Registration Dossier

Toxicological information

Genetic toxicity: in vitro

Currently viewing:

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 guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:
adopted July 1997
Qualifier:
according to
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:
May 2008
Qualifier:
according to
Guideline:
EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test
Version / remarks:
August 1998
Qualifier:
according to
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. certificate)
Remarks:
Hess. Ministerium für Umwelt, ländlicher Raum und Verbraucherschutz, Wiesbaden, Germany
Type of assay:
mammalian cell gene mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
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/ 106cells

induction factor

relative cloning efficiency I [%]

relative cloning efficiency II [%]

mutant colonies/106cells

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