Pentasodium (carboxylatomethyl)iminobis(ethylenenitrilo)tetraacetate

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

23 March - 17 July 1987

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: No data on batch number and composition; basic data given, comparable to guidelines/standards (max reliability score can be 2)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1987

Report date:

1987

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

yes

Remarks:

exposure duration with S9 mix was 2 hours rather than 3-6 hours

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Test material

Test material information

Constituent 1

Reference substance name:

7216-95-7

Cas Number:

7216-95-7

IUPAC Name:

7216-95-7

Details on test material:

- Name of test material (as cited in study report): GLYCINE N-N BIS [2-[BIS(CARBOXYMETHYL) AMINO] ETHYL] - PENTA
POTASSIUM SALT
- Physical state: white solid
- Analytical purity: not indicated
- Impurities (identity and concentrations): not indicated
- Lot/batch No.: not indicated
- Expiration date of the lot/batch: not indicated
- Storage condition of test material: room temperature in the dark
- Date of receipt: not indicated

Method

Target gene:

Structural chromosomal aberrations

Species / strain

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

- Type and identity of media: in Hams F12 medium supplemented with 5% foetal calf serum
- Properly maintained: yes, at 37 degr. C in a humid atmosphere
containing 5% CO2
- Periodically checked for Mycoplasma contamination: not indicated

Additional strain / cell type characteristics:

other: strain K1-BH4

Metabolic activation:

with and without

Metabolic activation system:

S9 mix (rat liver Aroclor 1254 induced)

Test concentrations with justification for top dose:

without S9 mix: 0, 1, 2, 4, 8 µg/ml
with S9 mix: 0, 25, 125, 200, 250 µg/ml

Vehicle / solvent:

Vehicle: sterile distilled water

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

distilled water

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

with S9 mix

Migrated to IUCLID6: 20 µg/ml

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

distilled water

True negative controls:

no

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

without S9 mix

Migrated to IUCLID6: 0.4 µg/ml

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: 24 hours
- Exposure duration: 20 hours (- S9 mix) 2 hours with test compound + 18 hours without (+ S9 mix)
- Expression time (cells in growth medium): 20 hours
- Fixation time (start of exposure up to fixation or harvest of cells): 23 hours

SPINDLE INHIBITOR (cytogenetic assays): colchicine (0.25 µg/ml), 3 hours before end of 20-h period
STAIN (for cytogenetic assays): 10% Giemsa

NUMBER OF REPLICATIONS: two cultures per concentration and for the positive control; four cultures for the solvent
control

NUMBER OF CELLS EVALUATED: ca. 100 metaphases for each culture (200 in total for test and positive control; 400 for solvent
control), with normally a maximum of 25 from each slide

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index

OTHER EXAMINATIONS:
- Determination of polyploidy: not reported
- Determination of endoreplication: not reported

Evaluation criteria:

Statistically sgnificant increase above control values.

Statistics:

Fisher's test

Results and discussion

Test results

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: none
- Effects of osmolality: none
- Evaporation from medium: none
- Water solubility: good
- Precipitation: none

RANGE-FINDING/SCREENING STUDIES: due to very high toxicity in the first test withouit S9 - mix, the test was repeated at lower
concentrations (see above).

Any other information on results incl. tables

Results

Compound	Concentration (µg/ml)	Number of cells examined	No. aberrant cells (excl. gaps) (%)	No. aberrant cells (incl. gaps) (%)	Mitotic index (mean %)
- S9 mix
Vehicle	0	400	4 (1)	4 (1)	9.1
Test	1 2 4 8	200 200 200 151	2 (1) 1 (0.5) 0 (0) 0 (0)	1 (0.5) 0 (0) 1 (0.5) 0 (0)	10.3 10.1 8.9 1.0
Mitomycin C	0.4	177	93 (52.5) ***	93 (52.5) ***
+ S9 mix
Vehicle	0	400	4 (1)	6 (1.5)	8.4
Test	25 125 200 250	200 200 200 200	2 (1) 1 (0.5) 0 (0) 0 (0)	2 (1) 2 (1) 0 (0) 0 (0)	9.2 6.8 8.4 8.8
Cyclophosphamide	20	200	49 (24.5) ***	49 (24.5) ***

Statistics: Fisher’s test; *** p0.001

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

The test compound did not show clastogenic activity in this in vitro cytogenetic system.

Executive summary:

Glycine N-N bis[2 -[bis(carboxymethyl)amino]ethyl]-pentapotassium salt was tested in vitro to determine whether it would cause chromosomal aberrations in a mammalian cell line derived from Chinese hamster ovary tissue. The cells were routinely grown and subcultured in tissue culture medium at 37'C in a humid atmosphere containing 5% carbon dioxide. They were incubated with the test compound both with and without supplementary metabolic activation (rat S-9 mix).

A preliminary toxicity test was carried out to assess the effect of the compound on the mitotic index of cultured CHO cells. In the absence of S-9 mix, all of the concentrations tested in the preliminary test proved extremely toxic. A second toxicity test was carried out using a lower range of concentrations. The results of this test indicated that the concentration required to reduce the mitotic index by 50% of control levels (EC50) was approximately 8 µg/ml. This concentration was chosen as the highest dose level for metaphase analysis, with additional dose levels of 1, 2 and 4 µg/ml. In the presence of S-9 mix, the results of the preliminary toxicity test suggested an EC50 of approximately 300 µg/ml. This concentration was used as the highest dose level for metaphase analysis, and additional cultures were also treated with 30, 150, 200 and 250 µg/ml. When slides from this test were examined, 300 µg/ml was found to be extremely toxic. The next two dose levels, 250 and 200 µg/ml, showed erratic toxicity and poor reproducibility between duplicate cultures. The test was repeated using concentrations of 250, 200, 125 and 25 µg/ml, which were subsequently analysed for chromosome damage.

Cultures treated with Glycine N-N bis[2-[bis(carboxymethyl)amino] ethyl]-pentapotassium salt showed no significant increases in the incidence of chromosomal aberrations at any dose level, in either the absence or presence of supplementary metabolic activation.

Both positive control compounds mitomycin C and cyclophosphamide, caused large, statistically highly significant increases in the incidence of chromosomal damage, thus demonstrating the sensitivity of the test system and the efficacy of the S-9 mix.

It is concluded that Glycine N-N bis[2-[bis(carboxymethyl)amino] ethyl]-pentapotassium salt has shown no evidence of clastogenic activity in this in vitro cytogenetic test system.