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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 based on grouping of substances (category approach)
Adequacy of study:
key study
Study period:
1998-06-25 to 1998-08-05
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was conducted according to the appropriate OECD test guideline, and in compliance with GLP.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1998
Report Date:
1998

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Dequest 2006 deflocculent and sequestrant

Method

Species / strain
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Metabolic activation system:
Aroclor induced rat liver S9
Test concentrations with justification for top dose:
up to 4400 µg active salt/ml
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: water
- Justification for choice of solvent/vehicle: none given in report
Controlsopen allclose all
Untreated negative controls:
yes
Remarks:
growth medium
Negative solvent / vehicle controls:
yes
Remarks:
water
True negative controls:
no
Positive controls:
yes
Positive control substance:
mitomycin C
Remarks:
without activation
Untreated negative controls:
yes
Remarks:
growth medium
Negative solvent / vehicle controls:
yes
Remarks:
water
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
with activation
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 3 hours (initial assay) 17.8 hours (confirmatory assay)
- Expression time (cells in growth medium): 16.8 hours (initial assay 2 hours (confirmatory assay)
- Fixation time (start of exposure up to fixation or harvest of cells): 20 hours

SPINDLE INHIBITOR (cytogenetic assays): Colcemid (present for last 2 hours of incubation)
STAIN (for cytogenetic assays): 5% Giesma solution

NUMBER OF REPLICATIONS: duplicate cultures

NUMBER OF CELLS EVALUATED:100 from each replicate culture where possible

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; other: assessment of percent confluence of cell monolayer and presence of mitotic or dead cells floating in medium.
OTHER EXAMINATIONS:
- Determination of polyploidy: yes
- Determination of endoreplication: yes
Evaluation criteria:
The test substance is considered positive for inducing chromosomal aberrations if there is significant dose-dependent increase (p<=0.01) in the number of cells with aberrations at one or more concentrations
Statistics:
Cochran-Armitage test for linear trend and Fisher's Exact test to compare percentage of cells with aberrations in treated cells with control results.

Results and discussion

Test results
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
other: Non cytotoxic for 3h treatments. <500 µg/ml for continuous treatments without activation.
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: pH was measured at 9.0 (culture medium pH 8.5)
- Precipitation: no precipitation was observed.

RANGE-FINDING/SCREENING STUDIES: no precipitate observed in the absence of cells at a concentration of 4400 µg/ml

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

ADDITIONAL INFORMATION ON CYTOTOXICITY: dead monolayers and approximately 85% reduction in monolayer confluence reported at 4400 µg/ml. Unhealthy monolayers and approximately 70% reduction reported at 3080 µg/ml. Unhealthy monolayers and approximately15% or 45% reduction in monolayer confluence at 2160 µg/ml. In absence of metabolic activation no cytotoxicity was seen with 3 hr treatment. With continuous treatment cytotoxicity was induced.  The top dose scored (250µg/ml) had a relative mitotic index of 60%.  The higher dose (500µg/ml) had a relative mitotic index of <10%. 

Any other information on results incl. tables

Table 1: Results of chromosome analysis Experiment 1 (3 h treatment, 20 incubation) without activation (total count from 2 cultures / 200 cells)

Treatment

Untreated

Solvent*

Control

Positive

Control

Low dose 1510 µg/ml

Mid dose 2160 µg/ml

Mid dose 3080 

µg/ml

High dose 4400 µg/ml

Cytotoxicity

-

-

-

no

no

no

no

Cell numbers

Percentage from 200 cells

Cells with aberrations (%)

3.0

1

52

3.5

0

3.0

0

Mitotic index (%)

14.8

20.8

NR

23.1

23.5

24.0

17.8

Polyploidy (%)

2.5

2.5

3.0

2.5

3.0

2.1

2.0

Endo reduplication (%)

2

1.5

1.0

2.0

1.5

0.5

2.5

 *Solvent control with water

NR not reported

 

 

Table 2: Results of chromosome analysis Experiment 2a (17.8 h treatment, 20 h incubation) without activation

Treatment

Untreated

Solvent*

Control

Positive

Control

Low dose 31.3 µg/ml

Mid dose 62.6 µg/ml

Mid dose 125 µg/ml

High dose 250 µg/ml

Cytotoxicity

-

-

-

no

no

no

no

Number of cells

Percentage from 200 cells

Cells with aberrations (%)

 

0

0

12.8

1.5

0

1.0

1.5

Mitotic index (%)

6.5

7.0

NR

4.0

6.9

4.0

4.2

Polyploidy (%)

9.5

0.5

2.5

1.5

0

0.5

0.5

Endo reduplication (%)

0

0

0

1.5

0

0

0

*Solvent control with water

NR not reported

 

Table 3: Results of chromosome analysis Experiment 1, (3 h treatment, 20 h incubation) with activation

Untreated

Solvent*

Control

Positive

Control

Low dose 1510 µg/ml

Mid dose 2160 µg/ml

Mid dose 3080 µg/ml

High dose 4400 µg/ml

Cytotoxicity

-

-

-

no

no

no

no

 

Percentage from 200 cells

Cells with aberrations

 

1.5

1.0

60.0

1.0

8.0

1.5

1.0

Mitotic index (%)

16.0

19.9

NR

21.9

15.7

16.9

11.7

Polyploidy (%)

3.0

2.5

2.5

2.0

2.0

2.0

1.0

Endo reduplication (%)

1.5

0

1.5

1.0

6.0

0

0

*Solvent control with water

NR not reported

 

Table 4: Results of chromosome analysis Experiment 2, (3 h treatment, 20 h incubation) with activation

 Treatment

Untreated

Solvent*

Control

Positive

Control

Low dose 1510 µg/ml

Mid dose 2160 µg/ml

Mid dose 3080 µg/ml

High dose 4400 µg/ml

Cytotoxicity

-

-

-

no

no

no

no

 Number of cells

Percentage from 200 cells

Cells with aberrations (%)

 

1.5

2.0

52

1.0

1.5

0.5

0.5

Mitotic index (%)

13.1

14.1

NR

12.8

13.8

13.3

13.3

Polyploidy (%)

2.0

1.5

2.0

2.0

3.0

2.0

2.5

Endo reduplication (%)

2.0

1.5

0.5

2.5

2.9

1.0

2.5

*Solvent control with water

NR not reported

Applicant's summary and conclusion

Conclusions:
The test substance did not induce chromosome aberrations in vitro when tested according to OECD TG 473 under GLP up to acceptable limits for this assay (10mM and cytotoxicity). It is concluded that ATMP, sodium salt does not cause chromosomal aberrations under the conditions of the test.