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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
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
Dec. 1989
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Comparable to guideline study, performed under GLP conditions. 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:
1990
Report date:
1990

Materials and methods

Test guideline
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
yes
Remarks:
(100 instead of 200 metaphases were scored; unusual exposure times)
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test

Test material

Constituent 1
Reference substance name:
Silicon dioxide
EC Number:
231-545-4
EC Name:
Silicon dioxide
Cas Number:
7631-86-9
IUPAC Name:
dioxosilane
Details on test material:
- Name of test material (as cited in study report): Cab-O-Sil EH-5: CAS-Name: Silica, amorphous, fumed, cryst.-free; CAS-No.: 112945-52-5
- Substance type: inorganic
- Physical state: solid
- Analytical purity: >99%
- Lot/batch No.: 1H049
- Stability under test conditions: stable

Method

Target gene:
not applicable
Species / strain
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):
- Type and identity of media: Ham´s F-12 medium without hypoxanthine with 5 % FBS, 1 % penicilin-streptomycin and 1 % L-glutamine
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: no data
- Periodically "cleansed" against high spontaneous background: no data
- Source: A. Hsie, Oak Ridge National Laboratories, directly received in frozen state
Metabolic activation:
with and without
Metabolic activation system:
Aroclor induced rat liver S9 (adult male SD rats)
Test concentrations with justification for top dose:
38, 75, 150, 300 µg/mL (without S9)
250, 500, 750, 1000 µg/mL (with S9)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
Controls
Untreated negative controls:
yes
Remarks:
untreated cells
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: Triethyleneamine without S9, cyclophosphamide with S9
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium; preincubation

DURATION
- Preincubation period: 16 - 24 hours at 37 +/-1 °C (5 x10^5 cell/25 ml flask in 5 +/- 1% CO2 atmosphere
- Exposure duration: 18 h (without S9); 2 h (with S9)
- Expression time (cells in growth medium): 11 h (for S9-activated cultures)
- Fixation time (start of exposure up to fixation or harvest of cells): Sum Exposure + expression time

SPINDLE INHIBITOR (cytogenetic assays): Colcemid (addition of 0.1 µg/mL 2 h prior to sampling of metaphase cells)
STAIN (for cytogenetic assays): 5 % Giemsa

NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED: 100

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cell cycle delay

OTHER EXAMINATIONS:
- Other: chromatid-type and chromosome-type aberrations (breaks, various exchange figures)
Chromatid and isochromatid gaps were recorded, but not included in the analysis.

OTHER: Mitotic index was recorded as percentage of cells in mitosis per 500 cells counted.
Evaluation criteria:
Dose-response with one or more concentrations elevated in relation to the solvent control (p =<0.05). A significant increase at the highest dose only with no dose-response was considered suspect. A significant increase at one dose level other than the high dose with no dose-response was considered equivocal.

Validity: The number of cells with chromosome aberrations in the negative and solvent control must be no greater than 6 %. The percentage of aberrations in the positve controls must be statistically increased (p=<0.05 Fisher´s exact test) relative to the untreated control.
Statistics:
Fisher´s exact test for pairwise comparison of the aberrations in test and control cultures. The Cochran-Armitage test was applied for measure dose-responsiveness.

Results and discussion

Test results
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
see "Additional information on results"
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
none mentioned

RANGE-FINDING/SCREENING STUDIES:
9 concentration between 0.1 - 1000 µg/ml
with and without S9

ADDITIONAL INFORMATION ON CYTOTOXICITY:

Mitotic index (MIx) and cell cycle kinetics (M1):
-S9 + S9
MIx[%] M1[%] MIx[%] M1[%]
__________________________________________________
DMSO control: 5 2 3.4 3
0.1 µg/L 3.8 2 2.6 3
10 µg/L 3.2 7 3.2 10
30 µg/L 2.6 17 2.8 7
100 µg/L 2.2 14 3.2 3
300 µg/L 1.2 27 1.0 24
__________________________________________________
Cell proliferation began to be inhibited at 30 µg/L (-S9) and 300 µg/L (+S9).
Simultaneously, the cell cycle became retarded with an accumulation of cells in the M1 phase.
Neither in the control nor in the treated cultures, cells were observed in the M3 phase (except 1 instance in the DMSO control).
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Summary of Results

Harvest time: 18 h (-S9); 15 h (+S9)

Total metaphase cells scored: 100 each

    

Treatment

S9 Activation

Mitotic Index

Cells with Aberrations

Untreated

4.8

2

DMSO

No

2.4

1

Cab-O-Sil EH-5 [µg/mL]

38

No

1.7

1

75

No

1.0

2

150

No

0.5

2

300

No

0.2

2

Triethylenemelamine, 0.5 µg/ml

No

1.6

34**

Untreated

Yes

7.0

0

DMSO

Yes

5.7

2

Cab-O-Sil EH-5 [µg/mL]

250

Yes

2.3

2

500

Yes

2.7

3

750

Yes

2.0

0

1000

Yes

2.1

1

Cyclophosphamide,

50 µg/ml

Yes

4.1

19**

** = statistically significant p =<0.01

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative