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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Neither the studies in Silicic acid, aluminum sodium salt (CAS 1344-00-9, NAS) nor that in the read-across substance Silicic acid, aluminum magnesium sodium salt (CAS 12040-43-6, SMAS) showed genotoxicity nor cytotoxicity.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
test procedure in accordance with national standard methods with acceptable restrictions
Qualifier:
no guideline followed
Principles of method if other than guideline:
Cytogenetic study to determine chromosomal damage: This is accomplished
by observing cells in anaphase. As the chromatids separate and move along the spindle, aberrations may occur.
GLP compliance:
not specified
Type of assay:
in vitro mammalian chromosome aberration test
Specific details on test material used for the study:
Sodium aluminosilicate (Compound FDA 71-45, Synthetic Silica, Lot Number SR-1621, as supplied by the Food and Drug Administration), from the study report it cannot be ascertained wether a crystalline or amorphous form was used
Species / strain / cell type:
mammalian cell line, other: human embryonic lung cultures (WI-38)
Metabolic activation:
without
Test concentrations with justification for top dose:
1, 10, 100 µg/ml
Vehicle / solvent:
saline
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
triethylenemelamine
Key result
Species / strain:
mammalian cell line, other: human embryonic lung cultures (WI-38)
Metabolic activation:
without
Genotoxicity:
not determined
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
The negative controls, medium level and low level tested exhibited 2, 0, and 1 percent acentric fragments, respectively. The high level had one cell with an acentric fragment and one cell with a bridge. This was not considered significant. The positive controls contained four cells with pulverization together with other aberrations.
Conclusions:
The compound produced no significant aberration in the anaphase chromosomes of human tissue culture cells when tested at the dosage levels employed in this study.
Executive summary:

This study investigated into the potential of Sodium aluminosilicate to produce cytotoxicity in human embryonic lung cultures (WI-38). No significant changes to the control were observed.

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Comparable to guideline study, comprehensive testing programme (NTP/USA)
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
4 instead of 5 strains
Principles of method if other than guideline:
Test protocol according to Haworth S et al. (1983): Salmonella mutagenicity results for 250 chemicals. Environ. Mutagen., 5(Suppl. 1), 3 - 142
GLP compliance:
not specified
Type of assay:
bacterial reverse mutation assay
Species / strain / cell type:
other: Salmonella typhimurium TA 98, TA100, TA1535, and TA 1537 (see Table 204 p. 95)
Metabolic activation:
with and without
Metabolic activation system:
from Arochlor-1254 induced livers (male SD rat and male Syrian hamster)
Test concentrations with justification for top dose:
0, 100, 333, 1000, 3333, and 4000 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: distilled water
- Justification for choice of solvent/vehicle: used as dispersant rather than solvent (very slight water solubility)
Negative solvent / vehicle controls:
yes
Remarks:
+ distilled water
Positive controls:
yes
Positive control substance:
other: -S9: sodium azide (TA 1535, TA100); 9-aminoacridine (TA97, TA1537); 4-nitro-o-phenylenediamine (TA98) // +S9: 2-aminoanthracene (all strains)
Details on test system and experimental conditions:
METHOD OF APPLICATION: preincubation, 37 °C

DURATION
- Preincubation period: 20 min
- Exposure duration: 48 h
- Expression time (on agar): 48 h at 37 °C


NUMBER OF REPLICATIONS: 3


DETERMINATION OF CYTOTOXICITY
- Method: clearing in the density of the background lawn of TA 100)


Evaluation criteria:
positive: reproducible, dose-related increase over the solvent control
Species / strain:
other: Salmonella typhimurium TA 98, TA100, TA1535, and TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Remarks:
Precipitation at the two highest doses
Vehicle controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Conclusions:
Interpretation of results (migrated information):
negative
Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
cytotoxicity and genotoxicity in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
experimental phase: Jun. 16, 2016; Sep. 19, 2017
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Reason / purpose for cross-reference:
reference to same study
Reason / purpose for cross-reference:
reference to same study
Reason / purpose for cross-reference:
reference to same study
Reason / purpose for cross-reference:
reference to same study
Qualifier:
according to guideline
Guideline:
other: Cytotoxicity: DB-ALM Protocol No. 17: MTT Assay; Genotoxicity: ASTM-E2186
Deviations:
not specified
GLP compliance:
yes
Type of assay:
in vitro mammalian cell micronucleus test
Specific details on test material used for the study:
Sodasil P95
Species / strain / cell type:
mammalian cell line, other: Human hepatoma HepG2
Species / strain / cell type:
mammalian cell line, other: Human lung carcinoma A549
Species / strain / cell type:
mammalian cell line, other: Human colorectal carcinoma CaCo-2
Metabolic activation:
without
Test concentrations with justification for top dose:
2.6, 8.1, 25.6, 81.0 and 256 μg/mL
Vehicle / solvent:
The culture medium used for growth of the cells was based in Eagle's minimum essential medium (MEM) supplemented with 10% fetal bovine serum (FBS) inactivated, 2 mM L-glutamine, 1% nonessential amino acids (NEAA), 1% sodium pyruvate, 100 μg/mL penicillin/streptomycin. The cells were maintained into an incubator at 37ºC at 5% CO2 in humidified atmosphere.
Negative solvent / vehicle controls:
yes
Remarks:
The solvent for disperse the test items 0.05% BSA in water at 10% v/v in culture medium was used as negative control.
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
other:
Details on test system and experimental conditions:
Seed process
Cells from a growing culture were used. They were seeded at a density between 1-3 x 10^5 cells/mL in 96-well plates. Cells were kept in the incubator for 20 hours at 35-37ºC and 5% CO2 for proper adhesion, before being exposed to the test items.

Treatment
The test items were mixed with medium according to concentration just prior to apply. Then, the growth medium of cells in 96-well plate was removed and 100 μL of treatment was added. Each treatment was done in duplicate and the exposure time was approximately 24 hours.
Rationale for test conditions:
Cytotoxicity

Upon completion of the treatment time:
o The culture medium was removed and cells were washed with 50 μL/well with PBS tempered at 37ºC.
o MTT working solution was prepared mixing 1 mL of MTT at 5 mg/mL in water with 9 mL of culture medium without FBS.
o A total 100 μL of the MTT working solution was applied to each well.
o The plates were incubated at 37°C in humid atmosphere and 5% CO2 for three hours.
o After incubation, the culture medium was removed and it was added 100 μL per well of dimethyl sulfoxide (DMSO).
o The plates were shaken for five minutes and the absorbance was read at 570 nm.


Genotoxicity

o After the treating time of the cells, the culture medium was removed and cells were washed with 50 μL/well with PBS tempered at 37ºC.
o A volume of 80 μL of the cells suspension were mixed with a suspension of 160 μL of low melting agarose, at 0.9%, and spread on slides precoated with a layer of 1% agarose. After solidification of the agarose, the slides were immersed in lysis buffer (2,5 M NaCl, 100 mM Na2·EDTA, 10 mM Tris, 1% of Triton X-100, 1% of Lauryl Sarcosine at pH 10) for 1 hour at 4ºC to break the membranes and sequester the proteins. Subsequently the slides were kept 40 minutes in lectrophoresis buffer (1 mMNa2·EDTA, 300 mM NaOH) at 4ºC to allow DNA unwinding and finally electrophoresis at 25 V and 300 mA (at 4°C) for 30 minutes was carried out. To adhere the DNA mobilized, three washes were performed with Tris buffer (1 M Tris at pH 7.5). The slides were dried and kept protected from light until analysis.
o For analysis, the slides were humidified in water and then stained with the
fluorochrome DAPI (4,6-Diamidin-2-phenylindole, at a concentration of 5 μg/mL).
Evaluation criteria:
Cytotoxicity assay
The means and standard deviations of absorbance were calculated for each condition. The percentage of viability was calculated comparing absorbance of treatments versus negative control. Means and standard deviations of each replica were calculated.

Validity criteria
Test results are acceptable if the following criteria are reached:
a) Viability of the solvent control is ≥80% with regard to the medium control.
b) The cell viability obtained with the positive control is within two standard deviations of the historical mean or between 0% and 5%.


Genotoxicity assay
For genotoxicity assay, the analysis of DNA breakage was carried out by image analyzer software Comet Assay IV (version 4.11). Tail intensity of the 50 nucleotides was evaluated for each sample.
The results were expressed as the intensity percentage of the tail versus to the total intensity of the DNA, called Tail Intensity. The median of each replicate was calculated. The mean and standard deviation of each treatment was calculated.

Validity criteria
Test results are acceptable if the tail intensity of the positive control is ≥60%.
Key result
Species / strain:
mammalian cell line, other: Human hepatoma HepG2
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
mammalian cell line, other: Human lung carcinoma A549
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
mammalian cell line, other: Human colorectal carcinoma CaCo-2
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid

In the cytotoxicity assay, no differences were observed in the effect of  Sodasil P95 at the concentration range assessed (2.6–256 μg/mL) on A549, HepG2 and CaCo-2 cell lines. The highest concentration allowed complete dissolution in ethanol:BSA under sonication.


The estimated IC30 value for Sodasil P95 is 446 μg/mL in CaCo-2 cells.


In the genotoxicity assay, no biologically relevant effects were observed for any compound. All tail intensities remained below 1.6% as compared to positive compounds that reach values clearly over 10%. Hence, all the test compounds are devoid of genotoxic effects.

Conclusions:
Sodasil P95 was non-cytotoxic when tested up to 256 μg/mL in human lung A549, human hepatoma HepG2 and human colorectal carcinoma CaCo-2 cells. Further, it did not show any genotoxic effect at 256 μg/mL by using the comet assay.
Executive summary:

The aim of this study was to carry out an in vitro test battery with different cell lines addressed to the determination of the basal cytotoxicity, genotoxicity, acute ocular irritation, sensitization and inflammation potentials and bioavailability of eight nanosilicates in order to provide a comprehensive knowledge of the safety profile of these test items. In this endpoint only the results on cytotoxicity and genotoxicity for Sodasil P95 are documented

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

All genetic toxicity studies in vivo were carried out with the test substance Silicic acid, aluminum sodium salt (CAS 1344-00-9, NAS). Their results were all negative.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vivo mammalian germ cell study: gene mutation
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Qualifier:
no guideline available
Principles of method if other than guideline:
The host organism is inoculated by intraperitoneal injection with a common indicator microorganism/tester strain before treatment with the test substance. After "incubation" in the host organism, the tester strain is withdrawn from the ascites and tested for mutation on minimal agar plate., e.g. according to Ames.
GLP compliance:
no
Type of assay:
other: Host mediated assay
Specific details on test material used for the study:
FDA-Compound 71-45, "sodium silicoaluminate", synthetic silica, Lot no. SR-1621, from the study report it cannot be ascertained wether a crystalline or amorphous form was used
Species:
mouse
Strain:
ICR
Sex:
male
Route of administration:
oral: gavage
Vehicle:
saline
Duration of treatment / exposure:
single administration ("acute") and repeated administration (5 times, "subacute")
Frequency of treatment:
1x and 5x (1x/d)
Remarks:
Doses / Concentrations:
4.25, 42.5 and 425 mg/kg bw, suspended in 0.85 % saline, administered 1x/d (Test I), 5000 mg/kg bw (Test II)
Basis:

Control animals:
yes, concurrent vehicle
Positive control(s):
Ethyl Methane Sulfonate: 350 mg/kg bw
Dimethyl Nitrosamine: 100 mg/kg bw
Key result
Sex:
male
Genotoxicity:
negative
Toxicity:
not specified
Vehicle controls validity:
valid
Positive controls validity:
valid

There was a high increase in mutants following oral
treatment with Dimethylnitrosamine(DMN), but no significant increases in mutation
rates at any dose and dose regimen.

Endpoint:
in vivo mammalian germ cell study: cytogenicity / chromosome aberration
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Comparable to guideline study, detailed documentation, acceptable for assessment
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 478 (Genetic Toxicology: Rodent Dominant Lethal Test)
GLP compliance:
no
Type of assay:
rodent dominant lethal assay
Specific details on test material used for the study:
FDA-Compound 71-45, "sodium silicoaluminate", synthetic silica, Lot no. SR-1621,
from the study report it cannot be ascertained wether a crystalline or amorphous form was used
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: no data
- Age at study initiation: 10 - 12 weeks
Route of administration:
oral: gavage
Vehicle:
- vehicle: 0.85 % saline
- Volume of the vehicle: no data
- Justification for choice of solvent/vehicle: no data
- Concentration of test material in vehicle: no data


MAXIMUM DOSE VOLUME APPLIED: no data
Duration of treatment / exposure:
single administration ("acute") and repeated administration (5 times, "subacute")
Frequency of treatment:
1x and 5x (1x/day)
Remarks:
Doses / Concentrations:
4.25, 42.5 and 425 mg/kg bw, suspended in 0.85 % saline, administered 1x/d (Test I), 5000 mg/kg bw (Test II)
Basis:

No. of animals per sex per dose:
10 males (treated) mated to virgin female rats (2 females/1 male)
Control animals:
yes, concurrent vehicle
yes, historical
Positive control(s):
triethylene melamine
- Route of administration: i.p.
- Doses / concentrations: 0.3 mg/kg bw
Tissues and cell types examined:
Fertility index = No. pregnant females / No. mated
Total No. of implantations
Total number of corpora lutea
Preimplantation losses
see Report p. 132 - 135:
Dead implants
Females with one or more dead implants
Dead implants per total implants
Statistics:
Chi-square test, Armitage´s trend test, regression analyses, Freeman-Tukey transformation, t-test (Report p. 128 - 132)
Sex:
male
Genotoxicity:
negative
Toxicity:
not specified
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid

For single and repeated dosage of 4.25, 42.5, and 425 mg/kg bw (Report p. 77 - 94) as well as single and repeated

dosage of 5000 mg/kg bw (Report p. 95 - 112), there was no dose-response and time-trend pattern of effects

following the silicate treatment: The values calculated for reproduction parameters (see "Examinations" above)

that related to the treated animals did not significantly vary from those obtained from the negative controls,

whereas TEM caused a significant preimplantation loss and embryo resorption during the first five weeks.

Conclusions:
Interpretation of results (migrated information): negative
Endpoint:
in vivo mammalian somatic cell study: cytogenicity / bone marrow chromosome aberration
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Comparable to guideline study, basic data given, acceptable for assessment
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 475 (Mammalian Bone Marrow Chromosome Aberration Test)
Principles of method if other than guideline:
Analysis of metaphase-chromosomes from isolated bone-marrow cells after colcemid-induced arrest of cell division in the metaphase
GLP compliance:
no
Type of assay:
mammalian bone marrow chromosome aberration test
Specific details on test material used for the study:
FDA-Compound 71-45, "sodium silicoaluminate", synthetic silica, Lot no. SR-1621, from the study report it cannot be ascertained wether a crystalline or amorphous form was used
Species:
rat
Sex:
male
Key result
Sex:
male
Genotoxicity:
negative
Toxicity:
not examined
Vehicle controls validity:
valid
Positive controls validity:
valid

Metaphase aberrations: single dose (from Report Table p. 74)

Silene

Dosage

[mg/kg bw]

Time

[h]

No. of cells

Mitotic

index1)

% cells with breaks

% cells with reunion

% cells with other aberr.2)

% cells aberr.

  4.25

6

250

11

2

0

0

2

 

24

250

11

1

0

0

1

48

250

11

3

0

0

3

42.5

6

250

8

0

0

0

0

   24  250 9  0  0  1
   48  250  10  0  0  0  0
 425  6  250  12  2  0  0  2
   24  250  6  0  0  0  0
                48  250  10  2  0  0  2
  Saline  6  150  10  2  0  0  2
   24  150  11 3  0  0  3
   48  150  9 3  0  0  3
 TEM (0.3)  48  250  3  32  12  5(a); 1(pp)  48

1)% cells in mitosis: 500 cells observed/animal

2)Cells with polyploidy, pulverisation (pp), or greater than 10 aberrations (a)

Metaphase aberrations: repeated dose (5x, 1x/d) (from Report Table p. 75)

Silene

Dosage

[mg/kg bw]

Time after last dose

[h]

No. of cells

Mitotic

index1)

% cells with breaks

% cells with reunion

% cells with other aberr.2)

% cells aberr.

  4.25

6

250

10

2

0

0

2

42.5

6

250

11

2

0

0

2

 425.0  6  250 8  3  0  0 3
  Saline  6  150  8  3  0  0 3

1)% cells in mitosis: 500 cells observed/animal

2)Cells with polyploidy, pulverisation (pp), or greater than 10 aberrations (a)

Conclusions:
Interpretation of results (migrated information): negative
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Justification for classification or non-classification

As no adverse effects were observed, there is no need for classification.