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Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / bone marrow chromosome aberration
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 10 SEP 1984 to 11 DEC 1984
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: comparable to guideline study (OECD 475) with some restrictions (insufficient number of metaphase cells (only 50 instead of 100) and cells for mitotic index (only 500 instead of 1000) examined)

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1985
Report date:
1985

Materials and methods

Test guideline
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 475 (Mammalian Bone Marrow Chromosome Aberration Test)
Deviations:
yes
Remarks:
insufficient number of metaphase cells (only 50 instead of 100) and cells for mitotic index(only 500 instead of 1000) examined
GLP compliance:
yes
Remarks:
according to US FDA
Type of assay:
micronucleus assay

Test material

Constituent 1
Chemical structure
Reference substance name:
Reaction mass of 7-azatridecane-1,13-diamine and hexamethylenediamine
EC Number:
907-605-7
Cas Number:
68815-47-4
Molecular formula:
C6H16N2 (HMD) C12H29N3 (BHT)
IUPAC Name:
Reaction mass of 7-azatridecane-1,13-diamine and hexamethylenediamine
Details on test material:
- Name of test material (as cited in study report): BHMT (Bis (Hexamethylene) Triamine)
- Physical state: brown solid
- Storage condition of test material: at room temperature

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Strain specifics: Crl:COBS; CD (SD)BR
- Source: Charles River breeding Laboratories Inc., Kingston, New York, USA
- Age at study initiation: 46 to 51 days
- Weight at study initiation: males: 295.8-339.6 g; females: 226.6-234.8 g
- Assigned to test groups randomly: yes
- Housing: individually
- Diet (ad libitum): Purina Certified Laboratory Chow No. 5001
- Water: tap water, ad libitum
- Acclimation period: 19 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22.2 +/- 3.3
- Humidity (%): 30-70
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: corn oil
- Justification for choice of solvent/vehicle: Because of the corrosive nature of the test material, administration was performed in corn oil to limit excessive mortality from gastric ulcerations.
- Amount of vehicle (if gavage or dermal):10ml/kg bw for all groups
- Lot/batch no. (if required):52500 of C.F. Sauer Company
- Purity:100 % assumed
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: test material was freshly prepared on the day of use. Dosing solutions were prepared on a weight per volume basis.
Duration of treatment / exposure:
single exposure
Frequency of treatment:
once
Post exposure period:
6, 24, or 48 hours (time intervall to scheduled death of the vehicle control and the treatment group; only one positive control group which was treated for 24 h)
Doses / concentrations
Remarks:
Doses / Concentrations:
0.770 mg submission substance/kg bw
Basis:
actual ingested
No. of animals per sex per dose:
15
Control animals:
yes, concurrent vehicle
Positive control(s):
cyclophosphamide at 40 mg/kg bw

Examinations

Tissues and cell types examined:
bone marrow cells
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: dose levels were chosen based on results of range finding experiment.

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):
At 6, 24 or 48 hours 5 male and 5 female animals were sacrificed in order to harvest bone marrow cells from vehicle control group or treatment group.
Bone marrow cells from 5 positive control animals per sex were harvested only 24 hours after compound administration.

DETAILS OF SLIDE PREPARATION:
Approx. 4, 22 or 24 hours after compound administration of the test and control substances the appropriate groups of animals received a single intraperitoneal injection of colchicin (2 mg/kg bw; dosing factor 5 ml/kg bw). Two hours thereafter the respective animals were sacrificed by CO2 asphyxation.
Immediately after sacrifice, bone marrow cells were collected from both femurs of each animal. Three adjacent washing steps with fixative (in methanol:acetic acid, 3:1) were conducted 25 minutes after cell preparation. After freesing of cells another washing step was included with fixative before dispersion onto precleanded glass micriscope slides. The cells on the slides were air-dried (2 slides/animal). The slides were stained with Giemsa stain, afterwards coverslipped and blinded for evaluation.

METHOD OF ANALYSIS: scoring visually under the microscope
50 cells in metaphase from each rat (if not possible: as many spreads as could be found)
Scanning via low power objective (10 and 25x) afterwards chromosome analysis via high power oil immersion (100x).
only cells in metaphase stage of mitosis were evaluated concerning the presence of cytogenetic abnormalities.
The following items were recorded for each animal: numbers and types of chromosomal aberrations, mitotic index, chromosome number for each metaphase and the vernier location of each metaphase containing damage.
Evaluation criteria:
classification of cytogenetic aberrations
- chromatid breaks - including fragments and deletions
- chromosome breaks - including acentric fragments, deletions and minutes
- chromatid and chromosome gaps
- exchanges - rings, dicentrics, translocations, quadriradials and triradials
- cells with >= 10 aberrations
- pulverised cells
Statistics:
mean mitotic index, mean chromosome number, percent aberrant cells and mean number of aberrations per cell for each group were statistically compared using Kruskal-Wallis nonparametric analysis of variance and nonparametric pairwise group comparisons (KW-ANOVA).
Body weight data was analysed by analysis of covariance (ANCOVA). All tests were evaluated at the one-tailed, 95% confidence interval.

Results and discussion

Test results
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Remarks:
A variety of clinical signs of toxicity were observed in treated rats (details given in additional information on results).
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Dose range:
1st experiment: 50, 150, and 500 mg BHMT pure substance/kg (i.e. 77, 230, and 770 mg submission substance/kg)
2nd experiment: 250, 500, 1000 and 2000 mg BHMT pure substance/kg (i.e. 385, 770, 1538, and 3077 mg submission substance/kg)
- Clinical signs of toxicity in test animals:
at 77 mg submission substance/kg: no abnormal signs were observed
at 230 mg submission substance/kg: slightly depressed animals
at 385 mg submission substance/kg: slightly depressed animals, soft feces, red stains around nose/eyes (effects marginal, each effect noted only in one animal)
at 770 mg submisson substance/kg: the toxic effects were more pronounced as evident in mor animals, soft feces, wheezing, and red stains around nose/eyes were evidence of toxicity, along with depressions in body weights.
-at higher doses: same effects but more pronounced
- Evidence of cytotoxicity in tissue analyzed: no mitotic delay was observed at 770 mg submission substance/kg
- Harvest times: 24 h
- Other: no excessive mortality was observed at 770 mg submission substance/kg

RESULTS OF DEFINITIVE STUDY
- Types of structural aberrations for significant dose levels (for Cytogenetic or SCE assay): No significant differences between the mean chromosome numbers or mean mitotic indices of negative controls versus the dosed compound groups were seen. The cyclophosphamide treated controls exhibited a significant increase in the average number of aberrations, percent of cells with aberrations, decreased mitotic index and decreased chromosome number, confirming the sensitivity of the assay to known mutagens.
- Appropriateness of dose levels and route: was verified in the range finding study
- Other: One male and one female died while on study after being given 770 mg submission substance/kg. A variety of clinical signs of toxicity were
observed in treated rats. These included depressed motor activity, labored breathing and wheezing, soft feces, and red stains around nose/eyes. Significant body weight depressions were found in treated rats of both sexes at 24 and 48 hours in comparison to controls. The positive control females also experienced a body weight depression.


Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative
Results obtained in this in vivo rat bone marrow chromosome aberration test demonstrated that the test item is not a cytogenetic active agent under the conditions tested.
Executive summary:

To investigate cytogenetic potential of the submission substance an in vivo rat bone marrow assay was performed comparable to OECD TG 475.

The submission substance dosed at 770 mg/kg did not produce any evidence of chromosome damage as measured by increases in chromosome aberrations, altered mitotic index (as measure for cytotoxic effects), or chromosome number as compared to concurrent controls in this assay. The positive control substance, cyclophosphamide, produced significant increase in the percent of cells displaying chromosome aberrations, average number of aberrations and decreased the mitotic index and chromosome number, confirming senstitivity of the test system applied. Under the conditions of this assay, the submisson substance is not cytogenetically active.