Formaldehyde

Endpoint summary

Description of key information

There is evidence that formaldehyde induces toxic effects only at the site of contact after oral, dermal or inhalation exposure. Toxicity is not evident at remote sites such that general signs of toxicity occur only secondary to these local lesions. In spite of some recent studies describing effects after inhalation of formaldehyde far of the portal of entry, this assessment is still upheld after comparing these studies with key guideline studies of high validity.
In chronic drinking water studies in rats local effects in the forestomach and stomach were induced, the NOAEC is 0.020-0.026% formaldehyde in drinking water. The NOAEL for systemic effects is 82 mg/kg bw/day in males and 109 mg/kg bw/day in females.
Studies on repeated dermal dose toxicity in compliance to current Guidelines are not available.
Local effects in the upper respiratory tract were induced after repeated inhalation exposure in experimental animals. The most sensitive site in rodents and monkeys following inhalation exposure is the respiratory epithelium in the anterior part of the nasal cavity. At higher exposure levels also the olfactory epithelium, larynx or trachea were affected. Rats are more sensitive than mice or hamsters. The LOAEC is 2 ppm in rats, 3 ppm in monkeys and 6 ppm in mice. The overall NOAEC for local effects in experimental animals is 1 ppm (1.2 mg/m³). The NOAEC for systemic effects not occurring at the site of first contact in long-term inhalation studies in rats and mice is 15 ppm.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Reference 1

Endpoint:

chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Nov 8, 1984 to Nov 8, 1986

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 453 (Combined Chronic Toxicity / Carcinogenicity Studies)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Specific details on test material used for the study:

TEST MATERIAL
- Source: Celanese Chemicals, Dallas, TX, USA
- Lot/Batch number: UN2213
- Paraformaldehyde prills 95 % (plus 5 % water)

Species:

rat

Strain:

Wistar

Remarks:

Cpb:WU, SPF

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: TNO Central institute for the breeding of laboratory animals, Zeist, the Netherlands
- Age at study initiation: 5 weeks old
- Weight at study initiation: 35 - 50 g
- Housing condition: 5 per cage, stainless-steel cages fitted with wire-mesh floor and front
- Diet: Institute's grain-based open-formula diet, ad libitum
- Water: drinking water, ad libitum
- Acclimatization period: 9 days

DETAILS OF FOOD AND WATER QUALITY
- The contaminants in basal diet and in drinking water are determined twice a year

ENVIRONMENTAL CONDITIONS
- Temperature: 20 - 24°C
- Humidity: 40 - 70 %
- Air changes: about 10 times per hour
- Photoperiod: 12 hours / 12 hours

Route of administration:

oral: drinking water

Vehicle:

unchanged (no vehicle)

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS
The test substance was administered to the rats in the drinking-water (tap-water), to provide target intake levels of 5, 25 and 125 mg test substance/kg body weight/day. Fresh solutions were prepared every week and stored in closed plastic containers in a room kept at 15°C. The concentrations of the test substance in the solutions were adjusted weekly for the first 12 weeks of the study on the basis of the estimated mean body weight and liquid consumption for the next week. Such adjustments were made every 4 week from week 12 to 52, on the basis of the estimated mean body weight and liquid consumption for the forthcoming 4 weeks. After week 52, the concentrations were kept constant, because of the considerable decrease in liquid consumption in the top-dose rats due to their rejection of the test solution. The concentrations of the test substance present in the various experimental solutions at the time of consumption were calculated to be on average 20, 260 or 1900 mg/L for the low-, mid- and high-dose groups, respectively.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The determination of formaldehyde in drinking water was conducted by iodometric titration according to Van der Linden and Visser (1968).

Duration of treatment / exposure:

24 months (interim sacrifice after 12 or 18 months)

Frequency of treatment:

daily, 7 days each week

Dose / conc.:

5 mg/kg bw/day (nominal)

Remarks:

1.2 mg/kg bw/day measured dose levels in males (mean)
1.8 mg/kg bw/day measured dose levels in females (mean)

Dose / conc.:

25 mg/kg bw/day (nominal)

Remarks:

15 mg/kg bw/day measured dose levels in males (mean)
21 mg/kg bw/day measured dose levels in females (mean)

Dose / conc.:

125 mg/kg bw/day (nominal)

Remarks:

82 mg/kg bw/day measured dose levels in males (mean)
109 mg/kg bw/day measured dose levels in females (mean)

No. of animals per sex per dose:

70, subgroups of 10 rats/sex/dose killed 12 or 18 months after start of exposure

Control animals:

yes, concurrent vehicle

Details on study design:

Concentrations adapted in weekly intervals to changing body weights up to week 52. Average concentration: 0, 20, 260, or 1900 mg/L (0, 0.002, 0.026, or 0.19% in water).

Positive control:

no

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS
- Time schedule: daily

CLINICAL OBSERVATIONS
- Time schedule: daily

BODY WEIGHT
- Time schedule: at the start of the study, weekly in the first 12 weeks, once every 4 weeks thereafter

FOOD CONSUMPTION
- Time schedule: weekly in the first 12 weeks, 2week periods every 3 months thereafter

WATER CONSUMPTION AND COMPOUND INTAKE
- Time schedule: weekly periods throughout the study
- Compound intake was calculated on the basis of intended levels of the test substance it the solutions, and liquid intake and body weight figures as measured in the corresponding weeks and after correction for the stability of diluted test substance solutions

OPHTHALMOSCOPIC EXAMINATION
- Cornea, conjunctivae, sclera, iris and fundus oculi
- Were made in all rats of the control and top-dose groups prior to the administration of the test substance and in week 52 end 103
- Eye examinations were carried out using a hand slit lamp after induction of medriasis by a 1 %solution of atropine sulphate

HAEMATOLOGY AND CLINICAL CHEMISTRY
- Blood samples were collected from the tail tips of ten rats/sex/group in week 26 and 103 and were examined for haemoglobin concentrations, packed cell volume and erythrocyte, leucocyte and thrombocyte counts
- Whole blood taken from ten rats/sex/group after overnight fasting in week 27, 52, 78 and 104 was examined for glucose
- Orbital blood samples taken from ten rats/sex/group in week 28 and abdominal blood samples taken from ten rats/sex/group in week 53, 79 and 105 were centrifuged at 1250 g for 15 min and then analyzed by conventional methods for alkaline phosphatase, aspartate aminotransferase, alanine aminotransferase, total protein, albumin, total bilirubin, urea, creatinine, cholesterol, y-glutamyl transferase and calcium, inorganic phosphate, chloride, sodium and potassium

URINALYSIS
- In week 27, 52, 78 and 104, ten rats/sex/group were deprived of water for 24 hour and of food for 16 hour
- Urine was collected during the last 16 hour of the deprivation period and its volume and density were determined
- In pooled urine samples collected in week 27 and 104 semi-quantitative observations of protein, glucose, occult blood, ketones, urobilinogen and bilirubin were made and the sediment was examined by microscopy
- pH determinations were carried out in week 27, 52, 78 and 104 in freshly voided 3-hour urine samples

Sacrifice and pathology:

Before the start of the study, two subsets each of 10 male and 10 female rats and one of 50 rats of each sex were defined in each group. The survivors of the first (10 rats/sex/group), second (10 rats/sex/group) and third (50 rats/sex/group) subsets were killed in week 53, 79 and 105, respectively. The rats were killed by exsanguination from the abdominal aorta, whilst under light ether anaesthesia, and a thorough autopsy was performed. The following organs of each rat were weighed and the organ to body weight ratios were calculated: adrenals, brain, heart, kidneys, liver, ovaries, pituitary, spleen, testes and thyroid. Samples of these organs and of the skin, skeletal muscle, mammary glands (females), Harderian and exorbital lachrymal glands, nose, lungs, aorta, parotid, submandibular and sublingual salivary glands, oesophagus, forestomach, glandular stomach, small and large intestine, pancreas, urinary bladder, epididymides, prostate, uterus, stemum, mesenteric and axillary lymph nodes, spinal cord, sciatic nerve and eyes were fixed in 10% neutral buffered formalin, embedded in paraffin wax, sectioned at 5 μm, and stained with haematoxylin and eosin. Detailed microscopic examinations were carried out on all mentioned organs of all rats of the control and the high-dose groups. The liver. lungs, stomach and nose of all rats of the low- and mid-dose groups were also examined. In addition, we examined the adrenals, kidneys, spleen, testes, thyroid, ovaries, pituitary and mammary glands (females) of the rats of subset three (killed in wk 105) of the low- and mid-dose groups. A thorough autopsy was also performed on rats that were found dead or were killed when moribund during the study. The organs of these animals were not weighed, but tissues were preserved if autolysis was not too advanced.

Statistics:

Data on body weight were evaluated by a one-way analysis of covariance, followed by Dunnett's multiple comparison tests. The laboratory determinations and organ weights were evaluated by a one-way analysis of variance, followed by Dunnett's multiple comparison tests, except for the differential white blood cell counts, which were analyzed by the Mann-Whitney U-test. Data on food and liquid intake were evaluated by analysis of variance, followed by least significant difference tests (experimental unit: the cage). The mortality incidences and the histopathological changes were examined by Fisher's exact probability test (two-sided).

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

No overt signs of toxicity due to the administration of the test substance were observed. The behaviour of the rats during the first year of the study was unremarkable. After 12 months, ageing symptoms developed in all groups and the number of unthrifty rats increased. The incidence of partly closed eyes was more frequently observed in the top-dose rats than in the rats of the other groups, including the controls. The incidences of all the other phenomena observed were about equally distributed among the groups; there were no consistent differences between groups in time of appearance of clinical signs. In general the animals remained in good health. Of the 12- and 18-month interim kill grossly visible or palpable masses were observed in a few rats only, without evidence of a relationship with the treatment. In the 24-month study grossly visible or palpable masses occurred frequently in all groups. In females of the top-dose group, both the total number of masses and the number of animals bearing masses was slightly higher than in controls, however, the differences were not statistically significant.
An incidental finding was the slight yellowing of the fur in the mid- and top-dose rats from week 3 - 6. The yellow colour was more intense on the back and flanks than on the belly and cheeks of the animals. The individual hairs were coloured from the skin till the top of the hair. The colour intensified to canary yellow in the course of the next 10 - 12 weeks and then did not change much in intensity. In the mid-dose rats the phenomenon was much less pronounced than in the top-dose rats. The low-dose rats did not show any yellow discolouration.

Mortality:

mortality observed, treatment-related

Description (incidence):

From the mortality data it appears that up to week 72 mortality was very low in both sexes. Thereafter, mortality gradually increased. Mortality rate was statistically significantly increased in males of the mid-dose group at the end of the study (p < 0.05), but increased mortality was not observed in the top-dose group. In females there were no significant differences in mortality amnong the groups. After 24 months mortality for males and females of the control group was 25 and 31 %, respectively.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Mean body weights of the top-dose males were statistically significantly decreased throughout the study. Mean body weights of the females of this group were invariably bower than those of the controls; the 5 differences reached the level of statistically significance only in week 1, 24 to 40 and 76 to 104.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Food intake of the top-dose males was statistically significantly diminished. In females of this group a similar effect was observed, but it was less pronounced. The differences with the controls reached the level of significance only incidentically.

Food efficiency:

effects observed, treatment-related

Description (incidence and severity):

Food conversion efficiency of the top-dose males calculated over the period of rapid growth (week 0 to 12) was generally lower than in controls, the differences being statistically significant in wneek 1, 7 and 10 (p < 0.001). In females no treatment-related differences in food conversion efficiency were observed amongst groups.

Water consumption and compound intake (if drinking water study):

effects observed, treatment-related

Description (incidence and severity):

Liquid intake showed a considerable decrease in the top-dose group in both sexes. The mid-dose group consumed slightly less liquid than did the controls, but the differences were generally not statistically significant. In the last week of the study there was a significant decrease in liquid intake in males of the low- and mid-dose groups. We have no explanation for this irregularity.

Ophthalmological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Ophthalmoscopic examination, carried out prior to the start of the study and week 52 and 103, did not reveal any difference between test rats and controls that suggested an effect of the test substance.

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Haematology did not show statistically significant differences among the various groups in week 26. In week 103 packed cell volume was decreased in the top-dose females (p < 0.05), but this phenomenon was not associated with significant changes in other red blood cell parameters and therefore, no toxicological significance was attached to this finding. White blood cell counts in week 103 were relatively low in all test groups in males, the differences with the controls being statistically significant in the mid- and top-dose groups (p < 0.05). However, there was no evidence of a dose-related response. In the top-dose females the percentage of neutrophils was increased and that of the lymphocytes decreased (p < 0.05).

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

In week 27/28 plasma alkaline phosphatase activity and total plasma protein content were slightly decreased at the mid- and top-dose level in both sexes; the differences with the controls were not always statistically significant in the mid-dose group. Total plasma protein content was also slightly decreased in the top-dose males in week 78/79. Plasma urea content was slightly increased in the top-dose males in week 27/28 only. In week 78/79 plasma cholesterol levels were slightly, though statistically significantly decreased in the mid- and top-dose groups in males. There was, however, no evidence of a dose-related response. Plasma potassium concentration was statistically significantly increased in females of the top-dose group. Clinical chemistry values -including the above mentioned parameters obtained in week 52/53 and 103/104 did not show statistically significant differences among the various groups. Therefore, no toxicological significance is attached to the slight changes seen in week
26/27 and 78/79.

Endocrine findings:

not examined

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

A statistically significant increase in density of the urine, accompanied by a tendency towards lower urine production was observed in the top-dose group in males in week 27 and 52 and in females in week 27. Mean urinary pH was increased in males of the low- and mid-dose group in week 27 and 78 and decreased in females of the top-dose group in week 27 and 78. The occurrence of occult blood in the urine was increased in all test groups in males in week 27 and in the mid- and top-dose group in females in week 104. However, there was no evidence of a dose-related response. Therefore, no toxicological significance was attached to this finding. There were no changes of any significance in the composition of the urine with respect to appearance, protein, glucose, ketones, urobilinogen, bilirubin or microscopy of the sediment either in week 27 or 104 .

Behaviour (functional findings):

no effects observed

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

The absolute weights of the heart and liver were statistically significantly decreased in males of the top-dose group in week 79 and 105, while testes and kidney weight were decreased in top-dose males in week 79 and 105, respectively. The decreases are most probably the result of the lower body weights in this group. Absolute organ weights of the rats killed after one year did not show statistically significant differences among the various groups. The relative weight of the kidneys in females was increased in the top-dose group at all stages, the differences with the controls being statistically significant only in week 53 and 105. The relative weight of the brain was statistically significantly increased in the top-dose group in males at all stages and in females in week 105. Relative testes weight was increased in the top-dose group in week 105 only. These increases in relative brain and testes weights are ascribed to the lower body weights of the rats in this group and the well-known inverse correlation between body weight and 1relative brain and testes weights.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

FIRST SUBSET (rats found dead, killed in extremis or killed in week 53)
Gross examination at autopsy revealed treatment-related changes in the stomach of both males and females. These changes comprised: i) a raised or thickened limiting ridge in one female of the low-dose group, in several mid-dose rats and in most top-dose animals. The severity of this alteration was positively correlated with the test substance level. ii) nodular thickenings and irregular areas of the mucosa of the fundus (glandular stomach) in one female of the bow-dose group and in a few animals of the mid- and top-dose groups.

SECOND SUBSET (rats found dead, killed in extremis or killed in week 79)
Gross examination at autopsy of the rats killed in week 79 revealed treatment-related abnormalities in the stomach only. The limiting ridge of the forestomach was raised and thickened in most animals of the top-dose group. Two females of the mid-dose group and one of the bow-dose group also showed slight thickening of the limiting ridge. In addition, slight surface irregularities in the glandular stomach were seen in 3 male and 6 female top-dose rats as well as in one female control animal. All other gross changes observed were about equally distributed among the various groups or occurred only in a single animal. Three female animals of this subset ware found dead or were killed in extremis before week 79. One control female bad a large polyp in the uterus and one female mid-dose rat bad a large tumour of the pituitary. The cause of death of the third animal (a mid-dose female) could not be established at autopsy.

THIRD SUBSET (rats found dead, killed in extremis or killed in week 105)
Gross examination at autopsy of the rats of the main study revealed treatment-related changes in the stomach only. The limiting ridge of the forestomach was raised and thickened in most male (36/50) and female (43/50) rats of the top-dose group. A few animals, both males and females, of the other groups, controls included, showed the same phenomenon. In addition, several animals of the top-dose group exhibited surface lesions in the forestomach (8 males and 6 females) and/or a strickingly smooth appearance of the glandular stomach (4 males and 13 females). Occasionally, the same changes were found in animals of the other
groups, controls included. The number of male top-dose rats showing gross signs of nephropathy (discolouration and irregular surface of the kidneys frequently accompanied by enlarged parathyroids) was significantly lower than the number of controls showing these lesions. Moreover, the number of male rats with atrophic testes was also remarkably low in the top-dose group as compared to controls (3 in the top-dose group versus 12 in the controls). The incidence of some other gross changes such as ovarian cysts, pituitary tumorous mass, hydrothorax and spotted appearance of the lungs varied considerably among the groups. Since, however, a distinct dose-response relationship was absent and large variations in incidence of these age-associated lesions are not uncommon, these differences are not considered to be related to treatment.

Neuropathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

FIRST SUBSET (rats found dead, killed in extremis or killed in week 53)
Treatment-related changes were found only in the stomach of top-dose animals. The changes included papillary epithelial hyperplasia in the forestomach and focal atrophic gastritis occasionally accompanied by ulceration of the fundic mucosa. The hyperplasia in the forestomach was seen as a thickenend layer of keratinized stratified squamous epithelium which was mainly found on the limiting ridge or in the vicinity of the limiting ridge. Cellular or nuclear atypia was not observed. In the glandular stomach focal atrophic gastritis occasionally accompanied by mucosal ulceration occurred to a varying degree in each of the top-dose males and females. In the affected areas the mucosa was reduced in width compared to an unaffected mucosa, and the lesions had bulky borders. Occasionally, bulky plugs of necrotic tissue, inflammatory exudate, mucus and feed particles were seen to be attached to the damaged mucosa. The inflammatory process was generally restricted to the lamina propria but in some cases involved the entire mucosa. In a few animals the defect in the wall was seen to extend to the muscularis mucosae and was diagnosed as ulceration. In one top-dose female a focus of glandular hyperplasia was present in affected mucosa. "Blinded" histopathological review of the stomach slides of all animals of the low- and mid-dose rats did not demonstrate microscopic changes even not in the additional sections of the rats with grossly visible mucosal alterations. lt is well-known from previous studies performed in our Institute that a thickened limiting ridge, which is clearly visible at gross examination is not always recognizable as such upon microscopic examination. The nodular irregularities grossly seen in the fundic mucosa of several rats of the mid-dose group and of one female of the low-dose group most probably represent aggregates of mucus and feed particles attached to the mucosa and formed as a result of fixation by the test substance. These aggregates may have disappeared during processing of the organ or may not be recognizable as such upon microscopy. However, in several top-dose rats such plugs of mucus and feed particles indeed were visible upon microscopy but in these cases they also contained inflammatory exudate and necrotic material; in addition in these animals the plugs were seen to be attached to the damaged mucosa. All other non-neoplastic histopathological changes obser-ved were commnon findings in rats of this age and strain or they occurred in one or a few animals and are therefore not considered to be treatrnent-related.

SECOND SUBSET (rats found dead, killed in extremis or killed in week 79)
Treatment-related changes were found only in the stomach of top-dose animals. The changes included focal hyperkeratosis of the forestomach epithelium and chronic atrophic gastritis of the glandular stomach. Focal ulceration in the forestomach or glandular stomach was observed in two male top-dose rats. The histopathological changes observed in the forestomach ware mainly seen as papillary epithelial hyperplasia frequently accompanied by hyperkeratosis located on the limiting ridge or in the vicinity of the limiting ridge. Frequently the mucosa showed an irregular layer of hyperplastic basal cells. Occasionally, similar changes but to a minimal degree ware observed in one or two animals of the other groups, controls included. In the glandular stomach chronic atrophic gastritis occurred to a varying degree in each of the top-dose animals. In the affected areas the mucosa was reduced in width compared to the unaffected mucosa, and the lesions had bulky borders. Occasionally, bulky plugs of necrotic tissue, inflammatory exudate, mucus and feed particles were seen to be attached to the damaged mucosa. The inflammatory process, characterized by infiltrations of lymphocytes and macrophages (or mononuclear cells) and fibrosis was restricted to the lamina propria but in some cases involved the entire mucosa. When the defect in the wall was seen to extend to the muscularis mucosae, the lesion was diagnosed as ulceration. The slight thickening of the limiting ridge seen in 3 female rats of the lower dose groups was not seen at microscopy. This phenomenon is not unexpected since it is well-known from previous studies performed in our Institute that a thickened limiting ridge, which is easily recognizable at gross examination is not always found as such upon microscopical examination. All other non-neoplastic histopathological changes observed were common findings in rats of this age and strain or they occurred in one or a few animals and are therefore not considered to be treatment-related.

THIRD SUBSET (rats found dead, killed in extremis or killed in week 105)
Microscopical examination of rats in the main study revealed test substance-related gastric and renal changes in animals of the top-dose group only. In the forestomach, an increased degree and/or incidence of the following histopathological changes were seen in the top-dose group: (i) Papillary epithelial hyperplasia frequently acconipanied by hyperkeratosis, and ii) Focal ulceration. Hyperkeratosis of a remarkably folded, slightly hyperplastic layer of keratinized stratified squamous epithelium was mainly seen on the limiting ridge or in the vicinity of the limiting ridge. Frequently, the mucosa showed an irregular layer of hyperplastic basal cells. An ulcer of the forestomach was found in 8 male and 5 female rats of the top-dose group; it invariably occurred near the limiting ridge.
Similar histopathological changes were occasionally seen in rats of the other groups, controls included and are known to occur in rats due to stress (e.g. starvation). They are not uncommon in historical control animals. In the glandular stomach the following compound-related changes were observed: chronic atrophic gastritis, in several animals accompanied by ulceration and/or glandular hyperplasia. Chronic atrophic gastritis occurred to a varying degree in each of the top-dose animals, except for one male rat. In the affected areas the mucosa was reduced in width as compared to the unaffected mucosa, and the lesions demonstrated bulky borders. In some cases the inflammatory process involved the entire mucosa arid was seen to extend to the whole muscularis mucosae meeting the criteria for the diagnosis ulceration. The incidence and/or degree of renal papillary necrosis was higher in males and females of the top-dose group than in the other groups, controls included. This renal change was seen as an early interstitial lesion located in the tip of the papilla. lt was characterized by patchy necrosis of interstitial cells, capillaries and loops of Henle, and in more severe cases the necrotic papilla appears as a rather homogeneous eosinophilic structure with ghost-like remnants of the normal architecture. Remarkably, nephrosis was less severe in males of the top-dose group than in males of the other groups. In females, however, the incidence of nephrosis was slightly higher in the test groups than in the controls, but a dose-response relationship was absent. The decrease in severity of nephrosis in males of the top-dose group was accompanied by a decrease in both the incidence of (bilateral) glandular hyperplasia of the parathyroids and the incidence of periarteritis in the testes. This relationship is not unexpected since these histopathological changes are all part of the well-known nephrotic syndrome in rats. The incidence of several other histopathological changes varied considerably among the various groups, but in no case a consistent or relevant relationship with doses was observed. Thus, there were no indications that the incidence or severity of any of these lesions was influenced by the exposure to the test substance. Moreover, the changes observed are age-associated and a considerable variation in their incidence and severity is not uncommon in rats of the strain used.

Histopathological findings: neoplastic:

effects observed, treatment-related

Description (incidence and severity):

FIRST SUBSET (rats found dead, killed in extremis or killed in week 53)
The following neoplastic lesions were observed in various groups: one fibroadenoma of the mammary glands, one haemorrhagic tumour of the pituitary gland, one mesenchymoma in the skin and two fibroznatous polyps in the uterus. Except for the mesenchymoma all tumours are commnon findings in the strain of rats used. The mesenchymoma found in a male top-dose animal is an uncommon finding; it is considered to be a fortuitous observation unrelated to the test compound.

SECOND SUBSET (rats found dead, killed in extremis or killed in week 79)
The following neoplastic lesions ware observed: 3 fibroadenomas of the mammary glands (all females; 2 controls and 1 top-dose rat); 1 fibroadenoma in the skin of a male control animal; 6 tumours of the pituitary gland (1 mid-dose male; 1 control, 1 low-dose and 3 top-dose females); 1 adenoma in the thyroid of a male control rat; 5 polyps in the uterus (2 controls, 2 low-dose and 1 mid-dose animal); 1 mesenchymal tumour (male mid-dose rat) and 1 lipoma (male low-dose rat) in the abdominal cavity. The tumours observed are common findings in the strain of rats used and moreover there was no indication that the incidence had been influenced by the test compound. Therefore, these tumours were not ascribed to the ingestion of the test substance.

THIRD SUBSET (rats found dead, killed in extremis or killed in week 105)
Apart from 2 (benign) papillomas, one in a male of the low-dose group and one in a female control rat, gastric tumours were not observed. All tumours are common neoplasms in the strain of rats used. In males of the top-dose group the total number of tumourous and the number of tumour-bearing rats were lower than in controls. Although differences in their incidence occurred between the groups, there was no indication that these differences ware related to the exposure to the test substance. They are considered to be related to normal ageing or to represent incidental findings without any toxicological significance.

Dose descriptor:

NOAEL

Effect level:

25 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

food consumption and compound intake

gross pathology

haematology

organ weights and organ / body weight ratios

urinalysis

water consumption and compound intake

Remarks on result:

other: actual doses: males 15 mg/kg, females 21 mg/kg

Critical effects observed:

yes

Lowest effective dose / conc.:

82 mg/kg bw/day (actual dose received)

System:

gastrointestinal tract

Organ:

stomach

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

yes

Conclusions:

NOAEL = 15 mg/kg bw/day

Executive summary:

In a reliable GLP-conform study similar to OECD TG 453, a combined chronic toxicity/carcinogenicity study, 70 male and 70 female Wistar rats per dose (subgroups of 10 rats/sex/dose killed 12 or 18 months after start of exposure) received via the drinking water 0, 1.2, 15, 82 mg/kg bw/day (males) or 0, 1.8, 21, 109 mg/kg bw/day (females) for 105 weeks (concentration: 0, 20, 260, or 1900 mg/L or 0, 0.002, 0.026, 0.19%).

At the high dose body weight gain was decreased in males & females and food and water consumption decreased. Other parameters (except pathology) were not altered.Pathological alterations in the kidney like the renal papillary necrosis detected in high dose males and females is discussed as an effect of the reduced water intake and is indirectly treatment related. Treatment related lesions were detected in the forestomach (focal papillary epithelial hyperplasia, ulceration and hyperkeratosis) and the glandular stomach (chronic atrophic gastritis, ulceration and hyperplasia) of males and females in the high dose group. No gastric tumours were induced. This study did not provide any evidence of carcinogenicity in rats after oral administration of formaldehyde.

Oral exposure via the drinking water induced local effects in the stomach of rats at a concentration of 0.19% corresponding to 82 mg/kg bw/d in males and 109 mg/kg bw/d in females; the NOAEC is 0.026% corresponding to 15 mg/kg bw/d in males and 21 mg/kg bw/d in females.

Reference 2

Endpoint:

chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

not specified

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Remarks:

partly limited documentation

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 452 (Chronic Toxicity Studies)

Deviations:

yes

Remarks:

low number of animals (interim sacrifice), no urinalysis, not all recommended organs examined histopathologically, limited hematology and clinical chemistry

GLP compliance:

not specified

Limit test:

no

Specific details on test material used for the study:

TEST MATERIAL
- Source: Wako Pure Chemical Ind. Ltd., Osaka, Japan
- Purity: 80 %
- Crystalline paraformaldehyde

Species:

rat

Strain:

Wistar

Remarks:

Slc

Details on species / strain selection:

not specified

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Shizuoka Lab Animals, Shizuoka, Japan
- Age at study initiation: 4 weeks
- Housing: singly in stainless-stell hanging cages
- Diet: commercial F-1 (Funabashi Animal Farm Co. Ltd., Chiba) diet
- Water: ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature: 21 - 25°C
- Humidity: 50 - 70 %

Route of administration:

oral: drinking water

Details on route of administration:

not specified

Vehicle:

unchanged (no vehicle)

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
- Crystalline para-formaldehyde was dissolved in distilled water for use twice weekly as the drinking water after mixing for 5 hours at 80 °C and being cooled

Analytical verification of doses or concentrations:

not specified

Details on analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

24 months, interim sacrifice after 12 and 18 months

Frequency of treatment:

daily, 7 days each week

Dose / conc.:

0.02 other: %

Remarks:

10 mg/kg bw/d (nominal)

Dose / conc.:

0.1 other: %

Remarks:

50 mg/kg bw/d (nominal)

Dose / conc.:

0.5 other: %

Remarks:

300 mg/kg bw/d (nominal)

No. of animals per sex per dose:

20 (6 rats sacrificed at each interim sacrifice after 12 and 18 month)

Control animals:

yes, concurrent no treatment

Details on study design:

TEMPLATE
- Dose selection rationale:
- Rationale for animal assignment (if not random):
- Fasting period before blood sampling for clinical biochemistry:
- Rationale for selecting satellite groups:
- Post-exposure recovery period in satellite groups:
- Section schedule rationale (if not random):
- Dose range finding studies:
- Other:

Positive control:

No

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS
- Time schedule: daily

BODY WEIGHT
- Time schedule: weekly or biweekly

FOOD CONSUMPTION
- Time schedule: weekly or biweekly

WATER CONSUMPTION
- Time schedule: weekly or biweekly

HAEMATOLOGY
- Time schedule: at necropsy
- Parameters: red blood cells, white blood cells, hematocrit, hemoglobin content

CLINICAL CHEMISTRY
- Time schedule: at necropsy
- Parameters: total protein, albumin, urea nitrogen, uric acid, total cholesterol, inorganic phosphorus, alkaline phosphatase, glutamic-oxaloacetic transaminase, glutamic-pyruvic transaminase

Sacrifice and pathology:

GROSS PATHOLOGY: Yes, including organ weights for brain, heart, lung, liver, kidney, spleen adrenals, testis, ovary, pituitary, thyroid

HISTOPATHOLOGY: Yes, including brain, heart, lung, liver, kidney, spleen adrenals, testis, ovary, pituitary, thyroid, forestomach, stomach, intestine, pancreas, uterus, lymph nodes, tumours

Statistics:

Mortality was analyzed by life-table techniques. Hematological and biochemical data as well as absolute and relative organ weights were analyzed statistically using Student's t-test.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Poor general state was observed.

Mortality:

mortality observed, treatment-related

Description (incidence):

Death was observed as early as 9 days after the start of the treatment. At 12 months, the mortality rates reached 45 % and 55 %, respectively, in males and females. All animals in the 0.50 % group bad died by 24 months.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Reduction of body weight gain in the highest dose was observed.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Reduction of food consumption in the highest dose was observed.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

effects observed, treatment-related

Description (incidence and severity):

Reduction of water consumption in the highest dose was observed.

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

There were some decreases of the red blood cells and hemoglobin content in the treated rats throughout the study, although not dose-related.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

At 12 months, significantly lowered levels of total protein, albumin and T-chol, and significantly higher levels of urea-N were observed in males and females of the 0.50 % group vs. controls. In male rats in the 0.10 % group, total protein level was significantly decreased at 24 months. At the same time, dose-dependent decreases in the levels of Pi were observed in males and females.

Endocrine findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

There were no dose-related significant changes in either absolute or relative weights of any organs examined.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Most non-neoplastic lesions related to formaldehyde treatment were found in the forestomach and glandular stomach of rats in the 0.50 % group which were sacrificed at 12 months. In those animals, erosions and/or ulcers and hyperplasia of the squamous epithelium with or without hyperkeratosis were observed in the forestomach. Basal cell hyperplasia with downward growth into the submucosa was observed in most cases. Also in rats given 0.50 % formaldehyde, erosions and/or ulcers accompanied by submucosal inflammatory cell infiltrates were seen in the glandular stomach. Regenerative changes of the glandular epithelium (glandular hyperplasia) were noticed in most cases along the limiting ridge of the fundic mucosa. Dilated gastric glands with clearly increased numbers of mucous neck cells were observed deep in the fundic mucosa. In the 0.10 % group, forestomach hyperkeratosis was observed in only 1 of 6 males and in 1 of 8 females which were killed at 18 and 24 months. On the other hand, there were no mucosal lesions of the glandular stomach in the 0.10 % group. No such lesions of the forstomach and glandular stomach were found in rats of the 0.02 % group. Various chronic nephropathic changes occured in both treated and control rats.

Neuropathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

See Description at "Gross pathological findings"

Histopathological findings: neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

Various types of tumors were observed in organs such as pituitary, thyroid, testios, adrenals, mammary gland and skin in all groups including the controls. Histologically the tumors were similar to those occuring spontaneously in this strain of rat. There were ni significant increases in the incidences of any tumors in any test groups as compared to control groups.

Dose descriptor:

NOAEL

Effect level:

10 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

Remarks on result:

other: 0.02 % in the drinking water

Critical effects observed:

yes

Lowest effective dose / conc.:

0.1 mg/kg bw/day (nominal)

System:

gastrointestinal tract

Organ:

stomach

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

yes

Conclusions:

NOAEL = 10 mg/kg bw/day

Executive summary:

In a reliable study similar to OECD TG 452, groups of 20 male and 20 female Wistar rats were given formaldehyde solution in their drinking water at concentrations of 0, 0.02, 0.1 and 0.5 % (0, 10, 50, 300 mg/kg bw/day) for 24 months.

In the high dose group, poor general state, reduction of body weight gain and both food and water consumption, increased mortality (ca. 50 % after 12 months, no rat alive after 24 months), and changes in various clinical parameters were recorded. Histopathology revealed local effects in the stomach/forestomach of the high dose group. Lesions of the forestomach in males and females were squamous and and basal cell hyperplasia, hyperkeratosis, erosions/ulcers, and submucosal cell infiltration. Glandular hyperplasia, erosions/ulcers, and submucosal cell infiltration in the glandular stomach were also found at the high dose level. A high incidence of renal papillary necrosis was observed in male and female animals (about 50 % versus 0 - 10 % in the other groups). This finding is ascribed to the dehydration caused by the considerable decrease of liquid consumption. No other treatment related lesions were reported. However, local effects were also detected at the mid dose level. Administration of 0.10 % resulted in forestomach hyperkeratosis in animals after 18 (1/6 males) and 24 months (1/8 females). According to the authors, the NOAEL was 10 mg/kg bw/day (NOAEC 0.02 %).

Local effects in the forestomach were detected in rats after chronic oral exposure to ≥ 0.10 % in the drinking water (50 mg/kg bw/day); the NOAEC was 0.02 % in the drinking water (10 mg/kg bw/day).

Reference 3

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

not specified

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Remarks:

partly limited documentation

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Deviations:

yes

Remarks:

no platelet counts and blood clotting

GLP compliance:

not specified

Limit test:

no

Specific details on test material used for the study:

TEST MATERIAL
- Source: Celanese Chemicals, Dallas, Texas, USA
- Batch Number: UN 2213
- Maximum content 60 ppm (v/v)
- Paraformaldehyde prills 95 % plus 5 % water

Species:

rat

Strain:

Wistar

Remarks:

Cpb : WU, SPF

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: TNO central institute for the breeding of laboratory animals, Zeist, Netherlands
- Age at study initiation: 5 weeks
- Housing condition: singly in the water-restricted control, 5 per cage in other groups, stainless-steel cages with wire-mesh florr and front
- Diet: Institute`s grain-based open-formula diet, ad libitum
- Water: tap water, ad libitum (except water-restricted controls)
- Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature: 20 - 24°C
- Humidity: 40 - 70 %
- Air changes per hour: 10
- Photoperiod: 12 hours / 12 hours

Route of administration:

oral: drinking water

Vehicle:

unchanged (no vehicle)

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS
- Fresh solutions were prepared every week and stored in closed plastic containers in a refrigerator at 4°C until they were used
- The concentrations required were adapted weekly to the changing body weights and liquid consumption, as measured over the preceding week
- Test solutions and tap-water were supplied in glass bottles which were filled daily and cleaned once a week

Analytical verification of doses or concentrations:

not specified

Details on analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

28 days

Frequency of treatment:

daily, 7 days each week

Dose / conc.:

5 mg/kg bw/day (actual dose received)

Dose / conc.:

25 mg/kg bw/day (actual dose received)

Dose / conc.:

125 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

test animals: 10, control animals 30

Control animals:

yes, concurrent vehicle

Details on study design:

- Rats were randomly assigned to eight groups
- Six treatment groups (10 per sex/group) received the various test solutions
- One control group (20 per sex) was given tap water without supplement
- A water-restricted group (10 per sex) received the same amount of water as the amount of liquid consumed by the group on the highest dose

Positive control:

no

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS
- Time schedule: daily

BODY WEIGHT
- Time schedule: weekly

FOOD AND LIQUID INTAKE
- Time schedule: weekly

HAEMATOLOGY
- Time schedule: week 4
- Blood samples were collected from the tip of the tail of all animals and were examined for haemoglobin concentration, packed-cell volume, and erythrocyte and leucocyte counts

CLINICAL CHEMISTRY
- Time schedule: week 4
- Parameters: whole blood taken from each of the animals after overnight fasting was examined for glucose; at autopsy, blood samples collected from the abdominal aorta of all rats were centrifuged at 1250 g for 15 min and then analyzed for alkaline phosphatase, aspartate aminotransferase, alanine aminotransferase, total protein, albumin, total bilirubin, urea, creatinine and calcium

URINALYSIS
- Time schedule: week 4
- Rats were deprived of water for 24 hours and of food for 16 hours
- Urine was collected during the last 16 hours of the deprivation period and its volume and density were determined

Sacrifice and pathology:

- Early in week 5
- Adrenals, brain, liver, heart, kidneys, spleen, testes, thymus, thyroid and ovaries were weighed and organ-to-body weight ratios were calculated
- Samples of the organs weighed, and of the lips, Iungs (with main-stem bronchi), pancreas, pharynx, stomach (non-glandular and glandular), nose, oesophagus, tongue, urinary bladder and
uterus, were fixed in 10% neutral buffered formalin
- Histopathological examination was restricted to the liver and kidneys, known to be the major target organs for systemic effects, and to the tongue, pharynx, oesophagus, stomach and nose (six standard cross-sections) as possible target organs for local effects
- Tissue samples of these organs from all controls, all water-restricted and all top-dose rats, and of the stomach of all low- and mid-dose rats were processed, embedded in paraffin wax, sectioned at 5 μm, stained with haematoxylin and eosin and examined microscopically

Statistics:

- Data on growth rate and organ weights, haematology, clinical chemistry and urinalysis were evaluated by a one-way analysis of variance, followed by Dunnett's multiple comparison tests (experimental unit: the rat)
- Data on food and liquid intake and food efficiency were evaluated by analysis of variance followed by LSD tests (experimental unit: the cage)

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

5 & 25 mg/kg bw: No treatment related effects were observed. 125 mg/kg bw: fur with yellowish discoloration from week 3 onwards. This discoloration is due to formaldehyde spilt on the fur from drinking bottles.

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

No effects in any treatment group but significant decrease in the water-restricted control males.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

In males and females of the high dose group significantly decreased food and water intake. In females of the other treatment groups the food intake was increased.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

effects observed, treatment-related

Description (incidence and severity):

In males and females of the high dose group significantly decreased food and water intake. In females of the other treatment groups the food intake was increased.

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

No effects except slightly higher packed-cell volume (m&f) and increased erythrocyte counts (m) in water-restricted controls.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

In water-restricted controls a number of parameters were altered. In high dose males total protein and albumin levels were significantly reduced (no data about historical controls).

Endocrine findings:

not examined

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

Tendency to increased urine density and decreased volume in the high dose group (m&f), but not significant like in water-restricted controls.

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

In water-restricted controls the relative weight of several organs was increased (presumably due to reduced body weight) in males and females. In high dose females the relative kidney weight was increased. No further effects.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

No effects except thickening of the limiting ridge of the forestomach in males and females of the high dose group accompanied by yellowish discoloration in some animals of this group.

Neuropathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

No effects were detected except lesions of the forestomach and glandular stomach (see Table below).

Histopathological findings: neoplastic:

no effects observed

Dose descriptor:

NOAEL

Effect level:

25 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

Critical effects observed:

yes

Lowest effective dose / conc.:

125 mg/kg bw/day (actual dose received)

System:

gastrointestinal tract

Organ:

stomach

Treatment related:

yes

Dose response relationship:

not specified

Relevant for humans:

not specified

Histopathology

Incidence of lesions in the forestomach and glandular stomach of rats after 4 week treatment via the drinking water
Type of lesion	Dose in mg/kg bw/day
	Control	5	25	125	Wr-control
Number of rats examined	20	10	10	10	10
Males
Focal hyperkeratosis of forestomach very slight slight moderate	3 1 0	0 0 0	0 0 0	0 4 6	0 0 0
Focal gastritis slight moderate	0 0	0 0	0 0	2 1	0 0
Dilated fundic gland	0	0	0	0	2
Submucosal mononuclear-cell infiltrate	0	0	0	1	0
Females
Focal hyperkeratosis of forestomach very slight slight moderate	6 0 0	0 0 0	0 0 0	2 2 6	0 0 0
Focal gastritis very slight slight moderate	0 0 0	0 0 0	0 0 0	1 2 1	0 0 0
Focal papillomatous hyperplasia	0	0	0	1	0
Leukocytic infiltrate	0	0	0	1	0
Wr: water-restricted

Conclusions:

NOAEL = 25 mg/kg bw/day

Executive summary:

In a reliable study comparable to OECD TG 407 groups of ten male and ten fenale 5-week-old rats received formaldehyde in the drinking-water over period of 4 weeks at dose levels of 5, 25 and 125 mg/kg body weight/day. A group of 20 males and 20 females served as controls and received unsupplemented drinking-water ad libitum. An additional group of ten males

and ten females was given unsupplemented drinking-water in an amount equal to the amount of liquid consumed by the group given the top.

Food and liquid intake were decreased in the groups on the top dose. Effects observed only in the top-dose group, were yellow discoloration of the fur, decreased protein and albumin levels in the blood plasma, thickening of the limiting ridge and hyperkeratosis in the forestomach, and focal gastritis in the glandular stomach.

It was concluded that in this study the no-observed-adverse-effect levels of formaldehyde were 25 mg/kg body weight/day.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

82 mg/kg bw/day

Study duration:

chronic

Experimental exposure time per week (hours/week):

168

Species:

rat

Quality of whole database:

similar to OECD TG 453, K1

System:

gastrointestinal tract

Organ:

stomach

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records

Reference

Endpoint:

repeated dose toxicity: inhalation, other

Type of information:

other: Review

Adequacy of study:

key study

Study period:

not specified

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

data from handbook or collection of data

Qualifier:

no guideline required

Principles of method if other than guideline:

The authors discuss the scientific literature available for cell replication, histopathological alterations and tumour response (polypoid adenomas) and present new statistical analyses for cell replication and tumour response (polypoid adenomas). Data is discussed in relation to the conclusion of the Committee for Risk Assessment (RAC) of the European Chemicals Agency which concluded that 2 ppm formaldehyde represent a Lowest Observed Adverse Effect Concentration (LOAEC) for polypoid adenomas, histopathological lesions and cell proliferation.

GLP compliance:

not specified

Limit test:

no

Species:

other: mammals

Route of administration:

inhalation

Details on results:

In this review the assessments of RAC is assessed and the following conclusions are drawn:
- PA (papillomas, polypoid adenomas) are not related to development of squamous cell carcinomas (SCC) and the data do not allow to define a LOAEC or NOAEC.
- For histopathological alterations a NOAEC of 1 ppm can be derived and lesions at 2 ppm are not related to SCC development.
- For cell replication the total database clearly justifies a NOAEC of 2 ppm and LOAECs of 3 ppm to 4 ppm.

Basis for effect level:

other: Reviewing the available data NOAECs for histopathological lesions and cell proliferation of 1 ppm and 2 ppm, respectively, are clearly indicated

Remarks on result:

not measured/tested

Critical effects observed:

not specified

Conclusions:

This reassessment of the extremely broad database does not support the assessments of RAC (2012) that 2 ppm FA represent a LOAEC for tumour development, histopathological lesions and cell proliferation. Rather, the available data clearly indicate NOAECs for histopathological lesions and cell proliferation of 1 ppm and 2 ppm, respectively.

Executive summary:

In this reliable review the authors discuss the scientific literature available for cell replication, histopathological alterations and tumour response (polypoid adenomas).

In 2012 the Committee for Risk Assessment (RAC) of the European Chemicals Agency concluded that 2ppm formaldehyde represent a Lowest Observed Adverse Effect Concentration (LOAEC) for polypoid adenomas, histopathological lesions and cell proliferation. An analysis of all data shows that a LOAEC of 2ppm it is not justified for cell proliferation and polypoid adenomas. Higher values are also supported by a new statistical analysis. For histopathological lesions a NOAEC of 1ppm may be defined but the lesions at 2ppm cannot be regarded as pre-stages for tumour development. One major uncertainty exists: the description of polypoid adenomas and the lesions at 2ppm often is insufficient and diagnostic uncertainties can only be resolved by a re-evaluation according to modern histomorphological standards. Although the discrepancy between our assessment and that of RAC may seem rather small we feel the LOAECs proposed by RAC must be challenged taking into consideration the broad data base for formaldehyde and the potential impact of any published RAC opinion on the present discussions about appropriate occupational and indoor exposure limits.

Endpoint conclusion

Dose descriptor:

NOAEC

1.2 mg/m³

Additional information

Repeated oral exposure

In a study comparable to OECD Guideline 453 (Til et al., 1989; combined chronic toxicity/ carcinogenicity study) 70 male and 70 female Wistar rats per dose (subgroups of 10 rats/sex/dose killed 12 or 18 months after start of exposure) received formaldehyde via the drinking water at dose levels of 0, 1.2, 15, 82 mg/kg bw/day (males) or 0, 1.8, 21, 109 mg/kg bw/day (females) for 105 weeks (concentration: 0, 20, 260, or 1900 mg/L or 0, 0.002, 0.026, 0.19%). At the high dose body weight gain was decreased in males & females as well as food and water consumption. Other parameters (except pathology) were not altered. Pathological alterations in the kidney, such as the renal papillary necrosis detected in high dose males and females, are attributed to a secondary effect arising from significantly reduced (~40%) water intake. Treatment related lesions were detected only in the forestomach (focal papillary epithelial hyperplasia, ulceration and hyperkeratosis) and the glandular stomach (chronic atrophic gastritis, ulceration and hyperplasia) of males and females in the high dose group. No gastric tumors were induced (see Section Carcinogenicity). In summary, oral exposure via the drinking water induced local effects in the stomach of rats at a concentration of 0.19% corresponding to 82 mg/kg bw/d in males and 109 mg/kg bw/d in females; the NOAEC is 0.026% corresponding to 15 mg/kg bw/d in males and 21 mg/kg bw/d in females.

Very similar results were presented by Tobe et al. (1989; comparable to OECD Guideline 452 with acceptable restrictions). In this drinking water study 20 Wistar rats per dose per sex were exposed to 0, 0.020, 0.10, 0.50% formaldehyde in the drinking water (0, 10, 50, 300 mg/kg bw/day) for up to 24 months (no post exposure observation period; 6 rats per sex per dose sacrificed at each interim sacrifice after 12 and 18 month). In the high dose group, poor general state, reduction of body weight gain and both food and water consumption, increased mortality (ca. 50% after 12 months, no rat alive after 24 months), and changes in various clinical parameters were recorded. Histopathology revealed local effects in the stomach/forestomach of the high dose group. Lesions of the forestomach in males and females were squamous and basal cell hyperplasia, hyperkeratosis, erosions/ulcers, and submucosal cell infiltration. Glandular hyperplasia, erosions/ulcers, and submucosal cell infiltration in the glandular stomach were also found at the high dose level. A high incidence of renal papillary necrosis was also observed in male and female animals (about 50% versus 0-10% in the other groups). This finding is ascribed by the authors to the dehydration caused by the considerable decrease of liquid consumption (significant decrease in the high dose group, data presented in a graph revealed a range of 10-20 mL/rat/day versus 25-35 mL/rat/day in the control). No other treatment related lesions were reported. However, local effects were also detected at the mid dose level: administration of 0.10% resulted in forestomach hyperkeratosis in animals after 18 (1/6 males) and 24 months (1/8 females). According to the authors, the NOAEL was 10 mg/kg/d (NOAEC 0.02%).

In a subacute study (Til et al., 1988) oral exposure of rats via the drinking water for 28 days induced local effects in the stomach at 125 mg/kg bw/day; the NOAEL was 25 mg/kg bw/day, however, no data are given on the formaldehyde concentration in the drinking water.

There are several relatively new oral gavage studies the interpretation of which is hampered by several important drawbacks: detailed information is missing on the dosing solution, histopathology of the stomach, the primary portal of entry, was not carried out, and only one dose level of FA was used such that a dose response relationship cannot be assessed. Furthermore, the relatively steep rise in blood levels obtained by gavage leads to some uncertainty in risk assessment for humans exposed via food or liquid intake. Finally, the authors generally failed to discuss their observations in relation to other prominent studies in rodents, like e.g. the chronic study of Til et al. (1989). Therefore, the findings of these studies will only be summarized without going into details. These studies concentrated on effects by combined exposure to FA and other substances and here we will only report on the parts dealing solely with FA.

Soni et al. (2013) described necrosis of the liver after daily exposure of male mice to 25 mg/kg bw/d over 8 weeks. The strain of mice and the source of FA are not given nor the FA concentrations or the dosing volume.

Abd-Elhakim et al. (2016) observed in male Swiss mice alterations in the hematogram, leukogram and in immunological parameters at 25 mg/kg bw/d over 60 consecutive days. A 40% solution of FA (possibly formalin) was diluted in water to “working stock solutions” without giving further details. Taking into account the dosing volume of 0.1 ml/mouse (20-25 g initial weight) a FA concentration in the range of 6000 mg/l can be estimated being by a factor of ~3 higher than the highest drinking water concentration used by Til et al. (1989). Under consideration of the bolus application severe effects on the stomach are to be expected with unknown systemic sequelae and missing data in this respect is a severe deficiency.

Mohamed et al. (2016) observed in Dawley Albino rats (3 months of age) adverse effects on biochemical liver and kidney parameters and histopathological alterations in these organs. The animals received 100 mg formalin/kg bw/d (concentration of FA not given) over 30 consecutive days. For a 40% formalin solution the dose would be 40 mg FA/kg bw/d. As the dosing volume was not specified the concentration of FA cannot be calculated.

Conclusion on repeated oral exposure:

The main effects in chronic drinking water studies with rats are local lesions in the forestomach and the glandular stomach at a concentration of 0.10% in the drinking water corresponding to 50 mg/kg bw/day (Tobe et al., 1989) or 0.19% corresponding to 82 mg/kg bw/day in males and 109 mg/kg bw/day in females (Til et al., 1989). In both studies the NOAEC for local effects is very similar: 0.026% formaldehyde in the drinking water corresponding to 15 mg/kg bw/day in males and 21 mg/kg bw/day in females (Til et al. 1989) or in the study of Tobe and coworkers (1989) 0.02% corresponding to 10 mg/kg bw/day. Til et al. (1989) as well as Tobe et al.(1989) reported an increased incidence in papillary necrosis of the kidney, but this effect was due to the reduced water intake and is only indirectly treatment related. The decrease in body weight is related to the reduced water & food consumption and/or is secondary to lesions of the stomach. The NOAEL for systemic effects is 82 mg/kg bw/day in males and 109 mg/kg bw/day in females (Til et al. 1989). The study of Tobe et al. (1989) is less suitable for final evaluation of systemic effects.

 

Repeated dermal exposure

Studies on repeated dermal dose toxicity in compliance to current Guidelines are not available. According to column 2 of REACH Annex VIII Section 8.6.1, the study does not need to be conducted as exposure to formaldehyde via inhalation is considered to be the main route of exposure.

The available data on this endpoint are of limited validity due to restricted documentation and methodological shortcomings. In the study of Iversen (1986) the NOAEC for skin effects in mice was 200 μL of 1% formaldehyde applied twice weekly for 60 weeks; at a concentration of 10% formaldehyde hyperplasia of the epidermis was found and a few mice had small ulcers and scratches (no further data given on origin, whether self-inflicted or caused directly by formaldehyde) of the skin; no treatment related tumors at remote tissues were detected (limited documentation; histopathology of the lung, nasal mucosa, brain and all tumors only in the high dose group).

In a subacute pilot study for an initiation/promotion study (Krivanek et al. 1983) daily dermal application of 100 μl 0.5% formaldehyde in acetone/water (50:50) solution resulted in reversible irritation of the skin in mice, no effects were detected at a concentration of 0.1%.

 

Repeated inhalation exposure

Monticello et al. (1991) presented an inhalation study in male F344 rats which is restricted to histopathology and autoradiography of the respiratory tract. Six rats per group were exposed 6 h/day for 1, 4, 9 days, or 6 weeks to 0, 0.7, 2, 6, 10, 15 ppm formaldehyde (whole body exposure was used in all studies described in detail in this section). Histopathology of the respiratory tract was performed and additionally, autoradiography of the nasal cavity (3H-thymidine injected 2 h prior to sacrifice for DNA labelling) for measurement of cell proliferation. No effects were detected at 0.7 and 2 ppm. At >= 6 ppm lesions of the respiratory epithelium in the nasal cavity were observed; no effects were noted in any other part of the respiratory tract. Effects in the nasal cavity were dose dependent and increased with exposure time. The lesions in nasal cavity exhibited an anterior-posterior severity gradient, more severe lesions were observed in the anterior part. In histoautoradiography significant effects were reported at a dose level of >= 6 ppm and exposure period >= 1 day. In conclusion, repeated inhalation exposure resulted in lesions of the respiratory epithelium in the nasal cavity of rats, the NOAEC was 2 ppm, an anterior-posterior severity gradient was shown and the lesions were associated with epithelial cell proliferation (Monticello et al.,1991).

Swenberg et al. (1983a) exposed rats to 15 ppm for 1-9 days (6h/d). After 1 day of exposure, acute degeneration of the respiratory epithelium with edema and congestion was evident. This was followed by ulceration, necrosis, and an influx of inflammatory exudates at days 3-9. Early squamous metaplasia was detected after as little as 5 days of exposure. Five days of exposure followed by 2 days without exposure led to considerable regeneration. These initial lesions were most severe on the tip of the maxilloturbinatesand nasoturbinates. By comparing these acute toxicity data with changes observed after prolonged exposure the authors conclude that adaptive changes occur.

In another short-term exposure study male rats were exposed for 1-4 days (6h/d) to 0.5, 2, or 6 ppm.At 0.5 or 2 ppm ultrastructural changes occurred at the cilia of the respiratory epithelial cells (blebbing of the membranes in some cilia) which are not considered to be an epithelial injury. At 6 ppm hypertrophy of the goblet and ciliated cells was noted after an exposure for 1, 2, or 4 days. Non-keratinysed squamous cells were seen as early 4 days after exposure to 6 ppm and at 1 and 2 days after exposure 15 ppm. Neutrophil infiltration was observed after an exposure to 6 ppm (Monteiro-Riviere & Popp, 1986).

Time dependency of early histopathological lesions can be seen from a study primarily designed to investigate the time and concentration dependency of formaldehyde-induced effects on mucociliary function in rats (Morgan et al., 1986). A single 6 h exposure to 15 ppm produced only minimal histopathological lesions in the anterior nasal passages. After 2 days of exposure, epithelial damage and inflammation were more severe with a more posterior extension and cellular proliferation with early squamous metaplasia after 4 days. After 9 days of exposure (6 h/d; 5 d/w) epithelial degeneration was severe with ulceration in some parts of the nasoturbinate. Similar but less severe changes were found at 6 ppm and no epithelial lesions in rats exposed to 0.5 or 2 ppm.

Cell proliferation in the middle portion of respiratory mucosa after a few days of exposure was investigated by Swenberg et al. (1986). A transient increase in cell proliferation was seen in rats exposed to 0.5 or 2 ppm for just one day. This increase disappeared after 3 and 9 days of exposure. At 6 ppm rats had a massive increase in labelling index after a single 6-h exposure, a lesser response after 3 exposures that returned to nearly the control rate after 9 exposures.

A sub-chronic inhalation study in Wistar rats was performed according to current guideline (OECD 413) with acceptable restrictions (haematology without blood clotting parameter & limited clinical chemistry; no ophthalmological examination). Ten male (m) and 10 female (f) rats were exposed for 13 weeks to 0, 1, 10, or 20 ppm formaldehyde (6 h/day for 5 days per week). At the high dose uncoordinated locomotion and excitation (m&f), growth retardation (m&f), decreased total protein and increased serum enzyme activity (ASAT, ALAT, and ALP in m; considered not to be ahepatotoxic effect because there were no effects on liver weight and no histopathological effects in the liver), squamous metaplasia of thelarynx (m) and olfactory epithelium (m&f), focal epithelial thinning (m&f) and keratinisation (m) of olfactory epithelium, and diffuse squamous metaplasia of nasal respiratory epithelium (m&f) were observed. At 10 ppm (LOAEC) focal squamous metaplasia, hyperplasia, and keratinization as well as epithelial disarrangement were seen in the nasal respiratory epithelium of males and females. No effects were detected in other organs. In summary, local effects in the respiratory epithelium of the nasal cavity were induced at 10 ppm (LOAEC) and at the higher dose of 20 ppm also in the olfactory epithelium and larynx. The NOAEC in this study is 1 ppm (NCF 1984).

In the study of Wilmer et al. (1989) it was shown that exposure concentration is more important than exposure duration. The study is limited to toxic effects in the nasal cavity of male Wistar rats after inhalation exposure. Rats were exposed a) continuously: 8 h/day, 5 days per week or b) intermittently: daily 8 x 30 min exposure separated by 30 min non-exposure periods, 5 days per week for 13 weeks in a) and b). Effects were studied by histopathology and histoautoradiography after thymidine-labelling. Intermittent exposure to a concentration of 4 ppm (product of concentration [4 ppm] x time [4 h/day] is 16 ppm x h/day) but not 2 ppm resulted in treatment related effects in the respiratory epithelium confined to the nasal cavity of rats which included: increased degree and incidence of epithelial disarrangement, squamous metaplasia with and without keratinisation, and basal cell hyperplasia. No effects were seen with continuous exposure to 1 or 2 ppm although the product of concentration (2 ppm) x time (8 h/day) is the same than with interrupted exposure to 4 ppm (16 ppm x h/day) suggesting that the concentration rather than the 'dose' is responsible for the effects. No significant, treatment related effects were found on cell proliferation. The authors suggested a NOAEC of 2 ppm for effects on the nasal epithelium.

The predominance of exposure concentration over cumulative dose can also be derived from a comparison of effects in rats observed after 6 months with different exposure schedules. Striking differences in the pathological response to 15 ppm formaldehyde (6h/d, 5d/week corresponding to 450 ppm x h/week) in comparison to 3 ppm (22h/d, 7d/week corresponding to 462 ppm x h/week). The first exposure scenario corresponded to that of Kerns et al. (1983) and the latter to that of Rusch et al. (1983). Although the exposure-time product was comparable, much less toxicity was observed by exposure to 3 ppm indicating that the concentration is more important than the total dose for the observed nasal injury.

Similar findings were also noted after short-term exposure when investigating the influence on cell proliferation by exposing rats to either 3 ppm x 12 h, 6 ppm x 6 h, or 12 ppm x 3 h (each concentration x time product = 36 ppm x h) over 3 or 10 days. In the most anterior part of the nose where mucociliary clearance is minimal, the total dose was more important than the concentration concerning cell proliferation. In contrast, at level II, where mucociliary clearance is prominent and where most of the tumors originate, cell proliferation was concentration dependent. Cell proliferation rates after 3 days of exposure (suggested combination of hyperplasia due to thickening of the epithelium and compensatory proliferation for cell death)were clearly higher than after 10 days of exposure (only compensatory proliferation; Swenberg et al., 1983b). These latter findings indicate to an adaptation as initial cell proliferation rates diminish with prolongation of exposure.

Mice are less sensitive than rats. Early experiments demonstrated that the enhanced sensitivity of rats compared to mice for tumor induction is also reflected by increased cell proliferation as measured by pulse labelling with [3H]-thymidine. A single 6-h exposure to 15 ppm caused a 13-fold increase in cell proliferation in rats and only an 8-fold increase in mice. The labelling index was further increased to 23-fold when rats were exposed to 15 ppm for 5 days, but only a small additional increase was noted for mice (Chang et al., 1983). 

In a sub-chronic inhalation study 10 male and 10 female B6C3F1 mice per group were exposed 6 h/day, 5 days per week for 13 weeks to 0, 2, 4, 10, 20, or 40 ppm formaldehyde. Lesions of the anterior part of the nasal cavity (squamous metaplasia [m&f] and rhinitis [m]) were reported at >= 10 ppm. Severity and site of lesions (also more posterior parts of the nasal cavity) increased with rising concentrations. Higher dose levels of >= 20 ppm in males & females also affected the larynx and trachea; lesions of the bronchus occurred at 40 ppm. No effects of toxicological relevance were detected at 4 ppm (distant site tissues not examined). Comparison with rat studies (see NCF, 1984 or Wilmer et al., 1989; or direct comparison by CIIT 1981) revealed that mice are less sensitive than rats. Conclusion: Inhalation exposure for 13 weeks induced local effects in the anterior nasal cavity of mice at 10 ppm, higher dose levels induced also effects in more proximal parts of the upper respiratory tract; the NOAEC was 4 ppm (Maronpot et al., 1986).

Rusch et al.(1983) compared toxic effects after inhalation exposure in 3 different species: rat, hamster, and monkey. The study was restricted to effects on the respiratory tract. F344 rats (20 m and 20 f per dose); Syrian golden hamster (10 m and 10 f per dose), and monkeys (Cynomolgus; 6 males per dose) were exposed to 0, 0.2, 1, or 3 ppm, 22 h per day, 7 days per week for 26 weeks. Rats revealed no treatment related effects at 0.2 and 1 ppm. At 3 ppm clinical symptoms were not reported but body weight was decreased in males. This level induced also effects in the epithelium of nasoturbinates in males and females including increased incidence in squamous metaplasia and hyperplasia at the middle section level of the nasoturbinates. The LOAEC was 3 ppm and the NOAEC was 1 ppm. Very similar results were found in monkeys. This species revealed no treatment related effects at 0.2 and 1 ppm. At 3 ppm clinical symptoms were reported. This level induced also effects in the nasoturbinates: squamous metaplasia and hyperplasia. No effects were detected in other tissues of the respiratory tract. In contrast, Syrian golden hamsters revealed no treatment related effects in any studied respiratory tract tissue (lung, trachea, nasal cavity) even at a level of 3 ppm. Hamsters seem to be a less sensitive animal model for formaldehyde exposure compared to rats.

Supporting evidence for a NOAEC of 1 ppm in rats came also from a study using transmission electron microscopy (SOCMA, 1980) after exposure for 26 weeks to 0 or 1 ppm (5 rats per sex, 22 h/day, 5 days/week). Rats exposed to 1 ppm did not show any ultrastructural alterations of the nasal epithelium, tracheal epithelium or epithelium of terminal bronchioles (SOCMA 1980).

In a long-term inhalation study restricted to the investigation of effects in the nasal cavity 90-147 male F344 rats per dose were exposed to 0, 0.7, 2, 6, 10, 15 ppm formaldehyde gas 6 h/day, 5 days per week for 24 months. Rats exposed to 0.7 or 2 ppm did not show any histopathological changes in the nasal cavity and no effects on cell proliferation index. Nonneoplastic effects were detected at a dose level of 6 ppm (mixed cell infiltrate, squamous metaplasia, hyperplasia in the anterior part of the nasal cavity) as well as a minimal carcinogenic response (incidence 1/90 versus 0/90 in control). A sharp increase in tumor incidences in the nasal cavity (mainly squamous cell carcinoma) was reported at 10 and 15 ppm. Dose dependent increases on cell proliferation were detected also only at >=10 ppm. In conclusion, inhalation exposure for 2 years induced local effects in the nasal cavity of male F344 rats at a dose level of 6 ppm and clearly carcinogenic effects at 10 ppm; the NOAEC was 2 ppm (Monticello et al., 1996).

In the inhalation study of Kamata et al. (1997; reliable with acceptable restrictions concerning local effects) 32 male F344 rats per dose were exposed to 0, 0.3, 2.2, or 15 ppm formaldehyde 6 h/day, 5 days per week for 28 months; 5 rats per dose were sacrificed after 12, 18, or 24 months of exposure (no post exposure observation period). Rats exposed to 0.3 ppm for 28 months did not show any histopathological changes in the nasal cavity. At 2.2 ppm a significant increase was noted in the incidence of squamous cell metaplasia with and without hyperplasia but no carcinogenic effects. Clear carcinogenic effects were noted at a level of 15 ppm but only in the nasal cavity; no tumors were found in non-site-of-contact tissues. There is some evidence that this study is less suitable for deriving a LOAEL (more variable exposure concentrations than in other studies). In summary, inhalation exposure for 28 months induced rats non-neoplastic effects in the respiratory epithelium of the nasal cavity at ≥ 2.2 ppm and neoplastic effects at 15 ppm.

In a long-term study comparable to OECD guideline 453 with acceptable restrictions (no histopathology of sternum, larynx and pharynx) the inhalation exposure of male and female F344 rats for 2 years at dose levels of 0, 2, 6, 15 ppm (120 rats per sex per dose; 24 months exposure, 6 h per day, 5 days per week; post exposure period 6 months; interim sacrifices) resulted exclusively in local effects of the nasal cavity and of the proximal trachea in males and females. Only non-neoplastic effects were reported in the high dose group in the proximal trachea but a high incidence in squamous cell carcinomas of the nasal cavity was observed. Carcinogenic effects in the mid dose group (squamous cell carcinomas in nasal cavity) are not of statistical significance but possibly of toxicological relevance. Non-neoplastic effects in the mid dose group like dysplasia and squamous cell metaplasia in the nasal cavity are considered to be treatment related since these effects occurred in more posterior parts or the nasal cavity. At 2 ppm purulent rhinitis, epithelial dysplasia, and squamous metaplasia were present in the anterior part of the nasal cavity. Dysplasia occurred earlier than metaplasia. Generally the location of the histopathological lesions moved from the anterior parts of the nose to more posterior sites with increasing exposure duration and concentration. There is some evidence that effects at the low dose level are less suitable for derivation of a LOAEC due to more variable exposure concentrations than in other studies particularly at the 2 ppm exposure level. In conclusion, clearly carcinogenic effects in the nasal cavity of rats were reported after inhalation exposure to 15 ppm formaldehyde for up to 2 years; local effects in the nasal cavity were found even at a level of 2 ppm. The exposure period of 24 months was followed by 3 months without exposure. At month 27 there was a significant decrease in the incidence of squamous metaplasia at all exposure concentrations regressing predominantly from the more posterior parts of the nose to the anterior sections (CIIT, 1981; Kerns et al. 1983).

In this study also B6C3F1 mice were exposed using the same experimental design. Formaldehyde exposure resulted only in local effects in the nasal cavity (males and females), predominantly in the respiratory epithelium; no tumors were seen in other organs. Rhinitis, dysplasia, squamous metaplasia were induced, but also atrophy of olfactory epithelium with increasing exposure periods. Except for reduced body weight no further effects were detected. The NOAEC was 2 ppm and the LOAEC 6 ppm. In contrast to rats only a few tumors were detected in the nasal cavity at 15 ppm. Generally the lesions in mice were less severe than in rats.At 27 months (after a 3 months observation period without exposure) dysplastic epithelial lesions were only present in the 15 ppm group. Squamous metaplasia was not present at this time interval and the low- and intermediate-exposure groups were free of treatment-related effects (CIIT, 1981; Kerns et al. 1983).The NOAEC for systemic effects in these long-term inhalation studies in rats and mice is 15 ppm.

Recently a subacute inhalation study in rats at exposure concentrations of 0, 0.7, 2, and 6 ppm up to 3 weeks (6 h/d; 5 d/week) was reported with focus on effects in the nasal cavity including genomic signatures (Andersen et al., 2008). Neither cell proliferation nor histopathological alterations were found in nasal cavity at 0.7 ppm. At 2 ppm a few animals exhibited epithelial hyperplasia that occurred in all animals exposed to 6 ppm. A significantly increased cell proliferation was only found at 6 ppm at the end of the first week (day 5) but not at the end of week 3. This is in line with former studies showing that cell proliferation decreases as exposure duration increases. Microarray analysis was conducted on respiratory epithelium of those nasal sites that receive a high formaldehyde flux corresponding to regions with a high tumor incidence in chronic studies. In microarray studies, no genes were altered at 0.7 ppm. At 2 ppm 15 genes were changed on day 5; 28 genes were changed at 6 ppm on day 5. No genes were changed at 2 ppm at day 15. The majority of changes was observed at 6 ppm. The pattern of gene changes at 2 and 6 ppm, with transient squamous metaplasia at day 5, indicated tissue adaptation and reduced tissue sensitivity by day 15 (compare with Swenberg 1983 a/b & 1986). In an acute part of this study rats were exposed to 15 ppm; in addition a group was included receiving a formaldehyde solution by nasal instillation (40 µLper nostril, 400 mM solution). Both instillation of 400 mM and inhalation of 15 ppm formaldehyde altered many more genes than were affected at 6 ppm. Three times more genes were affected by instillation than by inhalation exposure to 15 ppm. U-shaped dose responses were noted in the acute study for many genes that were also altered at 2 ppm on day 5. On the basis of cellular component gene ontology benchmark dose analysis, the most sensitive changes were for genes associated with extracellular components and plasma membrane. Generally,genomic markers provide, at best, only modestly more sensitive measures of tissue response compared with histology.

A summary of other reviewed data on repeated inhalation toxicity is presented in a supporting data record (BfR, 2006).

 

A literature search after the last IUCLID update was carried out up to May 2022.

Gelbke et al. (2014, key) assessed the NOAELs/LOAELs for the nose of rats after inhalation for histopathological lesions, cell proliferation, and the formation of polypoid adenomas. A new statistical analysis combined with a detailed review of the studies showed that the LOAEL is >2 ppm for cell proliferation and polypoid adenomas. Polypoid adenomas cannot be considered as pre-stages to the development of squamous cell carcinomas, the predominant tumor type in rats and mice after inhalation of formaldehyde. The NOAEL for histopathological lesions in the upper respiratory tract is 1 ppm but lesions observed at 2 ppm are not pre-stages to tumor development. It is noted that descriptions of histopathological lesions, including polypoid adenomas, in former studies often are insufficient and sometimes do not allow a final decision.

 

Immunological effects

These investigations warrant a separate consideration because of the known skin sensitisation by formaldehyde and its alleged implication in respiratory sensitisation. In addition, Rager et al. (2014, see Section Genetic toxicity in vitro) analysed miRNA profiles and noted transcriptional changes of mRNAs involved in the immune/inflammation system in response to changes of miRNAs.

Sapmaz et al. (2015, supporting) studied the effect of formaldehyde inhalation on humoral immunity in rats (5 and 10 ppm, 8 h/d, 5 d/week over 4 weeks). Exposure significantly increased IgA, IgM, and complement 3 levels in blood and decreased IgG dose dependently starting already at 5 ppm. These findings would need to be supported by an independent experiment.

Conclusions on repeated inhalation exposure toxicity:

Local effects in the upper respiratory tract were induced after repeated inhalation exposure in experimental animals. It has been shown in rats, mice, and monkeys that the respiratory epithelium in the nasal cavity is the most sensitive site. In rats and monkeys squamous metaplasia and hyperplasia were reported, in mice rhinitis, dysplasia and squamous metaplasia.

The LOAEC is 3 ppm in monkeys (NOAEC 1 ppm) and 6 ppm in mice (NOAEC 4 ppm). In rats, however, the LOAEC was considered to be 2 ppm (CIIT, 1981; Kamata et al., 1997), although this dose level was the NOAEC in other studies (Monticello et al., 1991; Wilmer et al., 1989; Monticello et al., 1996). These contradictory results most probably related to differences in the accuracy of constant exposure concentrations because formaldehyde induced lesions are predominantly concentration dependent and thereby fluctuations in concentration may become important, such as observed in the CIIT study.

Using the same experimental design mice are less sensitive than rats (CIIT, 1981); this might be related to the lower RD50 value in mice compared to rats (Section Actute Toxicity: inhalation) leading to a decreased minute volume for mice.

In chronic studies with rats as well as in subacute studies with only a few days duration (measuring cell proliferation; see supporting data record BfR, 2006) or weeks duration (measuring epithelial lesions; see data above) the NOAEC was the same range as that in long term studies. Effects were noted in the respiratory epithelium at a concentration of 2 ppm. At dose levels above the LOAEL the severity of the lesion in respiratory epithelium increased with the concentration and the duration of the exposure period (cf. Monticello et al., 1991).However, studies in rats (using the constant concentration x exposure duration) revealed that the concentration rather than the “total dose” is responsible for the effects (Wilmer et al., 1989). At high dose levels (10-20 ppm) no complete reversibility of lesions in the nasal cavity was reported in rats after 13 weeks of exposure and a post exposure observation period of up to 126 weeks (Feron et al., 1988; see supporting data record BfR, 2006). Not only the anterior part of the nasal cavity but also more proximal parts of the upper respiratory tract were affected with increasing formaldehyde concentrations in rats and mice (e.g. CIIT, 1981; Maronpot et al., 1986): olfactory epithelium, larynx, trachea, and bronchus. Increase in toxicity and severity is not linear but shows a sharp increase at 6-10 ppm. By microarray analysis no significant gene changes were observed at 0.7 ppm and minimal changes at 2 ppm only on day 5 that had returned to control level at day 15.The majority of changes were found at 6 ppm (highest exposure level for repeated exposure). However, authors concluded that genomic markers provide, at best, only modestly more sensitive measures of tissue response compared with histology (Andersen et al., 2008).

Related to the studies described above the overall NOAEC for non-neoplastic tissue changes based on chronic studies with rats, mice, hamsters and monkeys is 1 ppm. corresponding to 1.2 mg/m³.

A detailed review of former studies in rats confirmed that the NOAEL for regenerative cell proliferation is between 2 and 6 ppm and for histopathological lesions 1 ppm. Lesions observed at 2 ppm are not prestages to tumor development. Also the polypoid adenomas observed in some inhalation studies are not prestages to squamous cell carcinomas, the most prominent tumor type observed in the nose.

In the recent literature several non-guideline studies report lesions in the lower respiratory tract, in other organs apart from the respiratory tract, or claim indications for immunological effects. The evidence reported in these studies has to be weighed against findings in former guideline studies up to 2 years of exposure with large number of experimental animals, none of which gave indications for lesions apart from the nose. Therefore, by weighting the validity of the different studies, the conclusion is still upheld that formaldehyde inhalation will only lead to lesions in the upper respiratory tract.

Studies reporting immunological effects may warrant a separate evaluation because of the known skin sensitising properties of formaldehyde and indications that formaldehyde inhalation may lead to transcriptional changes by miRNA involved in the immune system. However, the majority of these non-guideline studies had to be disregarded due to missing dose response relationships, investigation of tissues far of the portal of entry, irrelevant exposure routes, or unclear exposure concentrations.

Effects in humans occupationally exposed via inhalation (supporting data record BfR, 2006): There is some evidence from a number of studies that formaldehyde exposure may also induce squamous cell metaplasia and hyperplasia in the respiratory epithelium of the nasal cavity in long-term exposed humans. However, the data base is not sufficient for conclusions on dose or time response relationships. None of the studies were conducted with sufficient methodological accuracy and no firm conclusion can be drawn (WHO, 2002; BfR, 2006).

Chronic respiratory effects of indoor formaldehyde exposures reported by  Krzyzanowski et al. 1990 were amongst other publications reviewed by the German UBA. Recently, German UBA (2016) performed a comprehensive review of epidemiological studies investigating the association between formaldehyde exposure and the induction or exacerbation of asthma in children including several of the studies mentioned above. UBA concluded that there is no clear association between formaldehyde exposure in the indoor environment and asthma in children. It was stated that the above mentioned epidemiological studies (e.g. Krzyzanowski et al., 1990; Annesi-Maesano et al. 2012 etc.) suffer from small sample size, implausible formaldehyde concentrations, and the fact that other substances or factors initiating asthma and asthma-like complaints were not adequately considered. Results derived from controlled human exposure studies as well as animal experiments support their opinion. It should also be noted that the Krzyzanowski et al. (1990) findings are inherently limited by the cross-sectional nature of their study and found that the study design is not sufficiently described in the published report.   

 

In the literature search up to April 20, 2015, following the last IUCLID update an epidemiological study was identified unrelated to potential cancer risks. Pinkerton et al. (2013, supporting) assessed mortality from amyotrophic lateral sclerosis (ALS) in a cohort of formaldehyde exposed garment workers that had already been studies for cancer incidence rates by Pinkerton et al. (2004), and Meyers et al. (2013). In their introduction they referred to two former studies related to ALS. The large American Cancer Society’s Cancer Prevention Study II cohort comprised about 1 million subjects with 22 exposed cases and 1120 unexposed. The rate ratio for self-reported formaldehyde exposure was 2.47 (95% CI 1.58, 3.86). The rate ratios were strongly associated with exposure duration. Reliance on self-reported exposure was a mojor limitation with no information on exposure frequency or intensity (Weisskopf et al., 2009). In a case control study with 109 cases and 253 controls, formaldehyde exposure inferred from occupation was not associated with ALS (Fang F, Quiulan P, Ye W, Bumer MK, Umbach DM,Sandler DP,et al. Work place exposures and the risk of amyotrophic lateral sclerosis. Environ Health Perspect,2009;117:1387-92). The study of Pinkerton et al. (2013) comprised 11098 employees exposed to formaldehyde for at least for 3 months. In the early 1980s the overall geometric mean formaldehyde concentration was 0.15 ppm. In total 8 ALS cases were observed with a SMR of 0.89 (95% CI 0.38, 1.75). No association for ALS with formaldehyde exposure was found when analysed for year of first exposure, duration of exposure, or time since first exposure. The main limitations of this study are the small number of cases and the low exposure levels.

Justification for classification or non-classification

According to EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008, classification and labelling is not needed for repeated dose toxicity. However, under consideration of the RAC (2012) proposal for classification and labelling, formaldehyde is classified as Carc. Cat 1B (H350) according to EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008, Annex VI.