Silicic acid, lithium salt

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

04 Nov 2008 - 08 Jan 2009

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

GLP-guideline study, tested with the source substance sodium silicate solution. According to the ECHA guidance document “Practical guide 6: How to report read-across and categories (March 2010)”, the reliability was changed from RL1 to RL2 to reflect the fact that this study was conducted on a read-across substance.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2009

Report date:

2009

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Version / remarks:

adopted July 1997

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Version / remarks:

May 2008

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test

Version / remarks:

August 1998

Test guideline 4

Qualifier:

according to guideline

Guideline:

other: Japanese Guidelines: Kanpoan No. 287, Environment Protection Agency; Eisei No. 127, Ministry of Health and Welfare; Heisei 09/10/31 Kikyoku No. 2, Ministry of International Trade and Industry

Principles of method if other than guideline:

first experiment 4 hours treatment with and without metabolic activation
second experiment 24 hours treatment without metabolic activation and 4 hours treatment with metabolic activation

GLP compliance:

yes (incl. QA statement)

Remarks:

Hess. Ministerium für Umwelt, ländlicher Raum und Verbraucherschutz, Wiesbaden, Germany

Type of assay:

mammalian cell gene mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

1344-09-8 (content: 36%)

IUPAC Name:

1344-09-8 (content: 36%)

Details on test material:

Identity: C-SAT 080094; Sodium silicate solution (weight ratio 3.35)
Tradename: Natronwasserglas 37/40PE (Sodium Silicate 37/40PE, aqueous Sodium
Silicate Solution WR 3.35)
Batch No.: 248908210
Molecular Weight: 263 g/mol
Purity: 36% active ingredient, 64% water
Stability in Solvent: Not indicated by the sponsor
Storage: At room temperature
Expiration Date: September 10, 2009

On the day of the experiment (immediately before treatment), the test item was dissolved in deionised water.
The final concentration of deionised water in the culture medium was 10% v/v.
In the pre-experiment the pH at the three highest concentrations was adjusted with 2N hydrochloric acid.

Method

Target gene:

HPRT locus

Species / strain

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

- Type and identity of media: MEM supplemented with 10% fetal calf serum
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes

Metabolic activation:

with and without

Metabolic activation system:

cofactor supplemented post-mitochondrial fraction (S9-mix), prepared from the livers of Wistar HsdCpb:WU rats treated with Phenobarbital/ß-Naphthoflavone

Test concentrations with justification for top dose:

Experiment I:
without S9 mix: 28.1, 56.3, 112.5, 225.0, 337.5 and 450 µg/mL (4h)
with S9 mix: 56.3, 112.5, 225.0, 450.0, 675.0 and 900.0 µg/mL (4h)
Experiment II:
without S9 mix: 28.1, 56.3, 112.5, 225.0, 450.0 and 675.0 µg/mL (24h)
with S9 mix: 112.5, 225.0, 450.0, 900.0, 1350.0 and 1800 µg/mL (4h)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: water
- Justification for choice of solvent/vehicle: solubility properties

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

water

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: ethylmethane sulfonate, 150 or 75 µg/mL in nutrient medium, without S9; 7,12-dimethylbenz(a)anthracene, 1.1 µg/mL in DMSO, with S9

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 1st experiment: 4 h exposure with and without S9-mix; 2nd experiment: 24 h exposure without S9-mix and 4 h exposure with S9-mix
- Expression time (cells in growth medium): 7 days
- Selection time (if incubation with a selection agent): 8 days

SELECTION AGENT (mutation assays): 11 µg/mL thioguanine (6-TG)

NUMBER OF REPLICATIONS: duplicates each in two independent experiments

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency (cloning efficiency I (survival): cloning efficiency determined immediately after treatment to measure toxicity); cloning efficiency II (viability): cloning efficiency determined after expression period to measure viability of cells without selection agent)

Evaluation criteria:

A test item is classified as positive if it induces either a concentration-related increase of the mutant frequency or a reproducible and positive response at one of the test points.
A test item producing neither a concentration- related increase of the mutant frequency nor a reproducible positive response at any of the test points is considered non-mutagenic in this system.
A positive response is described as follows:
A test item is classified as mutagenic if it reproducibly induces a mutation frequency that is three times above the spontaneous mutation fre¬quency at least at one of the concentrations in the experiment.
The test item is classified as mutagenic if there is a reproducible concentration-related increase of the mutation frequency. Such evaluation may be considered also in the case that a threefold increase of the mutant frequency is not observed.

However, in a case by case evaluation this decision depends on the level of the corresponding negative control data. If there is by chance a low spontaneous mutation rate in the range normally found (0.5 – 31.8 mutants per 10E6 cells) a concentration-related increase of the mutations within this range has to be discussed. The variability of the mutation rates of negative and solvent controls within all experiments of this study was also taken into consideration.

Statistics:

Linear regression analysis (least squares) to assess a possible dose dependent increase of mutant frequencies;
p<0.05

Results and discussion

Test results

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

1st exp.: at 337.5 µg/mL, -S9; 2nd exp.: at 675 µg/mL, -S9 and at 1350 µg/mL and above, +S9

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: In the pre-experiment the pH at the three highest concentrations was adjusted with 2N hydrochloric acid
- Effects of osmolality: In the pre-test the osmolarity of the medium was measured at the maximal concentration (301 mOsm) and the solvent control (280mOsm) and showed no relevant deviation.
- Precipitation: pre-test: Following 4 h treatment precipitation was observed at 1825, 3650 and 7300 μg/mL in the presence of s9-mix. Following continuous treatment precipitation occurred at the maximum concentration of 7300 μg/mL. Main experiments: No precipitation of the test item was observed up to the maximal concentration.

RANGE-FINDING/SCREENING STUDIES:
The highest concentration used in the pre-test was chosen with regard to the purity (36 % active ingredient) and the molecular weight of the test item (263 g/mol). Test item concentrations between 57.0 and 7300 μg/mL were used to evaluate toxicity in the presence (4 h treatment) and absence (4 h and 24 h treatment) of metabolic activation. Strong toxic effects occurred at 456.3 μg/mL in the absence of metabolic activation following 4 and 24 hours treatment. At higher concentrations the cell growth was completely inhibited. In the presence of metabolic activation the no cells survived at 912.5 μg/mL and above.
Based on the results of the pre-experiment, the concentration range of the main experiments was selected.

Remarks on result:

other: strain/cell type: V79

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Summary Table

	conc. µg/mL	S9mix	relative cloning efficiency I [%]	relative cloning efficiency II [%]	mutant colonies/ 10^6cells	induction factor	relative cloning efficiency I [%]	relative cloning efficiency II [%]	mutant colonies/10^6cells	Induction factor
column	1	2	3	4	5	6	7	8	9	10
Experiment I / 4 h treatment			culture I				culture II
Solvent control with water		-	100.0	100.0	18.4	1.0	100.0	100.0	17.1	1.0
Positive control with	150.0	-	76.2	94.8	137.7	7.5	84.7	86.2	132.3	7.7
Test item	28.1	-	98.4	114.2	15.4	0.8	92.7	104.2	13.0	0.8
Test item	56.3	-	100.0	110.3	12.0	0.7	97.1	98.0	16.8	1.0
Test item	112.5	-	102.8	101.8	16.7	0.9	88.2	96.3	15.5	0.9
Test item	225.0	-	87.2	102.0	21.5	1.2	76.9	95.5	19.9	1.2
Test item	337.5	-	0.2	98.4	14.6	0.8	8.8	89.2	20.5	1.2
Test item	450.0	-	0.0	culture was not continued#			0.0	culture was not continued#
Solvent control with water		+	100.0	100.0	24.6	1.0	100.0	100.0	17.9	1.0
Positive control with DMBA	1.1	+	43.9	89.0	636.4	25.9	36.6	99.5	620.9	34.7
Test item	56.3	+	94.4	culture was not continued##			97.3	culture was not continued##
Test item	112.5	+	89.5	94.1	16.9	0.7	95.7	105.9	22.3	1.2
Test item	225.0	+	94.1	89.8	17.6	0.7	94.2	85.8	20.6	1.2
Test item	450.0	+	84.7	90.5	16.3	0.7	94.5	81.5	20.9	1.2
Test item	675.0	+	88.5	114.3	13.2	0.5	93.6	92.6	17.9	1.0
Test item	900.0	+	85.0	85.7	27.8	1.1	92.9	96.4	15.5	0.9
Experiment II / 24 h treatment			culture I				culture II
Solvent control with water		-	100.0	100.0	13.7	1.0	100.0	100.0	14.1	1.0
Positive control with	75.0	-	92.5	97.7	132.5	9.7	102.2	93.2	105.6	7.5
Test item	28.1	-	104.7	culture was not continued##			101.8	culture was not continued##
Test item	56.3	-	102.5	95.7	16.9	1.2	102.2	69.3	18.8	1.3
Test item	112.5	-	88.0	94.5	17.4	1.3	102.7	97.8	9.0	0.6
Test item	225.0	-	95.1	88.7	23.2	1.7	102.0	71.3	17.4	1.2
Test item	450.0	-	94.1	84.2	23.6	1.7	101.4	65.3	17.7	1.3
Test item	675.0	-	43.3	90.9	16.2	1.2	102.9	63.5	28.4	2.0
Experiment II / 4 h treatment			culture I				culture II
Solvent control with water		+	100.0	100.0	13.7	1.0	100.0	100.0	16.1	1.0
Positive control with DMBA	1.1	+	55.9	73.5	809.9	59.1	51.2	78.8	595.5	36.9
Test item	112.5	+	114.6	78.2	20.1	1.5	95.6	90.4	22.6	1.4
Test item	225.0	+	91.3	101.5	24.6	1.8	107.2	98.0	14.5	0.9
Test item	450.0	+	95.5	109.1	22.9	1.7	99.1	83.9	20.5	1.3
Test item	900.0	+	111.7	99.4	26.1	1.9	112.6	80.5	20.8	1.3
Test item	1350.0	+	10.6	100.7	11.4	0.8	7.4	102.9	8.8	0.5
Test item	1800.0	+	0.0	culture was not continued#			0.0	culture was not continued#

#   culture not continued due to exceedingly strong toxic effects

## culture was not continued since a minimum of only four analysable concentrations is required

 

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative