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Toxicological information

Respiratory sensitisation

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Administrative data

Endpoint:
respiratory sensitisation: in vivo
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP study, according to scientifically sound standard test protocol

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2007
Report Date:
2007

Materials and methods

Principles of method if other than guideline:
24 male rats per group were induced either topically of by inhalation to aerosols of the test substance.
2 weeks later, 8 rats/group were each challenged (inhalation) with the test substance and the monomer for 30 min and then subjected to a methacholine bronchoprovocation challenge. The respiratory sensitisation potential was evaluated based on immediate-onset lung function measurements, methacholine challenge, bronchoalveolar lavage and immunological determinations. Furthermore the rats were sacrificed and e.g. the lung weights determined.
GLP compliance:
yes
Remarks:
Bayer AG

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent

Test animals

Species:
rat
Strain:
other: Brown Norway (BN/Crl BR)
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River, Sulzfeld, Germany
- Weight at study initiation: ca. 235 g (mean)
- Housing: singly in conventional Makrolon type III cages with bedding consisted of type BK 8/15 low-dust wood granulate from Ssniff, Soest, Germany
- Diet: standard fixed-formula diet (Kliba 3883 = Nafag 9441 pellets maintenance diet for rats and mice, Provimi Kliba SA, 4303 Kaiseraugust, Switzerland); ad libitum
- Water: tap water; ad libitum
- Acclimation period: approx. 1 week

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22
- Humidity (%): 40 - 60
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12

Test system

Route of induction exposure:
other: topical and inhalation
Route of challenge exposure:
inhalation
Remarks:
nose only
Vehicle:
other: AOO (acetone:olive oil (4+1))
Concentration:
- Induction, epidermal:
100 µL of 25% solution (day 0, trunk) and 50 µL of 12.5% solution (day 7, dorsum of both ears), skin areas were cleaned 1 day after administration

- Induction, inhalative:
52 mg HDI-BT/m³, 5 x 3 h/day
and 27 mg HDI/m³ 1 x 3 h/day (additional group of rats)

- Challenge, inhalative: 56 mg/m³ test substance no. 1 aerosol; 49 mg/m³ test substance no. 2 aerosol or 2.8 mg/m³ HDI monomer; 1 x 30 min
No. of animals per dose:
24 (+ 3 replacement animals)
Details on study design:
MAIN STUDY
A1. INDUCTION EXPOSURE (epidermal)
- No. of exposures: 2 (day 0 and 7)
- Exposure period: day 0; day 7: cleaning with saline after 24 h
- Test groups: 24 animals: test substance 1; 24 animals: test substance 2
- Control group: no treatment
- Site: back/flank, ca. 10 cm²
- Concentrations: day 0: 25%, day 7: 12.5%
- Vehicle: acetone:olive oil 4+1 (AOO)
or
A2. INDUCTION EXPOSURE (inhalative)
- No. of exposures: 5
- Exposure period: 3 h
- Test groups: 24 animals (test substance 1); 24 animals (test substance 2); 24 animals (HDI-monomer)
- Control group: 5 x 3 h air, 1 x 3h monomer
- Site: nose only inhalation
- Frequency of applications: daily
- Duration: 5 days
- Concentrations: test substance 1 and 2: approx. 51 and 52 mg/m³ (with 0.14 and 1.8 mg/m³ residual monomer, respectively); monomer: 27 mg/m³

B. CHALLENGE EXPOSURE
- No. of exposures: 1
- Day(s) of challenge: 21 +/- 3
- Exposure period: 30 min
- Test groups: 8 animals per group with different induction procedures/test substances
- Control group: same treatment
- Site: nose only inhalation
- Concentrations: test substance 1 and 2: approx. 50 mg/m³ (with 0.15 and 1.2 - 2.2 mg/m³ residual monomer, respectively); monomer: 3.5 mg/m³
- methacholine challenge (one day after hapten challenge)

OTHER:
Mode of inhalative exposure: Animals were exposed to the aerosolized test substance in restrainers made of Plexiglas. The design of the directed-flow inhalation chamber prevents rebreathing of the test atmosphere. Under dynamic conditions, the test substance was fed into the intake of the cylinddrical inhalation chamber. Dry conditioned air was fed through the liquid of the test substance contained in a glass bubbler (diameter: 5 cm, height of liquid level: 5 cm, content: 100 mL) using a calibrated flow meter, and was subsequently diluted with conditioned dry air to achieve the target concentrations. The glass bubbler containing the test compound was maintained at 40 °C using a thermostat. For challenge exposures smaller glass bubblers (ca. 20 mL ) were used.
Atmospheres of the aerosols for inhalation exposures were generated under dynamic conditions using a digitally controlled pump with a syringe containng the test substance. The test substance was nebulized using conditioned compressed air (dispersion pressure approx. 600 kPa, 10 µL test solution/min). The nozzle were maintained at approx. 40 °C. The increase of temperature within the nozzle resulted in a marked decrease in viscosity and hence increased reproducibility throughput of aerosol.
Inhalation chamber (aluminium): dimension of each segment: inner diameter: 14 cm; outer diameter: 35 cm, height: 25 cm, internal volume: 3.8 L
Flow rates: 30 L/min
Air conditioning: automatically by subsequent passage through a VIA compressed air dryer
A steady state was established. During the exposure period air flows were monitored continuously and, if necessary, readjusted to the conditions required. Air flows were measured with calibrated flow-meters and/or soap bubble meter and were checked for correct performance at regular intervals.
Treatment of exhaust air: purified via cotton-wool/HEPA filters and activated char coal.

- Analysis of the HDI (monomer) test atmospheres:
The nominal concentration was calculated taking into account the actually evaporated mass of HDI (difference of weight of the glass bubbler before and after exposure divided by total airflow through the chamber.
To determine the analytical concentration, samples were taken from the breathing zone using in sampling tubes containing N-4-nitrobenzyl-N-n-Proyplamine as a trap for intact HDI. Alternatively, noncoated PTFE filters were used and processed with nitroreagent postsampling. The resultant urea derivative was subsequently extracted and anayzed by HPLC.

- Analysis of homopolymer test atmosphere:
The nominal concentration was calculated from the ration of the total quantity of test item consumed during the exposure period and the total throughput of air through the inhalation chamber. For calculations the airflows used for dilutions were taken into account.
- Aerodynamic particle-size distribution analyzed using a Berner-type aeras low-pressure critical orifice cascade impactor (Kauke, Gmunden, Austria). Mass Median Aerodynamic Diameter (MMAD) and Geometric Standard Deviation (GSD) were calculated.
Respirability of the particles was demonstrated (MMAD of aerosol: 4.9 µm, GSD: 1.6µm).

Results and discussion

Results:
Only results of the animals sensitized and challenged with HDI-BT (0.1% and 2%) are reported:
- during dermal sensitization most rats exhibited local effects at the sites of induction. The rats of the inhalation induction groups displayed signs of respiratory distress during the exposure period which subsided within few days during the post-exposure period.
- Mortality did not occur in any exposure group.
- Clinical signs:
Group 1 (control): all rats appeared normal at all time points.
Group 2 and 3 (Biuret 0.1 or 2% HDI, inhalation induction): labored breathing patterns, irregular breathing patterns, stridor nasal discharge (serous).
Group 4 (Biuret 0.1% HDI, topical induction): application site reddened, encrustations, undefined skin responses, solidification, red encrustations/hemorrhagic, pinna: reddening.
Group 5 (Biuret 2% HDI, topical induction): application site reddened, red encrustations, undefined skin responses, solidification, hemorrhagic, red encrustations/hemorrhagic, pinna: reddening, urine: red discolorations.
(Group 6 (HDI induction): labored breathing patterns, dyspnoea, breathing sounds, bradypnea, stridor, nasal discharge (serous), high-legged gait, limp, motility reduced, nostrils: red encrustations, nose: reddened.)
- Body weight changes were within the normal range.
- Elucidation of respiratory hypersensitivity - HDI-BT-Challenge: none of the rats sensitized topically or by repeated inhalation exposures to the test substances displayed any consistent changes in breathing pattern following HDI-BT-challenge (0.1% or 2% residual HDI) that resembles an immediate-onset respiratory response.
- Elucidation of respiratory hypersensitivity - MCh-challenge: the baseline levels of Penh were not affected when compared to the control group. However, at higher MCh concentrations the incremental increase of Penh showed a more pronounced increase in rats induced by inhalation to TS2 (high monomer content).
- Bronchioalveolar lavage: the recovery of bronchoalveolar lavage fluid (BALF) was approx. 80 - 90% of the instilled volume and similar in all groups. No statistical significant changes were observed.
- Lung and lymph node weight:
sacrifice of rats one day after the MCh challenge revealed slightly increased lung weights in the group sensitized by inhalation to TS2, irrespective of the nature of the challenge (no statistical significance).
Induction or challenge related effects on lung-associated lymph nodes were not observed. The weight of auricular lymph nodes were slightly increased in topically sensitized animals and in the high level inhalation (TS2) group compared to sham control, but the changes were statistically not significant.
- IgE determination did not reveal any differences amongst the groups.
- Necropsy: gross pathological examinations showed roughly the same incidence of macroscopically apparent lung changes in all animals of this study. The most prominent findings were related to enlarged lymph-nodes. A dose-dependent increased incidence of enlarged Payers' patches was observed in intestine in rats sensitized topically.
- Histopathology: in rats of the groups 2 - 5 a slightly increased incidence of BALT activation occurred when challenged with either TS1 or HDI monomer. In lung-associated lymph nodes no differences between the groups could be detected. Regarding all other pulmonary findings no treatment related finding could be detected. The different protocols applied did not result in any treatment dependent differences of eosinophilic infiltration.

Any other information on results incl. tables

Table 1: Inhalation induction of animals/analysis of inhalation atmospheres

Group

1

2

3

6

Test substance

Control

No. 1

No. 2

HDI monomer

Target concentration (mg/m³)

0

50

50

30

Nominal concentration (mg/m³)

Test article

-

244.4

244.4

28.9

HDI monomer

-

0.49

5.38

28.9

Analytical concentration (mg HDI/m³)

Nitro-reagent glass tubes

-

0.143

1.809

26.77

PTFE filter and NR glass tubes (total)

-

0.133

1.546

-

Gravimetric concentration (mg/m³)

-

51.2

52.5

-

Total chamber air flow (L/min)

30

30

30

30

Temperature (°C)

26.2

22.8

23.5

26.7

Rel. Humidity (%)

5.2

8.8

5.4

5.9

MMAD (µm)

-

1.97

1.85

-

GSD

-

1.56

1.58

-

Aerosol Mass < 3 µm (%)

-

82.5

85.37

-

MMAD: Mass Median Aerodynamic Diameter, GSD: Geometric Standard Deviation, -: not applicable or not performed, PTFE: Polytetrafluorethylen

 

Table 2a: Inhalation challenge to test substance 1 / analysis of inhalation atmospheres

Group

1

2

3

4

5

6

Mean

Induction treatment

Control

TS1, inhal.

TS2, inhal.

TS1, topical

TS2, topical

HDI monomer, inhal.

 

Target concentration (mg/m³)

50

50

50

50

50

50

-

Nominal concentration (mg/m³)

244.4

244.4

244.4

244.4

244.4

244.4

-

Analytical concentration (mg HDI/m³)

 

 

 

 

Nitro-reagent glass tubes

0.148

0.118

0.132

0.128

0.171

0.141

0.14

PTFE filter and NR glass tubes (total)

0.120

0.163

0.161

0.194

0.144

0.128

0.15

Gravimetric concentration (mg/m³)

54.7

41.9

31.7

41.1

45.9

56.3

56.3

Total chamber air flow (L/min)

30

30

30

30

30

30

-

Temperature (°C)

22.6

23.2

23.0

23.7

25.9

22.7

-

Rel. Humidity (%)

< 5

< 5

< 5

< 5

< 5

< 5.5

-

TS1: test substance No. 1 with low monomer content; TS2: test substance No. 2 with high monomer content

 

Table 2b: Inhalation challenge to test substance 2 7 analysis of inhalation atmospheres

Group

1

2

3

4

5

6

Mean

Induction treatment

Control

TS1, inhal.

TS2, inhal.

TS1, topical

TS2, topical

HDI monomer, inhal.

 

Target concentration (mg/m³)

50

50

50

50

50

50

-

Nominal concentration (mg/m³)

244.4

244.4

244.4

244.4

244.4

244.4

-

Analytical concentration (mg HDI/m³)

 

 

 

 

Nitro-reagent glass tubes

1.505

1.755

1.303

1.542

1.792

1.607

1.58

PTFE filter and NR glass tubes (total)

1.202

1.401

2.279

1.260

1.502

1.319

1.49

Gravimetric concentration (mg/m³)

58.3

36.9

50.3

35.1

51.9

61.0

48.92

Total chamber air flow (L/min)

30

30

30

30

30

30

-

Temperature (°C)

22.8

23.8

23.0

23.1

23.0

21.6

-

Rel. Humidity (%)

< 5

< 5

< 5

< 5

< 5

< 6.2

-

TS1: test substance No. 1 with low monomer content; TS2: test substance No. 2 with high monomer content

 

Table 2c: Inhalation challenge to HDI monomer-vapour

Group

1

2

3

4

5

6

Mean

Induction treatment

Control

TS1, inhal.

TS2, inhal.

TS1, topical

TS2, topical

HDI monomer, inhal.

 

Target concentration (mg/m³)

3.5

3.5

3.5

3.5

3.5

3.5

-

Nominal concentration (mg/m³)

6.5

4.8

3.9

4.8

2.8

6.5

4.88

Analytical concentration (mg HDI/m³)

 

 

 

 

Nitro-reagent glass tubes

2.503

3.13

0.478

4.53

3.197

2.818

2.78

Total chamber air flow (L/min)

30

30

30

30

30

30

-

Temperature (°C)

21.2

22.7

22.7

23.0

21.7

20.5

-

Rel. Humidity (%)

8.9

< 5.3

< 5

< 5

< 5

< 5

-

TS1: test substance No. 1 with low monomer content; TS2: test substance No. 2 with high monomer content

 

Table 3: Toxicological results - induction: summary of morbidity and mortality

Group

Toxicological results

Duration of respiratory signs

Duration of skin inflammation

1

Control

0/0/27

-

-

2

TS1, inhal.

0/25/27

Day 1 – 10

-

3

TS2, inhal.

0/21/27

Day 1 - 7

-

4

TS1, topical

F: 0/20/27

E: 0/27/27

-

Day 1 – 24

Day 8 – 9

5

TS2, topical

F: 0/20/27

E: 0/03/27

-

Day 1 – 23

Day 9 – 11

6

HDI, inhal.

0/27/27

Day 0 - 14

-

TS1: test substance No. 1 with low monomer content; TS2: test substance No. 2 with high monomer content; F: flanks/trunk, E: ears

x/y/z: no. dead animals/no.Animals with signs after exposure/no. animals exposed

 

Table 4: Respiratory response upon inhalation challenge (intensity)

Parameter

Challenge

Induction – group No.

1

2

3

4

5

6

PEF

TS1

3

2

0

2

0

3

TS2

3

1

1

0

0

5

HDI

1

1

0

0

4

0

MV

TS1

2

1

1

4

2

3

TS2

2

4

1

2

4

4

HDI

3

1

3

0

5

1

RR

TS1

2

2

0

1

3

2

TS2

2

2

0

0

2

2

HDI

1

1

1

0

5

0

TS1: test substance No. 1 with low monomer content; TS2: test substance No. 2 with high monomer content; HDI: HDI-monomer; PEF: peak expiratory flow during tidal breathing; MV: respiratory miute volume; RR: respiratory rate.

Indexes represent the intensity of response as area under the curve.

Applicant's summary and conclusion

Interpretation of results:
not sensitising