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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:
23 June 1998 to 03 September1998
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

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

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
Deviations:
no
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test

Test material

Constituent 1
Chemical structure
Reference substance name:
heptan-2-yl [(5-chloroquinolin-8-yl)oxy]acetate
EC Number:
619-447-3
Cas Number:
99607-70-2
Molecular formula:
C18H22ClNO3
IUPAC Name:
heptan-2-yl [(5-chloroquinolin-8-yl)oxy]acetate
Details on test material:
- Name of test material (as cited in study report): CGA185072 technical
- Analytical purity: 97%
- Physical state: beige powder
- Expiration date of the lot/batch: reanalysis date 31 December 1999
- Stability: confirmed

Method

Target gene:
Chinese hamster ovary cells (ATCC CCL61)
Species / strain
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
Aroclor induced rat liver S9
Test concentrations with justification for top dose:
Cytotoxicity test: 0, 0.39, 0.78, 1.56, 3.13, 6.25, 12.50, 25 and 50 µg/mL
Experiments without metabolic activation: 0, 6.25, 12.50 and 25.00 µg/mL
Experiments with metabolic activation: 0, 12.50, 25.00 and 50.00 µg/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
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 S9 Migrated to IUCLID6: 0.2 µg/mL
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
with S9 Migrated to IUCLID6: 20.0 µg/mL
Details on test system and experimental conditions:
DURATION
- Preincubation period: approximately 24 hours
- Exposure duration: 21 or 45 hours (without S9); 3 hours treatment followed by 18 hours recovery or 3 hours treatment followed by 18 hours recovery (with S9)

SPINDLE INHIBITOR
- Two hours prior to harvesting, the cultures were treated with 0.4 µg/mL Colcemide to arrest cells in metaphase. After harvesting the cells were spread on glass slides and air dried.

NUMBER OF REPLICATIONS: 4

NUMBER OF CELLS EVALUATED:
- The percentage of mitotic suppression was determined by evaluating at least 2000 cells from each slide. Whenever possible two hundred well spread metaphase figures with 19 to 21 centromeres from two cultures (100 metaphases per replicate culture) in the vehicle control and in the treated groups were scored. At least fifty metaphases were scored in the positive controls (25 per replicate culture).
- The slides were examined for specific and unspecific structural aberrations.
- The percentages of mitotic suppression, in comparison with the controls, were evaluated by counting at least 2000 cells per slide for each treatment group.

DETERMINATION OF CYTOTOXICITY
- Determination of cloning efficiency: Cell cultures were exposed to the test substance for 3 hours in the presence of a metabolic activation system. Four concentrations of the test substance and the vehicle (DMSO) control were tested. The treatment was terminated by washing the cultures with phosphate buffered saline (PBS). The cells were then suspended by trypsinisation, pelleted, resuspended in fresh growth medium and counted with a haemocytometer. The cultures were diluted so that 100 cells were seeded per 9.6 cm² in 3 ml of growth medium. After seven to eight days of growth at 37°C the cultures were fixed and stained with Giemsa and the surviving colonies (cloning efficiency) were determined by eye.
Evaluation criteria:
Criterion for positive response based on an increased incidence of specific chromosomal aberrations.

Criteria for a positive test: the percentage of metaphases containing specific aberrations in a treatment group is higher than 6.0, and differs statistically from the respective negative control, with a demonstrable concentration-related response.

Criteria for a negative response: percentage of metaphases containing specific aberrations in a treatment group is less than or equal to 6.0, and does not differ statistically from the respective negative control.



Statistics:
Not applicable

Results and discussion

Test results
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Analysis for test article content of the lowest, by serial dilution prepared stock solutions used in the mutagenicity test revealed that the test article was stable in the vehicle used and that the cells were exposed to the intended concentrations. The actual concentrations ranged between 94.4 –95.6% of the nominal concentrations.

Cytotoxicity test - Concentrations higher than 50 µg/mL could not be tested, due to solubility limitations of the test substance in the vehicle DMSO. In the experiments with and without metabolic activation, marked cytotoxicity was observed at the highest concentrations.

In all experiments performed with or without metabolic activation no statistically significant increase in the number of metaphases containing specific chromosomal aberrations was observed.
Remarks on result:
other: strain/cell type: CHO cells ATCC CCL61
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Table 1: CGA185072: Mitotic Index (% of control) of CHO cells

 

 

Original study

Confirmatory study

Experiment

1

2

3

4

5

6

Metabolic Activation

-

+

-

+

-

+

Treatment (h)

21

3

21

3

45

3

Recovery (h)

-

18

-

18

-

42

CGA185072

 

 

 

 

 

 

50.00 µg/mL

0

95

0

138

1

79

25.00 µg/mL

20

107

19

106

50

95

12.50 µg/mL

103

96

89

93

89

99

6.25 µg/mL

96

 

71

 

128

 

3.13 µg/mL

109

 

94

 

87

 

1.56 µg/mL

 

 

 

 

 

 

0.78 µg/mL

 

 

 

 

 

 

0.39 µg/mL

 

103

 

101

 

94

 

Table 2: Percent incidence of specific chromosomal aberrations in CHO cells treated with CGA185072

 

Original study

Confirmatory study

Experiment

1

2

3

4

5

6

Metabolic Activation

-

+

-

+

-

+

Treatment (h)

21

3

21

3

45

3

Recovery (h)

-

18

-

18

-

42

Negative control

2.0

2.0

2.5

2.0

3.0

3.5

CGA185072

 

 

 

 

 

 

50.00 µg/mL

 

 

 

 

 

 

25.00 µg/mL

 

3.0

 

4.0

 

4.0

12.50 µg/mL

3.0

2.0

4.5

4.5

2.5

1.0

6.25 µg/mL

2.0

2.5

0.5

3.5

3.0

3.0

3.13 µg/mL

0.5

 

2.0

 

2.0

 

1.56 µg/mL

 

 

 

 

 

 

0.78 µg/mL

 

 

 

 

 

 

 

 

 

 

 

 

 

Positive controla,b

80.0***

60.0***

80.0***

70.0***

 

 

200 cells with well spread metaphase figures were scored, except for positive controls where 50 cells were scored.

-  no positive control

aCyclophosphamide, 20 µg/mL             bMitomycin-C, 0.2 µg/mL

* p ≥ 0.05; *** p ≤ 0.001 (Chi-square test)

Applicant's summary and conclusion

Conclusions:
negative with and without metabolic activation
Executive summary:

The clastogenicity of the test substance was tested under GLP to OECD TG 473 in an in vitro study. Chinese hamster ovary cells (ATCC CCL61) were incubated with several concentrations of cloquintocet-mexyl. Mitomycin C (0.2 µg/mL, without metabolic activation), and cyclophosphamide (20.0 µg/mL, with metabolic activation), were used as positive controls. Experiments without metabolic activation were conducted at 0, 6.25, 12.50 and 25.00 µg/mL in the main study, experiments with metabolic activation were conducted at 0, 12.50, 25.00 and 50.00 µg/mL in the main study.

In all experiments performed with or without metabolic activation, no statistically significant increase in the number of metaphases containing specific chromosomal aberrations was observed.

Cloquintocet-mexyl caused no clastogenic effects in Chinese hamster ovary cells in vitro.