Ethylenediaminetetraacetonitrile

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

November 2009 - January 2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Well performed and reported study according to GLP

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Type of assay:

micronucleus assay

Test material

Test material information

Constituent 1

Reference substance name:

Ethylenediaminetetraacetonitrile

EC Number:

227-290-3

EC Name:

Ethylenediaminetetraacetonitrile

Cas Number:

5766-67-6

Molecular formula:

C10H12N6

IUPAC Name:

2-({2-[bis(cyanomethyl)amino]ethyl}(cyanomethyl)amino)acetonitrile

Details on test material:

Batch number: CFC 9070
Purity: 99.8 +/- 0.5%
Expiry date: 13 January 2012

Test animals

Species:

mouse

Strain:

NMRI

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River, Sulzfeld, Germany
- Age at study initiation: 6-7 weeks at start of treatment
- Weight at study initiation: 37.6 +/- 1.9 g for males and 29.6 +/- 1.4 g for females
- Assigned to test groups randomly: no data
- Fasting period before study: 3-4 h prior to dosing. However, because of absence of toxicity, ip dosing was used in the main study
- Housing: 5 per sex per cage
- Diet (e.g. ad libitum): yes
- Water (e.g. ad libitum): yes
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.2-22.3
- Humidity (%): 43-73%
- Air changes (per hr): ca. 15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 23 NOvember 2009 To: 4 January 2010

Administration / exposure

Route of administration:

intraperitoneal

Vehicle:

- Vehicle(s)/solvent(s) used: corn oil
- Justification for choice of solvent/vehicle: not indicated
- Concentration of test material in vehicle: 312, 625 or 1250 mg/kg bw given at 10 mL/kg bw results in 31.2, 62.5 or 125 mg/mL
- Amount of vehicle (if gavage or dermal): 10 mL/kg
- Type and concentration of dispersant aid (if powder): not applicable
- Lot/batch no. (if required): not indicated
- Purity: not indicated

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: suspensions were blended to obtain a homogeneous suspension; suspensions were given within 3 h after preparation
In the RF study animals were first dosed by gavage; because no toxicity was observed, animals of the remainder of the RF study and of the main study were treated by ip injection

Duration of treatment / exposure:

Single adminstration

Frequency of treatment:

Single administration

Post exposure period:

Animals were necropsied 24 h or 48 h after treatment

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
0, 312, 625 or 1250 mg/kg bw (females)
Basis:
other: actual injected

Doses / concentrations 2

Remarks:

Doses / Concentrations:
0, 500, 1000, 2000 mg/kg bw (males)
Basis:
other: actual injected

No. of animals per sex per dose:

RF study (total number of 15 animals):
- 3 males / 3 females (oral)
- one group of 3 M and 1 F, two group with each 1 F, one group with 3 F
Main study (total number of 60 animals)
- vehicle control (24 h sampling time): 5 M + 5 F
- low dose group (24 h sampling time): 5 M + 5 F
- mid dose group (24 h sampling time): 5 M + 5 F
- two high dose groups (24 and 48 h sampling time): 10 M + 10 F
- positive control (48 h sampling time): 5 M + 5 F

Control animals:

yes, concurrent vehicle

Positive control(s):

cyclophosphamide
- Justification for choice of positive control(s): no
- Route of administration: ip
- Doses / concentrations: 40 mg/kg bw at 10 mL/kg bw

Examinations

Tissues and cell types examined:

Bone marrow (femurs)

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: RF study

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields): 24 and/or 48 h (see above)

DETAILS OF SLIDE PREPARATION: The cell suspension was collected and centrifuged at 1000 rpm (216 g) for 10 min. The supernatant was removed with a Pasteur pipette. A drop of serum was left on the pellet. The cells in the sediment were carefully mixed with the remaining serum. A drop of the cell suspension was placed on the end of a clean slide, which was previously immersed in a 1:1 mixture of 96% (v/v) ethanol (Merck, Darmstadt, Germany)/ether (Merck) and cleaned with a tissue. The slides were marked with the study identification number and the animal number. The drop was spread by moving another clean slide with round-whetted sides at an angle of approximately 45° over the slide with the drop of bone marrow suspension. The preparations were air-dried, fixed for 5 min in 100% methanol (Merck) and air-dried overnight. Two slides were prepared per animal. The slides were automatically stained using the "Wright-stain-procedure" in an "Ames" HEMA-tek slide stainer (Miles, Bayer Nederland B.V.). This staining is based on Giemsa. The dry slides were dipped in xylene (Klinipath, Duiven, The Netherlands) before they were embedded in Pertex (Klinipath) and mounted with a coverslip.

METHOD OF ANALYSIS: To prevent bias, all slides were randomly coded before examination. An adhesive label with the study identification number and random code was stuck over the marked slide. At first the slides were screened at a magnification of 100x for regions of suitable technical quality, i.e. where the cells were well spread, undamaged and well stained. Slides were scored at a magnification of 1000x. The number of micronucleated polychromatic erythrocytes was counted in 2000 polychromatic erythrocytes. The ratio of polychromatic to normochromatic erythrocytes was determined by counting and differentiating the first 1000 erythrocytes at the same time. Micronuclei were only counted in polychromatic erythrocytes. Averages and standard deviations were calculated.

OTHER:

Evaluation criteria:

The micronucleus test is considered acceptable if it meets the following criteria:
a) The incidence of micronucleated polychromatic erythrocytes in the positive control animals should be above the historical control data range.
b) The positive control substance induced a statistically significant (Wilcoxon Rank Sum Test, one-sided, p < 0.05) increase in the frequency of micronucleated polychromatic erythrocytes.
c) The incidence of micronucleated polychromatic erythrocytes in the control animals should reasonably be within the laboratory historical control data range.

Statistics:

Data evaluation and statistical procedures: Equivocal results should be clarified by further testing using modification of experimental conditions.

A test substance is considered positive in the micronucleus test if:
- It induced a biologically as well as a statistically significant (Wilcoxon Rank Sum Test, one-sided, p < 0.05) increase in the frequency of micronucleated polychromatic erythrocytes (at any dose or at any sampling time) in the combined data for both sexes or in the data for male or female groups separately and the number of micronucleated polychromatic erythrocytes in the animals was above the historical control data range.
A test substance is considered negative in the micronucleus test if:
- None of the tested concentrations or sampling times showed a statistically significant (Wilcoxon Rank Sum Test, one-sided, p < 0.05) increase in the incidence of micronucleated polychromatic erythrocytes either in the combined data for both sexes or in the data for male or female groups separately and the number of micronucleated polychromatic erythrocytes in the animals was within the historical control data range.

Results and discussion

Test results

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

not valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Dose range: at 2000 mg/kg orally no clinical signs and no mortality. Females dosed with 2000 and 1500 mg/kg bw ip died within 44 h after dosing.
- Solubility: OK
- Clinical signs of toxicity in test animals: yes at 2000, 1500, 1250 and 1000 mg/kg bw ip
- Evidence of cytotoxicity in tissue analyzed: not analyzed in RF study
- Rationale for exposure: ip route because of lack of toxicity via oral route
- Harvest times: not applicable to RF study
- High dose with and without activation: no, in vivo study
- Other:

RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei (for Micronucleus assay): no
- Ratio of PCE/NCE (for Micronucleus assay): normal
- Appropriateness of dose levels and route: OK
- Statistical evaluation: see above

Any other information on results incl. tables

See attached table.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information): negative
EDTN did not induce micronuclei in vivo following ip injection with EDTN.

Executive summary:

EDTN was tested in the Micronucleus Test in mice, to evaluate its genotoxic effect on erythrocytes in bone marrow. The study procedures described in this report were based on the most recent OECD and EC guidelines. The test substance was suspended in corn oil. In the dose range finding study 3 males and 3 females were dosed by oral gavage with 2000 mg EDTN per kg body weight. The animals showed no treatment related clinical signs or mortality after dosing. To maximize the chance of the test substance to reach the target tissue 3 males were dosed by intraperitoneal injection with 2000 mg EDTN per kg body weight. These males showed the following toxic signs after dosing: rough coat and a hunched posture. Females were successively dosed by intraperitoneal injection with 2000, 1500, 1000 and 1250 mg EDTN per kg body weight. The females dosed with 2000 and 1500 mg EDTN per kg body weight died within 44 hours after dosing. The females showed the following toxic signs after dosing with 1250 mg/kg body weight: lethargy, rough coat and a hunched posture. In the main study males were dosed by intraperitoneal injection with vehicle or with 2000, 1000 and 500 mg EDTN per kg body weight. Females were dosed by intraperitoneal injection with vehicle or with 1250, 625 and 312 mg EDTN per kg body weight. Positive control groups were dosed by intraperitoneal injection with 40 mg cyclophosphamide (CP) per kg body weight. In total 6 treatment groups per sex were used, each consisting of 5 males or 5 females. The males dosed with 2000 mg/kg body weight showed the following toxic signs after dosing: hunched posture and rough coat. The females dosed with 1250 mg/kg body weight showed the following toxic signs after dosing: lethargy, hunched posture and rough coat. Two females dosed with 625 mg/kg body weight had a hunched posture and a rough coat. No treatment related clinical signs or mortality were noted in any male animal treated with 1000 and 500 mg EDTN per kg body weight, in three females treated with 625 mg EDTN per kg body weight and in females treated with 312 mg EDTN per kg body weight or control animals receiving vehicle or cyclophosphamide. Bone marrow of the groups treated with EDTN was sampled 24 or 48 (highest dose of both sexes only) hours after dosing. Bone marrow of the negative and positive control groups was harvested 24 and 48 hours after dosing, respectively. No increase in the mean frequency of micronucleated polychromatic erythrocytes was observed in the bone marrow of animals treated with EDTN compared to the vehicle treated animals. The mean number of micronucleated polychromatic erythrocytes per 2000 polychromatic erythrocytes of each group was within the laboratory historical control data range. The incidence of micronucleated polychromatic erythrocytes in the bone marrow of all negative control animals was within the historical solvent control data range. Cyclophosphamide, the positive control substance, induced a statistically significant increase in the number of micronucleated polychromatic erythrocytes in both sexes. Hence, both criteria for an acceptable assay were met. The groups that were treated with EDTN showed no decrease in the ratio of polychromatic to normochromatic erythrocytes compared to the vehicle controls, which reflects a lack of toxic effects of this test substance on erythropoiesis. The groups that were treated with cyclophosphamide showed an expected decrease in the ratio of polychromatic to normochromatic erythrocytes compared to the vehicle controls, demonstrating toxic effects on erythropoiesis. It is concluded that EDTN is not clastogenic or aneugenic in the micronucleus test under the experimental conditions described in this report.