Tributyl phosphate

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1990

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1990

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Principles of method if other than guideline:

Tributyl phosphate was tested in the chromosome aberration assay using Chinese hamster ovary cells. The assay was conducted both in the absence and presence of an aroclor-induced S-9 activation system at dose levels of 0.013, 0.025, 0.05, 0.1 and 0.15 µl/ml for the non-activated study and 0.01, 0.019, 0.038, 0.075 and 0.15 µl/ml for the S-9 activated study. Metaphase cells were collected at 12 hours after treatment for microscopic evaluation.

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Test material

Test material information

Constituent 1

Reference substance name:

Tributyl phosphate

EC Number:

204-800-2

EC Name:

Tributyl phosphate

Cas Number:

126-73-8

Molecular formula:

C12H27O4P

IUPAC Name:

tributyl phosphate

Details on test material:

purity: 99% or greater

Method

Species / strain

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

CHO-K1

Cytokinesis block (if used):

yes, colcemid 10µg/mL

Metabolic activation:

with and without

Metabolic activation system:

S-9 mix
9000 x g supernatant of an Aroclor 1254 induced male Sprague Dawley rat liver homogenate

Test concentrations with justification for top dose:

Test concentrations were selected on the basis of a toxicity test. Metaphase preparations were prepared and stained for sister chromatid differentiation using a modified fluorescence plus Giemsa technique (Perry and Wolff, 1974). Slides were evaluated for the percentage of first, second and third-division metaphase cells for estimation of the test article effect on cell cydle kinetics. The mitotic index was determined for each treatment condition as the percentage of mitogic cells in a population of 500 cells scored. CHO cells were exposed to solvent alone and to nine concentrations of test article ranging from 0.0005 µL/mL to 5 µl/mL in the absence and presence of S9 mix for 6 hours

Based on the findings of the toxicity test, dose levels of
0.013, 0.025, 0.05, 0.1 and 0.15 µl/ml for the non-activated study and
0.01, 0.019, 0.038, 0.075 and 0.15 µl/ml for the S-9 activated study
were selected for the main experiments.
Due to a slight delay observed in cell cycle kinetics, the harvest time was set at 12 hours.

Vehicle / solvent:

DMSO

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

cyclophosphamide

other: Triethylenemelamine (TEM), 0.5 µg/mL

Evaluation criteria:

The toxic effects of treatment are based upon mitotic inhibition relative to the solvent-treated control and are presented for the toxicity and aberration study. The number and types of aberrations found are presented for each treatment group. The percentage of structurally damaged cells (percent aberrant cells) in the total population of cells examined was calculated for each group. The frequency of structural aberrations per cell (mean aberrations per cell) was also calculated and reported for each group. Chromatid and iso-chromatid gaps are presented in the data cut are not included in the total percentage of cells with one or more aberrations or in the frequency of structural aberrations per cell.
All conclusions were based on sound scientific basis; however, as a guide to interpretation of the data, the test article was considered to induce a positive response when the percentages of cells with aberrations are increased in a dose responsive
manner with one or more concentrations being statistically elevated relative to the solvent control group (p<0.05). A significant increase at the high dose only with no dose response was considered suspect. A significant increase at one dose level other than the high dose with no dose response was considered equivocal.

Statistics:

Statistical analysis of the percent aberrant cells was performed using the Fisher's exact test. Fisher's test was used to compare pairwise the percent aberrant cells of each treatment group with that of the solvent control. In the event of a positive Fisher's test at any test article dose level, the Cochran-Armitage test was used to measure dose-responsiveness.

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

Any other information on results incl. tables

The activity of Tributyl Phosphate in the induction of chromosome aberrations in CHO cells when treated in the absence of an exogenous source of metabolic activation is summarized by group in Table 5 (see Attachment). The test article was soluble in solvent and treatment medium at all concentrations tested. At the time of harvest, microscopic examination of the cell monolayer indicated slight toxicity at dose level 0.05 ul/ml and moderate to extreme toxicity at dose levels 0.1 and 0.15 ul/ml. No metaphase cells were located tor
analysis at test concentration 0.15 ul/ml. The mitotic index was markedly reduced relative to the solvent control at dose level 0.1 ul/ml. Due to excessive toxicity at test concentration 0.1 ul/ml, only 54 metaphase cells were located tor the analysis of chromosome aberrations. The percentage of cells with structural aberrations in the test article-treated groups was not significantly increased above that of the solvent control (p>0.05, Fisher’s exact test). The percentage of damaged cells in the TEM group was 28% (p<0.01, Fisher’s exact test).

The activity of Tributyl Phosphate in the induction of chromosome aberrations in CHO cells when treated in the presence of an S-9 reaction mixture is summarized by group in Table 5. The test article was soluble in solvent and treatment medium at all concentrations tested. At the time of harvest, dose levels 0.075 and 0.15 ul/ml were observed to be slightly toxic and toxic, respectively, based upon microscopic examination of the cell monolayer. No metaphase cells were located for analysis at test concentration 0.15 µL/mL. The percentage of cells with structural aberrations in the test article-treated groups was not statistically increased above that of the solvent control (p>0.05, Fisher's exact test). The percentage of damaged cells in the CP group was 13% (p<0.01, Fisher's exact test).

 

The four highest test concentrations with analyzable metaphase cells were evaluated for chromosome aberrations. No increase in chromosome aberrations was observed in either the non-activated or S-9 activated test system. Tributyl phosphate was concluded to be negative in the CHO cytogenetics assay.

Applicant's summary and conclusion

Executive summary:

Tributyl phosphate was tested in the chromosome aberration assay using Chinese hamster ovary cells. The assay was conducted both in the absence and presence of an Aroclor-induced S-9 activation system at dose levels of 0.013, 0.025, 0.05, 0.1 and 0.15 µl/ml for the non-activated study and 0.01, 0.019, 0.038, 0.075 and 0.15 µl/ml for the S-9 activated study. Metaphase cells were collected at 12 hours after treatment for microscopic evaluation.

Under the conditions of the assay described in this report, tributyl phosphate was concluded to be negative in the CHO cytogenetics assay.