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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
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: comparable to guideline study with acceptable restrictions

Data source

Referenceopen allclose all

Reference Type:
publication
Title:
Chromosome aberration and sister chromoatid exchange tests in Chinese hamster ovary cells in vitro III: Results with 27 chemicals
Author:
Gulati DK, Witt K, Anderson B
Year:
1989
Bibliographic source:
Environ. Mol. Mutagen. 13, 133-193
Reference Type:
publication
Title:
Tests for the induction of chromosomal aberrations and sister chromatid exchanges in cultured Chinese hamster ovary (CHO) cells.
Author:
Gulati DK, Sabharwal PS, Shelby MD
Year:
1985
Bibliographic source:
Progr. Mutat. Res. 5, 413-427

Materials and methods

Test guideline
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
GLP compliance:
not specified
Type of assay:
in vitro mammalian chromosome aberration test

Test material

Constituent 1
Reference substance name:
Bis(2-ethylhexyl) phthalate
EC Number:
204-211-0
EC Name:
Bis(2-ethylhexyl) phthalate
Cas Number:
117-81-7
IUPAC Name:
bis(2-ethylhexyl) phthalate
Details on test material:
- Name of test material (as cited in study report): Di-(2-ethylhexyl)phthalate DEHP
- Source : Fluka
- Purity : >99%

Batch 1 Lancaster Synthesis. Ltd. - DMSO (Ppt at >500 µg/ml)
Batch 2 Matheson Coleman & Bell 96% DMSO (Ppt at >500 µg/ml)
Batch 3 W. R. Grace & Co. 99.6% DMSO (Ppt at >500 µg/ml)

Method

Target gene:
Chromosomal aberrations
Species / strain
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):
- Type and identity of media: McCoy's 5A medium (modified) supplemented with L-glutamine (2 mM), antibiotics, and 10% fetal bovine serum (FBS)
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: no
- Periodically "cleansed" against high spontaneous background: no
Metabolic activation:
with and without
Metabolic activation system:
liver fraction (S9) prepared from Aroclor 1254-induced male Sprague Dawley rats (Microbiological Associates, Bethesda, MD)
Test concentrations with justification for top dose:
0, 5, 16, 50, 600, 1600, 2000, 3000, 4000, 5000 µg/ml
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: mitomycin C (-S9) and cyclophosphamide (+S9)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium;

DURATION
- Preincubation period: 24h
- Exposure duration: 10-11h
- Harvest time: 12-13.5 h
For chemicals that caused cell cycle delay, harvest times were extended, generally in 5 h increments, with colcemid present for the last 2 h. For chromosomal aberrations tests, harvest times were similarly extended based on the observation of cell cycle delay in the sister chromatic exchange trials.

SPINDLE INHIBITOR (cytogenetic assays): Colcemid (2-3h prior harvest)
STAIN (for cytogenetic assays): Giemsa 6%

NUMBER OF CELLS EVALUATED: 100 cells were scored for each dose in early studies and 200 cells per dose in later studies

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index;

OTHER EXAMINATIONS:
- Determination of polyploidy: yes
- Determination of endoreplication: yes

Statistics:
The percentage of cells with aberrations was analyzed. Both the dose-response curve and individual dose points were statistically analyzed. A statistically significant (P < 0.003) trend test or a significantly elevated dose point (P < 0.05) was sufficient to indicate a chemical effect.

Results and discussion

Test results
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Downloaded from the NTP site:

http://ntp-apps.niehs.nih.gov/ntp_tox/index.cfm?fuseaction=invitroca.cadata&study_no=923560&cas_no=117%2D81%2D7&endpointlist=CAB

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In Vitro Cytogenetics - Chromosome Aberrations
Info (Collapse/expand this info box)
Current Search Criteria
Chemical Name: Di(2-ethylhexyl) phthalate
CAS Number: 117-81-7
Study Type: Chromosome Aberrations
Study ID: 923560
Table Instructions and Notes:
Below is a summary of Chromosome Aberrations for the selected Study ID. Solvent control marked in white.
Study Result: Negative
Activation Trial Trial Call
No Activation 1 Negative
No Activation 2 Negative
Induced Rat Liver S9 3 Negative
Induced Rat Liver S9 4 Negative
Trial #:1   Activation: No Activation   Date: 12/10/1982   Harvest Time: 13.5 hrs   Trial Call: Negative  
  Dose Total Cells Examined Total Aberrations Complex Aberrations Simple Aberrations Other Abs.
µg/mL No. of Abs % Cells No. of Abs % Cells No. of Abs % Cells No. of Abs % Cells
  Abs. Per With Abs. Per With Abs. Per With Abs. Per With
    Cell Abs.   Cell Abs.   Cell Abs.   Cell Abs.
Abs: Aberrations
Vehicle Control: Negative 0          100 0 0.000 0.000 0 0.000 0.000 0 0.000 0.000 0 0.000 0.000
Vehicle Control: Dimethylsulfoxide 0          100 0 0.000 0.000 0 0.000 0.000 0 0.000 0.000 0 0.000 0.000
Positive Control: Mitomycin C  0.5        100 99 0.990 63.000 26 0.260 25.000 43 0.430 32.000 30 0.300 28.000
Test Chemical: Di(2-ethylhexyl)phthalate (DEHP) 50          100 0 0.000 0.000 0 0.000 0.000 0 0.000 0.000 0 0.000 0.000
160          100 0 0.000 0.000 0 0.000 0.000 0 0.000 0.000 0 0.000 0.000
500          100 0 0.000 0.000 0 0.000 0.000 0 0.000 0.000 0 0.000 0.000
1600          100 0 0.000 0.000 0 0.000 0.000 0 0.000 0.000 0 0.000 0.000
5000          100 0 0.000 0.000 0 0.000 0.000 0 0.000 0.000 0 0.000 0.000
  Trend: 0.000 0.000 0.000  
Probability: 0.000 0.000 0.000  
Trial #:2   Activation: No Activation   Date: 12/21/1982   Harvest Time: 12.0 hrs   Trial Call: Negative  
  Dose Total Cells Examined Total Aberrations Complex Aberrations Simple Aberrations Other Abs.
µg/mL No. of Abs % Cells No. of Abs % Cells No. of Abs % Cells No. of Abs % Cells
  Abs. Per With Abs. Per With Abs. Per With Abs. Per With
    Cell Abs.   Cell Abs.   Cell Abs.   Cell Abs.
Abs: Aberrations
Vehicle Control: Negative 0          100 2 0.020 2.000 1 0.010 1.000 1 0.010 1.000 0 0.000 0.000
Vehicle Control: Dimethylsulfoxide 0          100 2 0.020 2.000 2 0.020 2.000 0 0.000 0.000 0 0.000 0.000
Positive Control: Mitomycin C  0.5        100 76 0.760 48.000 25 0.250 21.000 34 0.340 24.000 17 0.170 13.000
Test Chemical: Di(2-ethylhexyl)phthalate (DEHP) 2000          100 3 0.030 3.000 1 0.010 1.000 2 0.020 2.000 0 0.000 0.000
3000          100 1 0.010 1.000 0 0.000 0.000 1 0.010 1.000 0 0.000 0.000
4000          100 1 0.010 1.000 0 0.000 0.000 1 0.010 1.000 0 0.000 0.000
5000          100 1 0.010 1.000 0 0.000 0.000 1 0.010 1.000 0 0.000 0.000
  Trend: -1.064 -2.126 0.308  
Probability: 0.856 0.983 0.379  
Trial #:3   Activation: Induced Rat Liver S9   Date: 12/10/1982   Harvest Time: 12.0 hrs   Trial Call: Negative  
  Dose Total Cells Examined Total Aberrations Complex Aberrations Simple Aberrations Other Abs.
µg/mL No. of Abs % Cells No. of Abs % Cells No. of Abs % Cells No. of Abs % Cells
  Abs. Per With Abs. Per With Abs. Per With Abs. Per With
    Cell Abs.   Cell Abs.   Cell Abs.   Cell Abs.
Abs: Aberrations
Vehicle Control: Negative 0          100 0 0.000 0.000 0 0.000 0.000 0 0.000 0.000 0 0.000 0.000
Positive Control: Cyclophosphamide 50          100 74 0.740 51.000 16 0.160 15.000 40 0.400 32.000 18 0.180 17.000
Vehicle Control: Dimethylsulfoxide 0          100 0 0.000 0.000 0 0.000 0.000 0 0.000 0.000 0 0.000 0.000
Test Chemical: Di(2-ethylhexyl)phthalate (DEHP) 50          100 0 0.000 0.000 0 0.000 0.000 0 0.000 0.000 0 0.000 0.000
160          100 0 0.000 0.000 0 0.000 0.000 0 0.000 0.000 0 0.000 0.000
500          100 0 0.000 0.000 0 0.000 0.000 0 0.000 0.000 0 0.000 0.000
1600          100 0 0.000 0.000 0 0.000 0.000 0 0.000 0.000 0 0.000 0.000
5000          100 0 0.000 0.000 0 0.000 0.000 0 0.000 0.000 0 0.000 0.000
  Trend: 0.000 0.000 0.000  
Probability: 0.000 0.000 0.000  
Trial #:4   Activation: Induced Rat Liver S9   Date: 12/21/1982   Harvest Time: 12.0 hrs   Trial Call: Negative  
  Dose Total Cells Examined Total Aberrations Complex Aberrations Simple Aberrations Other Abs.
µg/mL No. of Abs % Cells No. of Abs % Cells No. of Abs % Cells No. of Abs % Cells
  Abs. Per With Abs. Per With Abs. Per With Abs. Per With
    Cell Abs.   Cell Abs.   Cell Abs.   Cell Abs.
Abs: Aberrations
Vehicle Control: Negative 0          100 1 0.010 1.000 1 0.010 1.000 0 0.000 0.000 0 0.000 0.000
Positive Control: Cyclophosphamide 50          100 74 0.740 52.000 16 0.160 14.000 27 0.270 24.000 31 0.310 21.000
Vehicle Control: Dimethylsulfoxide 0          100 3 0.030 3.000 2 0.020 2.000 1 0.010 1.000 0 0.000 0.000
Test Chemical: Di(2-ethylhexyl)phthalate (DEHP) 1000          100 4 0.040 4.000 4 0.040 4.000 0 0.000 0.000 0 0.000 0.000
2000          100 4 0.040 3.000 3 0.030 2.000 1 0.010 1.000 0 0.000 0.000
3000          100 2 0.020 2.000 1 0.010 1.000 1 0.010 1.000 0 0.000 0.000
4000          100 2 0.020 2.000 0 0.000 0.000 2 0.020 2.000 0 0.000 0.000
  Trend: -0.768 -1.634 0.909  
Probability: 0.779 0.949 0.182  

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

DEHP did not induce any noteworthy increase in the number of cells with structural chromosome aberration, both with and without S9 mix, in either experiment or at either harvest time. 
Executive summary:

The potential of DEHP to induce structural chromosome aberrations in Chinese hamster ovary cells was evaluated with and without a metabolic activation system according a protocol similar to the OECD 473.

Cells were treated for about 10-11 h and colcemid was added 2-3 hr prior to tell harvest by mitotic shake-off.

The chromosome number was recorded for each cell and chromosome or chromatid type aberrations were classified into three categories: simple (breaks, fragments, double minutes), complex (interchanges, rearrangements), and other (pulverized, more than ten aberrations/cell).

Under these experimental conditions, DEHP did not induce any noteworthy increase in the number of cells with structural chromosome aberration, both with and without S9 mix, in either experiment or at either harvest time.