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Skin sensitisation

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Endpoint:
skin sensitisation: in vitro
Type of information:
experimental study
Adequacy of study:
key study
Study period:
experimental phase: Jul. 16, 2016 - Sep. 19, 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Cross-referenceopen allclose all
Reason / purpose for cross-reference:
reference to same study
Reference
Endpoint:
basic toxicokinetics in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
supporting 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
Objective of study:
bioaccessibility (or bioavailability)
Qualifier:
no guideline followed
Principles of method if other than guideline:
Bioavailability was tested in three human cell lines: Test items were exposed at fixed concentration of 256 μg/mL for 1, 4 and 24 hours. TEM images were analyzed for the presence or absence of the test item inside the cells.
GLP compliance:
yes
Specific details on test material used for the study:
Sodasil P95
Radiolabelling:
no
Dose / conc.:
256 other: µg/ml
Bioaccessibility (or Bioavailability) testing results:
Regarding the bioavailability assessment of the test items to penetrate into the three cell lines, all of them were able to penetrate into the cells. The main presence was generally seen into the cytoplasm, some of them showing crystallographic aggregates, other totally dispersed and other that appeared to be in process of vacuolization. However, no clear data could be reached from the current data designs to discern whether nanosilicates are externalized or
remain into the cells for a long time.
Conclusions:
Sodasil P95 was able to be internalized into the cytoplasm of the three cell lines.
Executive summary:

The bioavailability of Sodasil P95 was tested in three cell lines (human hepatoma HepG2, human lung carcinoma A549 and human colorectal carcinoma CaCo-2). It was able to be internalized into the cytoplasm of the three cell lines.

Reason / purpose for cross-reference:
reference to same study
Reference
Endpoint:
additional toxicological information
Type of information:
experimental study
Adequacy of study:
key study
Study period:
experimental phase: Jun. 6, 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:
read-across source
Reason / purpose for cross-reference:
reference to same study
Reason / purpose for cross-reference:
reference to same study
Type of study / information:
Inflammation potential in human lung carcinoma A549
Qualifier:
no guideline followed
Principles of method if other than guideline:
The assay focused on the measurement of different inflammatory mediators (IL1ß, IL4, IL6, IL10, IL12, IL17A, IFNg, TNFa, MIP-1a and MIP-1b) in human lung carcinoma A549 cells after applying the test item at two concentrations.
GLP compliance:
yes
Specific details on test material used for the study:
Sodasil P-95

In the inflammatory potential assay, where human lung carcinoma A549 cells were used, none of the test items showed a pro inflammatory signal in any of the inflammatory mediators tested: IL1ß, IL4, IL6, IL10, IL12, IL17A, IFNg, TNFa, MIP-1a and MIP-1b. However, while TGFb1 and MCP1
showed response in front of the cocktail of interleukins as positive control (PC) as compared to the kit negative control (NC), these parameters were unable to be used for the rest of test items due to the solvent interfered the signal by increasing the optical density. Hence, the test items did not show any pro- or anti-inflammatory potential under the experimental conditions assayed.

Conclusions:
Inflammatory mediators (pro- or anti-inflammatory and chemokines) determined in A549 cells were not altered by Sodasil P95, while the kit positive control did.
Executive summary:

Cells were exposed to the test items at to two concentrations, 81 and 256 μg/m. Inflammatory mediators (pro- or anti-inflammatory and chemokines) determined in A549 cells were not altered by any of the test items assayed.

Reason / purpose for cross-reference:
reference to same study
Reference
Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
experimental phase: Jun. 16, 2016 - Sep. 19, 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline 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
Reason / purpose for cross-reference:
reference to same study
Qualifier:
according to guideline
Guideline:
OECD Guideline 491 (Short Time Exposure In Vitro Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)
Deviations:
not specified
GLP compliance:
yes
Specific details on test material used for the study:
Sodasil P95
Species:
rabbit
Details on test animals or tissues and environmental conditions:
The Statens Serum Institute Rabbit Cornea cell line (SIRC) was used. SIRC cells were cultured at 37°C under 5% CO2 and humidified atmosphere in a culture flask containing a culture medium comprising MEM supplemented with 10% FBS, 2 mM L-glutamine, 50–100 U/mL penicillin and 50–100 μg/mL streptomycin.
Vehicle:
physiological saline
Remarks:
0.05% BSA in saline
Controls:
yes, concurrent vehicle
yes, concurrent positive control
Amount / concentration applied:
5% of test item in saline (w/v),
0.05% of test item in saline (w/v)
Duration of treatment / exposure:
5 minutes
Duration of post- treatment incubation (in vitro):
2-hour reaction time in an incubator
Number of animals or in vitro replicates:
3
Details on study design:
Seed process
Cells from a growing culture were used. They were seeded at a density between 10–12 x 10³ cells per well at 200 μL. Cells were kept in the incubator for three days reaching at confluence of more than 80% at 35–37ºC and 5% CO2, before
being exposed to the test items.

Treatment
The test items (200 μL of test solution/well) were tested at 5% and 0.05% at room temperature. The negative control was 0.05% BSA in saline, while Sodium lauryl sulfate (SLS) in saline at 0.01% in saline was used as a positive control in each plate of each repetition. A blank composed by phosphate buffer saline was also used to compensate the optical density background.
Each treatment was done in triplicate and the exposure time was approximately five minutes.

Evaluation
Cells cultured in the 96-well plate will be exposed to 200 μL/well of a 5% or a 0.05% concentration of the test item solution, at room temperature, for five minutes.
After exposure, cells will be washed twice with 200 μL of PBS and 200 μL of MTT solution (0.5 mg MTT/mL of culture medium) was added. After a 2-hour reaction time in an incubator (37°C at 5% CO2), the MTT solution was decanted, MTT formazan was then extracted by adding 200 μL of 0.04N hydrochloric acid-isopropanol that were kept under resting conditions in the dark at room
temperature for 60 minutes. The absorbance of the MTT formazan solution was measured at 570 nm with a plate reader spectrophotometer.
The optical density (OD) values obtained for each test item and positive control were used to calculate the cell viability relative to the solvent control, which was set at 100%.
Irritation parameter:
mean percent tissue viability 
Run / experiment:
0.5 mg/ml conc. (= 0.05% of test item in saline (w/v))
Value:
ca. 94.87 - ca. 114.31
Vehicle controls validity:
valid
Positive controls validity:
valid
Irritation parameter:
mean percent tissue viability 
Run / experiment:
50 mg/ml conc. (= 5% of test item in saline (w/v))
Value:
ca. -15.41 - ca. 2.87
Vehicle controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
In the SIRC assay, the viability and effect percentages were different regarding the doses tested (5 or 0.05%). The viability percentage was approximately 0% for Sodasil P95 at the highest concentration, however, at the lowest concentration, the test item showed viability values of approximately 100%.

Neither category nor prediction of irritation could be made or assigned to the test item Sodasil P95 by using the SIRC eye model, since cell viability was ≤ 70% at a 5% concentration and > 70% at 0.05% concentration.
Interpretation of results:
study cannot be used for classification
Conclusions:
Neither category nor prediction of irritation could be made or assigned to the test item Sodasil P95 by using the SIRC eye model, since cell viability was ≤ 70% at a 5% concentration and > 70% at 0.05% concentration.
Executive summary:

Eye irritation for the test item Sodasil P95 was assessed by using the SIRC eye model. According to OECD guideline 491 all test chemicals, like Sodasil P95, that induce a cell viability of ≤ 70% at a 5% concentration and > 70% at 0.05% concentration are subsequently be tested with other adequately validated in vitro test methods or, as a last option, in the in vivo rabbit eye test. As no other eye irritation study in Sodasil P95 is available, it is hereby refferred to the studies in the NASs Zeolex 23A and Zeolex 7.

Reason / purpose for cross-reference:
reference to same study
Reference
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

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
other: DB-ALM Protocol No. 36
Deviations:
not specified
GLP compliance:
yes
Type of study:
human Cell Line Activation Test (h-CLAT)

Test material

Constituent 1
Chemical structure
Reference substance name:
Silicic acid, aluminum sodium salt
EC Number:
215-684-8
EC Name:
Silicic acid, aluminum sodium salt
Cas Number:
1344-00-9
Molecular formula:
nSiO2*mAl2O3*zNa2O n = 2-4; m = 0.12-3.20; z = 0.11-4.5
IUPAC Name:
aluminium(3+) sodium bis(oxosilanebis(olate))
Test material form:
solid: nanoform, no surface treatment
Remarks:
crystalline-free
Specific details on test material used for the study:
Sodasil P95

In vitro test system

Details of test system:
other: THP-1 human monocytic leukemia cell line
Details on the study design:
THP-1 cellular line was cultured in RPMI 1640 medium with 2 mM glutamine, 10% FBS, 0.05 mM 2-Mercaptoethanol and 100 U/mL penicillin and 100 μg/mL streptomycin and cultured at 37ºC under 5% CO2 and humidified atmosphere.
The solvent for disperse the test items 0.05% BSA in saline was used as negative control.
A total five concentrations were prepared for a preliminary concentration-range finding assay and measurement of the CD86/CD54 expression. In this case, the maximum dose evaluated for the test item was the highest soluble concentration with a dilution factor 1:2.
Vehicle / solvent control:
saline [442E]
Positive control:
other: NiSO4 at a final concentration of 200 μg/mL was used as positive control for the preliminary assay and DNCB at a final concentration of 4 μg/mL was used as positive control for measurement of CD86/CD54 expression.

Results and discussion

In vitro / in chemico

Resultsopen allclose all
Key result
Group:
test chemical
Parameter:
RFI CD54>150 [442E]
Value:
0.61 µg/mL
Cell viability:
95.5 %
Vehicle controls validity:
valid
Positive controls validity:
valid
Key result
Group:
test chemical
Parameter:
RFI CD54>150 [442E]
Value:
0.73 µg/mL
Cell viability:
95.0 %
Vehicle controls validity:
valid
Positive controls validity:
valid
Key result
Group:
test chemical
Parameter:
RFI CD54>150 [442E]
Value:
0.88 µg/mL
Cell viability:
94.7 %
Vehicle controls validity:
valid
Positive controls validity:
valid
Key result
Group:
test chemical
Parameter:
RFI CD54>150 [442E]
Value:
1.06 µg/mL
Cell viability:
95.0 %
Vehicle controls validity:
valid
Positive controls validity:
valid
Key result
Group:
test chemical
Parameter:
RFI CD54>150 [442E]
Value:
1.27 µg/mL
Cell viability:
95.3 %
Vehicle controls validity:
valid
Positive controls validity:
valid
Group:
other: solvent control
Parameter:
RFI CD54>150 [442E]
Value:
0 µg/mL
Cell viability:
93.9 %
Vehicle controls validity:
not applicable
Positive controls validity:
valid
Key result
Group:
test chemical
Parameter:
RFI CD86>200 [442E]
Value:
0.61 µg/mL
Cell viability:
95.4 %
Vehicle controls validity:
valid
Positive controls validity:
valid
Key result
Group:
test chemical
Parameter:
RFI CD86>200 [442E]
Value:
0.73 µg/mL
Cell viability:
94.5 %
Vehicle controls validity:
valid
Positive controls validity:
valid
Key result
Group:
test chemical
Parameter:
RFI CD86>200 [442E]
Value:
0.88 µg/mL
Cell viability:
94.4 %
Vehicle controls validity:
valid
Positive controls validity:
valid
Key result
Group:
test chemical
Parameter:
RFI CD86>200 [442E]
Value:
1.06 µg/mL
Cell viability:
94.7 %
Vehicle controls validity:
valid
Positive controls validity:
valid
Key result
Group:
test chemical
Parameter:
RFI CD86>200 [442E]
Value:
1.27 µg/mL
Cell viability:
95.0 %
Vehicle controls validity:
valid
Positive controls validity:
valid
Group:
other: solvent control
Parameter:
RFI CD86>200 [442E]
Value:
0 µg/mL
Cell viability:
95.3 %
Vehicle controls validity:
not applicable
Positive controls validity:
valid

Any other information on results incl. tables

Concentrations tested were selected in accordance to the CV75 value. The assays focused on the h-CLAT method applying the test items on THP-1 cells allowed no apparent sensitization potential (i.e. negative response of CD86 and CD54) keeping an appropriate cell viability according to the mean data.

Applicant's summary and conclusion

Conclusions:
No sensitization capacity could be observed for Sodasil P95 when assayed under the experimental conditions.
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. Only the experiments in skin sensitisation with Sodasil P95 are documented here.
Sodasil P95 was unable to induce any sensitization signal on CD86 and CD54 in THP-1 cells after exposure at ranges comprised between 0.61 and 1.27 μg/mL.