Reaction mass of Dimethyl [3-[(hydroxymethyl) amino]-3-oxopropyl] phosphonate and Dimethyl (3-amino-3-oxopropyl)phosphonate

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2004

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2004

Report date:

2004

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

GLP (The Netherlands)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Reaction mass of Dimethyl [3-[(hydroxymethyl) amino]-3-oxopropyl] phosphonate and Dimethyl (3-amino-3-oxopropyl)phosphonate

EC Number:

701-402-5

Molecular formula:

C6H14NO5P.C5H12NO4P

IUPAC Name:

Reaction mass of Dimethyl [3-[(hydroxymethyl) amino]-3-oxopropyl] phosphonate and Dimethyl (3-amino-3-oxopropyl)phosphonate

Constituent 2

Reference substance name:

Dimethyl (3-amino-3-oxopropyl)phosphonate

EC Number:

219-765-9

EC Name:

Dimethyl (3-amino-3-oxopropyl)phosphonate

Cas Number:

2526-69-4

Molecular formula:

C5H12NO4P

IUPAC Name:

dimethyl (3-amino-3-oxopropyl)phosphonate

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 137244407
- Expiration date of the lot/batch: July 2004

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

Method

Species / strain

Species / strain / cell type:

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

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

rat liver S9-mix

Test concentrations with justification for top dose:

Trial 1: 0.3125, 0.625, 1.25, 2.5 and 5.0 µL/plate
Trial 2: 0.213, 0.470, 1.033, 2.272 and 5.0 µL/plate

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

TA 1537; In absence of metabolic activation

Controls 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

TA1535 and TA100; in absence of metabolic activation

Controls 3

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

TA98; in absence of metabolic activation

Controls 4

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cumene hydroperoxide

Remarks:

TA102; in absence of metabolic activation

Controls 5

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminofluorene

Remarks:

TA1537, TA 1535, TA98, TA100, TA102; in presence of metabolic activation

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: duplicate

METHOD OF TREATMENT/ EXPOSURE:
- Test substance added in medium; in agar (plate incorporation);

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable:
- Exposure duration/duration of treatment: Overnight

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method, relative survival (RS)
- Any supplementary information relevant to cytotoxicity:

Statistics:

Simple linear regression

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 4

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 5

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Any other information on results incl. tables

Solubility and Precipitation Tests

Solubility and precipitation tests were performed prior to cytotoxicity test. The solubility of Aflammit KWB was tested in distilled water and found to be a suitable vehicle for treatment as it completely dissolves in distilled water. Stock A (50 gL/mL) was prepared by addition of 50 ul of test substance to 950 PL of water. A volume of 100 ul of this stock solution was added to 2 mL of molten top agar to assess the precipitation. Precipitation was not observed at this concentration. Therefore 5 uL/plate has been selected as the highest concentration for cytotoxicity assay.

Cell Viability Test

Fresh cultures for the test were prepared by inoculating frozen permanent cultures to a flask containing 10 ml of sterile nutrient broth N^O2 (Oxoid). The flasks were then incubated at 37 ± 1 °C in an orbital shaking incubator for approximately 16 h up to early stationary or late exponential phase (approximately 10⁸to 10⁹cells/mL). After incubation period, the culture flasks were removed from the orbital shaking incubator. Aseptically, the cultures were diluted in distilled water and the optical density was measured at 660 nm using a Photoelectric Colorimeter (Erection & Instrumentation Engineerings India), Nutrient broth diluted in the same manner was used as control blank to set "0", Cell viability of tester strain was determined prior to treatment. The optical density was found to be in the acceptable range (Approx 0.4) and used for the study.

Genotype Confirmation Test

Fresh cultures for the test were prepared by inoculating frozen permanent cultures to a flask containing 10 mL of sterile nutrient broth N^O2 (Oxoid). The flasks were then incubated at 37 ± 1^oC in an orbital shaking incubator for approximately 16 h up to early stationary or late phase.

Cytotoxicity Test

Before commencing the mutagenicity study, Aflammit KWB was assessed for cytotoxicity to Salmonella typhimurium tester strain TA100. The experiment was conducted both in the presence and absence of metabolic activation (5 % v/v S9). A stock solution (50 µg/mL) of Aflammit KWB was prepared by addition of 75 µL of Aflammit KWB to 1.425 mL sterile distilled water (Stock A). Further stock solution of concentrations viz, 10.0, 2.0, 0.4, 0.08 and 0.016 µL/mL were prepared from the first stock solution. Six different concentrations viz., 0.0016, 0.008, 0.04, 0.2, 1.0 and 5.0 µL/plate were tested for cytotoxicity. Tubes containing 2 mL of molten top agar with 0.5 rnM histidine/biotine were maintained at 45 °C. A volume of 500 µL of 5% VIV S9 mix was added to one ofthe sets and 500 µL of 0.2 M Phosphate buffer was added to the second set. 100 uL of relevant stock solution of test substance or distilled water was to the tubes for treatment and negative control respectively. Finally 100 µl. of bacterial culture was added to the tubes and mixed. Details of culture preparation are provided under "Cell Viability Test" (cultures used were checked for cell viability prior to testing). This treatment mixture was poured on minimal glucose agar plates and allowed to solidify. Duplicate sets were used for each concentration and controls. The petriplates were incubated at 37 ± 1 °C for 48 hours and then examined to assess the state of the background bacterial growth. Cytotoxicity was assessed by clearance of background lawn. Background lawn inhibition was not observed up to 5.0 µl/plate in presence and absence of metabolic activation. Hence 5.0 uL/plate has been selected as the highest test concentration for mutagenicity test.

Mutagenicity Test

Mutagenicity test was conducted as two independent experiments. In both the trials, the treatment was given in the presence and absence of metabolic activation (5 % and 10 % v/v S9 mix in first and second trial respectively). The treatments were performed by plate in corporation technique as in cytotoxicity test. Duplicate plates were maintained for each test concentration of Aflammit KWB, negative and positive controls.

In the first trial the strains were exposed at 0.3125, 0.625, 1 .25, 2.5 and 5.0 µL/pIate (factor 2). First stock solution (Stock A) of test substance was prepared by addition of 300 µL Aflammit K WB in 5.7 mL of sterile distilled water (50.0 µL/mL). Further stock solutions viz 25 (Stock B), 12.5 (Stock C), 6.25 (Stock D) and 3.125 (Stock E) were prepared from the first stock solution by dilution. Volumes of 100 µLof these stock solutions (A-E) were used to obtain the required test concentrations. The number of revenant colonies was recorded after 48 h incubation period. Similarly a second trail was conducted to confirm the negative results obtained in the first trial. In the second trial, the concentration spacing was modified using a factor 2.2. The highest concentration being 5.0 µL/plate, four lower doses viz 0.213, 0.470, 1.033 and 2.272 µL/plate were tested. In the second trial the first stock solution of the test substance (Stock A) was prepared by addition of 300 µL Aflammit KWB in 5.7 mL of sterile distilled water (50.0 uL/mL). Further stock solutions viz 22.72 (Stock B), 10.33 (Stock C), 4.70 (Stock D) and 2.13 (Stock E) µL/mL were prepared from stock A by dilution. Volumes of 100 µL of these stock solutions (A-E) were used to obtain the required test concentrations. The number of revertant colonies was recorded after 48 h incubation period.

Treatment with 2-aminofluorene in the absence of metabolic activation was performed along with both the trials to confirm the efficiency of S9 fraction used in the study.

Evaluation Criteria

A positive result is defined as a statistically significant, dose-dependent increase in the numb histidine independent revertants with at least one dose level inducing a revertant frequency that is at least two-fold of the spontaneous solvent control value. If the test substance does not induce a statistically significant, dose-dependent increase in revertant frequency in both the replicates, but does induce revertant frequency at one dose level that is at least two-fold the spontaneous control value, the result is considered equivocal. A negative result is defined as the absence of statistically significant or dose-dependent increase in the number of histidine independent revenant.

Applicant's summary and conclusion

Conclusions:

DMPPA_701-402-5 is considered to be non-mutagenic in this Salmonella typhimurium reverse mutation assay.

Executive summary:

This study was performed to investigate the potential of DMPPA_701-402-5 to induce gene mutations in the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, and TA 100, and TA102 according to OECD 471 guideline. It can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used both with and without metabolic activation. Based on the study result, DMPPA_701-402-5 is considered to be non-mutagenic in this Salmonella typhimurium reverse mutation assay.