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Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2000
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2000
Report date:
2000

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Version / remarks:
1997
Qualifier:
according to guideline
Guideline:
other: ICH Guidance on Specific Aspects of Regulatory Genotoxicity Tests for Pharmaceuticals
Version / remarks:
1996
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test

Test material

Constituent 1
Chemical structure
Reference substance name:
Ionone, methyl-
EC Number:
215-635-0
EC Name:
Ionone, methyl-
Cas Number:
1335-46-2
Molecular formula:
C14H22O
IUPAC Name:
(1E)-1-(2,6,6-trimethylcyclohex-2-en-1-yl)pent-1-en-3-one

Method

Species / strain
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):
CELLS USED
- Source of cells: American Type Culture Collection, Manassas, VA
- Suitability of cells: working cell stocks were not used beyond passage 20 to assure karyotypic stability
- Cell cycle length: 10-14 hours
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254-induced rat liver S9 from male Sprague-Dawley rats
Test concentrations with justification for top dose:
12.5, 25, 50 µg/mL
Top dose based on toxicity test with the test item: cell viability at 75 µg/mL: 0%
Vehicle / solvent:
- solvent used: ethanol
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
mitomycin C
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium
- Cell density at seeding: 5 x 10^5 cells/25 cm^2 flask

DURATION
- Exposure duration: 4h (S9 activated); 4 or 20h (non-activated)
- Fixation time (start of exposure up to fixation or harvest of cells): 2h

NUMBER OF REPLICATIONS: 2

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED:
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. Slides were identified by the study number, date prepared and the treatment condition. The dried slides were stained with 5% Giemsa, air dried and permanently mounted.

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE: a minimum of 100 per duplicate flask (200 in total)

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index

OTHER EXAMINATIONS:
- Determination of cells: Polyploid and endoreduplicated cells were evaluated from each treatment flask per 100 metaphase cells scored.
- Methods, such as kinetochore antibody binding, to characterize whether micronuclei contain whole or fragmented chromosomes (if applicable):

Results and discussion

Test results
Key result
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
Toxicity of the test item (cell growth inhibition relative to the solvent control) in CHO cells when treated for 4 hours in the absence of S9-activation was 68% at 50 µg/mL, the highest test concentration evaluated for chromosome aberrations. The mitotic index at the highest dose level evaluated for chromosome aberrations, 50 µg/mL, was not reduced relative to the solvent control. The dose levels selected for microscopic analysis were 12.5, 25, and 50 µg/mL. The percentage of cells with structural aberrations in the test article-treated groups was not significantly increased above that of the solvent control (p>0.05, Fisher's exact test). The percentage of cells with numerical aberrations in the test article-treated groups was significantly increased above that of the solvent control at dose level 25 µg/mL (p<0.05, Fisher's exact test). The Cochran-Armitage test was negative for a dose response (p>0.05). The statistically significant increase in the percentages of numerically aberrant cells at dose level 25 µg/mL (5.0%) was within the range of the percentage of numerically aberrant cells observed with the historical solvent control (0.0-6.5%). Therefore, the statistically significant increase in numerically aberrant cells in the non-activated 4 hour exposure group were considered not biologically significant. The percentage of structurally damaged cells in the MMC group was found to be statistically significant (26.0%).
Toxicity of the test item (cell growth inhibition relative to the solvent control) in CHO cells when treated for 4 hours in the presence of S9-activation was 61% at 50 µg/mL, the highest test concentration evaluated for chromosome aberrations. The mitotic index at the highest dose level evaluated for chromosome aberrations, 50 µg/mL, was not reduced relative to the solvent control. The dose levels selected for microscopic analysis were 12.5, 25, and 50 µg/mL. The percentage of cells with structural aberrations in the test article-treated groups was statistically increased above that of the solvent control at dose level 50 µg/mL (p<0.05, Fisher's exact test). The Cochran-Armitage test was also positive for a dose response (p<0.05). However, the statistically significant increase in the percentage of structurally aberrant cells at dose level 50 (µg/mL (3.5%) was within the range of the percentage of structurally aberrant cells observed with the historical solvent control (0.0-6.0%). Therefore, the statistically significant increases in structurally aberrant cells in the S9 activated 4 hour exposure group were considered not biologically significant. The percentage of cells with numerical aberrations in the test article-treated groups was not significantly increased above that of the solvent control (p>0.05, Fisher's exact test). The percentage of structurally damaged cells in the CP group was found to be statistically significant (20.5%).
In the absence of a positive response in the non-activated 4 hour exposure group, slides from the non-activated 20 hour exposure group were evaluated for chromosome aberrations. Toxicity of the test item (cell growth inhibition relative to the solvent control) was 66% at 50 µg/mL, the highest test concentration evaluated for chromosome aberrations in the non-activated 20 hour continuous exposure group. The mitotic index at the highest dose level evaluated for chromosome aberrations, 50 µg/mL, was 30% reduced relative to the solvent control. The dose levels selected for microscopic analysis were 12.5, 25, and 50 µg/mL. The percentage of cells with structural aberrations in the test article-treated groups was significantly increased above that of the solvent control at dose levels 12.5 and 25 µg/mL (p<0.05 and 0.01, respectively, Fisher's exact test). The Cochran-Armitage test was negative for a dose response (p>0.05). The percentage of cells with numerical aberrations in the test article-treated groups was not significantly increased above that of the solvent control (p>0.05, Fisher's exact test). The percentage of structurally damaged cells in the MMC group was found to be statistically significant (37.0%). See table 1 for all results.

Any other information on results incl. tables

Tabel 1: Cytogenetic analysis of CHO cells treated with the test item in the presence and absence of exogenous metabolic activation

 

Treatment

S9

Activation

Treatment1Time (Hours)

Mitotic Index

Cells Scored

Aberrations Per Cell2(Mean ± SD)

Cells With Aberrations3(%)

Numerical

Structural

Ethanol

-

4

9.9

200

0.055 ± 0.229

1.5

5.5

Test item

 

12.5 µg/mL

-

4

11.9

200

0.080 ± 0.323

4.5

6.5

25 µg/mL

-

4

12.4

200

0.220 ± 1.319

5.0*

5.5

50 µg/mL

-

4

11.8

200

0.150 ± 0.800

3.0

8.0

MMC, 0.2 µg/mL

-

4

11.6

200

0.375 ± 0.943

2.5

26.0**

Ethanol

+

4

12.1

200

0.005 ± 0.071

1.5

0.5

Test item

 

12.5 µg/mL

+

4

10.5

200

0.000 ± 0.000

1.5

0.0

25 µg/mL

+

4

10.2

200

0.030 ± 0.171

4.0

3.0

50 µg/mL

+

4

12.8

200

0.035 ± 0.184

4.5

3.5*

CP, 10 µg/mL

+

4

6.4

200

0.440 ± 1.359

2.5

20.5**

Ethanol

-

20

8.4

200

0.055 ± 0.269

5.0

4.5

Test item

 

12.5 µg/mL

-

20

10.0

200

0.155 ± 0.777

7.5

10.0*

25 µg/mL

-

20

9.5

200

0.335 ± 1.233

3.0

14.5**

50 µg/mL

-

20

5.9

200

0.175 ± 1.025

7.5

8.5

MMC, 0.1 µg/mL

20

8.9

100

0.720 ± 1.577

4.5

37.0**

 

1Cells from all treatment conditions were harvested at 20 hours after the initiation of the treatments.

2Severely damaged cells were counted as 10 aberrations.

3*, p ≤ 0.05; **, p ≤ 0.01; Fisher's exact test.

Applicant's summary and conclusion

Conclusions:
The test item was concluded to be negative in both the absence and presence of S9 activation for the induction of numerical chromosome aberrations in Chinese hamster ovary (CHO) cells.
Executive summary:

The test item was tested in the chromosome aberration assay using Chinese hamster ovary (CHO) cells in both the absence and presence of an Aroclor-induced S9-activation system according to OECD 473. A preliminary toxicity test was performed to establish the dose range for the chromosome aberration assay. The chromosome aberration assay was used to evaluate the clastogenic potential of the test article. Ethanol was determined to be the solvent of choice based on the solubility of the test article and compatibility with the target cells. The test article was soluble in ethanol at a concentration of 500 mg/mL, the maximum concentration tested. In the preliminary toxicity assay, the maximum dose tested was 2060 µg/mL. This dose level represents the 10mM concentration based on a test article molecular weight of 206.30. Visible precipitate was observed in treatment medium at dose levels > 206 µg/mL in the non-activated and S9 activated 4 hour exposure groups, and at dose levels > 618 µg/mL in the non-activated 20 hour exposure group. Dose levels < 61.8 µg/mL in the 4 hour exposure groups, and < 206 µg/mL in the non-activated 20 hour exposure group were soluble in treatment medium. Selection of dose levels for the chromosome aberration assay was based on cell growth inhibition relative to the solvent control. Substantial toxicity, i.e., at least ~50% cell growth inhibition, was observed at dose levels of 61.8 µg/mL in both the non-activated and S9-activated exposure groups. Based on these findings, the doses chosen for the chromosome aberration assay ranged from 12.5 to 175 µg/mL for both the non-activated and the S9-activated exposure groups. In the chromosome aberration assay, the cells were treated for 4 and 20 hours in the non-activated test system and for 4 hours in the S9-activated test system, and all cells were harvested at 20 hours after treatment initiation. The test article was soluble in treatment medium at all concentrations tested. Toxicity (cell growth inhibition) was 68% and 66% at the highest dose level evaluated for chromosome aberrations, 50 µg/mL, in the non-activated 4 hour and 20 hour exposure groups, respectively. Toxicity (cell growth inhibition) was approximately 61% at the highest dose level evaluated for chromosome aberrations, 50 µg/mL, in the S9 activated study. Initially, the non-activated and S9-activated 4 hour exposure groups were scored for structural and numerical chromosome aberrations. No statistically significant increases in structural chromosome aberrations were observed in the non-activated 4 hour exposure group relative to the solvent control group, regardless of dose level (p>0.05, Fisher's exact test). A statistically significant increase in structural chromosome aberrations was observed in the S9-activated 4 hour exposure group relative to the solvent control group, at dose level 50 µg/mL (p<0.05, Fisher's exact test). The Cochran-Armitage test was also positive for a dose response (p<0.05). However, the statistically significant increase in the percentage of structurally aberrant cells at dose level 50 µg/mL (3.5%) was within the range of the percentage of structurally aberrant cells observed with the historical solvent control (0.0-6.0%). Therefore, the statistically significant increase in structurally aberrant cells in the S9 activated 4 hour exposure group was considered not biologically significant. A statistically significant increase in numerical chromosome aberrations was observed in the non-activated 4 hour exposure group relative to the solvent control group, at dose level 25 µg/mL (p<0.05, Fisher's exact test). The Cochran-Armitage test was negative for a dose response (p>0.05). The statistically significant increase in the percentages of numerically aberrant cells at dose level 25 (µg/mL (5.0%) was within the range of the percentage of numerically aberrant cells observed with the historical solvent control (0.0-6.5%). Therefore, the statistically significant increase in numerically aberrant cells in the non-activated 4 hour exposure group was considered not biologically significant. No statistically significant increases in numerical chromosome aberrations were observed in the S9-activated 4 hour exposure group relative to the solvent control group, regardless of dose level (p>0.05, Fisher's exact test). In the absence of a positive response in the non-activated 4 hour exposure group, the non-activated 20 hour continuous exposure group was evaluated for structural and numerical chromosome aberrations. Statistically significant increases in structural chromosome aberrations were observed in the non-activated 20 hour continuous exposure group relative to the solvent control group, at dose levels 12.5 and 25 µg/mL (p<0.05 and 0.01, respectively, Fisher's exact test). The Cochran-Armitage test was negative for a dose response (p>0.05). No statistically significant increases in numerical chromosome aberrations were observed in the non-activated 20 hour continuous exposure group relative to the solvent control group, regardless of dose level (p>0.05, Fisher's exact test). Based on the findings of this study, Methyl ionone was concluded to be positive in the absence of S9 activation for the induction of structural chromosome aberrations, negative in the presence of S9 activation for the induction of structural chromosome aberrations in Chinese hamster ovary (CHO) cells. Methyl ionone was concluded to be negative in both the absence and presence of S9 activation for the induction of numerical chromosome aberrations in Chinese hamster ovary (CHO) cells.