Registration Dossier

Administrative data

Endpoint:
acute toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report date:
2010

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 403 (Acute Inhalation Toxicity)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.2 (Acute Toxicity (Inhalation))
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.1300 (Acute inhalation toxicity)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Test type:
standard acute method

Test material

Constituent 1
Reference substance name:
Reaction product of 2,4/ 2,6 TDI (m-tolylidene diisocyanate, CAS 26471-62-5) in excess and m-TDA (diaminotoluene, CAS 25376-45-8) forming Biuret structures via non-isolated polyurea intermediates
IUPAC Name:
Reaction product of 2,4/ 2,6 TDI (m-tolylidene diisocyanate, CAS 26471-62-5) in excess and m-TDA (diaminotoluene, CAS 25376-45-8) forming Biuret structures via non-isolated polyurea intermediates
Details on test material:
- Name of test material (as cited in study report): Desmodur VP.PU 60WF14
- Physical state: liquid
- Purity: 100 % (as certified in Report No. 2009/0032/02 of Currenta GmbH)
- Lot/batch No.: P1DE000345

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Harlan GmbH, Horst, Netherlands
- Strain: HsdCpb:Wu (SPF)
- Age at study initiation: approx. 2-3 months
- Weight at study initiation: at the study start the variation of individual weights did not exceed ± 10 per cent of the mean for each sex
- Housing: singly in conventional Makrolon® Type IIIH cages
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: at least 5 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 3
- Humidity (%): 40-60
- Air changes (per hr): approx. 10
- Photoperiod (hrs dark / hrs light): 12 / 12

Administration / exposure

Route of administration:
inhalation: aerosol
Type of inhalation exposure:
nose only
Vehicle:
other: dry air
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Mode of exposure: Animals were exposed to the aerosolized test substance in Plexiglas exposure tubes applying a directed-flow nose-only exposure principle.
- Vehicle: The neat test article was aerosolized undiluted.
- Aerosol generation: Under dynamic conditions the test substance is atomized neat into a baffle (pre-separator) from which the substance was conveyed into the intake of the cylindrical inhalation chamber. The test substance is nebulized using a binary nozzle with conditioned compressed air (15 l/min); dispersion pressure approximately 600 kPa. With an injection of 6 to 16 µl test substance/min the resultant gravimetric concentrations of the atmospheres were between 55.3 and 211.9 mg/m3, respectively. Based on these results the liquid test substance is fed into the nozzle by a digitally controlled Harvard PHD 2000 infusion pump. Both higher concentrated test atmospheres were generated by direct entrainment into the inhalation chamber. The lower atmospheres (target 50 and 68 mg/m3) were revealed by air dilution of +7.0/-7.0 L/min or +3.0/-3.0 L/min compressed air, respectively.
- Inhalation chamber: The aluminium inhalation chamber has the following dimensions: inner diameter = 14 cm, outer diameter = 35 cm (two-chamber system), height = 25 cm (internal volume = about 3.8 L). Details of this modular chamber and its validation have been published previously (Pauluhn, Journal of Applied Toxicology 14: 55-62, 1994).
- Optimization of respirability: In order to increase the efficiency of the generation of respirable particles and to prevent larger particles from entering the chamber a preseparator/ baffle system was used.
- Conditioning the 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 (> 200 x, continuous generation of test atmosphere). Under such test conditions steady state is attained within the first minute of exposure (t99% = 4.6 x chamber volume/flow rate). The ratio between the air supplied and exhausted was chosen so that approximately 80-90 % of the supplied air is removed via the exhaust system. The remainder provides adequate dead-space ventilation for the exposure tubes. At each exposure port a minimal air flow rate of 0.75 I/min was provided. The test atmosphere can by no means be diluted by bias-air-flows.
- Exhaust air treatment: The exhaust air was purified via cotton-wool/HEPA filters. These filters were disposed of by Bayer Schering Pharma AG.
- Temperature and humidity measurements: Temperature and humidity control was performed using a computerized system (Hydra, Fluke-Philips). The test atmosphere temperature and humidity were measured at the exposure location using a FTF-sensor (Elka-Elektronik, Lüdenscheid). Data were recorded automatically at intervals of 5 min. The measured values were evaluated using spreadsheet software.

ANALYSIS OF TEST ATMOSPHERE
- Nominal concentration: The nominal concentration was calculated from the ratio of the total quantity of test item discharged during the exposure period and the total throughput of air through the inhalation chamber, taking into account the respective dilution ratio. The lower actual (gravimetric) concentrations compared with the nominal concentrations are attributed to effective removal of larger particles in the baffle/pre-separator system.
- Gravimetric concentration: The test-substance concentration was determined by gravimetric analysis (filter: Glass-Fiber-Filter, Sartorius, Göttingen, Germany; digital balance). The mass collected by the filter was converted to the test substance taking into account the concentration of constituents contained in it that are prone to evaporate subsequent to nebulization.
- Analytical concentration: HPLC nitro-reagent transition technique; this method based on the HPLC determination of the reaction products of the analyte 2,4-toluene-diisocyanate, 2,6-toluene-diisocyanate and TDI biuret with the nitro-reagent, respectively utilizing their specific reference substances. The sum of these individually determined concentrations refer to DESMODUR VP.PU 60WF14 mg/m³ air.
- Samples taken from breathing zone: yes
- Particle size distribution: The particle-size distribution was analyzed using a Berner cascade impactor (Hauke, Gmunden, Austria).

RESULTS OF PARTICLE-SIZE ANALYSES
- Particle size distribution: In the 50, 68, 100 and 250 mg/m3 exposure groups (target conc.) 80.4, 86.6, 87.2 and 85.8 % ,resp. of particles were < 3 µm.
- MMAD (Mass median aerodynamic diameter) / GSD (Geometric st. dev.): In the 50, 68, 100 and 250 mg/m3 exposure groups (target conc.) MMAD was 1.62, 1.35, 1.44 and 1.81 µm, resp. (GSD: 2.07, 2.06, 1.92 and 1.61, resp.).
Analytical verification of test atmosphere concentrations:
yes
Duration of exposure:
4 h
Concentrations:
analytical concentration (sum TDI-isomers): 75.7, 105.4, 114.9, 177.6 mg/m3
analytical concentration (total mass): 111.5, 154.1, 163.7, 288.7 mg/m3
target concentration: 50, 68, 100, 250 mg/m3
gravimetric concentration: 55.3, 80.8, 94.5, 211.9 mg/m3
No. of animals per sex per dose:
5 animals per sex and dose; 6 additional animals were applied for histopathology of male group 114.9 mg/m3
Control animals:
yes
Details on study design:
- Duration of observation period following administration: at least 3 weeks
- Frequency of observations and weighing: clinical signs were examined several times on the day of exposure and at least once daily therafter; body weights were measured before exposure, on days 1, 3 and 7, and weekly thereafter.
- Necropsy of survivors performed: yes
- Other examinations performed: rectaI temperatures were measured shortly after cessation of exposure (approx. within half an hour after the end of exposure); 6 additional animals from the male group 114.9 mg/m3 were applied for histopathology of the tissues/organs of the nasal passages, pharynx, larynx, trachea and lung (3 animals each on post-exposure days 1 and 12).
Statistics:
-Necropsy flndings: If specific findings occur from the respiratory tract of surviving rats they are evaluated statistically using the pairwise Fisher test after the R x C chi-squared test.
-Body weights: Means and single standard deviations of body weights are calculated. Since in acute studies individual group means may differ prior to commencement of the first exposure, the body weight gain was statistically evaluated for each group. For these evaluations a one-way ANOVA (vide infra) is used.
-Calculation of the LC50: If calculation of a median lethal concentration (LC50) is possible, it is performed by computer (PC) according to the method of AP. Rosiello, I.M. Essigmann, and G.N. Wogan (1977) as modified by Pauluhn (1983). This method is based on the maximurn-likelihood method of C.I. Bliss (1938). If only 2 pairs of values with greater than 0% lethality and less than 100% are available then the first linear approximation is based on these values and a homogeneity test is not performed. The interpolated concentration at 50% lethality in this case was designated at approximate LC50.
-Analysis of variance (ANOVA): This parametric method checks for normal distribution of data by comparing the median and mean The groups are compared at a confidence level of (1-alpha)= 95% (p=0.05) The test for the between-group homogeneity of the variance employed Box's test if more than 2 study groups were compared with each other. If the above F-test shows that the intra-group variability is greater than the inter-group variability, this is shown as "no statistical difference between the groups". If a difference is found then a pairwise post-hoc comparison is conducted (1- and 2-sided) using the Games and Howell modification of the Tukey-Kramer significance test.

Results and discussion

Effect levelsopen allclose all
Sex:
male/female
Dose descriptor:
other: approximate LC50
Effect level:
160 mg/m³ air (analytical)
Based on:
test mat.
Exp. duration:
4 h
Remarks on result:
other: NOAEL: <111.5 mg/m3
Sex:
male/female
Dose descriptor:
other: approximate LC50
Effect level:
112 mg/m³ air (analytical)
Based on:
other: sum of TDI isomers
Exp. duration:
4 h
Remarks on result:
other: NOAEL: <75.7 mg/m3
Mortality:
Mortality occurred in a concentration-dependent manner at 105.4 mg/m³ and above. A particular sex-difference in susceptibility was not apparent. Mortality appeared to be caused by acute lung edema and inflamatoric changes in the respiration tract, it occurred largely delayed in the second observation week.
Clinical signs:
other: Exposures to concentrations of 75.7 mg/m3 and above were followed by concentration-dependent signs indicative of respiratory tract irritation, such as bradypnea, labored breathing patterns, breathing irregular, breathing accelerated, dyspnea, breathing so
Body weight:
Body weight were significantly decreased at 75.7 mg/m3 and above.
Gross pathology:
Animals sacrificed at the end of the observation period: The macroscopic findings reached from indistinguishable from the control group up to severe findings such as less collapsed lungs and a trachea with whitish foamy content.

Animals succumbing during the observation period: Nose: red encrusted, yellowish discharge; pleural cavity: yellow clear liquid content; lungs: less collapsed, dark-red discoloration, hepatoid areas, mucous viscous content; larynx: mucous viscous content; trachea: white foamy content; stomach: bloated; intestines: bloated, mucosa reddened; liver: light discoloration; spleen: reduced in size, light discoloration; kidneys: bilaterally light discoloration.
Other findings:
-Histopathology of 6 additional male rats of the exposure group 114.9 mg/m3:
At the end of the exposure period in all evaluated rats and in all organs/tissues of the respiratory tract, acute inflammatory findings and severe necroses were seen. Furthermore, light brown material (probably test substance) and mucus and/or cells were detectable and in the lungs focal hemorrhages and interstitial edema. At the end of recovery period, almost all findings in the nasal cavities had recovered. In some locations, minimal or slight atrophy/degeneration of the olfactory epithelium as well as increased goblet cells was seen. Additionally, turbinate remodelling occurred in one rat. In the larynx, additionally to minimal inflammatory infiltrates minimal to slight epithelial metaplasia was present. In the lungs, thickening of the airway epithelium was obvious together with granulation tissue (minimal or slight), slight inflammation and alveolar edema. Summarizing the histopathological findings of this acute study with Desmodur VP.PU 60WF14 in rats, severe acute findings of the respiratory tract occurred at the end of the exposure period. These findings were generally recovered after the 12 day exposure-free period. A final assessment of the relevance of the turbinate remodelling can not be given

- Rectal temperature were significantly decreased at 75.7 mg/m3 and above.

- Reflex measurements on the first post-exposure day revealed reduced tonus and grip strength as well as an impairment of the righting response.

Any other information on results incl. tables

Table 1: Summary of acute inhalation toxicity (4 hrs, aerosol) of Desmodur VP.PU 60WF14

 Sex Analytical concentration(sum of TDI-isomers) (mg/m3) Toxicological results  Onset and duration of signs   Rectal temperature (°C)

 Onset and duration of mortality

 male 0 (Control) 0 / 0 / 5 ---

38.4

 ---
  75.7 0 / 5 / 5 0d - 21d 32.0 **  ---
  105.4 1 / 5 / 5 0d - 21d 32.1 **   14d 
  114.9 4 / 11 / 11 * 0d - 14d 30.7 **   1d, 10d, 11d, 12d

177.6 5 / 5 / 5  0d - 9d 30.2 **    1d, 2d, 9d 

female 

 0 (Control) 0 / 0 / 5  --- 

38.4

   --- 
  75.7 0 / 5 / 5 0d - 12d 34.2 **   --- 
  105.4 2 / 5 / 5 0d - 21d 32.8 **   9d, 10d
  114.9 2 / 5 / 5 0d - 21d 32.3 **   9d, 12d
 

177.6

5 / 5 / 5  0d - 14d 31.1 **  2d, 9d, 12d, 14d 

Toxicological results:

number of dead animals / number of animals with signs after cessation of exposure / number of animals exposed

* In this group 6 additional animals were applied for histopathology; 3 animals were sacrificed at each point of time (1d and 12d)

** p < 0.01

Applicant's summary and conclusion

Executive summary:
An acute inhalation toxicity study according to OECD TG 403 was conducted on male and female rats, which were nose-only exposed to liquid aerosol concentrations of 75.7, 105.4, 114.9 and 177.6 mg/m3 (analytical concentrations - sum TDI-isomers). The aerosol was of adequate respirability for the rats (MMAD 1.35 -1.81 µm / GSD 1.61 -2.07µm). Mortality occurred in a concentration-dependent manner at 105.4 mg/m³ and above. A particular sex-difference in susceptibility was not apparent. For both genders combined an approximate LC50 (4 hrs, aerosol) of 112 mg/m3 was calculated based on the TDI-isomers. The calculation of the LC50 value based on test substance concentration reveals an approximate LC50 of 160 mg/m3. Decreased body weights and clinical signs indicative of respiratory tract irritation were observed at 75.7 mg/m3 and above. Reflex assessment after exposure revealed reduced grip strength and abnormal righting response. Histopathology of additional males of the exposure group 114.9 mg/m3 revealed acute inflammatory findings and severe necroses of the respiratory tract. In these animals almost all histopathological findings in the nasal cavities had recovered 12 days after exposure. Early mortality appeared to be caused by acute lung edema and delayed mortality (around day 10) by inflamatoric changes in and obstruction of the respiration tract.