Reaction mass of diisopropyl hydrogen phosphate and {[hydroxy(propan-2-yloxy)phosphoryl]oxy}(propan-2-yloxy)phosphinic acid and isopropyl dihydrogen phosphate and methyl dihydrogen phosphate and phosphoric acid

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:

March - May 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

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)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

(propan-2-yloxy)phosphonic acid; [(diethoxyphosphoryl)oxy](ethoxy)phosphinic acid; bis(propan-2-yloxy)phosphinic acid; methoxyphosphonic acid; phosphoric acid

EC Number:

908-995-1

Molecular formula:

can not be determined for reaction masses

IUPAC Name:

(propan-2-yloxy)phosphonic acid; [(diethoxyphosphoryl)oxy](ethoxy)phosphinic acid; bis(propan-2-yloxy)phosphinic acid; methoxyphosphonic acid; phosphoric acid

Specific details on test material used for the study:

Batch: DEH 2035729
Purity: 100 %

Method

Target gene:

Histidine for Salmonella typhimurium
Tryptophan for E. Coli

Species / strain

Species / strain 1

Species / strain / cell type:

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

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A pKM 101

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

0.312, 0.625, 1.25, 2.5 and 5 mg/plate (according to guideline)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used:distilled water
- Justification for choice of solvent/vehicle: solubility

Controls

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

other: 2-aminoanthracene, 4-nitroquinoline 1-oxide

Details on test system and experimental conditions:

1. Initial Cytotoxicity Test for Dose Selection

Based on the results of solubility and precipitation test, an initial cytotoxicity test was conducted for the selection of test doses for the mutation assay. Salmonella typhimurium TA100 strain was exposed to vehicle control, 1, 2, 3, 4 and 5 mg/plate of test item. An initial cytotoxicity test was conducted using 15 hrs old culture of Salmonella typhimurium TA100 tester strain, both in the presence and the absence of metabolic activation, in triplicate, along with concurrent vehicle control (distilled water). Each concentration of test item was mixed with soft agar containing histidine and biotin, either S9 mix (for presence of metabolic activation) or phosphate buffer saline (for absence of metabolic activation), Salmonella typhimurium TA100 of cell density approximately 18×108 cells/mL and overlaid on to pre-labeled minimal glucose agar plates. The plates were incubated at 37°C for 48 hrs.

2. Plate Incorporation Method

In the first trial, which is a plate incorporation method, the bacterial suspensions are exposed to the test item, vehicle and the positive controls in the presence and absence of an exogenous metabolic activation system. These bacterial suspensions are then mixed with soft agar and plated immediately onto minimal glucose agar medium viz., his- for Salmonella typhimurium and try- for Escherichia coli.
Plate Incorporation method was carried out with concentrations of 0.312, 0.625, 1.25, 2.5 and 5 mg/plate; where the test item, vehicle, positive control and the tester strains along with S9/phosphate buffer saline were mixed with 2 mL soft agar and poured on to minimal glucose agar plates. Five concentrations of the test item were plated, with each of the following tester strains: Salmonella typhimurium TA98, TA100, TA1535, TA1537 and Escherichia coli WP2 uvrA (pKM101) with and without metabolic activation. Plates were incubated at 37°C for 48 hrs.

The condition of the bacterial background lawn was evaluated for evidence of the test item cytotoxicity using the code system and revertant colonies for each strain within the test item dilution series were counted manually.

3. Preincubation Method

The confirmatory trial or second experiment is a preincubation method. The test constituents are mixed with the bacteria inside a tube, incubated in an incubator shaker, mixed with soft agar and plated immediately onto minimal glucose agar medium his- for Salmonella typhimurium and try- for Escherichia coli.
After 48 to 72 hrs of incubation at 37±1ºC, revertant colonies are counted and compared to the number of spontaneous revertants on vehicle control plates.
Preincubation method was carried out with concentrations of 0.312, 0.625, 1.25, 2.5 and 5 mg/plate; where the test item, vehicle, positive control and the tester strains along with S9/Phosphate Buffer Saline were mixed with 2 mL soft agar and poured on to minimal glucose agar plates. Five concentrations of the test item were plated, with each of the following tester strains: Salmonella typhimurium TA98, TA100, TA1535, TA1537 and Escherichia coli WP2 uvrA (pKM101) with and without metabolic activation. Plates were incubated at 37°C for 48 hrs.

The condition of the bacterial background lawn was evaluated for evidence of the test item cytotoxicity using the code system and revertant colonies for each strain within the test item dilution series were counted manually.

Rationale for test conditions:

Based on solubility and cytotoxicity pretest; according to guideline

Evaluation criteria:

A test item is considered as a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA 98, TA 100, and WP2 uvrA) or thrice (strains TA 1535 and TA 1537) the colony count of the corresponding solvent control is observed.
A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration.
An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment.
A dose dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative and solvent controls such an increase is not considered biologically relevant.

Statistics:

The data (in the form of appendix) were verified with the raw data. The data of number of revertants were subjected to computer statistical processing using Graphpad prism software version 5 wherever possible. All individual data were summarized and presented as tables. All findings were presented in the report as per the standard reporting procedure. Data was analyzed for differences among vehicle control, treatment and positive control group using ANOVA. Differences between the vehicle control, treatment with different concentrations of test item and positive control groups were tested by one-way ANOVA with Dunnett test at a 5% level (p<0.05) of significance. The statistical significances are designated by superscripts as given below:

Results and discussion

Test results

Test results 1

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

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 3

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

Positive controls validity:

valid

Test results 4

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

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

E. coli WP2 uvr A pKM 101

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Applicant's summary and conclusion

Conclusions:

From the results obtained, the test item HORDAPHOS CC MIS is found to be non-mutagenic at and up to the highest concentration of 5 mg/plate in Bacterial Reverse Mutation Test in the tester strain Salmonella typhimurium TA98, TA100, TA1535, TA1537 and Escherichia coli WP2 uvrA (pKM101) in the presence and absence of metabolic activation in plate incorporation and preincubation methods under laboratory conditions tested.

Executive summary:

The test item HORDAPHOS CC MIS was assessed for its mutagenic effects using Salmonella typhimurium strains: TA98, TA100, TA1535, TA1537 and Escherichiacoli WP2 uvrA (pKM101). The test item was tested at the concentrations of 0.312, 0.625, 1.25, 2.5 and 5 mg/plate using distilled water as vehicle based on the precipitation and initial cytotoxicity test. The study was conducted, with and without the metabolic activation (S9 fraction). The S9 fraction was prepared from sodium phenobarbitone and β-naphthoflavone induced ratliver. Vehicle control and appropriate positive controls (2-nitrofluorene, sodium azide and 9-Aminoacridine,4-nitroquinoline 1-oxidefor trials “without metabolic activation” and 2-Aminoanthracene for trials “with metabolic activation”) were tested simultaneously. Two trials were carried out for this study in triplicates with plate incorporation method (Trial I) and preincubation method (Trial II). Data were statistically analyzed and expressed as mean ±SD.

From the experimental results obtained, the mean numbers of revertant colonies at the above mentioned concentrations were comparable to those of the dimethyl sulphoxide, in both the trials - plate incorporation method and preincubation method, in the presence and absence of metabolic activation. There was no significant increase in number of revertant colonies at any of the concentrations tested in both plate incorporation and preincubation method. The number of revertant colonies in the positive controls has shown 2.3 to 20.1 fold increase with statistical significance at a 5% level (p<0.05) underidenticalconditions.