Trientine

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:

March 10, 1987 - April 17, 1987

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

No information of substance purity or composition. In appendix I of the report analytical information has been added but that is an MS printout, purity 68.5%? The study followed GLP and was performed according to methods similar to OECD 474. 1000 instead of 2000 immature erythocytes were examined per animal. No bone marrow toxicity was observed; however, at higher dose levels as administered in this test mortality occurred.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1987

Report date:

1987

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

other: Environmental Protection Agency - Health Effect Test Guidelines, EPA Report 560/6-83-001

Test guideline 2

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Principles of method if other than guideline:

- 1000 instead of 2000 immature erythocytes were examined per animal.

GLP compliance:

yes

Type of assay:

micronucleus assay

Test material

Test material information

Constituent 1

Reference substance name:

Trientine

EC Number:

203-950-6

EC Name:

Trientine

Cas Number:

112-24-3

Molecular formula:

C6H18N4

IUPAC Name:

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

Details on test material:

TETA
I.D.# 36-ARD-31-10)
BRRC sample number: 49-459
Storge: room temperature

No further information. In appendix I of the report analytical information has been added but that is an MS printout.

Test animals

Species:

mouse

Strain:

Swiss Webster

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles Rivers Laboratories, Portage, MI
- Age at study initiation: 5 weeks
- Weight at study initiation: male 23.6 g to 26.6 g, female 20.5 g to 23.5 g.
- Assigned to test groups randomly: yes, under following basis: randomized by weight and animals outside a range of two standard deviations from the mean were not used.
- Fasting period before study: not applicable
- Housing: Five mice/sex/cage were housed in shoe-box type plastic cages, measuring 30 x 20 x 12.5 cm.
- Diet (e.g. ad libitum): ad libitum with a basic diet of Agway PROLAB@ Animal Diet
- Water (e.g. ad libitum): Municipal Authority of Westmoreland County (Greensburg, PA) and was available ad libitum.
- Acclimation period: 5-6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): - Humidity (%): In the opinion of the study director, there were no unacceptable variations in temperature or humidity during the testing periods which would adversely affect the quality or integrity of the study.
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: March 10, 1987 - April 17 1987

Administration / exposure

Route of administration:

intraperitoneal

Vehicle:

water

Details on exposure:

None

Duration of treatment / exposure:

na

Frequency of treatment:

single i.p. injection.

Post exposure period:

72 hours

Doses / concentrations

Remarks:

Doses / Concentrations:
185, 370, 600 mg/kg bw
Basis:
nominal conc.

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Positive control(s):

triethylenemelamine
- Justification for choice of positive control(s): standard positive control
- Route of administration: i.p.
- Doses / concentrations: 0.3 mg/kg bw

Examinations

Tissues and cell types examined:

Blood from the tail fo the mice

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: range finding study.

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields): Three dose levels of approximately 80%, 50% and 25% of the pooled LD50 value were evaluated for effects upon the incidence of micronuclei. A single i.p. injection was given. Blood samples were taken at 3 time periods at approximately 30, 48 and 72 hr after dosing.

DETAILS OF SLIDE PREPARATION: One or two blood smear slides were prepared for each animal sampling time. Micronuclei in peripheral blood, polychromatic erythrocytes were stained with Gurr's R-66 Giemsa diluted in phosphate buffer. Slides were coded by animal number only and read blindly to prevent bias.

METHOD OF ANALYSIS:
A minimum of 1000 polychromatic erythocytes was examined microscopically for each animal per sample time, unless cytotoxicity of the test material prevented this goal. The polychromatic:normochromatic erythrocyte ratio for approximately 1000 total cells was calculated and recorded and these data are summarized in the final report as an estimate, of cytotoxicity of the test agent.
Micronuclei were identified as darkly-stained, spherical, inclusions in polychromatic erythrocytes. Polychromatic, erythrocytes were identified by the pale-bluish staining of the cytoplasm in contrast to the lack of blue stain for normochromatic erythrocytes.

Statistics:

Data were compared for significant differences from the vehicle control frequencies using the Fisher's Exact Test (Sokal and Rohlf, 1981). Data for
males and female mice at each sample perfod were combined for statistical analyses because Analysis of Variance tests showed that there was.no
significant difference in micronuclei frequencies between sexes at each sample period. A positive result in the micronucleus test was concluded if at least one statistically significant (p ≤0.01) increase above the vehicle control was .observed with an indication of a dose-related effect of treatment. A test was considered to be inconclusive if only one dose produced effects statistically different from the control (0.05 ≥ p ≥ 0.01) and a dose-effect relationship was apparent. A test result was considered to be negative if no statistically significant differences were apparent between the vehicle control and groups of animals treated with TETA.

Results and discussion

Test results

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Remarks:

No bone marrow toxicity was observed however at higher dose levels as administered in this test mortality occurs.

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
Analysis of variance testing indicated that there was no significant difference between the mortality response of male and female mice treated with TETA, Thus, a combined LD50 value of approximately 740 mg/kg was calculated and used to determine the test doses for the definitive micronucleus test. The lower and upper 95% fiducial limits for the LD50 value were 651 mg/kg and 877 mg/kg, respectively.
The PCE/NCE ratio of the vehicle control and the highest test dose with adequate numbers of survivors (≥ 3) was quantified and compared to determine possible bone marrow cytotoxicity. At 48 hrs after dosing; the PCE/NCE ratios of both the male and female mice were similar to the vehicle control values, thus indicating the absence of bone marrow toxicity. Since no bone marrow toxicity was evident at this sample period, no additional blood smears were obtained at later time intervals

RESULTS OF DEFINITIVE STUDY
Micronucleus determinations were conducted using a minimum of 5 animals/sex/group. Additional animals were added to some groups because deaths were expected at the higher dosages. However, extra animals were assessed for micronucleus frequencies only as needed to assure that a total of five animals are evaluated. Three dose levels of 600 mg/kg, 370 mg/kg and 185 mg/kg were selected for testing in the definitive micronucleus test at approximately 80%, 50% and 25% of the combined male-female LD50 value, respectively.

No remarkable decreases in the PCE/NCE ratios relative, to the control values were observed in this study at any of the three sampling periods. PCE/NCE ratios of the male animals treated with TEM were lower than the concurrent negative control values which is an expected and typical finding because of the cytotoxicity and clastogenicity of this agent.

Analysis of variance (AOV) testing indicated that the data for male and female mice sampled at 30 hr, 48 hr or 72 hr after dosing were not statistically different; thus, values were pooled for Fisher's Exact analyses. No statistically significant or treatment-related increases in the numbers of micronuclei were observed with any of the treatment groups sampled at any of the sample intervals following injection of the test chemical.

TEM, used as a positive control agent for this study, produced highly significant increases in numbers of. micronuclei demonstrating the appropriate sensitivity of the test system. Numbers of micronuclei in the vehicle control animals were in a low and acceptable range for this test system at all sampling
times.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information): negative
Test results for this study showed that TETA was not an active agent in producing treatment-related increases in micronuclei in male and female
Swiss-Webster mice. Relatively high dosage levels of TETA were evaluated no treatment-related clastogenic activity was observed. TETA was considered to be inactive as a clastogenic agent in vivo under the conditions of the micronucleus test.

Executive summary:

Triethylenetetrarnine (TETA) was evaluated for potential clastogenic (chromosome-damaging) activity with the in vivo micronucleus test system employing both male and female Swiss-Webster mice. Test doses for the rnicronucleus test were chosen from data obtained in a preliminary toxicity study with mice. Five doses of TETA ranging from 434 mg/kg to 900 mg/kg were administered as a single intraperitoneal (i .p. ) injection, The LD50 dose was calculated ftom the cumulative mortality observed during a three day period after dosing. To select dose levels for the definitive micronucleus test, a combined LD50 value of approximately 740 mg/kg (651 to 877; 95% fiducial limits) was calculated by pooling the total number of deaths for males and females.

For the definitive micronucleus test, doses of 185 mg/kg, 370 mg/kg and 600 mg/kg were tested with both male and female Swiss-Webster mice. Concurrent positive (triethylenernelamine) and negative (water) control agents, administered by i.p, injection, were used to demonstrate the reliability and sensitivity of the micronucleus test system. Results from the micronucleus determination demonstrated that TETA did not produce positive or dose-related increases in the incidence of micronuclei in peripheral blood polychromatic erythrocytes of the test animals at any of the sample periods tested. Data from the positive and negative control groups of animals demonstrated the appropriate resbonses for the animals in the test system consistent with a valid test. The absence of positive effects of TETA upon the incidence of micronuclei indicates that TETA does not possess clastogenic activity in vivo under the conditions of the micronucleus test system.