L-Aspartic acid, N,N'-1,2-ethanediylbis-

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:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

Start date 26 March 1993, finish date not given

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Well documented study report. Protocol equivalent to international guidelines, to GLP; on related material

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1994

Report date:

1994

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Test material

Test material information

Constituent 1

Reference substance name:

Trisodium EDDS

IUPAC Name:

Trisodium EDDS

Constituent 2

Reference substance name:

l-aspartic acid, N,N'-1,2-ethanediylbis-, trisodium salt

IUPAC Name:

l-aspartic acid, N,N'-1,2-ethanediylbis-, trisodium salt

Constituent 3

Reference substance name:

178949-82-1

EC Number:

605-842-8

Cas Number:

178949-82-1

IUPAC Name:

178949-82-1

Constituent 4

Reference substance name:

-

EC Number:

416-530-4

EC Name:

-

IUPAC Name:

416-530-4

Details on test material:

- Name of test material (as cited in study report): trisodium EDDS (under code)
- Molecular formula (if other than submission substance): C10-H13-N2-O8. 3Na
- Molecular weight (if other than submission substance): 358
- Smiles notation (if other than submission substance): [Na+].[Na+].[Na+].OC(=O)[C@H](CC([O-])=O)NCCNC(CC([O-])=O)C([O-])=O
- Substance type: technical product
- Physical state: clear, colourless liquid
- Isomers composition: no data
- Purity test date: no data
- Lot/batch No.: no data
- Expiration date of the lot/batch: 1 January 1998
- Stability under test conditions: no data
- Storage condition of test material: room temperature, protected from light

Method

Target gene:

not applicable

Species / strain

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

- Type and identity of media: Ham's F-12 supplemented with 10% fetal bovine serum, L-glutamine and antibiotics (penicillin and streptomycin)
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9 derived from Aroclor 1254-induced rat liver S9

Test concentrations with justification for top dose:

0, 79, 157, 313, 625, 1250, 2500 and 5000 µg/mL without S9 (6 and 18 h exposure without S9; 6 h exposure with S9)
0, 5, 10, 20, 40, 79, 157, 313, 625 and 1250 µg/mL without S9 (42 h exposure)

Vehicle / solvent:

Distilled water

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: N-methyl-N'-nitro-N-nitrosoguanidine

Remarks:

2 ug/mL, without S9

Controls 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Remarks:

30 ug/mL, with S9

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
Exposure period (with and without metabolic activation): 6 h (with a 12 h recovery period)
Exposure period (without metabolic activation): 18 and 42 h

Fixation time: 18 or 42 h (from start of exposure to harvesting of the cells)

SPINDLE INHIBITOR (cytogenetic assays): colcemid added to the cultures 2 h before harvest
STAIN (for cytogenetic assays): Giemsa

NUMBER OF REPLICATIONS: duplicate flasks prepared

NUMBER OF CELLS EVALUATED: 200 per dose level (100/flask)

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cell viability determined by trypan blue exclusion

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

Evaluation criteria:

Gaps were noted, but not included in the total percentage of cells with aberrations or in the frequency of structural aberrations per cell. In the negative controls no cell must have more than two aberrations and the overall frequency must not be greater than 6%. A statistically significant frequency must be evident in the positive controls.

Statistics:

Fischer's exact test was used to compare the percent aberrant cells at each treatment level to that of the controls. The Cochran-Armitage trend test was used to determine dose responsiveness.

Results and discussion

Test results

Test results 1

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

625 µg/mL

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

625 µg/mL (study 1); 1250 µg/mL (study 2)

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 3

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

313 µg/mL (study 2)

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 4

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

without

Genotoxicity:

positive

Remarks:

numerical aberrations

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS:
none


RANGE-FINDING/SCREENING STUDIES: to determine cytotoxicity at 6, 18 and 42 h exposures without S9 and 6 h exposure with S9.


COMPARISON WITH HISTORICAL CONTROL DATA: yes (details not given)

Remarks on result:

other: other: 6 h exposure

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

There was a statistically significant increase in numerical aberrations (p < 0.025) at 20 and 40 ug/mL in study 1 and 2, respectively, after 42 h exposure. Although a statistically significant increase (p < 0.025) was also seen in structural aberrations in study 2 at 40 ug/mL at this exposure time, the percentage increase was within the acceptable range of historical control values, and therefore was considered by the investigators not to be biologically relevant.

Summary of cytogenetic analysis after 42 h exposure without metabolic activation

Treatment	No. cells scored	% Aberrant cells		Structural aberrations per cell
		Numerical	Structural
Study 1
Untreated	200	1.0	3.5	0.035 ± 0.184
Water (vehicle)	200	2.0	2.5	0.025 ± 0.157
Trisodium EDDS  5 ug/mL	200	1.0	1.0	0.010 ± 0.100
10 ug/mL	200	0.5	4.0	0.045 ± 0.231
20 ug/mL	200	27.5	5.5	0.070 ± 0.325
MNNG (2 ug/mL)	200	2.5	18.5	0.385 ± 1.193
Study 2
Untreated	200	0.5	1.0	0.020 ± 0.199
Water (vehicle)	200	0.5	0.0	0.000 ± 0.000
Trisodium EDDS 10 ug/mL	200	0.0	0.5	0.000 ± 0.000
20 ug/mL	200	6.5	0.0	0.000 ± 0.000
40 ug/mL	200	13.5	6.0	0.090 ± 0.416
MNNG (2 ug/mL)	200	1.0	42.0	2.370 ± 3.708

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
positive without metabolic activation after 42 h exposure
negative without metabolic activation after 6 or 18 h exposure
negative with metabolic activation after a 6 h exposure

In a GLP study, equivalent to OECD Guideline 473, trisodium EDDS induced a statisically significant increase in chromosome numbers and structural aberrations (although the latter effect was not considered biologically relevant) in cultured Chinese hamster ovary cells after a 42-h exposure in the absence of S9. No increases in numerical or structural chromosome aberrations were seen after 6 h or 18-h exposures without S9, or after a 6-h incubation in the presence of S9.

Executive summary:

In a GLP study, equivalent to OECD Guideline 473, trisodium EDDS was assessed for its ability to induce chromosome aberrations in Chinese hamster ovary (CHO) cells. CHO cells were incubated with seven or more dose levels of the test sustance for either 6, 18 or 42 h without S9, or for 6 h with S9. The concentration ranges were determined on the basis of cytotoxicity studies using the same exposure times; 5 mg/mL was selected as the highest dose for the 6 and 18 h exposures and 1.25 mg/mL for the 42 h exposure. After a total incubation period of either 18 or 42 h, cells were fixed and stained and the three highest doses with 200 scorable metaphases were selected for evaluation of chromosome aberrations.

A statistically significant increase in numerical aberrations was evident after an exposure of 42 h without S9. Although a statistically significant increase was also seen in structural aberrations at 40 ug/mL at this exposure time, the percentage increase was within the acceptable range of historical control values, and therefore was considered by the investigators not to be biologically relevant. No significant increase in either numerical or structural aberrations was detected after 6 or 18 h without S9, or after 6 h with S9.

Overall, trisodium EDDS caused chromosome aberrations in an in vitro mammalian cytogenicity assay.

[Data on trisodium EDDS is considered relevant to use for understanding the potential genotoxicity of EDDS acid, and is acceptable for using as read-across information.]