Disodium 2-hydroxyethyliminodi(acetate)

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From Feb. 20, 1986 to March 07, 1986

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Study well documented, followed methods comparable to guideline with deviation.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1986

Report date:

1986

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

No strain such as Salmonella typhimurium TA102 or E.coli WP2 was included to detect cross-linking mutagens

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Disodium 2-hydroxyethyliminodi(acetate)

EC Number:

205-187-4

EC Name:

Disodium 2-hydroxyethyliminodi(acetate)

Cas Number:

135-37-5

Molecular formula:

C6H9NO5.Na2

IUPAC Name:

disodium 2-hydroxyethyliminodi(acetate)

Constituent 2

Reference substance name:

Hydroxyethyliminodiacetic acid, disodium salt

IUPAC Name:

Hydroxyethyliminodiacetic acid, disodium salt

Test material form:

other: Yellow liquid

Details on test material:

- Name of test material: Hydroxyethyliminodiacetic acid
- TSIN: E-2748.03
- Substance type: Pure active substance
- Physical state: Yellow liquid
- Storage condition of test material: At room temperature
- Solubility: Soluble in water

Method

Target gene:

Histidine locus of following Salmonella Typhimurium strains:
TA 1535: his G46
TA 1537: his C3076
TA 1538: his D3052
TA 98: his D3052
TA 100: his G46

Species / strain

Species / strain 1

Species / strain / cell type:

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

Additional strain / cell type characteristics:

other: Faulty lipopolysaccharide envelope (rfa -), defective DNA repair capacity (uvr B -) and ampicllin resistance gene in TA 100 and TA 98

Species / strain 2

Species / strain / cell type:

S. typhimurium TA 1538

Additional strain / cell type characteristics:

other: Faulty lipopolysaccharide envelope (rfa -) and defective DNA repair capacity (uvr B -)

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254-induced rat liver S9

Test concentrations with justification for top dose:

Preliminary toxicity test: 0, 5, 50, 500 and 5000 µg/plate
Mutation test (initial and confirmatory): 0, 50, 150, 500, 1500, 5000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Water
- Justification for choice of solvent/vehicle: The test substance is water soluble

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

water

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

N-ethyl-N-nitro-N-nitrosoguanidine

Remarks:

Without metabolic activation (2-nitrofluorene for TA1538 and TA98 at 2 µg/plate and 1 µg/plate respectively; 9-aminoacridine at 80 µg/plate for TA1537; N-ethyl-N-nitro-N-nitrosoguanidine for TA100 and TA 1535 at 3 µg/plate and 5 µg/plate respectively )

Controls 2

Positive control substance:

other: 2-Aminoanthracene for strains TA1535 (at 2 µg/plate), TA1537 (at 2 µg/plate), TA1538 (at 0.5 µg/plate), TA98 (at 0.5 µg/plate) and TA100 (at 0.5 µg/plate)

Remarks:

With metabolic activation

Details on test system and experimental conditions:

MAINTAINENCE OF TESTER STRAIN: The salmonella tester strains TA98, TA100, TA1535, TA1537 and TA1538 were obtained from Dr. Ames, University of California, Berkeley. E. coli was received from the National Collection of Industrial and Marine Bacteria, Aberdeen, Scotland. The strains were sub cultured and stored as follows:
- Sub-cultures: For use in mutagenicity assay, sub-cultures were grown in Nutrient Broth (Oxoid) at 37˚C for 18 hours. This culture provided approximately 2 x 10(9) organisms/mL which was assessed by cell counting.
- Storage: The strain cultures were stored at -80°C or colder in approximately 8% DMSO.

METHOD OF APPLICATION:
- Preliminary toxicity assay and mutagenicity test (initial and confirmatory): Plate incorporation method

EXPERIMENTAL PROCEDURE AND DURATION: Following procedure was carried out on each tester strain:
- Initial assay:
0.5 mL of sterile 0.1 M sodium phosphate buffer (without metabolic activation) or liver homogenate S9 mix (with metabolic activation), 0.1 mL of bacterial suspension and 0.1 mL of vehicle or test material were added to each one set of sterile bijou bottles. 2 mL of histidine deficient agar was added to each bottles, thoroughly mixed and then overlaid onto previously prepared plates containing 20 mL of minimal agar.
After the overlay had solidified, the plates were inverted and incubated for 72 hours at 37°C.
- Second mutation assay:
The procedure outlined above in ‘initial assay’ was repeated at a later stage with same test concentrations and tester strains.

NUMBER OF REPLICATIONS: 3 replicates per dose level

NUMBER OF CELLS EVALUATED: 2 x 10 (9) cells/mL

DETERMINATION OF CYTOTOXICITY
- Method: A substantial reduction in revertant colony counts (compared to solvent control) or absence of a complete background bacterial lawn. Revertant colonies were counted using Biotran Automatic Colony Counter.

Evaluation criteria:

The mean number of revertant colonies for all treatment groups was compared with those for solvent and positive control groups. The effect of metabolic activation was assessed by comparing the results obtained both in the presence and absence of liver microsomal fraction for each treatment group.
The test substance was deemed to provide evidence of mutagenic potential if:
(1) A statistically significant dose-related increase in the number of revertant colonies was obtained in two separate experiments, and
(2) The increase in the number of revertant colonies was at least twice the concurrent solvent control value.

Statistics:

According to the OECD guideline 471, a statistical analysis of the data is not required.

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

Remarks:

initial and second mutation assay

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

S. typhimurium TA 1538

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

initial and second mutation assay

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES: A dose-range finding test was conducted in which four concentrations of test substance ranging from 5 to 5000 µg/plate were tested in each bacterial strain (TA1535, TA1537, TA1538, TA98, TA100) . The test substance was not toxic towards the tester strains at any concentration therefore, 5000 µg/plate was chosen as the top dose level in the mutation tests.

MAIN MUTAGENICITY STUDIES: No substantial increases in revertant colonies were observed in any tester strain following treatment with test material, either in the presence or absence of metabolic activation system.
Details on number of revertant colonies are provided in the study report.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:

Interpretation of results: negative

Hydroxyethyliminodiacetic acid was considered non-mutagenic in Salmonella typhimurium reverse mutation assay with and without metabolic activation system.

Executive summary:

The bacterial reverse mutation test of Hydroxyethyliminodiacetic acid was determined following method comparable to the OECD guideline 471 (Bacterial Reverse Mutation Test).

Plate incorporation method usingSalmonella typhimurium strains TA98, TA1535, TA100, TA1537 and TA1538 was used to evaluate mutagenicity potential of the test substance with and without S9 metabolic activation. 

A preliminary toxicity test was conducted at five test concentrations (0, 5, 50, 500 and 5000 µg/plate) using five Salmonella Typhimurium tester strains. Based on the results of preliminary toxicity assay, 5000 µg/plate was chosen as the maximum dose level for the main mutagenicity assay.

Mutagenicity assay (initial and repeat) was conducted at following test concentrations:

0 (solvent), 50, 150, 500, 1500 and 5000 µg/plate

Appropriate reference mutagens (positive controls) and distilled water (solvent control) were included in each assay, for all the strains, both with and without metabolic activation.

During the initial assay, no substantial increase in revertant colony numbers were observed at any dose level in any of the tester strains, either in the presence or absence of metabolic activation. The results were confirmed in a second mutagenicity assay conducted at same dose levels using the same tester strains.

Based on above, Hydroxyethyliminodiacetic acid was considered non- mutagenic inSalmonella typhimuriumm reverse mutation assay with and without metabolic activation system.

This bacterial reverse mutation test is classified as acceptable, and satisfies the guideline requirements of the OECD 471 method.