N,N'-bis(3-aminopropyl)ethylenediamine

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:

disregarded due to major methodological deficiencies

Reliability:

3 (not reliable)

Rationale for reliability incl. deficiencies:

other: scientifically acceptable study (TS presumably contained unspecified impurities, only one post-treatment sample (after 48 hours), 2 treatments at 48 hours intervals, limited documentation)

Data source

Reference

Reference Type:

publication

Title:

Unnamed

Year:

1992

Report date:

1992

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

yes

Remarks:

limited documentation, 2 treatments at 48 hours intervals, only one post-treatment sample (after 48 hours)

GLP compliance:

yes

Type of assay:

chromosome aberration assay

Test material

Test material information

Constituent 1

Reference substance name:

N,N'-bis(3-aminopropyl)ethylenediamine

EC Number:

234-147-9

EC Name:

N,N'-bis(3-aminopropyl)ethylenediamine

Cas Number:

10563-26-5

Molecular formula:

C8H22N4

IUPAC Name:

(3-aminopropyl)({2-[(3-aminopropyl)amino]ethyl})amine

Details on test material:

- Name of test material (as cited in study report): N-BAPED
- Physical state: yellow liquid
- Analytical purity: technical grade; 95%
- Impurities (identity and concentrations): not specified
- Lot/batch No.: 21955-60-1 (Aldrich Chem Co Inc catalog number: 23,939-9)

Test animals

Species:

mouse

Strain:

Balb/c

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: animals from a randomly bred closed colony purchased from Charles River Breeding Laboratories, Inc., Wilmington, MA
- Age at study initiation: not specified; adult male and female mice were used
- Weight at study initiation:
- Assigned to test groups randomly: [no/yes, under following basis: ]
- Fasting period before study: not specified
- Housing: group-housed, 7 mice/cage
- Diet: not specified (a commercial diet was used); ad libitum
- Water: not specified (“water”); ad libitum
No additional detail provided

Administration / exposure

Route of administration:

intraperitoneal

Vehicle:

- Vehicle(s)/solvent(s) used: A set of mice was dosed with the solvent (filter sterilized 0.99% saline; intraperitoneal [IP] injection), which served as a negative control.

Details on exposure:

Dosing consisted of 2 intraperitoneal (IP) administrations of the test substance / positive control substances / vehicle.

Duration of treatment / exposure:

48 hours

Frequency of treatment:

The initial dose was given at time-0 (i .e. immediately following the initial blood sampling). A second dose was given 48 hours thereafter.

Post exposure period:

24 hoours; the final blood sample was obtained at 96 hours after the first injection.

Doses / concentrations

Remarks:

Doses / Concentrations:
ca. 119, 238 and 475 mg/kg bw (calculated from 0.125, 0.25 and 0.5 µl/g bw assuming test substance density of 0.95 g/cm³)
Basis:
other: actual injected

No. of animals per sex per dose:

7

Control animals:

yes, concurrent vehicle

other: An initial (0 hour; before dosing began) blood sample from each mouse provides an internal control for comparison with dosed blood (96 hour samples). These initial blood samples provided a mouse-specific control blood sample.

Positive control(s):

methylmethanesulfonate (MMS)
- Route of administration: IP
- Doses / concentrations: 100 mg/kg bw

Examinations

Tissues and cell types examined:

polychromatic cell (PCE) of peripheral blood

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
The high dose was detetmined in a preliminary lethal dose study. In order to evaluate the lethal dose range, mice were dosed with the test material diluted in 0.9% sterile saline to the following concentrations: ca. 238, 594, 1188, 1782, and 2375 mg/kg bw (converted from 2.5, 1.875, 1.25, 0.625, and 0.25 μl/g bw). The mice were injected with 25 ml test substance/kg bw. Those animals which were injected with the 1.25 μl/g bw solution and higher did not survive, while all the others survived 96 hours. Therefore, the high dose was set at 0.5 μl/g bw (ca. 475 mg/kg bw), the medium dose at 0.25 μl/g bw (ca. 238 mg/kg bw) and the low dose at 0.125 μl/g bw (ca. 119 mg/kg bw).
A preliminary analysis of the PCE number was obtained in order to insure that stem cell toxicity was not a factor in the micronucleus assay.

DETAILS OF SLIDE PREPARATION:
Blood cells were obtained from the tail vein of each mouse and collected in heparin. The blood samples were fixed with alcohol and were stained with dye solutions of Hoechst 33258 (DNA of the micronucleus) and propidium iodide (RNA of PCEs). With this dye combination, it was possible to provide a quantitative analysis of the number of RBCs, PCEs and micronucleated cells that are present in each sample. A fixed and stained blood sample from a malaria infected mouse was used during the instrument setup in order to evaluate the resolution of the signals.

METHOD OF ANALYSIS:
- The 0 and 96 hour blood samples were quantitatively analyzed by high speed flow cytometry (FCM; DNA of a micronucleus emit blue fluorescence, and RNA of polychromatic cells red). The stained cells were then excited with a 363 nm beam from a 5 watt Argon Ion laser focused to 20 microns at the sample stream. The fluorescence from each cell was collected by photomultiplier tubes (PMTs) and the signal levels were stored in computer memory for later processing. In addition to obtaining fluorescence profiles of each cell, FCM simultaneously provided cell size information by determining the light scatter properties of each cell or combination of cells. By setting hardware or software gates, it was possible to exclude ("gate out") cells and/or debris from analysis and thus focus on those cells of specific interest (e.g. the number of micronucleated cells, PCEs, or RBCs present in a sample).
- For each mouse, the number of micronucleated cells was determined in 2000000 total blood cells 1000000 cells from the t-0 blood and 1000000 cells from the final blood [t-96] for each mouse. The MN cell count of each initial (0 hour) blood sample was compared with the corresponding 96 hour blood sample.

Evaluation criteria:

A mean and standard deviation (SD) of the micronucleated cell counts (t=0 and t=96 hours) was provided for each set (i .e. each treatment group) of mice. In addition, the change between the t=0 and the t=96 hours values were provided for the micronucleated cells and the PCEs. A comparison of the means was done by statistical analysis.

Statistics:

The test substance induced effects were analysed by ANOVA, and a t-test was used to highlight the source of the variation.

Results and discussion

Test results

Sex:

male/female

Genotoxicity:

positive

Remarks:

a significant increase in the number of micronucleated cells was observed with all doses for the females and with the high dose for the males, but the variations within the test groups were very high.

Toxicity:

yes

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Any other information on results incl. tables

Table 1: Summary of the results

Treatment group	Sex	Means of micronucleus (MNs) / polychromatic erythrocytes (PCEs)
		MN and changes (MN96 hours– MN0 hour)			PCEs
		0 hours value	96 hours value	Change	0 hours value	96 hours value
Negative control	Females	1929	1982	53	2340	5606
	Males	2665	2699	34	2828	5826
	Mean variation in both sexes			44
Low dose (119 mg/kg)	Females	2076	2356	280*	2660	5700
	Males	2714	2812	99	2960	5700
	Mean variation in both sexes			189
Medium dose (238 mg/kg)	Females	1997	2234	236*	3052	6171
	Males	2621	2777	156	2764	5681
	Mean variation in both sexes			196
High dose (475 mg/kg)	Females	2080	2338	258*	2914	6173
	Males	2670	2905	234	2925	5783
	Mean variation in both sexes			246*
Positive control	Females	2013	5225	3212*	2285	5298
	Males	2689	570	3071*	2510	4554
	Mean variation in both sexes			3142

 *: significant different from the negative control

 

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information): positive