Dimethyl methylphosphonate

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: DNA damage and/or repair

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: comparable to guideline standard (peer reviewed NTP protocol)

Data source

Reference

Reference Type:

publication

Title:

Unnamed

Year:

1987

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

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

Principles of method if other than guideline:

Mouse lymphma test; Mitchell et al. (1988), Evaluation of the L5178Y Mouse Lymphoma Cell Mutagenesis Assay: Methods Used and Chemicals Evaluated. Environ. Mol. Mutagen. 12 (Suppl. 13): 1-18.

GLP compliance:

no

Remarks:

well documented study peer reviewed study

Type of assay:

mammalian cell gene mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Dimethyl methylphosphonate

EC Number:

212-052-3

EC Name:

Dimethyl methylphosphonate

Cas Number:

756-79-6

Molecular formula:

C3H9O3P

IUPAC Name:

dimethyl methylphosphonate

Details on test material:

- Name of test material (as cited in study report): Dimethyl methylphosphonate; Synonyms: Fyrol DMMP; Methyl phosphonic acid, dimethyl ester; DMMP; Methanephosphonic acid dimethyl ester; Dimethyl methanephosphonate
- Molecular formula (if other than submission substance): C3H9O3P
- Molecular weight (if other than submission substance): 124.1
- Analytical purity: >98%
- Composition of test material, percentage of components: 0.11% water and 0.5-1.5% total impurities
- Lot/batch No.: EA113077
- Stability under test conditions and storage condition of test material: stability studies with the gas chromatographic indicated that dimethyl methylphosphonate was stable as a bulk chemical when kept for 2 weeks at temperatures of up to 60° C. The test substance at 0.6% in corn oil was stable when stored at room temperature for up to 7 days. A subsequent stability study performed at the study laboratory indicated that dimethyl methylphosphonate/corn oil mixtures are stable for 14 days under refrigeration. In the 2-year studies, dose mixtures were stored at 4° C for no longer than 13 days.

Method

Species / strain

Species / strain / cell type:

mouse lymphoma L5178Y cells

Additional strain / cell type characteristics:

other: mouse lymphoma L5178Y/tk+/-

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254-induced male Sprague Dawley rat liver S9 enzymes and cofactor mix

Test concentrations with justification for top dose:

0, 1100, 3670, and 11000 µg/ml (first experiment) or 0, 14300, 17600 and 22000 µg/ml (second experiment)

Vehicle / solvent:

water

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

water

True negative controls:

no

Remarks:

the test is regulary conducted in the testing facility

Positive controls:

yes

Positive control substance:

other: methyl methanesulfonate (MMS; without activation) and methylcholanthrene (MCA; with activation) were used as positive controls

Details on test system and experimental conditions:

Not provided; the NTP tests were generally conducted as followed:
METHOD OF APPLICATION: in medium

All study chemicals tested by the NTP in this assay were supplied as coded aliquots from Radian Corporation. The highest dose of test chemical was determined by solubility or toxicity, and did not exceed 5 mg/kg in the absence of dose-limiting toxicity. Mouse lymphoma L5178Y TK+/- cells were maintained at 37° C as suspension cultures in Fischer's medium supplemented with 2 mM l-glutamine, 110 ug/mL sodium pyruvate, 0.05% luronic F68, antibiotics, and heat-inactivated horse serum; normal cycling time was approximately 10 hours. To reduce the number of spontaneously occurring trifluorothymidine (TFT) resistant cells, subcultures were exposed once to medium containing thymidine, hypoxanthine, methotrexate, and glycine for one day; to thymidine, hypoxanthine, and glycine for one day; and to normal medium for 3 to 5 days. For cloning, horse serum content was increased and Noble agar was added. All treatment levels within an experiment, including concurrent positive and solvent controls, were replicated. Treated cultures contained 6 x 10 6 cells in 10 mL of medium. This volume included the S9 fraction in those experiments performed with metabolic activation.

DURATION
- Preincubation period: 10 hours; see above
- Exposure duration: incubation with the test chemical continued for 4 hours,
- Expression time (cells in growth medium): after incubation, the medium plus chemical was removed and the cells were resuspended in 20 mL of fresh medium and incubated for an additional 2 days to express the mutant phenotype. Cell density was monitored so that log phase growth was maintained.
- Selection time (if incubation with a selection agent): 12 days (see below)

SELECTION AGENT (mutation assays): after the 48-hour expression period, 3 x 106 cells were plated in medium and soft agar supplemented with TFT for selection of TFT-resistant cells (TK-/-) and in nonselective medium and soft agar to determine cloning efficiency. Plates were incubated at 37 C. in 5% CO2 for 10 to 12 days.

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency

OTHER:
At the end of incubation, colonies were counted with an automated counter. The test was initially performed without S9. If a clearly positive response was not obtained, the test was repeated using freshly prepared S9 from the livers of either Aroclor 1254-induced or non-induced male Fischer 344 rats.

Typically, 4 solvent control cultures and 3 positive control cultures were included in each experiment. Two or three cell cultures were used for each concentration of test chemical that was studied in a single experiment. Five to six concentration levels were tested per experiment. All experiments were replicated at least once.

Evaluation criteria:

Quality control factors (minimum criteria for accepting an experiment as valid) included such factors as cloning efficiencies within acceptable parameters for control and treated cultures, relative total growth of treated cultures above 1%, absence of test chemical precipitate, and two or more acceptable cultures per dose set. Both statistic responses had to be significant (P < 0.05) for a chemical to be considered capable of inducing TFT resistance; a single significant response led to a "questionable" conclusion, and the absence of both a trend and a peak response resulted in a "negative" call.

Statistics:

All data were evaluated statistically for trend and peak responses.

Results and discussion

Test results

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

positive

Remarks:

see Table 1

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

see Table 1

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Table 1: Summary of the mouse lymphoma test

Nonactivation Trial 1: Experiment Call:Positive
		Conc. µL/ml	Cloning Efficiency	Relative Total Growth	Mutant Colonies	Mutant Frequency	AVG Mutant Frequency
Vehicle Control	H2O	0	72	100	82	38	43
			63	98	86	45
			56	88	85	51
			79	114	91	39
Test Chemical		0.25	36	80	52	48	36
			82	128	67	27
			68	112	68	34
		0.5	64	98	92	48	49
			67	87	105	52
			60	78	86	48
		1	88	79	129	49	58
			58	96	98	56
			48	52	99	69
		1.5	58	51	152	87	76*
			60#	51	122	68
			93	81	199	71
		3	86	90	192	75	83*
			63	65	175	92
			75	73	188	83
		5	89	80	199	74	90*
			73	70	240	110
			74	57	190	85
Positive Control	MMS	5	71	30	640	303	387*
			35	22	546	522
			58	58	590	336
Nonactivation Trial 2: Experiment Call:Positive
		Conc. µL/ml	Cloning Efficiency	Relative Total Growth	Mutant Colonies	Mutant Frequency	AVG Mutant Frequency
Vehicle Control	H2O	0	86	90	58	23	23
			82	93	54	22
			72	86	70	32
			103	132	53	17
Test Chemical		0.25	85#	84	75	30	40*
			61#	70	112	61
			83	95	76	31
		0.5	39	59	59	50	49*
			79	76	97	41
			57#	86	94	55
		1	56	62	101	61	50*
			62#	83	74	40
		2	63#	61	109	58	53*
			75#	69	111	50
			62#	72	95	51
		3	62	63	129	69	63*
			51	51	79	51
			70#	73	142	68
		5	80	53	210	87	77*
			93	44	186	67
Positive Control	MMS	5	84	44	359	143	145*
			64	53	288	151
			70#	54	299	142
Trials Notes	Asterisks (*) indicate significant responses. r = rejected value due to quality control criteria; # = reduced sample size because of the loss of one culture dish due to contamination; MMS = methyl methanesulfonate; MCA = methylcholanthrene; DMSO = dimethylsulfoxide (solvent)