Dioctyl phosphonate

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:

From May 4th to Jun 1st, 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

July, 1997

Deviations:

no

Test guideline 2

Qualifier:

according 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

Reference substance name:

Dioctyl phosphonate

EC Number:

217-315-6

EC Name:

Dioctyl phosphonate

Cas Number:

1809-14-9

Molecular formula:

C16H35O3P

IUPAC Name:

dioctyl phosphonate

Test material form:

liquid

Method

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

Metabolic activation:

with and without

Metabolic activation system:

S9 tissue fraction prepared from livers of Sprague-Dawley rats pretreated with Phenobarbital and 5,6-benzoflavone.

Test concentrations with justification for top dose:

The concentration used in the first experiment (plate incorporation method) was selected considering the results of preliminary toxicity test as follows:
TA1535 (with and without S9): 2.50, 1.25, 0.625, 0.313, 0.156, 0.0781 μl/plate
TA1537 (without S9): 1.25, 0.625, 0.313, 0.156, 0.0781 μl/plate
TA1537 (with S9): 5.00, 2.50, 1.25, 0.625, 0.313, 0.156 μl/plate
TA98, WP2 uvrA (with and without): 5.00, 2.50, 1.25, 0.625, 0.313 μl/plate
TA100 (with and without S9): 0.625, 0.313, 0.156, 0.0781, 0.0391, 0.0195 μl/plate

The concentration used in the second experiment (pre-incubation method) was modified taken in account the results of first assay as follows:
TA1535 (with and without S9): 0.625, 0.313, 0.156, 0.0781, 0.0391 μl/plate
TA1537 (without S9): 0.313, 0.156, 0.0781, 0.0391, 0.0195 μl/plate
TA1537 (with S9): 2.50, 1.25, 0.625, 0.313, 0.156 μl/plate
TA98, WP2 uvrA (with and without): 5.00, 2.50, 1.25, 0.625, 0.313 μl/plate
TA100 (without S9): 0.156, 0.0781, 0.0391, 0.0195, 0.00977, 0.00488 μl/plate
TA100 (with S9): 0.313, 0.156, 0.0781, 0.0391, 0.0195 μl/plate

Vehicle / solvent:

- Solvents used: Dimethylsulfoxide (DMSO) for test item; DMSO or water for injection to dissolve positive controls.
- Justification for choice of solvent/vehicle : solubility of the test item was evaluated in a preliminary trial using DMSO. This solvent was selected since it is compatible with the survival of the bacteria and the S9 metabolic activity. The test item was found to be fully miscible at a concentration of 10% v/v. This result permitted a maximum concentration of 5 µl/plate to be used in the toxicity test.

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

methylmethanesulfonate

Remarks:

Without S9

Controls 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

With S9

Details on test system and experimental conditions:

BACTERIA STRAIN:
Four strains of Salmonella typhimurium (TA1535, TA1537, TA98 and TA100) and a strain of Escherichia coli (WP2 uvrA) were used in this study. Permanent stocks of these strains are kept at -80 °C in the laboratory. Overnight subcultures of these stocks were prepared for each day’s work. Bacteria were taken from vials of frozen cultures, which had been checked for the presence of the appropriate genetic markers, as follows:
- Histidine requirement: No Growth on Minimal plates + Biotin and Growth on Minimal plates + Biotin + Histidine.
- Tryptophan requirement: No Growth on Minimal agar plates and Growth on Minimal plates + Tryptophan.
- uvrA, uvrB: Sensitivity to UV irradiation.
- rfa: Sensitivity to Crystal Violet.
- pKM101: Resistance to Ampicillin.

MEDIA:
- Nutrient Broth: Oxoid Nutrient Broth No. 2 was prepared at a concentration of 2.5% in distilled water and autoclaved prior to use. This was used for the preparation of liquid cultures of the tester strains.
- Nutrient Agar: Oxoid Nutrient Broth No. 2 (25 g) and Difco Bacto-agar (15 g) were added to distilled water(1 litre) and autoclaved. The solutions were then poured into 9 cm plastic Petri dishes and allowed to solidify and dry before use. These plates were used for the non-selective growth of the tester strains.
- Minimal Agar: Minimal medium agar was prepared as 1.5% Difco Bacto-agar in Vogel-Bonner Medium E, with 2% Glucose, autoclaved and poured into 9 cm plastic Petri dishes.
- Top Agar: "Top Agar" (overlay agar) was prepared as 0.6% Difco Bacto-agar + 0.5% NaCl in distilled water and autoclaved. Prior to use, 10 ml of a sterile solution of 0.5 mM Biotin + 0.5 mMHistidine (or 0.5 mM tryptophan) was added to the top agar (100 ml).

S9 TISSUE:
One batch of S9 tissue fraction was used in this study and had the following characteristics:
- Species: Rat
- Strain: Sprague Dawley
- Tissue: Liver
- Inducing Agents: Phenobarbital – 5,6-Benzoflavone
The mixture of S9 tissue fraction and cofactors (S9 mix) was prepared as follows (for each 10 ml): S9 tissue fraction 1.0 ml+ NADP (100 mM) 0.4 ml+G-6-P (100 mM) 0.5 ml+ KCl (330 mM) 1.0 ml+ MgCl2 (100 mM) 0.8 ml+ Phosphate buffer (pH 7.4, 200 mM) 5.0 ml+ Distilled Water 1.3 ml.

PRELIMINARY TOXICITY TEST:
A preliminary toxicity test was undertaken in order to select the concentrations of the test item to be used in the Main Assays. In this test a wide range of dose levels of the test item, set at half-log intervals, were used. Treatments were performed both in the absence and presence of S9 metabolism using the plate incorporation method; a single plate was used at each test point and positive controls were not included. Toxicity was assessed on the basis of a decline in the number of spontaneous revertants, a thinning of the background lawn or a microcolony formation.

MAIN ASSAY
Two Main Assays were performed including negative and positive controls in the absence and presence of an S9 metabolising system. Three replicate plates were used at each test point.
In addition, plates were prepared to check the sterility of the test item solutions and the S9 mix and dilutions of the bacterial cultures were plated on nutrient agar plates to establish the number of bacteria in the cultures.
The first experiment was performed using a plate-incorporation method. The components of the assay (the tester strain bacteria, the test item and S9 mix or phosphate buffer) were added to molten overlay agar and vortexed. The mixture was then poured onto the surface of a minimal medium agar plate and allowed to solidify prior to incubation. The overlay mixture was composed as follows: Overlay agar (held at 45°C) 2.0 ml, Test or control item solution 0.1ml, S9 mix or phosphate buffer (pH 7.4, 0.1M) 0.5 ml, Bacterial suspension 0.1ml.
The second experiment was performed using a pre-incubation method. The components were added in turn to an empty test-tube: Bacterial suspension 0.1 ml, Test item solution or control item solution 0.05 ml, S9 mix or phosphate buffer (pH 7.4, 0.1 M) 0.5ml. The incubate was vortexed and placed at 37°C for 30 minutes. Two ml of overlay agar was then added and the mixture vortexed again and poured onto the surface of a minimal medium agar plate and allowed to solidify.

INCUBATION AND SCORING
The prepared plates were inverted and incubated for approximately 72 hours at 37°C. After this period of incubation, plates from the preliminary toxicity test were held at 4°C for approximately 24 hours before scoring, while plates from the Main Assays were immediately scored by counting the number of revertant colonies on each plate.

METHOD OF APPLICATION: plate incorporation in the first experiment and pre-preincubation in the second experiment.

ACCEPTANCE CRITERIA:
The assay was considered valid if the following criteria were met:
1.Mean plate counts for untreated and positive control plates should fall within 2 standard deviations of the current historical mean values.
2. The estimated numbers of viable bacteria/plate should fall in the range of 100 – 500 millions for each strain.
3. No more than 5 % of the plates should be lost through contamination or other unforeseen event.

Evaluation criteria:

For the test item to be considered mutagenic, two-fold (or more) increases in mean revertant numbers must be observed at two consecutive dose levels or at the highest practicable dose level only. In addition, there must be evidence of a dose-response relationship showing increasing numbers of mutant colonies with increasing dose levels.

Statistics:

The regression analysis fits a regression line to the data by the least squares method, after square root transformation of the plate counts to satisfy normal distribution and homosce-dasticity assumptions. The regression equation is expressed as: y= a+bx

where:
y = transformed revertant numbers
a = intercept
b = slope value
x = dose level (in the units given).
The regression line does not include the untreated control data, but includes the solvent control data.
Regression lines are calculated using a minimum of the three lowest dose levels, and then including the further dose levels in turn. The correlation co-efficient (r),the value of students "t" statistic, and the p-value for the regression lines are also given.

Results and discussion

Test results

Key result

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Toxicity as indicated by mild reduction of revertant colonies and/or thinning of the background lawn was observed at higher dose levels, both in the absence and presence of S9 metabolism, with all tester strains with the exception of TA98 and WP2 uvrA

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

MAIN ASSAYS
Two main assay were performed.
Based on the results obtained in the preliminary toxicity test, the maximum concentration of the test item to be used in Main Assay I was selected as the concentration which elicits a moderate toxicity. The number of lower dose-levels included in each treatment series of Main Assay I was selected in order to have a sufficient number of analysable concentrations (at least three concentrations without toxicity). Toxicity as indicated by mild reduction of revertant colonies and/or thinning of the background lawn was observed at higher dose levels, both in the absence and presence of S9 metabolism, with all tester strains with the exception of TA98 and WP2 uvrA in Main Assay I.
As no relevant increase in revertant numbers was observed at any concentration tested, a Main Assay II was performed including a pre-incubation step for all treatments. The dose range used was modified to take into account the toxicity observed in the first assay.
Toxicity ranging from slight to complete toxicity (microcolony formation) was observed with all tester strains with the exception of TA98 and WP2 uvrA. Treatments in the absence of S9 metabolic activation proved to be more toxic than treatments in its presence, showing toxicity over all concentrations tested with TA1535, TA1537 and TA100 tester strains.
No precipitation of the test item was observed at the end of the incubation period at any concentration, in any experiment.
No relevant increase in the number of revertant colonies was observed in the plate incorporation or pre-incubation assay, at any dose level, with any tester strain, in the absence or presence of S9 metabolism.
The sterility of the S9 mix and of the test item solutions was confirmed by the absence of colonies on additional agar plates spread separately with these solutions. Marked increases in revertant numbers were obtained in these tests following treatment with the positive control items, indicating that the assay system was functioning correctly.

TOXICITY PRELIMINARY TEST
The test item was assayed in the toxicity test at a maximum concentration of 5.00 µl/plate and at four lower concentrations spaced at approximately half-log intervals: 1.58, 0.500, 0.158 and 0.0500 µl/plate. No precipitation of the test item was observed at the end of the incubation period at any concentration. Dose-related toxicity, as indicated by reduction in revertant colonies and/or thinning of the background lawn,was observed at higher dose levels both in the absence and presence of S9 metabolic activation, with all tester strains with the exception of TA98 and WP2 uvrA. No increase in revertant colonies was observed with any tester strain/activation condition combinations.

ACCEPTANCE CRITERIA AND EVALUATION:
Results show that mean plate counts for untreated and positive control plates fell within the normal range based on historical control data.
The estimated numbers of viable bacteria/plate (titre) fell in the range of 100 - 500 million for each strain. No plates were lost through contamination or cracking. The study was accepted as valid.
The test item did not induce two-fold increases in the number of revertant colonies in the plate incorporation or pre-incubation assay, at any dose level, in any tester strain, in the absence or presence of S9 metabolism. However, it should be noted that in pre-incubation experiment with TA1535, TA1537 and TA100 tester strains in the absence of S9 metabolism high toxicity was observed for all tested concentrations.

Remarks on result:

other: The highest toxicity was observed without S9 for TA 1535, TA 1537 and TA100.

Applicant's summary and conclusion

Conclusions:

The test item Dioctyl phosphonate does not induce reverse mutation in Salmonella typhimurium or Escherichia coli in the absence or presence of S9 metabolism.

Executive summary:

The test item Dioctyl phosphonate was examined for the ability to induce gene mutations in tester strains of Salmonella typhimurium and Escherichia coli, as measured by reversion of auxotrophic strains to prototrophy. The five tester strains TA1535, TA1537, TA98, TA100 and WP2 uvrA were used. Experiments were performed both in the absence and presence of metabolic activation, using liver S9 fraction from rats pre-treated with phenobarbital and 5,6-benzoflavone.

Toxicity test

The test tem was assayed in the toxicity test at a maximum concentration of 5.00µl/plate and at four lower concentrations space data approximately half-log intervals: 1.58, 0.500, 0.158 and 0.0500 µl/plate. No precipitation of the test item was observed at the end of the incubation period at any concentration. Dose related toxicity was observed with TA1535,TA1537 and TA100 tester strains at higher dose levels both in the absence and presence of S9 metabolic activation. No relevant increase in revertant colonies was observed, with any tester strain, at any dose level, in the absence or presence of S9 metabolism.

Main assays

On the basis of toxicity test results, in Main Assay I, using the plate incorporation method, the test item was assayed at the following dose levels:

TA1535 (with and without S9): 2.50, 1.25, 0.625, 0.313, 0.156, 0.0781 μl/plate

TA1537 (without S9): 1.25, 0.625, 0.313, 0.156, 0.0781 μl/plate

TA1537 (with S9): 5.00, 2.50, 1.25, 0.625, 0.313, 0.156 μl/plate

TA98, WP2 uvrA (with and without): 5.00, 2.50, 1.25, 0.625, 0.313 μl/plate

TA100 (with and without S9): 0.625, 0.313, 0.156, 0.0781, 0.0391, 0.0195 μl/plate

Toxicity was observed at higher dose levels, both in the absence and presence of S9 metabolism,with all tester strains with the exception of TA98 and WP2uvrA.

As no relevant increase in revertant numbers was observed at any concentration tested, a Main Assay II was performed including a pre-incubation step for all treatments. The dose range used was modified to take into account the toxicity observed in the first assay and the following dose levels were used:

TA1535 (with and without S9): 0.625, 0.313, 0.156, 0.0781, 0.0391 μl/plate

TA1537 (without S9): 0.313, 0.156, 0.0781, 0.0391, 0.0195 μl/plate

TA1537 (with S9): 2.50, 1.25, 0.625, 0.313, 0.156 μl/plate

TA98, WP2 uvrA (with and without): 5.00, 2.50, 1.25, 0.625, 0.313 μl/plate

TA100 (without S9): 0.156, 0.0781, 0.0391, 0.0195, 0.00977, 0.00488 μl/plate

TA100 (with S9): 0.313, 0.156, 0.0781, 0.0391, 0.0195 μl/plate

Toxicity ranging from slight to severe was observed with all tester strains with the exception of TA98 and WP2 uvrA. Treatments in the absence of S9 metabolic activation proved to be more toxic than treatments in its presence,showing toxicity over all concentrations tested with TA1535, TA1537 and TA100 tester strains.

No precipitation of the test item was observed at the end of the incubation period at any concentration in any experiment.

The test item did not induce two-fold increases in the number of revertant colonies in the plate incorporation or pre-incubation assay, at any dose level, in any tester strain, in the absence or presence of S9 metabolism.However, it should be noted that in pre-incubation experiment with TA1535, TA1537 and TA100 tester strains in the absence of S9 metabolism high toxicity was observed for all tested concentrations.

 

Conclusion

It is concluded that no positive increase in revertant numbers was observed after treatment with Dioctyl phosphonate in Salmonella typhimurium or Escherichia coli in the absence or presence of S9 metabolism, under the reported experimental conditions.