1H-Pyrazole-5-carboxylic acid, 3-bromo-1-(3-chloro-2-pyridinyl)-

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

2007-04-11 to 2007-07-19

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study was performed according to OECD guideline 473 and GLP.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2007

Report date:

2007

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

Remarks:

The study was conducted according to the guideline in effect at time of study conduct.

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

481-150-8

EC Name:

-

Cas Number:

500011-86-9

Molecular formula:

C9H5BrClN3O2

IUPAC Name:

3-bromo-1-(3-chloropyridin-2-yl)-1H-pyrazole-5-carboxylic acid

Constituent 2

Reference substance name:

DBC80

IUPAC Name:

DBC80

Constituent 3

Reference substance name:

1H-Pyrazole-5-Carboxylic Acid, 3-Bromo-1-(3-Chloro-2-Pyridinyl)-

IUPAC Name:

1H-Pyrazole-5-Carboxylic Acid, 3-Bromo-1-(3-Chloro-2-Pyridinyl)-

Details on test material:

- Name of test material (as cited in study report): IN-DBC80
- Physical state: Tan solid
- Analytical purity: 96.7 %
- Lot/batch No.: IN-DBC80-008

Method

Species / strain

Species / strain / cell type:

primary culture, other: human peripheral blood lymphocytes

Details on mammalian cell type (if applicable):

- Type and identity of media: RPMI 1640 medium (serum free medium for + S9 activated cultures)

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254 induced rat liver S9 (10 %)

Test concentrations with justification for top dose:

4 hour exposure without activation - 1000, 2000, and 3025 ug/mL.
4 hour exposure with activation - 500, 1000 and 2000 ug/mL.
22 hour exposure without activation - 250, 500, and 1000 ug/mL.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The vehicle was determined to be the solvent of choice based on the solubility of the test substance and compatibility with the target cells. This vehicle permitted preparation of the highest workable/soluble stock concentration. The test substance was soluble in the vehicle at 302.5 mg/ml (1 M), the highest stock concentration that was prepared for use on this study. Under the conditions of the test substance, the final concentration of DMSO in the treatment medium did not exceed 1 % of the treatment medium.

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

Without S9 activation; Positive controls were not used for numerical aberrations

Migrated to IUCLID6: 0.2 ug/mL (4 and 22 hour exposure periods)

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

With S9 activation; Positive controls were not used for numerical aberrations

Migrated to IUCLID6: 5 ug/mL (4 hour exposure period, no 22 hour exposure performed)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 4 and 22 hours in the non-activated test condition and 4 hours in the S9 activated test condition. Colcemid was added 19 hours after initiation of exposure.
- Expression time (cells in growth medium): There was no expression period for the 22 hour exposure and approximately a 15 hour expression period for the 4 hour exposures.
- Fixation time (start of exposure up to fixation or harvest of cells): Cells were harvested about 22 hours (time represents approximately 1.5 normal cell cycles, and is determined to ensure assessment of clastogenicity in first-division metaphase cells) after initiation of treatment. Colcemid was present during the last 3 hours.

SPINDLE INHIBITOR (cytogenetic assays): Colcemid
STAIN (for cytogenetic assays): Giemsa

NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED: 200 metaphase cells total (when available) per group (the number of metaphases evaluted per duplicate flask was less if 10 or more aberrant cells were observed among the first 25 cells scored).

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index

OTHER EXAMINATIONS:
- Determination of polyploidy: yes
- Determination of endoreplication: yes
- Other: A visual evaluation of all cultures was made at the beginning and end of the treatment period to assess both pH and precipitation.

OTHER: Whole venous blood was drawn from selected healthy, male and/or female donor(s) less than 50 years old without previous chemotherapy or radiotherapy and without recent (within the last 6 months) viral disease, use of prescription drugs, or X-ray exposure. Approximately 10 to 20 mL of blood per donor was collected into heparinzied containers. The lymphocyte cultures were initiated in 15 mL centrifuge tubes by inoculating 0.5 mL of heparinized blood to 9.5 mL of complete medium (RPMI 1640 medium containing approximately 15 % fetal bovine serum, 2 mM L-glutamine, 100 units penicillin/mL, and 100 ug streptomycin/mL) supplemented with 1 % phytohemagglutinin-M. Cultures were incubated at 37 degrees Celcius +/- 2 degrees in a humidified atmosphere of 5 +/- 2 % CO2 in air. Approximately 48 hours after culture initiation, the whole blood cultures were centrifuged and the culture medium was discarded and replaced with treatment medium.

Evaluation criteria:

An assay was considered acceptable for evaluation if the following were satisfied:
1. Vehicle Control- The frequency of cells with structural chromosome aberrations was in the frequency range of the historical control.
2. Positive Controls- The % of cells with structural chromosome aberrations was statistically significantly greater (p<0.05, Fisher's exact test) than the vehicle control.

The clastogenic potential of the test substance was assessed based on its ability to induce structural chromosome aberrations.  The % of cells with structural aberrations was used.  Statistical analysis was used as a guide to determine if there was a positive response.  Interpretation relied on additional considerations including the magnitude of the observed response relative to the vehicle control response and the presence of a dose response trend.

The following conditions were used as a guide to determine a positive response:
1. A statistically significant increase (p<0.05, Fisher's exact test) in the % of cells with structural aberrations was seen in one or more treatment groups relative to the vehicle control.
2. The observed increased frequencies were accompanied by a concentration-related increase.
3. A statistically significant increase was observed at the highest dose only.
Note: Statistically significant values that did not exceed the historical control range for the vehicle control may be judged as not being biologically significant.

The following condition was used as a guide to determine an equivocal response:
1. Results observed in any of the assays resulted in statistically significant elevations in structural chromosome aberrations at more than one test concentration level, except the highest dose, without demonstrating a dose-response.

The test substance was judged negative if the following condition was met:
1. There was no statistically significant increase in the % of cells with structural aberrations in any treatment group relative to the vehicle control.

Statistics:

Statistical analysis consisted of a Fisher's exact test to compare the percentage of cells with structural or numerical aberrations (or the percentage of cells with more than one aberration, if required) in the test substance treated groups with the vehicle control response. A Cochran-Armitage test for dose responsiveness was conducted only on values that were statistically significant based on the Fisher's exact test.

Results and discussion

Test results

Species / strain:

primary culture, other: human periperal blood lymphocytes

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at highest dose levels tested at 4 and 22 hours with metabolic activation, 2000 ug/mL and 1000 ug/mL respectively

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: In the range-finding assay the pH of the highest test substance concentration in media was similar to the pH of the vehicle control and the pH did not change during the treatment period. In the non-activated test system, the measured pH for the highest test substance concentration in media was 7.25 compared to 8.15 for the vehicle control. In the S9-activated test system, the measured pH for the highest test substance concentration in media was 7.04 compared to 7.79 for the vehicle control. In the chromosome aberration assays the pH of the treatment medium of the highest concentrations at the beginning and end of treatment periods was also similar to the pH of the vehicle control and the pH did not change during the treatment periods.
- Effects of osmolality: Range-Finding assay- The osmolality of the highest concentration tested in treatment medium was 412 and 426 mmol/kg in the non-activated and activated test system, respectively. The osmolality of the vehicle in the treatment medium was 445 and 446 mmol/kg in the non-activated and activated test system, respectively. The observed changes in osmolality were <= 20 % and were not considered significant.
- Precipitation: Range-Finding assay and Chromosome Aberration assay- A visible precipitate was observed in the treatment medium at 3025 ug/mL in the beginning and the end of the 4-hour treatment periods. At the end of the 22 hour treatment period, no visible precipitate was observed at any concentration level.
- Other confounding effects: The test substance formed a dark brown solution in DMSO at 302.5 mg/mL (the highest stock solution used in the study), and was soluble at all concentrations tested in the preliminary toxicity and chromosome aberration assays.


RANGE-FINDING/SCREENING STUDIES: Human peripheral blood lymphocytes were treated with the test substance for 4 and 22 hours in the absence of S9 and for 4 hours in the presence of S9 at concentrations ranging from 25 to 3025 ug/mL. After completion of the 4-hour exposure periods only, the cells were collected by centrifugation and the treatment medium was replaced with complete RPMI 1640 culture medium and incubated until cell harvest. All cells were harvested 22 hours after treatment initiation, with Colcemid added 3 hours prior to harvest. The highest concentration was selected for the chromosome aberration assay that induced greater than a 50 % reduction of the mitotic index relative to the vehicle control. Four additional dose levels, demonstrating limited toxicity or no toxicity were also evaluated. In cases where there was little or no cytotoxicity, but a precipitate was observed the lowest dose level demonstrating a precipitate and two other lower dose levels were selected for analysis.
The osmolality and pH of the vehicle control, as well as the highest soluble test substance concentration, were determined. An increase of <= 20 % relative to the vehicle control in the osmolality of the test substance in the treatment medium was considered acceptable. The pH of the treatment medium and precipitation were evaluated both at the beginning and end of the treatment period.
No substantial toxicity (at least a 50 % reduction in mitotic index relative to the vehicle control) was observed at any concentration level in the 4-hour non-activated test system. Substantial toxicity was observed at 3025 ug/mL in the 4-hour activated test system and at concentrations >= 1000 ug/mL in the 22 hour non-activated test system. The mitotic inhibition relative to the vehicle control for the 4 hour non-activated test system at 3025 ug/mL was 12 %. The mitotic inhibition relative to the vehicle control for the 4-hour S9 activated test system at 3025 ug/mL was 71.4 %. The mitotic inhibition relative to the vehicle control for the 22 hour non-activated test system at 1000 ug/mL was 51 %. Based on these findings, the highest concentration chosen for the chromosome aberration assay was noted to be 3025 ug/mL for the 4 hour non-activated and activated test systems and 2000 ug/mL for the 22 hour non-activated test system. The information presented on the actual chromosomal aberration study indicate that the highest concentrations chosen were 3025 ug/mL for the 4-hour non-activated, 2000 ug/mL for the 4-hour activated test system, and 1000 ug/mL for the 22-hour non-activated test system

COMPARISON WITH HISTORICAL CONTROL DATA: Solvent and positive control values for % structural and numerical aberrations fell within the historical control range for all assays except the positive control of the 4 hour treatment period in the presence of S9, which had a mean value of 13.5 % structural aberrations. The historical control range was 14-58 %.

ADDITIONAL INFORMATION ON CYTOTOXICITY AND CHROMOSOME ABERRATIONS:
Non-activated 4-hour assay- The mitotic index for the highest test concentration evaluated microscopically for chromosome aberrations, 3025 ug/mL, was 6.8 %, compared with 11.9 % for the vehicle control. This represented a 42.9 % mitotic inhibition in relation to the vehicle control. The concentrations selected for microscopic analysis of chromosome aberrations were 1000, 2000, and 3025 ug/mL. The percentage of cells with structural or numerical aberrations in the test substance groups was not significantly increased above that of the vehicle control at any concentration (p>=, Fisher's exact test). The percentage of cells with structurally damaged chromosomes in the positive control group (16.5%) was statistically significant (p< 0.05, Fisher's exact test).

S9-activated 4-hour assay- The mitotic index for the highest test concentration evaluated microscopically for chromosome aberrations, 2000 ug/mL, was 5.3 %, compared with 12.5 % for the vehicle control. This represented a 57.6 % mitotic inhibition in relation to the vehicle control. The concentrations selected for microscopic analysis of chromosome aberrations were 500, 1000, and 2000 ug/mL. The percentage of cells with structural or numerical aberrations in the test substance treated group was not significantly increased above that of the vehicle control at any concentration (p>= 0.05, Fisher's exact test). The percentage of cells with structurally damaged chromosomes in the positive control group (13.5 %) was statistically significant (p< 0.05, Fisher's exact test).

Non-activated 22 hour assay- The mitotic index for the highest test concentration evaluated microscopically for chromosome aberrations, 1000 ug/mL, was 5.7 %, compared with 12.3 for the vehicle control. This represented a 53.7 % mitotic inhibition in relation to the vehicle control. The concentrations selected for microscopic analysis of chromosome aberrations were 250, 500, and 1000 ug/mL. The percentage of cells with structural or numerical aberrations in the test substance-treated group was not significantly increased above that of the vehicle control at any concentration (p>=0.05 %, Fisher's exact test). The percentage of cells with structurally damaged chromosomes in the positive control treatment group (42 %) was statistically significant (p<0.05 %, Fisher's exact test).

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

The test substance was evaluated in the in vitro chromosome aberration assay for clastogenic potential. All criteria for a valid study were met. Under the conditions of the study, the test substance was not found to induce structural or numerical chromosome aberrations in the in vitro mammalian chromosome aberration test in human peripheral blood lymphocytes in either the non-activated or S9 activated test systems. It was concluded that the test substance was negative in the in vitro chromosome aberration assay.