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Repeated dose toxicity: inhalation

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Endpoint:
sub-chronic toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
experimental phase: Feb. 12, 2018 - May 29, 2019; study completion date: September, 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
Please see also attached communication resp:
ECHA decision dated 11 March 2015 [decision number: SEV-D- 2114297905-32-01/F],
as amended by the Board of Appeal in its decision dated 30 June 2017 (A-015-2015)
Cross-reference
Reason / purpose:
reference to other study
Reference
Endpoint:
sub-chronic toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
other information
Study period:
experimental phase: Feb. 12, 2018 - May 29, 2019; study completion date: September, 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
Please see also attached communication resp:
ECHA decision dated 11 March 2015 [decision number: SEV-D- 2114297905-32-01/F],
as amended by the Board of Appeal in its decision dated 30 June 2017 (A-015-2015)
Reason / purpose:
reference to other study
Qualifier:
according to
Guideline:
OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)
Version / remarks:
2017
Deviations:
yes
Principles of method if other than guideline:
Deviations from the guideline: This study was conducted according to an ECHA decision (dated June, 30 2017). This decision skipped the endpoints clinical pathology and ophthalmology. Gross pathology and histopathology shall be conducted on the lungs, trachea, naso-pharyngeal tissues, nasal-associated lymphoid tissue (NALT) and larynx; other organs and tissues were excluded from examination. As an addition, collagen was analysed.
GLP compliance:
yes
Specific details on test material used for the study:
Test material 1
Name: SAS1 - High BET; BET: Approx. 400 m²/g (high surface area); Purity: > 99.8%; Molecular formula: SiO2; Molecular weight: 60.08 g/mol;
Manufacturer: CABOT Corporation, Tuscola IL, USA; CAS number: 7631-86-9, 112945-52-5; CAS name: Silica, amorphous, fumed, crystalline-free
EC number: 231-545-4; Date of delivery: January 4, 2018; Expiration date: September 19, 2019
Species:
rat
Strain:
Wistar
Details on species / strain selection:
Male and female Wistar rats [strain Crl:WI(Han)] were purchased from Charles River Deutschland (Sulzfeld, Germany).
Wistar rats are commonly used in subchronic and chronic inhalation toxicity studies. They fulfil the criteria stated by a U.S. EPA Workshop (Vu et al., 1996) such as (i) a low background rate of neoplasia, (ii) a low background rate of pulmonary disease, (iii) longevity, and (iv) a history of laboratory use.
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland (Sulzfeld, Germany).
- Females (if applicable) nulliparous and non-pregnant: [yes/no]
- Age at study initiation: approx. 8-9 weeks
- Weight at study initiation: approx. 250 gram for males and approx. 175 gram for females
- Fasting period before study:
- Housing: Makrolon (polycarbonate) cages type IV, two rats of the same sex per cage, and were maintained under conventional laboratory conditions. Cages and absorbing softwood bedding material (Lignocel BK8-15) were changed twice a week or more often, if necessary.
- Diet: commercial chow in pellet form was used, identified as Ssniff V1534, purchased from Ssniff-Spezialdiäten (Soest, Germany). The diet was stored in room T1.015 at a temperature of max. 18 °C and a relative humidity of max. 70 %. No batch of diet was used after 6 months have elapsed from manufacturing date. The diet was offered fresh weekly or more often, if necessary.
- Water: Tap water from the Hannover city water supplier was offered fresh weekly or more often, if necessary, in a Makrolon bottle fitted with a stainless steel nipple top with a hole approximately 0.5 mm in diameter.
- Acclimation period: for a period of at least 3 weeks prior to exposure animals were trained to become accustomed to nose-only tubes


ENVIRONMENTAL CONDITIONS
In the cages the rats find paperboard crinklets as nesting material and wooden blocks for nibbling. The temperature and the relative humidity of the animal room were monitored electronically and recorded on a continuous basis
- Temperature (°C): 22 °C ± 2 °C
- Humidity (%): 55 % ± 15 % for relative humidity
- Air changes (per hr): at least 10 times per hour light/dark cycle
- Photoperiod (hrs dark / hrs light): A 12-hour light/dark cycle was used controlled by an automatic timing device

IN-LIFE DATES: From: To:
Route of administration:
inhalation: aerosol
Type of inhalation exposure:
nose only
Vehicle:
clean air
Mass median aerodynamic diameter (MMAD):
<= 3 µm
Geometric standard deviation (GSD):
3.53
Remarks on MMAD:
The photometer gives a scattering light signal which is proportional to the particle concentration, if the particle size distribution is constant.
Using this system a mass median aerodynamic diameter (MMAD) of ≤ 3 µm was achieved as required by the ECHA decision.
Details on inhalation exposure:
The two test item aerosols were generated by dispersing the dry powder. Dispersion was achieved by a feeding system and pressurized air dispersion nozzle (dynamic system). For each nose-only exposure unit (total of 8 units for groups 2 - 9), the aerosol was generated by a pneumatic disperser. The disperser was fed with the test item under computerized control, i.e. with a feedback loop to a scattering signal from an aerosol photometer which reflects the actual aerosol concentration.
The aerosol was given to the rats by a flow-past nose-only inhalation exposure system which was used for previous particle and fiber inhalation studies at Fraunhofer ITEM. In this system, aerosols were supplied to each rat individually, and exhaled air is immediately exhausted. The airflow to each rat was approximately 1 L/min which is calculated to be laminar. Therefore measurement of the oxygen concentration is not necessary. Prior to the 90-day exposure of rats, technical trials to adjust particle size distributions and exposure levels were conducted.
For exposure to the test item the rats were restrained in acrylic tubes with a flexible stopper. The exposure tubes are arranged around a cylinder capable to take up 16 animals per level on four levels. The rat nose is located at the front end of a tube connected to the inner cylinder of the exposure unit delivering the aerosol. Through thin pipes, the aerosol is supplied to each rat nose individually and exhaled air is drawn off immediately by a cylinder surrounding the aerosol delivering cylinder. The position of individual rat at the cylinder is changed daily according to a rotation plan to minimize exposure differences due to geometry. The exposure units (including the clean air control: 9 units) are located each under a separate hood to prevent cross contamination among the different dose groups.
The duration of exposure was 6 hours/day, 5 days/week for 13 weeks.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Filter samples of the aerosols were taken at least twice per week to control the aerosol concentrations and to adjust the aerosol photometer readings. These samples were collected at a port of the nose-only exposure unit, thus, under the same conditions the rats are inhaling the aerosol and will be analysed gravimetrically.
Duration of treatment / exposure:
13 weeks
Frequency of treatment:
6 h / d
5 d / w
Dose / conc.:
0.5 mg/m³ air (nominal)
Dose / conc.:
1 mg/m³ air (nominal)
Dose / conc.:
2.5 mg/m³ air (nominal)
Dose / conc.:
5 mg/m³ air (nominal)
No. of animals per sex per dose:
10 (sacrified 1 day after treatment);
5 each (sacrified 90, 180 or 360 days after treatment) [cf. table 3 below, for details on dose groups]
Control animals:
yes, concurrent vehicle
Details on study design:
The test item was administered to the test animals by nose-only inhalation. This administration route is preferred compared to the intratracheal instillation route because it is the more physiological way of particle deposition. Nose-only inhalation is preferred to whole-body inhalation because it provides a specific model of inhalation exposure, restricting ingestion via grooming activity.
To specify adequate aerosol concentrations in this 90-day study, a preceding 90-day study (6 hrs/day on 5days/week) was used as a basis (Fraunhofer ITEM study # 02G11018; conducted with another SAS, i.e. NM-200; JRC code). Based on this study 0.5, 1.0, 2.5 and 5 mg/m³ were used for the test item in the very low, low, mid and high dose groups, respectively. Upon cessation of exposure, animals were investigated for various endpoints (see Table 3) on days 1, 90, 180 and 360 of a subsequent post-exposure observation period. Sacrifice dates at 180 and 360 days post-exposure would have been skipped if no adverse findings were observed after 90 or 180 days. - Dose selection rationale:
- Rationale for animal assignment (if not random):
- Fasting period before blood sampling for clinical biochemistry:
- Rationale for selecting satellite groups:
- Post-exposure recovery period in satellite groups:
- Section schedule rationale (if not random):
- Other:
Observations and examinations performed and frequency:
Cf. also table 5 below for details on examinations!

DETAILED CLINICAL OBSERVATIONS: Yes
All animals were clinically observed in their cages at least once a day. Once a week, they were inspected outside their home cages and carefully examined for clinical symptoms, i.e. abnormalities concerning their general condition. This includes inspection of skin, fur, eyes, visible mucous membranes, examination for pathomorphological changes (e.g. unusual breathing pattern, masses, nodules), abnormal behaviour and central nervous symptoms (e.g. changes in gait, posture or grooming activity, unusual response to handling, secretion/excretion abnormalities, clonic/tonic movements, stereotypies) and/or other clinical abnormalities. The observations were recorded daily with the PROVANTIS system. On the exposure days, the animals were clinically observed before, during and after exposure.

BODY WEIGHT: Yes
Individual body weights were recorded to the nearest 0.1 g twice a week for the first month and reduced to once a week throughout the remainder of the study provided there is no significant body weight effect in the first month (including post-exposure observation period) for all animals.
All body weight data were collected using electronic balances, interfaced with a computer and programmed for direct on-line data acquisition (Provantis).

FOOD CONSUMPTION
Food consumption was recorded weekly during the study period (including post-exposure observation period) using 10 male animals per dose group.

WATER CONSUMPTION: Yes
Water consumption was recorded weekly during the study period (including post-exposure observation period) using 10 male animals per dose group. CAGE SIDE OBSERVATIONS: Yes / No / Not specified

OPHTHALMOSCOPIC EXAMINATION: No
- skipped by ECHA decision (dated June, 30 2017)

HAEMATOLOGY: No
- skipped by ECHA decision (dated June, 30 2017)

CLINICAL CHEMISTRY: No
- skipped by ECHA decision (dated June, 30 2017)

URINALYSIS: No
- skipped by ECHA decision (dated June, 30 2017)

OTHER:
This decision skipped the endpoints clinical pathology and ophthalmology. Gross pathology and histopathology shall be conducted on the lungs, trachea, naso-pharyngeal tissues, nasal-associated lymphoid tissue (NALT) and larynx; other organs and tissues were excluded from examination. As an addition, collagen was analysed.
Sacrifice and pathology:
Cf. also tables 5 and 6 below for details on examinations!

All animals were subjected to a complete necropsy, which includes careful examination of the external surface of the body, all orifices, and the cranial, thoracic and abdominal cavities and their contents.
The rats were anesthetized with an overdose pentobarbital sodium (Narcoren) and killed by cutting the vena cava caudalis.
The abdominal cavity was opened and the diaphragm was cut carefully allowing the lungs to collapse. Heart, oesophagus, upper half of trachea, thymus and lung associated lymph nodes (LALN; mediastinal and tracheobronchial) will be removed from the lung convolution.
The lung and the lower half of the trachea were weighed and used for BAL (right lobes # 1-4) and histopathology (left lobe #5). For histopathology the left lung lobe # 5 was inflated under a pressure of about 20 cm water with formalin and was fixed by immersion for a minimum of two hours, and used for histopathology.
The following organs were trimmed and wet weights will be recorded:
Liver, kidneys, adrenals, testes, epididymides, thymus, spleen, brain, thyroid, lung and heart. The respiratory tract was preserved as follows: Nasal passages (including nasal-associated lymphoid tissue-NALT), larynx, trachea, lungs, and LALN (mediastinal and tracheobronchial). All tissues listed in OECD Guideline No. 413 excluding those in brackets and the seminal vesicles were prepared for histopathology. Tissues in brackets were preserved only.
Other examinations:
Bronchoalveolar Lavage (BAL)
Bronchoalveolar lavage was performed in 5 rats per time point and group, i.e. after end of exposure (day 1 of post-exposure) and after 3, 6 and 12 months of the post-observation period. The method of Henderson et al. (1987) was used with minor modifications.
Cytokines (e.g. IL-6, IL-8) will be measured in the BAL of 5 animals per sex and group on day 1, 90, 180 and 360 post exposure.
Collagen will be determined in lung tissue following an acidic hydrolysis with 37 % HCl. A hydroxyproline analysis will be done according to the method of Creemers & Jansen (1997). Alternatively, the analysis could be done on lungs starting with a homogenisation of the lung tissue. The collagen analysis in BALF will be done under non-GLP conditions.

Histopathology
Lungs were embedded in paraffin, sectioned, and stained with hematoxylin and eosin (H & E). The following histopathological examinations were performed in 10 or 5 animals per sex and group after end of exposure:
• Histopathology on the respiratory tract in all animals of the clean air control group (dose group 1), the Synthetic Amorphous Silica high dose groups (dose groups 5 and 9) and of all animals that died or were killed moribund during the study. In the very low, low and mid dose groups (dose groups 2-4; 6-8), the respiratory tract may be analysed after the results in the high dose groups will have been completed.
• The respiratory tract includes lung lobes, with bronchi and the lung-associated lymph nodes (LALN, mediastinal and tracheobronchial), trachea, larynx, pharynx and the nasal cavities (including NALT) in all animals of all groups (groups 1, 5, 9). Other organs than the respiratory tract were included only if macroscopical findings occurred.
• Trimming of lungs: 3 sections; nose 4 sections.
For the animals sacrificed 3, 6 and 12 months post exposure, all tissues were preserved but only those showing lung changes on day 1 were examined histopathologically.
Statistics:
Differences between groups were considered statistically significant at p < 0.05. Data were analyzed using analysis of variance. If the group means differ significantly by the analysis of variance the means of the treated groups were compared with the means of the control groups using Dunnett's test.
The statistical evaluation of the histopathological findings was done with the two-tailed Fisher test by the PROVANTIS system. If necessary, further statistical procedures were applied upon agreement with the sponsor.
Clinical signs:
no effects observed
Mortality:
mortality observed, non-treatment-related
Description (incidence):
All animals survived their scheduled study period.
In the 13-week recovery (R1) period one female in group 5 (5.0 mg/m3) and one male (group 2, 0.5 mg/m3) were found dead. The female died due to astrocytoma in the brain.The cause of death of the male was mesenchymal tumor of the kidneys.
In the 52-week recovery (R3) one female (group 4, 2.5 mg/m3) was sacrified due to poor condition by malignant lymphoma. One male (group 4, 5.0 mg/m3) was sacrified due to poor condition by malignant schwannoma in the body cavity.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
Relevant statistically significant changes were not observed in the treatment groups as compared to controls.
The enlarged abdomen with increased body weight and enlarged spleen and foci in liver from one male (group 2, 0.5 mg/mg3) during the 13-week recovery period was due to yolk sac carcinoma.
CAB-O-SIL S-17D induced a statistically significant increase of the absolute and relative lung wet weights in the female high dose group at 1 day post-exposure only. This effect had disappeared at 3 months post-exposure.
All other statistically significant changes detected at other organs than lungs are considered as incidental findings.

Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
no relevant statistically significant changes as compared to concurrent controls
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
no relevant statistically significant changes as compared to concurrent controls
Ophthalmological findings:
no effects observed
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Lung weights: statistically significant increase of the absolute and relative lung wet weights in the female high dose group at 1 day post-exposure. This effect had disappeared at 3 months post-exposure.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Upon necropsy, enlarged lung-associated lymph nodes (LALN) were observed in the high dose group. This is a particle-specific finding at lung overload conditions. Other test item- or dose-related macroscopical findings were not detected.
Neuropathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
In nasal cavities, the major lesions consisted of:
- Goblet cell proliferation in levels 1 and 2 and nasopharyngeal duct at the end of treatment and after 13 weeks recovery in all dose groups.
- Hyaline inclusions in olfactory mucosa at higher incidences and severity with increased incidences during the course of the study.
- Chitinase-positive crystals in olfactory mucosa in nasal cavity levels 2-4 up to 26-week recovery without any further injury in olfactory mucosa, mainly in CAB-O-SIL S-17D treated animals.

In lungs, the findings consisted of:
- End of treatment:
- increased perivascular infiltration in groups low dose to high dose ≤ 1.0 mg/m3 groups.
- increased alveolar macrophages and macrophage aggregations, as well as macrophage type II hyperplasia dose-dependently for CAB-O-SIL S-17D and granulomas at the bronchio-alveloar junctions, granulomatous inflammation at a minor severity was noted in single animals from groups very low dose and low dose, and in most animals from all other test item treated groups mid dose to high dose.
- bronchio-alveolar hyperplasia in single animals from groups low dose up to high dose group.
- minimal macrophage agglomeration in the BALT of a few animals at ≤ 1.0 mg/m3 but all animals at 5.0 mg/m3.
- reversible BALT fibrogenesis at an increased incidence at higher doses.
- 13 weeks recovery:
- macrophage aggregates in animals at ≤ 1.0 mg/m3.
- single cases of granulomas at the alveolar-bronchiolar junctions in CAB-O-SIL S-17D-treated groups.
- Fibrogenesis due to inflammatory processes in one animal per sex at 0.5 mg/m3.
- 26 weeks recovery: similar findings than observed after 13 weeks recovery.
- 52 weeks recovery:
- no findings except the presence of macrophage agglomeration.

In lymph nodes, the gross lesions consisted of:
- End of treatment: enlarged lymph nodes at increased incidences in animals at >0.5 mg/m3, whereby almost all males at 5.0 mg/m3 were affected. After 13-, 26- and 52 weeks recovery gross lesions were similar.
- Histologically, the findings consisted of:
- End of treatment:
- granulomas in lymph nodes >0.5 mg/m3
- related granulomatous inflammation at a minor severity in single males >1.0 mg/m3
- lymphoid hyperplasia in most affected lymph nodes
- 13 weeks recovery:
- granulomas and single cases of granulomatous inflammation in animals at 5.0 mg/m3
- Fibrogenesis in one female 1.0 mg/m3.
- 26 weeks recovery:
- CAB-O-SIL S-17D: only single cases of lymphoid hyperplasia and granulomas in a few animals at 2.5 and 5.0 mg/m3
- 52 weeks recovery:
- no findings due to reversibility of effects
Histopathological findings: neoplastic:
no effects observed
Other effects:
effects observed, treatment-related
Description (incidence and severity):
Bronchoalveolar Lavage (BAL)
- Cytological and Biochemical Parameters
Mean values are shown in Figures 10-17 and Tables 43-58 in attached document BALF_Cytological amd Biochemical Parameters.
At day 1 post-exposure statistically significant increases of polymorphonuclear neutrophils (PMN) were detected in the mid and high dose groups of both sexes. In both dose groups a full recovery was detected at 3 months post-exposure.
For lactic dehydrogenase (LDH), ß-glucuronidase (GLU) and total protein (TP) no statistically significant increases were detected in all groups at all 4 sacrifice dates (but for total protein in the female high dose group at day 1).
Key result
Dose descriptor:
NOAEC
Effect level:
5 mg/m³ air (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
histopathology: non-neoplastic
other: BALF, lymph nodes
Remarks on result:
other: end of recovery; local lung effect; not substance specific, particle-related effect; no systemic effects
Remarks:
Overall, the effects depicted through these studies show a similar picture of pathology for both non-surface treated SAS and surface treated SAS. In some studies, at the end of exposure dose-dependent particle related local inflammation in lung and lung associated tissues (lymph nodes) were observed accompanied by corresponding changes of inflammatory marker in the bronchoalveolar fluid. Any clinical effects or morphological changes of other tissues indicating systemic toxicity are not associated with SAS exposure. There are no substantial different pathological findings when comparing different SAS grades. Test item related changes in lungs are dose-dependent and characterized by increased perivascular infiltration, alveolar macrophages and macrophage aggregations, as well as macrophage type II hyperplasia. Accordingly, reactive changes were observed in BALT (bronchus-associated lymphoid tissues) and regional lymph nodes. These effects are not substance-specific, all respirable particles will show the same response directly after exposure1 (day 1). At day 1 the lowest-observed effect concentrations (LOECs) were typically in the range of 0.5 to 50 mg/m3. Based on the biosolubility of SAS reversibility of effects is demonstrated when recovery groups are included in the study with periods of up to one year. Accordingly, in these recovery groups a LOEC and NOAEC are higher. For some SAS grades regional lung lymph nodes show as result of inflammation morphological changes which are not accompanied by any other findings. There are a number of repeated dose studies for SAS available showing a range of LOAEC/NOAEC at the end of recovery. Considering the most conservative values, the LOAEC for SAS is 2.5 mg/m³ at the end of recovery for lung (90-day inhalation toxicity study low specific surface (BET), Fraunhofer ITEM, 2019).
Dose descriptor:
NOAEC
Remarks:
Local lung inflammation
Effect level:
1 mg/m³ air (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
histopathology: non-neoplastic
Remarks on result:
other: end of exposure; local lung effect; not substance specific, particle-related effect; no systemic effects
Remarks:
Overall, the effects depicted through these studies show a similar picture of pathology for both non-surface treated SAS and surface treated SAS. In some studies, at the end of exposure dose-dependent particle related local inflammation in lung and lung associated tissues (lymph nodes) were observed accompanied by corresponding changes of inflammatory marker in the bronchoalveolar fluid. Any clinical effects or morphological changes of other tissues indicating systemic toxicity are not associated with SAS exposure. There are no substantial different pathological findings when comparing different SAS grades. Test item related changes in lungs are dose-dependent and characterized by increased perivascular infiltration, alveolar macrophages and macrophage aggregations, as well as macrophage type II hyperplasia. Accordingly, reactive changes were observed in BALT (bronchus-associated lymphoid tissues) and regional lymph nodes. These effects are not substance-specific, all respirable particles will show the same response directly after exposure1 (day 1). At day 1 the lowest-observed effect concentrations (LOECs) were typically in the range of 0.5 to 50 mg/m3. Based on the biosolubility of SAS reversibility of effects is demonstrated when recovery groups are included in the study with periods of up to one year. Accordingly, in these recovery groups a LOEC and NOAEC are higher. For some SAS grades regional lung lymph nodes show as result of inflammation morphological changes which are not accompanied by any other findings. There are a number of repeated dose studies for SAS available showing a range of LOAEC/NOAEC at the end of recovery. Considering the most conservative values, the LOAEC for SAS is 2.5 mg/m³ at the end of recovery for lung (90-day inhalation toxicity study low specific surface (BET), Fraunhofer ITEM, 2019).
Dose descriptor:
NOAEC
Effect level:
0.5 mg/m³ air (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: lymph nodes
Remarks on result:
other: end of exposure / not substance specific, particle-related effect; no systemic effects
Remarks:
Overall, the effects depicted through these studies show a similar picture of pathology for both non-surface treated SAS and surface treated SAS. In some studies, at the end of exposure dose-dependent particle related local inflammation in lung and lung associated tissues (lymph nodes) were observed accompanied by corresponding changes of inflammatory marker in the bronchoalveolar fluid. Any clinical effects or morphological changes of other tissues indicating systemic toxicity are not associated with SAS exposure. There are no substantial different pathological findings when comparing different SAS grades. Test item related changes in lungs are dose-dependent and characterized by increased perivascular infiltration, alveolar macrophages and macrophage aggregations, as well as macrophage type II hyperplasia. Accordingly, reactive changes were observed in BALT (bronchus-associated lymphoid tissues) and regional lymph nodes. These effects are not substance-specific, all respirable particles will show the same response directly after exposure1 (day 1). At day 1 the lowest-observed effect concentrations (LOECs) were typically in the range of 0.5 to 50 mg/m3. Based on the biosolubility of SAS reversibility of effects is demonstrated when recovery groups are included in the study with periods of up to one year. Accordingly, in these recovery groups a LOEC and NOAEC are higher. For some SAS grades regional lung lymph nodes show as result of inflammation morphological changes which are not accompanied by any other findings. There are a number of repeated dose studies for SAS available showing a range of LOAEC/NOAEC at the end of recovery. Considering the most conservative values, the LOAEC for SAS is 2.5 mg/m³ at the end of recovery for lung (90-day inhalation toxicity study low specific surface (BET), Fraunhofer ITEM, 2019).
Critical effects observed:
yes
Lowest effective dose / conc.:
5 mg/m³ air (analytical)
System:
respiratory system: lower respiratory tract
Organ:
lungs
Treatment related:
yes
Dose response relationship:
not specified
Relevant for humans:
yes
Conclusions:
Under the conditions of this test at the end of recovery the Now-Observed-Adverse-Effect-Concentration (NOAEC) for the lung is based on histopathology and inflammatory marker 5 mg/m³ for the high surface area and the Low-Observed-Adverse-Effect-Concentration (LOAEC) for the low surface area SAS is 2.5 mg/m³.
The determination of the LOAEC for the low surface area SAS is a conservative approach due to the fact that it is based on minor morphological inflammatory changes only. These morphological changes were not accompanied by significant changes of inflammatory marker or any systemic effects. Therefore, these morphological findings can be evaluated as non-adverse since they can be considered as local physiological adaptive response to foreign material. At the end of exposure dose-dependent particle related local inflammation in lung and lung associated tissues (lymph nodes) were observed accompanied by corresponding changes of inflammatory marker in the bronchoalveolar fluid. Any clinical effects or morphological changes of other tissues indicating systemic toxicity are not associated with SAS exposure. Test item related changes in lungs are dose -dependently and characterized by increased perivascular infiltration, alveolar macrophages and macrophage aggregations, as well as macrophage type II hyperplasia. Accordingly, reactive changes were observed in BALT (bronchus-associated lymphoid tissues) and regional lymph nodes. These effects are not substance-specific, all respirable particles will show the similar response directly after exposure (day 1). Based on the biosolubility of SAS reversibility of effects are demonstrated for both SAS grades examined. Most likely based on the lower solubility, low surface SAS-exposed animals showed still morphological inflammatory changes after 1-year recovery in all dose groups. These lymph node findings were not accompanied by any other findings up to a dose of 2.5 mg/m³ at the end of recovery. The lower lung and lymph node effect concentration of low surface SAS compared to high surface SAS is most likely related to the lower biosolubility of this SAS grade resulting in an extension of the lung clearance.
Executive summary:

Discussion and conclusion

The aim of this present study was to investigate the toxicity of two Synthetic Amorphous Silicas (High surface (BET) and low surface SAS in a 90-day nose-only inhalation study and to analyse effects also after an additional post-inhalation recovery period (up to 12 months); to establish exposure-dose-response relationships of the inhaled test item in rats after sub-chronic exposure and to use an experimental design adapted from OECD guideline 413 with additional endpoints (bronchoalveolar lavage, collagen content in lungs and lymph nodes).

Two-hundred twenty-five male and 225 female Wistar rats [strain Crl:WI (Han)] were used for this study and allocated to 9 treatment groups each: Clean air control, very low (0.5 mg/m3), low (1 mg/m3), mid (2.5 mg/m3) and high (5 mg/m3). The two test item aerosols were generated by dispersing the dry powder. Dispersion was achieved by a feeding system and pressurized air dispersion nozzle (dynamic system) developed by Fraunhofer ITEM (Koch, 1998). Using this system a mass median aerodynamic diameter (MMAD) of≤3 µm could be achieved as required by the ECHA decision. In the test item high dose groups a very slight lung overload situation (no volumetric overload, however, toxic impact due to surface chemistry) will be induced whereas in the low and mid dose groups the physiological lung clearance will not be retarded.

The observed differences in severity of the similar pathological effects are most likely caused by test substance differences (particle size distribution, surface area, number of silanols, density, volume, agglomeration status and biosolubility characteristics). At the end of exposure dose-dependent particle related local inflammation in lung and lung associated tissues (lymph nodes) was observed accompanied by corresponding changes of inflammatory markers in the bronchoalveolar fluid. Any clinical effects or morphological changes of other tissues indicating systemic toxicity are not associated with SAS exposure. Test item related changes in lungs are dose-dependent and characterized by increased perivascular infiltration, alveolar macrophages and macrophage aggregations as well as macrophage type II hyperplasia. Accordingly, reactive changes were observed in the bronchus-associated lymphoid tissues (BALT) and regional lymph nodes. These effects are not substance-specific, all respirable particles will show the similar response directly after exposure (day 1). Based on the varying biosolubility of SAS, reversibility of effects was demonstrated for both SAS grades examined.

These new studies with and low surface area pyrogenic SAS showed no lung fibrosis and accordingly no increase of collagen.

In association with lung clearance SAS accumulates in the regional lymph nodes associated with increase of lymph node size and inflammatory changes at the end of exposure. High surface SAS-exposed animals showed reversibility of lymph node effects in all affected dose groups at the end of recovery. Most likely based on the lower solubility, low surface SAS-exposed animals showed still morphological inflammatory changes after 1-year recovery in all dose groups. These lymph node findings were not accompanied by any other findings up to a dose of 2.5 mg/m³ at the end of recovery. The lower lung and lymph node effect concentration of low surface SAS compared to high surface SAS is most likely related to the lower biosolubility of this SAS grade resulting in an extension of the lung clearance.

The results of the lymph nodes are indicating that solubility of low surface SAS requires a longer period of time compared to high surface SAS. Final assessment of reversibility of lung lymph node effects for low surface SAS was not possible in this study. Further examinations of the lymph nodes are running to examine the lung lymph nodes of low surface SAS-exposed animals. SAS content in lymph nodes at the end of exposure will be compared with the results at the end of recovery by ashing and EDX examination and will be attached as amendment to this study report.

Indications for irreversible effects (lung fibrosis) have been published in literature and were based on study materials of one of the applicants' studies according to OECD TG 413 (Sub-chronic inhalation toxicity, Reuzel et al., 1987). A pathology working group reviewed this finding using the same (original) tissue slides according to the highest current standards and concluded the rapid clearance of the synthetic amorphous silica products will not lead to persistent inflammation and epithelial cell proliferation and therefore will not result in fibrosis/lung tumour induction (PWG Publication, Weber et al. 2018).The new studies performed on request by ECHA with high and low surface area pyrogenic SAS confirmed the PWG results and showed also no lung fibrosis and accordingly no increase of collagen.

Conclusion

Under the conditions of this test at the end of exposure dose-dependent particle related local inflammation in lung and lung associated tissues (lymph nodes) were observed accompanied by corresponding changes of inflammatory marker in the bronchoalveolar fluid. Any clinical effects or morphological changes of other tissues indicating systemic toxicity are not associated with SAS exposure. Test item related changes in lungs are dose-dependently and characterized by increased perivascular infiltration, alveolar macrophages and macrophage aggregations, as well as macrophage type II hyperplasia. Accordingly, reactive changes were observed in BALT (bronchus-associated lymphoid tissues) and regional lymph nodes. These effects are not substance-specific, all respirable particles will show the similar response directly after exposure (day 1). Based on the biosolubility of SAS reversibility of effects are demonstrated for both SAS grades examined. Most likely based on the lower solubility, low surface SAS-exposed animals showed still morphological inflammatory changes after 1-year recovery in all dose groups. These lymph node findings were not accompanied by any other findings up to a dose of 2.5 mg/m³ at the end of recovery. The lower lung and lymph node effect concentration of low surface SAS compared to high surface SAS is most likely related to the lower biosolubility of this SAS grade resulting in an extension of the lung clearance. 

At the end of recovery the Now-Observed-Adverse-Effect-Concentration (NOAEC) for the lung is based on histopathology and inflammatory marker 5 mg/m³ for the high surface area and the Low-Observed-Adverse-Effect-Concentration (LOAEC) for the low surface area SAS is 2.5 mg/m³.

Data source

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

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)
Version / remarks:
2017
Deviations:
yes
Remarks:
This study was conducted according to an ECHA decision (dated June, 30 2017).
Principles of method if other than guideline:
Deviations from the guideline: This study will be conducted according to an ECHA decision (dated June, 30 2017). This decision skipped the endpoints clinical pathology and ophthalmology. Gross pathology and histopathology shall be conducted on the lungs, trachea, naso-pharyngeal tissues, nasal-associated lymphoid tissue (NALT) and larynx; other organs and tissues will be excluded from examination. As an addition, collagen will be analysed.
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: nanoform, no surface treatment
Details on test material:
Pyrogenic,
Surface area / BET [m2/g]: 35-65

Specific details on test material used for the study:
Test material 2
Name: SAS2 - Low BET; BET: Approx. 40-50 m²/g (low surface area); Purity: > 99.8%; Molecular formula: SiO2; Molecular weight: 60.08 g/mol;
CAS number: 7631-86-9, 112945-52-5; CAS name: Silica, amorphous, fumed, crystalline-free
EC number: 231-545-4; Date of delivery: January 4, 2018; Expiration date: December 10, 2019

Test animals

Species:
rat
Strain:
Wistar
Details on species / strain selection:
Male and female Wistar rats [strain Crl:WI(Han)] purchased from Charles River Deutschland (Sulzfeld, Germany).
Wistar rats are commonly used in subchronic and chronic inhalation toxicity studies. They fulfil the criteria stated by a U.S. EPA Workshop (Vu et al., 1996) such as (i) a low background rate of neoplasia, (ii) a low background rate of pulmonary disease, (iii) longevity, and (iv) a history of laboratory use.
Sex:
male/female
Details on test animals and environmental conditions:
For a period of at least 3 weeks prior to exposure animals were trained to become accustomed to nose-only tubes. The age of the animals at the start of exposure was approx. 8-9 weeks and the body weight approx. 250 gram for males and approx. 175 gram for females. Rats will be exposed to the test item by nose-only inhalation.
Animals were housed in Makrolon (polycarbonate) cages type IV, two rats of the same sex per cage, and were maintained under conventional laboratory conditions. Cages and absorbing softwood bedding material (Lignocel BK8-15) were changed twice a week or more often, if necessary. Tap water from the Hannover city water supplier was offered fresh weekly or more often, if necessary, in a Makrolon bottle fitted with a stainless steel nipple top with a hole approximately 0.5 mm in diameter. As diet a commercial chow in pellet form was used, identified as Ssniff V1534, purchased from Ssniff-Spezialdiäten (Soest, Germany). The diet was stored in room T1.015 at a temperature of max. 18 °C and a relative humidity of max. 70 %. No batch of diet was used after 6 months have elapsed from manufacturing date. The diet was offered fresh weekly or more often, if necessary.
In the cages the rats find paperboard crinklets as nesting material and wooden blocks for nibbling. The temperature and the relative humidity of the animal room was monitored electronically and recorded on a continuous basis. The limits was set at 22 °C ± 2 °C for temperature and 55 % ± 15 % for relative humidity. A 12-hour light/dark cycle was used controlled by an automatic timing device. The air exchange rate was at least 10 times per hour light/dark cycle was used controlled by an automatic timing device.

Administration / exposure

Route of administration:
inhalation: aerosol
Type of inhalation exposure:
nose only
Vehicle:
clean air
Mass median aerodynamic diameter (MMAD):
<= 3 µm
Geometric standard deviation (GSD):
3.53
Remarks on MMAD:
The photometer gives a scattering light signal which is proportional to the particle concentration, if the particle size distribution is constant.
Using this system a mass median aerodynamic diameter (MMAD) of ≤ 3 µm was achieved as required by the ECHA decision.
Details on inhalation exposure:
The two test item aerosols were generated by dispersing the dry powder. Dispersion is achieved by a feeding system and pressurized air dispersion nozzle (dynamic system). For each nose-only exposure unit (total of 8 units for groups 2 - 9), the aerosol was generated by a pneumatic disperser. The disperser was fed with the test item under computerized control, i.e. with a feedback loop to a scattering signal from an aerosol photometer which reflects the actual aerosol concentration.
The aerosol was given to the rats by a flow-past nose-only inhalation exposure system which was used for previous particle and fiber inhalation studies at Fraunhofer ITEM. In this system, aerosols was supplied to each rat individually, and exhaled air is immediately exhausted. The airflow to each rat was approximately 1 L/min which is calculated to be laminar. Therefore measurement of the oxygen concentration is not necessary. Prior to the 90-day exposure of rats, technical trials to adjust particle size distributions and exposure levels was conducted.
For exposure to the test item the rats were restrained in acrylic tubes with a flexible stopper. The exposure tubes are arranged around a cylinder capable to take up 16 animals per level on four levels. The rat nose is located at the front end of a tube connected to the inner cylinder of the exposure unit delivering the aerosol. Through thin pipes, the aerosol is supplied to each rat nose individually and exhaled air is drawn off immediately by a cylinder surrounding the aerosol delivering cylinder. The position of individual rat at the cylinder is changed daily according to a rotation plan to minimize exposure differences due to geometry. The exposure units (including the clean air control: 9 units) are located each under a separate hood to prevent cross contamination among the different dose groups.
The duration of exposure will be 6 hours/day, 5 days/week for 13 weeks.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Filter samples of the aerosols were taken at least twice per week to control the aerosol concentrations and to adjust the aerosol photometer readings. These samples were collected at a port of the nose-only exposure unit, thus, under the same conditions the rats are inhaling the aerosol and will be analysed gravimetrically.
Duration of treatment / exposure:
13 weeks
Frequency of treatment:
6 h / d
5 d / w
Doses / concentrationsopen allclose all
Dose / conc.:
0.5 mg/m³ air (nominal)
Dose / conc.:
1 mg/m³ air (nominal)
Dose / conc.:
2.5 mg/m³ air (nominal)
Dose / conc.:
5 mg/m³ air (nominal)
No. of animals per sex per dose:
10 (sacrified 1 day after treatment);
5 each (sacrified 90, 180 or 360 days after treatment) [cf. table 3 below, for details on dose groups]
Control animals:
yes, concurrent vehicle
Details on study design:
The test item were administered to the test animals by nose-only inhalation. This administration route is preferred compared to the intratracheal instillation route because it is the more physiological way of particle deposition. Nose-only inhalation is preferred to whole-body inhalation because it provides a specific model of inhalation exposure, restricting ingestion via grooming activity.
To specify adequate aerosol concentrations in this 90-day study, a preceding 90-day study (6 hrs/day on 5days/week) was used as a basis (Fraunhofer ITEM study # 02G11018; conducted with another SAS, i.e. NM-200; JRC code). Based on this study 0.5, 1.0, 2.5 and 5 mg/m³ were used for the test item in the very low, low, mid and high dose groups, respectively. Upon cessation of exposure, animals were investigated for various endpoints (see Table 3) on days 1, 90, 180 and 360 of a subsequent post-exposure observation period. Sacrifice dates at 180 and 360 days post-exposure may be skipped if no adverse findings will be observed after 90 or 180 days.

Examinations

Observations and examinations performed and frequency:
Cf. also table 5 below for details on examinations!

Clinical Observations
All animals were clinically observed in their cages at least once a day. Once a week, they were inspected outside their home cages and carefully examined for clinical symptoms, i.e. abnormalities concerning their general condition. This includes inspection of skin, fur, eyes, visible mucous membranes, examination for pathomorphological changes (e.g. unusual breathing pattern, masses, nodules), abnormal behaviour and central nervous symptoms (e.g. changes in gait, posture or grooming activity, unusual response to handling, secretion/excretion abnormalities, clonic/tonic movements, stereotypies) and/or other clinical abnormalities. The observations were recorded daily with the PROVANTIS system. On the exposure days, the animals were clinically observed before, during and after exposure.

Body Weight
Individual body weights were recorded to the nearest 0.1 g twice a week for the first month and reduced to once a week throughout the remainder of the study provided there is no significant body weight effect in the first month (including post-exposure observation period) for all animals.
All body weight data were collected using electronic balances, interfaced with a computer and programmed for direct on-line data acquisition (Provantis).

Food Consumption
Food consumption were recorded weekly during the study period (including post-exposure observation period) using 10 male animals per dose group.

Water Consumption
Water consumption were recorded weekly during the study period (including post-exposure observation period) using 10 male animals per dose group.
Sacrifice and pathology:
Cf. also tables 5 and 6 below for details on examinations.

All animals were subjected to a complete necropsy, which includes careful examination of the external surface of the body, all orifices, and the cranial, thoracic and abdominal cavities and their contents.
The rats were anesthetized with an overdose pentobarbital sodium (Narcoren) and killed by cutting the vena cava caudalis.
The abdominal cavity were opened and the diaphragm were cut carefully allowing the lungs to collapse. Heart, oesophagus, upper half of trachea, thymus and lung associated lymph nodes (LALN; mediastinal and tracheobronchial) will be removed from the lung convolution.
The lung and the lower half of the trachea were weighed and used for BAL (right lobes # 1-4) and histopathology (left lobe #5). For histopathology the left lung lobe # 5 were inflated under a pressure of about 20 cm water with formalin and were fixed by immersion for a minimum of two hours, and used for histopathology.
The following organs were trimmed and wet weights will be recorded:
Liver, kidneys, adrenals, testes, epididymides, thymus, spleen, brain, thyroid, lung and heart. The respiratory tract were preserved as follows: Nasal passages (including nasal-associated lymphoid tissue-NALT), larynx, trachea, lungs, and LALN (mediastinal and tracheobronchial). All tissues listed in OECD Guideline no. 413 excluding those in brackets and the seminal vesicles were prepared for histopathology. Tissues in brackets were preserved only.
Other examinations:
Bronchoalveolar Lavage (BAL)
Bronchoalveolar lavage were performed in 5 rats per time point and group, i.e. after end of exposure (day 1 of post-exposure) and after 3, 6 and 12 months of the post-observation period. The method of Henderson et al. (1987) were used with minor modifications.
Cytokines (e.g. IL-6, IL-8) were measured in the BAL of 5 animals per sex and group on day 1, 90, 180 and 360 post exposure.
Collagen were determined in lung tissue following an acidic hydrolysis with 37 % HCl. A hydroxyproline analysis was done according to the method of Creemers & Jansen (1997). Alternatively, the analysis could be done on lungs starting with a homogenisation of the lung tissue. The collagen analysis in BALF was done under non-GLP conditions.

Histopathology
Lungs were embedded in paraffin, sectioned, and stained with hematoxylin and eosin (H & E). The following histopathological examinations were performed in 10 or 5 animals per sex and group after end of exposure:
• Histopathology on the respiratory tract in all animals of the clean air control group (dose group 1), the Synthetic Amorphous Silica high dose groups (dose groups 5 and 9) and of all animals that died or were killed moribund during the study. In the very low, low and mid dose groups (dose groups 2-4; 6-8), the respiratory tract may be analysed after the results in the high dose groups will have been completed.
• The respiratory tract includes lung lobes, with bronchi and the lung-associated lymph nodes (LALN, mediastinal and tracheobronchial), trachea, larynx, pharynx and the nasal cavities (including NALT) in all animals of all groups (groups 1, 5, 9). Other organs than the respiratory tract were included only if macroscopical findings occurred.
• Trimming of lungs: 3 sections; nose 4 sections.
For the animals sacrificed 3, 6 and 12 months post exposure, all tissues were preserved but only those showing lung changes on day 1 were examined histopathologically.
Statistics:
Differences between groups were considered statistically significant at p < 0.05. Data were analyzed using analysis of variance. If the group means differ significantly by the analysis of variance the means of the treated groups were compared with the means of the control groups using Dunnett's test.
The statistical evaluation of the histopathological findings was done with the two-tailed Fisher test by the PROVANTIS system. If necessary, further statistical procedures were applied upon agreement with the sponsor.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Relevant statistically significant changes were not observed in the treatment groups as compared to controls.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
no relevant statistically significant changes as compared to concurrent controls
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
no relevant statistically significant changes as compared to concurrent controls
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
Bronchoalveolar Lavage (BAL)
- Cytological and Biochemical Parameters
Mean values are shown in Figures 10-17 and Tables 43-58 in attached document BALF_Cytological and Biochemical Parameters.
At day 1 post-exposure statistically significant increases of PMN were detected in all dose groups of both sexes. Full recovery was detected in the very low dose group at 3 months, in the low dose group at 6 months and in the mid and high dose groups at 12 months post-exposure.
In the mid and high dose groups, statistically significant increases of lactic dehydrogenase (LDH), ß-glucuronidase (GLU) and total protein (TP) were observed at 1 and 90 days post-exposure; these effects returned to normalisation mostly at 6 and 12 months post-exposure.
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
AEROSIL® OX 50 induced statistically significant increases of the absolute and relative lung wet weights in the low, mid and high dose groups at 1 day post-exposure (both sexes). Lung weights recovered at 3 months post-exposure; the high dose group only showed a persistent statistically significant increase at 6 and 12 months post-exposure.
All other statistically significant changes detected at other organs than lungs are considered as incidental findings.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Upon necropsy, enlarged lung-associated lymph nodes (LALN) were observed in the high dose group. This is a particle-specific finding at lung overload conditions. Other test item- or dose-related macroscopical findings were not detected.
Neuropathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
In nasal cavities, the major lesions consisted of:
- Goblet cell proliferation in levels 1 and 2 and nasopharyngeal duct at the end of treatment and after 13 weeks recovery in all high surface SAS CAB-O-SIL S-17D and low surface SAS AEROSIL® OX 50 groups.
- Hyaline inclusions in olfactory mucosa at higher incidences and severity with increased incidences during the course of the study.
- Chitinase-positive crystals in olfactory mucosa in nasal cavity levels 2-4 up to 26-week recovery without any further injury in olfactory mucosa, mainly in CAB-O-SIL S-17D treated animals.

In lungs, the findings consisted of:
- End of treatment: discoloration or discoloured foci in lungs from animals treated at ≤ 1.0 mg/m3 AEROSIL® OX 50 associated with inflammatory lesions that increased in incidence and/or severity in test item-treated groups.
- increased perivascular infiltration in all AEROSIL® OX 50-treated groups.
- increased alveolar macrophages and macrophage aggregations, as well as macrophage type II hyperplasia dose-dependently for AEROSIL® OX 50 associated with interstitial inflammation, granulomas at the bronchio-alveloar junctions, granulomatous inflammation at a minor severity was noted in most animals from all other test item treated groups very low dose up to high dose (AEROSIL® OX 50).
- bronchio-alveolar hyperplasia in single animals from groups very low dose up to high dose of AEROSIL® OX 50.
- hyperplasia in the BALT in one group 6 male and one group 8 female (0.5 and 2.5 mg/m3 AEROSIL® OX 50).
- minimal macrophage agglomeration in the BALT in almost all animals treated with AEROSIL® OX 50 and granulomatous inflammation in the BALT in animals treated with AEROSIL® OX 50.
- reversible BALT fibrogenesis in all doses of AEROSIL® OX 50 with increasing incidence.
- 13 weeks recovery: foci in the lungs mainly in groups treated with AEROSIL® OX 50.
- increased perivascular infiltration in AEROSIL® OX 50-treated groups.
- increased in incidence and severity of alveolar macrophages in AEROSIL® OX 50-treated groups and macrophage aggregates in all groups treated with AEROSIL® OX 50.
- Increased incidence of macrophage type II hyperplasia and interstitial inflammation in AEROSIL® OX 50.
- Increased alveolar-bronchiolar hyperplasia in AEROSIL® OX 50-treated groups without clear dose-dependency.
- BALT macrophage agglomeration was noted in a few animals from AEROSIL® OX 50 groups associated with some cases of granulomatous inflammation. The latter caused fibrogenesis in the BALT in single cases of AEROSIL® OX 50-treated animals.
- Fibrogenesis due to inflammatory processes in most animals treated with AEROSIL® OX 50.
- 26 weeks recovery: similar findings than observed after 13 weeks recovery.
- 52 weeks recovery: still increased discoloured foci in lungs from animals at ≤ 1.0 mg/m3 in AEROSIL® OX 50-treated groups.
- AEROSIL® OX 50-treated groups: still a few inflammatory lesions present, mainly in animals ≤ 1.0 mg/m3.
- AEROSIL® OX 50-treated groups: no BALT inflammation present any longer, however, in a few animals at >1.0 mg/m3, there was a minimal BALT macrophage agglomeration.
- AEROSIL® OX 50-treated groups: inflammatory changes in the lungs at increased incidence in both sexes >1.0 mg/m3 AEROSIL® OX 50 due to still ongoing inflammatory processes.

In lymph nodes, the gross lesions consisted of:
- End of treatment: enlarged lymph nodes at increased incidences in animals at ≥0.5 mg/m3. After 13-, 26- and 52 weeks recovery, gross lesions were similar.
Histologically, the findings consisted of:
- End of treatment:
- granulomas in lymph nodes in all AEROSIL® OX 50 groups
- related granulomatous inflammation in a high number of animals from all groups
- lymphoid hyperplasia in most affected lymph nodes
- fibrogenesis in the lymph nodes from several animals from all AEROSIL® OX 50-treated groups, and fibrosis in one female at 0.5 mg/m3 AEROSIL® OX 50, and in both sexes at ≤ 1.0 mg/m3 AEROSIL® OX 50.
- 13 weeks recovery:
- Fibrogenesis at high incidence at minor severities in all groups.
- 26 weeks recovery:
- increased in incidence and severity of lymphoid hyperplasia in all groups and granulomas in all groups
- granulomatous inflammation and dose-dependent increased severity of fibrogenesis and fibrosis.
- 52 weeks recovery:
- granulomas or granulomatous inflammation
- increased incidence of lymphoid hyperplasia in animals at >1.0 mg/m3
- fibrogenesis or fibrosis in a few animals.
Histopathological findings: neoplastic:
no effects observed
Other effects:
effects observed, treatment-related
Description (incidence and severity):
Bronchoalveolar Lavage (BAL)
- Cytological and Biochemical Parameters
At day 1 post-exposure statistically significant increases of PMN were detected in all dose groups of both sexes. Full recovery was detected in the very low dose group at 3 months, in the low dose group at 6 months and in the mid and high dose groups at 12 months post-exposure.
In the mid and high dose groups, statistically significant increases of lactic dehydrogenase (LDH), ß-glucuronidase (GLU) and total protein (TP) were observed at 1 and 90 days post-exposure; these effects returned to normalisation mostly at 6 and 12 months post-exposure.

Effect levels

open allclose all
Key result
Dose descriptor:
LOAEC
Effect level:
2.5 mg/m³ air (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
histopathology: non-neoplastic
Remarks on result:
other: end of recovery; local lung effect; not substance specific, particle-related effect; no systemic effects. Minor morphological inflammatory changes were not accompanied by significant changes of inflammatory marker.
Remarks:
The determination of the LOAEC is a conservative approach due to the fact that it is based on minor morphological inflammatory changes only. These morphological changes were not accompanied by significant changes of inflammatory marker or any systemic effects. Therefore, these morphological findings can be evaluated as non-adverse since they can be considered as local physiological adaptive response to foreign material.
Dose descriptor:
LOEC
Effect level:
0.5 mg/m³ air (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
gross pathology
histopathology: non-neoplastic
Remarks on result:
other: end of recovery; not substance specific, particle-related effect; no systemic effects
Remarks:
Overall, the effects depicted through these studies show a similar picture of pathology for both non-surface treated SAS and surface treated SAS. In some studies, at the end of exposure dose-dependent particle related local inflammation in lung and lung associated tissues (lymph nodes) were observed accompanied by corresponding changes of inflammatory marker in the bronchoalveolar fluid. Any clinical effects or morphological changes of other tissues indicating systemic toxicity are not associated with SAS exposure. There are no substantial different pathological findings when comparing different SAS grades. Test item related changes in lungs are dose-dependent and characterized by increased perivascular infiltration, alveolar macrophages and macrophage aggregations, as well as macrophage type II hyperplasia. Accordingly, reactive changes were observed in BALT (bronchus-associated lymphoid tissues) and regional lymph nodes. These effects are not substance-specific, all respirable particles will show the same response directly after exposure1 (day 1). At day 1 the lowest-observed effect concentrations (LOECs) were typically in the range of 0.5 to 50 mg/m3. Based on the biosolubility of SAS reversibility of effects is demonstrated when recovery groups are included in the study with periods of up to one year. Accordingly, in these recovery groups a LOEC and NOAEC are higher. For some SAS grades regional lung lymph nodes show as result of inflammation morphological changes which are not accompanied by any other findings. There are a number of repeated dose studies for SAS available showing a range of LOAEC/NOAEC at the end of recovery. Considering the most conservative values, the LOAEC for SAS is 2.5 mg/m³ at the end of recovery for lung (90-day inhalation toxicity study low specific surface (BET), Fraunhofer ITEM, 2019).
Dose descriptor:
LOAEC
Effect level:
0.5 mg/m³ air (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
histopathology: non-neoplastic
Remarks on result:
other: end of exposure; not substance specific, particle-related effect; no systemic effects
Remarks:
Overall, the effects depicted through these studies show a similar picture of pathology for both non-surface treated SAS and surface treated SAS. In some studies, at the end of exposure dose-dependent particle related local inflammation in lung and lung associated tissues (lymph nodes) were observed accompanied by corresponding changes of inflammatory marker in the bronchoalveolar fluid. Any clinical effects or morphological changes of other tissues indicating systemic toxicity are not associated with SAS exposure. There are no substantial different pathological findings when comparing different SAS grades. Test item related changes in lungs are dose-dependent and characterized by increased perivascular infiltration, alveolar macrophages and macrophage aggregations, as well as macrophage type II hyperplasia. Accordingly, reactive changes were observed in BALT (bronchus-associated lymphoid tissues) and regional lymph nodes. These effects are not substance-specific, all respirable particles will show the same response directly after exposure1 (day 1). At day 1 the lowest-observed effect concentrations (LOECs) were typically in the range of 0.5 to 50 mg/m3. Based on the biosolubility of SAS reversibility of effects is demonstrated when recovery groups are included in the study with periods of up to one year. Accordingly, in these recovery groups a LOEC and NOAEC are higher. For some SAS grades regional lung lymph nodes show as result of inflammation morphological changes which are not accompanied by any other findings. There are a number of repeated dose studies for SAS available showing a range of LOAEC/NOAEC at the end of recovery. Considering the most conservative values, the LOAEC for SAS is 2.5 mg/m³ at the end of recovery for lung (90-day inhalation toxicity study low specific surface (BET), Fraunhofer ITEM, 2019).
Dose descriptor:
LOEC
Effect level:
0.5 mg/m³ air (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
gross pathology
histopathology: non-neoplastic
Remarks on result:
other: end of exposure; not substance specific, particle-related effect; no systemic effects
Remarks:
Overall, the effects depicted through these studies show a similar picture of pathology for both non-surface treated SAS and surface treated SAS. In some studies, at the end of exposure dose-dependent particle related local inflammation in lung and lung associated tissues (lymph nodes) were observed accompanied by corresponding changes of inflammatory marker in the bronchoalveolar fluid. Any clinical effects or morphological changes of other tissues indicating systemic toxicity are not associated with SAS exposure. There are no substantial different pathological findings when comparing different SAS grades. Test item related changes in lungs are dose-dependent and characterized by increased perivascular infiltration, alveolar macrophages and macrophage aggregations, as well as macrophage type II hyperplasia. Accordingly, reactive changes were observed in BALT (bronchus-associated lymphoid tissues) and regional lymph nodes. These effects are not substance-specific, all respirable particles will show the same response directly after exposure1 (day 1). At day 1 the lowest-observed effect concentrations (LOECs) were typically in the range of 0.5 to 50 mg/m3. Based on the biosolubility of SAS reversibility of effects is demonstrated when recovery groups are included in the study with periods of up to one year. Accordingly, in these recovery groups a LOEC and NOAEC are higher. For some SAS grades regional lung lymph nodes show as result of inflammation morphological changes which are not accompanied by any other findings. There are a number of repeated dose studies for SAS available showing a range of LOAEC/NOAEC at the end of recovery. Considering the most conservative values, the LOAEC for SAS is 2.5 mg/m³ at the end of recovery for lung (90-day inhalation toxicity study low specific surface (BET), Fraunhofer ITEM, 2019).

Target system / organ toxicity

Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
2.5 mg/m³ air (nominal)
System:
respiratory system: lower respiratory tract
Organ:
lungs
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
yes

Applicant's summary and conclusion

Conclusions:
Under the conditions of this test at the end of recovery the Now-Observed-Adverse-Effect-Concentration (NOAEC) for the lung is based on histopathology and inflammatory marker 5 mg/m³ for the high surface area and the Low-Observed-Adverse-Effect-Concentration (LOAEC) for the low surface area SAS is 2.5 mg/m³.

The determination of the LOAEC for the low surface area SAS is a conservative approach due to the fact that it is based on minor morphological inflammatory changes only. These morphological changes were not accompanied by significant changes of inflammatory marker or any systemic effects. Therefore, these morphological findings can be evaluated as non-adverse since they can be considered as local physiological adaptive response to foreign material. At the end of exposure dose-dependent particle related local inflammation in lung and lung associated tissues (lymph nodes) were observed accompanied by corresponding changes of inflammatory marker in the bronchoalveolar fluid. Any clinical effects or morphological changes of other tissues indicating systemic toxicity are not associated with SAS exposure. Test item related changes in lungs are dose -dependently and characterized by increased perivascular infiltration, alveolar macrophages and macrophage aggregations, as well as macrophage type II hyperplasia. Accordingly, reactive changes were observed in BALT (bronchus-associated lymphoid tissues) and regional lymph nodes. These effects are not substance-specific, all respirable particles will show the similar response directly after exposure (day 1). Based on the biosolubility of SAS reversibility of effects are demonstrated for both SAS grades examined. Most likely based on the lower solubility, low surface SAS-exposed animals showed still morphological inflammatory changes after 1-year recovery in all dose groups. These lymph node findings were not accompanied by any other findings up to a dose of 2.5 mg/m³ at the end of recovery. The lower lung and lymph node effect concentration of low surface SAS compared to high surface SAS is most likely related to the lower biosolubility of this SAS grade resulting in an extension of the lung clearance.
Executive summary:

Discussion and conclusion

The aim of this present study was to investigate the toxicity of two Synthetic Amorphous Silicas (High surface (BET) and low surface SAS in a 90-day nose-only inhalation study and to analyse effects also after an additional post-inhalation recovery period (up to 12 months); to establish exposure-dose-response relationships of the inhaled test item in rats after sub-chronic exposure and to use an experimental design adapted from OECD guideline 413 with additional endpoints (bronchoalveolar lavage, collagen content in lungs and lymph nodes).

Two-hundred twenty-five male and 225 female Wistar rats [strain Crl:WI (Han)] were used for this study and allocated to 9 treatment groups each: Clean air control, very low (0.5 mg/m3), low (1 mg/m3), mid (2.5 mg/m3) and high (5 mg/m3). The two test item aerosols were generated by dispersing the dry powder. Dispersion was achieved by a feeding system and pressurized air dispersion nozzle (dynamic system) developed by Fraunhofer ITEM (Koch, 1998). Using this system a mass median aerodynamic diameter (MMAD) of≤3 µm could be achieved as required by the ECHA decision. In the test item high dose groups a very slight lung overload situation (no volumetric overload, however, toxic impact due to surface chemistry) will be induced whereas in the low and mid dose groups the physiological lung clearance will not be retarded.

The observed differences in severity of the similar pathological effects are most likely caused by test substance differences (particle size distribution, surface area, number of silanols, density, volume, agglomeration status and biosolubility characteristics). At the end of exposure dose-dependent particle related local inflammation in lung and lung associated tissues (lymph nodes) was observed accompanied by corresponding changes of inflammatory markers in the bronchoalveolar fluid. Any clinical effects or morphological changes of other tissues indicating systemic toxicity are not associated with SAS exposure. Test item related changes in lungs are dose-dependent and characterized by increased perivascular infiltration, alveolar macrophages and macrophage aggregations as well as macrophage type II hyperplasia. Accordingly, reactive changes were observed in the bronchus-associated lymphoid tissues (BALT) and regional lymph nodes. These effects are not substance-specific, all respirable particles will show the similar response directly after exposure (day 1). Based on the varying biosolubility of SAS, reversibility of effects was demonstrated for both SAS grades examined.

These new studies with and low surface area pyrogenic SAS showed no lung fibrosis and accordingly no increase of collagen.

In association with lung clearance SAS accumulates in the regional lymph nodes associated with increase of lymph node size and inflammatory changes at the end of exposure. High surface SAS-exposed animals showed reversibility of lymph node effects in all affected dose groups at the end of recovery. Most likely based on the lower solubility, low surface SAS-exposed animals showed still morphological inflammatory changes after 1-year recovery in all dose groups. These lymph node findings were not accompanied by any other findings up to a dose of 2.5 mg/m³ at the end of recovery. The lower lung and lymph node effect concentration of low surface SAS compared to high surface SAS is most likely related to the lower biosolubility of this SAS grade resulting in an extension of the lung clearance.

The results of the lymph nodes are indicating that solubility of low surface SAS requires a longer period of time compared to high surface SAS. Final assessment of reversibility of lung lymph node effects for low surface SAS was not possible in this study. Further examinations of the lymph nodes are running to examine the lung lymph nodes of low surface SAS-exposed animals. SAS content in lymph nodes at the end of exposure will be compared with the results at the end of recovery by ashing and EDX examination and will be attached as amendment to this study report.

Indications for irreversible effects (lung fibrosis) have been published in literature and were based on study materials of one of the applicants' studies according to OECD TG 413 (Sub-chronic inhalation toxicity, Reuzel et al., 1987). A pathology working group reviewed this finding using the same (original) tissue slides according to the highest current standards and concluded the rapid clearance of the synthetic amorphous silica products will not lead to persistent inflammation and epithelial cell proliferation and therefore will not result in fibrosis/lung tumour induction (PWG Publication, Weber et al. 2018).The new studies performed on request by ECHA with high and low surface area pyrogenic SAS confirmed the PWG results and showed also no lung fibrosis and accordingly no increase of collagen.

Conclusion

Under the conditions of this test at the end of exposure dose-dependent particle related local inflammation in lung and lung associated tissues (lymph nodes) were observed accompanied by corresponding changes of inflammatory marker in the bronchoalveolar fluid. Any clinical effects or morphological changes of other tissues indicating systemic toxicity are not associated with SAS exposure. Test item related changes in lungs are dose-dependently and characterized by increased perivascular infiltration, alveolar macrophages and macrophage aggregations, as well as macrophage type II hyperplasia. Accordingly, reactive changes were observed in BALT (bronchus-associated lymphoid tissues) and regional lymph nodes. These effects are not substance-specific, all respirable particles will show the similar response directly after exposure (day 1). Based on the biosolubility of SAS reversibility of effects are demonstrated for both SAS grades examined. Most likely based on the lower solubility, low surface SAS-exposed animals showed still morphological inflammatory changes after 1-year recovery in all dose groups. These lymph node findings were not accompanied by any other findings up to a dose of 2.5 mg/m³ at the end of recovery. The lower lung and lymph node effect concentration of low surface SAS compared to high surface SAS is most likely related to the lower biosolubility of this SAS grade resulting in an extension of the lung clearance. 

At the end of recovery the Now-Observed-Adverse-Effect-Concentration (NOAEC) for the lung is based on histopathology and inflammatory marker 5 mg/m³ for the high surface area and the Low-Observed-Adverse-Effect-Concentration (LOAEC) for the low surface area SAS is 2.5 mg/m³.