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Repeated dose toxicity: inhalation

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Administrative data

Endpoint:
sub-chronic toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
21.02.2013-31.08.2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Robust study with silicon according to OECD test guidelines.
Cross-referenceopen allclose all
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2014
Report Date:
2014

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)
Deviations:
yes
Remarks:
No ophthamolgy or urine analysis.
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
- Name of test material (as cited in study report): Jetmilled Silicon

- Physical state: Grey-brown powder
- Analytical purity: > 99.5% Si
:
- Lot/batch No.: J-257
- Expiration date of the lot/batch: Dec 13, 2018

- Storage condition of test material: Room temperature, at a dark place
- Other:
Particle size distribution: d10=1.3, d50=3, d90=7.4 (laser diffraction)
BET surface area 4.5-5.1 m2/g

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:

- Diet (e.g. ad libitum): standard laboratory pellet diets, commercial chow in pellet form used, identified as ssniff "V1534", purchased from ssniff Spezialdiäten GmbH, Soest, Germany
- Water (e.g. ad libitum): tap water ad libidum
- Acclimation period: Rats exposed to the test item by nose-only inhalation. For a period of 2 - 3 weeks prior to exposure animals will be trained to become accustomed to nose-only tubes.

TEST ANIMALS
- Source: Wistar rats, strain Crl:WI (Han), purchased from Charles River Deutschland (Sulzfeld, Germany)
- Age at study initiation: approximately 8 weeks
- Weight at study initiation: males approximately 250 grams, females approximately 175 grams
- Fasting period before study: no
- Housing: Makrolon® (polycarbonate) cages type III, two rats per cage.Lignocel BK 8-15' absorbing softwood bedding material, changed twice a week or more often if necessary.
- Diet: commercial chow in pellet form: Ssniff "V1534", purchased from ssniff Spezialdiäten GmbH, Soest, Germany ad libitum
- Water (e.g. ad libitum): Tap water ad libitum
- Acclimation period: The animals were first allowed to adjust and become acclimatized to the environment for one day. During 2-3 weeks prior to exposure start, all rats were trained to the 6-h restraint in nose-only tubes.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2 C
- Humidity (%): 55% ± 15%
- Photoperiod (hrs dark / hrs light): 12 h light/dark

IN-LIFE DATES: From: 1 day to 365 days

Administration / exposure

Route of administration:
inhalation: aerosol
Type of inhalation exposure:
nose only
Vehicle:
air
Remarks on MMAD:
MMAD / GSD: Particle size of silicon < 50 µm; PSD (particle size distribution) (µm) of silicon =d10 = 1.3; d50 = 3; d90 = 7.4 (laser diffraction).
MMAD approx. 2.6 µm. Mean GSD 1.9.

MMAD approx. 1.4 µm (GSD: 1.8) for positive control Quartz DQ12
Details on inhalation exposure:
- Exposure apparatus: The test item was administered to the test animals by nose-only inhalation, using a flow-past nose-only inhalation exposure system.

- Method of holding animals in test chamber: The rat nose is located at the front end of a tube being connected to a cylinder delivering the aerosol. Through the thin pipes, the aerosol was supplied to each rat nose individually and exhaled air was drawn off immediately by a cylinder surrounding the aerosol delivering cylinder.
The exposure tubed were arranged around a cylinder capable to take up 16 tubes per platform. The position of exposuretubes of tats at the cylinder was changed daily to minimize exposure differences due to geometry.

- System of generating particulates/aerosols: The particulate sample aerosols were generated by dispersing the dry powder. Dispersion was achieved by a feeding system and a high-pressure, high-velocity pressurized air dispersion nozzle. For each nose-exposure unit, the aaerosols were generated by a high-pressure pneumatic disperser, fed with the silicon powder under computerized control.

- Temperature, humidity, pressure in air chamber: 22° C + 2° C for temperature and 55 % + 15 % for relative humidity

- Air flow rate: laminar airflow to each rat of approximately 1 l/min
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The actual aerosol concentrations were measured daily using a photometer (gravimetric analysis of filter samples). The mean concentrations were very close to the target concentrations, being 104%, 100% and 101% in the low, mid, and high dose groupes, respectively.
Duration of treatment / exposure:
13 weeks of exposure




Frequency of treatment:
6 hours/day, 5 days/week
Doses / concentrations
Remarks:
Doses / Concentrations:
1, 4 and 16 mg/m3 for silicon and 1 mg/m³ for the positive control
Basis:
nominal conc.
No. of animals per sex per dose:
15 per dose (incl. control) per sex, 15 per recovery period (3, 6, 12 months) for highest dose and positive control
Control animals:
yes
Details on study design:
- Dose selection rationale: Doses were selected based on a range-finding study (Fraunhofer 2013)
- Rationale for animal assignment: The animals were allocated to groups based on body weight, in order to ensure that all animals in the groups had body weights deviating not more than approximately 20% from the mean weight of that group (male/female) at that time.

- Examinations of all groups 1 day after the cessation of the 90-day exposure period.
- Post-exposure recovery period in satellite groups: Animals from the high-dose silicon group (16 mg/m3) and the positive control group were followed up during one year; the sacrifice time-points being 91, 182 and 365 days upon cessation of the 90-day exposure.
Positive control:
- Quartz DQ12
- Obtained from Bergbauforschung, Essen, Germany; lot #DQ12<5µm/AB/1985
- Expiration date 31 Dec 2015
- 87% crystallinity
- MMAD approximately 1.4 µm
- Exposure: 1 mg/m3 for 90 days, following the same protocol as for silicon-groups. Follow up: 91, 182 and 365 days.

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least once daily


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once per week

BODY WEIGHT: Yes
- Time schedule for examinations: Twice a week in the first 4 weeks (only rats that were sacrificed day 1 after end of exposure) and once a weeke thereafter throughout the studt for all animals.

FOOD CONSUMPTION:
- Food consumption was recorded weekly during the whole study- and follow-up period using 10 male animals per dose group.

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION: Yes
- Time schedule for examinations: weekly during the whole study- and follow-up period using 10 male animals per dose group.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Day 1 after the end of exposure
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes, 16 h
- How many animals: 10
- Parameters checked
Red blood cells (RBC) - T/l Total white blood cells (WBC) - G/l
Hemoglobin (HB) - mmol/l Differential white cell count (% and *absolute)
Hematocrit (HCT) - % Platelets (PTL) - G/l
Reticulocytes (RET) - G/l
* Mean cell volume (MCV) - fl
* Mean hemoglobin/erythrocyte (MCH) -fmol
* Mean hemoglobin concentration/erythrocyte (MCHC) - mmol/l
Prothrombin time (PT) sec

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Day 1 after the end of exposure
- Animals fasted: Yes, 16 h
- How many animals: 10
- Parameters checked
Aspartate aminotransferase (AST) EC 2.6.1.1.-U/l
Alanine aminotransferase (ALT) EC 2.6.1.2.-U/l
Alkaline phosphatase (AP) - U/l
γ-Glutamyl transpeptidase, (GGT) EC 2.3.2.2.-U/l
Urea - mmol/l
Creatinine (CREA) -µmol/l
Total protein (TP) -g/l
Albumin (ALB) -g/l
*Globulin (GLB) -g/l
*ALB/GLB (A/G)
Glucose (GLUC) -mmol/l
Cholesterol (CHOL) - mmol/l
Sodium (Na) -mmol/l
Calcium (Ca) -mmol/l
Potassium (K) -mmol/l
Phosphorus (PO4) -mmol/l
Chloride (CL) -mmol/l

URINALYSIS: No


NEUROBEHAVIOURAL EXAMINATION: No


OTHER:
BRONCHOALVEOLAR LAVAGE (BAL)
5 male and 5 female rats per group and sex at days 1, 91, 182 and 365 post-exposure
Cytological parameters:
-total cell count
- differential cell count
Biochemical parameters:
- LDH
- B-glucuronidase
- TGF-B1
- IL-8

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
-external surface of the body
- all orifices
- cranial, thoracic and abdominal cavities

HISTOPATHOLOGY: Yes
- 10 animals per group and sex at days 1, 91, 182 and 365.
- All animals of all groups: histopathology of left lung lobes, including bronchi and the lung-associated lymph nodes, trachea, larynx, pharynx and the nasal cavities.
- Only for the control group (air only), 16 mg/m3 silicon group, and positive control (quartz DQ12), at 1, 91, 182 and 365 days histopathological examination only if needed, e.g. as indicated by macroscopical findings:
adrenal gland
aorta
bone marrow
cecum
cerebellum
cerebrum
coagulating gland
colon
duodenum
ear
esophagus
femur
forestomach
glandular stomach
heart
hematopoietic tissue
ileum
jejumum
joint
kidney
liver
lymph node, mandibular
lymph node, mesentheric
mammary gland
ovary
pancreas
parathyroid gland
peripheral nerve
pituitary gland
prostate
rectum
salivary gland, nos
seminal vesicle
sceletal muscle
skin
spinal cord
spleen
sternum
testis
thymus
thyroid gland
urinary bladder
uterus
Other examinations:
Retention of the Test/Reference Item (non-GLP)
Lungs of the male and female groups (right lobes) in all exposure groups underwent a chemical analysis to verify the predicted retained mass of the test item and the reference item Quartz DQ12.

Hydroxyproline analysis
For collagen anaöysis the hydroxyproline concentration in lung tissue was determined.
Statistics:
Data analyzed using analysis of variance. Differences between groups considered statistically significant at p < 0.05.
The means of the treated groups were compared with the means of the control groups using Dunnett's test (if the group means differed significantly by the analysis of variance).
Satistical evaluation of the histopathological findings with two-tailed Fisher test by the PROVANTIS system.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
increased lung weight
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
very mild inflammation
Histopathological findings: neoplastic:
effects observed, treatment-related
Description (incidence and severity):
All findings considered as incidental and unrelated to silicon exposure
Details on results:
CLINICAL SIGNS AND MORTALITY
No significant effects on clinical parameters. No mortality.

BODY WEIGHT AND WEIGHT GAIN
No significant effects.

FOOD CONSUMPTION
No significant effects

WATER CONSUMPTION
No significant effects

HAEMATOLOGY
No significant effects

CLINICAL CHEMISTRY
No significant effects

ORGAN WEIGHTS
Lung weights significantly increased on day 1 after exposure in the 16 mg/m3 Si and the quartz group among males and in the 4 and 16 mg/m3 Si and the quartz group among females. During the recovery period, significantly increased values were only seen in the quartz group.

GROSS PATHOLOGY
Test-item or dose-related macroscopical findings were not observed.

HISTOPATHOLOGY: NON-NEOPLASTIC
- No statistically significant effects in the 1 and 4 mg/m3 groups at the end of the exposure period. There was no follow-up of these groups.

- 16 mg/m3 group, after exposure (day 1): Alveolar granulocyte infiltration, interstitial mononuclear cell infiltration (very slight (minimal) inflammatory effects)

- 16 mg/m3 group, 13 weeks (91 days) of recovery: Interstitial mononuclear cell infiltration (very slight (minimal) inflammatory effects). In addition, two cases of very slight interstitial fibrosis in the lungs (not statistically significant from control group).

- 16 mg/m3 group, 26 weeks (192 days) of recovery: Interstitial mononuclear cell infiltration observed in all animals (very slight (minimal) inflammatory effects). In addition, six cases (out of 20 examined rats) of very slight interstitial fibrosis in the lungs (not statistically significant from control group).

- 16 mg/m3 group, 52 weeks (365 days) of recovery: Interstitial mononuclear cell infiltration observed in all animals (very slight (minimal) inflammatory effects). Chronic granulomatous inflammation and fibrosis (not statistically significant) of the bronchus-associated lymphoid tissue. Interstitial fibrosis (very slight) of the lung (significant) including the pleura (not statistically significant). Chronic granulomatous inflammation of the lung-associated lymph nodes. Fibrosis (very slight) in the lung-associated lymph nodes.

- Common spontaneous non-neoplastic changes were observed in various organs of silicon-exposed animals (as well as in controls and quartz-exposed animals) of the 91, 182 and 365 day follow up groups. All those were regarded incidental and usual for rats of this strain and age and unrelated to particle-exposure.

HISTOPATHOLOGY: NEOPLASTIC
In the follow-up after 365 days, three malignant tumours were observed among animals exposed to silicon (16 mg/m3): one malignant lymphoma in a male rat arsising from the lung-associated lymph nodes, one malignant scwannoma originating in the mediastinum and one hemangiosarcoma if a meseteric lymph node.
Two types of benign tumour were found in the 16 mg/m3 silicon group (365 days): three animals with adenomas of the pars distalis in the pituitary gland and 2 animals with endometrial stromal polyps of the uterus.
All neoplastic findings were considered incidental and usual for rats of this strain and age and unrelated to particle-exposure.


OTHER FINDINGS
BAL fluid: increased amounts of polymorphonuclear cells (neutrophils) and lymphocytes in 16 mg/m3 silicon group at days 1 and 91 after exposure, but not at later time points. The effects were not considered as adverse.

The inhalation exposure resulted in a moderate lung overload situation (for details, see 7.1 Toxicokinetics).

Effect levels

open allclose all
Dose descriptor:
NOAEC
Effect level:
4 mg/m³ air
Sex:
male/female
Basis for effect level:
other: histopathology
Dose descriptor:
LOAEC
Effect level:
16 mg/m³ air
Sex:
male/female
Basis for effect level:
other: histopathology

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
Repeated inhalation of silicon particles for 90 days did not induce any severe adverse effects in rats. Alveolar granulocyte infiltration and interstitial mononuclear cell infiltration, indicating very slight inflammatory effects, were however seen at the highest dose level (16 mg/m3). The effects were persistent during the one-year follow-up period. Furthermore, a progressive granulomatous inflammation and fibrosis were observed in the 16 mg/m3 silicon group during the long-term follow-up. 4 mg/m3 was identified as the NOAEC. The effects observed with silicon were significantly milder than in the positive control group exposed to quartz DQ12.
Executive summary:

A 90-day nose-only inhalation study with silicon particles (MMAD 2.6 um) was performed in Wistar rats according to OECD 413 (Fraunhofer ITEM 2014), with exposures 6 hours/day, 5 days/week during 13 weeks. The doses used in the test were 1, 4 and 16 mg/m3 of silicon and they were selected on the basis of a range-finding study. Quartz DQ12 (1 mg/m3) was used as a positive control. The rats were exposed nose-only six hours/day, five days/week during 13 weeks. All groups were examined on day 1 after end of exposure (day 1). In addition, animals exposed to 16 mg/m2 silicon or 1 mg/m3 quartz were examined after recovery periods of 13 weeks (day 91), 26 weeks (day 182) and 52 weeks (day 365). The highest silicon exposure concentration (16 mg/m3) resulted in moderate lung overload.

The results showed no effects indicating systemic toxicity in animals exposed to silicon and all statistically significant local effects observed were of minimal/very slight severity. Silicon caused no significant effects on body weight, haematology or clinical chemistry. Lung weights were significantly increased on day 1 in males of the 16 mg/m3 silicon group and in the quartz group and among females in the 4 and 16 mg/m3 silicon groups and in the quartz group. During the recovery period, significantly increased lung weights were only seen in the quartz group.

No statistically significant effects were observed in the 1 and 4 mg/m3 groups at the end of the exposure period.

One day after the end of exposure, alveolar granulocyte infiltration and interstitial mononuclear cell infiltration, indicating very slight (minimal) inflammatory effects, were observed in the 16 mg/ m3 group. The inflammation persisted during the recovery period.

A statistically significant increase of polymorphonuclear neutrophils in bronchoalveolar lavage fluid was observed in animals of the 16 mg/m3 group at the end of exposure. 13 weeks later, neutrophils were still observed, but at decreasing concentrations. In addition, upon 13 weeks of recovery, two cases of very slight interstitial fibrosis were observed in the lungs of rats from the 16 mg/m3 group. After 26 weeks of recovery, fibrosis was detected in six rats. One year after the end of the exposures, the statistically significant findings in the 16 mg/m3 recovery group included interstitial mononuclear cell infiltration (observed in all animals; very slight (minimal) inflammatory effects), interstitial fibrosis (very slight) of the lungs, granulomatous inflammation of the bronchus-associated lymphoid tissue (very slight to slight) and of the lung-associated lymph nodes (slight to severe), and fibrosis of the lung-associated lymph nodes (slight to moderate). In addition, very sight pleural fibrosis was observed in five animals (out of 20 examined rats; not statistically significant from control group).

The effects observed in the quartz DQ12 group (1 mg/m3) were much more severe than in the silicon 16 mg/m3 group. Fibrosis, hypertrophy, and alveolar lipoproteinosis occurred already at day 1 after the end of the exposure, and progressed within the one-year recovery period. In addition to the effects detected earlier, necrosis was observed in the lung-associated lymph nodes of animals examined 26 and 52 weeks after the end of the exposure.

The NOAEC for silicon was 4 mg/m3and the LOAEC 16 mg/m3.