Registration Dossier

Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / bone marrow chromosome aberration
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:
8 March - 24 June 1994
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Reliable study, well documented, to GLP, using a protocol equivalent to international guidelines and according to published protocols (available at the time); on related material
Cross-reference
Reason / purpose:
reference to other study

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:
OECD Guideline 475 (Mammalian Bone Marrow Chromosome Aberration Test)
Deviations:
no
GLP compliance:
yes
Type of assay:
chromosome aberration assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Type:
Constituent
Type:
Constituent
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, pale yellow, slightly viscous liquid
- Purity test date: no data
- Lot/batch No.: no data
- Expiration date of the lot/batch: no data
- Stability under test conditions: no data
- Storage condition of test material: at room temperature, protected from light

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Sprague-Dawley, Frederick, Maryland, USA
- Age at study initiation: 8-10 weeks
- Weight at study initiation: males: 284-312 g; females: 191-220 g
- Assigned to test groups randomly: yes, under following basis: distributed according to body weight
- Fasting period before study: no data
- Housing: plastic cages on woodchip bedding
- Diet (e.g. ad libitum): conventional, ad libitum
- Water (e.g. ad libitum): tap water, ad libitum
- Acclimation period: at least 5 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23 ± 3
- Humidity (%): 50 ± 20
- Air changes (per hr): "controlled environment"
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
Distilled water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: prepared immediately before use
Duration of treatment / exposure:
single dose
Frequency of treatment:
once
Post exposure period:
10, 16, 28 h for all test groups and vehicle control; also at 40 h for the vehicle control and 2000 mg/kg bw groups only
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 200, 670 and 2000 mg/kg bw
Basis:
nominal conc.
No. of animals per sex per dose:
5/sex/dose/bone collection time (i.e. 15 animals/sex/dose at 200 and 670 mg/kg bw and 20/sex/dose at 0 and 2000 mg/kg bw)
Control animals:
yes, concurrent vehicle
Positive control(s):
cyclophosphamide (structural aberrations); vinblastine sulphate (numerical aberrations)
- Justification for choice of positive control(s): cyclophosphamide is included in the list of recommended substances
- Route of administration: oral gavage
- Doses / concentrations: cyclophosphamide, 20 mg/kg bw; vinblastine sulphate, 6 mg/kg bw

Examinations

Tissues and cell types examined:
Femur bone marrow cells
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: based on the acute oral toxicity in rats; as the test material was nontoxic, the top dose was set at 2 g/kg bw and the mid and low doses were set at 1/3 and 1/10 of this.


TREATMENT AND SAMPLING TIMES (in addition to information in specific fields): rats were dosed with a volume of 10 mL/kg bw about 2 h after subcutaneous implantation of bromodeoxyuridine tablets (to allow differentiation of first- and second-division metaphases). Colchicine was given by intraperitoneal injection about 2 h before sacrifice.


DETAILS OF SLIDE PREPARATION: bone marrow cells were flushed out of the femurs, washed and fixed in methanol:acetic acid (3:1) and stored overnight at 2-6oC. The resuspended cells were dropped onto slides, air-dried, and stained by fluorescence plus Giemsa stain.


METHOD OF ANALYSIS: slides were randomly coded. A minimum of 100 first-division metaphases per animal containing 42 ± 1 centromeres were scored for numerical and structural aberrations and endoreduplication in the 10 and 16 h exposure groups. Where possible 100 second-division metaphases per animal were scored for total chromosome numbers and endoreduplication in the 28 and 40 h exposure groups.
Evaluation criteria:
A positive response was interpreted as a statistically significant dose-related increase in aberrations. Gaps were reported in the data but were not included in the total percentage of cells with aberrations, or in the average number of aberrations per cell. The percentage of cells in the vehicle controls having aberrations should not exceed 4%.
Statistics:
Fisher's exact test was used to compare the incidence of numerical or structural aberrations in the treated and vehicle control groups. Evidence of a dose-response was analysed using the Cochran-Armitage trend test.

Results and discussion

Test results
Sex:
male/female
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY in male rats
- Dose range: 2000 mg/kg bw
- Solubility: soluble in water
- Clinical signs of toxicity in test animals: no data
- Evidence of cytotoxicity in tissue analyzed: no, cell cycle times similar to controls
- Rationale for exposure: performed to determine appropriate harvest times
- Harvest times: 24 h


RESULTS OF RANGE-FINDING STUDY in Chinese hamster ovary cells
- Dose range: 20 and 40 ug/mL, with and without metabolic activation
- Solubility: soluble
- Evidence of cytotoxicity: cell cycle delay was noted in the 40 ug/mL dose level only without S9
- Rationale for exposure: performed to assist in selection of metaphase collection times in the in vivo assay
- Harvest times: 24 h


RESULTS OF DEFINITIVE STUDY
- Types of structural aberrations for significant dose levels: no structural aberrations detected in treated or vehicle control groups.
- Appropriateness of dose levels and route: appropriate doses based on acute toxicity data
- Statistical evaluation: no statistically significant increase in structural aberrations (p>0.025) or numerical aberrations (p>0.025) at any tested dose or harvest time
- Clinical signs of toxicity in test animals: no adverse effect on body weight gain was evident. In the high dose group, 15/20 males and 1/19 females had diarrhoea; one female at the high dose died during the study from undetermined causes.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative
In a GLP study equivalent to OECD Guideline 475, trisodium EDDS showed no evidence of induction of numerical or structural chromosome aberrations when administered as a single oral dose at up to 2000 mg/kg bw in a bone marrow cytogenetic assay in male and female rats.
Executive summary:

In a GLP study equivalent to OECD Guideline 475, trisodium EDDS was assessed for its ability to induce chromosome aberrations in the bone marrow cells of Sprague-Dawley rats following single oral administration. Twenty animals of each sex were given a single dose of 0 or 2000 mg/kg bw and 15/sex were administered 200 or 670 mg/kg bw by gavage (in water). Cells were harvested (from 5 animals/dose/sex) at 10, 16, 28 or (for the control and top dose groups only) 40 h after exposure. Bone marrow cells were flushed from the femurs and after fixation and staining, 100 metaphases per animal were scored for both numerical and structural aberrations.

The test substance produced no treatment-related increases in structural or numerical chromosomal aberrations at any tested dose or harvest time.

In conclusion, trisodium EDDS showed no evidence of induction of numerical or structural chromosome aberrations when administered as a single oral dose at up to 2000 mg/kg bw in a bone marrow cytogenetic assay in rats.

[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.]