[nitrilotris(methylene)]trisphosphonic acid, ammonium salt

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

migrated information: read-across based on grouping of substances (category approach)

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study was conducted according to a test protocol that is comparable to the appropriate OECD test guideline, and in compliance with GLP. Read-across to the registered substance is considered scientifically justified.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1986

Report date:

1988

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

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

Deviations:

yes

Remarks:

: not tested in the absence of metabolic activation

Principles of method if other than guideline:

Method: Clive et al

GLP compliance:

yes

Type of assay:

mammalian cell gene mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Nitrilotrimethylenetris(phosphonic acid)

EC Number:

229-146-5

EC Name:

Nitrilotrimethylenetris(phosphonic acid)

Cas Number:

6419-19-8

IUPAC Name:

[nitrilotris(methylene)]tris(phosphonic acid)

Method

Species / strain

Species / strain / cell type:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Metabolic activation system:

Aroclor induced rat liver S9

Test concentrations with justification for top dose:

0-1.2 µl Dequest 2000/ml (calculated by previous reviewer as 0-0.78 µl active acid/ml). These concentrations were positive in the previous experiment.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: water
- Justification for choice of solvent/vehicle: no information

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

3-methylcholanthrene

Remarks:

with activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 4 h
- Expression time (cells in growth medium): 2 days
- Selection time (if incubation with a selection agent): 11-12 days
- Fixation time (start of exposure up to fixation or harvest of cells): 2 days


SELECTION AGENT (mutation assays): 5ug/ml trifluorothymidine

NUMBER OF REPLICATIONS: duplicate cultures

NUMBER OF CELLS EVALUATED: 6x10E06 (1st experiment); 9x10E06 (2nd experiment)

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth (RTG)

Evaluation criteria:

Positive result: Dose-related increase in number of mutant colonies and mutant frequency at one or more concentrations of greater than or equal to twice control levels with an RTG of greater than 10%.

Statistics:

No statistical analysis of results was carried out.

Results and discussion

Test results

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not determined

Remarks:

Concentrations based on data from previous experiment

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: test solution was neutralised to eliminate pH effects. Slight drop in pH seen at highest concentration but approximate fall in 0.08pH units only
- Precipitation: white precipitate reported at concentrations greater than 0.61µl/ml

RANGE-FINDING/SCREENING STUDIES: not conducted as this is a follow-up experiment

COMPARISON WITH HISTORICAL CONTROL DATA: no data

Any other information on results incl. tables

In the first experiment all treated cultures had higher RTGs than the control culture and a dose-related increase in mutant frequency was seen (see Table 1). The experiment was discounted because of the low control growth.

Table 1: Results of Mammalian Mutagenicity assay 1 with tester strain L5178Y in presence of metabolic activation

Concentration µl/ml	Mutant frequency x10E-06	RTG %
0*	23	100
0.49	22	195
0.51	33	134
0.77	48	149
0.96	59	133
1.2	58	121

In the second experiment no increases in mutant frequency and no cytotoxicity were seen. It had been proposed that a selective advantage of mutants in the presence of test substance was the reason for the increase in revertants observed in the previous experiment. No effects on levels of spontaneous mutants were seen when cells were plated immediately after treatment. This finding does not support the proposed reason for the previously observed positive result.

Table 2: Results of Mammalian Mutagenicity assay 2 with tester strain L5178Y in presence of metabolic activation

Concentration µl/ml	Mutant frequency x10E-06	RTG %
0*	101	100
0.61	91	100
0.77	112	74
0.96	98	92
1.2	100	88
1.5	112	103
Positive control	659	46

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative with metabolic activation

A neutralized solution of ATMP acid was tested for mutagenicity to mammalian cells under GLP. No increase in the number of revertants was observed in the presence of metabolic activation when tested up to the solubility limit. The test results indicate that the positive findings in the previous experiment with un-neutralised ATMP acid were an artefact of pH. It is concluded that ATMP acid is not mutagenic to mammalian cells under the conditions of this test.