Methylphosphonic acid

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2013-01-21 to 2013-02-25

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

The following alteration from the guidelines was performed:
The treatment of lymphocytes was started approx. 72 hours after mitogenic stimulation.

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Test material

Test material information

Constituent 1

Reference substance name:

Methylphosphonic acid

EC Number:

213-607-2

EC Name:

Methylphosphonic acid

Cas Number:

993-13-5

Molecular formula:

CH5O3P

IUPAC Name:

methylphosphonic acid

Details on test material:

- Name of test material (as cited in study report): Methyl phosphonic acid 70%
- Molecular weight (if other than submission substance): 96.02 g/mol
- Analytical purity: 70 % in water
- Lot/batch No.: 0021634600
- Expiration date of the lot/batch: November 27, 2015
- Stability under test conditions: Stable in water
- Storage condition of test material: At room temperature
- CAS Number: 993-13-5

Method

Species / strain

Species / strain / cell type:

lymphocytes: human

Details on mammalian cell type (if applicable):

- Type and identity of media: Dulbeccos's modified Eagle's medium/Ham's F12 medium
- Properly maintained: yes

Metabolic activation:

with and without

Metabolic activation system:

rat liver S9

Test concentrations with justification for top dose:

With metabolic activation:
Experiment I: 8.9, 15.6, 27.3, 47.8, 83.6, 146.3, 256.0, 448.0, 784.0, 1372.0 µg/mL
Experiment II: 83.6, 146.3, 256.0, 448.0, 784.0, 1372.0 µg/mL

Without metabolic activation:
Experiment I: 8.9, 15.6, 27.3, 47.8, 83.6, 146.3, 256.0, 448.0, 784.0, 1372.0 µg/mL
Experiment II: 8.9, 15.6, 27.3, 47.8, 83.6, 146.3, 256.0, 448.0, 784.0, 1372.0 µg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Deionised water
- Justification for choice of solvent/vehicle: solubility and relatively low cytotoxicity in accordance to the OECD Guideline 473

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

cyclophosphamide

Details on test system and experimental conditions:

Two independent experiments were performed. In Experiment I the exposure period was 4 hours with and without metabolic activation. In Experiment II the exposure period was 4 hours with S9 mix and 22 hours without S9 mix. The chromosomes were prepared 22 hours after start of treatment with the test item. Evaluation of two cultures per dose group.

METHOD OF APPLICATION: in culture medium

DURATION
- Exposure duration: 4 hours (+/- S9 mix) and 22 hours (- S9 mix)
- Fixation time (start of exposure up to fixation or harvest of cells): 22 hours

SPINDLE INHIBITOR (cytogenetic assays): Colcemid
STAIN (for cytogenetic assays): Giemsa

NUMBER OF REPLICATIONS: about 1.5

NUMBER OF CELLS EVALUATED: 100 per culture, except for the positive control in Experiment I in the presence of S9 mix, where only 50 metaphases were evaluated.

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index

Evaluation criteria:

Evaluation of the cultures was performed (according to standard protocol of the "Arbeitsgruppe der Industrie, Cytogenetik") using NIKON microscopes with 100x oil immersion objectives. Breaks, fragments, deletions, exchanges, and chromosome disintegrations were recorded as structural chromosome aberrations. Gaps were recorded as well but not included in the calculation of the aberration rates. 100 well spread metaphases per culture were scored for cytogenetic damage on coded slides, except for the positive control in Experiment I in the presence of S9 mix, where only 50 metaphases were evaluated.
Only metaphases with characteristic chromosome numbers of 46 ± 1 were included in the analysis. To describe a cytotoxic effect the mitotic index (% cells in mitosis) was determined.

Statistics:

Statistical significance was confirmed by means of the Fisher´s exact test (p <0.05).

Results and discussion

Test results

Species / strain:

lymphocytes:

Metabolic activation:

with and without

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

The test item methylphosphonic acid , dissolved in deionised water, was assessed for its potential to induce chromosomal aberrations in human lymphocytes in vitro in the absence and presence of metabolic activation by S9 mix. Two independent experiments were performed. In Experiment I the exposure period was 4 hours with and without S9 mix. In Experiment II the exposure period was 4 hours with S9 mix and 22 hours without S9 mix. The chromosomes were prepared 22 hours after start of treatment with the test item. In each experimental group two parallel cultures were analysed. 100 metaphases per culture were scored for structural chromosomal aberrations, except for the positive control in Experiment I in the presence of S9 mix, where only 50 metaphases were evaluated. 1000 cells were counted per culture for determination of the mitotic index. The highest treatment concentration in this study, 1372.0 µg/mL (approx. 10 mM) was chosen with regard to the molecular weight and the purity (70 % in water) of the test item and with respect to the OECD Guideline for in vitro mammalian cytogenetic tests. No visible precipitation of the test item in the culture medium was observed. No relevant influence on osmolarity or pH value was observed. In this study, in the absence as well as in the presence of S9 mix, no biologically relevant cytotoxicity indicated by clearly reduced mitotic indices could be observed. In both experiments, in the absence and presence of S9 mix, no biologically relevant increase in the number of cells carrying structural chromosome aberrations was observed. The aberration rates of the cells after treatment with the test item (0.5 – 1.5 % aberrant cells, excluding gaps) were similar to the range of the solvent control values (0.5 – 1.5 % aberrant cells, excluding gaps) and within the range of the laboratory historical solvent control data. No evidence of an increase in polyploid metaphases was noticed after treatment with the test item as compared to the control cultures. In both experiments, either EMS (550.0 or 770.0 µg/mL) or CPA (15.0 or 20.0 µg/mL) were used as positive controls and showed distinct increases in cells with structural chromosome aberrations.

Remarks on result:

other: strain/cell type: human lymphocytes

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Summary of results of the chromosomal aberration study with Methyl phosphonic acid 70%

Exp.		Preparation			Test item	Mitotic indices	Aberrant cells
		interval			concentration	in %	in %
					in µg/mL	of control	incl. gaps*	excl. gaps*	carrying exchanges
	Exposure period 4 hrs without S9 mix
I		22 hrs			Solvent control^1	100.0	1.0	0.5	0.0
					Positive control^2	51.6	24.5	24.0^S	11.5
					448.0	91.3	0.5	0.5	0.0
					784.0	74.6	1.0	0.5	0.0
					1372.0	95.6	2.0	1.5	0.0
			Exposure period 22 hrs without S9 mix
II		22 hrs			Solvent control^1	100.0	0.5	0.5	0.0
					Positive control^3	46.6	20.5	20.0^S	4.5
					448.0	82.5	1.0	1.0	0.0
					784.0	107.7	1.5	1.5	0.0
					1372.0	112.3	1.0	1.0	0.0
				Exposure period 4 hrs with S9 mix
I		22 hrs			Solvent control^1	100.0	1.5	1.5	0.0
					Positive control^4#	49.9	18.0	18.0^S	1.0
					448.0	93.4	1.0	1.0	0.0
					784.0	96.7	0.5	0.5	0.0
					1372.0	94.5	1.5	1.5	0.0
II		22 hrs			Solvent control^1	100.0	1.5	1.0	0.0
					Positive control^5	49.6	14.5	13.5^S	2.5
					448.0	85.4	1.0	1.0	0.0
					784.0	115.4	2.5	1.5	0.0
					1372.0	90.0	1.5	1.5	0.0

*  Including cells carrying exchanges

^#   Evaluation of 50 metaphases per culture due to strong clastogenic effects

^S  Aberration frequency statistically significant higher than corresponding control values

¹   Deionised water 10.0 % (v/v)

²     EMS     550.0 µg/mL

³        770.0 µg/mL

⁴     CPA       20.0 µg/mL

⁵   CPA       15.0 µg/mL

 

Applicant's summary and conclusion

Conclusions:

Methylphosphonic acid 70 % is considered to be non-clastogenic in this chromosome aberration test, when tested up to the highest required concentration.

Executive summary:

The test item methylphosphonic acid 70%, dissolved in deionised water, was assessed for its potential to induce structural chromosomal aberrations in human lymphocytes in vitro in two independent experiments. The following study design was followed:

	Without S9 mix		With S9 mix
	Exp. I	Exp. II	Exp. I & II
Exposure period	4 hrs	22 hrs	4 hrs
Recovery	18 hrs	-	18 hrs
Preparation interval	22 hrs	22 hrs	22 hrs

In each experimental group two parallel cultures were analysed. Per culture 100 metaphases were evaluated for structural chromosomal aberrations, except for the positive control in Experiment I in the presence of S9 mix, where only 50 metaphases were evaluated. The highest applied concentration in this study (1372.0 µg/mL of the test item, approx. 10 mM) was chosen with regard to the molecular weight and the purity (70 % in water) of the test item and with respect to the current OECD Guideline 473. Dose selection of the cytogenetic experiment was performed considering the toxicity data in accordance with OECD Guideline 473. In the absence and presence of S9 mix, no cytotoxicity was observed up to the highest applied concentration. In both independent experiments, neither a statistically significant nor a biologically relevant increase in the number of cells carrying structural chromosomal aberrations was observed after treatment with the test item. No evidence of an increase in polyploid metaphases was noticed after treatment with the test item as compared to the control cultures. Appropriate mutagens were used as positive controls. They induced statistically significant increases in cells with structural chromosome aberrations.