had developed squamous-cell carcinomas, whereas no tumours were found at the other dose levels. At 14.3 ppm a variety of non-neoplastic lesions in the nasal cavities were commonly found. (IARC 1995).

Fischer rats (120 animals of each sex per dose groups) were exposed to formaldehyde at 2, 5.6, 14.3 ppm (2.5, 6.9, 17.6 mg/m³) 6 hours/ day, 5 days/week for up to 24 months (Kerns et al. 1983, IARC 1995).

The histopathological findings with respect to neoplastic lesions are given in Table 4.1.

Other histopathologic findings from the study were:

2.0 ppm: after 12 months of exposure purulent rhinitis, epithelial dysplasia and squamous metaplasia in the anterior part of the nasal turbinates were observed. Three months post-exposure (month 27) there was a significant decrease in the frequency of metaplasia.

5.6 ppm and 14.3 ppm: exposure related increase in intensity of the effects observed at 2.0 ppm. The lesions were observed also in the deeper parts of the nasal cavities (Kerns et al. 1983).

Monticello et al. (1996) exposed groups of 90-147 male F344 rats to 0, 0.7, 2, 6, 10, and 15 ppm of formaldehyde (0.9, 2.5, 7.4, 12, and 18 mg/m³) 6 hours/day, 5 days/week for up to 24 months. Six rats in each groups were interim killed at 3, 6, 12 and 18 months. In the 6, 10 and 15 ppm groups squamous cell carcinoma in the nasal cavities were found in 1/90, 20/90, and 69/147 of the animals, respectively, while no nasal tumours occurred at the lower exposure levels.

In the study, cell proliferation in different parts of the nasal mucosa was determined using a DNA labelling technique. From these data significant increases in cell proliferation occurred only at the two highest exposure levels indicating a threshold at about 6 ppm for the induction of cell proliferation. Furthermore, the occurrence of tumours in different part of the nasal mucosa was correlated to the degree of increased labelling index for the cell populations in the specific areas. The authors suggested that the cell proliferation induced by high formaldehyde exposure would make the tissue more vulnerable for the genotoxic effects of formaldehyde.

In another study, 100 Sprague Dawley rats were exposed to 14.2 ppm formaldehyde (17.5 mg/m³) 6 hours/day, 5 days/week during lifetime. At the end of the experiment 38 squamous-cell carcinomas of the nasal cavities and 10 polyps or papillomas were observed compared to no neoplastic changes in a group of 99 control animals (Sellakumar et al. 1985, IARC 1995).

In another study nine groups of 43-45 Wistar rats were exposed to 10 ppm (12.3 mg/m³) and 20 ppm (25 mg/m³) formaldehyde 6 hours/day, 5 days/week for 4, 8 and 13 weeks. The animals were observed for 117-126 weeks after exposure. The authors concluded that nasal tumours were only induced to a significant extent in the 20 ppm groups, where 6 exposure related cases of nasal tumours occurred among a total of 132 animals. Thus, after 4,8 and 13 weeks exposure 1, 1 and 4 nasal tumours in the 20 ppm groups were found. (IARC 1995).

Three long term oral carcinogenicity studies have been performed with rats:

Groups of 50 male and 50 female Sprague Dawley rats were administered formaldehyde in drinking water at concentration levels of 10, 50, 100, 500, 1000, and 1500 ppm (mg/l) for 104 weeks (Soffritti et al. 1989). [Corresponding to 1, 5, 10, 50, 100, 150 mg/kg b.w. per day according to WHO (1996)]. Two control groups received either pure drinking water or drinking water containing 15 ppm of methanol (the formaldehyde contained 0.3% of methanol as stabiliser).

In the treated groups, dose related increased incidences of leukaemia (leukaemia included observed cases of: lymphoblastic leukaemia and lymphosarcomas plus immunoblastic lymphosarcomas plus other haemolymphoreticular neoplasia) was observed at rates of 3%, 9%, 9%, 12%, 13%, 18% in the dosed groups versus 3.5% in the water control group and 8% in the water/ methanol group.

(Due to differences in the occurrence of spontaneous leukaemia between sexes, it seems relevant to separate the cases on the individual sexes. Furthermore, the cases consisting of "other haemolymphoreticular neoplasia" is not described in more details in the paper and should therefore not without further explanation be added to the cases of lymphoblastic leukaemia and lymphosarcomas and the cases of immunoblastic lymphosarcomas. Taking these consideration into account significant increased incidences of leukaemia still persisted in the two highest dose groups of females and in the highest dose group of males).

Further, increased incidences of benign and malignant neoplasia in the gastro-intestinal tract were observed, especially at the highest dose level (at 1500 ppm: 3/100 having adenoma + adenocarcinoma and 5/100 having leiomyomas + leiomyosarcomas, compared to 0% in each of the two control groups consisting of a total of 300 animals. Further, the rare occurrence of these type of tumours in historical controls were emphasised).

In a two-year study with dosing of drinking water containing 2500 ppm formaldehyde to rats and their offspring only the parent rats showed increased incidence of leukaemia (11% compared to 2.5% in the controls), whereas 5.6% of the parent rats and 17.8% of the offspring developed gastrointestinal tract neoplasia compared to 0% in comparable control groups (Soffriti et al. 1989).

In contrast two other studies showed no evidence of formaldehyde induced carcinogenicity:

Til et al. (1989) exposed groups of 70 male and 70 female Wistar rats to formaldehyde in drinking water. The males/females received doses of 0/0, 1.2/1.8, 15/21, 82/109 mg/kg b.w. per day for up to 24 months (formaldehyde content in drinking water of 0, 20, 260, and 1900 mg/l). Treatment related hyperplastic lesions, ulceration and atrophy were found in the forestomach and glandular stomach. No increased mortality occurred and the incidences of tumours did not differ significantly between the groups. (Til et al. 1989, IARC 1995).

In the third study groups of only 20 Wistar rats of each sex were exposed to drinking water containing 0, 200, 1000, and 5000 ppm for 24 months. Non-neoplastic lesions consisting of squamous- and base-cell hyperplasia, erosion and ulceration were seen in the forestomach and stomachs at the highest dose level. There were no significant differences in the incidence of tumours among the groups. The sensitivity of this study, however, was significantly impaired because of early death in the highest dose group, as only 50% of the animals survived one year and none of the animals survived the 2 year dosing period (Tobe 1989, IARC 1995).

4.6 Combination effects

Irritation

In a study where respiratory tract irritation were correlated to decrease in respiratory frequency in rats, it was found that combination exposure to the three aldehydes formaldehyde, acetaldehyde and acrolein resulted in additive response if the response was fitted to a model of competitive receptor binding kinetics (Cassee et al. 1995).

In another study where combination exposure to the same three aldehydes was performed the lesions in the nasal mucosa of rats were determined. At the lowest levels (formaldehyde 1.2 mg/m³; acetaldehyde 1350 mg/m³; acrolein 0.6 mg/m³, which were levels near the individual NOAELs) no additive effects were found, however, at higher levels (formaldehyde 3.9 mg/m³; acetaldehyde 2700 mg/m³; acrolein 1.5 mg/m³) the combined exposure resulted in more than additive effects (Feron et al. 1995).

Carcinogenicity

Combined exposure has been conducted with formaldehyde plus hydrochloric acid and formaldehyde plus wood dust in experimental animal studies (IARC 1995). In both studies only minor differences in effects and responses were found between the combination exposure group and the formaldehyde exposure group. Thus, no clear conclusion can be drawn with respect to the nature of possible combination effects from these studies.

Tumour promoting effect of formaldehyde has been demonstrated in both mice and rats. Thus oral exposure to both N-nitrosodimethylamine (carcinogenic initiator) and formaldehyde resulted in significantly increased tumourigenic response in mice (tumours in liver, kidney and lung) and rats (papillomas of the forestomach) compared to treatment with either of the substances alone. (IARC 1995).

In an experiments with a single dermal exposure to dimethylbenz(a)anthracene in mice followed by dermal exposures to formaldehyde, the application of formaldehyde was shown to enhance the rate at which skin tumours occurred (IARC 1995).

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