Pentapotassium ({[(hydroxyphosphinato)methyl](phosphonatomethyl)nitroryl}methyl)phosphonate

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:

1994-02-22 to 1994-05-12

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1994

Report date:

1994

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

1981

Deviations:

yes

Remarks:

experiment was not repeated to confirm negative result, exposure period without metabolic activation 20 hours

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:

pentapotassium {[(hydrogen phosphonomethyl)(phosphonomethyl)-oxo-$l^{5}-azanyl]methyl}phosphonate

EC Number:

700-903-6

Cas Number:

255830-15-0

Molecular formula:

C3H7K5NO10P3

IUPAC Name:

pentapotassium {[(hydrogen phosphonomethyl)(phosphonomethyl)-oxo-$l^{5}-azanyl]methyl}phosphonate

Details on test material:

- Name of test material (as cited in study report): Briquest 3010-25K; Potassium salt of N-oxide nitrilotris(methylene phosphonic acid)
- Substance type: solution of monoconstituent substance
- Physical state: pale straw-coloured liquid
- Analytical purity: 40%
- Storage condition of test material: room temperature

Method

Species / strain

Species / strain / cell type:

lymphocytes: peripheral human

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254 - induced rat liver S9

Test concentrations with justification for top dose:

393.75-1575 µg/ml (without metabolic activation), 787.5-3150 µg/ml (with metabolic activation). This is equivalent to an active acid content of approximately 250-1000 500-2000 µg/ml and 500-2000 µg/ml.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: water. Hepes buffer was added to maintain pH balance.

- Justification for choice of solvent/vehicle: none given in report.

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

without metabolic activation: 500 µg/ml

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

without metabolic activation: 25 µg/ml

Details on test system and experimental conditions:

ACTIVATION: Aroclor induced rat liver S9 mix included NADP, G6P, KCl and MgCl₂ in Phosphate buffer, final concentration S9 mix in cultures was 1%

METHOD OF APPLICATION: in medium;

DURATION
- Preincubation period:
- Exposure duration: 4 hours (with metabolic activation),
- Expression time (cells in growth medium): 16 hours (with metabolic activation),
- Fixation time (start of exposure up to fixation or harvest of cells): 20 hours

SPINDLE INHIBITOR (cytogenetic assays): demecolcine (2 h before harvest)
STAIN (for cytogenetic assays): Giesma

NUMBER OF REPLICATIONS: duplicate cultures for each dose level

NUMBER OF CELLS EVALUATED: 100 well-spread metaphases from each culture were scored for gaps, breaks or rearrangements. 2000 lymphocyte cell nuclei were counted and the number of cells in metaphase recorded and expressed as mitotic index and percentage of vehicle control values.

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; other: percentage of vehicle control values

OTHER EXAMINATIONS:
- Determination of polyploidy: yes
- Determination of endoreplication: no

Evaluation criteria:

A positive response was recorded if the percentage of cells with aberrations markedly exceeded the concurrent vehicle control. For modest increases a dose-response relationship is generally required.

Statistics:

Fisher's exact test.

Results and discussion

Test results

Species / strain:

lymphocytes: peripheral human

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

1575 µg/ml (without metabolic activation)

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: pH was controlled with buffer
- Effects of osmolality: not recorded

COMPARISON WITH HISTORICAL CONTROL DATA: The vehicle control cultures gave values of aberrations within the range of historical controls.

Any other information on results incl. tables

Results of Chromosome Aberration Assay

Test material: BRIQUEST 3010-25K

Harvest time: 20 hours

 

Treatment Group	Replicate Identification	No. of Cells Scored	Total Gaps	Chromatid		Chromosome		Others	Total Aberrations		Aberrant Cells		Mean Mitotic Index
				Breaks	Exchanges	Breaks	Exchanges	X	(+Gaps)	(-Gaps)	(+Gaps)	(-Gaps)
Without Metabolic Activation
Solvent Control	Total	200	3	1	0	0	0	0	4	7	4	1	8.05
393.75 (µg/ml)	Total	192	1	0	0	0	0	0	1	0	1	0	6.15
787.5 (µg/ml)	Total	200	3	0	0	0	1	0	4	1	4	1	4.73
1575 (µg/ml)	Total	200	1	0	0	1	0	0	2	1	2	1	3.25
Positive Control EMS 500 (µg/ml)	Total	200	30	41	0	14	0	0	85	55	57***	38***	4.58
With Metabolic Activation
Solvent Control	Total	200	3	2	0	0	0	0	5	2	5	2	5.05
787.5 (µg/ml)	Total	200	4	0	0	1	0	0	5	1	5	1	4.15
1575 (µg/ml)	Total	200	4	2	0	0	0	0	6	2	6	2	4.15
3150 (µg/ml)	Total	200	0	0	0	1	0	0	1	1	1	1	4.03
Positive Control CP 25 (µg/ml)	Total	200	24	13	1	4	0	1	42	18	30***	17***	1.70

X = > 10 aberrations per cell (not included in total aberrations)

*** = p < 0.001

Applicant's summary and conclusion

Conclusions:

In an in vitro cytogenicity / chromosome aberration study in mammalian cells, ATMP-N-oxide-xK was tested according to OECD TG 473 and in compliance with GLP. No increase in the number of cells with aberrations was observed when peripheral human lymphocytes were tested up to cytotoxic concentrations. Exposure lasted for 4 hours in the presence of metabolic activation and for 20 hours in the absence of metabolic activation. Appropriate solvent and positive controls were included and gave expected results. It is concluded that the test substance is negative for the induction of chromosome aberrations under the conditions of the test.