2,3,5,6-tetrachloropyridine

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:

29 May 1991 to 24 February 1992

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1992

Report date:

1992

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

GLP compliance:

yes

Type of assay:

micronucleus assay

Test material

Test material information

Constituent 1

Reference substance name:

2,3,5,6-tetrachloropyridine

EC Number:

219-283-9

EC Name:

2,3,5,6-tetrachloropyridine

Cas Number:

2402-79-1

Molecular formula:

C5HCl4N

IUPAC Name:

2,3,5,6-tetrachloropyridine

Details on test material:

Reference: AGR 0289294
Batch number: WP-900914-748
Purity = 98.6%

Test animals

Species:

mouse

Strain:

CD-1

Sex:

male/female

Details on test animals or test system and environmental conditions:

Male and female CD-1 (ICR) BR (abbreviated as CD-1) mice, approximately 8 weeks old, were used in the study. The mice were ordered from Charles River Laboratories, Inc., of Wilmington, MA. This strain of mouse was selected because of its (1) suitability for use in the micronucleus test, (2) general acceptance for short-term testing, and (3) availability of spontaneous micro¬nucleus rates. Upon arrival, the animals were examined by a veterinarian and found to be in good health. The mice were housed singly in suspended wire-bottom cages with computer generated, unique identification numbers. The mice were eartagged with identification numbers. Dustless, neomycin-impregnated cage boards were used to retard formation of unpleasant odors and to aid in the maintenance of a clean environment. Upon receipt, the mice were allowed to acclimatize for at least a week in a room designed to maintain adequate environ¬mental conditions with regard to temperature and humidity. The animals were maintained in the same environment during the remainder of the study. The mice were fed on Purina Certified Rodent Chow #5002 (Purina Mills, Inc., Richmond, IN). The chow is certified to contain no more than the specified maximum concentrations of heavy metals, organo¬phosphates, chlorinated hydrocarbons, PCB's, aflatoxins, and to have been manufactured in a plant where antibiotics and synthetic estrogens are prohibited. Municipal water was available to the animals at all times via pressure-activated water outlets. Records of periodic water analysis and feed certification are maintained at the laboratory.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

Corn oil

Details on exposure:

All doses of the test material and positive control substance cyclophosphamide (CP) were administered by single oral gavage. In both the range-finding test and the micronucleus assay, the test material was administered in aliquots of 10 ml/kgBW. Negative control mice received 10 ml/kgBW of corn oil. CP was administered in aliquots of 10 ml/kgBW.

Duration of treatment / exposure:

All doses of the test material and CP were administered by single oral gavage. Groups of animals were sacrificed at three intervals, viz., 24, 48, and 72 hours after treatment. Positve control mice treated with CP were sacrificed after 24 hours.

Frequency of treatment:

All doses of the test material and CP were administered by single oral gavage.

Post exposure period:

Groups of animals were sacrificed at three intervals, viz., 24, 48, and 72 hours after treatment. Positve control mice treated with CP were sacrificed after 24 hours.

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
22.5, 75.0, 225.0 mg/kg bw (males)
Basis:
actual ingested
oral gavage

Doses / concentrations 2

Remarks:

Doses / Concentrations:
93.0, 310.0, 930.0 mg/kg bw (females)
Basis:
actual ingested
oral gavage

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Positive control(s):

Cyclophosphamide (CP, CAS No. 50-18-0) purchased from Sigma, St. Louis, MO, was used as a positive control chemical at a dose level of 120 mg/kgBW. This dose level was based upon the experience of this laboratory (Gollapudi et al., 1985) and was selected to induce an unequivocal positive response.

Examinations

Tissues and cell types examined:

Bone marrow.

Details of tissue and slide preparation:

At the end of the specified intervals following dosing, the animals were sacrificed by cervical dislocation. Bone marrow samples were obtained from both femurs in the following way. After separating the bone from the adjoining muscles, the distal end of the femur was severed to expose the marrow cavity. A 25-gauge needle was used to aspirate the bone marrow into a 3 ml syringe containing approximately 0.5 ml of fetal bovine serum (GIBCO, Grand Island, New York). After aspiration, the contents of the syringe were transferred into a 1.5 ml centrifuge tube containing 0.5 ml of serum. The cells were resuspended in the serum by gentle aspiration using the syringe and needle. The tubes were centrifuged at about 1000 rpm for approximately 5 minutes in a table-top centrifuge. The supernatant was discarded leaving a small amount of serum covering the pellet. The cell pellet was resuspended using a disposable transfer pipet. Wedge smears were prepared on microscope slides using small portions of the cell suspension. The slides were allowed to air dry and stained with Wright-Giemsa using a Hematek automatic slide stainer.

The slides were coded and scored blindly by a single investigator. One thousand polychromatic erythrocytes were examined from each animal and the number of micronucleated polychromatic erythrocytes (MN-PCE) was recorded. Micronuclei were identified as darkly stained bodies with sharp contours and varying shapes such as round, almond, or ring (Schmid, 1976). The ratio of PCE-NCE in the bone marrow was determined by examining 1000 erythrocytes per animal. The ratio was expressed as PCEx100/PCE+NCE.

Evaluation criteria:

Statistically significant increase in the fequencies of micronuceated bone marrow polychromatic eryhtrocytes.

Statistics:

The raw data on the counts of MN-PCE for each animal was first transformed by adding 1 to each count and then taking the natural log of the adjusted number. The transformed MN-PCE data and the data on percent PCE were analyzed separately by a three-way analysis of variance (sex, dose, and time) assuming the three-way interaction to be zero. From these initial analyses, the two-way interactions were reviewed for significance. Depending on these reviews, the data were analyzed by either one-, two-, or three-way analysis of variance looking at main effects only. Pairwise com¬parisons of treated vs. control groups were done, if necessary, by Dunnett's t-tests, one-sided (upper) for MNPCE and two-sided for percent PCE. The alpha level at which all the tests were conducted was 0.01.

The final interpretation of biological significance of the responses was based on both statistical outcome and scientific judgment.

Results and discussion

Test results

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

The dose levels for the micronucleus assay were based on the outcome of a range finding assay in which toxicity was observed.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information): negative
Based upon the results, it was concluded that the test chemical did not induce a significant increase in the frequencies of micronucleated bone marrow polychromatic erythrocytes when given as a single oral dose to male and female CD-1 mice. Hence, under the experimental conditions used, the test chemical was judged negative in the mouse bone marrow micronucleus test.

Executive summary:

A GLP compliant study conducted according to OECD, EEC and US EPA guidelines was conducted in order to assess the mutagenic potential of the test substance. Based upon the results, it was concluded that the test chemical did not induce a significant increase in the frequencies of micronucleated bonemarrow polychromatic erythrocytes when given as a single oral dose to maleand female CD-1 mice. Hence, under the experimental conditions used, the test chemical was judged negative in the mouse bone marrow micronucleus test.