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Genetic toxicity in vitro

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Reference
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:
28 February 2006 - 30 March 2006
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Test was conducted according to OECD Test Guideline No. 473, under GLP Standards, and QA.
Reason / purpose:
reference to same study
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes
Remarks:
Statement of Compliance
Type of assay:
in vitro mammalian chromosome aberration test
Target gene:
Not relevant
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):
- Type and identity of media: Complete medium (McCoy's 5A medium)
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254-induced rat liver S9
Test concentrations with justification for top dose:
- Preliminary Toxicity Test: 50, 350, 750, 1000, 1500, 2000, 2500, 3000, and 3905 ug/ml.
- Concurrent Toxicity Test: 15, 30.5, 61, 488, 1952.5 , 3905 ug/ml.
- Chromosome Aberration Assay: 30.5, 61, and 3905 ug/ml.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Dimethyl sulfoxide (DMSO)
- Justification for choice of solvent/vehicle: DMSO was the solvent of choice based on the solubility of the test article and compatibility with the target cells. The test article was soluble in DMSO at a concentration of approximately 500 mg/ml, the maximum concentration tested for solubility.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: Without S9 mix: Mitomycin C (direct-acting mutagen); with S9 mix: Cyclophosphamide (promutagen)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: with S9-mix: 4 hours; without S9-mix: 4 hours or 20 hours continuous exposure
- Fixation time (start of exposure up to fixation or harvest of cells): approx. 20 hours

SPINDLE INHIBITOR (cytogenetic assays): Colcemid, 0.1 μg/ml medium, last 2 hours of incubation
STAIN (for cytogenetic assays): Giemsa solution, 5 %

NUMBER OF REPLICATIONS: duplicate cultures for each treatment

NUMBER OF CELLS EVALUATED: 100 metaphase chromosome spreads from each culture

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index

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

OTHER: A preliminary toxicity assay was performed for the purpose of selecting dose levels for the chromosome aberration assay and consisted of an evaluation of test article effect on cell growth. The osmolality of the solvent and of the highest and lowest concentration of dosing solution in the treatment medium was measured. The pH of the highest concentration of dosing solution in the treatment medium was measured. The presence of test article precipitate was assessed using the unaided eye. Cell viability was determined. The cell counts and percent viability were used to determine cell growth inhibition relative to the solvent control.
Evaluation criteria:
The test article was considered to induce a positive response when the percentage of cells with aberrations is increased in a dose-responsive manner with one or more concentrations being statistically significant (p ≤ 0.05). However, values that are statistically significant but do not exceed the range of historic solvent controls may be judged as not biologically significant. Test articles not demonstrating a statistically significant increase in aberrations will be concluded to be negative.
Statistics:
Statistical analysis of the percent aberrant cells was performed using the Fisher's exact test. In the event of a positive Fisher's exact test at any test article dose level, the Cochran-Armitage test was used to measure dose-responsiveness.
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
RANGE-FINDING/SCREENING STUDIES: In the preliminary toxicity assay, the maximum dose tested was 3905 µg/ml (10 mM). The test article was soluble in DMSO at all 9 dose levels (50, 350, 750, 1000, 1500, 2000, 2500, 3000, and 3905 ug/ml) tested. Visible precipitate was observed in treatment medium at all dose levels tested at the beginning of the treatment period. At the conclusion of the treatment period, visible precipitate was observed in treatment medium at dose levels ≥ 350 µg/ml, and dose level 50 µg/ml was soluble in treatment medium.

COMPARISON WITH HISTORICAL CONTROL DATA: The number of cells with chromosome aberrations found in the solvent control cultures was within the laboratory historical control data range. The number of polyploid cells and cells with endoreduplicated chromosomes in the solvent control cultures was within the laboratory historical control data range. The percentage of cells with numerical aberrations observed in the non-activated 20-hour exposure group (4.0%) was within the historical solvent control range of 0.0% to 7.5%.

ADDITIONAL INFORMATION ON CYTOTOXICITY: Selection of dose levels for the chromosome aberration assay was based upon a dose-responsive increase in cell growth (cell growth inhibition) relative to the solvent control. Substantial toxicity (i.e., at least 50% cell growth inhibition, relative to the solvent control) was not observed at any dose level in all 3 exposure groups. Based on these findings, the doses chosen for the chromosome aberration assay ranged from 15 to 3905 µg/ml for all 3 exposure groups.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Conclusions:
Interpretation of results:
negative with metabolic activation
negative without metabolic activation

The percentage of cells with structural or numerical aberrations in the non-activated and the S9-activated 4-hour exposure groups was not significantly increased above that of the solvent control at any dose level. The percentage of cells with structural aberrations in the non-activated 20-hour exposure group was not significantly increased above that of the solvent control at any dose level. The percentage of cells with numerical aberrations in the test article-treated group was significantly increased above that of the solvent control at 3905 µg/ml (p ≤ 0.05, Fisher's exact test). The Cochran-Armitage test was negative for a dose response (p>0.05). However, the percentage of cells with numerical aberrations in the test article-treated group (4.0%) was within the historical solvent control range of 0.0% to 7.5%. Therefore, it is not considered to be biologically significant. Based on the findings of this study, Isodecyl diphenyl phosphate was concluded to be negative for the induction of structural and numerical chromosome aberrations in CHO cells in both the non-activated and the S9-activated test systems.
Executive summary:
The test article, Isodecyl diphenyl phosphate, was tested in the chromosome aberration assay using Chinese hamster ovary (CHO) cells in both the absence and presence of an Aroclor-induced S9 activation system. The test was performed according to OECD Guideline 473. A preliminary toxicity test was performed to establish the dose range for the chromosome aberration assay. The chromosome aberration assay was used to evaluate the clastogenic potential of the test article. DMSO was the solvent of choice based on the solubility of the test article and compatibility with the target cells. The test article was soluble in DMSO at a concentration of approximately 500 mg/ml, the maximum concentration tested for solubility. In the preliminary toxicity assay, the maximum dose tested was 3905 µg/ml (10 mM). The test article was soluble in DMSO at all 9 dose levels (50, 350, 750, 1000, 1500, 2000, 2500, 3000, and 3905 ug/ml) tested. Visible precipitate was observed in treatment medium at all dose levels tested at the beginning of the treatment period. At the conclusion of the treatment period, visible precipitate was observed in treatment medium at dose levels ≥ 350 µg/ml, and dose level 50 µg/ml was soluble in treatment medium. Selection of dose levels for the chromosome aberration assay was based upon a dose-responsive increase in cell growth (cell growth inhibition) relative to the solvent control. Substantial toxicity (i.e., at least 50% cell growth inhibition, relative to the solvent control) was not observed at any dose level in all 3 exposure groups. Based on these findings, the doses chosen for the chromosome aberration assay ranged from 15 to 3905 µg/ml for all 3 exposure groups. In the chromosome aberration assay, the cells were treated for 4 and 20 hours in the non-activated test system, and for 4 hours in the S9-activated test system. All cells were harvested 20 hours after treatment initiation. The test article was soluble in DMSO at all dose levels tested. Visible precipitate was observed in treatment medium at dose levels ≥ 61 µg/ml, and dose levels ≤ 30.5 µg/ml were soluble in treatment medium at the beginning and conclusion of the treatment period. The top dose (3905 ug/ml) selected for microscopic analysis was based on dose dependent increase in toxicity observed consistently in the preliminary toxicity and the definitive assays. The mid-dose (61 ug/ml) was selected based on the lowest precipitating dose. The low dose (30.5 ug/ml) was the highest soluble dose. The percentage of cells with structural or numerical aberrations in the non-activated and the S9-activated 4-hour exposure groups was not significantly increased above that of the solvent control at any dose level. The percentage of cells with structural aberrations in the non-activated 20-hour exposure group was not significantly increased above that of the solvent control at any dose level. The percentage of cells with numerical aberrations in the test article-treated group was significantly increased above that of the solvent control at 3905 µg/ml (p ≤ 0.05, Fisher's exact test). The Cochran-Armitage test was negative for a dose response (p>0.05). However, the percentage of cells with numerical aberrations in the test article-treated group (4.0%) was within the historical solvent control range of 0.0% to 7.5%. Therefore, it is not considered to be biologically significant. Based on the findings of this study, Isodecyl diphenyl phosphate was concluded to be negative for the induction of structural and numerical chromosome aberrations in CHO cells in both the non-activated and the S9-activated test systems.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

For this endpoint 3 in vitro tests, performed similar to OECD test guidelines, were selected as key studies:

- One Bacterial Reverse Mutation Assays (Ames test).The concentrations used were 0.01, 0.1, 1.0, 5.0, and 10 ul/plate. The results of the tests conducted on IDDPP in the absence and in the presence of a metabolic activation system were all negative. IDDPP did not demonstrate genetic activity in any of the assays conducted in this evaluation and was considered not mutagenic under these test conditions.

- In the in vitro mammalian Chromosome Aberration test, CHO cells were treated at 30.5, 61, and 3905 ug/ml for 4 and 20 hours in the non-activated test system, and for 4 hours in the S9-activated test system. All cells were harvested 20 hours after treatment initiation. The percentage of cells with structural or numerical aberrations in all exposure groups was not (biologically) significantly increased above that of the solvent control at any dose level. Based on these results IDDPP was concluded to be negative for the induction of structural and numerical chromosome aberrations in CHO cells in both the non-activated and the S9-activated test systems.

- In the in vitro Mammalian Cell Gene Mutation Test cells derived from the mouse lymphoma cell line L5178Y were grown in culture media containing IDDPP at concentrations of 0.004, 0.008, 0.016, 0.031, and 0.063 ul/ml, with and without microsomal activation. IDDPP did not induce an increase in mutations at the TK locus in these cells at all concentrations tested with and without microsomal activation. As no evidence of mutagenic activity was obtained in this study, IDDPP was not mutagenic for mouse lymphoma L51784, TK+/- cells under the conditions of this evaluation.

One Bacterial Reverse Mutation Assays (Ames test) was considered a supporting study. Dose levels used in the mutation assays were: 10000, 3333, 1000, 333 and 100 ug/plate. No substantial increases in revertant colony numbers of any of the test strains were observed following treatment with IDDPP at any dose level, in the presence or absence of S-9 mix, in this mutation test. It is concluded that IDDPP shows no evidence of mutagenic activity in this bacterial system.


Short description of key information:
- Gene mutation in bacteria (Bacterial Reverse Mutation Assay/Ames) (similar to OECD 471): not mutagenic.
- In vitro Mammalian Chromosome Aberration test (similar to OECD 473): negative.
- In vitro Mammalian Cell Gene Mutation Test (similar to OECD 476): not mutagenic.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

In all genetic toxicity in vitro studies (3 key and 1 supporting) IDDPP did not show any genotoxic potential. Therefore, it can be concluded that the substance is not mutagenic, and does not need to be classified for mutagenicity according to the criteria outlined in Annex I of 1272/2008/EC and Annex VI of 67/548/EEC