Nitrilotriacetonitrile

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

May-August 2009

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Well conducted and reported study, according to GLP

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2009

Report date:

2009

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Nitrilotriacetonitrile

EC Number:

230-804-9

EC Name:

Nitrilotriacetonitrile

Cas Number:

7327-60-8

Molecular formula:

C6H6N4

IUPAC Name:

2-[bis(cyanomethyl)amino]acetonitrile

Details on test material:

Appearance: off-white crystalline powder
Purity: 99.6 +/- 1.5%

Method

Target gene:

reverse mutations at the histidine locus and tryptophan locus

Species / strain

Species / strain 1

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254-induced rat liver homogenate

Test concentrations with justification for top dose:

In initital test: 1.60, 5.00, 16.0, 50.0, 160, 500, 1600, 5000 µg/plate with and without S9-mix
In confirmatory test: 50.0, 160, 500, 1600, 5000 µg/plate with and without S9-mix

Vehicle / solvent:

DMSO, purity 99.94%

Controls

Controls 1

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Positive control substance:

other: 2-nitrofluorene, sodium azide, ICR-191, 4-nitroquinoline-N-oxide

Remarks:

without S9-mix

Controls 2

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

no

Remarks:

benzo(a)pyrene, 2-aminoanthracene

Remarks:

with S9-mix

Details on test system and experimental conditions:

Plating Procedures: Each plate was labeled with a code that identified the test article, test phase, tester strain, activation condition and dose level. The S9 mix and dilutions of the test article were prepared immediately prior to their use. Treatments were performed by adding 100 µL tester strain and 50 µL test or control
article to 2.5 mL molten selective top agar (maintained at 45 ± 2°C). After the required components had been added, the mixture was vortexed and overlaid onto the surface of 25 mL minimal bottom agar in a 15 x 100 mm petri dish. After the overlay solidified, the plates were inverted and incubated for 52 ± 4 hours at 37 ± 2°C. Cultures were treated in the presence of S9 in an identical manner, except using 2.0 niL undiluted molten selective top agar and adding 500 µL S9 mix.

Scoring the Plates: Plates which were not evaluated immediately following the incubation period were held at >0 to 10°C until such time that colony counting and bacterial background lawn evaluation could take place.

Bacterial Background Lawn Evaluation: The condition of the bacterial background lawn was evaluated macroscopically and microscopically (using a dissecting microscope) for indications of cytotoxicity and test article precipitate. Evidence of cytotoxicity was scored relative to the vehicle control plate and was recorded along with the revertant counts for all plates at that dose level. Lawns were scored as normal (N), reduced (R), obscured by precipitate (0), macroscopic precipitate present (P), absent (A), or enhanced (E); contaminated plates (C) were also noted.

Counting Revertant Colonies: Revertant colonies were counted by automated colony counter and by hand.

Evaluation criteria:

See below at remarks

Statistics:

Not done

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

NTAN was found to form a transparent, non-viscous, colorless solution in DMSO at a concentration of approximately 100 mg/mL, which was the highest dose
formulation prepared for treatment; it remained freely soluble at all succeeding lower dilutions prepared assay.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

Under the conditions of this test, NTAN did not show mutagenic effects.

Executive summary:

The objective of this study was to evaluate the test article, NTAN (nitrilotriacetonitrile; CAS No.: 7327-60-8), and/or its metabolites for their ability to induce reverse mutations at the histidine locus in several strains of Salmonella (TA98, TAI00, TA1535, and TA1537), and at the tryplophan locus of E. coli strain WP2uvrA, in the presence or absence of an exogenous mammalian metabolic activation system (S9).

NTAN was evaluated in the initial mutagenicity assay, in all five tester strains, at doses of 1.60, 5.00, 16.0, 50.0, 160, 500, 1600, and 5000 µg/plate with and without S9. All doses of the test article, as well as the concurrent positive and vehicle controls were evaluated in duplicate plates. Normal growth was observed in all five tester strains, and the test article was found to be freely soluble in the aqueous top agar, at all doses evaluated with and without S9. Revertant frequencies for all doses ofNTAN, in all tester strains with and without S9, approximated those observed in the concurrent vehicle control cultures.

NTAN was re-evaluated in the confirmatory mutagenicity assay at doses of 50.0, 160, 500, 1600, and 5000 µg/plate with and without 89. All doses of the test article, as well as the concurrent positive and vehicle controls, were evaluated in triplicate plates. Normal growth again was observed in all five tester strains, and the test article again was found to be freely soluble, at all doses evaluated with and without S9. Revertant frequencies for all doses of NTAN, in all tester strains with and without 89, again approximated control values. All positive and vehicle control values were within acceptable ranges, and all criteria for a valid study were met.

These results indicate NTAN was negative in the Bacterial Reverse Mutation Assay with a Confirmatory Assay under the conditions, and according to the criteria, of the test protocol.