Registration Dossier

Administrative data

Description of key information

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2002
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.26 (Sub-Chronic Oral Toxicity Test: Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
Source: Charles River Deutschland, D-97633 Sulzfeld
Acclimatization period: 9 d
age at 1st dose: m 33 d, f 35 d; body weight m 75-82 g and f 75-81 g upon receipt.
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on oral exposure:
Dose: 0, 30, 90, 270 mg/kg bw
Vehicle: Corn oil
Concentration in vehicle: 0, 6, 18, 54 mg/ml (0, 0.6, 1.8, 5.4%)
Total volume applied: 5 ml/kg bw (prepared freshly every day)
Analytical verification of doses or concentrations:
yes
Duration of treatment / exposure:
90 days
Frequency of treatment:
Once daily, 7 days per week
Remarks:
Doses / Concentrations:
0, 30, 90, 270 mg/kg bw ; Concentration in vehicle: 0, 6, 18, 54 mg/ml (0, 0.6, 1.8, 5.4%)
Basis:
nominal in diet
No. of animals per sex per dose:
10 m and 10 f per dose level
Control animals:
yes, concurrent vehicle
Observations and examinations performed and frequency:
Clinical signs: At least twice daily each rat was observed for clinical signs, additionally immediately after each oral application. Detailed clinical observations were made in all animals once a week starting before the 1st application in a standard arena and included changes in skin, mucous membranes, autonomic activity, gait, posture, response to handling, as well the presence of abnormal secretions, abnormal movements or behaviour. At week 13 these observations were performed prior to any laboratory test.At the end of the exposure period (>= week 11) functional observations were recorded including sensory reactivity to different types of stimuli (auditory, visual, proprioreceptive), assessment of positive geotropism, limb rotation, grip strength and motor activity. Body temperature was measured.

Mortality: Mortality was checked twice daily and premortal symptoms recorded.

Body weight: Measured once weekly starting day 0.

Food consumption: Calculated for each treatment week by the total amount of food given to and left by each rat in this week.

Water consumption: Monitored daily.

Ophthalmoscopic examination: Complete examination performed before exposure starts and prior to termination in all groups.

Haematology: Blood samples taken from all surviving rats at day 90. Measured parameters: Haematocrit, haemoglobin concentration, erythrocyte count, total and differential leukocyte count, platelet count, activated partial thromboplastin time, thromboplastin time.

Clinical Chemisty: Blood samples taken from all surviving rats at day 90. Parameters: sodium, potassium, plasma glucose, total cholesterol, urea, creatinine, total plasma protein, plasma albumin, alanine aminotransferase (ALAT), aspartate aminotransferase (ASAT), alkaline phosphatase (aP).

Urinanalysis: Not done, but only optional according to OECD 408.
Sacrifice and pathology:
The organ weights of liver, kidneys, adrenals, testes, epididymis, uterus, ovaries, thymus, spleen, brain, heart were determined before fixation. Same procedure with animals which died during exposure period but these data were not included in mean value comparison.
Other examinations:
All organs which are listed in the OECD guideline 408 were fixed in 7% formalin. Histopathological examinations of these organs were done in tissues of controls and animals of the high dose group including animals which died during the exposure period.
Due to substance related changes also stomach and bone marrow of low and mid dose rats were examined histopathologically.
Statistics:
Suitable statistical methods were used and the level of significance was p<0.01 except in histopathology (p<0.05; Fisher exact test).
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
30 mg/kg bw: no treatment related effects were observed in the low dose groups.
90 mg/kg bw: In the mid dose group all males and females showed long-lasting piloerection from day 16 onwards (0/20 at day 15 but 20/20 at day 16 and up to day 90). Salivation was noted in all males starting at day 64 (0/10 at day 63 but 10/10 at day 64).
270 mg/kg bw: all males and females showed long-lasting piloerection from day 16 onwards (0/20 at day 13 but 20/20 at day 14 and up to day 90). Salivation was noted in all animals starting at day 27 (0/20 at day 26 but 20/20 at day 27).
Comment: it is unusual that effects start in all animals at the same day of the study.
Functional observation battery (week 13)
30 mg/kg bw: No treatment related effects were recorded at week 13.
90 mg/kg bw: Reduced pupil size was detected in 7/9 male and 7/10 female survivors. A negative pupil response was noted in 5/9 males. Further parameters were within the normal range.
270 mg/kg bw: Reduced pupil size was detected in 8/9 male and 4/9 female survivors. A negative pupil response was noted in 4/9 males. A reduction in the number of static and stereotype movements in males and females was detected as well as a slight reduction in active locomotion. Further parameters were within the normal range.
Mortality:
mortality observed, treatment-related
Description (incidence):
No mortality in the low dose groups.
1 male of the mid dose group died at day 81.
High dose group: 1 male died day 74 and 2 females at day 81 & 89.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
No effect on body weight gain was detected in the low and mid dose group. From test week 1 onwards the body weight of males in the high dose group was decreased. The difference to controls was -10 to -31% (p<0.01). Less decreased body weights were measured in females. The difference to controls was -5 to -10% (not significant).
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
The food consumption was not influenced by the test substance treatment.
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
Drinking water consumption was not influenced by the treatment.
Ophthalmological findings:
no effects observed
Description (incidence and severity):
No treatment related effects. Effects on pupils see above.
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
Test substance related changes were presented in the Table below.
No effects were recorded at 30 mg/kg bw.
90 mg/kg bw: An increase in the thromboplastin time (TPT) in females was recorded but no such effect was detected in the high dose group (not treatment related). Slight but not significant (p<0.01) effects on leucocyte counts and differential blood counts were reported (only males; compare with high dose group).
270 mg/kg bw: In males and females an increase in leucocyte counts was demonstrated as well as a shift in differential blood count. Effects on leucocyte counts and alteration in differential blood count might be treatment related and a consequence of the lesions detected in the stomach.
No other parameter was treatment related altered. However, no historical control data of this lab are available.
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
No effects were detected at 30 and 90 mg/kg bw.
In rats of the high dose group a reduction in glucose (m), plasma albumin (f) and total protein (f) was measured. These effects were considered by the authors to be treatment related. But no data are available on historical controls of this lab.
Urinalysis findings:
not specified
Behaviour (functional findings):
not specified
Immunological findings:
not specified
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
No effects on relative or absolute organ weights were detected in the low and mid dose groups.
270 mg/kg bw: No effects were reported in female rats.
Male survivors revealed an significant increase (p<0.01) in relative organ weight of brain (+37%), gonads (+39% [left] and +33% [right]), adrenal (+47% left [not significant] and 103% right).
The absolute organ weights in males of the kidney (left -28%, right -25%), heart (-26%), thymus (-49%), liver (-27%), epididymis (left -31%, right -18%) were decreased (no data on statistical significance).
Authors comment: effects on relative and absolute organ weight are due to the severe body weight reduction in males.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Macroscopical findings
30 mg/kg bw: No treatment related effects were recorded in the low dose group.
90 mg/kg bw: In males and females alterations in the stomach were observed. Greenish or blackish discoloured indurations/nodules were detected in the stomach of 7/10 males and 2/10 females. Ulcerous lesion of the stomach was noted in one male and haemorrhagic foci in 3/10 females. Alterations in other organs were not considered to be treatment related.
270 mg/kg bw: Treatment related effects were noted in the stomach of all males and females: discoloured foci, nodules or deposits (10/10 males & 9/10 females), ulcer (1 male and 1 female), haemorrhagic indurations (1 female). The liver was adhered to the stomach in 4/10 males and 3/10 females as well as the spleen (2/10 males and 1/10 females). Other effects were not considered to be treatment related.
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Histopathology
30 mg/kg bw: The treatment did not induce any histopathological alterations in the 2 organs examined in this group (stomach and bone marrow) except a chronic multifocal ulcerative gastritis/peritonitis in one male rat. This finding is not discussed by the authors but it is considered to be of spontaneous nature (single observation).
90 mg/kg bw: Local effects in the stomach and granulocytopoesis in bone marrow (inflammatory response, see Table below) were observed. Only these 2 organs were investigated.
270 mg/kg bw: Treatment related histopathological effects were seen only in the stomach and bone marrow. Further changes in various organs were within the normal control range for animals of this strain and age.
Conclusion: The treatment induced chronic ulcerative gastritis and peritonitis and/or hyperplastic-hyperceratototic epithelial reactions with bacterial infection especially of the proventricular part of the stomach in 10 males and 5 females of the mid dose group and all males and females of the high dose group. As physiological response to the inflammatory reaction in the stomach most of these animals showed an increased granulocytopoiesis in the bone marrow.
Histopathological findings: neoplastic:
no effects observed
Key result
Dose descriptor:
NOAEL
Effect level:
30 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: local effects
Dose descriptor:
LOAEL
Effect level:
90 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: local effects
Key result
Critical effects observed:
no
Conclusions:
At dose level of ≥ 90 mg/kg bw and a concentration of ≥ 1.8% in corn oil the test substance induced local effects in the stomach after repeated administration via gavage.
Executive summary:

The study is conducted according to OECD guideline 408 with GLP. 10 male and 10 female Sprague-Dawley rats received via gavage once daily, 7 days per week for 90 days 0, 30, 90, 270 mg/kg bw in corn oil (concentration 0, 0.6, 1.8, 5.4%). The test substance induced local effects in the stomach at a dose level of ≥ 90 mg/kg bw. Such effect is expected of a substance releasing formaldehyde in situ. Other reported effects like reduced body weight gain and some decrease in motor activity in the high dose groups and alteration in haematology like increased leucocyte counts, shift in the differential blood count at the high dose level are considered to be a consequence of the chronic ulcerative gastritis & peritonitis. The reduced pupil size detected in males and females and the negative pupil response in males of the mid and high dose group (detected in functional observation battery at week 13) might be a systemic effect of the test substance but the toxicological relevance is unclear.

Altered clinical chemistry parameters in the high dose group are recognized but evaluation is limited without historical control data of the same laboratory.

Presumably higher LOAELs can be expected if the test substance is applied via drinking water or diet and not via gavage (bolus effect). 

Minor deficiencies include no data about the purity of the test substance (responsibility of the sponsor, minor restriction); blood urea nitrogen not determined in clinical chemistry and no data on significance at the level p<0.05 (minor deficiencies).

Additional information

Data on formaldehyde

Repeated oral exposure

The main effects in chronic drinking water studies with rats are local lesions at a concentration of 0.19% (Til et al. 1989) or 0.1% (Tobe et al. 1989). These concentration-dependent effects included papillary epithelial hyperplasia, hyperkeratosis and ulceration of the forestomach and the glandular stomach.The NOAEC is 0.026% (Til et al. 1989) or 0.02% (Tobe et al. 1989).Til 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 in chronic studies is 82 mg/kg bw/day in males and 109 mg/kg bw/day in females (Til et al. 1989; higher validity than Tobe et al., 1989). No carcinogenic effects were reported (Til et al. 1989, Tobe et al. 1989). Similar results concerning local effects were presented in a subacute study, the NOAEL was 25 mg/kg bw/day (no data given about the concentration; Til et al. 1988).

In the subchronic drinking water study in rats (Johannsen et al., 1986) no local and no systemic effects were detected at a dose levels up to 150 mg/kg bw/day. Concerning the missing local effects at the high concentration of 0.1% in water the results are in agreement with the chronic drinking water study of Tobe et al. (1989). Local effects in the stomach could be clearly demonstrated at a concentration of 0.5%. At a concentration of 0.1% only 1 out of 20 male and 1 out of 20 female Wistar rats revealed these local effects (Tobe et al., 1989). The concentration of formaldehyde in the subchronic drinking water of Johannsen et al. (1986) was probably too low to induce local effects in the gastro-intestinal tract of rats.

In a subchronic study no local and no systemic effects were reported in dogs exposed via the diet to dose levels up to 100 mg/kg bw/day.The NOAEL for systemic effects is 100 mg/kg bw/day.No data were given on the concentration, however, it can be assumed thatthe concentration of formaldehyde in the diet was not sufficient for induction of local effects in the gastro-intestinal tract.

In conclusion, the overall NOAEC for the oral route based on chronic studies with rats is 0.026% in water (LOAEC 0.1%). The NOAEL for systemic effects in rats is 82 mg/kg bw/day in males and 109 mg/kg bw/day in females.

Repeated dermal exposure

Data available on this endpoint are of limited validity due to restricted documentation. In mice only local skin effects and no systemic toxicity were induced after topical application of formaldehyde at a concentration of 10% and an amount of 200 µL. Slight hyperplasia of the epidermis was found and a few mice had small ulcers and scratches (Iversen 1986). In another study there is some evidence for local irritation at a concentration of >= 0.5%.

Repeated inhalation exposure

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. Carcinogenicity data are evaluated in a separate chapter.

The NOAEL is 2 ppm in rats (e.g. Monticello et al. 1996), and 1 ppm in monkeys (Rusch et al. 1983). Mice are less sensitive, the NOAEL in a subchronic study is 4 ppm (Maronpot et al. 1986); this might be related to the higher RD50value in mice compared to rats. Lowest sensitivity was reported for hamsters, no local or systemic effects were noted at 3 ppm (Rusch et al. 1983) and slight effects at 10 ppm.

Studies with rats have shown that the NOAEL and the LOAEL are independent of the exposure duration. In chronic studies as well as in subacute studies with only a few days (measuring cell proliferation) or weeks duration (measuring epithelial lesions) the NOAEL was the same or even lower than 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).

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, mice and monkeys: olfactory epithelium, larynx, trachea, and bronchus. Increase in toxicity and severity is not linear but shows a sharp increase at ca. 10 ppm.

Based on the studies described above the overall NOAEL based on chronic studies with rats, mice, hamsters and monkeys is 1 ppm corresponding to 1.2 mg/m³.

Effects in humans occupationally exposed via inhalation

There is some evidence from a number of survey 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 (IARC 1995, Greim 2000, BfR 2006). 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).

Justification for classification or non-classification