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Toxicological information

Genetic toxicity: in vivo

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Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2000
Report date:
2000

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
adopted in 1997
GLP compliance:
yes (incl. QA statement)
Remarks:
BASF SE, Experimental Toxicology and Ecology
Type of assay:
other: Erythrocyte Micronucleus Assay

Test material

Constituent 1
Chemical structure
Reference substance name:
Disodium dihydrogen ethylenediaminetetraacetate
EC Number:
205-358-3
EC Name:
Disodium dihydrogen ethylenediaminetetraacetate
Cas Number:
139-33-3
Molecular formula:
C10H16N2O8.2Na
IUPAC Name:
disodium dihydrogen 2,2',2'',2'''-(ethane-1,2-diyldinitrilo)tetraacetate
Test material form:
solid

Test animals

Species:
mouse
Strain:
NMRI
Sex:
male
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland GmbH
- Weight at study initiation: mean 29 g
- Housing: Makrolon cages type Mill, in groups of 5
- Diet: Kliba, standardized pelleted feed, Provimi Kliba SA, Kaiseraugst, Switzerland, ad libitum
- Water: ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24
- Humidity (%): 30 - 70
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
- Vehicle used: water
Details on exposure:
- The low dose group was given 500 mg test substance/kg body weight or 20 mL/kg body weight of a solution with a concentration of 2.5 g/100 mL.
- The intermediate dose group was given 1000 mg test substance/kg body weight or 20 mL/kg body weight of a solution with a concentration of 5.0 g/100 mL.
- The top dose group was given 2000 mg test substance/kg body weight or 20 mL/kg body weight of a solution with a concentration of 10.0 g/100 mL.
Duration of treatment / exposure:
48 h (animals were treated twice at 24 h intervals)
Frequency of treatment:
twice
Doses / concentrationsopen allclose all
Dose / conc.:
500 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
Dose / conc.:
2 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Positive control(s):
20 mg of cyclophosphamide (CPP)/kg bw or 0.15 mg of vincristine sulphate (VCR)/kg bw, both, dissolved in purified water, were administered to male animals once, orally each in a volume of 10 mL/kg bw.

Examinations

Tissues and cell types examined:
bone marrow
Details of tissue and slide preparation:
METHOD OF ANALYSIS:
2000 polychromatic erythrocytes (PCE) from each of the male animals of every test group are evaluated and investigated for micronuclei (MN). The normochromatic erythrocytes (NCE) which occur are also scored. The following parameters have been evaluated:
- Number of polychromatic erythrocytes
- Number of polychromatic erythrocytes containing micronuclei
- Number of normochromatic erythrocytes
- Number of normochromatic erythrocytes containing micronuclei
- Ratio of polychromatic to normochromatic erythrocytes
- Number of small micronuclei (d < D/4) and of large micronuclei (d > D/4) (d = diameter of micronucleus, D = cell diameter )
Evaluation criteria:
The test chemical is to be considered positive in this assay if the following criteria are met:
- A dose-related and significant increase in the number of micronucleated polychromatic erythrocytes was observed .
- The proportion of cells containing micronuclei exceeded both the values of the concurrent negative control range and the negative historical control range .

A test substance is generally considered negative in this test system if:
- There was no significant increase in the number of micronucleated polychromatic erythrocytes at any dose above concurrent control frequencies.
- The frequencies of cells containing micronuclei were within the historical control range.
Statistics:
The statistical evaluation of the data was carried out using the program system MUKERN. A comparison of the dose group with the vehicle control was carried out using the Wilcoxon test for the hypothesis of equal medians.

Results and discussion

Test results
Key result
Sex:
male
Genotoxicity:
negative
Toxicity:
no effects
Remarks:
The only adverse effect observed was piloerrection.
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
- After two administrations of the highest dose of 2000 mg/kg body weight, 0.4% of polychromatic erythrocytes containing micronuclei were found after 24 hours.
- In the two lower dose groups, rates of micronuclei of about 0.6% (1000 mg/kg group) and 0.7% (500 mg/kg group) were detected.
- With 12.7% the positive control substance cyclophosphamide for clastogenicity led to the expected increase in the number of polychromatic erythrocytes containing exclusively small micronuclei at a dose level of 20 mg/kg body weight.
- With 81.7% the positive control vincristine sulphate for induction of spindle poison effects also led to a clearly enhanced number of polychromatic erythrocytes containing micronuclei with the expected amount of large micronuclei, i .e. 10.5%.
- The number of normochromatic erythrocytes containing micronuclei did not differ to any appreciable extent in the negative control or in the various dose groups

Thus, the test item did not lead to any increase in the rate of micronuclei. The number of normochromatic or polychromatic erythrocytes containing small micronuclei (d < D/4) did not deviate from the vehicle control value and was within the historical control range. Nor were large micronuclei (d > D/4) observed either in the negative control group or in the three test item dose groups.

No inhibition of erythropoiesis, induced by the treatment of mice with the test item was detected; the ratio of polychromatic to normochromatic erythrocytes was always in the same range as that of the control values in all dose groups.

Applicant's summary and conclusion

Conclusions:
No increase in frequency of micronucleated polychromatic erythrocytes was found after a two oral gavage doses of 500, 1000 and 2000 mg/kg bw (24 h interdosing interval) and a bone marrow sampling at 24 hours after the second treatment from each of 5 male animals as compared to vehicle and positive control (cyclophosphamide and vincristine sulphate) animals.