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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:
experimental study
Adequacy of study:
key study
Study period:
from January 7, 2008 to April 18, 2008
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: This study was carried out in accordance with internationally valid GLP principles.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2008
Report Date:
2008

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
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): Humic acids, potassium salts
- Molecular formula: not known - UVCB substance
- Molecular weight: not known - UVCB substance
- Substance type: technical product
- Physical state: solid
- Lot/batch No.: 16. 5. 2007/R
- Expiration date of the lot/batch: 05/2022
- Stability under test conditions: stable
- Storage condition of test material: dry conditions

Method

Species / strain
Species / strain / cell type:
lymphocytes:
Metabolic activation:
with and without
Metabolic activation system:
The post-mitochondrial fraction (S9) from rat liver and a mixture of cofactors
Test concentrations with justification for top dose:
Priliminary test: 0.5, 1.0, 2.0, 2.5, 3.0 and 5.0 mg/mL of final culture
Main test: 0.5, 1.0 and 2.5 mg/mL of final culture
Vehicle / solvent:
- Vehicle used: sterile water
- Justification for choice of solvent/vehicle: Solubility in water
Controlsopen allclose all
Untreated negative controls:
yes
Remarks:
water
Negative solvent / vehicle controls:
yes
Remarks:
water
True negative controls:
yes
Remarks:
water
Positive controls:
yes
Positive control substance:
cyclophosphamide
Untreated negative controls:
yes
Remarks:
water
Negative solvent / vehicle controls:
yes
Remarks:
water
True negative controls:
yes
Remarks:
water
Positive controls:
yes
Positive control substance:
other: Thiotepa (Triethylenethiophosphoramide)
Details on test system and experimental conditions:
Test System:
Cells: Human peripheral blood lymphocytes (25 mL whole blood treated with heparin)
Donor: Healthy adult female volunteer
Instructions for storage: 4-8ºC
Lymphocyte Mitogen: Phytohaemagglutinin
Normal Cell Cycle Time: 16-20 hours
Modal Chromosome Number: 46 chromosomes
Evaluation criteria:
Evaluation of Results
The result is considered positive if the test substance increases the average % frequency of aberrant cells to more than twice that of the negative control value (solvent=water). The values of the historical negative controls are among 0-5% of aberrant cells. The criteria for positive results must also include the two-fofld rule (increase in the number of chromosomal aberration) , the dose-response relationship and reproducibility of the results in repeated test.
Positive results in the vitro cytogenetic assay indicate that under the conditions used the test substance induces chromosomal aberrations in cultured mammalian somatic cells. Negative results indicate that the substance does not induce significant number of chromosomal aberrations in mammalian somatic cells.

Results and discussion

Test results
Species / strain:
lymphocytes: Human peripheral blood lymphycytes
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Remarks:
doses above 3 mg/mL highly toxic
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:

COMPARISON WITH HISTORICAL CONTROL DATA:
The data of positive controls and negative control (water) are in agreement with historical control data.

Remarks on result:
other: strain/cell type:
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Preliminary test

Exposition: 4 hours

A-    mg/mL

% AB.B.

A+  mg/mL

% AB.B.

5.0

*

5.0

*

3.0

*

3.0

*

2.5

3.0

2.5

1.0

2.0

1.0

2.0

2.0

1.0

3.0

1.0

2.0

0.5

4.0

0.5

4.0

Ko

1.0

Ko A

3.0

Ko

4.0

Ko A

2.0

TT

12.0

CP A

11.0

TT

8.0

CP A

12.0

Main test

1st Test (4 hours)

2nd Test (26 hours)

A-    mg/mL

% AB.B.

A+  mg/mL

% AB.B.

A-    mg/mL

% AB.B.

2.5

2.0

2.5

2.0

2.5

0.5

1.0

1.0

1.0

2.0

1.0

2.5

0.5

2.5

0.5

1.0

0.5

0.5

Ko

1.0

Ko A

2.0

Ko

1.0

Ko

1.0

Ko A

2.0

Ko

3.0

TT

15.0

CP A

8.0

TT

14.0

TT

15.0

CP A

10.0

TT

11.0

% AB.B. Frequency of aberrant cells

A- Without activation

A+            With activation

*              High toxicity

Ko            Negative control

Ko A         Negative control with activation

TT           Thiotepa

CP A        Cyclophosphamide

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative with metabolic activation
negative without metabolic activation

The potential clastogenicity of the test substance was tested by In Vitro Mammalian Chromosome Aberration Test. The test was carried out in human peripheral blood lymphocytes with and without metabolic activation system in two separate assays using three concentration levels of 0.5, 1.0 and 2.5 mg/mL of culture.
The negative results obtained indicate that, under the test conditions, the test substance does not induce structural chromosome aberrations in cultured mammalian somatic cells.
Executive summary:

The clastogenicity potential of the test substance was determined using In Vitro Mammalian Chromosome Aberration Test.

The study was performed acocording to the OECD Guideline for the Testing of Chemicals No. 473, 1977.

The test was carried out in human peripheral blood lymphocytes with and without metabolic activation system in two separate assays.

A range of six concentrations of test substance in water solution was used in the preliminary testing with and without metabolic activation: 0.5, 1, 2, 2.5, 3 and 5 mg/mL of culture. The doses 2.0, 2.5 mg/mL partially inhibited cell activation, doses above 3 mg/mL of culture were evaluated as highly toxic.

Three concentrations - 0.5, 1.0 and 2.5 mg/mL of test substance were used in the main test. At predetermined intervals (4 and 26 hours) after exposure of cell cultures to the test substance, cells were arrested at metaphase with colchicine, harvested and slides stained. Metaphase cells were analysed microscopically for the presence of chromosome aberrations. A total of 200 well-spread metaphases were examined per concentration on coded slides. Concurrent positive (Cyclophosphamide, Thiotepa) and negative (water) controls were included in each experiment.

Under the test conditions the test substance does not induce increase of structural chromosome aberrations in cultured human peripheral blood lymphocytes.