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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1988-06-27 to 1988-09-15
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1988
Report date:
1988

Materials and methods

Test guideline
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
GLP compliance:
yes
Type of assay:
other: Chromosomal Aberration Assay

Test material

Constituent 1
Chemical structure
Reference substance name:
α,α,α-trifluoro-4-nitro-m-cresol
EC Number:
201-818-2
EC Name:
α,α,α-trifluoro-4-nitro-m-cresol
Cas Number:
88-30-2
Molecular formula:
C7H4F3NO3
IUPAC Name:
α,α,α-trifluoro-4-nitro-m-cresol
Test material form:
solid
Specific details on test material used for the study:
OTHER SPECIFICS: black liquid
RT#975297, FCL#1409 6/3/88

Method

Species / strain
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):
CELLS USED
- Source of cells: laboratory of Dr. S. Wolff, University of California, San Francisco, USA
- Cell cycle length: 12 - 14 h
- Modal number of chromosomes: 21


MEDIA USED
- Type and identity of media including CO2 concentration if applicable: McCoy's 5a culture medium which was supplemented with 10 % fetal calf serum (FCS), 1 % L-glutamine, and 1 % penicillin and streptomycin, at ~ 37 °C, in an atmosphere of ~5 % CO2 in air.
Cytokinesis block (if used):
Colcemid
Metabolic activation:
with and without
Metabolic activation system:
S9 mix, derived from the liver of male Sprague-Dawley rats which had been previously treated with Aroclor 1254.
Test concentrations with justification for top dose:
- S9 mix: 49.9, 99.2, 149.0, and 198 µg/mL
+ S9 mix: 384.0, 765.0, 1150.0, and 1540.0 µg/mL
Based on the results of the range finding assay
Vehicle / solvent:
- Solvent used: DMSO
- Justification for choice of solvent: homogenous distribution of the test substance.
Controlsopen allclose all
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
mitomycin C
Remarks:
without S9 mix
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
with S9 mix
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium
- Cell density at seeding:
20 h test: 1.2E6 / 75 cm² flask
10 h test: 1.5E6 / 75 cm² flask

DURATION
- Exposure duration:
17.25 h without S9 mix
2 h with S9 mix
- Expression time: 7.75 h with S9 mix

SPINDLE INHIBITOR: 2.5 h colcemid (0.1 µg/mL)

STAIN: Giemsa staining (5 %)

NUMBER OF REPLICATIONS: 2

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: air dried slides, the slides were stained in pH 6.8 buffered 5% Giemsa solution

NUMBER OF CELLS EVALUATED: 100 from each duplicate

OTHER EXAMINATIONS:
Cells with aberrations, but not gaps, were recorded on the data sheets by the microscope stage location. Chromatid and isochromatid gaps, if observed, were noted in the raw data and were tabulated. They were not, however, considered in the evaluation of the ability of the test article to induce chromosomal aberrations since they may not represent true chromosomal breaks.

Evaluation criteria:
The following factors were taken into account in the evaluation of the chromosomal aberrations data:
1. The overall chromosomal aberration frequencies.
2. The percentage of cells with any aberrations.
3. The percentage of cells with more than one aberration.
4. Any evidence for increasing amounts of damage with increasing dose, i.e., a positive dose response.
5. The estimated number of breaks involved in the production of the different types of aberrations which were observed, i.e. complex aberrations may have more significance than simple breaks.
Statistics:
Statistical analysis employed the Fisher's Exact Test with an adjustment for multiple comparisons (Sokal and Rohlf, 1981) to compare the percentage of cells with aberrations in each treatment group with the results from the pooled solvent and negative controls (the solvent and negative controls were statistically evaluated for similarity prior to the pooled evaluation using Fisher's Exact Test). Test article significance was established where p < 0.01.

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
not examined
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
not examined
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: Solubility of the test article was evaluated in McCoy's 5a culture medium and dimethyl sulfoxide (DMSO). Uneven dark emulsions were obtained in McCoy's 5a culture medium at concentrations of 500, 300, 100, and 50 mg/mL. Dark even suspensions were obtained in DMSO at 500, 300, 200, and 100 mg/mL.
- Precipitation: Subsequent 1:100 dilutions (see above) were made from these stocks in McCoy's 5a culture medium to evaluate precipitation. Small black globules precipitated and adhered to the dilution tube at final concentrations of 5000 and 3000 µg/mL. A slight clouding was observed immediately upon dosing at 2000 µg/mL, which disappeared as the compound went into solution with no precipitate visible. DMSO was the solvent of choice at a stock concentration of 200 mg/mL. A slight clouding was observed immediately upon dosing at 2000 µg/mL, which disappeared as the compound went into solution with no precipitate visible. DMSO was the solvent of choice at a stock concentration of 200 mg/mL.


RANGE-FINDING/SCREENING STUDIES:
Rangefinding Assay Without Metabolic Activation
A precipitate was visible at the time of dosing at 2000 µg/mL. An unhealthy cell monolayer was observed at this concentration with floating dead cells and an accurate assessment could not be made on the presence of mitotic cells. There was no visible precipitate at the subsequent concentrations of 667 and 200 µg/mL and complete cellular toxicity (total absence of metaphase cells) was observed at these dose levels with no cell monolayer remaining. An unhealthy cell monolayer with floating dead cells was observed at 66.7 µg/mL. Results were evaluated at 0.667, 2.00, 6.67, 20.0, and 66.7 µg/mL. Severe cell cycle delay was observed at 6.67, 20.0, and 66.7 µg/mL. This delay in cell cycle persisted at 2.00 µg/mL. An extended 20 hour aberrations assay was selected for testing a dose range of 4.00, 6.00, 8.00, 20.0, 40.0, 60,0, and 80.0 µg/mL.
Rangefinding Assay With Metabolic Activation
A precipitate was visible at the time of dosing at 667 and 2000 µg/mL and complete toxicity (total absence of metaphase cells) was observed at these concentrations. There was no discernible toxicity at the subsequent concentration of 200 µg/mL. Results were analyzed at 20.0, 66.7, and 200 µg/mL. There was no significant cell cycle delay at these test concentrations. A regular 10 hour harvest was selected for the aberrations assay testing a dose range of 34.9, 52.4, 69.9, 175, 349, 524, and 699 µg/mL.

CYTOKINESIS BLOCK: Colcemid

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: Prior to the harvest of the cultures visual observations of toxicity were made. These observations included an assessment of the percent confluence of the cell monolayer within the culture flasks. The cultures were also evaluated for the presence of mitotic (large rounded cells) or dead cells floating in the medium.

Any other information on results incl. tables

Metabolic Activation: - S9 Mix

Number and Type of Aberration

 

 

 

 

 

 

Not computed

simple

Complex

 

 

 

 

Conc. (µg/mL)

Cells Scored

TG

SG

UC

TB

SB

IM

ID

TR

QR

CR

D

R

CI

Other GT

No. of Aberrations/Cell

% Cells with Aberrations

% Cells with >1 Aberrations

Controls, Negative and Solvent

 

200

3

1

 

1

1

 

 

1

 

 

2

 

 

 

0.03

2.5

0.0

Positive: MMC

0.08

25

2

1

 

4

2

 

1

2

3

 

 

 

2

 

0.60

36.0*

120.*

Test Substance

99.2

200

6

1

 

5

4

 

 

 

 

 

 

1

 

 

0.05

3.0

0.5

Test Substance

149

200

18

1

 

15

6

 

 

 

 

 

3

1

 

 

0.13

6.5

3.0

Test Substance

198

200

17

1

 

14

4

 

 

 

 

 

5

 

 

1

>0.16

8.5* 

2.5

* Significantly greater than the pooled negative and solvent controls, p < 0.01

Metabolic Activation: - S9 Mix

 

Number and Type of Aberration

 

 

 

 

 

 

Not computed

simple

 

Complex

 

 

 

 

Conc. (µg/mL)

Cells Scored

TG

SG

UC

TB

SB

IM

ID

TR

QR

CR

D

R

CI

DF

Other GT

No. of Aberrations/Cell

% Cells with Aberrations

% Cells with >1 Aberrations

Controls, Negative and Solvent

 

200

7

5

 

 

1

 

 

 

 

 

 

 

 

 

 

0.01

0.5

0.0

Positive:  CP

12.5

25

1

 

 

4

3

 

1

2

1

 

 

 

3

 

 

0.56

40.0*

16.0*

Test substance

384

200

8

4

 

 

 

 

 

 

 

 

1

 

 

 

 

0.01

0.5

0.0

Test Substance

769

200

15

1

 

2

1

 

 

1

 

1

 

 

 

1

 

0.03

2.5

0.5

Test Substance

1150

200

20

5

 

6

6

 

2

7

3

 

1

 

1

 

 

0.13

11.0*

2.0

Test Substance

1540

100

10

4

 

10

43

 

1

1

3

 

 

 

 

 

 

0.58

41.0*

13.0*

* Significantly greater than the pooled negative and solvent controls, p < 0.01

 

DEFINITIONS OF CHROMOSOME ABERRATIONS FOR GIEMSA STAINED CELLS:

 

NOT COMPUTED

TG Chromatid Gap: ("tidgap"). An achromatic (unstained) region in one chromatid, the size of which is equal to or smaller than the width of a chromatid. These are noted but not usually included in final totals of aberrations as they may not all be true breaks.

SG Chromosome Gap: ("isochromatidgap, IG"). Same as chromatid gap but at the same locus in both sister chromatids.

UC Uncoiled Chromosome Failure of chromatin packing. Probably not a true aberration.

PP Polyploidcell: A cell containing multiple copies of the haploid number (n) of chromosomes. Only indexed if very common.Not counted in the cells scored for aberrations.

E Endoreduplication: 4n cell in which separation of chromosome pairs has failed. Only indexed if very common. Not counted in the cells scored for aberrations.

 

SIMPLE

TB Chromatid Break: An achromatic region in one chromatid, larger than the width of a chromatid. The associated fragment may be partially or completely displaced.

SB Chromosome Break: Chromosome has a clear break, forming anabnormal (deleted) chromosome with an acentric fragment that is dislocated. This classification now includes the acentric fragment (AF). The AF was different from the SB only in that it was not apparently related to any specific chromosome.

Applicant's summary and conclusion

Conclusions:
In this in vitro chromosome aberration assay (CA) similar to OECD guideline 473, the test substance showed genotoxic properties.
Executive summary:

In this study, cultured CHO cells were exposed to the test substance at concentrations of 49.6, 99.2, 149, or 198 µg/mL for 17.25 h in absence of the S9 metabolic activation. In the presence of the S9 activation, the CHO cells were exposed to the test substance at concentrations of 115, 384, 769, 1150, or 1540 µg/mL for 2 h. After exposure to the test substance, the treated cells were washed with buffered saline, and complete McCoy’s a medium containing 0.1 µg/mL colcemid was added to the washed cells. The cells were then incubated for 2.5 h (without S9) or 7.5 h (with S9). The metaphase cells were then harvested, and slides prepared for analysis. The results showed that without S9 activation, the test substance at concentrations of 149 and 198 µg/mL induced chromosomal aberrations, consisting mainly of simple chromatid breaks. In the presence of S9 activation, 1150 and 1540 µg/mL of the test substance caused a statistically significant and dose-related increase in chromosomal aberrations, consisting of simple chromatid and chromosome breaks.

The test substance was tested up to cytotoxic concentrations, based on the results of a rangefinding study. The positive controls (without S9 mix: 0.08 µg/mL mitomycin C, with S9 mix: 12.5 µg/mL cyclophosphamide) did induce the appropriate response. There was a concentration related positive response of induced chromosomal aberrations compared to untreated and solvent control.

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