Tributyl phosphate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In all relevant in vitro genetic toxicity assays (Ames test, cytogenetic test, CHO/HGPRT test) and in the in-vivo micronucleus test tributyl phosphate showed negative results.

Link to relevant study records

Reference

Reference 1

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

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

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

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.

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

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.

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.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1990

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: scientific acceptable and well documented

Principles of method if other than guideline:

Tributyl phosphate was tested in the CHO/HGPRT mutation assay in the absence and presence of metabolic activation with Aroclor-induced rat liver S-9. The assay was conducted at dose levels of 0.11, 0.09, 0.08, 0.07 and 0.05 µl/ml in the non-activated study and at 0.15, 0.125, 0.10, 0.08 and 0.06 µl/ml in the presence of S-9.

GLP compliance:

yes

Type of assay:

mammalian cell gene mutation assay

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

CHO-K1-BH4

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S-9 mix

Test concentrations with justification for top dose:

0.11, 0.09, 0.08, 0.07 and 0.05 µl/ml in the non-activated study and at
0.15, 0.125, 0.10, 0.08 and 0.06 µl/ml in the presence of S-9.

Vehicle / solvent:

DMSO

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

ethylmethanesulphonate

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration (single, duplicate, triplicate): triplicate

For information on test system and experimental conditions see report excerpt attached.

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

greater 0.15 µl/ml

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Under the conditions of these mutagenicity tests, test article tributyl phosphate was negative in both the absence and presence of exogenous metabolic activation.

For information on results see report excerpt attached.

Executive summary:

Tributyl phosphate was tested in the CHO/HGPRT mutation assay in the absence and presence of metabolic activation with Aroclor-induced rat liver S-9. The assay was conducted at dose levels of 0.11, 0.09, 0.08, 0.07 and 0.05 µl/ml in the non-activated study and at 0.15, 0.125, 0.10, 0.08 and 0.06 µl/ml in the presence of S-9 mix.

Under the conditions of this mutagenicity test, tributyl phosphate was negative in both the absence and presence of exogenous metabolic activation.

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

other: only 2 S.typimurium strains (TA 102 and TA 2638) were used

Principles of method if other than guideline:

compounds were tested for mutagenicity using the plate incorporation method with and without meatabolic activation, essentially as described by Maron and Ames (Maron DM and Ames BM (1983) Revised methods for the Salmonella mutagenicity test, Mutation Res, 113, 173-215)

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Target gene:

no data

Species / strain / cell type:

E. coli WP2 uvr A pKM 101

Species / strain / cell type:

other: S. typhimurium TA2638

Species / strain / cell type:

S. typhimurium TA 102

Metabolic activation:

with and without

Metabolic activation system:

S9 mix
For metabolic activation an S9 mix was used which contained 10% of S9 fraction (purchased from Kikkoman Co., Chiba, Japan and Oriental Yeast Co., Tokyo, Japan, by 7 and 3 laboratories, respectively), which was prepared from livers of Sprague-Dawley rats induced by phenobarbital and 5,6-benzoflavone.

Test concentrations with justification for top dose:

up to 5000 µg/plate

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

not specified

True negative controls:

not specified

Positive controls:

yes

Remarks:

Mitomycin C, 2-(2-Furyl)-3-(5-nitro-2-furyl)acrylamide, 2-Aminoanthracene

Positive control substance:

other: Mitomycin C, 2-(2-furyl)-3-(5-nitro-2-furyl)acrylamide, 2-Aminoanthracene

Details on test system and experimental conditions:

Positive controls were included in each experiment. Mitomycin C (MMC) was tested in the S. typhimurium strains without metabolic activation at a dose level of 0.05 µg/plate (TA102) or 0.1 µg/plate (TA2638). 2-(2-Furyl)-3-(5-nitro-2-furyl) acrylamide was tested in the E. coli strains without metabolic activation at a dose level of 0.1 µg/plate (WP2/pKM101) or 0.01 µg/ plate (WP2 uvrA/pKMlO1). 2-Aminoanthracene was used at a dose level of 5 ixg/plate (TA102) or 10 µg/plate (TA2638 and WP2/pKM101) or 1 µg/plate (WP2 uvrA/pKMlO1) with metabolic activation.

Statistics:

The results were analyzed for statistical significance using a linear regression test. This statistical analysis recommended by UKEMS [9] was conducted based on the dose-response relationship and carried out at the 1% significance level. Doses with observed cytotoxi- city, which was judged by a toxicity to the back- ground lawn and/or a reduction in the number of revertant colonies, were excluded from the statistical analysis.

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

not specified

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

valid

tributyl phospahte was negative in TA 102, TA 2638, and in E.coli WP2/pKM101, WP2 uvrA/pKM101

Executive summary:

The compound was tested for mutagenicity using the plate incorporation method with and without metabolic activation.

Tributyl phosphate was negative in S. typhimurium TA 102 and TA 2638, as well as in E. coli WP2/pKM101, and WP2 uvrA/pKM101 with and without metabolic activation.

Reference 4

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1985

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Principles of method if other than guideline:

Ames Test (according to Ames et al., Proc. nat. Acad. Sci. 70, 1973a; Mutation Res. 31, 1975)

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Target gene:

histidine gene

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9 mix
obtained from the livers of male SD rats, induced with Aroclor 1254 (5 days before sacrifice)

Test concentrations with justification for top dose:

test concentrations in the first trial were 20 to 12500 µg/plate. The concentrations in the second trial were determined with15 to 480 µg/plate based on toxicity in the first experiment.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: Natriumazid (10 µg/plate) - for TA 1535 Nitrofurantoin (0.2 µg/plate) - for TA 100 4-Nitro-o-phenylene diamine - 10 µg/plate for TA 1537 4-Nitro-o-phenylene diamine - 0.5 µg/plate for TA 98 2-Aminoanthacene - 3 µg/plate for all strains with S9 mix

Details on test system and experimental conditions:

Ames test

Evaluation criteria:

a reproducable dose-dependent increase of mutant frequency (about twice the negative control) in at least one strain is considered a positive result

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at concentrations above 120 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

For detailed information see Tables 1-4 (experiment 1) and Tables 5-8 (experiment 2) attached

Genotoxicity: negative

Conclusions:

Interpretation of results: negative

Executive summary:

The test substance Tri-n-butylphosphate was investigated in the Salmonella/microsome test for point-mutagenic effects in doses up to 12500 µg per plate on four Salmonella typhimurium LT2 mutants. These were the histidine-auxotropic strains TA 1535, TA 100; TA 1537 and TA 98.

Doses up to and including 120 µg per plate did not cause any bacteriotoxic effects. The total bacteria counts remained unchanged. No inhibition of growth was observed. In doses above this the substance had a strong bacteriotoxic effect specific to strain, so that this range can only be used to a limited extent up to 500 µg per plate for evaluation purposes.

Evidence of mutagenic activity for the test substance was not found. Neither a dose-related doubling of the mutant count nor a biologically relevant increase in the same, in comparison with the negative controls, was observed.

The positive controls sodiumazide, nitrofurantoine, 4 -nitro-o-phenylenediamine and 2 -aminoanthracene had a marked mutagenic effect, as can be seen from the biologically relevant increase of mutant colonies, compared with the corresponding negative control.

The test substance was found to be negative for genotoxicity.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Link to relevant study records

Reference

Endpoint:

in vivo mammalian germ cell study: cytogenicity / chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1991

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: well documented and scientifically acceptable

Principles of method if other than guideline:

Male and female sprague-Dawley rats were treated by gavage with 300, 600, or 1200 mg Tributyl phosphate/kg body weight which was given as single administration. The high dose level was set at the maximum tolerated dose. Bone marrow cells, arrested in metaphase and collected 12, 24 and 36 hours after treatment, were examined microscopically for structural chromosome aberrations.

GLP compliance:

yes

Type of assay:

mammalian bone marrow chromosome aberration test

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan Sprague Dawley Inc., Frederick, MD
- Age at study initiation: 6-8 weeks
- Weight at study initiation: 197 to 241 g (males), 166 to 209 g (females)
- Housing: single housed
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: at least 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 74+-6 °F
- Humidity (%): 50 +-20%
- Air changes (per hr):
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: 1991

Route of administration:

oral: gavage

Vehicle:

corn oil; 10 mL/kg bw

Details on exposure:

For dose selection, tributyl phosphate was adminstered to male and female rats at 4 treatment levels and a vehicle control: 463, 833, 1500, and 2700 mg/kg bw. Mortality occurred within 2 days after dosing: 2/5 males and 4/5 females receiving 1500 mg/kg bw and 4/5 males and 5/5 males receiving 2700 mg/kg bw. A single dose of 1200 mg/kg bw was estimated to be the maximum tolerated dose.

all rats in the experimental and control groups were weighed immediately prior to dose administration and the dose volume was based in individual body weights. Colchicine, used to arrest dividing cells at metaphase, was administed i.p. at 2.5 mg/kg bw to all rats 2-4 hours prior to sacrifice.

Duration of treatment / exposure:

single administration

Frequency of treatment:

single administration

Post exposure period:

12, 24 or 36 hours

Dose / conc.:

300 mg/kg bw/day (actual dose received)

Dose / conc.:

600 mg/kg bw/day (actual dose received)

Dose / conc.:

1 200 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Positive control(s):

Cyclophosphamide at 20 mg/kg bw

Tissues and cell types examined:

bone marrow cells

Evaluation criteria:

Mitotic index and total number and types of aberrations found in each animal are presented. Gaps are counted but not included in the results.

Statistics:

Fischer's exact test

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Remarks:

1200 mg/kg was the maximum tolerated dose

Vehicle controls validity:

valid

Negative controls validity:

not specified

Positive controls validity:

valid

A reduction in the rate of body weight gain was observed in males and females at 1200 mg/kg bw and in males only at 600 mg/kg bw. One male and 4 females receiving 1200 mg/kg bw died during the study period and were replaced at the time of bone marrow collection with animals from a replacement group which had also been dosed with 1200 mg/kg bw. One male and 1 female died from this replacement group.

The % damaged cells in the total poöulation of cells scored and the number of aberrations per cell for each treatment group are presented for male and female rats in tables 3 and 4 attached. The percentage of damaged cells was not statistically increased in the test article treated animals, regardless of sex, dose or sacrifice time (p>0.05). Cyclophosphamide induced a significant increase in cells containing one or more aberrations (p<0.01). 

Executive summary:

Male and female sprague-Dawley rats were treated by gavage with 300, 600, or 1200 mg Tributyl phosphate/kg body weight which was given as single administration. The high dose level was set at the maximum tolerated dose established in the toxicity study. Mortality occurred in the high dose group. Bone marrow cells, arrested in metaphase and collected 12, 24 and 36 hours after treatment, were examined microscopically for structural chromosome aberrations.

No statistically significant increases in % aberrant cells were observed in the test item groups. Under the conditions of the assay described in this report, tributyl phosphate, was negative in the acute cytogenetics assay using male and female sprague-Dawley rats.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Tributyl phosphate was investigated in the Salmonella/microsome test for point-mutagenic effects in doses up to 12500 µg per plate on four Salmonella typhimurium LT2 mutants (TA 1535, TA 100; TA 1537 and TA 98). Doses of above 120 µg per plate did cause bacteriotoxic effects. Evidence of mutagenic activity for the test substance was not found. Neither a dose-related doubling of the mutant count nor a biologically relevant increase in the same, in comparison with the negative controls, was observed. The positive controls had a marked mutagenic effect, as can be seen from the biologically relevant increase of mutant colonies, compared with the corresponding negative control. The test substance was found to be negative in this Ames Test.
A further Ames test is available for S. typhimurium TA 102 and TA 2638, as well as E. coli strains WP2/pKM101, and WP2 uvrA/pKM101. Tributyl phosphate was negative in these strains with and without metabolic activation.

Tributyl phosphate was tested in the mammalian cell CHO/HGRT gene mutation assay in the absence and presence of metabolic activation with Aroclor-induced rat liver S-9 mix. The assay was conducted at dose levels of 0.11, 0.09, 0.08, 0.07 and 0.05 µl/ml in the non-activated study and at 0.15, 0.125, 0.10, 0.08 and 0.06 µl/ml in the presence of S-9 mix. Under the conditions of this mutagenicity test, tributyl phosphate was negative in both the absence and presence of exogenous metabolic activation.
Additionally, tributyl phosphate was tested in the chromosome aberration assay using the mammalian cell line Chinese hamster ovary. 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.

Also an in-vivo micronucleus assay no indication for a mutagenic effect was observed.
Male and female Sprague-Dawley rats were treated by gavage with 300, 600, or 1200 mg Tributyl phosphate/kg body weight which was given as single administration. The high dose level was set at the maximum tolerated dose established in the toxicity study. Mortality occurred in the high dose group. Bone marrow cells, arrested in metaphase and collected 12, 24 and 36 hours after treatment, were examined microscopically for structural chromosome aberrations. No statistically significant increases in % aberrant cells were observed in the test item groups. Under the conditions of the assay, tributyl phosphate was negative in the bone marrow micronucleus assay using male and female Sprague-Dawley rats.

Justification for classification or non-classification

Tributyl phosphate was shown to be negative in all relevant test systems (Ames Test, HPRT test, in vitro CAb test und in vivo bone marrow CAb test. Therefore a classification is no justified.