Tris[2-[[2,4,8,10-tetra-tert-butyldibenzo[d,f][1,3,2]dioxaphosphepin-6-yl]oxy]ethyl]amine

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:

Jan 1994

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1994

Report date:

1994

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

26 May 1983

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Tris[2-[[2,4,8,10-tetra-tert-butyldibenzo[d,f][1,3,2]dioxaphosphepin-6-yl]oxy]ethyl]amine

EC Number:

279-459-6

EC Name:

Tris[2-[[2,4,8,10-tetra-tert-butyldibenzo[d,f][1,3,2]dioxaphosphepin-6-yl]oxy]ethyl]amine

Cas Number:

80410-33-9

Molecular formula:

C90H132NO9P3

IUPAC Name:

2-[(2,4,8,10-tetra-tert-butyldibenzo[d,f][1,3,2]dioxaphosphepin-6-yl)oxy]-N,N-bis{2-[(2,4,8,10-tetra-tert-butyldibenzo[d,f][1,3,2]dioxaphosphepin-6-yl)oxy]ethyl}ethanamine

Test material form:

solid

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The test substance was suspended in dimethylsulfoxide at room temperature. The highest concentration was 50 mg/ml. In the toxicity test the three highest concentrations were weighed out separately and the three lowest concentrations were prepared by appropriate dilution of the third highest concentration with dimethylsulfoxide. In the mutagenicty test all concentrations of the test substance were weighed out separately.

FORM AS APPLIED IN THE TEST (if different from that of starting material): suspended in dimethylsulfoxide (DMSO)

Method

Target gene:

his, trp

Species / strain

Species / strain 1

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

S9 mix (supplemented with cofactors) derived from Aroclor 1254 (i.p.) induced rat liver

Test concentrations with justification for top dose:

312.5; 625; 1250; 2500; 5000 µg/plate

3 plates per test substance concentration and controls

2 independent experiments were performed

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: dimethylsulfoxide (DMSO)
- Justification for choice of solvent/vehicle: Due to the insolubility of the test substance in all common solvents used for this test system, the solvent dimethylsulfoxide was chosen according to an internal SOP of the test facility.

Controls

Untreated negative controls:

other: = solvent control

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

2-nitrofluorene

sodium azide

cyclophosphamide

mitomycin C

other: 2-aminoanthracene (2.5 - 50 μg/plate, dissolved in DMSO; with S9 mix)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: about 48 h

DETERMINATION OF CYTOTOXICITY
- Toxicity detected by a
• decrease in the number of revertants
• clearing or diminution of the background lawn (= reduced his- or trp- background growth)
was recorded for all test groups both with and without S9 mix in all experiments.

Rationale for test conditions:

In a range finding test 6 concentrations of the test substance ranging from 20.6 to 5000 µg/plate were tested with strain Salmonella typhimurium TA100 and strain Escherichia coli WP2uvrA to determine the highest concentration to be used in the mutagenicity assay. The experiments were performed with and without metabolic activation. Normal background growth was observed with both strains. The numbers of revertant colonies were not reduced. From the results obtained, the highest concentration suitable for the mutagenicity test was selected to be 5000 µg/plate with and without metabolic activation.

Evaluation criteria:

Acceptance criteria:
A test is considered acceptable if the mean colony counts of the negative control values of all strains are within the acceptable ranges and if the results of the positive controls meet the criteria for a positive response.

Criteria for a positve response:
The test substance was considered to be positive in the test system if one or both of the following conditions are met:
• At least a reproducible doubling of the mean number of revertants per plate above that of the negative control at any concentration for one or more of the following strains: TA 98, TA 1535, TA 1537 and E. coli WP2 uvrA.
• A reproducible increase of the mean number of revertants per plate above that of the negative control by at least a factor of 1.5 for any concentration at least for one of the following strains: TA 100 and TA 102.
Generally a concentration-related eflfect should be demonstrable.

Statistics:

A statistical analysis was not performed.

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium TA 1535

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:

valid

Remarks:

= vehicle control

Positive controls validity:

valid

Test results 2

Species / strain:

S. typhimurium TA 100

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:

valid

Remarks:

= vehicle control

Positive controls validity:

valid

Test results 3

Species / strain:

S. typhimurium TA 1537

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:

valid

Remarks:

= vehicle control

Positive controls validity:

valid

Test results 4

Species / strain:

S. typhimurium TA 98

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:

valid

Remarks:

= vehicle control

Positive controls validity:

valid

Test results 5

Species / strain:

E. coli WP2 uvr A

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:

valid

Remarks:

= vehicle control

Positive controls validity:

valid

Test results 6

Species / strain:

S. typhimurium TA 102

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:

valid

Remarks:

= vehicle control

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
There were no known circumstances or occurrences in this study that were considered to have aflfected the quality or integrity of the test data.

Applicant's summary and conclusion

Conclusions:

Based on the results of these experiments and on standard evaluation criteria, it is concluded that the test substance and its metabolites did not induce gene mutations in the strains of S. typhimurium and E. coli used.

Executive summary:

The test substance, a solid and white powder, purity > 98%, was tested for mutagenic eflfects in vitro in histidine-requiring strains of Salmonella typhimurium and in a tryptophan-requiring strain of Escherichia coli. The following strains were used: S. typhimurium TA 98, TA 100, TA 102, TA 1535, TA 1537 and E. coli WP2 uvrA. The test was performed with and without the addition of rat-liver post mitochondrial supernatant (S9 fraction) as an extrinsic metabolic activation system. The compound was suspended in dimethylsulfoxide and tested at five concentrations in the range of 312.5 to 5000 µg/plate in the presence and absence of a metabolic activation system. In order to confirm the results, the experiments were repeated with the same concentrations. Each strain was additionally tested in the presence and in the absence of a metabolic activation system with a suitable, known mutagen as positive control.

In both experiments, performed with and without metabolic activation, none of the tested concentrations of the test substance led to an increase in the incidence of either histidine- or tryptophanprototrophic mutants by comparison with the negative control.