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Respiratory sensitisation

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respiratory sensitisation: in vivo
Type of information:
migrated information: read-across based on grouping of substances (category approach)
Adequacy of study:
key study
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Method validation details were published elsewhere: Pauluhn, J. (2006). Review. Animal Models of Respiratory Allergy. Target Organ Series – Toxicology of the Lung, Editors: D.E. Gardner, J.D. Crapo, and E.J. Massaro. Raven Press, New York (pp.419-460). Pauluhn, J. (2008). Brown Norway rat asthma model of diphenyl­methane-4,4'-diiso­cya­­nate (MDI): the elicitation dose-response relationship. Toxicological Sciences 104: 320-331. Pauluhn, J., Woolhiser, M.R., and Bloemen, L. (2005). Repeated inhalation challenge with diphenyl­methane-4,4'-diiso­cya­­nate in Brown Norway rats leads to a time-related increase of neutrophils bronchoalveolar lavage after topical induction. Inhalation Toxicology 17: 67-78.

Data source

Reference Type:

Materials and methods

Test guideline
equivalent or similar to
other: OECD-GD 39 (inhalation exposure methodology)
GLP compliance:

Test material

Details on test material:
- Name of test material (as cited in the publication): Polymeric methylenediphenyl-4,4'-diisocyanate. The test substance was supplied by Bayer AG. - Composition of test material, percentage of components: The content of monomeric MDI was 38.1% with higher oligomeric MDI as balance.The free isocyanate (NCO) content was 31.1%. - Stability under test conditions and storage condition: During handling and storage the headspace of MDI containers was purged with dry nitrogen to remove air and humidity to prevent its decomposition.

Test animals

Brown Norway
Details on test animals and environmental conditions:
TEST ANIMALS - Strain: BN/Crl BR - Source: Charles River, Sulzfeld, Germany. - Weight at study initiation: ~230g - Age: 2-3 months - Housing: individually, in polycarbonate cages containing bedding material (low-dust wood shavings) - Temperature/Humidity:approximately 22ºC with relative humidity at 40–60% - Light Dark Cycle:12-h light cycle beginning at 06:00h - Diet: ad libitum except during inhalation exposure periods (a standard fixed-formula diet (NAFAG No. 9439W10 pellets) - Water: ad libitum except during inhalation exposure periods (municipality tap water) - Acclimation period: at least 5 days

Test system

Route of induction exposure:
Route of challenge exposure:
unchanged (no vehicle)
Induction doses: Daily exposures of 97.1, 475, or 936 mg/m3 (×10 min x 5 days) or 2.9, 14.9, or 29.9 mg/m3 (×360 min x 5 days).
Challenge dose: 40mg/m3 x 30 min x 4 (at 2 week intervals)

Induction dose: 936 mg/m3 ×10 min x 5 (daily)
Challenge dose: 40mg/m3 x 30 min x 3 (at 2 week intervals); 4th challenge at 5, 15, or 40 mg/m3 ×30 min

Each inhalation exposure was accompanied by the determination of actual concentrations as promulgated by OECD GD-39.
No. of animals per dose:
8 male rats per group
Details on study design:
Phase I:
Eight Groups of 8 male rats: 2 naïve control groups (1 challenged, 1 unchallenged) and six groups sensitized by inhalation exposure.
The inhalation induction to MDI-aerosol was by daily directed-low nose-only inhalation using either a 5×10 min/day (breathing zone concentrations: 97.1, 475, and 936 mg/m3) or a 5×360 min/day (breathing zone concentrations: 2.9,14.9, and 29.9 mg/m3).
This was followed by four inhalation challenge exposures on target days 20, 35, 50, and 65 (tolerance 61–66 days due to multiple examinations at the last challenge) to 40mg/m3 for 30 min.

Phase II:
Six Groups of 8 male rats: 3 naïve control groups and 3 groups sensitized by inhalation exposure of 936mg/m3 x10 minutes daily for 5 days.
This was followed by three booster challenge exposures on target days 20, 35, and 50 to 40mg/m3 for 30 min. Two weeks later, the fourth challenge was a stepped dose-escalation to groups of naïve control and sensitized rats to 5, 15, or 40mg/m3 for 30 minutes (one control group and one sensitized group/challenge step).

Shortly after the last challenge, all rats (unrestrained and spontaneously breathing) were monitored for changes in lung function delayed in onset for ~20 h in pre-calibrated barometric whole-body plethysmographs (volume: 4.2L, airflow rate:2L/min). Equipment used for data collection: ACQ7700XE, software: Ponemah version 4.80 (DSI Ponemah, Valley View Ohio, USA). Based on previous experience, data analysis focused on "enhanced pause" (Penh).

One day after the fourth challenge, all rats were anaesthetized using sodium pentobarbital (Narcoren®; 120mg/kg body weight, intraperitoneal injection). Complete exsanguination was achieved by severing the aorta abdominalis. The excised lungs and lung-associated lymph nodes (LALN) of the lung hilus region were weighed followed by bronchoalveolar lavage (BAL) of the excised lungs.

EXPOSURE ATMOSPHERE: Atmospheres of MDI for inhalation exposures were generated under dynamic conditions using a digitally controlled Harvard PHD 2000 pump and a modified Schlick-nozzle Type 970, form-S 3 (Schlick GmbH, Coburg, Germany). MDI was atomized using conditioned (dry, oil-free) compressed air (dispersion pressure approximately 600kPa, 15l/min and inhalation chamber segment). The nozzle was maintained at approximately 40°C using a water jacket connected to a digitally controlled JULABO thermostat. The increase of temperature within the nozzle resulted in a marked decrease in viscosity and increased the respirability of aerosol. Targeted concentrations were achieved by pull/push dilution cascades. The equilibrium concentration (t95) was attained in less than 1 min with air flow rates of 0.75L/min at each exposure port. The test atmospheres were characterized by gravimetric analyses (filter: Glass-Fibre-Filter, Sartorius. Gottingen. Germany). The temporal stability of the aerosol generation and exposure system was measured by using real-time aerosol photometers (RAS-2 MIE, Bedford. MA. USA). The mass median aerodynamic diameter (MMAD) and geometric standard deviation (GSD) was determined using an 8-stage critical orifice cascade impactor. Throughout all exposures the aerosols size was in a similar range (MMAD 1.5-1.9µm, GSD 1.6-1.9). For both filter and particle size analyses air samples were collected from the breathing area.

Lung and lung-associated lymph nodes (LALN) were weighed; BAL fluid was analysed for total protein, lactate dehydrogenase (LDH), cell counts, and polymorphonuclear lymphocytes (PMN). Absolute counts of PMNs were calculated based on the total number of cells in BAL and the respective percentage obtained by cytodifferentiation.

Organ weights and BAL data were analyzed by one-way analysis of variance (ANOVA) followed by a multiple comparison Tukey–Kramer post hoc test. Statistical differences of Penh-AUC and PMN differentials between the challenged control and MDI groups were evaluated by a Mann–Whitney Rank Sum Test (SigmaPlot 11, Systat Software, Point Richmond,CA). For all tests the criterion for statistical significance was set at P < 0.05.

Results and discussion

At the dose-levels selected for inhalation induction the no-observed-adverse effect level (NO(A)EL) or NEL (ECHA, 2008) was 5 mg MDI/m3 at 30 min exposure duration.

The respective threshold of PMNs was lower and than of Penh-AUC8h. Therefore, the BAL-PMN endpoint was taken as lowest dose descriptor, the most sensitive endpoint to probe for induction-dependent differences upon challenge.

The endpoints examined in the non-challenged (C-) and challenged (C+) naive control groups were minimally elevated in the challenged C+ group relative to the C- group. This finding provides supporting evidence that the Cxt used for challenge exposures is minimally irritating and its extent does not interfere with the interpretation of study.

This concentration-response relationship was additionally analyzed using the US-EPA benchmark (HMD) approach (US EPA, 2010). Based on the curve-fitting, the BMD lower bound was 8.6 mg/m3 (confidence level: 0.95). This demonstrates that the applied analysis is implicitly conservative.

Applicant's summary and conclusion

Interpretation of results:
This BN rat MDI respiratory allergy study demonstrates the existence of a threshold for the elicitation of respiratory sensitization following inhalation sensitization encounters. The derived elicitation threshold C×t appears to be plausible relative to human evidence. The close association of C×t products triggering an elicitation response in asthmatic rats with the acute pulmonary irritation threshold C×t is intriguing and supports the view that for this class of chemicals portal of entry related allergic responses appear to be linked with pulmonary and/ or lower airway irritation. Accordingly, high concentrations delivered to the respiratory tract during short exposure periods appear to bear a higher sensitizing potency than equal C×t products during longer exposure periods.
Executive summary:

This study describes a 5-day inhalation exposure (days 0–4) of BN rats to two concentration x exposure time (C×t) relationships of 1000, 5000, and 10,000 mg MDI/m3 ×min at exposure durations of either 10 or 360 min. This was followed by four 30 min inhalation challenges to 40 mg MDI/m3 on target days 20, 25, 50, and 65. After the last challenge, changes in breathing patterns delayed in onset were recorded and allergic lung inflammation was probed by bronchoalveolar lavage (BAL). In a subsequent study groups of rats were sensitized using the 10 min C×t protocol and challenged 3 times at 40 mg MDI/m3. At the fourth challenge a dose escalation regimen was used to determine the elicitation threshold on ‘asthmatic’ rats. The most sensitive endpoints characterizing an allergic pulmonary inflammation were BAL-neutrophils and physiological measurements showing respiratory changes delayed in onset. The dose escalation challenge yielded an elicitation threshold (NOAEL) of 5 mg MDI-aerosol/m3 at 30 min challenge duration.

In summary, the results suggest the C×t product of MDI-aerosol that triggers an elicitation response in ‘asthmatic’ rats is slightly below of that causing acute pulmonary irritation in naïve rats. The high concentration delivered to the respiratory tract during the10 min exposure period elicited a more vigorous response than the similar C×t at 360 min. Therefore, short high level exposure patterns appear to bear a higher sensitizing potency than equal C×t products at longer exposure periods. Taking into account the respective differences in exposure intensities, the comparison of elicitation thresholds of BN rats sensitized by inhalation exposure did not demonstrate essential differences.