Diphenyl methylphosphonate

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2012-07-02 to 2012-08-20

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Version / remarks:

new OECD Guideline for this test: OECD Guideline 490 (issued in 2015)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Diphenyl methylphosphonate

EC Number:

231-388-1

EC Name:

Diphenyl methylphosphonate

Cas Number:

7526-26-3

Molecular formula:

C13H13O3P

IUPAC Name:

diphenyl methylphosphonate

Test material form:

solid: compact

Details on test material:

- Name of test material (as cited in study report): Diphenyl methylphosphonate
- Substance type: organophosphate
- Physical state: solid (if pure)
- Analytical purity: 99.88 %
- Stability in solvent: not indicated by the sponsor
- Storage condition of test material: at room temperature, under nitrogen

Method

Target gene:

thymidine kinase (TK) locus

Species / strain

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

- Type and identity of media: RPMI 1640 complete
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes

Additional strain / cell type characteristics:

other: high proliferation rate (doubling time 10 - 12 h in stock cultures) and cloning efficiencies of untreated cells of usually more than 50 %; a stable karyotype with a near diploid (40 ± 2) chromosome number

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/β-naphthoflavone induced Wistar rat liver microsome preparations (S9 mix)

Test concentrations with justification for top dose:

Pre-experiment: 19.4, 38.8, 77.5, 155, 310, 620, 1240 and 2480 µg/mL
Main Experiment I: 9.7, 19.4, 38.8, 77.5, 155 and 310 mg/mL (without S9 mix); 19.4, 38.8, 77.5, 155, 310, 465 and 620 µg/mL (with S9 mix);
Main Experiment II: 9.7, 19.4, 38.8, 77.5, 116.3 and 155 mg/mL (without S9 mix); 19.4, 38.8, 77.5, 155, 310, 387.5 and 465 µg/mL (with S9 mix);

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: [DMSO]. The final concentration of DMSO in the culture medium will be 0.5 % (v/v).
- Justification for choice of solvent/vehicle: the substance is well soluble in DMSO.

Controls

Untreated negative controls:

yes

Remarks:

solvent control

Negative solvent / vehicle controls:

yes

Remarks:

negative control

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

methylmethanesulfonate

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: thawed stock cultures were propagated in RPMI 1640 complete culture medium and the cells were subcultured two times prior to treatment (until density of 1x10E7 and 3x10E6 during 4h and 24 h exposure, respectively);
- Exposure duration: 4 and 24 hours in the experiments I and II, respectively;
- Expression time (cells in growth medium): 48 hours;
- Selection time (if incubation with a selection agent): not reported

SELECTION AGENT (mutation assays): TFT (Trifluorothymidine).


NUMBER OF REPLICATIONS: The cell density was determined each day and adjusted to 3x10E5 cells/mL, if necessary.

NUMBER OF CELLS EVALUATED: 3x10E5 cells/mL for relative suspension growth; 4x10E3 cells in selective medium; 2 cells per well into microtiter plate for cloning efficiency.

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency was determined by seeding about 2 cells per well into microtiter plates (same medium without TFT). The plates were incubated at 37 °C ± 1.5 °C in 4.5 % CO2/95.5 % water saturated air for 10 - 15 days. Then the plates were evaluated. The relative total growth (RTG) is calculated by the RSG multiplied by the viability.
- relative suspension growth (RSG) of the treated cell cultures was calculated at the end of the growth period by the day 1 fold-increase in cell number multiplied by the day 2 fold-increase in cell number according to the method of Clive and Spector:
D. Clive, J.F.S. Spector
Laboratory procedure for assessing specific locus mutation at the TK locus in cultured L5178Y mouse lymphoma cells
Mutation Research 31, 17-29, 1975.

Evaluation criteria:

A test item is classified as mutagenic if the induced mutation frequency reproducibly exceeds a threshold of 126 colonies per 10E6 cells above the corresponding solvent control.
A relevant increase of the mutation frequency should be dose-dependent.
A mutagenic response is considered to be reproducible if it occurs in both parallel cultures.
However, in the evaluation of the test results the historical variability of the mutation rates in the solvent con¬trols of this study are taken into consideration.
Results of test groups are generally rejected if the relative total growth is less than 10 % of the vehicle control unless the exception criteria specified by the IWGT recommendations are met.

Whenever a test item is considered mutagenic according to the above mentioned criteria, the ratio of small versus large colonies is used to differentiate point mutations from clastogenic effects. If the increase of the mutation frequency is accompanied by a reproducible and dose dependent shift in the ratio of small versus large colonies clastogenic effects are indicated.
A test item is classified as non-mutagenic if the induced mutation frequency does not reproducibly exceed a threshold of 126 colonies per 106 cells above the corresponding solvent control.
A test item not meeting the conditions for a classification as mutagenic or non-mutagenic will be considered equivocal in this assay and may be considered for further investigation

Statistics:

A linear regression (least squares) was performed to assess a possible dose dependent increase of mutant frequencies using SYSTAT11 (SYSTAT Software, Inc., 501, Canal Boulevard, Suite C, Richmond, CA 94804, USA) statistics software. The number of mutant colonies obtained for the groups treated with the test item was compared to the solvent control groups. A trend is judged as significant whenever the p-value (probability value) is below 0.05. However, both, biological relevance and statistical significance were considered together.

Results and discussion

Test results

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at the two highest concentrations in experiment I (465 and 620 µg/L) and II (387.5 and 465 µg/L) with metabolic activation

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no effects
- Effects of osmolality: no effects
- Precipitation: The test medium (main experiments) was checked for precipitation or phase separation visible to the naked eye at the end of the 4 hours treatment just before the test item was removed. Neither phase separation nor precipitation occurred up to the maximum concentration with and without metabolic activation.
- Other confounding effects: no

RANGE-FINDING/SCREENING STUDIES: The pre-experiment was performed in the presence (4 h treatment) and absence (4 h and 24 h treatment) of metabolic activation. Test item concentrations between 19.4 µg/mL and 2480 µg/mL (equal to a molar concentration of approximately 10 mM) were used. The highest concentration in the pre-experiment was chosen with regard to the purity (99.88%) and the molecular weight (248 g/mol) of the test item.
Toxic effects leading to RSG values below 50 % were observed following 4 hours treatment at 310.0 µg/mL and above with and without metabolic activation. After 24 hour treatment without metabolic activation toxic effects were noted at 155.0 µg/mL and above.
The test medium was checked for precipitation or phase separation at the end of the treatment period (4 and 24 hours) just before the test item was removed. Phase separation was observed following 4 hour treatment at 310.0 µg/mL and above without metabolic activation and at 620 µg/mL and above with metabolic activation.
Both, pH value and osmolarity was determined in the pre-experiment at the highest concentration of the test item and in the solvent control without metabolic activation. There was no relevant shift of both parameters. To overcome problems with possible deviations in toxicity and solubility the main experiments were started with more than four concentrations (see above).

COMPARISON WITH HISTORICAL CONTROL DATA: yes

ADDITIONAL INFORMATION ON CYTOTOXICITY: Relevant cytotoxic effect indicated by a relative total growth of less than 50 % of survival was observed in the first experiment at 155.0 µg/mL without metabolic activation (both cultures) and at 465.0 µg/mL with metabolic activation (culture II only, culture I was not analysable due to exceedingly severe cytotoxic effects). In the second experiment cytotoxic effects as described above occurred at 116.3 µg/mL and above without metabolic activation and at 310.0 µg/mL and above with metabolic activation. The recommended cytotoxic range of approximately 10-20 % RTG was covered with and without metabolic activation. The data generated in experiment I at 310 µg/mL without metabolic activation (both cultures) are not considered valid since the Relative Total Growth (RTG) remained far below the threshold of 10 % in both parallel cultures.

Any other information on results incl. tables

Pre-experiment

The osmolarity and the pH-value were determined in culture medium of the solvent control and of the maximum concentration in the pre-experiment without metabolic activation:

	Solvent control	Diphenyl methylphosphonate 2480 µg/mL
Osmolarity mOsm	369	340
pH-value	7.50	7.52

Main experiment

The assay was performed in two independent experiments, using two parallel cultures each. The first main experiment was performed with and without liver microsomal activation and a treatment period of 4 h. The second experiment was solely performed in the absence of metabolic activation with a treatment period of 24 hours. According to the results of the pre-test at least four adequate concentrations were chosen for the mutation experiment. However, to overcome problems with possible deviations in toxicity and solubility the main experiments were started with more than four concentrations (see above).

Following the expression phase of 48 hours the cultures at the lowest concentration in experiment I and II without metabolic activation were not continued since a minimum of only four analysable concentrations is required by the guidelines. The cultures at the two highest concentrations in experiment I and II with metabolic activation were not continued due to exceedingly strong toxic effects.

Therefore, the main experiments were evaluated at the following concentrations:

Experiment I:

without S9 mix:                                19.4; 38.8; 77.5; 155.0; and 310.0 µg/mL
with S9 mix:                                     19.4; 38.8; 77.5; 155.0; and 310.0 µg/mL

Experiment II:

without S9 mix:                                19.4; 38.8; 77.5; 116.3; and 155.0 µg/mL
with S9 mix:                                     19.4; 38.8; 77.5; 155.0; and 310.0 µg/mL

No substantial and reproducible dose dependent increase of the mutation frequency was observed with and without metabolic activation. The mutation frequency did not reach or exceed the threshold of 126 above the corresponding solvent control.

A linear regression analysis (least squares) was performed to assess a possible dose dependent increase of mutant frequencies using SYSTAT^â11 statistics software. No significant dose dependent trend of the mutation frequency indicated by a probability value of <0.05 was determined in any of the experimental groups.

In this study the range of the solvent controls was from 48 up to 78 mutant colonies per 10⁶cells; the range of the groups treated with the test item was from 25 up to 86 mutant colonies per 10⁶cells. The lowest solvent control value (48 colonies per 10⁶cells) fell just short of the recommended 50 – 170 x 10⁶control range as stated under paragraph 8.12,acceptability of the assay of this report. The data are acceptable however, as the mutant frequency in the parallel culture (55 colonies per 10⁶cells) and the mean of both parallel cultures (51.5 colonies per 10⁶cells) is fully acceptable.

MMS (19.5 µg/mL in experiment I and 13.0 µg/mL in experiment II) and CPA (3.0 µg/mL and 4.5 µg/mL in both main experiments) were used as positive controls and showed a distinct increase in induced total mutant colonies at acceptable levels of toxicity with at least one of the concentrations of the controls. Table 1: p-values

experimental group	p-value
experiment I, culture I without S9 mix	0.735
experiment I, culture II without S9 mix	0.069
experiment I, culture I with S9 mix	0.597
experiment I, culture II with S9 mix	0.391
experiment II, culture I without S9 mix	0.372
experiment II, culture II without S9 mix	0.801
experiment II, culture I with S9 mix	0.276
experiment II, culture II with S9 mix	0.993

Table 2: Summary of results

	Conc (µg/mL)	S9 mix	Relative total growth	Mutant colonies/10^6cells	Threshold	Relative total growth	Mutant colonies/10^6cells	Threshold
Experiment I (4h treatment)			Culture I			Culture II
DMSO		-	100.0	63	189	100.0	78	204
Positive control (MMS)	19.5	-	34.0	275	189	40.5	291	204
Test item	9.7	-	Culture was not continued#			Culture was not continued#
	19.4	-	139.9	25	189	133.3	45	204
	38.8	-	103.7	41	189	130.2	52	204
	77.5	-	109.3	39	189	103.5	42	204
	155.0	-	25.1	41	189	19.7	26	204
	310.0	-	0.4	71	189	0.1	0	204
Experiment I (4h treatment)			Culture I			Culture II
DMSO		+	100.0	52	178	100.0	51	177
Positive control (CPA)	3.0	+	92.6	107	178	95.8	75	177
Positive control (CPA)	4.5	+	31.7	367	178	27.5	289	177
Test item	19.4	+	136.6	59	178	Culture was not continued#
	38.8	+	116.7	81	178	79.3	51	177
	77.5	+	99.6	54	178	115.8	42	177
	155.0	+	79.6	86	178	89.9	33	177
	310.0	+	67.3	41	178	81.7	31	177
	465.0	+	Culture was not continued##			10.6	80	177
	620.0	+	Culture was not continued##			Culture was not continued##
Experiment II 24 h treatment			Culture I			Culture II
DMSO		-	100.0	68	194	100.0	64	190
Positive control (MMS)	13.0	-	34.3	260	194	29.0	311	190
Test item	9.7	-	Culture was not continued#			Culture was not continued#
	19.4	-	75.8	45	194	95.0	46	190
	38.8	-	66.6	46	194	94.2	42	190
	77.5	-	63.3	55	194	75.4	55	190
	116.3	-	15.8	60	194	28.0	48	190
	155.0	-	18.8	36	194	13.0	53	190
Experiment II 24 h treatment			Culture I			Culture II
DMSO		+	100.0	55	181	100.0	48	174
Positive control (CPA)	3.0	+	40.2	271	181	49.8	198	174
Positive control (CPA)	4.5	+	25.4	523	181	53.0	310	174
Test item	19.4	+	82.5	52	181	59.5	46	174
	38.8	+	49.0	77	181	90.8	40	174
	77.5	+	130.4	47	181	98.8	55	174
	155.0	+	71.2	56	181	87.8	54	174
	310.0	+	11.3	41	181	7.3	44	174
	387.5	+	Culture was not continued##			Culture was not continued##
	465.0	+	Culture was not continued##			Culture was not continued##

threshold = number of mutant colonies per 10⁶cells of each solvent control plus 126

#    culture was not continued since a minimum of only four analysable concentrations is required

##   culture was not continued due to exceedingly severe cytotoxic effects

The values printed in bold are judged as invalid, since the acceptance criteria are not met (RTG < 10 % in both parallel cultures).

Applicant's summary and conclusion

Conclusions:

In conclusion it can be stated that during the mutagenicity test described and under the experimental conditions reported the test item did not induce mutations in the mouse lymphoma thymidine kinase locus assay using the cell line L5178Y in the absence and presence of metabolic activation. Therefore, Diphenyl methylphosphonate is considered to be non-mutagenic in this mouse lymphoma assay.

Executive summary:

The study was performed to investigate the potential of Diphenyl methylphosphonate to induce mutations at the mouse lymphoma thymidine kinase locus using the cell line L5178Y.

The assay was performed in two independent experiments, using two parallel cultures each. The first main experiment was performed with and without liver microsomal activation and a treatment period of 4 h. The second experiment was performed with a treatment period of 24 hours in the absence of metabolic activation and 4 hours in the presence of metabolic activation.

The highest concentration (2480 µg/mL) applied in the pre-experiment was chosen with regard to the molecular weight of the test item corresponding to a molar concentration of about 10 mM. The concentration range of the main experiments was limited by cytotoxic effects of the test item. The concentrations which have been evaluated were between 19.4 µg/mL and 310 µg/mL (experiment I, with and without metabolic activation and experiment II with metabolic activation) and between 19.4 and 155 µg/mL (experiment II without metabolic activation).

No substantial and reproducible dose dependent increase in mutant colony numbers was observed in both main experiments. No relevant shift of the ratio of small versus large colonies was observed up to the maximal concentration of the test item.

Appropriate reference mutagens were used as positive controls and showed a distinct increase in induced mutant colonies, indicating that the tests were sensitive and valid.

Conclusion

In conclusion it can be stated that during the mutagenicity test described and under the experimental conditions reported the test item did not induce mutations in the mouse lymphoma thymidine kinase locus assay using the cell line L5178Y in the absence and presence of metabolic activation.

Therefore, Diphenyl methylphosphonate is considered to be non-mutagenic in this mouse lymphoma assay.

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