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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:
1990-08-30 to 1990-12-03
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: GLP guideline study, read-across

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1990
Report Date:
1991

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
Details on test material:
- Name of test material (as cited in study report): Phthalic acid, di-n-octyl, n-decyl ester
- Substance type: product
- Physical state: liquid
- Storage condition of test material: stored in the dark at room temperature
Other: Chemical names: di-n-C8-C10-alkylphthalate; octyl-decylphthalate

Method

Species / strain
Species / strain:
lymphocytes: obtained from human blood
Details on mammalian cell lines (if applicable):
- Type and identity of media: RPMI 1640 tissue culture medium
- Properly maintained: not applicable
- Periodically checked for Mycoplasma contamination: not applicable
- Periodically checked for karyotype stability: not applicable
- Periodically "cleansed" against high spontaneous background: not applicable
Additional strain characteristics:
other: stable karyotype with 46 chromosomes, stimulated to cell division in vitro by addition of the mitogen PHA (phytohaemogglutinin)
Metabolic activation:
with and without
Metabolic activation system:
rat liver S9 of Arochlor 1254 induced Sprague-Dawley rats
Test concentrations with justification for top dose:
Range finding: 0.4, 0.8, 1.6, 3.2, 6.3, 12.5, 25, 50, 100, 200 µg/mL
Dose levels selected for metaphase analysis in the main tests: 25, 100 and 200 µg/mL
Vehicle:
- Vehicle(s)/solvent(s) used: ethanol
- Justification for choice of solvent/vehicle: The test substance was miscible with ethanol at 500 mg/mL, all final concentrations greater than 200 µg/mL were unmiscible with the tissue culture medium. 200 µg/mL was, therefore, regarded as the maxium achievable concentration.
Controlsopen allclose all
Negative controls:
no
Solvent controls:
yes
Remarks:
ethanol, 1% in tissue culture medium
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
without metabolic activation

Migrated to IUCLID6: 750 µg/mL; dissolved in dimethylsulphoxide
Negative controls:
no
Solvent controls:
yes
Remarks:
ethanol, 1% in tissue culture medium
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
with metabolic activation

Migrated to IUCLID6: 15 µg/mL, dissolved in sterile distilled water
Details on test system and conditions:
METHOD OF APPLICATION: in medium
Cells were suspended at a concentation of 1x10E6 cells/mL in culture medium with addition of 20 % foetal bovine serum and 5 mL aliquots were incubated in multiwell plates at 37°C with 5 % CO2 for approx. 48 hours. The cells were treated with different test substance concentrations or positive controls and for tests with metabolic activation system the rat liver S9 mix were added. Three hours after dosing the cultures containing metabolic activation were resuspended with fresh medium and incubated for further 21 hours. 22 hours after treatment with test item mitotic activity was arrested by addition of colchicine (final concentration 0.25 µL/mL). After 2 hours cells were removed from the culture dishes, incubated under hypotonic conditions for 10 minutes. The cell suspensions were centrifuged and the cell pellets fixed by addition of fresh methanol/glacial (3:1 v/v). Prior to slide preparation the pellets were aspirated through a hypodermic needle, then centrifugated and finally resuspended in a small volume of fixative. The cells suspensions were dropped onto each of five slides where they allowed to air-dry and stained afterwards with Giemsa solution, dried and mounted in DPX. The prepared slights were examined by light microscopy.


DURATION
- Preincubation period: not applicable
- Exposure duration: 3 h in the presence of S9 mix; 24 h in the absence of S9 mix
- Expression time (cells in growth medium): not applicable
- Selection time (if incubation with a selection agent): not applicable
- Fixation time (start of exposure up to fixation or harvest of cells): 24 h


SELECTION AGENT (mutation assays): not applicable
SPINDLE INHIBITOR (cytogenetic assays): Colchicine (final concentration 0.25 µL/mL)
STAIN (for cytogenetic assays): Giemsa solution


NUMBER OF REPLICATIONS: two cultures per dose level, four replicates for control


NUMBER OF CELLS EVALUATED: 200 metaphases per dose level (with normal a maximum of 25 from each slide)


DETERMINATION OF CYTOTOXICITY
- Method: The proportion of metaphase figures were examined. From these results the dose level causing a decrease in mitotic index of 50 - 80 % of the solvent control value or, if there was no decrease, the maximum achievable (or advisable) concentration was used as the highest dose level for the metaphase analysis. The intermediate and low doses were 50 % and 12.5 % of the highest concentration.


OTHER EXAMINATIONS:
- Determination of polyploidy: no
- Determination of endoreplication: no
- Other: Cells were examined for the presence of the following aberrations:
Gaps (chromatid and isochromatid), breaks (chromatid and isochromatid), interchanges, dicentric chromosomes, acentric chromosomal fragments, chromosome rings, complete metaphase pulverisation


OTHER: no repeat experiment performed
Evaluation criteria:
not specified
Statistics:
Statistical analysis used was Fisher's test.

Results and discussion

Test resultsopen allclose all
Species / strain:
lymphocytes: human peripheral blood lymphocytes
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity:
no, but tested up to precipitating concentrations
Vehicle controls valid:
yes
Negative controls valid:
not examined
Positive controls valid:
yes
Species / strain:
lymphocytes: human peripheral blood lymphocytes
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity:
no, but tested up to precipitating concentrations
Vehicle controls valid:
yes
Negative controls valid:
not examined
Positive controls valid:
yes
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no data
- Effects of osmolality: no data
- Evaporation from medium: not applicable
- Water solubility: no data
- Precipitation: All final concentrations greater than 200 µg/mL were immiscible with the tissue culture medium
- Other confounding effects: none


RANGE-FINDING/SCREENING STUDIES: No cytotoxicity at concentrations up to 200 µg/ml in the presence and absence of metabolic activation.


COMPARISON WITH HISTORICAL CONTROL DATA: no data


ADDITIONAL INFORMATION ON CYTOTOXICITY: none

Any other information on results incl. tables

Table #1: Summary of data obtained in chromosomal aberration test              

    Treatment                Aberrant Cell Frequency   
  Conc. [µg/mL]   S9 Mix Mitotic index[%] No. of CellsScored    Including Gaps          Excluding Gaps
           %  Judge  %  Judge
Vehicle Control  1% Ethanol  +  8.9 400   0.0  -  0.0  -
Test substance  25 +  7.3 200  0.0 neg.  0.0  neg.
Test substance   100 +  8.0 200   0.5 neg.  0.0  neg.
Test substance 200 + 9.4 200   0.0  neg.  0 .0  neg.
 Cyclophosphamide 15 +  n.d. 151  15.9 pos. ***  15.9  pos.***
 Vehicle Control 1% Ethanol  -  8.5 400   1.5  -  1.5  -
Test substance  25 - 6.3 200   2.0  neg.  2.0  neg.
Test substance  100 8.1 200  1.0  neg.  1.0  neg.
Test substance  200 -  6.0 200   1.5  neg.  1.5  neg.
Ethylmethanesulphonate 750  -  n.d. 200   12.5   pos. ***  12.5  pos.***

neg. = negative aberration frequency pos.*** = positive aberration frequency, statistically significant (p<0.001) n.d. = not determined

neg. = negative aberration frequency pos.*** = positive aberration frequency, statistically significant (p<0.001)

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative
Executive summary:

It was concluded that the structurally related substance 1,2-Benzenedicarboxylic acid, di-C8-10-alkyl esters had shown no evidence of clastogenic activity in this in-vitro cytogenetic test system.