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Genetic toxicity: in vitro

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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:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted per OECD Test Guideline 473 following GLP.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2001
Report date:
2001

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test

Test material

Constituent 1
Chemical structure
Reference substance name:
4-(α,α-dimethylbenzyl)phenol
EC Number:
209-968-0
EC Name:
4-(α,α-dimethylbenzyl)phenol
Cas Number:
599-64-4
Molecular formula:
C15H16O
IUPAC Name:
4-(2-phenylpropan-2-yl)phenol
Constituent 2
Reference substance name:
290-968-0
IUPAC Name:
290-968-0
Details on test material:
Phenol, p-(alpha-, alpha-dimethylbenzyl)(p-Cumylphenol (PCP, CAS #599-64-4); Purity: >99%; received as white solid.

Method

Species / strain
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254-induced rat liver S9
Test concentrations with justification for top dose:
3.75 to 60 µg/mL
Vehicle / solvent:
DMSO was the chosen vehicle as it provided the preparation of the highest workable stock solution.
Controls
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
Positive controls:
yes
Remarks:
Mitomycin C (0.1 and 0.2 µg/mL) or Cyclophosphamide (10 and 20 µg/mL)
Details on test system and experimental conditions:
Description of test procedure: A preliminary toxicity assay was performed for the purpose of selecting dose levels for the chromosome aberration assay and consisted of an evaluation of test article effect on cell growth. CHO cells were seeded for each treatment condition at approximately 5 x 10^5 cells/25 cm2 flask and were incubated at 37 +/- 1 degree C in a humidified atmosphere of 5 +/- 1% CO2 in air for 16-24 hours. Treatment was carried out by refeeding the flasks with 5 mL complete medium (McCoy's 5A medium supplemented with 10% fetal bovine serum (FBS), 100 units penicillin and 100 ug streptomycin/mL, and 2 mM L-glutamine) for the non-activated study or S9 reaction mixture (4 mL serum-free medium plus 1 mL of 5xS9 mix) for the activated study, to which was added 50 µL dosing solution of test article in solvent or solvent alone. The osmolality of the highest concentration of dosing solution in the treatment medium was measured. The pH of the highest concentration of dosing solution in the treatment medium was measured using test tape. The cells were treated for 4 hours with and without S9, and continuously for 20 hours without S9. At completion of the 4 hour exposure period, the treatment medium was removed, the cells washed with calcium and magnesium-free phosphate buffered saline (CMF-PBS), refed with 5 mL complete medium and returned to the incubator for a total of 20 hours from the initiation of treatment. At 20 hours after the initiation of treatment the cells were harvested by trypsinization and counted using a Coulter counter. The presence of test article precipitate was assessed using the unaided eye. Cell viability was determined by trypan blue dye exclusion. The cell counts and percent viability were used to determine cell growth inhibition relative to the solvent control. In the preliminary toxicity assay, the maximum dose tested was 2123 µg/mL. Visible precipitate was observed at concentrations greater than or equal to 212.3 µg/mL and dose levels less than or equal to 63.7 were soluble. Selection of doses for the chromosome aberration assay was based on cell growth inhibition relative to the solvent control. Toxicity, i.e., at least 50% cell growth inhibition, was observed at any dose greater thn or equal to 63.7 µg/mL in the non activated 4- and 20-hour exposure groups and in the S9-activated 4 hour exposure group. Based on these findings, the doses chosen for the chromosome aberration assay ranged from 3.75 to 60 µg/mL for the non-activated 4- and 20-hour exposure groups and for the S9 activated 4 hour exposure group.

For the chromosome aberration assay, CHO cells were seeded at approximately 5 x 10^5 cells/25 cm2 flask and were incubated at 37 +/- 1 degree C in a humidified atmosphere of 5 +/- 1% CO2 in air for 16 to 24 hours. Treatment was carried out by refeeding duplicate flasks with 5 mL complete medium (McCoy's 5A medium supplemented with 10% FBS, 100 units penicillin and 100 µg streptomycin/mL, and 2 mM L-glutamine) for the non-activated study or 5 mL S9 reaction mixture for the S9 activated study, to which was added 50 µL of dosing solution of test or control article in solvent or solvent alone. The osmolality of the highest concentration of dosing solution in the treatment medium was measured. The pH of the highest concentration of dosing solution in the treatment medium was measured using test tape.

In the non-activated study, the cells were exposed to the test article continuously for 4 or 20 hours. After the exposure period for the 4 hour exposure group, the treatment medium was removed, the cells washed with CMF-PBS, refed with complete medium and returned to the incubator. Two hours prior to the scheduled cell harvest, Colcemid was added to duplicate flasks for each treatment condition at a final concentration of 0.1 µg/mL and the flasks returned to the incubator until cell collection. In the S9 activated study, the cells were exposed for 4 hours. After the exposure period, the treatment medium was removed, the cells washed with CMF-PBS, refed with complete medium and returned to the incubator. Two hours prior to the scheduled cell harvest, Colcemid was added to duplicate flasks for each treatment condition at a final concentration of 0.1 µg/mL and the flasks were returned to the incubator until cell collection. Two hours after the addition of Colcemid, metaphase cells were harvested for both the non-activated and S9 activated studies by trypsinization. Cells were collected approximately 20 hours after initiation of treatment. The cells were collected by centrifugation at approximately 800 rpm for 5 minutes. The cell pellet was resuspended in 2-4 mL 0.075 M potassium chloride (KCl) and allowed to stand at room temperature for 4-8 minutes. The cells were collected by centrifugation, the supernatant aspirated and the cells fixed with two washes of approximately 2 mL Carnoy's fixative (methanol:glacial acetic acid, 3:1, v/v). The cells were stored overnight or longer in fixative at approximately 2-8 degrees C. To prepare slides, the fixed cells were centrifuged at approximately 800 rpm for 5 minutes, the supernatant was aspirated, and 1 mL fresh fixative was added. After additional centrifugation (at approximately 800 rpm for 5 minutes) the supernatant fluid was decanted and the cells resuspended to opalescence in fresh fixative. A sufficient amount of cell suspension was dropped onto the center of a glass slide and allowed to air dry. The dried slides were stained with 5% Giemsa, air dried and permanently mounted.
Evaluation criteria:
Evaluation of metaphase cells: To ensure that a sufficient number of metaphase cells were present on the slides, the percentage of cells in mitosis per 500 cells scored (mitotic index) was determined for each treatment group. Metaphase cells with 20 ± 2 centromeres were examined under oil immersion without prior knowledge of treatment groups. Initially, the non-activated and S9 activated 4-hour exposure groups were evaluated for chromosome aberrations and since a negative result was obtained in the non-activated 4-hour exposure group, the non-activated 20-hour continuous exposure group was then also evaluated for chromosome aberrations. A minimum of 200 metaphase spreads (100 per duplicate flask) were examined and scored for chromatid-type and chromosome-type aberrations. Chromatid-type aberrations include chromatid and isochromatid breaks and exchange figures such as quadriradials (symmetrical and asymmetrical interchanges), triradials, and complex rearrangements. Chromosome-type aberrations include chromosome breaks and exchange figures such as dicentrics and rings. Fragments (chromatid or acentric) observed in the absence of any exchange figure were scored as a break (chromatid or chromosome). Fragments observed with an exchange figure were not scored as an aberration but instead were considered part of the incomplete exchange. Pulverized chromosome(s), pulverized cells and severely damaged cells (>=10 aberrations) were also recorded. Chromatid and isochromatid gaps were recorded but not included in the analysis. The XY coordinates for each cell with chromosomal aberrations were recorded using a calibrated microscope stage. Polyploid and endoreduplicated cells were evaluated from each treatment flask per 100 metaphase cells scored.

Results and discussion

Test resultsopen allclose all
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with
Genotoxicity:
negative
Vehicle controls validity:
valid
Positive controls validity:
valid
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
without
Genotoxicity:
positive
Vehicle controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Summary of Test Results

Treatment

(µg/mL)

S9

Activation

Treatment

Time

(hours)

Mean

Mitotic

Index

Cells

Scored

Aberrations

Per Cell

(Mean ± SD)

Cells with Numerical

Aberrations

(%)

Cells with Structural Aberrations

(%)

Vehicle (DMSO)

-

4

7.0

200

0.015 ± 0.122

4.0

1.5

Phenol, p-(alpha-, alpha-dimethylbenzyl)

7.5

-

4

6.0

200

0.045 ± 0.289

1.0

3.0

15

-

4

4.5

200

0.080 ± 0.405

3.5

5.0

45

-

4

2.7

200

0.120 ± 0.476

5.0

8.0

Positive control (MMC)

0.2

-

4

3.7

200

0.360 ± 1.117

0.5

22.0

Vehicle (DMSO)

+

4

7.8

200

0.045 ± 0.208

1.5

4.5

Phenol, p-(alpha-, alpha-dimethylbenzyl)

3.75

+

4

6.4

200

0.070 ± 0.275

3.0

6.5

7.5

+

4

5.1

200

0.075 ± 0.264

3.5

7.5

15

+

4

3.7

200

0.095 ± 0.311

7.5

9.0

Positive control (CP)

10

+

4

5.0

200

0.250 ± 0.574

1.0

19.5

Vehicle (DMSO)

-

20

7.2

200

0.050 ± 0.279

1.5

4.0

Phenol, p-(alpha-, alpha-dimethylbenzyl)

7.5

-

20

6.1

200

0.060 ± 0.356

3.0

4.0

15

-

20

5.9

200

0.105 ± 0.380

5.0

8.0

30

-

20

3.5

200

0.135 ± 0.357

6.0

13.0

Positive control (MMC)

0.1

-

20

4.7

100

0.720 ± 1.518

2.5

41.0

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
positive without metabolic activation

In conclusion, p-Cumylphenol was positive for the induction of structural aberrations in CHO cells in a non-metabolically activated system. However, p-Cumylphenol was concluded to be negative in the induction of structural aberrations in a metabolically activated system and did not induce numerical aberrations in either system.