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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2003-10-16 to 2003-11-28
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2004
Report Date:
2004

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test

Test material

Reference
Name:
Unnamed
Type:
Constituent

Method

Species / strain
Species / strain / cell type:
other: Chinese hamster ovary (CHO-K1) cells
Metabolic activation:
with and without
Metabolic activation system:
Aroclor induced rat liver S9, NADP as cofactor
Test concentrations with justification for top dose:
170.3, 340.6, 681.2 and 1362.4 µg/ml. The cells treated with 170.3 µg/ml were not evaluated for chromosome aberrations.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: based information provided by sponsor and compatibility with the target cells.
Controlsopen allclose all
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
mitomycin C
Remarks:
without activation
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
with activation
Details on test system and experimental conditions:
ACTIVATION: 1 ml S9 mix containing 20 µl Aroclor induced rat liver S9, NADP as cofactor, added to 4 ml of medium
METHOD OF APPLICATION: in medium;
DURATION
- Preincubation period: none
- Exposure duration: 4 or 20 hours (without activation), 4 hours with activation
- Expression time (cells in growth medium): 0 or 16 hours
- Fixation time (start of exposure up to fixation or harvest of cells): 20 hours

SPINDLE INHIBITOR (cytogenetic assays): Colcemid added 2 h prior to harvest
STAIN (for cytogenetic assays): Giesma

NUMBER OF REPLICATIONS: duplicate cultures per concentration

NUMBER OF CELLS EVALUATED: mitotic index in 500 cells, aberrations in 200 cells (100 per duplicate flask)

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; other: cell count and percentage viability

OTHER EXAMINATIONS:
- Determination of polyploidy: yes
- Determination of endoreplication:yes
Evaluation criteria:
A test substance is considered positive if the percentage of cells with aberrations is increased in a dose-responsive manner with one or more concentrations being statistically significant. (p
Statistics:
Fisher's exact test; in the event of a positive result, Cochran Armitage test used to measure dose-responsiveness

Results and discussion

Test results
Key result
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
positive
Remarks:
with activation
Cytotoxicity / choice of top concentrations:
other: > 1362.4 μg/ml
Additional information on results:
RANGE-FINDING/SCREENING STUDIES: cell growth inhibition was between 7% and 17% in the highest dose tested.

COMPARISON WITH HISTORICAL CONTROL DATA: control values were within historical data.

ADDITIONAL INFORMATION ON CYTOTOXICITY:

Any other information on results incl. tables

The percentage of cells with structural or numerical aberrations in the  non-activated 4 and 20 hour exposure groups was not significantly  increased above that of the solvent control at any dose level. The  percentage of cells with structural aberrations in the S9 activated 4  hour exposure group was significantly increased above that of the solvent  control at 1362.4 ug/mL. The Cochran-Armitage test was also positive for  a dose response. The percentage of cells with numerical aberrations in  the test article-treated group was not significantly increased above that  of the solvent control at any dose level. The results of the assay are  summarized in the following table:

WITHOUT S9:
Treatment time: 4 hr
Recovery time: 16 hr
Harvest time: 20 hr
Toxicity* at highest dose scored: 16%
Mitotic Index Reduction**: None
LED for Structural Aberrations (µg/mL): None
LED for Numerical Aberrations (µg/mL): None

WITHOUT S9:
Treatment time: 20 hr
Recovery time: 0 hr
Harvest time: 20 hr
Toxicity* at highest dose scored: 4%
Mitotic Index Reduction**: 9%
LED for Structural Aberrations (µg/mL): None
LED for Numerical Aberrations (µg/mL): None

WITH S9:
Treatment time: 4 hr
Recovery time: 16 hr
Harvest time: 20 hr
Toxicity* at highest dose scored: 22%
Mitotic Index Reduction**: 16%
LED for Structural Aberrations (µg/mL): 1362.4
LED for Numerical Aberrations (µg/mL): None


Where: 
* Cell growth inhibition
** Relative to solvent control at high dose evaluated for
chromosome aberrations
LED = Lowest Effective Dose

Table 1: 4 h treatment without activation (totals and percentages from 200 cells)

Treatment

Solvent Control

Positive control*

340.6 µg/ml

681.2 µg/ml

1362.4

µg/ml

Cytotoxicity

no

no

no

no

no

Chromatid aberrations

gaps

1

3

0

2

2

breaks

1

14

1

0

0

interchanges

0

13

1

1

.

Chromosome aberrations

breaks

0

0

0

0

0

dicentric

0

0

1

2

3

ring

0

0

0

0

0

Percent aberrant cells

Numerical

2.5

1.5

2

3

3

Structural

0.5

29.0

1.5

1.5

2

Mitotic index

7.3

8.1

8.4

7.5

7.6

* 100 cells evaluated

 

Table 2: 4 h treatment with activation (totals and percentages from 200 cells)

Treatment

Solvent Control

Positive control*

340.6 µg/ml

681.2 µg/ml

1362.4

µg/ml

Cytotoxicity

no

yes

no

no

no

Chromatid aberrations

gaps

1

4

0

0

2

breaks

1

30

0

0

7

interchanges

0

19

0

0

13

Chromosome aberrations

breaks

0

0

0

0

0

dicentric

0

3

2

2

2

ring

0

0

0

0

0

Percent aberrant cells

Numerical

2

1.5

1.5

3.5

4.0

Structural

0.5

22.5

1.0

3.0

9.5

Mitotic index

9.3

8.6

8.4

8.2

7.8

* 100 cells evaluated

 

Table 3: 20 h treatment without activation (totals and percentages from 200 cells)

Treatment

Solvent Control

Positive control*

340.6 µg/ml

681.2 µg/ml

1362.4

µg/ml

Cytotoxicity

no

no

no

no

no

Chromatid aberrations

gaps

1

6

0

1

1

breaks

1

28

0

0

0

interchanges

0

18

0

1

0

Chromosome aberrations

breaks

0

0

0

0

0

dicentric

0

2

3

4

4

ring

0

1

0

0

0

Percent aberrant cells

Numerical

2

1.5

2

1.5

1.5

Structural

0.5

20

1.5

2.5

2.0

Mitotic index

7.9

7.4

6.8

7.0

6.3

* 100 cells evaluated

Applicant's summary and conclusion

Conclusions:
Trimethoxy(methyl)silane has been tested in a valid study conducted according to OECD 473 and in compliance with GLP. The test substance induced a statistically significant dose related increase in the number of structural aberrations in Chinese hamster ovary (CHO-K1) cells in the presence of activation. No test-substance related genotoxicity was observed in the absence of metabolic activation. Appropriate positive and solvent controls were included and gave expected results. It is concluded that the test substance is positive for the induction of chromosome aberrations in the presence of activation under the conditions of the study.