2,4,6-tri-n-propyl-2,4,6-trioxo-1,3,5,2,4,6-trioxatriphosphorinane

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1995-05-16 to 1995-05-26

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1995

Report date:

1995

Materials and methods

Test guideline

Test guideline 1

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

T

Deviations:

no

Test guideline 2

Qualifier:

equivalent or similar to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: PPA 4/94
- Expiration date of the lot/batch: September 1995

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: darkness, appr. 20 °C
- Stability under test conditions: stable

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: On the day of the experiment the test substance was dissolved in DMSO at appropriate concentrations.

Method

Target gene:

Salmonella typhimurium: Histidine operon
E. Coli: trpE gene, uvrA gene

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: Salmonella typhimurium strains were obtained from B. N. Ames, Biochemistry Department, University of California, Berkeley, California 94720, USA, in April 1987. Escherichia coli strain was obtained from M. H. L. Green, MRC. Cell mutation, University of Sussex, Falmer Brighton, BNI 90G (England), in April 1987.

MEDIA USED
- Type and identity of media: Bacteria were grown overnight in nutrient broth (25 g Oxoid Nutrient Broth No. 2 /liter). Inoculation was performed with stock cultures which had been stored at approx. -80 °C.

Metabolic activation:

with and without

Metabolic activation system:

S9-mix from rat liver, Aroclor 1254 induced

Test concentrations with justification for top dose:

Concentration range in the main test (with metabolic activation): 0, 4, 20, 100, 500, 2500, 5000 µg/plate
Concentration range in the main test (without metabolic activation): 0, 4, 20, 100, 500, 2500, 5000 µg/plate

Vehicle / solvent:

- Vehicle solvent used: DMSO

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

other: N-Methyl-N-nitro-N-nitrosoguanidine, (without metabolic activation, WP2uvrA); 2-Aminoanthracene (with metabolic activation, all strains)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 h

SELECTION AGENT (mutation assays): histidine (Salmonella strains) and tryptophan (E. coli)

NUMBER OF REPLICATIONS:2

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth

Rationale for test conditions:

The test compound did not precipitate on the plates up to the highest investigated dose of 5000 microgram/plate and cytotoxicity was observed at the two highest doses.

Evaluation criteria:

A test article is classified as mutagenic if it has either of the following effects:
a) a test article produces at least a 2-fold increase in the mean number of revertants per plate of at least one of the tester strains over the mean number of revertants per plate of the appropriate vehicle control at complete bacterial background lawn
b) a test article induces a dose-related increase in the mean number of revertants per plate of at least one of the tester strains over the mean number of revertants per plate of the appropriate vehicle control in at least two to three concentrations of the test article at complete bacterial background lawn.
The test results must be reproducible.

Statistics:

not applicable

Results and discussion

Test results

Key result

Species / strain:

other: all strains tested

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

in the experiment for cytotoxicity conducted with strain TA100 the number of revertants at the dose groups 2500 and 5000 µg/plate was reduced compared to the lower dose groups

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: The test compound did not precipitate on the plates up to the highest investigated dose of 5000 microgram/plate.



ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: number of revertants

Any other information on results incl. tables

For details on results, please refer to the attached document.

Applicant's summary and conclusion

Conclusions:

The test substance was not mutagenic in bacterial cells with and without metabolic activation in this assay.

Executive summary:

The test substance was tested for mutagenicity with the strains TA 100, TA 1535, TA 1537 and TA 98 of Salmonella typhimurium and Escherichia coli WP2uvrA.

The mutagenicity studies were conducted in the absence and in the presence of a metabolizing system derived from rat liver homogenate. The test substance was dissolved in DMSO and a dose range of 6 different doses from 4 microgram/plate to 5000 microgram/plate was used (0, 4, 20, 100, 500, 2500, 5000 µg/plate). An experiment for cytotoxicity conducted with strain TA100 revealed a decrease in the number of revertants at the two highest doses compared to the lower doses, indicating cytotoxicity. In the main test this was not confirmed.

Control plates without mutagen showed that the number of spontaneous revertant colonies was similiar to that described in the literature. All the positive control compounds gave the expected increase in the number of revertant colonies.

In the absence of the metabolic activation system the test compound did not show a dose dependent increase in the number of revertants in any of the bacterial strains. Also in the presence of a metabolic activation system, treatment of the cells with the test compound did not result in relevant increases in the number of revertant colonies.

Summarizing, it can be stated that the test compound is not mutagenic in these bacterial test systems either with or without exogenous metabolic activation at the dose levels investigated.