Registration Dossier

Diss Factsheets

Administrative data

Endpoint:
sub-chronic toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
May 2018 - August 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2019
Report date:
2019

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)
Version / remarks:
07 September 2009
Deviations:
yes
Remarks:
see Any other information on materials and methods
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Constituent 1
Reference substance name:
Slimes and Sludges, blast furnace and steelmaking
EC Number:
266-006-2
EC Name:
Slimes and Sludges, blast furnace and steelmaking
Cas Number:
65996-73-8
Molecular formula:
not applicable
IUPAC Name:
Slimes and sludges, blast furnace and steelmaking
Constituent 2
Reference substance name:
266-066-2
IUPAC Name:
266-066-2
Details on test material:
- Physical state: solid
- Composition of test material, percentage of components: Fe (total) 54.29% (mainly as oxides), CaO 10.17%, Zn 5.25%, MgO 4.32%, C 2.06%, SiO2 1.64%, Mn 0.69%, K2O 0.126%, Na2O 0.239%, Al2O3 0.16%
- Lot/batch No.: 21.10.2009
- Expiration date of the lot/batch: unlimited
- Stability under test conditions: unlimited
- Storage condition of test material: stored in PE container at room temperature
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 20160418
- Purity test date: considered as 100%

Test animals

Species:
rat
Strain:
Wistar
Details on species / strain selection:
Preferred species of choice as historically they have been used for safety evaluation studies and they are specified by the appropriate regulatory authorities.
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH, Sandhofer Weg 7, D-97633 Sulzfeld
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 8 weeks
- Weight at study initiation: males: 215-257 g / females: 158-192 g
- Fasting period before study:
- Housing: group caging (in groups of 3 or 2 per cage, by sex), polycarbonate solid floor cages (type III) with stainless steel mesh lids.
- Diet (e.g. ad libitum): ssniff® SM R/M-Z+H "Autoclavable complete feed for rats and mice – breeding and maintenance" (ssniff Spezialdiäten GmbH, D-59494 Soest, Germany) ad libitum
- Water (e.g. ad libitum): drinking water, routinely analysed, ad libitum
- Acclimation period: 12 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.8 – 26.7°C
- Humidity (%): 30 - 76 %
- Air changes (per hr): at least 15 air exchanges per hour
- Photoperiod (hrs dark / hrs light): 12 hours of continuous artificial light in each 24 h period (from 6.00 a.m. to 6.00 p.m.)

Administration / exposure

Route of administration:
inhalation: dust
Type of inhalation exposure:
nose only
Vehicle:
air
Mass median aerodynamic diameter (MMAD):
>= 2.41 - <= 2.65 µm
Geometric standard deviation (GSD):
2.05
Remarks on MMAD:
The aerosol fraction was measured and characterized using a 7-stage cascade impactor of Mercer style (TSE Systems GmbH, Bad Homburg, Germany). Such devices employ an inertial separation technique to isolate particles into discrete aerodynamic size ranges. Samples were collected weekly at each concentration tested and measured gravimetrically. Samples were collected from a vacant animal exposure port (animals breathing zone) and the resulting data used to calculate the mass median aerodynamic diameter (MMAD), Geometric Standard Deviation (GSD) and percentage <3 µm (considered to be inhalable in the rat).
Details on inhalation exposure:
The animals were treated by the inhalation route using a nose-only exposure unit, in a TSE Rodent Exposure System with each individual concentration or control group in a dedicated tower. Four identical, modular multilevel flow-past, nose only exposure units (towers) were used. The exposure unit consisted of two, concentric anodised aluminium cylinders, the inner plenum and the outer chamber with 20 circularly arranged exposure ports.
The equipment was supported by a computer control system incorporating pressure detectors, mass flow controllers as well as temperature/RH, O2 and CO2 sensors or other similar equipment.
The exposure units were placed in closed hoods in order to avoid cross-contamination and contamination of the laboratory environment.
The animals were held in polycarbonate restraint tubes located around the chamber which allowed only the animals’ nares to enter the exposure port.
Atmosphere generation was dynamic. Fresh test atmosphere from the generation system was constantly supplied to the inner plenum (distribution chamber) of the exposure system from where was distributed to the individual exposure ports. After passing through the animal’s breathing zone, spent test atmosphere entered the outer cylinder from where it was exhausted through a suitable filter system.
Airflows and relative pressures within the system were constantly monitored and controlled by the computer system thus ensuring a uniform distribution and constant flow of fresh test atmosphere to each exposure port (breathing zone). The flow of air through each port was approximately 0.5 L/min. This flow rate is considered adequate to minimise re-breathing of the test atmosphere and maintained oxygen concentrations at greater than 19% and a carbon dioxide concentration not exceeding 1%.
Homogeneity of the test atmosphere within the test chamber and amongst the exposure ports was determined during the technical trial. However, to avoid any possible differences, the position of the animals was rotated on an exposure session basis.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The actual (achieved) concentration of generated atmospheres was measured gravimetrically at regular intervals during an exposure by pulling a suitable, known volume of test atmosphere, from the exposure chamber, TE35 PTFE membrane filters (Whatman, Germany). Sampling was normally performed each day shortly after chamber equilibration and then at regular intervals during the exposure, and samples were collected from a vacant animal exposure port (animals breathing zone). The actual sampling schedule employed was dependent on the required sample volume in order to obtain adequate quantities of test item (exposure period).
The average concentration of the test item in the test atmosphere during filter sampling (CAS, in mg/L) was calculated by the following formula:
CAS = M / (tS x FS)
M = Mass of the active substance on the sorbent (mg)
tS = Duration of sampling (min)
FS = Sampling flow rate (L/min)
From the samples collected during the animal exposure for concentration, supporting analysis of Fe, Zn, Mg, Mn, Si, Al (and if possible, Ca, K, Na) was performed using validated X-ray fluorescent analysis (FPBSTUDY-182-VAL1). The filter analysis was performed weekly during the exposure period.
Duration of treatment / exposure:
90 days
Frequency of treatment:
6 hour/day, 5 day/week
Doses / concentrationsopen allclose all
Dose / conc.:
0.03 mg/L air (nominal)
Remarks:
0.031 mg/L mean of daily samples of achieved gravimetric concentration
Dose / conc.:
0.1 mg/L air (nominal)
Remarks:
0.103 mg/L mean of daily samples of achieved gravimetric concentration
Dose / conc.:
0.3 mg/L air (nominal)
Remarks:
0.297 mg/L mean of daily samples of achieved gravimetric concentration
No. of animals per sex per dose:
10 male and 10 female animals/dose
Control animals:
yes, concurrent no treatment
Details on study design:
INHALATION EXPOSURE
The animals were exposed to an atmosphere of the test item for 6 hours per day for a period of 90 days on a 5 days/week basis. The atmosphere was generated according to the system and flow rates determined during the technical trials. The exposure did not start until theoretical chamber concentration equilibration has been reached. During the exposure period, changes were made to airflow and test material input rates in order to achieve the required concentration (documented).
ANIMAL EXPOSURE CONDITIONS
The animals were treated by the inhalation route using a nose-only exposure unit, in a TSE Rodent Exposure System with each individual concentration or control group in a dedicated tower. Four identical, modular multilevel flow – past, nose only exposure units (towers) were used. The exposure unit consisted of two, concentric anodised aluminium cylinders, the inner plenum and the outer chamber with 20 circularly arranged exposure ports.
The equipment was supported by a computer control system incorporating pressure detectors, mass flow controllers as well as temperature/RH, O2 and CO2 sensors or other similar equipment.
The exposure units were placed in closed hoods in order to avoid cross-contamination and contamination of the laboratory environment.
The animals were held in polycarbonate restraint tubes located around the chamber which allowed only the animals’ nares to enter the exposure port.
Atmosphere generation was dynamic. Fresh test atmosphere from the generation system was constantly supplied to the inner plenum (distribution chamber) of the exposure system from where was distributed to the individual exposure ports. After passing through the animal’s breathing zone, spent test atmosphere entered the outer cylinder from where it was exhausted through a suitable filter system.
Airflows and relative pressures within the system were constantly monitored and controlled by the computer system thus ensuring a uniform distribution and constant flow of fresh test atmosphere to each exposure port (breathing zone). The flow of air through each port was approximately 0.5 L/min. This flow rate is considered adequate to minimise re-breathing of the test atmosphere and maintained oxygen concentrations at greater than 19% and a carbon dioxide concentration not exceeding 1%.
Homogeneity of the test atmosphere within the test chamber and amongst the exposure ports was determined during the technical trial. However, to avoid any possible differences, the position of the animals was rotated on an exposure session basis.
JUSTIFICATION FOR CONCENTRATION LEVELS
The animals were exposed to the test atmosphere at three target concentration levels of 0.03, 0.1 and 0.3 mg/L, corresponding to the Low, Mid and High Doses, respectively, and an additional Filtered Air Control Group.
The concentration levels were suggested by the Study Director as a best advice based on previous data available, including the results of a preliminary dose range finding (DRF) study in the rat (Citoxlab Hungary study code :16/317-212PE). The dose selection was approved by the Study Monitor. The DRF study was performed in three target atmosphere concentrations (0.1 mg/L, 0.05mg/L and 0.025 mg/L) and 5 male and 5 female Wistar rats Crl:WI (Han) were exposed to the test atmospheres for 6 hours per day on a 5 day per week basis for a period of 4 weeks (total study duration of 26 days). The mean actual concentrations were 0.1023 mg/L, 0.0481 mg/L and 0.0244 mg/L. Based on the results of the DRF study, no significant adverse effect was observed at the target 0.1 mg/L concentration, although clear accumulation was observed in the lungs. Therefore, it is considered that a higher maximum concentration should be tested in the main study, in order to try to meet the guideline objective of inducing some significant toxicity at the High dose.
Positive control:
no

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily, immediately after exposure and approximately one hour after exposure

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: in all animals before treatment and in the control and High Dose animals on Week 13

HAEMATOLOGY: No

CLINICAL CHEMISTRY: No

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: day 77 - 78
- Dose groups that were examined: all animals
- Battery of functions tested: sensory activity, grip strength, motor activity

VAGINAL SMEARS: Yes
- Time schedule for examinations: prior to necropsy
- Dose groups that were examined: all female animals
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes
Statistics:
Group mean and standard deviation were calculated for numerical data. Statistical analysis was performed using SAS 9.2 (built in Provantis System).
The following decision tree was automatically applied within the validated Provantis system for statistical evaluation of numeric data:
The normality and heterogeneity of variance between groups were checked by Shapiro-Wilk and Levene tests using the most appropriate data format (log-transformed when justified). Where both tests show no significant heterogeneity, an Anova / Ancova (one-way analysis of variance) test was carried out. If the obtained result was positive, Dunnett’s (Multiple Range) test was used to assess the significance of inter-group differences; identifying differences of <0.05 or <0.01 as appropriate. This parametric analysis was the better option when the normality and heterogeneity assumptions implicit in the tests are adequate.
If either of the Shapiro-Wilk or Levene tests showed significance on the data, then the ANOVA type approach was not valid and a non-parametric analysis was required. A Kruskal-Wallis analysis of variance was used after Rank Transformation. If there was a positive result, the inter-group comparisons were performed using Dunn test; identifying differences of <0.05 or <0.01 as appropriate
For non-continuous data, the Cochran-Armitage test for trend was applied and the Chi-squared test was used for statistical differences relative to control.
For pathology data (macroscopic and microscopic data) the Cochran-Armitage test for trend was applied, then if appropriate, the Chi-squared test homogeneity test. If significance was plausible based on a user-defined value (0.05), a pairwise test of each treatment group versus the control group was made. If the group size was <5 then Fisher’s Exact Test was used, if the group sizes were bigger then the Chi-squared test was used; identifying differences of <0.05, <0.01 or <0.001 as appropriate.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Description (incidence and severity):
No toxicologically relevant clinical signs were recorded during the study.
Fur staining, fur thin, alopecia or scar were recorded. These observations were considered to be related to the restraint and exposure procedures and, in isolation, were considered not to be of toxicological relevance.
Mortality:
no mortality observed
Description (incidence):
There was no mortality in the study.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
There was a minimal, but statistically significant test item related effects on terminal body weight in Mid and High Dose males and High Dose females. The High Dose males and females had 7.1% and 8.0% reduction respectively in terminal body weight compared to controls. In Mid Dose males the terminal body weight reduction was 6.2% compared to controls.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Food intakes reflected the body weight gain difference in both sexes, showing reduced food consumption in Mid and High doses.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Description (incidence and severity):
No test item related changes compared to pre-treatment or/and the Control were noted at ophthalmoscopy examination. All examined animals were found to be normal.
Haematological findings:
no effects observed
Description (incidence and severity):
There were no toxicologically significant effects in investigated haematology parameters between test item treated animals and the controls.
There was no significant change in the blood clotting parameters.
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
In the males in the High Dose group statistically significant differences were seen calcium and in the chloride level, but they were in the normal range. There were no differences that were clearly related to treatment.
Urinalysis findings:
no effects observed
Description (incidence and severity):
There was no significant change in the urinalysis parameters.
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
There was no treatment related effect in the Landing Foot Splay, Irwin Test or locomotor activity in any dose group. Although, a significant change was measured in the Grip Strength in the Mid Dose females, this change was within the historical control range and not considered to be test item related adverse effect.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
When compared to the controls, weight changes were observed in the lungs and thymus in test item exposed animals which were considered to be test item-related.
The absolute and relative (to body and brain weights) lung weights were statistically significantly higher in High and Mid Dose males and females. The absolute and relative (to body and brain weights) lung weights were also higher in Low Dose males and females, with statistical significance in absolute and relative to body weights only. The increase in lung weights in test item exposed animals showed a dose dependent trend and correlated with the histopathological findings.
The absolute and relative (to body and brain weights) thymus weights were lower in all dose groups in females and Mid and High Dose males, attaining statistical significance in Mid and High Dose females, but they were within the historical control range.
These decrease in thymus weights in Mid and High Dose animals correlated with the macroscopic and microscopic findings.
Statistical differences in other organ weight were relatively small differences, without any corresponding macroscopic or microscopic findings and correlated with the slightly lower body weight of test item exposed animals, and were considered unrelated to the direct test item effect.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Diffuse black or multifocal to diffuse grey discolouration of all lobes of lungs was observed in majority of test item exposed animals.
Diffuse black or grey discolouration of the mediastinal lymph nodes was observed in majority of test item exposed animals. Enlargement of the lymph node was observed in majority of High Dose males and occasional High Dose females and Mid Dose males.
Small thymus was noted in 1/10 Mid and High Dose males, and High Dose females. Correlating microscopic changes were seen in these animals along with few other High Dose and Mid Dose animals.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
The test item related changes were observed in the lung, nasal cavity, mediastinal lymph node and thymus.
In the lung, macrophages loaded with a black pigment, which is considered most likely the test item was seen distributed throughout the parenchyma with a predominance around the centroacinar region at all dose levels in both sexes. Pigments were also seen outside the macrophages within the alveolar lumen, especially in High Dose animals. This microscopic finding correlated with macroscopic black or grey discolouration in the lung observed at necropsy and increase in the lung weight. There was no degenerative or inflammatory reaction around the pigmented macrophages. Uncomplicated increases in the size or number of alveolar macrophages in response to inhaled pharmaceutical materials are considered non adverse (Nikula et al 2014). Thus, the pigmented macrophages in the lung observed in the current study was considered as a result of normal non adverse physiological response to phagocytose the inhaled test item.
Pigments were also seen in the BALT and mediastinal lymph node, indicating drainage of the test item into the lymphatic system. Mediastinal lymph node also showed increase in cellularity in Mid and High Dose animals suggesting an immune response against the test item.
The test item exposed animals also showed multifocal alveolar epithelial hyperplasia mainly in the alveolar wall surrounding the collection of pigmented macrophages. A few Mid and High Dose animals also had peribronchiolar/perivascular infiltration of eosinophils, which was considered as a secondary response.
In the nasal cavity, eosinophilic droplets were seen at minimal or slight severity in the respiratory and/or olfactory epithelium at all dose groups without a dose dependent trend in incidence. Eosinophilic droplets in the respiratory epithelium are reported in low numbers in untreated control rats, especially older rats but at an increased incidence and severity in association with toxic effects which perhaps constitute an adaptive response (Monticello et al 1990; Renne et al 2007). The eosinophilic droplets observed in the respiratory/olfactory epithelium in the current study were considered as non-adverse.
Hyperplasia of the mucous cells in the respiratory epithelium mainly affecting the nasal septum was seen in all test item exposed animals at a higher incidence and severity compared to Controls. The hyperplasia of the mucous cells is considered as a response to clear off the test item. Minimal focal to multifocal atrophy of the olfactory epithelium (level 4) mainly covering the distal end of the nasal septum at the junction with respiratory epithelium, was seen in Mid and High Dose animals. The incidence of eosinophilic droplets in the respiratory / olfactory epithelium was also around this site at level 3. The minimal atrophy of the olfactory epithelium was considered non adverse.
A slightly higher incidence/severity of reduced size/cellularity of the thymus was observed in Mid and High Dose animals. This finding in thymus was not associated with increase in apoptotic lymphocytes or tingible body macrophages as normally seen in stress induced atrophy of the thymus, which are seen in severe body weight reduction observed in some toxicity studies. In the current study, the body weight reduction in Mid and High Dose males, and High Dose females were minimal in the range of –6.2 % to –8.0 % and considered not to cause stress response in these animals. Thus, the effect observed in the thymus in the current study was considered as a reflection of reduction of the body weight and a non-adverse effect.
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed
Description (incidence and severity):
There were no adverse or the test Item related observations in the animal oestrus cycle evaluated prior to necropsy and the animals showed the normal distribution of the oestrus phases.

Effect levels

Key result
Dose descriptor:
NOAEC
Effect level:
>= 0.3 mg/L air (nominal)
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects

Target system / organ toxicity

Key result
Critical effects observed:
no

Applicant's summary and conclusion

Conclusions:
The exposure to the test item Slimes and Sludges, blast furnace and steelmaking to Hannover Wistar rats for 90 days for 6 hours/day on a 5 day per week basis at dose levels 0.03 to 0.3 mg/L did not cause any clinical sign. There were no statistically significant adverse effects on body weight, food consumption, ophthalmology, clinical pathology or oestrus cycle. There was a minimal, but statistically significant test item related effects on terminal body weight in Mid and High Dose males and High Dose females.
The lung weights were increased and the thymus weights were decreased in the test item exposed animals in a dose dependent manner and correlated with histopathological findings in both organs.
In histopathology, test item-related changes were seen in the lung, in the nasal cavity and in the mediastinal lymph nodes at all dose levels. A higher incidence/severity of reduced size/cellularity of thymus was observed in Mid and High Dose animals. The histopathology changes were considered non-adverse, adaptive changes.
The test item related changes were considered non-adverse and hence the NOAEL is 0.3 mg/L.
Executive summary:

The objective of this 90-day study was to obtain information on the toxicity of the test item when administered to Wistar (Han) rats via inhalation route for at least 90 days with the aim of inducing toxic effects but no death or suffering at the highest concentration, and little or no evidence of toxicity at the lowest concentration level.

Ten male and 10 female Wistar rats Crl:WI (Han) in each groups were exposed by a 6 hour nose-only exposure to filtered air or to three fixed aerosol concentrations for consecutive 5 days/week to cover a wide range of doses. The target concentrations were 0.03, 0.1 and 0.3 mg/L, plus control. The duration of the study was at least 90 days. The animals were sacrificed on the day following the last exposure on day 91.

The concentration levels were suggested by the Study Director as a best advice based on previous data available, including the results of a preliminary dose range finding study in the rat. The dose selection was approved by the Study Monitor.

The animals were exposed to the test atmosphere using a nose-only exposure system. The test item was generated using a Dust Generator acc. to Budiman connected to pressurized air supply. The atmosphere concentrations of 0.031 mg/L, 0.103 mg/L and 0.297 mg/L were achieved in the respective groups, the target concentrations being 0.03 mg/L, 0.1 mg/L and 0.3 mg/L. The treatment was performed in 4 inhalation towers parallel, one tower for each treatment groups and an additional tower for the filtered air control group.

Parameters monitored for the animals during the study included mortality, clinical observations, body weight, food consumption and clinical pathology evaluation (haematology, coagulation and clinical chemistry), ophthalmology, functional observation battery (FOB). Gross macroscopic examination was performed at necropsy and selected organs were weighed.

Full histopathology was performed in Control and High dose groups and the histopathology of the lungs and other tissues affected in the High dose group was in the Low and Mid dose groups.

The test atmosphere concentration was monitored based on the gravimetric and X-ray fluorescence analysis. The results of the test atmosphere characterization were considered suitable for the study purposes.

No mortality was observed in the study. No toxicologically relevant clinical sign was recorded during the study. There were no statistically significant differences in ophthalmology, in neurological assessment or in the oestrus cycle. There was a minimal, but statistically significant test item related effects on terminal body weight in Mid and High Dose males and High Dose females. There was no significant difference in the food consumption. There were no treatment related changes in clinical pathology.

When compared to the controls, weight changes were observed in the lungs and thymus in test item exposed animals which were considered to be test item-related. The absolute and relative (to body and brain weights) lung weights were statistically significantly higher in High and Mid Dose males and females. The absolute and relative (to body and brain weights) lung weights were also higher in Low Dose males and females, with statistical significance in absolute and relative to body weights only. The increase in lung weights in test item exposed animals showed a dose dependent trend and correlated with the histopathological findings. The absolute and relative (to body and brain weights) thymus weights were lower in all dose groups in females and Mid and High Dose males, attaining statistical significance in Mid and High Dose females. These decrease in thymus weights in Mid and High Dose animals correlated with the macroscopic and microscopic findings.

In histopathology, test item-related changes were seen in the lung, in the nasal cavity and in the mediastinal lymph nodes at all dose levels. A higher incidence/severity of reduced size/cellularity of thymus was observed in Mid and High Dose animals. The histopathology changes were considered non-adverse, adaptive changes.