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Toxicological information

Respiratory sensitisation

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Administrative data

respiratory sensitisation: in vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
not specified
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment

Data source

Reference Type:
Acute airway effects of airborne formaldehyde in sensitized and non-sensitized mice housed in a dry or humid environment
Larsen ST, Wolkoff P, Hammer M, Kofoed-Sørensen V, Clausen PA, Nielsen GD
Bibliographic source:
Toxicology and Applied Pharmacology 268 (2013) 294–299

Materials and methods

Test guideline
no guideline available
Principles of method if other than guideline:
Male Balb/C mice were exposed to the test substance and the role of air humidity and allergic sensitization on the acute airway response were investigated.
GLP compliance:
not specified

Test material

Constituent 1
Chemical structure
Reference substance name:
EC Number:
EC Name:
Cas Number:
Molecular formula:
Details on test material:
- Name of test material (as cited in study report): formaldehyde
Specific details on test material used for the study:
- Formaldehyde was supplied by Kin-Tek (TX, USA) as a certified Trace Source™ permeation tube with paraformaldehyde

Test animals

Details on test animals or test system and environmental conditions:
- Source: Taconic, Denmark
- Housing: Polypropylene cages (380 × 220 × 150 mm) with pinewood sawdust bedding
- Diet: Altromin no. 1324, Altromin, ad libitum
- Water: ad libitum

- Temperature: 20 - 24°C
- Humidity: 30 - 50 %
- Photoperiod: 12 hours / 12 hours

Test system

Route of induction exposure:
other: intraperitoneal and inhalation
Route of challenge exposure:
other: air
Target concentrations: 0.4, 1.8 and 7 ppm, Measured concentrations: similar under dry and humid environments both at the low (0.42 ± 0.01 ppm) and medium (1.8 ± 0.09 ppm) test substance levels, at the highest test substance level, the concentrations were 4.0 ppm and 5.7 ppm in the dry and humid environments, respectively
No. of animals per dose:
Details on study design:
- Mice (n = 30) were immunized to OVA by intraperitoneal (i.p.) injections of 1 μg OVA in combination with 270 μg Al(OH)3 in 100 μL 0.9 % saline on day 0
- Mice were boosted i.p. on days 14 and 21 with 0.1 μg OVA in 100 μL 0.9 % saline
- Finally, the animals were exposed 20 min to an aerosol of 0.2 % OVA on days 28 and 29 using a Pari Star nebulizer
- For the non-sensitized control mice (n = 30), saline (0.9 %) was used for the three i.p. injections and the aerosol exposures
- On days 29 and 30, the sensitized and non-sensitized mice were housed at either low (b10) or high (85 - 89) % relative humidity

- On day 31, the mice were exposed to the test substance
- Test substance was generated from a Kin-Tek (TX, USA) gas standard generator (Model 491MB) by use of a permeation tube and dry air, and led to a 24 L exposure chamber
- The airflow rates in the chamber were set at 18.8 - 23.2 L/min
- The chamber exposure concentrations of the test substance were monitored pre- and post-test substance exposures and every 10 min during exposures by 10 min air sampling of 4.4 L on dinitrophenylhydrazine (DNPH) sampling cartridges
- Samples were analyzed by HPLC using a diode array detector
- Mice were inserted into body plethysmographs that were connected to the exposure chamber
- The respiratory parameters were obtained for each mouse from a pneumotachograph connected to each head-out plethysmograph that allows continuously monitoring of the parameters
- The exposures were preceded by a period that allowed the mice to adapt to the plethysmographs
- A 15 min period was used to establish baseline (control) values of the respiratory parameters
This period was followed by a 60 min exposure period and a 30 min recovery period

- Bioassay: the respiratory effects were studied in a mouse bioassay, which allows the detection of respiratory effects on the upper airway (sensory) irritation as well as effects in the conducting airways and at the alveolar level
- Respiratory parameters: continuous computerized monitoring of the breathing pattern of unanesthetized mice was used to study the following effects
- Sensory irritation pattern: sensory irritants decrease the respiratory rate (f, breaths per min) in mice due to a reflex causing a break at the end of the inspiratory phase
- Airflow limitation: narrowing of the bronchi causes increased resistance that reduces the expiratory flow (VD, mL/s)
- Pulmonary irritation: may be quantified by “time of a pause” (TP, s), which is characterized by a short break at the end of expiration
- Assessment of airway inflammation: bronchoalveolar lavage (BAL) was performed to assess whether the exposures caused inflammation in the lungs
Challenge controls:
Positive control substance(s):
not specified
Negative control substance(s):
not specified

Results and discussion

Lung inflammation:
No effect was seen on test substance exposure and humidity on the degree of lung inflammation. Only the sensitization procedure increased the number of BAL cells.

Airway effects of FA in sensitized mice:
A) Humid environment:
- In the humid environment, little effect of allergic sensitization was seen in the upper respiratory tract, as suggested by the superimposed Time of Break (TB) responses. As typical for nose irritation, the maximum response was reached within the first 20 min of exposure period and thereafter gradually reduced during the remaining part of the period. After cessation of exposure (at time = 60 min), the TB response rapidly approached the baseline level. No apparent difference was revealed by comparison of the AUCs from the OVA and saline sensitized mice, respectively. Similar results were seen at the two lowest FA concentrations.
- Sensitization to OVA amplified the effect of FA in the conducting airways at the highest FA concentration but not at lower concentrations.
- The stronger response from the lungs at the higher FA concentrations was also to some extent reflected by a marginally elongated Time of Pause.
B) Dry environment:
- Saline control mice had a high, short-lasting peak in TB, suggesting a vigorous, transient nose irritation
- OVA-sensitization reduced the airway responsiveness to FA in the conducting airways compared to the saline control mice. Thus, both at the medium and high FA exposure concentrations, the saline sensitized control mice showed a more pronounced reduction in VD compared to the OVA-sensitized mice.

Effect of relative air humidity on airway response to FA
A) Saline control mice: No effect of humidity was apparent on nasal irritation in the non-sensitized mice even at high concentrations of FA
B) The humidity had an impact on the conducting airways at FA >1.8 ppm. Hence, mice housed in a dry environment had a more pronounced decrease in expiratory flow at the highest FA concentration compared to mice housed in the humid environment. The amplifying effect of a dry environment on lower airway effects was also apparent from the TP; it showed that the highest FA concentration induced a stronger pulmonary irritation in the dry environment compared to the humid environment.
Positive control results:
not specified
Negative control results:
not specified

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Formaldehyde induced airway irritation was both influenced by air humidity and prior allergic airway inflammation, but only at high concentrations of ≥ 1.8 ppm. There was no interaction at 0.4 ppm, a concentration relevant for occupational and indoor exposures.
Executive summary:

In a reliable study, the role of air humidity and allergic sensitization on the acute airway response to inhaled formaldehyde vapor was investigated in mice. Mice were sensitized to the immunogen ovalbumin (OVA) by three intraperitoneal injections followed by two aerosol challenges. Once sensitized, the mice were housed at high (85–89%) or low (<10%) relative humidity, respectively for 48 h prior to a 60-min exposure to either 0.4, 1.8 or about 5 ppm formaldehyde. Before, during and after exposure, breathing parameters were monitored.

Formaldehyde induced airway irritation was both influenced by air humidity and prior allergic airway inflammation, but only at high concentrations of ≥ 1.8 ppm. There was no interaction at 0.4 ppm, a concentration relevant for occupational and indoor exposures.