Silicic acid, aluminum sodium salt

Endpoint summary

Description of key information

Oral

No adverse effects were seen with the test substance Silicic acid, aluminum sodium salt (CAS 1344-00-9, NAS) in studies in mice and rats at very high doses of up to 100,000 ppm (10% in the diet) after 14 days of administration.

The 28- and 90-day studies in the read-across substance silicon dioxide (SAS) also showed no adverse effects up to the highest or limit dose level applied, respectively, which was in both cases 1000 mg/kg bw/d.

 

Inhalation

There is only a 5-day study in the test substance Silicic acid, aluminum sodium salt (CAS 1344-00-9, NAS) available, but an expert statement claims that this type of study is as expressive as 90-day ones for testing nanomaterials.

In the mentioned 5-day study no adverse effects were observed in Wistar rats up to inhalation values of 21.93 mg/m³, which is above the GHS threshold level for repeated dose inhalation. All observed effects either did not differ from the control group or were at least regarded as not adverse, being without biological and toxicological relevance. Hence, the NOAEL was assessed as equal to or greater than 21.93 mg/m³.

As there is no 28- or 90-day study in the test substance Silicic acid, aluminum sodium salt (CAS 1344-00-9, NAS) itself, a read-across approach was conducted via the structurally related substance silicon dioxide (SAS).

Here, in the study performed by Creutzenberg (2014, OECD 413), under the conditions of this test, a LOAEL = 1 mg/m³ was derived (decisive endpoint: histopathology: mucous cell hyperplasia in nasal cavities). An experimental local NOAEL could not be derived (i.e. NOAEL < 1 mg/m³). But all effects showed either full reversible or showed a tendency to full reversibility.
The systemic NOAEL was 5 mg/m³ effectively. At this dose level, no systemic effects were observed. The nominal systemic concentration applied higher, and can be calculated by dividing the stated nominal concentration of 16.13 mg/m³ air with the aerosol generation efficiency (effective vs. nominal concentration) of 31%: 16.13 mg/m³ air / 0.31 = 5 mg/m³ air.

LOAEL (local): 1 mg/m³ (effective)
LOAEL (local): 3.23 mg/m³ (nominal)
No local NOAEL was identified, but all effects showed either full reversible or showed a tendency to full reversibility.

NOAEL (systemic): > 5 mg/m³ (effective)
NOAEL (systemic): > 16.13 mg/m³ (nominal)

Creutzenberg, 2014, OECD 413:
The target aerosol concentrations of 1, 2.5 and 5 mg / m³ were achieved to 104%, 100% and 101%, respectively. Calculation of the aerosol generation efficiency (actual vs. nominal concentration) resulted in 31%; this low value demonstrates that experimental conditions are different to any handling and use conditions of the tested silicon dioxide.

 - Due to the particle size distribution in the test medium the nominal concentration was 3.23 mg/m³, to achieve 1 mg/m³ aerosol test concentration.

 - Due to the particle size distribution in the test medium the nominal concentration was 8.06 mg/m³, to achieve 2.5 mg/m³ aerosol test concentration.

 - Due to the particle size distribution in the test medium the nominal concentration was 16.13 mg/m³, to achieve 5 mg/m³ aerosol test concentration.

This results in a NOAEL > 5 mg/m³ air corresponding to > 16.13 mg/m³ nominal concentration in air for the tested silicon dioxide.
For local effects an experimental NOAEL could not be derived (i.e. NOAEL < 1 mg/m³) due to mucous cell hyperplasia in nasal cavities observed in all dose groups. But according to GHS (2019) 3.9.2.8 d this effect does not support classification, because it is an adaptive response not considered toxicologically relevant.
Also, the increased lung weights in the medium and high dose groups do not support classification according to GHS (2019) 3.9.2.8 c, because no evidence of organ dysfunction was reported.

Therefore, for the assessment of classification, no sufficient critical effects were observed up to the highest dose employed, hence no hazard was identified and a NOAEL of equal or greater than 16.13 mg/m³ is applicable for local effects when assessing classification. 

Remark:

#1

Under normal handling and use of commercial products, the silicas tend to form agglomerates of high mass median aerodynamic diameters that are not respirable (MMAD >> 10 μm). The respirable fraction (=< 10 μm) comprises less than 1 wt% (see Stintz 2003). Under experimental conditions, the MMADs may range below 10 μm, the respirable fractions accounting for more than 80 %.
Reference: ECETOC (2006): Synthetic Amorphous Silica (CAS No. 7631-86-9). JACC Report No. 51, European Centre for Ecotoxicology and Toxicology of Chemicals, Brussels, September 2006

#2

Particle properties and relevance for specific hazards:
Toxicological inhalation studies were performed typically by using a homogeneous atmosphere of the test substance (what is gained under application of high shear forces) with particles sizes that are much smaller compared to particle size distributions of the bulk material.
As the inflammatory response in the lung will be determined by particle size and morphology rather than by particle composition: This means that in experimental studies due to the high shear forces applied, the toxicologically relevant particle fraction is assumed to be much higher than under workplace conditions. From this point, the results of the inhalation studies must be seen as a worst-case scenario that appears unlikely under usual workplace conditions.

 

Dermal

No dermal repeated dose study is available, but based on the high acute tolerance and the inert inorganic nature of silica, silicates and aluminium one also does not seem to be necessary.

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Reference 1

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

May 2 - Jun. 28, 1979; experimental phase: 21 May - 7 June 1979

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

no guideline available

Principles of method if other than guideline:

range-finding study to a sub-chronic one: 14-day administration, only reduced number of animals for histopathological examination

GLP compliance:

not specified

Limit test:

no

Specific details on test material used for the study:

Sodium Aluminosilicate, Lot No. B71778, Batch No. 01, received from Midwest Research Institute

Species:

rat

Strain:

Fischer 344

Sex:

male/female

Details on test animals or test system and environmental conditions:

Thirty-five (35) male and thirty-five (35) female Fischer 344 Rats (R-9, undeterminable source) were received for test on May 4, 1979. The rats were individually ear-tagged and placed into a sanitized quaran¬tine room for seventeen days. The rats were housed five per cage, per sex in polycarbonate cages (50.88 square inches, 7 inches) each equipped with a stainless steel cage top.
The feed was placed Into stainless steel feed hoppers equipped with a moveable lid that limited spillage and contamination of the feed. The volume of each feed hopper was 650 cubic centimeters.
The room temperature was maintained at 74 +/- 2°F, 50 +/- 5% relative humidity and had a fluorescent light cycle of 12 hours dark and 12 hours light. Ten to fifteen air changes per hour were maintained.
The rats were assigned to the six test groups (5 per sex/group) on day 0 of the test by, assigning each rat a random number and then assigning each rat by use of a random numbers table from rats grouped into several weight classes.

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on oral exposure:

On May 21, 1979, the test material was prepared for the initiation of the fourteen day repeated-dose test. The dosage mixtures were prepared by the chemist within the diet prep room. Four kilograms of dosed-feed were prepared for each dose level. The control feed consisted of the basal diet without.the incorporation of the test material. All preparations were made with NIH-07 Rat and Mouse Ration Mash. Each preparation was hand-mixed in a plastic tub for one minute. The preparation was then placed into a Vortex mixer and mixed with the intensifier bar for five minutes. The intensifier bar was turned off, and the preparation was mixed for fifteen additional minutes. After mixing, each preparation was collected in a plastic bag, labelled and stored in a metal container. Enough feed was mixed to provide sufficient feed for a one week period. On May 28, 1979, three kilograms of each dose level were again prepared in the same manner as described above. The dose-level preparations were mixed at the following concentra¬tions: 0, 0,625, 1,25, 2,5. 5 and 10% Sodium Aluminosilicate (w/w), Administration of Test Material:
On May 21, 1979, each feed hopper was filled with the corresponding dose level preparation per cage of five rats. The filled feed hopper was weighed. Whenever the feed level in the hopper was insufficient to supply 24 hours of feed, it was removed from the cage and weighed. After weighing, the residual feed in the hopper was discarded and the feed hopper was washed in the cage washing machine. A sanitized feed hopper was filled with the corresponding dosed-feed, weighed and placed into the cage, The feed was given to the rats for fourteen consecutive days. On the fifteenth day, the dosed-feed was removed and replaced with NIH-07 basal diet (control). The rats were observed for pharmacotoxic signs on day 15 and killed by Co2 asphyxiation on days 16 and 18 for gross necropsy observations.

Analytical verification of doses or concentrations:

no

Duration of treatment / exposure:

14 d

Frequency of treatment:

continuous in the diet

Dose / conc.:

100 000 ppm

Dose / conc.:

50 000 ppm

Dose / conc.:

25 000 ppm

Dose / conc.:

12 500 ppm

Dose / conc.:

6 250 ppm

No. of animals per sex per dose:

5

Control animals:

yes, plain diet

Details on study design:

Post-exposure period: 1 d (day 15)

Observations and examinations performed and frequency:

Each rat was observed daily for pharmacotoxic signs by a trained animal technician by removing the mouse from the cage and observing it. The rats were observed individually and the observations recorded for fifteen (15) consecutive days. Body weights were recorded on days 0, 7 and 14, Food consumption was measured on days 0, 7 and 14 and whenever the feed had to be replenished due to an inadequate feed supply.

Sacrifice and pathology:

The rats were observed for pharmacotoxic signs on day 15 and killed by CO2 asphyxiation on days 16 and 18 for gross necropsy observations.

Any rats found dead during the fifteen (15) day period were subjected to a gross autopsy examination, and all rats surviving the fourteen (14) day test period were killed by CO2 asphyxiation and subjected to a gross autopsy examination. At necropsy, the following tissues were examined in-situ:
Skin, Mandibular Lymph Node, Mammary Gland, Salivary Gland, Thigh Muscle, Sciatic Nerve, Bone Marrow, Costochondral Junction (Rib), Thymus, Larynx, Trachea, Lungs and Bronchi, Heart, Thyroids, Parathyroids, Esophagus, Stomach, Duodenum, Brain, Eyes, Spinal Cord, Jejunum, Ileum, Colon, Rectum, Mesenteric Lymph Node, Liver, Gall Bladder, Pancreas, Spleen, Kidneys, Adrenals, Bladder, Seminal Vesicles (males), Prostate (males), Testes (males), Ovaries (females), Uterus (females), Nasal Cavity, Pituitary, External and Middle Ears

Clinical signs:

no effects observed

Description (incidence and severity):

No abnormal external physical appearances, abnormal behavior or pharmacotoxic signs were observed at any time during thetest period, including the observation day, The feces of the males and females of the 5% and 10% dose groups were abnormally lighter in color than the feces of the rats in the control and three other dose groups, The fecal discoloration at the 5 and 10% levels was observed on the third day of consumption of the test diets and continued through the entire study and was still detectable during the 24-hour observation period.

Mortality:

no mortality observed

Description (incidence):

None of the test animals or controls died during the 15 day test period.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

The initial (day 0) mean body weights of all five dose groups of both sexes were comparable with those of the control group. On day 7, the mean body weight of 10% male dose group was lower (201 g) than the control male group (218 g). The mean body weights of either sex of the remaining four dietary groups were comparable with those of the controls. On day 14, the 10% dose group mean body weight of males was again lower (215 g) than the control male group (243 g). However, the 10% female dose group mean body weight was identical as the control female group (both 178 g). The day 14 body weights of the other dietary groups were comparable with the control group values.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

During weeks one and two the 10% dose group and the control group rats of both sexes consumed similar amounts of ood, The similarity in food consumption between the control and highest dose group indicates that the high dose feed was palatable and gives an assurance that the rats consumed the test material at the desired level. The 0,625 and 1,25% dose group males did not consume less food than the control group during the first week. During week 2, the 0,625, 1.25 and 2,5% dose group males also did not ingest as much food as the control group. The females of the five dose groups consumed equal amounts of food as the control group females during weeks one and two.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Endocrine findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

The changes observed at necropsy were confined to the lungs and were described as discoloredor congested. These changes in the lungs might have resulted from a direct effect of the C02 asphyxiation method used for killing the rats. One female rat of the 10% dose group exhibited an enlarged heart. All other rats appeared normal at necropsy, and no evidence of gross tissue/organ changes were found that could be attributed to the ingestion of sodium aluminosilicate.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

Histopathologic evaluation of tissues of three randomly chosen control and three randomly chosen 10% group rats, showed no obvious trend of tissue change attributable to the ingestion of the test material.
The pulmonary congestions in control and treated rats were interpreted as being agonal and secondary to stress of euthanasia. Squamous metaplasia of the Harderian Gland in one control rat is an uncommon finding and when present, may suggest a potential problem. No obvious trend was detected among the treated and control animals.

Histopathological findings: neoplastic:

not examined

Key result

Dose descriptor:

NOAEL

Effect level:

>= 100 000 ppm

Sex:

male/female

Basis for effect level:

body weight and weight gain

clinical signs

gross pathology

histopathology: non-neoplastic

mortality

Key result

Critical effects observed:

no

The highest concentration of 10% in the diet (100,000 ppm) leads also to very high values when expressed as g/kg bw/d: c. 44.55 g/kg bw/d for males and c. 34.66 g/kg bw/d for females

Conclusions:

Based on body weights, food consumption and gross necropsy findings, the ingestion of sodium aluminosilicate by Fischer 344 male and female rats for fourteen (14) consecutive days at dietary levels of 0.625, 1.25, 2.5, 5 and 10% revealed no marked signs of toxicity.

Executive summary:

Male and female Fischer 344 Rats that ingested sodium aluminosilicate for fourteen consecutive days at levels of 0.625, 1.25, 2,5, 5 and 10% ex­hibited no abnormal physical appearance, abnormal behavior, pharmacotoxic signs or mortalities

Reference 2

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

May 2 - Jun. 28, 1979; experimental phase: 21 May - 6 June 1979

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

no guideline available

Principles of method if other than guideline:

range-finding study to a sub-chronic one: 14-day administration, only reduced number of animals for histopathological examination

GLP compliance:

not specified

Limit test:

no

Specific details on test material used for the study:

Sodium Aluminosilicate, Lot No. B71778, Batch No. 01, received from Midwest Research Institute

Species:

mouse

Strain:

B6C3F1

Sex:

male/female

Details on test animals or test system and environmental conditions:

Thirty-six (36) male and thirty-five (35) female B6C3F1 Hybrid mice (M-9, undeterminable source) were received for test on May 4, 1979. The mice were individually ear-tagged and placed into a sanitized quarantine room for seventeen (17) days. The mice were housed five (5) per cage per sex in polycarbonate cages (62.01 square inches, 4.88 inches high), each equipped with a stainless steel cage top.
The feed was placed into stainless steel feed hoppers equipped with a moveable lid that limited spillage and contamination of the feed. The volume of each feed hopper was 650 cubic centimeters.
The room temperature was maintained at 74 +/- 2°F, 50 +/- 5% relative humidity and had a fluorescent light cycle of 12 hours dark and 12 hours light. Ten (10) to fifteen (15) air changes per hour were maintained.
The mice were assigned to the six (6) test groups (5 per sex/ group) on day 0 of the test by assigning each mouse a random number and then assigning each mouse by use of a random numbers table, from mice grouped into several weight classes.

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on oral exposure:

Four (4) kilograms of dosed feed were prepared for each dose level. The control feed consisted of the basal diet without incorporation of the test material. All preparations were made with NIH-07 Rat and Mouse Ration Mash. Each preparation was hand-mixed in a plastic tub for one minute. The preparation was then placed into a Vortex Feed Mixer and mixed with the intensifier bar for five (5) minutes. The intensifier bar was turned off and the preparation was mixed for fifteen (15) additional minutes. After mixing, each preparation was collected in a plastic bag, labeled and stored in a metal container. Enough feed was mixed to provide sufficient feed for a one week period. On May 28, 1979, three (3) kilograms of each dose level were again prepared in the same manner as described above.
On May 21, 1979, each feed hopper was filled with the corresponding dose level preparation per cage of five (5) mice. The filled feed hopper was weighed. Whenever the feed level in the hopper was insufficient to supply 24 hours of feed, it was removed from the cage and weighed. After weighing, the residual feed in the hopper was discarded and the feed hopper was washed in the cage washing machine. A sanitized feed hopper was filled with the corresponding dosed feed, weighed and placed into the cage. The feed was given to the mice for fourteen (14) consecutive days. On the fifteenth (15th) day, the dosed feed was removed and replaced with NIH-07 basal diet (control).

Analytical verification of doses or concentrations:

no

Duration of treatment / exposure:

14 d

Frequency of treatment:

continuous in the diet

Dose / conc.:

100 000 ppm

Dose / conc.:

50 000 ppm

Dose / conc.:

25 000 ppm

Dose / conc.:

12 500 ppm

Dose / conc.:

6 250 ppm

No. of animals per sex per dose:

5

Control animals:

yes, plain diet

Details on study design:

Post-exposure period: 1 d (day 15)

Observations and examinations performed and frequency:

Each mouse was observed daily for pharmacotoxic signs by a trained animal technician by removing the mouse from the cage and observing it. The mice were observed individually and the observations recorded for fifteen (15) consecutive days. Body weights were recorded on days 0, 7 and 14, Food consumption was measured on days 0, 7 and 14 and whenever the feed had to be replenished due to an inadequate feed supply.

Sacrifice and pathology:

The mice were observed for pharmacotoxic signs on day 15 and killed by CO2 asphyxiation on days 16 and 17 for gross necropsy observations.

Any mice found dead during the fifteen (15) day period were subjected to a gross autopsy examination, and all mice surviving the fourteen (14) day test period were killed by CO2 asphyxiation and subjected to a gross autopsy examination. At necropsy, the following tissues were examined in-situ:
Skin, Mandibular Lymph Node, Mammary Gland, Salivary Gland, Thigh Muscle, Sciatic Nerve, Bone Marrow, Costochondral Junction (Rib), Thymus, Larynx, Trachea, Lungs and Bronchi, Heart, Thyroids, Parathyroids, Esophagus, Stomach, Duodenum, Brain, Eyes, Spinal Cord, Jejunum, Ileum, Colon, Rectum, Mesenteric Lymph Node, Liver, Gall Bladder, Pancreas, Spleen, Kidneys, Adrenals, Bladder, Seminal Vesicles (males), Prostate (males), Testes (males), Ovaries (females), Uterus (females), Nasal Cavity, Pituitary, External and Middle Ears

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

No abnormal external physical appearances, abnormal behavior or pharmacotoxic signs were observed at any time during the test period, including the observation day, for any female mice of the five (5) dose groups or the control group. However, at least one male from each of the five (5) dose groups and the control group exhibited some type of abnormal external appearance. The most widely observed abnormality was ulcerations on the "rump" of the dorsum, Other abnormalities included lacerations on the hindlegs, edematous hindpaws, lacerations at the base of the tail and scabbed areas on the dorsum. All of the abnormal observations were caused by fighting among the five (5) male mice within the cage. The abnormalities were not confined to one specific dose group and the effects of fighting were also observed in the control group.
The feces of all the mice of the 5% and 10% dose groups were abnormally lighter in color than the feces of the mice of the control group and 0.625, 1.25 and 2.5% dose groups.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

One male mouse of the 0.625% dose group was accidentally crushed to death in its cage, but no other mortalities occurred. The mouse’s body was found pinched between the feed hopper and the cage top lid.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

The ingestion of sodium aluminosilicate did not cause any pattern of dose dependent negative or positive differential weight gain relative to the control group.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Because of the spillage of food from the feeders by the mice, an accurate measurement of food consumption could not be made, but all mice gained weight.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Endocrine findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

cf. Clinical signs above

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Necropsies of the mice that ingested sodium aluminosilicate for fourteen (14) consecutive days indicated no evidence of gross tissue / organ changes which could be attributed to the ingestion of the test material.

In most cases, the internal changes observed at necropsy were confined to the lungs. The changes in the lungs were observed as discolorations (dark red and pale red) and might have resulted from a direct effect of the CO2 asphyxiation method used for killing the mice. Incidences of discolored lungs were observed in the control group and all dose groups, except the 0,625% group. One control group female had a collapsed gall bladder. One female of the 1.25% dose group exhibited a slightly enlarged mesenteric lymph node. One male of the 5% dose group had pinhead sized depressed greyish foci on the median surface of the median lobe of the liver (1-2 min.). The 0.625% dose group male that was crushed to death on day 9 had light colored patches on the undersurface of all lobes of the liver. All other mice appeared normal at necropsy and no evidence of gross tissue/organ changes were found that could be attributable to the ingestion of sodium aluminosilicate.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

Histopathologic evaluation of tissues of three randomly chosen control and three randomly chosen 10% group mice, showed no obvious trend of tissue change attributable to the ingestion of the test material.
Pulmonary congestions in one treated and one control mouse were interpreted as being agonal and secondary to stress of euthanasia. Ulcerative dermatitis in one control mouse was interpreted as an incidental finding. No obvious trend was detected among treated and control animals.

Histopathological findings: neoplastic:

not examined

Key result

Dose descriptor:

NOAEL

Effect level:

>= 100 000 ppm

Sex:

male/female

Basis for effect level:

body weight and weight gain

clinical signs

gross pathology

histopathology: non-neoplastic

mortality

Key result

Critical effects observed:

no

Because of the spillage of food from the feeders by the mice, an accurate measurement of food consumption could not be made. Therefore, a reliable value expressed in mg/kg bw/d can also not be calculated.

Conclusions:

Based upon body weight gain, food consumption and gross necropsy findings, the ingestion of sodium aluminosilicate by B6C3F1 Hybrid mice for fourteen (14) consecutive days at dietary levels of 0.625, 1.25, 2.5, 5 and 10% revealed no marked signs of toxicity.

Executive summary:

Female B6C3F1 Hybrid Mice that ingested sodium aluminosi1icate for fourteen (14) consecutive days at levels of 0.625, 1.25, 2.5, 5 and 10% exhibited no abnormal physical appearance, abnormal behavior, pharmacotoxic signs or mortalities.

Reference 3

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Oct. 31, 2019 - Oct. 28, 2020; experimental phase: Nov. 19, 2019 - Mar. 17, 2020

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Justification for type of information:

No validated in vitro method is available for assessing systemic toxicity after repeated exposure.

Qualifier:

according to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Version / remarks:

Jun. 25, 2018

Deviations:

yes

Remarks:

minor ones, not influencing the quality or integrity of the study

GLP compliance:

yes (incl. QA statement)

Limit test:

yes

Specific details on test material used for the study:

Name: Sipernat 22 S
CAS No.: 112916-00-8, 7631-86-9
Chemical Name: synthetic amorphous silica
Production route: precipitation
E-Number (Food Permission): E 551
Batch No.: 199021523
Physical State: solid powder
Colour: white
Storage Conditions: at room temperature, protected from moisture
Active Components: >97% (impurities were not taken into account for final formulation)
Date of Manufacture: 15 February 2019
Date of Analysis: 25 March 2019
Expiry Date: 14 February 2021

Species:

rat

Strain:

Wistar

Details on species / strain selection:

Species/strain: healthy Wistar rats, Crl: WI(Han) (Full Barrier)
Source: Charles River, 97633 Sulzfeld, Germany

Sex:

male/female

Details on test animals or test system and environmental conditions:

- age at the start of the treatment period: approx. 4-6 weeks old
- body weight at the allocation of the animals to the experimental groups:
males: 148 – 181 g (mean: 162.3 g, ± 20% = 129.9 – 194.8 g)
females: 114 – 151 g (mean: 128.2 g, ± 20% = 102.6 – 153.8 g)

- Full barrier in an air-conditioned room
- Temperature: 22 +/- 3 °C
- Relative humidity: 55 +/- 10%
- Artificial light, sequence being 12 hours light, 12 hours dark
- Air change: 10 x / hour
- Free access to Altromin 1324 maintenance diet for rats
- Free access to tap water in bottles, sulphur acidified to a pH of approximately 2.8 (drinking water, municipal residue control, microbiological controls at regular intervals). The purpose of the acidification was to provide additional protection against microbial contamination. This is a standard practice at BSL Munich and should not be associated with any risk for long-term studies.
- The animals were kept in groups of 5 animals / sex / group / cage in IVC cages (type IV, polysulphone cages) on Altromin saw fibre bedding

Route of administration:

oral: gavage

Vehicle:

water

Remarks:

Name: aqua ad injectionem Manufacturer: Delta Medica Batch No.: 906243 Physical State: liquid Storage Conditions: room temperature Expiry Date: May 2022

Details on oral exposure:

The test item formulation or vehicle was administered daily to the animals as a single dose by oral gavage. The application volume for all groups was 7.5 mL/kg body weight. For each animal the individual dosing volume was calculated on the basis of the body weight most recently measured.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Before the treatment period, formulation samples were prepared and analysed in order to obtain knowledge about concentration (±10%) of the test item in the selected vehicle. Investigation of stability was performed in the first study week. Investigation of homogeneity and substance concentration was included on samples from various levels (top, middle and bottom) of prepared formulations study weeks 1, 5, 9 and 13.

Dose Formulation Analysis
Recoveries of reference substance Sipernat 22 S were found between 88 and 95%. Samples of sampling week 5 and 13 fulfilled the requirement of ± 10% nominal test item concentration. Recoveries of study week 1 and 9 reached 88%.

Duration of treatment / exposure:

90 days

Frequency of treatment:

daily

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

10 (+ 5 in satellite group for recovery studies)

Control animals:

yes, concurrent vehicle

Details on study design:

additional 28 days of recovery for the satellite group

Positive control:

none

Observations and examinations performed and frequency:

Body Weight and Food Consumption
The body weight was recorded once before the assignment to the experimental groups, on the first day of administration and weekly during the treatment and recovery period.
Food consumption was measured weekly during the treatment and recovery period.

Clinical Observations
All animals were observed for clinical signs during the entire treatment period of 90 days. The recovery animals were observed for an additional period of 28 days following the last administration.
General clinical observations were made at least once a day, preferably at the same time each day and considering the peak period of anticipated effects after dosing. The health condition of the animals was recorded. Twice daily all animals were observed for morbidity and mortality except on weekends and public holidays when observations were made once daily.
Detailed clinical observations considering spontaneous activity, lethargy, recumbent position, convulsions, tremors, apnoea, asphyxia, vocalisation, diarrhoea, changes in skin and fur, eyes and mucous membranes (salivation, discharge), piloerection and pupil size were made outside the home cage in a standard arena once before the first administration and weekly thereafter.

Functional Observations and Ophthalmology
Once before the first exposure and in the last week of the exposure period as well as in the last week of the recovery period multiple detailed behavioural observations were made outside the home cage using a functional observational battery of tests. These tests were conducted in all animals.
Ophthalmological examinations, using an ophthalmoscope were made on all animals before the first administration and in the last week of the treatment period as well as at the end of the recovery period in the recovery animals.

Haematology
Haematological parameters were examined at the end of the treatment and recovery period prior to or as part of the sacrifice of the animals. After overnight fasting, blood from the abdominal aorta of the animals was collected in EDTA-coated tubes.

Sacrifice and pathology:

Blood Coagulation
Coagulation parameters were examined at the end of the treatment and recovery period. After overnight fasting, blood was collected from the abdominal aorta of the animals during terminal anaesthesia in citrate tubes.

Clinical Biochemistry
Parameters of clinical biochemistry were examined at the end of the treatment and recovery period prior to or as part of the sacrifice of the animals.
After overnight fasting, blood from the abdominal aorta of the animals was collected in serum separator tubes.

Urinalysis
One day after the last administration, urinary parameters from all surviving animals to be subjected to necropsy at the end of the treatment or recovery period were analysed. Urine colour and appearance were recorded. Parameters were measured using qualitative indicators (Henry Schein Urine Stripes URI 10SL).

Pathology
One day after the last administration (study day 91) all surviving animals of the treatment period and 4 weeks after the last administration all surviving animals of the recovery period (study day 119) were sacrificed using anaesthesia (ketamine/xylazin) and were subjected to a detailed gross necropsy which includes careful examination of the external surface of the body, all orifices and the cranial, thoracic and abdominal cavities and their contents. Vaginal smears were examined on the day of necropsy to determine the stage of oestrous
cycle.

Organ Weight
The wet weight of the organs of all sacrificed animals was recorded as soon
as possible. Paired organs were weighed together. Weight of thyroid/parathyroid glands was measured after fixation. Organ weights of animals found dead or euthanised for animal welfare reasons were not recorded.

Histopathology
The weighted organs were examined histopathologically after preparation of
paraffin sections and haematoxylin-eosin staining for the animals of the control and high dose groups sacrificed at the end of the treatment period and any animal found dead or euthanised before the planned day of sacrifice.

Statistics:

A statistical assessment of the results of the body weight, food consumption, parameters of haematology, blood coagulation and clinical biochemistry and absolute and relative organ weights was performed for each gender by comparing values of dosed with control animals using either a parametric one-way ANOVA and a post-hoc Dunnett Test or a nonparametric Kruskal-Wallis Test and a post-hoc Dunn’s Test, based on the results of homogeneity and normality tests. Furthermore, statistical comparisons of data acquired during the recovery period were performed with a Student’s t-Test or Mann-Whitney U-Test when appropriate. These statistics were performed with Ascentos 1.3.4 software or
GraphPad Prism V.6.01 software (p < 0.05 was considered as statistically significant).

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Few animals showed abnormal breathing. Piloerection was seen in 12 of 30 animals, but this was regarded rather a reaction of discomfort to application and not considered as test item related.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

2/30 in total (one male and one female) in treated and recovery groups
No test item related mortality occurred during the treatment or recovery period of the study.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

No toxicological relevant effects on mean body weight and body weight development were observed during the treatment or recovery phase of the study in the dose groups.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

No toxicological relevant effects on food consumption were determined during the
treatment or recovery phase of the study in the dose groups.

Food efficiency:

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Description (incidence and severity):

No toxicological relevant effects on parameters of haematology or blood coagulation were detected at the end of the treatment or recovery period in the dose groups.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

No toxicological relevant effects on hormones or parameters of clinical
biochemistry were detected at the end of the treatment or recovery period in the
dose groups.

Endocrine findings:

not examined

Urinalysis findings:

no effects observed

Description (incidence and severity):

No toxicological relevant effects on urinary parameters were detected at the end of the treatment or recovery period in the dose groups.

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

No toxicological relevant observations were made during the functional observation
battery during the treatment or recovery phase of the study in dose groups.

Immunological findings:

no effects observed

Description (incidence and severity):

Enhanced immune system evaluation did not reveal indicators for an affection of the immune system by the test substance.

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

Mean thyroid/parathyroid weights, thyroid/parathyroid to body weight ratios as well as thyroid/parathyroid to brain weight ratios of Sipernat 22 S treated males were slightly increased compared to controls, with differences in thyroid/parathyroid to body weight ratios reaching statistical significance.
Mean weight: 0.045 g vs. 0.033 g in controls
Organ/Body: 0.0116% vs. 0.0075% in controls
Organ/Brain: 2.13% vs. 1.56% in controls
As effects were small and not observed in females, these findings were considered as incidental.

Test item treated males had statistically significantly decreased epididymides to brain weight ratios (77.40%) compared to controls (83.38%). Mean epididymides weights were also decreased, yet not significantly.
Mean weight: Control: 1.74 g, Sipernat 22 S: 1.65 g
As effects were small and not observed in females, these findings were considered as incidental.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Only three of thirty animals treated with Sipernat 22 S showed pathological abnormalities:
- The thymus of male no. 12 had an abnormal brown/red colour, caused by congestion, which was considered to be incidental.
- The lungs of prematurely euthanized male no. 17 had an abnormal spotted colour, failed to collapse at necropsy and were fluid filled and spongy.
Histopathological analyses revealed that in the lungs, there was an intrabronchial foreign material associated with a bronchial/bronchiolar inflammation, a consequence of test item aspiration.
- Prematurely euthanized female no. 120 died of oesophageal perforation by misgavage. Consequently, the thoracic cavity was filled with a white, viscous fluid. Histopathological analysis revealed inflammation in the oesophagus, the
heart and the lung, leading to an adhesion between heart and lung. The thymus also exhibited inflammation and hence was enlarged and had an abnormal brown/red colour. The duodenum, jejunum and ileum were filled with a yellow fluid, a finding which was without a histopathological correlate. The thyroid gland also had an abnormal red colour, which was considered as incidental.

Neuropathological findings:

no effects observed

Description (incidence and severity):

No pathological change was recorded in the nervous system or the intestinal segments, and specifically, no unusual findings were noted in the Peyer’s patches.

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

There were no treatment-related gross lesions or histological findings in any of the organs and tissues examined that could be attributed to treatment with the test item. There were no findings that distinguished controls from test item-treated animals.

Histopathological findings: neoplastic:

no effects observed

Description (incidence and severity):

There were no treatment-related gross lesions or histological findings in any of the organs and tissues examined that could be attributed to treatment with the test item. There were no findings that distinguished controls from test item-treated animals.

Key result

Dose descriptor:

NOAEL

Effect level:

>= 1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

behaviour (functional findings)

body weight and weight gain

clinical biochemistry

clinical signs

food consumption and compound intake

gross pathology

haematology

histopathology: neoplastic

histopathology: non-neoplastic

immunology

mortality

neuropathology

organ weights and organ / body weight ratios

urinalysis

Key result

Critical effects observed:

no

No test item related mortality occurred during the treatment or recovery period of the study. No toxicologically relevant clinical findings were observed in the dose groups during the treatment or recovery period of the study.
Detailed clinical observations and the functional observation battery revealed no noticeable or toxicological relevant findings in the dose groups during the treatment or recovery period of the study.
No toxicological relevant effects on body weight or food consumption were observed during the treatment or recovery phase of the study in the dose groups.
No toxicological relevant effects regarding haematology, clinical biochemistry, hormones, blood coagulation, urine, organ weights or vaginal smears were detected at the end of the treatment or recovery period in the dose groups.
No test item related macroscopic findings were observed during necropsy.
There were no treatment-related gross lesions or histological findings in any of the organs and tissues examined that could be attributed to treatment with the test item. No pathological change was recorded in the nervous system or the intestinal segments, and specifically, no unusual findings were noted in the Peyer’s patches. In addition, enhanced immune system evaluation did not reveal indicators for an affection of the immune system.
Therefore, the oral application of Sipernat 22 S for 90 days at a dose of 1000 mg/kg was deemed to be toxicologically not harmful.

Conclusions:

The oral application of Sipernat 22 S for 90 days at a dose of 1000 mg/kg was deemed to be toxicologically not harmful.

Executive summary:

The aim of this study was to assess the possible health hazards which could arise from repeated exposure of Macrosorb F60, Zeofree 5133 and Sipernat 22 S via oral administration to rats over a period of 90 days. Only the results on the latter are documented here, since Sipernat 22 S (a synthetic amorphous silica) is a structurally related read-across substance to Silicic acid, aluminum sodium salt (CAS 1344-00-9, NAS). No adverse effects were found with the fixed dose of 1000 mg/kg bw/d.

Reference 4

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

Jan, 20 - Oct. 13, 2011; experimental phase: Jan. 26 - May 3, 2011

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Version / remarks:

2008

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

SAS (Synthetic Amorphous Silica PR-A-02 ): NM-200
supplied by JRC, Ispra, on behalf of the sponsor, CEFIC, The European Chemical Industry Council

Species:

rat

Strain:

Wistar

Details on species / strain selection:

purchased from: Charles River Deutschland, Sulzfeld, Germany

Sex:

male

Details on test animals or test system and environmental conditions:

Animals were housed in groups of 5 per cage in Makrolon Type IV cages in animal room T1.044 in the conventional area. Absorbent softwood was used as bedding material in the cages (Lignocel BK 8-15, ssniff GmbH, Soest, Germany). Drinking water from the Hannover city water supplier was offered fresh weekly, in Makrolon bottles (approximately 300 ml), ad libitum. Food was offered ad libitum fresh weekly. The diet used (ssniff R/M-H) was supplied by ssniff GmbH, Soest, Germany. The temperature in the animal room was set at 22 ± 2oC and the rel. humidity at 30 - 70%. The animal room lighting was a 12-hour light/dark cycle controlled by an automatic timing device.

Route of administration:

oral: gavage

Vehicle:

other: a solution of methylhydroxypropylcellulose (0.5 %) in deionised water (Milli-Q, Millipore)

Duration of treatment / exposure:

28 d

Frequency of treatment:

daily

Dose / conc.:

100 mg/kg bw/day (nominal)

Dose / conc.:

300 mg/kg bw/day (nominal)

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Remarks:

recovery group

No. of animals per sex per dose:

5 (males only)

Control animals:

yes, concurrent vehicle

Details on study design:

14-day observation phase for some animals (5 treated and 5 control)

Observations and examinations performed and frequency:

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 included 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.

Sacrifice and pathology:

Gross necropsy was performed on all rats.
Histopathological examination of the cerebrum, cerebellum, peripheral nerve, trachea, thyroid, lung, mandibular lymphnode, mesenteric lymphnode, thymus, heart, skeletal muscle, liver, spleen, spinal cord, adrenals, forestomach, glandular stomach, duodenum, jejunum, ileum, cecum, colon, rectum, kidneys, urinary bladder, eyes with optic nerve, testes, epididymis, coagulation glands, seminal vesicles, prostate, femur with bone marrow and joint was performed in 5 males of the high dose group 4 (1000 mg/kg bw Synthetic Amorphous Silica) and the control group 1 (vehicle). Gross findings of other organs were examined in addition.

Other examinations:

Locomotor Activity
During the last week of treatment, spontaneous locomotor activity over 60 minutes using the „Motitest“ computerized light-beam system (TSE, Homburg/Ts., Germany) was determined. The data were analyzed in 15-minutes intervals. In addition, the total values for distance, time in rest, time in movement, rearing time, and number of rearings were determined. Additional testing for spontaneous locomotor activity in the two recovery groups was not performed in the second recovery week, because no significant statistical differences between the dose groups and the control group were determined in the first testing.

Hematological, clinical chemistry analyses (hematology and serum chemistry) were performed before final sacrifice for all animals:
- Erythrocyte count, hemoglobin, hematocrit, mean erythrocyte volume, mean erythrocyte hemoglobin mass, mean erythrocyte hemoglobin concentration, total and differential leukocyte count, platelet count, prothrombin time, and thromboplastin time were recorded.
- Aspartate aminotransferase (EC 2.6.1.1), alanine aminotransferase (EC 2.6.1.2), gammaglutamyl transferase (EC 2.3.2.2), alkaline phosphatase (EC 3.1.3.1), sorbitol dehydrogenase (EC 1.1.1.14), cholinesterase (EC 3.1.1.8), creatin kinase (EC 2.7.3.2), total bilirubin, urea, creatinine, total protein, albumin, cholesterol, glucose, sodium, potassium, calcium, chloride, inorganic phosphate and triglyceride were measured. Globulin and
albumin/globulin ratio were calculated from the albumin and total protein data.

Analysis of Silicon in tissue (blood, kidney and liver) was performed by ICP-MS.

Statistics:

Differences between groups were considered case by case as statistically significant for p<0.05. Data were analyzed using analysis of variance. If the group means differed significantly according to the analysis of variance, the means of the treatment groups were compared with the means of the control group, using DUNNETT's modification of the t-test. Kruskal-Wallis ANOVA and Mann-Whitney U-test were applied in the case of non-homogeneous data. The statistical evaluation of the histopathological findings was done with the two-tailed FISHER test using the PROVANTIS system. For comparisons of semiquantitative data, the Chi-square test was used.

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

The only statistically significant difference in body weight gain was observed in the mid-dose group between day 7 and 14. It was not dose dependent and transient and therefore considered to be incidental and of no toxicological relevance.

Food consumption and compound intake (if feeding study):

no effects observed

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

A slight but significant decrease was found in the partial thromboplastin time (PTT) in the mid dose group. The white blood cell count (WBC) and the lymphocyte count (LYMC) were significantly decreased in the mid dose group. However, the relative lymphocyte count (LYM) was unchanged. As all values were within the normal range, these changes are considered to be of minor toxicological relevance.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

Cholinesterase (CHE) and glucose (GLUC) levels were marginally decreased in the mid dose group. However, all values were within the normal range and the changes reported above are thus considered to be incidental.

Behaviour (functional findings):

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

No substance-related findings were observed.

Two animals (one male of group 2 (low dose), one male of group 5 (recovery control)) displayed an unilateral exophthalmus, probably caused by the previous retro-boulbous blood sampling.
One male of group 3 (mid dose) displayed a slight reduction in size of the prostate and the seminal vesicle. This alteration is considered to be an incidental finding, occasionally occurring in this strain.
One male of group 6 (recovery high dose) displayed a slight unilateral reduction in size of the testes and epididymis. This alteration is considered to be an incidental finding, occasionally occurring in this strain.

Neuropathological findings:

no effects observed

Description (incidence and severity):

No influence on locomotor activity was observed.

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

Substance-related findings were not observed in the histologically examined organs of males of the control and high dose group.

Lungs: Findings in the lungs occurred only in single animals of the control group like very slight focal bronchiolo-alveolar hyperplasia, slight alveolar histiocytosis (1/5), very slight pulmonary hemorrhage (1/5) and very slight/slight focal inflammatory cell infiltration (2/5).
Kidney: The finding of unilateral focal very slight tubular basophilia with nuclear crowding was seen in 1/5 males of the control group. Other degenerative changes of the kidneys such as unilateral slight focal subcapsular mononuclear cell infiltration associated with focal very slight fibrosis occurred in another control male, which showed a bilateral multifocal slight tubular dilatation in addition.
Liver: (Multi)focal very slight microgranulomas were diagnosed in 3/5 males of the control and in 5/5 males of the high dose group.
Prostate: 4/5 of the control animals and 5/5 of the high dose group animals showed very slight/slight (multi)focal inflammatory lesions.
Testes, Epididymis: Very slight/slight/moderate multifocal degeneration and depletion of germ cells was seen in 4/5 males of the control, and 4/5 males of the high dose group. Very slight/slight multifocal tubular atrophy was seen in 1/5 males of the control and in 2/5 males of the high dose group in addition. Intraluminal exfoliated degenerated germ cells were detected in the corresponding epididymis of 1/5 male of the control and 1/5 of the high dose group. A slight focal spermatic granuloma was noted in the epididymis of one control male. Very slight (multi)focal interstitial mononuclear cell infiltration was recorded in the epididymis of 1/5 males of the control and in 1/5 males of the high dose group. These alterations are
considered to be incidental findings.
Other organs: Several other findings in various organs of the examined experimental groups were noted which also occurred incidentally and were not unusual for this strain and age.

Histopathological findings: neoplastic:

no effects observed

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

Determination of silicon ion concentration: The ion concentrations in kidney, liver and blood were comparable in control animals (group 1) and high dose animals (group 4) and no treatment related changes were observed.

Key result

Dose descriptor:

NOAEL

Effect level:

>= 1 000 mg/kg bw/day (nominal)

Sex:

male

Basis for effect level:

behaviour (functional findings)

body weight and weight gain

clinical biochemistry

clinical signs

food consumption and compound intake

gross pathology

haematology

histopathology: neoplastic

histopathology: non-neoplastic

mortality

neuropathology

organ weights and organ / body weight ratios

other: determination of silicon ion concentration

Key result

Critical effects observed:

no

No death occurred during the study and no adverse clinical symptoms were observed. No effects on food consumption or body weight were seen. The measurements of the spontaneous locomotor activity and the functional observational battery displayed no influence by the treatment. Evaluation of hematological and clinical chemistry parameters did not reveal any treatment related effects. Decreases of the partial thromboplastin time (PTT), white blood cell count (WBC) and lymphocyte count (LYMC) as well as cholinesterase (CHE) and glucose (GLUC) in group 3 (mid dose) after 28 d of exposure were considered not treatment related. Creatinin kinase (CK) and blood urea (UREA) concentration were mildly decreased in group 6 (high dose recovery) after a two week recovery period. All values were within the normal range and the changes were considered not treatment related and to be due to interindividual variability.

During necropsy, no substance-related findings were observed. No effects were seen on organ weights or the organ weight to bodyweight ratio. During histopathological examination, no substance-related findings were observed in the examined organs of males of the control and high dose group.

Conclusions:

Synthetic Amorphous Silica did not cause any substance-related effects in doses of up to 1000 mg/kg bodyweight after oral exposure for 28 days in male Wistar (WU) rats. Therefore, the highest dose tested (1000 mg/kg bw/d) was determined as the NOAEL in this study.

Executive summary:

The objective of this study was to evaluate the possible toxicity of Synthetic Amorphous Silica (NM-200) after oral administration (gavage) in rats for 28 days and an additional 14-day recovery period.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Study duration:

subacute

Experimental exposure time per week (hours/week):

168

Species:

other: rat, mouse

Quality of whole database:

reliability 2

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records

Reference

Reference 1

Endpoint:

short-term repeated dose toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

experimental phase (acute & sub-acute study): Jun. 5, 2018 - Apr. 17, 2019

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

test procedure in accordance with national standard methods with acceptable restrictions

Remarks:

only 5 day-test instead of a 28- or 90 day one (but cf. Landsiedel 2014)

Justification for type of information:

Cf. the endpoint: Literature, Landsiedel, 2014, Statement on 5-day studies. It claims that a 5-day study design can be as expressive as a 90-day study for testing nanoparticles.

Reason / purpose for cross-reference:

other: justification information

Reason / purpose for cross-reference:

reference to same study

Qualifier:

no guideline followed

Principles of method if other than guideline:

subacute inhalation: 4-hour exposure daily for 5 days, 3 dose levels, 13-week observation period

GLP compliance:

yes

Limit test:

no

Specific details on test material used for the study:

Test item identification: Sodasil P95
Description: Amorphous silicate of sodium and aluminum
Formula/Chemical group: Nanosilicates
Chemical Abstract No.: 1344-00-9
EINECS No.: 215-684-8
Aspect: White powder
Storage condition: Room temperature

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

Age (at treatment start): 10 weeks old
Body-weight range(at treatment start): 206.9 – 277.8 g (females) and 314.7 – 490.9 g (males)
Temperature: 20.0ºC – 24.0ºC (Mean 22.0ºC)
Humidity: 40.0% – 80.0% (Mean 60.0%)
Room air renovation cycles: 30
Photoperiod: 12 hours of light: 12 hours of darkness
Feeding: Irradiated certified laboratory dry diet RM1 (P) QC Reference 831193 (batch number 3077, Dietex SDS) was offered with unlimited supply.
Drinking: Autoclaved mineral water was offered ad libitum.

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

whole body

Vehicle:

other: 0.05% BSA (CAS No. 9048-46-8), 30 μL of absolute alcohol and sterile water

Mass median aerodynamic diameter (MMAD):

392.8 nm

Geometric standard deviation (GSD):

14.3

Remarks on MMAD:

Mass dispersion and size, as well as concentration in the air were assessed in the extraction tube from the inhalation chamber by using an optical particle sizer (ITENE). Sodasil P95 concentration measured allowed the observation of different dispersion grades of nanoparticles below 1 and 6 μm, being the resulting mean + standard deviation diameter value of 392.8 + 14.3 nm. According to these results there is a relative relevant difference with regard to the theoretical dose estimated. In contrast measurements made by using two air samples from the inhalation chamber, once upon achieved the steady-state demonstrated that 94–96% of nanoparticles are also ranged at mean values of 252 to 388 nm. Accordingly, actual doses applied to the animals were slightly lower than theoretical ones (i.e., 0.88, 4.39, 21.97 mg/m³ for the subchronic study). Hence, nanoparticles content in the air allowed to confirm the appropriate dose regime in order to assess the impact of subcacute treatments in the in vivo studies.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

s. above (Remarks on MMAD)

Duration of treatment / exposure:

4 hours per day for 5 days

Frequency of treatment:

daily

Dose / conc.:

21.97 mg/m³ air (analytical)

Remarks:

theoretical (cut-off dose): 28.5 mg/m³

Dose / conc.:

4.39 mg/m³ air (analytical)

Remarks:

theoretical (cut-off dose): 5.7 mg/m³

Dose / conc.:

0.88 mg/m³ air (analytical)

Remarks:

theoretical (cut-off dose): 1.1 mg/m³

No. of animals per sex per dose:

5

Control animals:

yes

Details on study design:

Subchronic systemic toxicity of Sodasil P95 was tested in male and female Wistar rats (n=5/sex/dose). Animals were subject to a daily 4-hour inhalation exposure for 5 days at theoretical dose levels of 1.1, 5.7 and 28.5 mg/m³. Actual doses applied to the animals were slightly lower than theoretical ones, i.e., 0.88, 4.39, 21.97 mg/m³.
The administration methods used were based on the procedures described in the OECD TG 436
Acute Inhalation Toxicity – Acute Toxic Class Method and the study design published by Arts et al. (2007) in Food Chem. Toxicol., 45: 1856-1867 to assess subchronic systemic toxicity following subacute exposure of the test item by using an inhalation Scireq chamber.
The observation period was 13 weeks.

Observations and examinations performed and frequency:

The animals were weighed and observed daily for the assessment of toxic effects for 90-94 days (subchronic systemic toxicity study).
Throughout the study periods, food and water were also weighed regularly to monitor changes in food and water intake.
Clinical findings were checked daily by observing any toxic effects.

Sacrifice and pathology:

Blood samples were drawn from the cava vein at the end of the experiment (Day 95) of all animals under light isoflurane anesthesia. The animals were not fasted before blood sampling but allowed access to water ad libitum. The samples were collected in the working day to reduce biological variation caused by circadian rhythms.
In addition, a bronchoalveolar lavage was performed by intubation of the trachea. A total 6 mL of PBS were perfused slowly for 1 minute and the solution was kept inside for 5 minutes before collection. The collected lavage was centrifuged at 1500 g for 10 minutes and then the pellet was resuspended in 0.5 mL of PBS to allow haematological and biomarker analyses.
Bioavailability were assessed in five tissues (blood, lung, liver, kidney and spleen).

At the end of the observational period: 13 weeks (subchronic) after treatment
initiation, all animals were necropsied and a macroscopic examination was performed. A full necropsy was performed on all main study and recovery animals. The necropsy included the examination of the external surface of the body, all orifices, cranial, thoracic and abdominal cavities and the observation of the organs both in situ and after evisceration.
At necropsy, several organs were subjected to microscopic examination:
Lungs, Spleen, Heart, Liver, Kidneys and Thoracic lymph nodes

Statistics:

For continuous data, a parametric analysis was performed to assess if variables follow a normal distribution and variances are homogeneous. If so, a one-way analysis of variance (ANOVA) followed by Dunnett’s test was performed. On the contrary, the Kruskal-Wallis ANOVA followed by the Mann-Whitney U-test was applied if Kolmogorov-Smirnov normality test was significant.
For organ weight data, parametric statistical comparison was done with absolute organ weights and those rated by the terminal body weight of each animal, unless non-parametric methods are applied when normality and variances homogeneity is not reached.

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Repeated administration for 5 days did not induce any change in bodyweight or bodyweight gain in females. The pattern was comparable in males, although those treated at the intermediate and high dose levels showed higher body weight gain as compared to controls. This effect was considered null of relevance from a toxicological point of view.

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:

effects observed, treatment-related

Description (incidence and severity):

Hematological analysis at the end of the observational period, where just occasional statistical differences (p<0.05, one-way ANOVA followed by Dunnett’s test or Kruskal-Wallis followed by Mann-Whitney U-test) could be seen: MCHC and monocytes count in males and, MCHC and MCH in females, both compared to control animals. However, these changes had no relevance since the final values were within the normal range of values for this animal species. Overall, the hematological profile in bronchoalveolar lavage (BAL) was not significant, although monocytes count was significantly lower (p<0.05, one-way ANOVA followed by Dunnett’s test) at the highest dose levels as compared to the control group.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Blood chemistry analysis in serum and in BAL were also performed. Overall there were no significant effects on any parameter: creatinine, urea, total bilirubin, GPT/ALT, GOT/AST, alkaline phosphatase (serum) and lactic dehydrogenase, albumin and N-acetyl-glucosamine (NAG) (BAL), but total proteins (p<0.05) and alkaline phosphatase (p=0.001, one-way ANOVA followed by Dunnett’s test) were significantly increased in male animals at the low and intermediate dose levels and at the highest level of Sodasil P95, as compared to control animals. These changes were considered effects not biologically relevant from a toxicological point of view.

Collagen concentration was estimated in dry lungs by means of quantification of the hydroxyproline levels. Mean tissue levels of collagen tended to increase with the dose increase in males (4, 18 and 35-fold) and females (34, 171 and 183-fold) at the three dose levels compared to control animals (mean of 2.1 µg/g tissue and

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

Macroscopic examination and organ weights did not allow the observation of any event or trend in male and female animals related to dose administration.

Gross pathological findings:

no effects observed

Description (incidence and severity):

Macroscopic examination and organ weights did not allow the observation of any event or trend in male and female animals related to dose administration.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

Histopathological examination of six relevant tissues (heart, lungs, liver, spleen, kidneys and thoracic lymph nodes) did not allow the registration of any relevant event. All findings recorded were considered to be within the range of normal background lesions that may be seen in rats of this strain and age and under the experimental conditions used in this study.

Histopathological findings: neoplastic:

no effects observed

Other effects:

effects observed, treatment-related

Description (incidence and severity):

Bioavailability was assessed in five tissues (blood, lung, liver, kidney and spleen) by TEM.
Small nanosilicates (< 160 nm) were seen in all tissues with the exception of whole blood,
where no conclusive observations could be made. Nanoparticles were confined, as expected,
inside the reticuloendothelial system in lysosomes into macrophages in lungs, Küpffer cells in
liver, proximal tubular cells (close to the glomerulus) in kidneys and in spleen. No nanosilicates
were seen in the nucleus nor in mitochondria. The lysosomal location is commonly known for
all nanoparticles, most of them devoid of any toxicological relevance, and there is no reason
to expect adverse effects here.

Key result

Dose descriptor:

NOAEL

Effect level:

>= 21.97 mg/m³ air

Sex:

male/female

Basis for effect level:

behaviour (functional findings)

body weight and weight gain

clinical biochemistry

clinical signs

food consumption and compound intake

gross pathology

haematology

histopathology: neoplastic

histopathology: non-neoplastic

Remarks on result:

other: The theoretical cut-off dose was 28.5 mg/m³. The actual dose applied to the animals was slightly lower: 21.97 mg/m³.

Key result

Critical effects observed:

no

There were no clinical signs/behavioral changes registered throughout the study and body weight gain and food and water intake of male and female animals treated with Sodasil P95, at the different dose levels, were similar to that registered in control animals.

Overall, no effects could be observed on hematological, serum biochemistry nor biomarkers measured in the bronchoalveolar lavage (BAL) and lung tissue at the end of the experimental period, although monocytes in whole blood, total proteins and alkaline phosphatase in BAL and content of collagen in lungs tended to increase with dose in a significant manner. All these changes were considered as effects (not adverse events) without biological relevance from a safety point of view, they might have been residual repair mechanisms in front of intensive inhalation. Hence, no adverse events could be established based on the hematological, clinical biochemistry in serum and in BAL at any dose level assayed

As expected, the bioavailability measured in five tissues (blood, lung, liver, kidney and spleen) allowed the observation of remnant nanosilicates inside lysosomes of the reticuloendothelial system from the different tissues (lungs, liver, kidneys and spleen), with the exception of whole blood, following the usual pattern seen for other nanoparticles.

Neither macroscopic changes nor clear significant trends in organ weights from animals treated at the different dose levels with Sodasil P95 could be recorded.

Finally, the histopathology examination of six relevant tissues (heart, lungs, liver, spleen, kidneys, and thoracic lymph nodes) did not allow the registration of any relevant effect.

 

Conclusions:

Sodasil P95 administered by 5-day repeated inhalation (and an observation period of 13 weeks) to the Wistar rat showed a No-Observed-Adverse-Event-Level (NOAEL) of 21.97 mg/m³, expressed as actual dose, according to the subchronical assessment.

Executive summary:

Subchronic systemic toxicity of Sodasil P95 was tested in male and female Wistar rats (n=5/sex/dose). Animals were subject to a daily 4-hour inhalation exposure for 5 days at theoretical dose levels of 1.1, 5.7 and 28.5 mg/m³. Actual doses applied to the animals were slightly lower than theoretical ones, i.e. 0.88, 4.39, 21.97 mg/m³. The observation period lasted 13 weeks. Although some effects were observed, none of these were assessed as adverse.

Reference 2

Endpoint:

sub-chronic toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Dec. 11, 2011 - May 18, 2014

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)

Version / remarks:

2009-09-07

Deviations:

yes

Remarks:

additional endpoints; males only

Principles of method if other than guideline:

OECD guideline 413 with additional endpoints (bronchoalveolar lavage, cell proliferation, immunological parameters, oxidative stress analysis, electron microscope analysis, toxicokinetics) to address nanoparticle-specific aspects of toxicity

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

UPAC Name: Silicon Dioxide
Common Name, Synonyma: Synthetic Amorphous Silica, NM-200
CAS #: 12926-00-8 (CAS # for precipitated synthetic amorphous silica)
Information note: CAS # 7631-86-9 (general CAS # for silica including synthetic amorphous silica)
Batch: NM-200 (Master-Batch: JRC, Ispra)
Expiry Date: 12/2012 (Stable particle sample)
Purity: 96.5% (value given by JRC, Ispra)
Molecular weight (g/mol): 60.74

Species:

rat

Strain:

Wistar

Remarks:

Wistar WU

Details on species / strain selection:

purchased from Charles River Deutschland (Sulzfeld, Germany)

Sex:

male

Details on test animals or test system and environmental conditions:

The age of the animals at the start of exposure was approx. 9-10 weeks and the weight approx. 280 gram, two rats housed per cage, tap water from the Hannover city water supplier was offered fresh weekly or more often, if necessary. As diet a commercial chow in pellet form was used, identified as Ssniff "V1534". 22 +/- 2 °C, 40 - 70% humidity, 12-hour light/dark.

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

nose only

Vehicle:

clean air

Mass median aerodynamic diameter (MMAD):

ca. 2.16 - ca. 3.12 µm

Remarks on MMAD:

The MMADs were determined by two methods: impactor and SEM analysis.
low dose: 2.16 µm, GSD: 0.09
mid dose: 2.94 µm, GSD: 0.2
high dose: 3.12 µm, GSD: 0.06

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The target aerosol concentrations of 1, 2.5 and 5 mg NM-200/m³ were achieved to 104%, 100% and 101%, respectively. Calculation of the aerosol generation efficiency (actual vs. nominal concentration) resulted in 31%; this low value demonstrates that experimental conditions are different to any handling and use conditions of NM-200.

- Due to the particle size distribution in the test medium the nominal concentration was
3.23 mg/m³, to achieve 1 mg/m³ aerosol test concentration.

- Due to the particle size distribution in the test medium the nominal concentration was
8.06 mg/m³, to achieve 2.5 mg/m³ aerosol test concentration.

- Due to the particle size distribution in the test medium the nominal concentration was
16.13 mg/m³, to achieve 5 mg/m³ aerosol test concentration.

Duration of treatment / exposure:

6 h / d

Frequency of treatment:

5 d / w for 90 days (= 65 exposure days)

Dose / conc.:

1 mg/m³ air

Remarks:

effective concentration (corresponds to 3.23 mg/m³ nominal concentration)

Dose / conc.:

2.5 mg/m³ air

Remarks:

effective concentration (corresponds to 8.06 mg/m³ nominal concentration)

Dose / conc.:

5 mg/m³ air

Remarks:

effective concentration (corresponds to 16.13 mg/m³ nominal concentration)

No. of animals per sex per dose:

55 (males only)

Control animals:

yes, concurrent vehicle

Details on study design:

90-day post-inhalation recovery period

Observations and examinations performed and frequency:

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 included 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.
Individual body weight was recorded to the nearest 0.1 g twice a week for the first month and once a week throughout the remainder of the study (including post-exposure observation period) for all animals.

Sacrifice and pathology:

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 lung and the lower half of the trachea were weighed, and used for BAL or histopathology. The following organs were trimmed and wet weights were recorded: liver, kidneys, adrenals, testes, epididymides, thymus, spleen, brain, 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 given in brackets were prepared for histopathology.

Other examinations:

Cytological parameters
• total cell count (recruitment of lung leukocytes)
• viability test (giving percentage of alive leukocytes among the total number of cells)
• differential cell count (inflammatory (PMNs) or immunological (lymphocytes) reactions)
Biochemical parameters
• lactic dehydrogenase (LDH = cytosolic marker enzyme; increased permeability of membranes, cell damage and lysis)
• β-glucuronidase (measure of phagocytic activity of macrophages; lysis of macrophages)
• total protein (marker of transsudation; damage of epithelial cells).
Oxidative Stress and Immunotoxicological Parameters:
TNF-a, IL-8 (CINC-1), IL-6, TGF-b

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.

Clinical signs:

no effects observed

Mortality:

mortality observed, non-treatment-related

Description (incidence):

only 2 / 220
Effects indicating systemic toxicity were not observed.

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

Statistically significant increase of food comsumption as compared to controls was observed on several dates in all NM-200 dose groups. Due to inconsistency in dose-dependency and only small absolute differences observed these findings are considered as incidental.

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Statistically significant changes were observed for hemoglobin and for segmented neutrophils (calculated) in mid dose group. This findings are considered as incidental, not-treatment related findings.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

• Statistically significant increases of polymorphonuclear neutrophils, lymphocytes and lactic dehydrogenase, ß-glucuronidase and total protein levels/activity and decrease of macrophages were detected in the mid and high dose groups 1 day after end of exposure. In the low dose group a statistically significant increase of polymorphonuclear neutrophils was detected. However, all these effects were reversible and had returned to control levels at the 3-month post-exposure sacrifice date. Thus, NM-200 dust showed a strong acute response, however, rapid recovery upon cessation of exposure.
• The analysis of the oxidative stress related secretion of reactive oxygen intermediates (ROI) showed a decrease with and without zymosan stimulation in the high dose group 14 days after end of exposure as compared to clean air controls but not at 1 day postexposure. A significantly increased concentration of the stimulatory cytokine CINC-1 was observed 14 days after end of exposure but not at 1 day postexposure. For tumour necrosis factor-a and interleukin-6 no significant changes in treatment groups compared to controls were detected.
• In the NM-200 high dose group SiO2 particles (confirmation by EDX) were found within the cytoplasm of intraalveolar macrophages. The agglomerates of particles seem to be more densely packed 90 days post-exposure than 29 days post-exposure.

But all these reported effects were assessed as non-relevant by the authors, who concluded: "Haematology, clinical chemistry and urine analysis data did not show any relevant changes as compared to concurrent controls."

Urinalysis findings:

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

The absolute organ wet weights showed statistically significant changes as compared to controls for the absolute lung weights (high dose group only) 1 day after end of exposure and after 3 months of recovery (mid and high dose group). The relative lung weights were statistically significantly increased 1 day after exposure end (mid and high dose group).

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Histopathological examination 1 day after end of exposure revealed
- (multi)focal mucous (goblet) cell hyperplasia and epithelial hyaline droplets, accompanied with multifocal epithelial (mixed) inflammatory cell infiltration, were diagnosed dose-dependently in 10 out of 10 rats in the nasal cavity (statistically significantly increased) in all NM-200 treated groups.
- a significant increase of alveolar infiltration of granulocytes in lungs was detected in the mid and high dose groups /interstitial macrophage infiltration and of (multi)focal very slight alveolar granulocyte infiltration in the lungs of the high dose group.
Histopathological examination 3 months after end of exposure revealed a full recovery of all treatment-related effects in lungs. In nasal cavities the effects persisted.

Datailed results on nasal cavity
1. Animals sacrificed after 90 days of treatment
(Multi)focal mucous (goblet) cell hyperplasia was diagnosed in a single male (slight) of the control group and dose-dependently in 10/10 males each of the NM-200 low- (all slight) and mid-dose groups (8/10 slight, 2/10 moderate) as well as in 9/9 males of ther high-dose group (2/9 slight, 7/9 moderate). Mucous (goblet) cell hyperplasia was predominantly observed in the respiratory epithelial lining of the nasal septum and the ventral nasal meatus in levels I to III of the 4 nasal cavity sections. Two of 10, 5/10 and 9/9 males of the NM-200 low, mid- and high-dose group, respectively, showed (multi)focal slight hyperplasia of the respiratory epithelium in addition to mucous cell hyperplasia.
Another exposure-related effect was the dose-dependent occurrence of very slight (minimal) to moderate epithelial hyaline droplets (eosinophilic inclusions), mainly within the respiratory epithelium of the nasal septum und ventral nasal meatus at levels II - III of the nasal cavity sections. While in the clean air control group, 4/10 (3/10 very slight, 1/10 slight) males revealed very slight focal hyaline droplets, this change showed a multifocal distribution in the particle exposure groups at incidences of 10/10 each in the NM-200 low- (all slight) and mid-dose groups (9/10 slight, 1/10 moderate) as well as in 9/9 males of the high-dose group (all moderate). In a single male of the control group (very slight) and in 10/10 (all very slight), 10/10 (6/10 very slight, 4/10 slight) and 9/9 (2/9 very slight, 7/9 slight) males of the NM-200 low-, mid- and high-dose group, respectively, the epithelial hyaline droplets were associated with multifocal epithelial (mixed) inflammatory cell infiltration.
Incidental findings such as very slight to slight focal subepithelial corpora amylacea and very slight focal subepithelial mononuclear cell infiltration were unrelated to exposure and observed in single rats of different groups.

2. Animals sacrificed at the end of the recovery period (day 182)
Focal mucous (goblet) cell hyperplasia was observed at a slight degree in 3/10 males of the control group. In the particle-exposure groups, multifocal mucous cell hyperplasia occurred dose-dependently at incidences of 8/10 (3/10 very slight, 3/10 slight, 2/10 moderate), 10/10 (all slight) and 10/10 (6/10 slight, 3/10 moderate, 1/10 severe) in rats of the NM-200 low-, mid- and high-dose group, respectively. In comparison to the main subset, the overall degree of severity of this change had slightly decreased. This was not the case for (multi)focal respiratory epithelial hyperplasia, which was diagnosed in 5/10 (1/10 very slight, 4/10 slight), 7/10 (2/10 very slight, 5/10 slight) and 10/10 (4/10 very slight, 6/10 slight) rats of the NM-200 low-, mid- and high-dose group, respectively. The incidences were slightly higher than in the main subset, however, not the effect as such.
(Multi)focal epithelial hyaline droplets (eosinophilic inclusions) were observed in 7/10 control animals (all very slight) and in 10/10 males each of the NM-200 low- (9/10 slight, 1/10 moderate), mid- (all slight) and high-dose group (2/10 slight, 7/10 moderate, 1/10 severe), respectively. Incidence, degree of severity and distribution of this change were comparable to the ones seen in the main subset. (Multi)focal epithelial (mixed) inflammatory cell infiltration associated with the epithelial hyaline droplets was diagnosed in 2/10 (all very slight), 8/10 (6/10 very slight, 2/10 slight), 9/10 (8/10 very slight, 1/10 slight) and 10/10 (8/10 very slight, 2/10 slight) rats of the control-, NM-200 low-, mid- and high-dose group, respectively. Although the incidences were about the same as in the main subset, there was a remarkable decrease in the degree of severity of the inflammatory infiltrates in all particle exposure groups towards the end of the 90-day recovery period. As a further exposure-related finding, a single male of the NM-200 high-dose group revealed a slight multifocal (chronic) inflammation of the nasal submucosal glands.
Incidental findings included very slight to slight focal subepithelial mononuclear cell infiltration in 2/10 control rats and slight focal subepithelial corpora amylacea in a single rat of the NM-200 mid-dose group.

Histopathological findings: neoplastic:

no effects observed

Description (incidence and severity):

In all treatment groups, on day 8 and 91 postexposure no statistically significant increases in the proliferation index were detected

Details on results:

Lung burdens of 91, 172 and 307 µg were analysed on day 1 after end of exposure in the low, mid and high dose groups, respectively. Data at 3 months postexposure revealed that, in addition to the approx. 70 days of physiological clearance, a dissolution effect is responsible for the low values of 12, 21 and 34 µg/lung, respectively.
All test animals but two survived treatment and were euthanized at scheduled dates. Effects indicating systemic toxicity were not observed.

Key result

Dose descriptor:

NOAEL

Remarks:

local effects

Effect level:

< 1 other: mg/m³ effective concentration

Based on:

test mat.

Sex:

male

Basis for effect level:

histopathology: non-neoplastic

Remarks on result:

not determinable

Remarks:

due to adverse effects at lowest tested concentration level

Key result

Dose descriptor:

NOAEL

Remarks:

local effects

Effect level:

< 3.23 mg/m³ air (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

histopathology: non-neoplastic

Remarks on result:

other: nominal concentration: calculated by dividing the stated concentration with the aerosol generation efficiency (effective vs. nominal concentration) of 31% (3.23 mg/m³ air / 0.31 = 1 mg/m³ air

Key result

Dose descriptor:

LOAEL

Remarks:

local effects

Effect level:

1 other: mg/m³ effective concentration

Based on:

test mat.

Sex:

male

Basis for effect level:

histopathology: non-neoplastic

Key result

Dose descriptor:

LOAEL

Remarks:

local effects

Effect level:

3.23 mg/m³ air (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

histopathology: non-neoplastic

Remarks on result:

other: nominal concentration: calculated by dividing the stated concentration with the aerosol generation efficiency (effective vs. nominal concentration) of 31% (3.23 mg/m³ air / 0.31 = 1 mg/m³ air

Key result

Dose descriptor:

NOAEL

Remarks:

systemic effects

Effect level:

>= 5 other: mg/m³ effective concentration

Based on:

test mat.

Sex:

male

Remarks on result:

other: value mentioned in study report

Key result

Dose descriptor:

NOAEL

Remarks:

systemic effects

Effect level:

>= 16.13 mg/m³ air (nominal)

Based on:

test mat.

Sex:

male

Remarks on result:

other: nominal concentration: calculated by dividing the stated concentration with the aerosol generation efficiency (effective vs. nominal concentration) of 31% (16.13 mg/m³ air / 0.31 = 5 mg/m³ air

Key result

Dose descriptor:

NOAEL

Remarks:

local effects, but only taking into account effects supporting classification according to GHS (2019) 3.9.2.7.3 and 3.9.2.8

Effect level:

>= 16.13 mg/m³ air (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

histopathology: non-neoplastic

organ weights and organ / body weight ratios

Remarks on result:

other: nominal concentration: calculated by dividing the stated concentration with the aerosol generation efficiency (effective vs. nominal concentration) of 31% (16.13 mg/m³ air / 0.31 = 5 mg/m³ air

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

1 other: mg/m³ air effective concentration

System:

respiratory system: upper respiratory tract

Organ:

nasal cavity

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

yes

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

3.23 mg/m³ air (nominal)

System:

respiratory system: upper respiratory tract

Organ:

nasal cavity

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

yes

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

2.5 other: mg/m³ air effective concentration

System:

respiratory system: lower respiratory tract

Organ:

lungs

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

yes

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

8.06 mg/m³ air (nominal)

System:

respiratory system: lower respiratory tract

Organ:

lungs

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

yes

The actual (=effective) systemic concentration applied was calculated by dividing the stated nominal concentration of 5 mg/m³ air with the aerosol generation efficiency (effective vs. nominal concentration) of 31%:

16.13 mg/m³ air / 0.31 = 5 mg/m³ air

 

The local effects seen (mucous cell hyperplasia in nasal cavities) were adaptive responses not considered toxicologically relevant. According to GHS (2019) 3.9.2.8 d this effect does not support classification.

Also the increased lung weights in the medium and high dose groups do not support classification according to GHS (2019) 3.9.2.8 c, because no evidence of organ dysfunction was reported.
Therefore, for the assessment of classification, no sufficient critical effects were observed up to the highest dose employed, hence no hazard was identified and a NOAEL of equal or greater than 16.13 mg/m³ is applicable for local effects when assessing classification. 

Conclusions:

Conclusion on NOAEL: Under the conditions of this test an LOAEL = 1 mg/m³ was derived (decisive endpoint: histopathology: mucous cell hyperplasia in nasal cavities). An experimental local NOAEL could not be derived (i.e. NOAEL < 1 mg/m³). But all effects showed either full reversible or showed a tendency to full reversibility.
The systemic NOAEL was 5 mg/m³ effectively. At this dose level no systemic effects were observed. The nominal systemic concentration applied higher, and can be calculated by dividing the stated nominal concentration of 16.13 mg/m³ air with the aerosol generation efficiency (effective vs. nominal concentration) of 31%: 16.13 mg/m³ air / 0.31 = 5 mg/m³ air.

The observed local effects (mucous cell hyperplasia in nasal cavities) and the increased lung weights do not support classification according to GHS (2019) 3.9.2.8. Therefore, for the assessment of classification, no sufficient critical effects were observed up to the highest dose employed, hence no hazard was identified and a NOAEL of >= 16.13 mg/m³ is also applicable for local effects when assessing classification. 

Executive summary:

A 3-month inhalation study with a structurally related SAS (NM-200) followed by a 90-day recovery period was performed in male rats. Calculation of the aerosol generation efficiency (effective vs. nominal concentration) resulted in 31%. Therefore, two effect levels are stated each, the effective concentration stated in the study report and the nominal concentration applied to achieve the target concentration (= effective concentration). A further, third, NOAEL is provided for the assessment of classification, because no sufficient critical effects were observed supporting it according to GHS (2019) criteria.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Study duration:

subchronic

Experimental exposure time per week (hours/week):

30

Species:

rat

Quality of whole database:

reliability 1

Repeated dose toxicity: inhalation - local effects

Link to relevant study records

Reference

Reference 1

Endpoint:

short-term repeated dose toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

experimental phase (acute & sub-acute study): Jun. 5, 2018 - Apr. 17, 2019

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

test procedure in accordance with national standard methods with acceptable restrictions

Remarks:

only 5 day-test instead of a 28- or 90 day one (but cf. Landsiedel 2014)

Justification for type of information:

Cf. the endpoint: Literature, Landsiedel, 2014, Statement on 5-day studies. It claims that a 5-day study design can be as expressive as a 90-day study for testing nanoparticles.

Reason / purpose for cross-reference:

other: justification information

Reason / purpose for cross-reference:

reference to same study

Qualifier:

no guideline followed

Principles of method if other than guideline:

subacute inhalation: 4-hour exposure daily for 5 days, 3 dose levels, 13-week observation period

GLP compliance:

yes

Limit test:

no

Specific details on test material used for the study:

Test item identification: Sodasil P95
Description: Amorphous silicate of sodium and aluminum
Formula/Chemical group: Nanosilicates
Chemical Abstract No.: 1344-00-9
EINECS No.: 215-684-8
Aspect: White powder
Storage condition: Room temperature

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

Age (at treatment start): 10 weeks old
Body-weight range(at treatment start): 206.9 – 277.8 g (females) and 314.7 – 490.9 g (males)
Temperature: 20.0ºC – 24.0ºC (Mean 22.0ºC)
Humidity: 40.0% – 80.0% (Mean 60.0%)
Room air renovation cycles: 30
Photoperiod: 12 hours of light: 12 hours of darkness
Feeding: Irradiated certified laboratory dry diet RM1 (P) QC Reference 831193 (batch number 3077, Dietex SDS) was offered with unlimited supply.
Drinking: Autoclaved mineral water was offered ad libitum.

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

whole body

Vehicle:

other: 0.05% BSA (CAS No. 9048-46-8), 30 μL of absolute alcohol and sterile water

Mass median aerodynamic diameter (MMAD):

392.8 nm

Geometric standard deviation (GSD):

14.3

Remarks on MMAD:

Mass dispersion and size, as well as concentration in the air were assessed in the extraction tube from the inhalation chamber by using an optical particle sizer (ITENE). Sodasil P95 concentration measured allowed the observation of different dispersion grades of nanoparticles below 1 and 6 μm, being the resulting mean + standard deviation diameter value of 392.8 + 14.3 nm. According to these results there is a relative relevant difference with regard to the theoretical dose estimated. In contrast measurements made by using two air samples from the inhalation chamber, once upon achieved the steady-state demonstrated that 94–96% of nanoparticles are also ranged at mean values of 252 to 388 nm. Accordingly, actual doses applied to the animals were slightly lower than theoretical ones (i.e., 0.88, 4.39, 21.97 mg/m³ for the subchronic study). Hence, nanoparticles content in the air allowed to confirm the appropriate dose regime in order to assess the impact of subcacute treatments in the in vivo studies.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

s. above (Remarks on MMAD)

Duration of treatment / exposure:

4 hours per day for 5 days

Frequency of treatment:

daily

Dose / conc.:

21.97 mg/m³ air (analytical)

Remarks:

theoretical (cut-off dose): 28.5 mg/m³

Dose / conc.:

4.39 mg/m³ air (analytical)

Remarks:

theoretical (cut-off dose): 5.7 mg/m³

Dose / conc.:

0.88 mg/m³ air (analytical)

Remarks:

theoretical (cut-off dose): 1.1 mg/m³

No. of animals per sex per dose:

5

Control animals:

yes

Details on study design:

Subchronic systemic toxicity of Sodasil P95 was tested in male and female Wistar rats (n=5/sex/dose). Animals were subject to a daily 4-hour inhalation exposure for 5 days at theoretical dose levels of 1.1, 5.7 and 28.5 mg/m³. Actual doses applied to the animals were slightly lower than theoretical ones, i.e., 0.88, 4.39, 21.97 mg/m³.
The administration methods used were based on the procedures described in the OECD TG 436
Acute Inhalation Toxicity – Acute Toxic Class Method and the study design published by Arts et al. (2007) in Food Chem. Toxicol., 45: 1856-1867 to assess subchronic systemic toxicity following subacute exposure of the test item by using an inhalation Scireq chamber.
The observation period was 13 weeks.

Observations and examinations performed and frequency:

The animals were weighed and observed daily for the assessment of toxic effects for 90-94 days (subchronic systemic toxicity study).
Throughout the study periods, food and water were also weighed regularly to monitor changes in food and water intake.
Clinical findings were checked daily by observing any toxic effects.

Sacrifice and pathology:

Blood samples were drawn from the cava vein at the end of the experiment (Day 95) of all animals under light isoflurane anesthesia. The animals were not fasted before blood sampling but allowed access to water ad libitum. The samples were collected in the working day to reduce biological variation caused by circadian rhythms.
In addition, a bronchoalveolar lavage was performed by intubation of the trachea. A total 6 mL of PBS were perfused slowly for 1 minute and the solution was kept inside for 5 minutes before collection. The collected lavage was centrifuged at 1500 g for 10 minutes and then the pellet was resuspended in 0.5 mL of PBS to allow haematological and biomarker analyses.
Bioavailability were assessed in five tissues (blood, lung, liver, kidney and spleen).

At the end of the observational period: 13 weeks (subchronic) after treatment
initiation, all animals were necropsied and a macroscopic examination was performed. A full necropsy was performed on all main study and recovery animals. The necropsy included the examination of the external surface of the body, all orifices, cranial, thoracic and abdominal cavities and the observation of the organs both in situ and after evisceration.
At necropsy, several organs were subjected to microscopic examination:
Lungs, Spleen, Heart, Liver, Kidneys and Thoracic lymph nodes

Statistics:

For continuous data, a parametric analysis was performed to assess if variables follow a normal distribution and variances are homogeneous. If so, a one-way analysis of variance (ANOVA) followed by Dunnett’s test was performed. On the contrary, the Kruskal-Wallis ANOVA followed by the Mann-Whitney U-test was applied if Kolmogorov-Smirnov normality test was significant.
For organ weight data, parametric statistical comparison was done with absolute organ weights and those rated by the terminal body weight of each animal, unless non-parametric methods are applied when normality and variances homogeneity is not reached.

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Repeated administration for 5 days did not induce any change in bodyweight or bodyweight gain in females. The pattern was comparable in males, although those treated at the intermediate and high dose levels showed higher body weight gain as compared to controls. This effect was considered null of relevance from a toxicological point of view.

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:

effects observed, treatment-related

Description (incidence and severity):

Hematological analysis at the end of the observational period, where just occasional statistical differences (p<0.05, one-way ANOVA followed by Dunnett’s test or Kruskal-Wallis followed by Mann-Whitney U-test) could be seen: MCHC and monocytes count in males and, MCHC and MCH in females, both compared to control animals. However, these changes had no relevance since the final values were within the normal range of values for this animal species. Overall, the hematological profile in bronchoalveolar lavage (BAL) was not significant, although monocytes count was significantly lower (p<0.05, one-way ANOVA followed by Dunnett’s test) at the highest dose levels as compared to the control group.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Blood chemistry analysis in serum and in BAL were also performed. Overall there were no significant effects on any parameter: creatinine, urea, total bilirubin, GPT/ALT, GOT/AST, alkaline phosphatase (serum) and lactic dehydrogenase, albumin and N-acetyl-glucosamine (NAG) (BAL), but total proteins (p<0.05) and alkaline phosphatase (p=0.001, one-way ANOVA followed by Dunnett’s test) were significantly increased in male animals at the low and intermediate dose levels and at the highest level of Sodasil P95, as compared to control animals. These changes were considered effects not biologically relevant from a toxicological point of view.

Collagen concentration was estimated in dry lungs by means of quantification of the hydroxyproline levels. Mean tissue levels of collagen tended to increase with the dose increase in males (4, 18 and 35-fold) and females (34, 171 and 183-fold) at the three dose levels compared to control animals (mean of 2.1 µg/g tissue and

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

Macroscopic examination and organ weights did not allow the observation of any event or trend in male and female animals related to dose administration.

Gross pathological findings:

no effects observed

Description (incidence and severity):

Macroscopic examination and organ weights did not allow the observation of any event or trend in male and female animals related to dose administration.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

Histopathological examination of six relevant tissues (heart, lungs, liver, spleen, kidneys and thoracic lymph nodes) did not allow the registration of any relevant event. All findings recorded were considered to be within the range of normal background lesions that may be seen in rats of this strain and age and under the experimental conditions used in this study.

Histopathological findings: neoplastic:

no effects observed

Other effects:

effects observed, treatment-related

Description (incidence and severity):

Bioavailability was assessed in five tissues (blood, lung, liver, kidney and spleen) by TEM.
Small nanosilicates (< 160 nm) were seen in all tissues with the exception of whole blood,
where no conclusive observations could be made. Nanoparticles were confined, as expected,
inside the reticuloendothelial system in lysosomes into macrophages in lungs, Küpffer cells in
liver, proximal tubular cells (close to the glomerulus) in kidneys and in spleen. No nanosilicates
were seen in the nucleus nor in mitochondria. The lysosomal location is commonly known for
all nanoparticles, most of them devoid of any toxicological relevance, and there is no reason
to expect adverse effects here.

Key result

Dose descriptor:

NOAEL

Effect level:

>= 21.97 mg/m³ air

Sex:

male/female

Basis for effect level:

behaviour (functional findings)

body weight and weight gain

clinical biochemistry

clinical signs

food consumption and compound intake

gross pathology

haematology

histopathology: neoplastic

histopathology: non-neoplastic

Remarks on result:

other: The theoretical cut-off dose was 28.5 mg/m³. The actual dose applied to the animals was slightly lower: 21.97 mg/m³.

Key result

Critical effects observed:

no

There were no clinical signs/behavioral changes registered throughout the study and body weight gain and food and water intake of male and female animals treated with Sodasil P95, at the different dose levels, were similar to that registered in control animals.

Overall, no effects could be observed on hematological, serum biochemistry nor biomarkers measured in the bronchoalveolar lavage (BAL) and lung tissue at the end of the experimental period, although monocytes in whole blood, total proteins and alkaline phosphatase in BAL and content of collagen in lungs tended to increase with dose in a significant manner. All these changes were considered as effects (not adverse events) without biological relevance from a safety point of view, they might have been residual repair mechanisms in front of intensive inhalation. Hence, no adverse events could be established based on the hematological, clinical biochemistry in serum and in BAL at any dose level assayed

As expected, the bioavailability measured in five tissues (blood, lung, liver, kidney and spleen) allowed the observation of remnant nanosilicates inside lysosomes of the reticuloendothelial system from the different tissues (lungs, liver, kidneys and spleen), with the exception of whole blood, following the usual pattern seen for other nanoparticles.

Neither macroscopic changes nor clear significant trends in organ weights from animals treated at the different dose levels with Sodasil P95 could be recorded.

Finally, the histopathology examination of six relevant tissues (heart, lungs, liver, spleen, kidneys, and thoracic lymph nodes) did not allow the registration of any relevant effect.

 

Conclusions:

Sodasil P95 administered by 5-day repeated inhalation (and an observation period of 13 weeks) to the Wistar rat showed a No-Observed-Adverse-Event-Level (NOAEL) of 21.97 mg/m³, expressed as actual dose, according to the subchronical assessment.

Executive summary:

Subchronic systemic toxicity of Sodasil P95 was tested in male and female Wistar rats (n=5/sex/dose). Animals were subject to a daily 4-hour inhalation exposure for 5 days at theoretical dose levels of 1.1, 5.7 and 28.5 mg/m³. Actual doses applied to the animals were slightly lower than theoretical ones, i.e. 0.88, 4.39, 21.97 mg/m³. The observation period lasted 13 weeks. Although some effects were observed, none of these were assessed as adverse.

Reference 2

Endpoint:

sub-chronic toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Dec. 11, 2011 - May 18, 2014

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)

Version / remarks:

2009-09-07

Deviations:

yes

Remarks:

additional endpoints; males only

Principles of method if other than guideline:

OECD guideline 413 with additional endpoints (bronchoalveolar lavage, cell proliferation, immunological parameters, oxidative stress analysis, electron microscope analysis, toxicokinetics) to address nanoparticle-specific aspects of toxicity

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

UPAC Name: Silicon Dioxide
Common Name, Synonyma: Synthetic Amorphous Silica, NM-200
CAS #: 12926-00-8 (CAS # for precipitated synthetic amorphous silica)
Information note: CAS # 7631-86-9 (general CAS # for silica including synthetic amorphous silica)
Batch: NM-200 (Master-Batch: JRC, Ispra)
Expiry Date: 12/2012 (Stable particle sample)
Purity: 96.5% (value given by JRC, Ispra)
Molecular weight (g/mol): 60.74

Species:

rat

Strain:

Wistar

Remarks:

Wistar WU

Details on species / strain selection:

purchased from Charles River Deutschland (Sulzfeld, Germany)

Sex:

male

Details on test animals or test system and environmental conditions:

The age of the animals at the start of exposure was approx. 9-10 weeks and the weight approx. 280 gram, two rats housed per cage, tap water from the Hannover city water supplier was offered fresh weekly or more often, if necessary. As diet a commercial chow in pellet form was used, identified as Ssniff "V1534". 22 +/- 2 °C, 40 - 70% humidity, 12-hour light/dark.

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

nose only

Vehicle:

clean air

Mass median aerodynamic diameter (MMAD):

ca. 2.16 - ca. 3.12 µm

Remarks on MMAD:

The MMADs were determined by two methods: impactor and SEM analysis.
low dose: 2.16 µm, GSD: 0.09
mid dose: 2.94 µm, GSD: 0.2
high dose: 3.12 µm, GSD: 0.06

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The target aerosol concentrations of 1, 2.5 and 5 mg NM-200/m³ were achieved to 104%, 100% and 101%, respectively. Calculation of the aerosol generation efficiency (actual vs. nominal concentration) resulted in 31%; this low value demonstrates that experimental conditions are different to any handling and use conditions of NM-200.

- Due to the particle size distribution in the test medium the nominal concentration was
3.23 mg/m³, to achieve 1 mg/m³ aerosol test concentration.

- Due to the particle size distribution in the test medium the nominal concentration was
8.06 mg/m³, to achieve 2.5 mg/m³ aerosol test concentration.

- Due to the particle size distribution in the test medium the nominal concentration was
16.13 mg/m³, to achieve 5 mg/m³ aerosol test concentration.

Duration of treatment / exposure:

6 h / d

Frequency of treatment:

5 d / w for 90 days (= 65 exposure days)

Dose / conc.:

1 mg/m³ air

Remarks:

effective concentration (corresponds to 3.23 mg/m³ nominal concentration)

Dose / conc.:

2.5 mg/m³ air

Remarks:

effective concentration (corresponds to 8.06 mg/m³ nominal concentration)

Dose / conc.:

5 mg/m³ air

Remarks:

effective concentration (corresponds to 16.13 mg/m³ nominal concentration)

No. of animals per sex per dose:

55 (males only)

Control animals:

yes, concurrent vehicle

Details on study design:

90-day post-inhalation recovery period

Observations and examinations performed and frequency:

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 included 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.
Individual body weight was recorded to the nearest 0.1 g twice a week for the first month and once a week throughout the remainder of the study (including post-exposure observation period) for all animals.

Sacrifice and pathology:

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 lung and the lower half of the trachea were weighed, and used for BAL or histopathology. The following organs were trimmed and wet weights were recorded: liver, kidneys, adrenals, testes, epididymides, thymus, spleen, brain, 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 given in brackets were prepared for histopathology.

Other examinations:

Cytological parameters
• total cell count (recruitment of lung leukocytes)
• viability test (giving percentage of alive leukocytes among the total number of cells)
• differential cell count (inflammatory (PMNs) or immunological (lymphocytes) reactions)
Biochemical parameters
• lactic dehydrogenase (LDH = cytosolic marker enzyme; increased permeability of membranes, cell damage and lysis)
• β-glucuronidase (measure of phagocytic activity of macrophages; lysis of macrophages)
• total protein (marker of transsudation; damage of epithelial cells).
Oxidative Stress and Immunotoxicological Parameters:
TNF-a, IL-8 (CINC-1), IL-6, TGF-b

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.

Clinical signs:

no effects observed

Mortality:

mortality observed, non-treatment-related

Description (incidence):

only 2 / 220
Effects indicating systemic toxicity were not observed.

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

Statistically significant increase of food comsumption as compared to controls was observed on several dates in all NM-200 dose groups. Due to inconsistency in dose-dependency and only small absolute differences observed these findings are considered as incidental.

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Statistically significant changes were observed for hemoglobin and for segmented neutrophils (calculated) in mid dose group. This findings are considered as incidental, not-treatment related findings.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

• Statistically significant increases of polymorphonuclear neutrophils, lymphocytes and lactic dehydrogenase, ß-glucuronidase and total protein levels/activity and decrease of macrophages were detected in the mid and high dose groups 1 day after end of exposure. In the low dose group a statistically significant increase of polymorphonuclear neutrophils was detected. However, all these effects were reversible and had returned to control levels at the 3-month post-exposure sacrifice date. Thus, NM-200 dust showed a strong acute response, however, rapid recovery upon cessation of exposure.
• The analysis of the oxidative stress related secretion of reactive oxygen intermediates (ROI) showed a decrease with and without zymosan stimulation in the high dose group 14 days after end of exposure as compared to clean air controls but not at 1 day postexposure. A significantly increased concentration of the stimulatory cytokine CINC-1 was observed 14 days after end of exposure but not at 1 day postexposure. For tumour necrosis factor-a and interleukin-6 no significant changes in treatment groups compared to controls were detected.
• In the NM-200 high dose group SiO2 particles (confirmation by EDX) were found within the cytoplasm of intraalveolar macrophages. The agglomerates of particles seem to be more densely packed 90 days post-exposure than 29 days post-exposure.

But all these reported effects were assessed as non-relevant by the authors, who concluded: "Haematology, clinical chemistry and urine analysis data did not show any relevant changes as compared to concurrent controls."

Urinalysis findings:

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

The absolute organ wet weights showed statistically significant changes as compared to controls for the absolute lung weights (high dose group only) 1 day after end of exposure and after 3 months of recovery (mid and high dose group). The relative lung weights were statistically significantly increased 1 day after exposure end (mid and high dose group).

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Histopathological examination 1 day after end of exposure revealed
- (multi)focal mucous (goblet) cell hyperplasia and epithelial hyaline droplets, accompanied with multifocal epithelial (mixed) inflammatory cell infiltration, were diagnosed dose-dependently in 10 out of 10 rats in the nasal cavity (statistically significantly increased) in all NM-200 treated groups.
- a significant increase of alveolar infiltration of granulocytes in lungs was detected in the mid and high dose groups /interstitial macrophage infiltration and of (multi)focal very slight alveolar granulocyte infiltration in the lungs of the high dose group.
Histopathological examination 3 months after end of exposure revealed a full recovery of all treatment-related effects in lungs. In nasal cavities the effects persisted.

Datailed results on nasal cavity
1. Animals sacrificed after 90 days of treatment
(Multi)focal mucous (goblet) cell hyperplasia was diagnosed in a single male (slight) of the control group and dose-dependently in 10/10 males each of the NM-200 low- (all slight) and mid-dose groups (8/10 slight, 2/10 moderate) as well as in 9/9 males of ther high-dose group (2/9 slight, 7/9 moderate). Mucous (goblet) cell hyperplasia was predominantly observed in the respiratory epithelial lining of the nasal septum and the ventral nasal meatus in levels I to III of the 4 nasal cavity sections. Two of 10, 5/10 and 9/9 males of the NM-200 low, mid- and high-dose group, respectively, showed (multi)focal slight hyperplasia of the respiratory epithelium in addition to mucous cell hyperplasia.
Another exposure-related effect was the dose-dependent occurrence of very slight (minimal) to moderate epithelial hyaline droplets (eosinophilic inclusions), mainly within the respiratory epithelium of the nasal septum und ventral nasal meatus at levels II - III of the nasal cavity sections. While in the clean air control group, 4/10 (3/10 very slight, 1/10 slight) males revealed very slight focal hyaline droplets, this change showed a multifocal distribution in the particle exposure groups at incidences of 10/10 each in the NM-200 low- (all slight) and mid-dose groups (9/10 slight, 1/10 moderate) as well as in 9/9 males of the high-dose group (all moderate). In a single male of the control group (very slight) and in 10/10 (all very slight), 10/10 (6/10 very slight, 4/10 slight) and 9/9 (2/9 very slight, 7/9 slight) males of the NM-200 low-, mid- and high-dose group, respectively, the epithelial hyaline droplets were associated with multifocal epithelial (mixed) inflammatory cell infiltration.
Incidental findings such as very slight to slight focal subepithelial corpora amylacea and very slight focal subepithelial mononuclear cell infiltration were unrelated to exposure and observed in single rats of different groups.

2. Animals sacrificed at the end of the recovery period (day 182)
Focal mucous (goblet) cell hyperplasia was observed at a slight degree in 3/10 males of the control group. In the particle-exposure groups, multifocal mucous cell hyperplasia occurred dose-dependently at incidences of 8/10 (3/10 very slight, 3/10 slight, 2/10 moderate), 10/10 (all slight) and 10/10 (6/10 slight, 3/10 moderate, 1/10 severe) in rats of the NM-200 low-, mid- and high-dose group, respectively. In comparison to the main subset, the overall degree of severity of this change had slightly decreased. This was not the case for (multi)focal respiratory epithelial hyperplasia, which was diagnosed in 5/10 (1/10 very slight, 4/10 slight), 7/10 (2/10 very slight, 5/10 slight) and 10/10 (4/10 very slight, 6/10 slight) rats of the NM-200 low-, mid- and high-dose group, respectively. The incidences were slightly higher than in the main subset, however, not the effect as such.
(Multi)focal epithelial hyaline droplets (eosinophilic inclusions) were observed in 7/10 control animals (all very slight) and in 10/10 males each of the NM-200 low- (9/10 slight, 1/10 moderate), mid- (all slight) and high-dose group (2/10 slight, 7/10 moderate, 1/10 severe), respectively. Incidence, degree of severity and distribution of this change were comparable to the ones seen in the main subset. (Multi)focal epithelial (mixed) inflammatory cell infiltration associated with the epithelial hyaline droplets was diagnosed in 2/10 (all very slight), 8/10 (6/10 very slight, 2/10 slight), 9/10 (8/10 very slight, 1/10 slight) and 10/10 (8/10 very slight, 2/10 slight) rats of the control-, NM-200 low-, mid- and high-dose group, respectively. Although the incidences were about the same as in the main subset, there was a remarkable decrease in the degree of severity of the inflammatory infiltrates in all particle exposure groups towards the end of the 90-day recovery period. As a further exposure-related finding, a single male of the NM-200 high-dose group revealed a slight multifocal (chronic) inflammation of the nasal submucosal glands.
Incidental findings included very slight to slight focal subepithelial mononuclear cell infiltration in 2/10 control rats and slight focal subepithelial corpora amylacea in a single rat of the NM-200 mid-dose group.

Histopathological findings: neoplastic:

no effects observed

Description (incidence and severity):

In all treatment groups, on day 8 and 91 postexposure no statistically significant increases in the proliferation index were detected

Details on results:

Lung burdens of 91, 172 and 307 µg were analysed on day 1 after end of exposure in the low, mid and high dose groups, respectively. Data at 3 months postexposure revealed that, in addition to the approx. 70 days of physiological clearance, a dissolution effect is responsible for the low values of 12, 21 and 34 µg/lung, respectively.
All test animals but two survived treatment and were euthanized at scheduled dates. Effects indicating systemic toxicity were not observed.

Key result

Dose descriptor:

NOAEL

Remarks:

local effects

Effect level:

< 1 other: mg/m³ effective concentration

Based on:

test mat.

Sex:

male

Basis for effect level:

histopathology: non-neoplastic

Remarks on result:

not determinable

Remarks:

due to adverse effects at lowest tested concentration level

Key result

Dose descriptor:

NOAEL

Remarks:

local effects

Effect level:

< 3.23 mg/m³ air (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

histopathology: non-neoplastic

Remarks on result:

other: nominal concentration: calculated by dividing the stated concentration with the aerosol generation efficiency (effective vs. nominal concentration) of 31% (3.23 mg/m³ air / 0.31 = 1 mg/m³ air

Key result

Dose descriptor:

LOAEL

Remarks:

local effects

Effect level:

1 other: mg/m³ effective concentration

Based on:

test mat.

Sex:

male

Basis for effect level:

histopathology: non-neoplastic

Key result

Dose descriptor:

LOAEL

Remarks:

local effects

Effect level:

3.23 mg/m³ air (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

histopathology: non-neoplastic

Remarks on result:

other: nominal concentration: calculated by dividing the stated concentration with the aerosol generation efficiency (effective vs. nominal concentration) of 31% (3.23 mg/m³ air / 0.31 = 1 mg/m³ air

Key result

Dose descriptor:

NOAEL

Remarks:

systemic effects

Effect level:

>= 5 other: mg/m³ effective concentration

Based on:

test mat.

Sex:

male

Remarks on result:

other: value mentioned in study report

Key result

Dose descriptor:

NOAEL

Remarks:

systemic effects

Effect level:

>= 16.13 mg/m³ air (nominal)

Based on:

test mat.

Sex:

male

Remarks on result:

other: nominal concentration: calculated by dividing the stated concentration with the aerosol generation efficiency (effective vs. nominal concentration) of 31% (16.13 mg/m³ air / 0.31 = 5 mg/m³ air

Key result

Dose descriptor:

NOAEL

Remarks:

local effects, but only taking into account effects supporting classification according to GHS (2019) 3.9.2.7.3 and 3.9.2.8

Effect level:

>= 16.13 mg/m³ air (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

histopathology: non-neoplastic

organ weights and organ / body weight ratios

Remarks on result:

other: nominal concentration: calculated by dividing the stated concentration with the aerosol generation efficiency (effective vs. nominal concentration) of 31% (16.13 mg/m³ air / 0.31 = 5 mg/m³ air

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

1 other: mg/m³ air effective concentration

System:

respiratory system: upper respiratory tract

Organ:

nasal cavity

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

yes

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

3.23 mg/m³ air (nominal)

System:

respiratory system: upper respiratory tract

Organ:

nasal cavity

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

yes

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

2.5 other: mg/m³ air effective concentration

System:

respiratory system: lower respiratory tract

Organ:

lungs

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

yes

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

8.06 mg/m³ air (nominal)

System:

respiratory system: lower respiratory tract

Organ:

lungs

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

yes

The actual (=effective) systemic concentration applied was calculated by dividing the stated nominal concentration of 5 mg/m³ air with the aerosol generation efficiency (effective vs. nominal concentration) of 31%:

16.13 mg/m³ air / 0.31 = 5 mg/m³ air

 

The local effects seen (mucous cell hyperplasia in nasal cavities) were adaptive responses not considered toxicologically relevant. According to GHS (2019) 3.9.2.8 d this effect does not support classification.

Also the increased lung weights in the medium and high dose groups do not support classification according to GHS (2019) 3.9.2.8 c, because no evidence of organ dysfunction was reported.
Therefore, for the assessment of classification, no sufficient critical effects were observed up to the highest dose employed, hence no hazard was identified and a NOAEL of equal or greater than 16.13 mg/m³ is applicable for local effects when assessing classification. 

Conclusions:

Conclusion on NOAEL: Under the conditions of this test an LOAEL = 1 mg/m³ was derived (decisive endpoint: histopathology: mucous cell hyperplasia in nasal cavities). An experimental local NOAEL could not be derived (i.e. NOAEL < 1 mg/m³). But all effects showed either full reversible or showed a tendency to full reversibility.
The systemic NOAEL was 5 mg/m³ effectively. At this dose level no systemic effects were observed. The nominal systemic concentration applied higher, and can be calculated by dividing the stated nominal concentration of 16.13 mg/m³ air with the aerosol generation efficiency (effective vs. nominal concentration) of 31%: 16.13 mg/m³ air / 0.31 = 5 mg/m³ air.

The observed local effects (mucous cell hyperplasia in nasal cavities) and the increased lung weights do not support classification according to GHS (2019) 3.9.2.8. Therefore, for the assessment of classification, no sufficient critical effects were observed up to the highest dose employed, hence no hazard was identified and a NOAEL of >= 16.13 mg/m³ is also applicable for local effects when assessing classification. 

Executive summary:

A 3-month inhalation study with a structurally related SAS (NM-200) followed by a 90-day recovery period was performed in male rats. Calculation of the aerosol generation efficiency (effective vs. nominal concentration) resulted in 31%. Therefore, two effect levels are stated each, the effective concentration stated in the study report and the nominal concentration applied to achieve the target concentration (= effective concentration). A further, third, NOAEL is provided for the assessment of classification, because no sufficient critical effects were observed supporting it according to GHS (2019) criteria.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Study duration:

subchronic

Species:

rat

Quality of whole database:

reliability 1

Repeated dose toxicity: dermal - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: dermal

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

other:

Critical effects observed:

not specified

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: dermal

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

other:

Critical effects observed:

not specified

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

There is no need for classification:
- No oral adverse effects were seen with the test substance Silicic acid, aluminium sodium salt at doses of up to 100,000 ppm (10% in the diet) after 14 days of administration.
- By inhalation route no (local or systemic) adverse effects were observed with the test substance itself in a 5-day study. In an 90-day study with the read-across substance silicon dioxide (NM-200) no systemic effects were observed. The local effects seen were adaptive responses not considered toxicologically relevant. According to GHS (2019) 3.9.2.8 d this effect does not support classification.
Therefore, for the assessment of classification, no sufficient critical effects were observed up to the highest dose employed, hence no hazard was identified and a NOAEL of equal or greater than 16.13 mg/m³ is applicable for local effects when assessing classification. 
- Dermally no repeated dose study is available, but based on the high acute tolerance and the inert inorganic nature of silica and silicates, no hazard was identified.