Registration Dossier

Toxicological information

Respiratory sensitisation

Currently viewing:

Administrative data

Endpoint:
respiratory sensitisation: in vivo
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Study well documented, meets generally accepted principles, acceptable for assessment: investigative study; up to date no guideline for respiratory sensitisation studies available
Cross-reference
Reason / purpose:
reference to other study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1990
Report Date:
1990

Materials and methods

Principles of method if other than guideline:
Specific, internationally harmonized test procedures for experiments to assess the lung sensitization potential of low- or high-molecular weight compounds are not yet existing. In this respiratory sensitisation study guinea pigs were exposed on 5 consecutive days for 3 hours daily to different concentrations of the test substance aerosol. After a treatment free period of at least 2 weeks a challenge experiment with exposure to the test substance aerosol (= hapten) was performed. After one further exposure-free week a re-challenge with a synthetic protein-conjugate of the test substance (= antigen) was conducted. Lung function measurements were performed to investigate immediate or delayed effects after challenge. Additionally blood was sampled before challenge and investigated for specific IgG1-antibodies related to the substance.
The study was carried out in accordance with OECD TG 403 for technical exposure criteria. The principle of the method was based on Karol et. al., Fundam. Appl. Toxicol. 5, 1985, 459-472; Karol et. al., Am. Ind. Hyg. Assoc. 39, 1978, 546-556; Mullin et. al., Toxicol. Appl. Parmacol. 71, 1983, 113-122; Deceaurriz et. al., Toxicology 43, 1987, 93-101; Barrow et. al., Archives of Environmental Health, 1977, 68-76; Botham et. al., Toxicology Letters 41, 1988, 159-173.
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent

Test animals

Species:
guinea pig
Strain:
Dunkin-Hartley
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Strain: Iva:PDH (Pirbright - White - Dunkin - Hartley)
- Source: Ivanovas, Kisslegg, Germany
- Age at study initiation: according to weight 1 - 2 months
- Weight at study initiation: 300 +/- 25 g
- Housing: in groups of 5 in Makrolon type IV-cages (based on A. Spiegel and R. Goennert, Zschr. Versuchstierkunde, 1, 38 (1961) and G. Meister, Zschr. Versuchstierkunde, 7, 144-153 (1965))
- Diet and water: ad libitum
- Acclimation period: at least 1 week

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 2
- Humidity (%): approx. 50
- Air changes (per hr): approx. 10
- Photoperiod (hrs dark / hrs light): 12/12

Test system

Route of induction exposure:
inhalation
Remarks:
head/nose
Route of challenge exposure:
inhalation
Remarks:
head/nose
Vehicle:
other: The test-substance was nebulised in 10 % acetone; for nebulisation of the test substance-protein-conjugate 0.9 % aqueous saline solution was used.
Concentration:
inhalative induction (5x3 h): 3, 16, 49, and 261 mg/m³ test substance (target conc. 2, 10, 50, and 200 mg/m³)
challenge (1x30 min): 97 mg/m³ test substance
re-challenge (1x30 min): 27 mg/m³ test substance-protein-conjugate
No. of animals per dose:
15 test animals/15 controls
Details on study design:
RANGE FINDING TESTS: An irritation threshold concentration was preliminarily determined; see report no. 18393, Bayer AG, 1989. As target concentrations 2 mg/m³ was chosen as the lowest dose, because it was determined as the serologic NOEL, 200 mg/m³ was chosen as the highest dose where slight toxic effects could be expected.

MAIN STUDY
Inhalative induction (Day 0-4): The animals were simultaneously exposed to various concentrations of the test substance aerosol or to the vehicle acetone (control) on 5 consecutive days for 3 h daily followed by an exposure-free period of at least 2 weeks.

Challenge (Day 21-25): Before the challenge experiment blood was sampled from the retroorbital plexus of 7 animals for serological investigations. For first challenge the animals were exposed for 30 min. to the test substance aerosol at 97 mg/m³. Lung function measurements (immediate type) were performed for technical reasons only on 2x4 animals/group. For 4 of these animals also delayed effects on lung function were investigated.

Re-challenge (Day 30-38): Before the re-challenge experiment blood was sampled from the retroorbital plexus of 7 animals for serological investigations. For re-challenge the animals were exposed for 30 min. to test substance-guinea pig albumin-conjugate at 27 mg/m³. The loading density of test substance-guinea pig albumin-conjugate was approx. 10 mol HDI isocyanurate/mol guinea pig albumin. Lung function measurements (immediate type) were performed for technical reasons only on 2x4 animals/group. For 4 of these animals also delayed effects on lung function were investigated.

Lung function measurements: At first, animals were adapted to the exposure chambers for at least 10 min. During the adaption phase animals were only exposed to conditioned air. Then the basal lung function parameters were recorded for 30-45 min. During challenge exposure to the aerosolised test substance whole-body plethysmography was performed up to 45 minutes after end of challenge exposure at maximum. The respiratory rate of 4 animals was measured over 20 hours for the detection of delayed effects. The following lung function parameters were measured: peak expiratory flow, tidal volume, respiratory rate, minute volume, inspiration time, expiration time.

Serological examinations: The titer of IgG1-antibodies against test substance-guinea pig albumin-conjugate was determined via ELISA.

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Mode of exposure: Animals were head-nose exposed to the aerosolised test article in restrainers made of plexiglas.
- Generation of atmospheres: Atmospheres were generated under dynamic conditions with a binary nozzle and conditioned compressed air (p = 700 kPa).
- Generation of aerosol: To achieve an aerosol 10 % acetone was added to the test substance. The test substance-protein conjugate was nebulised in isotonic saline solution. In order to prevent larger particles from entering the chamber a preseparator/ baffle system was used (Tillery et. al., Environmental Health Perspectives, 16, 1976, 25). The inhalation chamber had the following dimensions: inner diameter = 30 cm, height = 28 cm (internal volume about 20 L). The inhalation chambers are commercially available (Rhema Labortechnik, Hofheim, Germany) and its validation has been published in detail (Pauluhn, in U. Mohr (ed.), Inhalation Toxicology, The Design and Interpretation of Inhalation Studies and their Use in Risk Assessment, Springer Verlag, 1988, 87-102; Pauluhn, P. Grosdanoff, R. Bass, U. Hackenberg, D. Henschler, D. Mueller, H.-J. Klimisch (eds.), Problems of Inhalatory Toxicity Studies, BGA-Schriften, MMV Medizin Verlag Muenchen, Vol. 5, 1984, 59-68).
- Conditioning of compressed air: Compressed air was supplied by Boge compressors and was conditioned (i.e. freed from water, dust, and oil) automatically by a VIA compressed air dryer. Adequate control devices were employed to control supply pressure.
- Inhalation chamber steady-state concentration: The test atmosphere generation conditions provide an adequate number of air exchanges per hour (ca. 30 x, continuous generation of test atmospheres). Under such test conditions steady state is attained 6 min. at maximum. The ratio between the air supplied and exhausted was chosen so that approx. 80% of the supplied air is removed via the exhaust system.
- Exhaust air treatment: The exhaust air was purified via filter systems.
- Temperature and humidity measurements were performed during inhalative induction (temp. approx. 23 °C, humidity approx. 15-30 % - a slightly lower humidity was chosen to diminish hydrolysis of the test substance).

TEST ATMOSPHERE
- The integrity and stability of the aerosol generation and exposure system was measured by using a RAS-2 aerosol photometer (MIE, Bedford, Massachusetts, USA).
- Samples taken from breathing zone: yes
- Brief description of analytical method used: by gravimetric analysis of filter samples (filter: Glass-Fibre-Filter, Sartorius, Goettingen, Germany; digital balance) and HPLC analysis (HPLC-method: Nitro-reagent-treated glass fibres were exposed to the test atmosphere. The content of isocyanate was analytically detected via HPLC.). Gravimetrically determined concentrations were essentially similar to concentrations determined by nitro-reagent derivatisation technique.
- Particle size distribution: The particle-size distribution was analysed using a Aerodynamic Particle Sizer with laser-velocimeter (TSI-APS 3300). The Aerosol mass - MMAD (Mass median aerodynamic diameter): The respirability of the aerosol was adequate, i.e. the mass median aerodynamic diameter (MMAD) was for induction exposure 1.61 at 2 mg/m³, 1.41 at 10 mg/m³, 1.40 at 50 mg/m³, and 1.49 at 200 mg/m³; GSD (Geometric st. dev.) approximately 1.4.
For 1st challenge MMAD approx. 1.5 µm, GSD approx. 1.5; for 2nd conjugate-challenge MMAD 3.7 µm, GSD 2.1;
Positive control substance(s):
trimellitic anhydride (TMA)
Negative control substance(s):
other: vehicle (acetone)

Results and discussion

Results:
All dose groups tolerated the induction exposure up to 261 mg/m³ without observable toxicological effects. No effect on body weight or body weight gain was observed compared to controls.

Lung function tests: No evidence of a lung sensitizing potential was seen after 1st and 2nd challenge. No immediate hypersensitivity reactions could be detected. A moderate to heavy increase in respiratory rate observed at 1 animal of the highest challenge concentration group (261 mg/m³ ) and 4 animals of the control group is regarded as an effect induced by pulmonary irritation. Some animals showed delayed increase in respiratory rate after protein conjugate challenge, but non dose-correlated.

Antibody determination: No specific test substance-guinea pig albumin-conjugate antibodies could be detected in animals that were induced with 3 mg/m³. Dose group 16 mg/m³ showed marginal increase, dose groups 49 and 261 mg/m³ noticeable concentration-related increase of specific IgG1 anti-conjugate antibody count. 2/7 animals with inhalative induction at 261 mg/m³ showed antibody counts > 2000.

At necropsy no treatment related effects were reported for induction concentration up to 49 mg/m³. Animals of the 261 mg/m³ group showed macroscopically an increase in lung findings (bloated lungs with superficial redness) . Only slight effects on lung weights were detected in the 261 mg/m³ group. Histopathological investigations revealed in this group inflammatory changes of the lung. No evidence of recruitment of eosinophilic granulocytes in the airway walls.
Positive control results:
The sensitivity of the test system was shown in studies with trimellitic anhydride after inhalative and intradermal induction (Bayer AG, 1990), and also in a method validation study with ovalbumin (Bayer AG, 1989).

Any other information on results incl. tables

For respiratory sensitisation a read across to HDI oligomers, isocyanurate type (EC 931 -274 -8) is applied. This substance is a close structural analogue to HDI oligomers, iminooxadiazindione type, also derived from catalytic oligomerisation of 1,6 -hexamethylene diisocyanate (HDI; CAS 822 -06 -0) and also belonging to the CAS number 28182-81-2 (Hexane, 1,6 - diisocyanato-, homopolymer).The read across is based on physicochemical and toxicological similarity. In fact, comparison of the toxicological endpoints, that are available for both of the two substances (Acute oral toxicity, Acute inhalation toxicity, Skin and Eye Irritation/Corrosion, Skin Sensitisation, Bacterial mutagenicity (Ames)) reveal good correlation. With respect to Inhalation Toxicity an expert statement is available justifying the read across (Pauluhn, Comparison of pulmonary irritation potency..., Bayer HealthCare AG, 2008).

Therefore, test results obtained for HDI oligomers, isocyanurate type can be transferred to HDI oligomers, iminooxadiazindione type and the data on respiratory sensitisation of HDI oligomers, isocyanurate type are also valid for HDI oligomers, iminooxadiazindione type. This approach is in accordance with Annex XI, section 1.5 of the REACH Regulation (Regulation (EC) No 1907/2006).

The study for HDI oligomers, isocyanurate type revealed no evidence for a respiratory sensitisation potential after repeated inhalative exposure to the test substance. This is not contradictive to the dose-related increase of specific test substance related IgG1 antibody counts observed, since only divalent reagins can mediate an asthmatic reaction. Nevertheless, the increase of specific IgG1 antibody titer give evidence that the aerosol was of respirability for the guinea pigs.

Applicant's summary and conclusion

Executive summary:

A study on Respiratory Sensitization was conducted, principally based on the method published by Karol et.al. (for instance Fundam. Appl. Toxicol. 5, 1985, 459). In this study groups of guinea pigs were exposed on 5 consecutive days for 3 hours daily to different concentrations of the test substance aerosol (0 (vehicle control), 3, 16, 49, and 261 mg/m³; MMAD approx. 1.5 µm, GSD approx. 1.4). After a treatment free period of at least 2 weeks a challenge experiment with exposure to the test substance aerosol (= hapten; 97 mg/m³; MMAD approx. 1.5, GSD approx. 1.5) was performed. After one further exposure free week a re-challenge with a synthetic protein-conjugate of the test substance (= antigen; 27 mg/m³; MMAD 3.7 µm, GSD 2.1; approx. 10 mol HDI isocyanurate/mol guinea pig albumin) was conducted. Lung function measurements were performed to investigate immediate or delayed effects after challenge. Additionally blood was sampled before challenge and investigated for specific IgG1-antibodies related to the substance.

All dose groups tolerated the induction exposure up to 261 mg/m³ without observable toxicological effects. No effect on body weight or body weight gain was observed compared to controls.

Lung function tests revealed no evidence of a lung sensitizing potential. No immediate or delayed hypersensitivity reactions could be detected neither after challenge with the test substance aerosol nor after conjugate challenge.

Animals of the 261 mg/m³ group showed slight increase in lung weights. Histopathology revealed in this group inflammatory changes in the lung; these were seen as causally related to the primary irritation potential of the substance. No evidence of recruitment of eosinophilic granulocytes in the airway walls was observed.

A concentration-related increase of specific test substance related IgG1 antibody counts demonstrated that the test substance aerosol was of adequate respirability for the animals.

In conclusion the study result shows with respect to the conditions of the test, that the test substance aerosol has no lung sensitizing potential after repeated exposure to the respiratory tract.