Reaction mass of hydroxyethyl-DETA, DETA and dihydroxyethyl-DETA

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:

Not applicable

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Comparable to a guideline study and followed GLP.

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

reference to same study

Cross-reference 2

Reason / purpose for cross-reference:

reference to other study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1988

Report date:

1988

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes

Type of assay:

micronucleus assay

Test material

Test material information

Constituent 1

Reference substance name:

Diethylenetriamine (DETA)

IUPAC Name:

Diethylenetriamine (DETA)

Constituent 2

Reference substance name:

2,2'-iminodi(ethylamine)

EC Number:

203-865-4

EC Name:

2,2'-iminodi(ethylamine)

Cas Number:

111-40-0

Molecular formula:

C4H13N3

IUPAC Name:

N-(2-aminoethyl)ethane-1,2-diamine

Test material form:

other: Clear yellow liquid

Details on test material:

- Name of test material (as cited in study report): Diethylenetriamine (DETA)
- Physical state: Clear yellow liquid
- Analytical purity: 98.9%
- Lot/batch No.: F286E6QL79
- Expiration date of the lot/batch: 31 July 2016
- Storage condition of test material: Room temperature, protected from light, with nitrogen

Test animals

Species:

mouse

Strain:

CD-1

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River, Wilmington, MA,
- Age at study initiation: Approximately 8-10 week old
- Weight at study initiation:
- Assigned to test groups randomly: yes
- Fasting period before study:
- Housing:housed singly in suspended wire-bottom cages
- Diet: Purina Certified Rodent Chow #5002 (Ralston Purina Company, Richmond, IN)
- Water: ad libitum
- Acclimation period:acclimatize for at least one week


ENVIRONMENTAL CONDITIONS
- Temperature (°F): 72 +/-5
- Humidity (%): 40-60
- Air changes (per hr): 12
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water (negative control)

Details on exposure:

The test material and CP were dissolved in distilled water. Freshly prepared solutions (less than 2 hours old) were used for dosing the animals.

All doses of the test material and CP were administered by single oral gavage in aliquots of 10 ml/kg b.w. Negative control mice received 10 ml/kg b.w. distilled water. The animals were weighed the day prior to dosing and the dosing volumes were based upon individual body weights.

Duration of treatment / exposure:

Exposure period: 24, 48, and 72 hours

Frequency of treatment:

Once

Post exposure period:

Not applicable

Doses / concentrations

Remarks:

Doses / Concentrations:
85, 283 and 850 mg/kg bw
Basis:
actual ingested

No. of animals per sex per dose:

Five

Control animals:

other: positive control chemical or water (negative control)

Positive control(s):

cyclophosphamide
- Justification for choice of positive control(s): This dose level was based upon the experience of this laboratory and was selected to induce an unequivocal positive response.
- Route of administration: oral gavage
- Doses / concentrations:120 mg/kg b.w.

Examinations

Tissues and cell types examined:

Bone marrow
micronucleated polychromatic erythrocytes

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: The highest dose for the micronucleus test was selected to be approximately 60% of the LD50 while the middle and low doses were one-third and one-tenth of the highest dose, respectively.


TREATMENT AND SAMPLING TIMES: At the end of the specified intervals following dosing, the animals were weighed and sacrificed by cervical dislocation . Bone marrow samples were obtained from both femurs in the following way.


DETAILS OF SLIDE PREPARATION: Bone marrow sampling: Cell smears were prepared on microscope slides using small portions
o f the cell suspension. The slides were allowed to air dry, fixed in methanol and stained in 5% Giemsa (Gollapudi and Kamra, 1979;
Heddl e and Sal amone, 1981) .
Slide scoring: The slides were coded and scored blindly. One thousand polychromatic erythrocytes were examined from each animal and the number o f micronucleated polychromatic erythrocytes (MN-PCE) was recorded. Micronuclei were i d e n t i f i e d as darkly stained bodies with sharp contours and varying shapes such as round, almond, or ring (Schmid, 1976).


METHOD OF ANALYSIS: The r a t i o o f PCE-NCE i n the bone marrow was determined by examining 200 erythrocytes. The r a t i o was expressed
as PCExlOO/PCE+NCE.


OTHER:

Evaluation criteria:

Bone marrow erythrocytes are especially suitable for scoring micronuclei because of the following reasons. The erythroblasts, the precursors of
erythrocytes, expel their main nuclei shortly (5 h) after their last division.. However, if an erythroblast contains a micronucleus, it will not be expelled along with the main nucleus; and hence micronuclei can be scored in a cell population devoid of the main nucleus. Young erythrocytes stain differently from older forms due to the presence of ribosomal RNA. The process of maturation of the young polychromatic erythrocyte (PCE) into the red staining normochromatic erythrocyte (NCE) takes approximately 24 h (Schmid, 1976). Thus, sampling of bone marrow cells at about 24 h following treatment allows enough time for the erythroblasts to undergo at least one post-treatment mitosis, to expel their main nucleus, and to mature into erythrocytes. However, if the treatment causes any mitotic delays, a longer interval between treatment and sampling time may be necessary to allow the treated cell population to recover from the cell cycle delay.

Statistics:

The raw data on the counts of MN-PCE for each animal were first transformed by adding 1 to each count and then taking natural log of the adjusted number. The transformed MNPCE data and the data on percent PCE were analyzed by a three-way analysis of variance (sex, dose, and time), assuming the three-way interaction to be zero. From this initial analysis, the two-way interactions were reviewed for significance. Depending upon this review, the data were analyzed by either one, two, or three-way analysis of variance looking only at main effects. Pairwise comparisons of treated vs. negative control groups were done, if necessary, by a t-test using Bonferroni correction for multiple comparisons. The alpha level at which all the tests were conducted was 0.01.

Results and discussion

Test results

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

There were no significant differences in MN-PCE frequencies between the groups treated with the test material and the negative controls. The ratios of PCE to NCE (expressed as % PCE) observed in the groups treated with the test material were not significantly different from those of the negative control mice.

The positive control chemical, CP, induced a significant increase in the frequencies of micronucleated polychromatic erythrocytes in the bone marrows of both males and females

Any other information on results incl. tables

Table 1 SUMMARY OF THE DATA ON THE FREQUENCIES OF MICRONUCLEATED POLYCHROMATIC ERYTHROCYTES (MN-PCE) IN THE BONE MARROW OF MICE TREATED WITH THE TEST MATERIAL OR CYCLOPHOSPHAMIDE (CP)

	24 h sacrifice		48 h sacrifice		72 h sacrifice
Treatment	MN-PCE	% PCE	MN-PCE	% PCE	MN-PCE	% PCE
Males
Water (neg. control)	0.4 + 0.5	63.6 + 6.4	0.6 + 0.5	67.8 + 4.2	0.6 + 0.9	64.0 + 7.1
DETA 85 mg/kg	0.0 + 0.0	65.7 + 7.0	1.0 + 1.0	62.0 + 7.2	0.8 + 0.4	66.4 + 6.1
DETA 283 mg/kg	0.8 + 0.8	65.7 + 7.2	0.4 + 0.5	62.6 + 8.8	0.6 + 0.9	66.5 + 8.3
DETA 850 mg/kg	0.8 + 1.3	62.9 + 3.5	0.2 + 0.4	62.1 + 7.3	0.6 + 0.9	69.2 + 4.7
CP 120 mg/kg (pos control)	44.8 + 15.2*	45.3 + 6.6*	ND	ND	ND	ND
Females
Water (neg control)	0.2 + 0.4	61.6 + 8.9	0.4 + 0.5	61.8 + 5.3	0.4 + 0.9	63.9 + 10.7
DETA 85 mg/kg	1.0 + 1.0	58.4 + 14.3	0.2 + 0.4	69.7 + 6.6	0.8 + 1.3	64.7 + 2.9
DETA 283 mg/kg	1.0 + 0.7	56.4 + 9.9	0.8 + 0.8	64.5 + 9.9	2.2 + 1.8	69.9 + 4.2
DETA 850 mg/kg	1.2 + 1.3	62.5 + 5.4	0.2 + 0.4	71.1 + 8.8	0.8 + 1.1	70.6 + 5.9
CP 120 mg/kg (pos control)	56.0 + 17.9*	47.0 + 10.3	ND	ND	ND	ND

5 male and 5 female mice were examined at each dose level; 1000 PCE were examined from each animal.

Data expressed as means and standard deviations.

* Significantly different (alpha < 0.01) from negative control

ND No data

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information): negative
The test did not significantly increase the frequency of micronucleated polychromatic erythrocytes and was therefore considered negative in the mouse bone marrow micronucleus test.

Executive summary:

Diethylenetriamine (DETA) was evaluated in the mouse bone marrow micronucleus test. The micronucleus test is capable of detecting agents causing chromosomal aberrations and spindle malfunction. The test material was mixed with water and administered to CD-1 (ICR) BR mice by single oral gavage at dose levels of 0 (negative control), 85, 283 and 850 mg/kg body weight (b.w.). Groups of animals were sacrificed at three intervals, viz., 24, 48, and 72 h after treatment. Mice treated with 120 mg/kg b.w. cyclophosphamide and sacrificed at 24 h served as positive controls. There were five animals per sex per dose level per sacrifice time. One thousand polychromatic erythrocytes (PCE) were evaluated from each animal and the frequencies of micronucleated polychromatic erythrocytes (MNPCE) were recorded. There were no significant increases in the frequencies of MN-PCE in groups treated with the test chemical compared to negative controls. The positive control mice showed significant increases in MN-PCE. Hence, under the experimental conditions used, the test chemical was considered negative in the mouse bone marrow micronucleus test.