Disodium 2-hydroxyethyliminodi(acetate)

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From March 20, 1986 to May 22, 1986

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Well documented study, followed guideline with deviation.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1986

Report date:

1986

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

yes

Remarks:

(One dose level was tested against the guideline requirement of 3 dose levels; Only 1000 polychromatic erythrocytes/animal were examined for the presence of micronuclei against the guideline requirement of 2000 polychromatic erythrocytes/animal)

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)

Deviations:

yes

Remarks:

(One dose level was tested against the guideline requirement of 3 dose levels; Only 1000 polychromatic erythrocytes/animal were examined for the presence of micronuclei against the guideline requirement of 2000 polychromatic erythrocytes/animal)

GLP compliance:

yes

Type of assay:

micronucleus 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:

2,2'-[(2-hydroxyethyl)imino]diacetic acid, disodium salt

IUPAC Name:

2,2'-[(2-hydroxyethyl)imino]diacetic acid, disodium salt

Test material form:

not specified

Details on test material:

- Name of test material: Hydroxyethyliminodiacetic acid, disodium salt
- TSIN: E-2748.03
- Substance type: Pure active substance
- Solubility: Soluble in water

Test animals

Species:

mouse

Strain:

CD-1

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Obtained from Charles River U.K. Limited, Margate, Kent, Englandon 7, 14 and 27 March 1986
- Age at study initiation: 3 - 4weeks old
- Weight at study initiation: 18 to 21 grams (on arrival)
- Assigned to test groups randomly: Yes
- Fasting period before study: Animals were fasted "overnight" prior to dosing
- Housing: Animals of both sexes were housed separately in groups of 2 or 5 in plastic disposable cages.
- Diet: Scientific Feeds rodent diet LAD-1 was provided ad libitum except during the overnight fasting period prior to dosing
- Water: tap water, ad libitum
- Acclimation period: 10 days

ENVIRONMENTAL CONDITIONS
- Temperature : 22 °C
- Humidity : Not reported
- Air changes: 30 air changes per hour
- Photoperiod : 12 hours dark/12 hours artificial light

EXPERIMENT INITIATION DATE: March 20, 1986

EXPERIMENT COMPLETION DATE: May 22, 1986

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

- Vehicle used: Deionised water
- Concentration of test material in vehicle: 97.2 mg/mL
- Amount of vehicle : 16 mL/kg bw

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: Solutions of test substance were prepared just prior to use in deionised water.

Duration of treatment / exposure:

48 h

Frequency of treatment:

Single treatment

Doses / concentrations

Remarks:

Doses / Concentrations:
1555.2 mg /kg bw
Basis:
actual ingested

No. of animals per sex per dose:

Vehicle control and test group: 15 animals/sex
Positive control group: 5 animals/sex

Control animals:

yes, concurrent vehicle

Positive control(s):

Mitomycin C
- Route of administration: Oral gavage (intragastric gavage)
- Doses / concentrations: 8 mg mitomycin C/kg bw . It was prepared as a solution in sterile 0.9% saline at a concentration of 0.5 mg/mL.

Examinations

Tissues and cell types examined:

Tissue: Femoral bone marrow
Cell types: Polychromatic erythrocytes

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: The dose for the micronucleus test was based on a preliminary toxicity test. Details are provided on the study report

TREATMENT AND SAMPLING TIMES: Bone marrow was collected from the negative control and test treatment group at 18, 24 and 48 h post dosing. 5 animals/sex were sacrificed at each sampling time.. A sampling time of 24 hours was used for the positive control group.

DETAILS OF SLIDE PREPARATION: Animals were killed by cervical dislocation and both femurs dissected out from each animal. A direct smear of bone marrow was made onto a slide containing a drop of calf serum. One smear was prepared from each femur. The prepared smears were air-dried and fixed in methanol. After fixation, the smears were air-dried and stained in Giemsa's stain, rinsed in pH 6.8 buffered distilled water, air-dried, and mounted with coverslips.

METHOD OF ANALYSIS: Coded slides were examined by light microscopy and 1000 polychromatic erythrocytes per animal were examined for the presence of micronuclei. The ratio of polychromatic to normochromatic erythrocytes was assessed by examination of at least 1000 erythrocytes per animal. The results for the treated group were compared with the results obtained with the concurrent vehicle control group using non-parametric statistical methods.

Evaluation criteria:

A positive result was defined as follows: either a statistically significant dose-related increase or a reproducible and statistically significant positive response for at least one of the dose levels was obtained.

Statistics:

The results obtained for the treatment group in the main test were compared with the results obtained with the concurrent vehicle control group using non-parametric statistical methods (e.g., Wilcoxon's sum of ranks test). In the preliminary toxicity test, the mortality data were subjected to a probit analysis.

Results and discussion

Test results

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Remarks:

(Slight piloerection and slight hunched posture were observed between 30 minutes and 2 hours post-dosing.)

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
The results of the preliminary study indicated that a dosage of 1555.2 mg/kg bw would be expected to kill approximately 1% of the animals within 6 days of dosing. Details are provided in the study report.

RESULTS OF DEFINITIVE STUDY
- Signs and mortalities: No animals died after treatment with the test substance in the main study. Clinical signs such as pilo-erection and hunched posture were observed at 30 min, 1 and 2 h post-dosing.
- Induction of micronuclei (for Micronucleus assay): At the 24 hour sampling time slight, but just statistically significant (p=0.045 using Wilcoxon's sum of ranks test) increases in the number of micronucleated polychromatic erythrocytes were reported in test substance-treated animals. The individual values for this group were within the laboratory historical control range and the group mean was comparable with the historical control mean. Consequently these increases were not considered to be evidence of a treatment-related effect. At the 18 and 48 hour sampling times, no such significant increases were reported. Also, the test substance did not cause any substantial increases in the incidence of micronucleated normochromatic erythrocytes at any of the three kill times.
- Ratio of PCE/NCE: The test substance failed to cause any significant decreases in the ratio of polychromatic to normochromatic erythrocytes (p>0.05 using Wilcoxon's sum of ranks test).

RESULTS OF POSITIVE CONTROL
- Positive control caused large, highly significant increases in the frequency of micronucleus polychromatic erythrocytes after 24 h exposure (P<0.001). Also, it did not significantly decrease the polychromatic to normochromatic erythrocytes ratio at the 24 h sampling time (P>0.05).
- Statistical evaluation: Non-parametric statistical methods (e.g., Wilcoxon's sum of ranks test, 1-sided probabilities) were used to compare results of treated group with results obtained with the concurrent vehicle control group.

Applicant's summary and conclusion

Conclusions:

Interpretation of results: negative
Hydroxyethyliminodiacetic acid when administered orally at 1555.2 mg /kg bw was non-mutagenic in the micronucleus test with bone marrow cells of the mouse.

Executive summary:

The purpose of this study was to assess the mutagenic potential of Hydroxyethyliminodiacetic acid in the micronucleus assay. The study was performed according to OECD guideline 474 and EU Method B.12.

CD-1 male and female mice weighing 18 to 21 grams from Charles River U.K. Limited, Margate, Kent, England were acclimated for 10 d. The food used was Scientific Feeds rodent diet LAD-1 (ad libitum). The animals of both sexes were housed separately in groups of 2 or 5 in plastic disposable cages and maintained under standard laboratory conditions (temperature: 22 °C; artificial light: 12-h cycle; Air changes: 30 air changes per hour).15 animals/sex were used in the test and vehicle group while 5 animals/sex were employed in positive control group.

The test substance was prepared in deionised water, which was also used as vehicle control. 1555.2 mg/kg bw of test substance (dose volume: 16 mL/kg bw) was administered to 15 animals/sex. This dose level was estimated from pre-experiment.

 

Positive control, Mitomycin C (8 mg/kg bw.), was administered orally to 5 animals/sex.

Bone marrow was collected from the negative control and test treatment group at 18, 24 and 48 h post dosing. 5 animals/sex were sacrificed per sampling time. A sampling time of 24 h was used for the positive control group.

At the 24-hour sampling time, slight statistically significant (p = 0.045, using Wilcoxon’s sum of ranks test) increases in the number of micronucleated polychromatic erythrocytes were observed in test substance-treated animals. The individual animal values for this group were within the laboratory historical control range and the group mean was comparable with the historical control mean. Consequently, these increases were not considered to be evidence of a treatment-related effect. At the 18and 48h sampling times, no such significant increases were obtained. The test substance did not cause any substantial increases in the incidence of micronucleated normochromatic erythrocytes at any of the three sampling times.

The positive control, Mitomycin C, caused highly significant increases in the frequency of micronucleus polychromatic erythrocytes after 24 h exposure (P<0.001).

In conclusion, Hydroxyethyliminodiacetic acid when administered orally at 1555.2 mg /kg bw was non-mutagenic in the micronucleus test with bone marrow cells of the mouse.

This Mammalian Erythrocyte Micronucleus Test is classified as acceptable, and satisfies the guideline requirements of the OECD 474 method.