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Toxicological information

Repeated dose toxicity: inhalation

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Administrative data

Endpoint:
short-term repeated dose toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2014 - 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report Date:
2015

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 412 (Subacute Inhalation Toxicity: 28-Day Study)
Deviations:
yes
Remarks:
will be decided
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
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: Han Wistar IGS rats from Charles River UK Limited, Margate, Kent, UK.
Acclimatization: for a period of 12 days before the commencement of dosing. For 5 days before commencement of dosing, all the animals were conditioned to the restraint procedures used for snout only exposure by placing the animals in the restraint tubes for gradually increasing periods of restraint time up to the maximum duration used on study.
Age and weight: at the initiation of dosing, the animals were approximately 10-11weeks old and weighed between 244-322 g for males and 166-223 g for females.
Number and groups: 6 animals will be used at each dose (3 animals/sex/group).
Identification: subcutaneously implanted microchip.
Animal housing: Animals will be housed 3 per cage by sex in appropriately sized suspended polycarbonate/polypropylene cages with stainless steel grid tops and solid bottoms.
Animal Enrichment: Animals were socially housed for psychological/environmental enrichment and were provided with items such as a hiding device (play tunnel) and a chewing object, except when interrupted by study procedures/activities.
Temperature: 19°C - 23°C
Relative humidity: 37 - 81 %,
Dark/light cycle : 12 h/12 h
Ventilation: at least 10 air changes/h
Feeding: SDS Rat and Mouse (modified) No. 1 Diet SQC Expanded.
Water: water ad libitum from the public supply from water bottles.
Veterinary care was available throughout the course of the study; however, no examinations or treatments were required.

Administration / exposure

Route of administration:
inhalation: dust
Type of inhalation exposure:
nose only
Vehicle:
clean air
Remarks on MMAD:
MMAD / GSD: Mass median aerodynamic (MMAD) and geometric standard deviation (GSD) results.
Gravimetric Particle Size Distribution
Group/ Aerosol Mass <2.8 µm MMAD (GSD)
Treatment (%) (µm)
2 Low Dose 41.9-48.4 2.75 (2.125) to 3.30 (1.919)
3 Intermediate Dose 53.0-68.4 1.96 (2.272) to 2.51 (1.854)
4 High Dose 30.3-45.5 3.06 (2.038) to 3.89 (1.968)
Particle size distribution data showed that the test aerosols were within the MMAD target range (1-3 µm) for 2/5 Group 2 and all Group 3 samples. For 3/5 Group 2 and all Group 4 samples the MMAD was estimated to be slightly higher than target. In these samples the mass aerosol was 41.9 to 44.0% and 30.3 to 45.5% smaller than 2.8 µm for Groups 2 and 4, respectively. Accordingly, a sizeable fraction of the test aerosol would be likely to deposit in the lower respiratory tract and although the particle size was larger, was reasonably comparable with the aerosols generated within the stated target range. Therefore, it was considered that the generated aerosols were respirable to the test species in all GGBS groups.
Details on inhalation exposure:
Inhalation exposures will be conducted in a room adjacent to, but separate from, the animal holding room. Exposures to the test aerosols will be performed using appropriately sized modular snout only stainless steel flow past systems. This exposure technique allows a continuous supply of test aerosol to be delivered to each animal; the biased flow ensures that there is no re-breathing of the test aerosol. Control animals will receive dry, filtered air only.
Separate inhalation exposure systems will be used for the delivery of control and test aerosols. Each inhalation exposure system will be located in an extract booth (for the protection of personnel and to prevent cross-group contamination) and will be operated to sustain a dynamic air flow sufficient to ensure an evenly distributed exposure aerosol. All air flow rates (delivered and extracted) will be monitored visually using calibrated flow meters. Chamber air flow rates, temperature and relative humidity levels will be monitored and recorded at regular intervals during each daily exposure period.
For inhalation exposure, the rats will be restrained in clear, tapered, polycarbonate tubes with an adjustable back-stop to prevent the animals from turning in the tubes. The animals’ snouts will protrude through the anterior end of the restraint tubes which will be connected to the exposure chamber by way of a push-fit through an inhalation port in the chamber wall. This exposure technique will be used to minimise concurrent exposure by the oral and dermal routes.
On the 2 days per week when animals are not dosed there will be no restraint procedures used.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The mass aerosol concentration of test aerosol in the animals’ breathing zone will be measured gravimetrically for all treated groups at regular intervals during each daily exposure period.
The aerodynamic particle size distribution (PSD) of the generated GGBS aerosols was assessed using a Marple 296 Cascade Impactor. Measurements were undertaken at least once per week over the dosing period of the study.
The mass median aerodynamic diameter (MMAD) and geometric standard deviation (GSD) of the test aerosols will be derived by Probit analysis (Finney, 1971; Ref 1).
Duration of treatment / exposure:
Target 6 hours
Frequency of treatment:
5 consecutive days / week for 4 weeks (5 or 6 days in Week 4) (20 or 21 exposures)
Doses / concentrations
Remarks:
Doses / Concentrations:
4 mg/m³, 8 mg/m³, 24 mg/m³
Basis:
nominal conc.
No. of animals per sex per dose:
12 males and 12 females (6 males and 6 females for main study, 6 males and 6 females for recovery)
Control animals:
yes
Details on study design:
The exposure concentrations were based on results obtained in previous rat toxicity studies of GGBS. A limit test of GGBS in male and female Wistar rats (acute inhalation toxicity study using the limit test design) given concentrations of 5235 mg GGBS/m³ (respirable solid aerosol of MMAD = 3.1 µm, GSD = 1.8) over 4 hours via inhalation did not lead to mortality (LC50 >5235 mg GGBS/m³). In a GLP-compliant pilot inhalation experiment, groups of male Wistar rats (strain Hsd Cpb:WU (SPF bred); N = 48/group) were nose-only exposed to high concentrations of GGBS once for 6 hours (Day 0). Exposure concentrations were 0, 10 and 250 mg/m³, i.e. 0, 10.3 and 230.1 mg/m³ as actual aerosol concentrations. The particle size distributions of the inhaled GGBS showed that the substance consisted mainly of respirable dust. The special feature of the acute inhalation study of GGBS was a target post-exposure period of 90 days with 3 interim sacrifices on post-exposure Days 1, 7 and 28. No animal was found dead during the course of the study and inhalation exposure to GGBS induced no clinical signs or body weight changes. After the single exposure to 230.1 mg/m³, there was already evidence for particle overload based on changes of cell counts in BAL fluid of GGBS-treated animals and organ weight changes. At the single exposure concentration of 10.3 mg/m³, there were also changes in BAL (on Days 1, 7 and 28) after the single dose (Bayer HealthCare Study No T3081617) but no evidence of particle overload.
Based on these results, a high exposure concentration of 24 mg/m³ was chosen for this study with repeated daily dosing (5 days a week) and a total of 20 exposure days to GGBS.
The high concentration of 24 mg/m³ represents 8.0 times the lung exposure in humans at the occupational exposure ‘limit value’ for respirable particles (in Europe the lowest valid value) of 3 mg/m³ of air at the workplace (average value per shift) for the respirable fraction of poorly soluble dusts or 2.4-times the lung exposure in humans at the general limit for inhalable dust (10 mg/m³ of air). With respect to the interspecies comparison of the lung burden, that is the dose of GGBS being deposited in the deep lungs, the high dose of 24 mg/m³ translates to a lung burden of approximately 0.168 mg GGBS/g lung tissue in rats and thus represents 6.0-times the estimated lung burden in humans (approximately 0.028 mg/g human lung tissue) at the 3 mg/m³ ‘limit value’ for poorly soluble dusts at the workplace.
Along with all poorly soluble particles, GGBS was expected to have a long retention half-time. In general, lung pathology related to undissolved material depends on the quantity of material deposited in the lungs over the time. As GGBS is a relatively inert particulate matter there may be accumulation of GGBS in the lungs of rats after repeated exposure towards the end of the 28-day treatment period. The cumulative exposure concentration at 24 mg/m3 is 480 mg/m³ after 20 exposure days, corresponding to 3.36 mg/g lung tissue. Particle-overloading conditions, defined as >1 mg/m³ particle mass per gram lung (Jones and Neef 2012, Pauluhn 2009), may be attained at exposure levels of 24 mg/m³ and above; however, severe toxicities or mortality were not expected to occur at the high exposure concentration of 24 mg/m³.
The low exposure concentration of 4 mg/m³, that translates to 0.028 mg/g lung tissue (cumulative exposure concentration of 80 mg/m³ after 20 exposure days, corresponding to 0.56 mg/g lung tissue) in rats, represents the lung exposure in humans at the ‘above described value’ of 4 mg/m³ of air at the workplace for the respirable fraction of poorly soluble dusts.
The mid exposure concentration of 8 mg/m³ is the geometric mean of the high and the low exposure concentration. It translates to 0.056 mg/g lung tissue (cumulative dose of 160 mg/m³ after 20 exposure days and corresponds to 1.12 mg/g lung tissue in rats.
Detailed clinical observations were carried out weekly during the pre-trial, exposure and recovery periods commencing from pre-trial Week 1. Animals were removed from their housing and examined.
On exposure days all animals were examined for reaction to treatment once before start of exposure, intermittently during exposure (approximately once every hour), on completion of exposure (up to 30 min post dose) and approximately 1-2 h post exposure. The onset, intensity and duration of these signs were recorded (if appropriate).
On non-exposure days (2 days per week) all animals were removed from their housing and examined at least once

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Mortality/moribundity checks were perfomed twice a day.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Detailed clinical observations were carried out weekly during the pre-trial, exposure and recovery periods commencing from pre-trial Week 1. Animals were removed from their housing and examined.

BODY WEIGHT: Yes
- Time schedule for examinations: Body weights were recorded once pre-trial (Day -7) and weekly during the exposure and recovery periods.

FOOD CONSUMPTION:
- Food consumption was quantitatively measured weekly during the exposure and recovery periods.

WATER CONSUMPTION: Yes
- Time schedule for examinations: Water consumption was monitored by visual inspection of the water bottles on a regular basis through the study period.

OPHTHALMOSCOPIC EXAMINATION: Yes
The eyes were examined using an indirect ophthalmoscope following application of a mydriatic agent (1% Tropicamide, Mydriacyl®). The anterior, lenticular and fundic areas were examined for all animals pre-trial and for Group 1 and 4 animals during Week 4.

HAEMATOLOGY and CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Blood samples were collected from all main study animals via orbital sinus on the same day of, but prior to, necropsy. Blood samples were collected from recovery study animals intravenously on the same day of, but prior to necropsy following the recovery period
- How many animals: 48 males and 48 females (6 males and 6 females for main study per dose, 6 males and 6 females for recovery per dose)
- Parameters checked in table [2] and [3] were examined.

URINALYSIS: Yes
- Time schedule for collection of urine: Urine samples were collected overnight over a period of approximately 16-18 hours from all main study animals which were individually housed in metabolism cages with access to food and water.
- Parameters checked in table [4] were examined.

OTHER: Bronchoalveolar Lavage Sample Analysis
- How many animals: Samples were collected from all animals.
Sacrifice and pathology:
All main study and recovery animals were subjected to a complete necropsy examination, which included evaluation of the carcass and musculoskeletal system; all external surfaces and orifices; cranial cavity and external surfaces of the brain; and thoracic, abdominal, and pelvic cavities with their associated organs and tissues. Scheduled necropsy examinations were conducted by a trained technician and consisted of an external and internal examination and recording of observations for all animals. A veterinary pathologist was available for consultation during normal working hours.
Statistics:
Means and standard deviations were calculated for selected inhalation parameters.
Unless otherwise stated, all statistical tests were two-sided and performed at the 5% significance level using in-house software. Males and females were analysed separately.
Pairwise comparisons were performed against the control group (Group 1).
Body weight, haematology, coagulation, clinical chemistry, selected urinalysis and BAL data were analysed for homogeneity of variance using the ‘F Max' test. If the group variances appear homogeneous, a parametric ANOVA was used and pairwise comparisons were made using Fisher’s F protected LSD method via Student's t test i.e. pairwise comparisons were made only if the overall F test is significant. If the variances are heterogeneous, log or square root transformations were used in an attempt to stabilise the variances. If the variances remained heterogeneous, then a Kruskal-Wallis non-parametric ANOVA was used and pairwise comparisons were made using chi squared protection (via z tests, the non-parametric equivalent of Student's t test).
In circumstances where it was not possible to perform the F Max test due to zero standard deviation in at least one group, the non-parametric ANOVA results were reported.
Organ weights were analysed using ANOVA as above and by analysis of covariance (ANCOVA) using terminal kill body weight as covariate. In addition, organ weights as a percentage of terminal body weight were analysed using ANOVA as above as an exploratory analysis. The results of this analysis were only presented in the study report if required to aid interpretation.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Description (incidence and severity):
No premature deaths were noted on study.
Mortality:
no mortality observed
Description (incidence):
No premature deaths were noted on study.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
No body weight changes were noted which could be attributed to exposure to GGBS.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
No changes in food consumption were noted which could be attributed to exposure to GGBS.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
No visually notable difference between control and groups exposed to GGBS were noted during the study.
Ophthalmological findings:
no effects observed
Description (incidence and severity):
No ophthalmic changes were noted in Week 4 which could be attributed to exposure to GGBS. No significant findings were noted which differed from pre-trial findings.
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
A statistically significant dose level related increase in neutrophil counts was noted in males.
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
Any differences in clinical chemistry parameters were considered due to individual variation, lacked a true dose-level relationship or exhibited a low magnitude or incidence and were therefore considered unrelated to exposure to GGBS.
Urinalysis findings:
no effects observed
Description (incidence and severity):
Any differences in urinalysis parameters were considered due to individual variation, lacked a true dose-level relationship or exhibited a low magnitude or incidence and were therefore considered unrelated to exposure to GGBS.
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
No test item-related lung or other organ weight changes were noted.
Gross pathological findings:
no effects observed
Description (incidence and severity):
No test item-related gross findings were noted.
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Results can be found in: "Any other information on results incl. tables"
Details on results:
HAEMATOLOGY
At Day 27, a statistically significant dose level related increase in neutrophil counts was noted at ≥8 µg/L in males (x 1.62 and x 1.77 of control, respectively).
After 13 weeks of recovery, an increase in neutrophil counts was still evident in at ≥4 µg/L in males (x 1.59, x 1.59 and x 1.65 of control, respectively). This increase was not seen in females.
Any other differences in haematology parameters were considered due to individual variation, lacked a true dose-level relationship or exhibited a low magnitude or incidence and were therefore considered unrelated to exposure to GGBS.

OTHER FINDINGS
Coagulation
Any differences in coagulation parameters were considered due to individual variation, lacked a true dose-level relationship or exhibited a low magnitude or incidence and were therefore considered unrelated to exposure to GGBS.

Bronchoalveolar lavage analysis
Following 4 weeks of exposures, the following findings were noted in bronchoalveolar lavage samples: A statistically significant increase in white blood cell count and neutrophil counts were seen at 24 µg/L in males and females when compared with controls. Neutrophil counts were raised at 8 µg/L in males and females, which reached statistical significance in females. An increase in macrophage levels were also seen at 24 µg/L in males (statistically significant) and females (x 3.3 and x 3.1 of controls respectively). No other differences from controls were noted in BAL fluid.
Changes still evident following 13 week recovery period were: increased white blood cell count at 24 µg/L in males and females (x 2.3 and x 2.6 of controls), increased neutrophil counts at ≥ 8 µg/L in males and females (statistically significant). An increase in macrophage levels were also seen at 24 µg/L in males and females (x 2.0 and x 2.2 of controls respectively). These changes were evident but of a lesser magnitude following the recovery period. A statistically significant increase was noted in total protein ≥ 8 µg/L in females, however due to variation in individual values over all groups these differences were considered unrelated to exposure with GGBS.
No other differences from controls were noted in BAL fluid.

Effect levels

Dose descriptor:
NOAEL
Effect level:
24.9 mg/m³ air
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: clinical observations; mortality; body weight; food consumption; water consumption; ophthalmoscopic examination; haematology; coagulation; clinical chemistry; urinalysis; bronchoalveolar lavage analysis; gross pathology; organ weights; histopathology

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

HISTOPATHOLOGY

Summary Microscopic Findings – Scheduled Euthanasia Animals (Day 27/28)

 

Males

Females

Group

1

2

3

4

1

2

3

4

Target Concentration (mg/L)

0

4

8

24

0

4

8

24

No. animals examined

6

6

6

6

6

6

6

6

Lung (No. examined)

6

6

6

6

6

6

6

6

Inflammatory cell infiltration, mononuclear cell

1

0

1

6

0

1

1

3

         Minimal

1

0

1

5

0

1

1

3

         Mild

0

0

0

1

0

0

0

0

Macrophage aggregates

0

1

0

3

0

0

0

1

         Minimal

0

1

0

2

0

0

0

1

      Mild

0

0

0

1

0

0

0

0

Pigmented macrophages

0

0

0

1

0

0

0

0

      Minimal

0

0

0

1

0

0

0

0

Other microscopic findings observed were considered incidental, of the nature commonly observed in this strain and age of rat, and/or were of similar incidence and severity in control and treated animals and, therefore, were considered unrelated to inhalation administration of GGBS.

Summary Microscopic Findings – Scheduled Euthanasia Animals (Day 120/121)

 

Males

Females

Group

1

2

3

4

1

2

3

4

Target Concentration (mg/L)

0

4

8

24

0

4

8

24

No. animals examined

6

6

6

6

6

6

6

6

Lung (No. examined)

6

6

6

6

6

6

6

6

Inflammatory cell infiltration, mononuclear cell

1

1

2

3

1

1

0

2

         Minimal

1

1

2

3

1

1

0

2

Macrophage aggregates

1

0

2

2

1

0

2

1

         Minimal

1

0

2

2

1

0

2

1

Pigmented macrophages

0

5

3

2

0

1

2

2

The increased incidence of minimal to mild inflammatory cell infiltration composed of mononuclear cells could be considered as part of background spontaneous pathology. Given the absence of significant inflammation and tissue destruction and the lack of a clear dose related effect macrophage aggregates and pigmented macrophages could be considered as an adaptive process (Nikula et al., 2014). 

Other microscopic findings observed were considered incidental, of the nature commonly observed in this strain and age of rats, and/or were of similar incidence and severity in control and treated animals and, therefore, were considered unrelated to inhalation administration of GGBS.

Applicant's summary and conclusion

Conclusions:
In conclusion, exposure of rats to aerosolised GGBS for 4 weeks (6 hours (nose-only) exposure per day for 5 days per week (5 or 6 days in Week 4)) was associated with increased neutrophil counts in blood, increased neutrophil, white blood cell and macrophage counts in BAL and microscopic findings in the lung (inflammatory cell infiltration, macrophage aggregates, and pigmented macrophages) at a measured aerosol exposure concentration of 24.9 µg/L (target concentration 24 µg/L). Changes observed were partially reversible following a 13 week recovery period.
Executive summary:

Male and female rats were exposed to solid GGBS aerosol at measured concentrations of 4.3, 9.5 or 24.9 µg/L for 6 hours per day for 5 days per week over 4 weeks (5 or 6 days in Week 4). Particle size distribution measurements indicated that the test aerosols were within the respirable range for rats and that the experiment could be considered valid. 

Elevated neutrophil counts seen in blood and BAL fluid and elevated white blood cell and macrophages in BAL at 24mg/L could be correlated with the microscopic findings (inflammatory cell infiltration, macrophage aggregates, and pigmented macrophages) observed in the lung. 

As haematology, BAL fluid and microscopic changes were also evident following 13 weeks of recovery (albeit to a lesser magnitude), this suggests an adaptive response which was partially reversible at the intermediate and high dose levels. Pigmented macrophages in the lung, observed in the low dose group following recovery, were not accompanied by any inflammatory findings in blood, BAL or other histopathology and their presence was therefore considered to be adaptive and non-adverse. 

No evidence of potential cell injury (lactate dehydrogenase), effects on the alveolar-capillary barrier (total protein), lysosomal instability (b-N-acetyl-glycosaminidase) were observed in the BAL fluid which would indicate any toxicity to exposure to GGBS. 

Overall, data suggests that there is no likely acute inhalation hazard due to exposure to GGBS up to 24 µg/L. There were no deaths or adverse clinical signs during the study period. No evidence of lung injury was seen. Minimal to mild changes noted in BAL fluid indicative of macrophage-mediated particle clearance and unspecific findings in the lung reflect the time-dependent physiological response of the body to inhaled mineral particulates of low solubility. There was a trend towards reversibility. At 24 µg/L the histopathological changes in the lung resembled pulmonary macrophage clearance, suggesting that the inhaled particulate is processed by alveolar macrophages and mononuclear inflammatory cells at the high level concentration.