Registration Dossier

Administrative data

Endpoint:
acute toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1997-04-21 to 1997-05-05
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
GLP study reliable without restrictions The following information were missing in the study report, but that had no effect on the results of this study. - the stability of the test material was missing in the study report. Deviations from OECD 403 (2009) - if animals were acclimatised to the exposure apparatus was not stated. - individual housing of animals instead of group housing - during a limit test only 3 males and 3 females should be used. In this study 5 males and 5 females were used. - animals were not weighed during acclimatisation and on day 1 after exposure. - the number of exposure ports per chamber was not stated. - rationale for target concentration selection and test concentration was missing in the study report.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1997
Report Date:
1997

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
EU Method B.2 (Acute Toxicity (Inhalation))
Version / remarks:
1992
Qualifier:
according to
Guideline:
OECD Guideline 403 (Acute Inhalation Toxicity)
Version / remarks:
(1981-05-12)
Deviations:
yes
Remarks:
please refer to "Rationale for reliability incl. deficiencies" above
GLP compliance:
yes
Test type:
standard acute method
Limit test:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- CAS name: 1H-Benzimidazole-4,6-disulfonic acid-2,2´-(1,4-diphenylene) bis, disodium salt
- Molecular formula: C20H12N4O12S4Na2
- Molecular weight: 674g/mol
- physical state: solid

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan-Winkelmann GmbH, Borchen, Germany
- Age at study initiation: approximately 2 months old
- Weight on exposure day: males: 176 - 190 g; females: 174 - 186 g
- Housing: during acclimatization and study periods the animals were housed singly in conventional Makrolon® Type II cages (based on A. Spiegel and R. Gönnert, Zschr. Versuchstierkunde, 1, 38 (1961) and G. Meister, Zschr. Veruschstierkunde, 7, 144 - 153 (1965)). Bedding consisted of type S 8/15 low-dust wood granulate from Ssniff, Soest/Westfalen, Germany.
- Diet (ad libitum): ration consisted of a standard fixed-formula diet (Altromin® 1324 pellets maintenance diet for rats and mice, Altromin GmbH, Lage)
- Water (ad libitum): drinking quality tap water
- Acclimation period: at least 5 days before use

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 2°C
- Relative humidity: approximately 50%
- Ventilation: approximately 10 air changes per hour
- Photoperiod (hrs dark / hrs light): 12/12 (light intensity: approximately 14 watt/m^2 floor area)

Administration / exposure

Route of administration:
inhalation: dust
Type of inhalation exposure:
nose only
Vehicle:
clean air
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: animals were exposed to the aerosolized test substance in Plexiglas exposure tubes applying a directed-flow nose-only exposure principle. Tubes were chosen that accommodated the animals size. These restrainers were designed so that the rat's tail remained outside the tube, thus restrained-induced hyperthermia can be avoided. The chambers used are commercially available (TSE, 61348 Bad Homburg) and the performance of this type of chamber has been published (Pauluhn, 1984; Pauluhn, 1988; Pauluhn, 1994).

- Exposure chamber volume: the aluminum 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 I). Details of this modular chamber and its validation with regard to spatial homogeneity of material distribution have been published (Pauluhn, 1994).

- Source and rate of 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.
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; McFarland, 1976). 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 1/min was provided. The test atmosphere can by no means be diluted by bias-air-flows from outside the chamber. The inhalation chamber was operated in a well ventilated chemical fume hood.

- System of generating particulates/aerosols: the test atmosphere of test compound was generated by an EXACTOMAT 4200 (TSE, 61348 Bad Homburg, Germany). For dry powder dispersion, conditioned compressed air (28 liters/min) was used. The principle performance of the EXACTOMAT 4200 dust generating system can be described as follows: The test substance was filled into a glass reservoir (approximately 1/2 kg). From this reservoir it was fed (by suction) into the orifice of a venturi tube. The airborne powder was then entrained into the inner cylinder of the inhalation chamber. Reproducible and temporally stable dosing into the orifice was achieved by an oscillating orifice. The orifice size was adjusted manually in order to obtain the targeted flow of powder. Stirring of the reservoir was performed using a minimum number of revolutions per unit of time. Thus the flowability of the test compound was maintained without imposing a potential vertical inhomogeneity of active ingredient.

Under dynamic conditions the test substance was fed into the intake of the cylindrical Inhalation chamber.

- Method of particle size determination: the samples for the analysis of the particle-size distribution were taken in the vicinity of the breathing zone. During each exposure two samples were taken. The particle-size distribution was analyzed using an ANDERSEN-Type cascade impactor. The mass
sampled was evaluated by the determination of weight of each glass stage before and alter sampling. Gravimetric analyses were made using a digital balance.

- Treatment of exhaust air: the exhaust air was purified via cotton-wool/activated charcoal and HEPA filters.

- Temperature, humidity, pressure in air chamber: 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. Their proper function has been validated by soap bubble meter measurements (Gilibrator, Ströhlein Instruments, Kaarst) and were checked for correct performance at regular intervals.
The temperature and humidity measurements were made using a computerized system (Leybold Heraeus). The values were recorded at intervals of 10 min (computerized recording). The test atmosphere temperature and humidity were measured at the exposure location (temperature: 22°C for control and test group; relative humidity: 3% (mean) and 15% (mean) for control and test group, respectively). Details of this monitoring system have been reported elsewhere (Pauluhn, 1986).
The relative humidity was measured with a humidity sensor (CCH capacitor). The humidity-sensor is protected from particles by a double sintered metal filter with an interposed Teflon membrane (pore size approximately 1 pm). This sensor was calibrated using saturated salt solutions according to Greenspan (1977). The temperature sensors were calibrated with a calibration thermometer. The measured values were recorded and evaluated with an Apple 2e computer and an MDP 8240/45 analogue-digital converter using an IEEE 488 interface.

TEST ATMOSPHERE
- Brief description of analytical method used: the test-substance concentration was determined by gravimetric analysis (filter: Cellulose-Acetate-Filter, Sartorius, Göttingen, Germany; balance: Mettler AE 100).
The number of samples taken was sufficient to characterize the test atmosphere and was adjusted so as to accommodate the sampling duration and/or the need to confirm specific concentration values. Optimally, samples were collected after the equilibrium concentration had been attained in hourly intervals. All analytical concentrations reported refer to mg of test substance/m3 air.

- Samples taken from breathing zone: yes

- Nominal concentration: a nominal concentration was not calculated since the construction and the heavy weight of the dust generators and their reservoirs, respectively, used did not allow for a precise measurement of the powder aerosolized.

TEST ATMOSPHERE
- MMAD (Mass median aerodynamic diameter) / GSD (Geometric st. dev.): MMAD 2.8 µm (mean of analyses) / GSD: 2.0
- Aerosol mass < 3 µm (%)
- Mass recovered: 1679 mg/m^3

RESPIRABILITY: the particle-size distribution achieved is adequate to reach all potential target structures of the respiratory tract.

STABILITY OF THE TEST ATMOSPHERE:
Attempts were made (during pre-trials) to monitor the integrity and stability of the aerosol generation and exposure system the stability of the exposure atmosphere was measured by using a RAS-2 real-time aerosol photometer (MIE, Bedford, Massachusetts, USA). Samples were taken continuously from the vicinity of the breathing zone. However, due to an obstruction of orifices, the tubing system and/or the sensing unit of the photometer this technique could not be used for this examination.
This chamber monitoring allows for an overall survey of toxicologically relevant technical parameters (inlet and exhaust flows as well as atmosphere homogeneity, temporal stability, and generation performance). Interruptions in exposure (e.g. resulting from obstruction of orifices or other technical mishaps) are recorded and, if applicable, a commensurate interval is added to the exposure duration for compensation.

COLLECTION EFFICIENCY: the sampling equipment was adjusted with calibrated flow meters to internationally recognized standards (ACGIH, 1978; Section I "Calibration of Air Sampling Instruments").
The conditions for generating the test atmosphere are optimized to provide maximum aerosol respirability to rats (Raabe, 1982; Snipes, 1989; SOT-Commentary, 1992).

References:
- GREENSPAN, L. (1977). Humidity Fixed Points of Binary Saturated Aqueous Solutions, Journal of Research of the National Bureau of Standards, Vol. 81 A, no. 1, Jan.-Febr. 1977.
- McFARLAND, H.N. (1976). Respiratory Toxicology - Essays in Toxicology, Vol. 7, pp. 121-154, Academic Press Inc., New York, San Francisco, London.
- PAULUHN, J. (1984). Head-only and nose-only exposure in P. Grosdanoff, R. Baß, U. Hackenberg, D. Henschler, D. Müller, H.-J. Klimisch (eds.), Problems of lnhalatory Toxicity Studies, BGA-Schriften, MMV Medizin Verlag München, Vol. 5, pp. 59-68.
- PAULUHN, J. (1986). Study to Determine Temperature and Humidity Data in Inhalation Chambers; BAYER AG Report No. 15007 dated August 22.
- PAULUHN, J. (1988). Different Methods used in Acute and Subchronic Inhalation Studies of Potential Lung Irritants with Particular Attention to Lung Function Measurements. In U. Mohr (ed.), Inhalation Toxicology - The Design and Interpretation of Inhalation Studies and their Use in Risk Assessment. Springer Verlag, pp. 87-101.
- PAULUHN, J. (1994). Validation of an improved nose-only exposure system for rodents. Journal of Applied Toxicology, 14:55-62.
- RAABE, O.G. (1982). Deposition and Clearance of Inhaled Aerosols in H. Witschi and P. Nettesheim - Mechanisms in Respiratory Toxicology Vol. 1, pp. 27-76, CRC Press, Inc. Boca Raton, Florida.
- SNIPES, M.B. (1989). Long-Term Retention and Clearance of Particles Inhaled by Mammalian Species. Critical Reviews in Toxicology, Vol. 20, pp. 175-211.
- SOT-COMMENTARY (1992). Recommendations for the Conduct of Acute Inhalation Limit Tests, prepared by the Technical Committee of the Inhalation Specialty Section, Society of Toxicology. Fundam. Appl. Toxicol. 18, pp. 321-327.
Analytical verification of test atmosphere concentrations:
yes
Remarks:
please refer "details on inhalation exposure" above
Duration of exposure:
4 h
Concentrations:
Target concentration: 2000 mg/m^3 (2.0 mg/L; calculated from value stated in the study report)
Actual concentration: 1870 mg/m^3 (1.87 mg/L; calculated from value stated in the study report)
MMAD 2.8 µm (GSD 2µm)
No. of animals per sex per dose:
5 males / 5 females
Control animals:
yes
Details on study design:
- Duration of observation period following administration: 14 days
- Frequency of observations and weighing: body weights were measured before exposure, on days 3 and 7, and weekly thereafter. Individual weights are also recorded at death, if applicable. The appearance and behavior of each rat were examined carefully several times on the day of exposure and at least once daily thereafter. Weekend assessments were made once a day (morning). Assessments from restraining tubes were made only if unequivocal signs occurred. The time of death is recorded as precisely as possible, if applicable. Since clinical signs can only be assessed adequately from freely moving animals, no specific assessment was performed during exposure while animals were restrained.
- Necropsy of survivors performed: yes; all surviving rats were sacrificed at the end of the observation period using sodium pentobarbital (Nembutal®) (approximately 300 mg/kg body weight, intraperitoneal injection). All rats, irrespective of the day of death, were given a gross-pathological examination. Consideration was given to performing a gross necropsy on animals as indicated by the nature of toxic effects, with particular reference to changes related to the respiratory tract. All gross pathological changes were recorded and evaluated.
- Other examinations performed: the rectal temperatures were measured directly after cessation of exposure (approximately within 1/2 hour after the end of exposure) using a Digimed digital thermometer with a rectal probe for rats.
Statistics:
Please refer to "Any other information on materials and methods incl. tables" below.

Results and discussion

Effect levelsopen allclose all
Sex:
male/female
Dose descriptor:
LC50
Effect level:
> 1 870 mg/m³ air (analytical)
Based on:
test mat.
Exp. duration:
4 h
Remarks on result:
other: approximate LC50
Sex:
male/female
Dose descriptor:
LC50
Effect level:
> 1.87 mg/L air (analytical)
Based on:
test mat.
Exp. duration:
4 h
Remarks on result:
other: approximate LC50 (calculated from the value given in the study report)
Mortality:
No mortality occurred during the study period.
Clinical signs:
All rats tolerated the exposure without specific signs.
Body weight:
Comparisons between the air control and test substance exposed groups did not show any effect on body weights.
Gross pathology:
In rats exposed to the test compound a conclusively increased incidence of macroscopic findings could not be observed.
Other findings:
- Rectal temperature: statistical comparison between groups did not indicate an effect on body temperature (temperature control group (m/f): 38.3 - 38.4°C; temperature test group (m/f): 38.3°C) .

Applicant's summary and conclusion

Interpretation of results:
not classified
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
LC50: > 1.87 mg/L air (analytical concentration). The aerosolised test substance proved to have virtually no acute inhalation toxicity to rats.
Executive summary:

Exposure to an aerosol concentration of 1870 mg/m3 were tolerated without mortality and clinical signs. With regard to the respirablility of the aerosol generated internationally recognized recommendations such as of SOT (1992) were fulfilled, i.e. the MMAD was <4µm. No empasize was made to test higher than the limit concentration of 2000 mg/m3 air (SOT, 1992, Pauluhn et al., 1996).