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Acute Toxicity: inhalation

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Administrative data

Endpoint:
acute toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2010
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 guideline
Qualifier:
according to
Guideline:
OECD Guideline 403 (Acute Inhalation Toxicity)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Test type:
standard acute method
Limit test:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Details on test material:
GGBS = Ground granulated slags, ferrous metal, blast furnace

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
Species: SPF-bred Wistar rats of the strain HsdCpb:WU from Laboratory animal breeder Harlan Nederland (NL), Kreuzelweg 53, 5960 AD Horst.
Acclimatization: at least 5 days before use. Acclimatization to the restraining tubes at least 1 x 2h, 2 x 4h).
Health status: Historical data is available. The breeding colony is routinely spot-checked for the main specific pathogens.Only healthy rats free of pathological signs will be used. Animals are not vaccinated or treated with anti-infective agents either before their arrival or during the acclimatization
or study periods. The females are nulliparous and not pregnant.
Age and weight: 2-3 months old. Weight range at the exposure day – males: 160 – 210 gram; - females: 150 – 200 gram
Number and groups: 6 animals will be used at each dose (3 animals/sex/group). When the test article is diluted, it may be necessary to use generic control animals (5 animals/sex) to identify exposure related effects
Identification: individual colour-marking and cage-labels.
Randomization: Prior to exposure, standard randomization procedure applied (computerized list of random numbers serves to assign animals at random to the treatment groups)
Animal housing: singly in Makrolon® Type IIIH cages (based on A. Spiegel and R. Gönnert, Zschr. Versuchstierkunde, 1, 38 (1961) and G. Meister, Zschr. Versuchstierkunde, 7, 144-153 (1965)). Cages are changed twice a week while unconsumed feed and water bottles are changed once per
week. The legal requirements of Directive 86/609 EEC were followed.
Bedding: Bedding consisted of type Lignocel BK 8-15 low-dust wood granulate from Rettenmaier. The wood granulate is randomly checked for harmful constituents at the request of the Laboratory Animal Services, Bayer HealthCare AG.
Temperature: 22 ± 3 °C
Relative humidity: 40 - 60 %,
Dark/light cycle : 12 h/12 h; artificial light from 6.00 a.m. to 6.00 p.m., light intensity: maximum 200 Lux
Ventilation: at least 10 air changes/h
Cleaning, disinfection, and pest control: The animal room is regularly cleaned and disinfected once a week with an aqueous solution of TEGOÒ 2000 VT25.
Pest control measures: Cleaning. Cockroach traps
Feeding: Ration consist of a standard fixed-formula diet (KLIBA 3883 = NAFAG 9441) pellets; PROVIMI KLIBA SA, 4303 Kaiseraugst, Switzerland) and tap water (drinking bottles). Both food and water is available ad libitum. The pelletized feed is contained in a rack in the stainless-steel wire cage
cover. Specification of ingredients and contaminants according to recommendations of GV SOLAS (August, 2001).
Water: Drinking quality municipality tap-water (current version of the Drinking Water Decree) is provided ad libitum in polycarbonate bottles containing approximately 300 ml (based on A. Spiegel and R. Gönnert, Zschr. Versuchstierkunde, 1, 38 (1961) and G. Meister, Zschr. Versuchstierkunde, 7, 144-153 (1965)).
Body weights: Body weights are recorded before exposure and 1, 3, 7, and 14 days (or similar time increments, if applicable) after exposure. Individual weights are also recorded at death, if applicable (onset of mortality ³ first postexposure day). As a rule, for data evaluation, the exposure day is
defined as day 0 (relative).
Clinical observations: The appearance and behaviour of each rat are examined carefully several times on the day of exposure and at least once daily thereafter. Weekend assessments are made once a day (morning). Assessments from restraining tubes are made only if unequivocal signs occur (e.g.
spasms, abnormal movements, and severe respiratory signs). Following exposure, observations are made and recorded systematically; individual records are maintained for each animal. Cage-side observations included, but are not limited to, changes in the skin and fur, eyes, mucous membranes,
respiratory, circulatory, autonomic and central nervous system, and somatomotor activity and behaviour pattern. Particular attention is directed to observation of tremors, convulsions, salivation, diarrhea, lethargy, somnolence and prostration. The time of death is recorded as precisely as
possible, if applicable. Since these signs can only be assessed adequately from freely moving animals, no specific assessment is performed during exposure while animals are restrained.
On the first postexposure day, each rat is first observed in its home cage and then individually examined. The following reflexes are examined, based on recommendations made by Irwin (Comprehensive Observational Assessment: Ia: A Systematic, Quantitative Procedure for Assessing the Behavioral and Physiologic State of the rats. Psychopharmacologica 13, pp. 222-257 (1968)):

Administration / exposure

Route of administration:
inhalation: dust
Type of inhalation exposure:
nose only
Vehicle:
clean air
Details on inhalation exposure:
Inhalation Chamber: An aluminium inhalation chamber with the following dimensions has to be used: inner diameter = 14 cm, outer diameter = 35 cm (two-chamber system), height = 25 cm (internal volume = about 3.8 l). If feasible, multiple segments of this chamber can be used, however, the respective air-flows have to be adapted to the larger chamber volume.
Optimization of respirability of aerosol (if applicable): In order to increase the efficiency of the generation of respirable particles and to prevent larger particles from entering the inhalation chamber, a pre-separator/baffle and/or elutriator/cyclone systems which favours the formation of fine particles is used.
Conditioning the compressed air: Compressed air is supplied by Boge compressors and is conditioned (i.e. freed from water, dust, and oil) automatically by a VIA compressed air dryer.
Inhalation chamber steady-state concentration: The test atmosphere generation conditions provide an adequate number of air exchanges per hour (approximately 200-times or more). Under such test conditions steady state is commonly attained within the first minute of exposure (t99% = 4.6 x
chamber volume/flow rate). The ratio between the air supplied and exhausted is 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 L/min must be provided.
Air flows: During the exposure period air-flows are monitored continuously and, if necessary, readjusted to the conditions required. Air-flows are measured with calibrated flow-meters and/or soap bubble meter (Gilibrator) and are checked for correct performance at regular intervals by other
equipment (e.g., spirometer).
Analytical verification of test atmosphere concentrations:
yes
Duration of exposure:
6 h
Remarks on duration:
2 weeks post exposure observation
Concentrations:
5235 mg/m3
No. of animals per sex per dose:
3 female and 3 males in exposure groups, 5 males in control group
Control animals:
yes
Details on study design:
Analysis of particle-size distributions:
Sampling in the vicinity of the breathing zone, two samples per exposure.
ANDERSEN- or an AERAS low-pressure critical orifice cascade impactor. The individual impactor stages are covered by an adhesive stage coating (silicone spray) in order to prevent particle bounce and re-entrainment. If not advisable due to the specific conditions of the test coating can be omitted, or other measures may be taken.
Determined Mass Median Aerodynamic Diameter (MMAD)
Calculation of Geometric Standard Deviation (GSD): GSD = 84.1% mark / 50% mark.
To verify graphically that the aerosol is in fact unimodal and log-normally distributed the normalized mass per stage (fH') is evaluated as a histogram. Calculate the histogram
Calculate the log-normal mass distribution y'(Dae) = 1/Nf x y(Dae) as a function of the aerodynamic diameter
Statistics:
Necropsy findings: Fisher test after the R x C chi-squared test if required
Body weights: Means and single standard deviations, one-way ANOVA (vide infra)
Physiological data: ANOVA procedure (vide infra).
Calculation of the LC50: according to the method of Rosiello ROSIELLO, A.P., ESSIGMANN, J.M., and WOGAN, G.N. (1977). Rapid and Accurate Determination of the Median Lethal Dose (LD50) and its Error with Small Computer. J. Tox. and Environ. Health 3, pp. 797-809).
Analysis of variance (ANOVA): Checks for normal distribution of data by comparing the median and mean. The groups are compared at a confidence level of (1-a) = 95% (p = 0.05). The test for the between-group homogeneity of the variance employed Box's test if more than 2 study groups are compared with each other. If the above F-test shows a difference then a pair-wise post-hoc comparison is conducted (1- and 2-sided) using the Games and Howell modification of the Tukey-Kramer significance test.

Results and discussion

Preliminary study:
as no mortality occured at the highest technically attainable concentration, no other test was done
Effect levelsopen allclose all
Sex:
male/female
Dose descriptor:
LC50
Effect level:
> 5 235 mg/m³ air (analytical)
Based on:
test mat.
Exp. duration:
4 h
Sex:
male/female
Dose descriptor:
LC0
Effect level:
>= 5 235 mg/m³ air (analytical)
Based on:
test mat.
Exp. duration:
4 h
Mortality:
No mortality occurred and the 4 h-LC50 inhalation (powder, aerosol) was > 5235 mg/m3.
Clinical signs:
The following clinical signs were observed, some of them up to the sixth (females; males forth) postexposure day: labored breathing patterns, irregular breathing patterns, hair coat ungroomed, piloerection, motility reduced, limp, high-legged gait, nasal discharge (serous), hypothermia, and decreased body weights. The 4 h-NO(A)EL was < 5235 mg/m3.
Body weight:
means of control males: d 0 (postexposure day 0): 183.2 g/animal, d 1: 181.4 g/animal, d 3: 200.0 g/animal, d 7: 230.4 g/animal, d 14: 285.2 g/animal
means of exposure group males: d 0 (postexposure day 0): 169.3 g/animal, d 1: 155.0 g/animal, d 3: 174.0 g/animal, d 7: 206.0 g/animal, d 14: 248.7 g/animal
means of control females: d 0 (postexposure day 0): 180.0 g/animal, d 1: 176.8 g/animal, d 3: 187.6 g/animal, d 7: 197.6 g/animal, d 14: 211.6 g/animal
means of exposure group females: d 0 (postexposure day 0): 168.3 g/animal, d 1: 152.7 g/animal, d 3: 171.7 g/animal, d 7: 188.0 g/animal, d 14: 206.3 g/animal
Gross pathology:
Macroscopic findings of animals sacrificed at the end of the observation period were essentially indistinguishable amongst exposure and control groups apart from lungs with gray areas
Other findings:
Hyperthermia: rectal temperatures were decreased from mean 37.9 °C to 34.3 °C in males and 38.4 °C to 32.7 °C in females as typical for "overloading" of the upper airways with dry, respirable dust

Applicant's summary and conclusion

Interpretation of results:
practically nontoxic
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
no classification required
Executive summary:

To determine the acute inhalation toxicity of slags, ferrous metal, blast furnace (ground, granulated – GGBS), a test was conducted with rats in accordance to OECD Technical Guideline 403 (2009) and Directive 92/69/EEC. Annex V Method B.2. GGBS was applied as a powder aerosol nose-only to one group of rats for an exposure duration of 4 hours at a concentration of 5235 mg/m3 which was also the maximum technically attainable concentration. The aerosol was respirable, i.e. the mass median aerodynamic diameter (MMAD) was 3.1 µm, and the geometric standard deviation was 1.8 µm. The exposure was followed by a postexposure observation period of 2 weeks.

No mortality occurred and the 4 h-LC50 inhalation (powder, aerosol) was > 5235 mg/m3. The following clinical signs were observed, some of them up to the sixth postexposure day: labored breathing patterns, irregular breathing patterns, hair coat ungroomed, piloerection, motility reduced, limp, high-legged gait, nasal discharge (serous), hypothermia, and decreased body weights. The 4 h-NO(A)EL was < 5235 mg/m3.

Slags, ferrous metal, blast furnace (ground, granulated – GGBS) are not toxic to rats via the inhalation route.