5,7-dichloro-4-(4-fluorophenoxy)quinoline;quinoxyfen (ISO)

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

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference 1

Reference Type:

study report

Title:

Unnamed

Year:

1994

Report date:

1994

Reference 2

Reference Type:

other: Supplemental report

Title:

Unnamed

Year:

2001

Report date:

2001

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OTS 798.5395 (In Vivo Mammalian Cytogenics Tests: Erythrocyte Micronucleus Assay)

Deviations:

no

GLP compliance:

yes

Type of assay:

other: bone marrow micronucleus test

Test material

Test material information

Constituent 1

Reference substance name:

Reference substance 001

Cas Number:

124495-18-7

Test material form:

solid: particulate/powder

Specific details on test material used for the study:

- Substance ID: TSN 100097
- Name of substance: XDE-795
- Purity: 97.4%

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 Laboratories, Inc., Portage, Michigan
- Assigned to test groups randomly: Yes
- Housing: Individually housed
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: At least 7 days

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

Corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: The test substance was mixed with corn oil for dosing. Cyclophosphamide (CP) was dissolved in distilled water. Freshly prepared solutions were used for dosing the animals. The concentrations of the test substance in the dosing solutions were verified by high pressure liquid chromatography (HPLC).

Duration of treatment / exposure:

24, 48, or 72 h after treatment

Frequency of treatment:

single

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

1 250 mg/kg bw/day

Doses / concentrations 2

Dose / conc.:

2 500 mg/kg bw/day

Doses / concentrations 3

Dose / conc.:

5 000 mg/kg bw/day

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Positive control(s):

Cyclophosphamide
- Justification for choice of positive control: Cyclophosphamide was selected to induce an unequivocal positive response
- Route of administration: Oral
- Doses / concentrations: 120 mg/kg bw (10 mL/kg)

Examinations

Tissues and cell types examined:

Bone marrow cells

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: The highest dose level of 5000 mg/kg bw was based upon the results of a range-finding test and it is also the generally accepted limit dose for relatively non-toxic test substances.

BONE MARROW SAMPLING: 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 disposable plastic syringe containing approximately 0.5 ml of fetal bovine serum. 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 1000 rpm (approximately 80 g) 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 pipette. 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.

SLIDE SCORING: The slides were coded and scored blindly. 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 smooth contours and varying shapes such as round, almond, or ring. The ratio of PCE-NCE in the bone marrow was determined by examining 1000 erythrocytes. The ratio was expressed as PCEx100/PCE+NCE.

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 MN-PCE 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 Dunnett's t-tests, one-sided (upper) for MN-PCE 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

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Dose range: 2500 and 5000 mg/kg bw
- Mortality: 10 of 10 animals treated with 5000 mg/kg bw of the test substance survived. At the 2500 mg/kg bw dose level, one female died on day two of observation. Necropsy of the dead animal indicated no evidence of gavage error. The cause of death was undetermined.
- Clinical signs of toxicity in test animals: All animals appeared normal immediately after dosing. At three hours post-dosing, all animals were exhibiting signs of lethargy. Six hours after dosing, one female and three males in the 2500 mg/kg bw group and two females and all males in the 5000 mg/kg bw group appeared to be lethargic. On day two of observation, one female in the 2500 mg/kg bw dose group was recorded as jumping violently and displaying erratic behavior. This animal died the same day. Two males in the 2500 mg/kg bw group remained lethargic on day 2 of observation. On day three of observations, two males were recorded as extremely lethargic and not eating as evidence of lack of fecal droppings under their cages. One of these males also had perineal staining.

RESULTS OF DEFINITIVE STUDY
- Clinical signs of toxicity: Lethargy was observed in most of the male mice treated with the test material two hours after dosing. At four hours post dosing, males in the 1250 mg/kg bw dose group appeared somewhat lethargic. Those in the 2500 mg/kg bw dose group were expressing varying degrees of lethargy and 7 of the 10 males in the 5000 mg/kg bw dose group appeared lethargic. At the 24-hour sacrifice, all males treated with the test materials had varying degrees of perianal staining. Lethargy was also observed in the 5000 mg/kg bw male group. The remaining males appeared to return to normal on day three of observation. At the 72 h sacrifice, a 1250mg/kg bw male group could not open its eyes due to ocular discharge.
- Induction of micronuclei: There were no significant differences in MN-PCE frequencies between the groups treated with the test substance and the negative controls. The positive control chemical, CP, induced a significant increase in the frequencies of micronucleated polychromatic erythrocytes. The percent PCE values observed in the test substance-treated animals were not significantly different from the negative control values at any dose level tested. However, the %PCE values in the test substance treated males tended to be lower than the negative controls especially in the groups harvested at 48 and 72 h after treatment. The biological significance of this trend, nevertheless, is uncertain. The positive control chemical (CP), on the other hand, had a significant effect on the ratio of PCE to NCE.

Applicant's summary and conclusion

Conclusions:

Negative mouse micronucleus test

Executive summary:

The test substance was evaluated in the mouse bone marrow micronucleus test following OECD guideline 474 and US EPA 798.5395. The micronucleus test is capable of detecting agents causing chromosomal aberrations and spindle malfunction. The test substance was administered to CD-1 mice by single oral gavage at dose levels of 0 (negative control), 1250, 2500, and 5000 mg/kg body weight. The highest dose level of 5000 mg/kg bw was based. upon the results of a range-finding test and it is also the generally accepted limit dose for relatively non-toxic test substances. The concentrations of the test substance in the dosing solutions were verified by analytical methods. Groups of animals were sacrificed at 24, 48, or 72 h after treatment. Mice treated with 120 mg/kg bw 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 surviving animal and the frequencies of micronucleated polychromatic erythrocytes (MN-PCE) were recorded. There were no statistically significant increases in the frequencies of MN-PCE in groups treated with the test substance as compared to negative controls. The positive control mice showed significant increases in MN-PCE. Hence, under the experimental conditions used, the test substance was considered to be negative in the mouse bone marrow micronucleus test.