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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:
weight of evidence
Study period:
2003-05-21 to 2003-08-09
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2004
Report date:
2004

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Qualifier:
according to guideline
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test

Test material

Constituent 1
Chemical structure
Reference substance name:
3,3,5-trimethylcyclohexan-1-one
EC Number:
212-855-9
EC Name:
3,3,5-trimethylcyclohexan-1-one
Cas Number:
873-94-9
Molecular formula:
C9H16O
IUPAC Name:
3,3,5-trimethylcyclohexan-1-one
Details on test material:
- Name of test material (as cited in study report): 3,3,5-Trimethylcyclohexanone of Atofina
- Analytical purity: 99.718 %
- Impurities (identity and concentrations): 0.157 % 3,3,5-trimethylcyclohexanol, 0.076 % isophorone, 0.027 % water
- Lot/batch No.: FP010101

Method

Species / strain
Species / strain / cell type:
other: Cultured human lymphocytes
Details on mammalian cell type (if applicable):
Cultured human lymphocytes with a stable karyotype with 46 chromosomes and an average cell cylcle time of 12-14 hours,
prepared from whole blood samples from two healthy donors
Metabolic activation:
with and without
Metabolic activation system:
S9-mix from the liver of rats treated with Arochlor 1254 intraperitoneal
Test concentrations with justification for top dose:
First experiment (+/- S9): 22, 44, 88, 175, 351, 701, 1052, 1402 mg/l (0.16 - 10 mM)
Second experiment (- S9): 44, 88, 175, 351, 701, 1402 mg/l (0.31 - 10 mM)
Second experiment (+ S9): 88, 175, 351, 701, 1052, 1402 mg/l (0.63 - 10mM)
Vehicle / solvent:
Dimethylsulfoxide DMSO, batch No. K30379650
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: cyclophosphamide (+S9); mitomycin C (-S9)
Details on test system and experimental conditions:
SYSTEM OF TESTING
- Species/cell type: Human lymphocytes (average cell cycle time 12-14  hours) from whole blood samples from two healthy donors (1 male, 1  
female).
- Metabolic activation system: S9 mix based on S9 fraction from Aroclor  1254 induced rat liver (Moltox, Boone, NC/USA)
- No. of metaphases analyzed: 100 / culture = 200 / experimental point;  only 50 / culture in cases of >= 10 % structural chromosomal aberrations
ADMINISTRATION: 
- Dosing:    
Experiment 1: 0.16; 0.31; 0.63; 1.25; 2.5; 5; 7.5; 10 mM   = 22; 43; 88; 175; 351; 701; 1052; 1402 mg/l (+/- metabolic activation)   
Experiment 2 (- metabolic activation): 0.31; 0.63; 1.25; 2.5; 5; 10 mM   = 43; 88; 175; 351; 701; 1402 mg/l   
Experiment 2 (+ metabolic activation): 0.63; 1.25; 2.5; 5; 7.5; 10 mM   = 88; 175; 351; 701; 1052; 1402 mg/l   
Selected for scoring:    5; 7.5; 10 mM = 701; 1052; 1402 mg/l for 3 hour exposure and 20 hour  harvest time (+/- metabolic activation)   
0.63; 1.25; 2.5 mM = 88; 175; 351 mg/l for 20 hour exposure (-  metabolic activation)   2.5 mM = 351 mg/l for 44 hour exposure 
(- metabolic activation)   10 mM = 1402 mg/l for 44 hour harvest time (+ metabolic activation)
- Number of replicates: 2
- Application: Solution of 280.44 mg/l in vehicle dimethyl sulfoxide  (DMSO, CAS RN 67-68-5)   Initial culturing at 37 °C for 48 hours   
3 hours exposure followed by rinsing; exception:    
continous exposure without rinsing in experiment 2 (- S9)   Addition of 10 mg colcemid/l 1.5 hours before harvest   Harvest at 20 hours 
(approx. 1.5 cell cycles); additional harvest time  44 hours in experiment 2   Hypotonic treatment (0.075 M KCl)   
Fixation in methanol / acetic acid (3:1 v/v)   Spreading on glass, staining with Giemsa.
- Positive and negative control groups and treatment:    
positive, without metabolic activation: 3 or 0.2 mg mitomycin C (MMC)/l   
positive, with metabolic activation: 12.5 or 25 mg cyclophosphamide  (CPA)/l   
negative: vehicle (DMSO)
Evaluation criteria:
CRITERIA FOR EVALUATING RESULTS:    
(1) Statistically significant (p < 0.05) increase in chromosomal  aberrations (excl. gaps) at one or more concentrations or harvest times   
(2) Positive results must be reproduced in an independent experiment.
Statistics:
For each test and for each harvest time, the frequency of cells with structural chromosome aberrations in treated cultures was compared to that of
the vehicle control cultures. If necessary, the comparison was performed using chi² test, in which p = 0.05 was used as the lowest level of significance

Results and discussion

Test results
Species / strain:
other: Cultured human lymphocytes
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
other: >= 0.63 mM (88 mg/l) (- S9) / 10 mM (1402 mg/l) (+ S9)
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
CONTROLS:   
The positive controls were functional.   
The negative (vehicle) controls revealed low chromosomal aberration frequencies  (excluding gaps: 0 to 1.0 %).
PRECIPITATION CONCENTRATION:   
Complete solubility in DMSO at 280.44 g/l.    
Slight emulsion at 1402 mg/l in culture medium   pH (7.1 vs. 7.1) and osmolality (350 vs. 384 mOsm/kg H2O) equivalent to  vehicle control at this 
latter concentration.
Remarks on result:
other: other: Cultured human lymphocytes
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Mitotic Index (M.I.) and Chromosomal Aberrations (Chr. Ab.) (excl. gaps):

Concentration

M.I. (%, mean)

% Chr.Ab.

- Experiment 1, -S9, 3/20 hours treatment/harvest

neg. control

4.45

1.0

22 mg/l

3.05

-

43 mg/l

3.75

-

88 mg/l

2.15

-

175 mg/l

2.55

-

351 mg/l

2.20

-

701 mg/l

2.25

2.0

1052 mg/l

2.35

0.0

1402 mg/l

2.15

2.5

3 mg/l MMC

0.65

30.0 ***

- Experiment 2, -S9, 20/20 hours treatment/harvest

neg. control

5.50

0.5

43 mg/l

7.00

-

88 mg/l

5.95

0.5

175 mg/l

6.00

1.0

351 mg/l

3.90

1.5

701 mg/l

0.00

-

1402 mg/l

0.00

-

0.2 mg/l MMC

4.05

11.3 ***

- Experiment 2, -S9, 44/44 hours treatment/harvest

neg. control

5.25

0.0

43 mg/l

3.75

-

88 mg/l

4.35

-

175 mg/l

4.75

-

351 mg/l

2.90

1.0

701 mg/l

0.60

-

1402 mg/l

0.00

-

- Experiment 1, +S9, 3/20 hours treatment/harvest

neg. control

3.85

0.5

22 mg/l

3.60

-

43 mg/l

3.00

-

88 mg/l

2.90

-

175 mg/l

2.35

-

351 mg/l

3.55

-

701 mg/l

2.50

2.0

1052 mg/l

2.70

1.5

1402 mg/l

4.20

4.5 *

25 mg/l CPA

0.60

-

12.5 mg/l CPA

1.30

24.0 ***

- Experiment 2, +S9, 3/20 hours treatment/harvest

neg. control

6.15

1.0

88 mg/l

5.55

-

175 mg/l

5.10

-

351 mg/l

5.40

-

701 mg/l

5.15

2.5

1052 mg/l

5.85

2.5

1402 mg/l

3.45

7.5 **

12.5 mg/l CPA

3.25

31.0 ***

- Experiment 2, +S9, 3/44 hours treatment/harvest

neg. control

2.90

0.0

88 mg/l

2.00

-

175 mg/l

2.05

-

351 mg/l

2.10

-

701 mg/l

2.85

-

1052 mg/l

3.75

-

1402 mg/l

1.75

3.0 *

Significance: * p<0.05; ** p<0.01; *** p<0.001
MMC: mitomycin C
CPA: cyclophosphamide

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
positive with metabolic activation

3,3,5-Trimethylcyclohexanone induced a slight increase in the frequency of cells with structural chromosome aberrations in cultured human lymphocytes. This effect was limited to the highest concentration tested under metabolic activation (S9 mix). At this concentration a marked decrease in the mitotic index was observed.
Executive summary:

In a mammalian cell cytogenetic assay [Chromosome aberration], human lymphocyte cultures were exposed to 3,3,5-Trimethylcyclohexanone (99.7%), dissolved in DMSO, at concentrations of 0 – 10 mM with and without metabolic activation.

3,3,5-Trimethylcyclohexanone was tested up to cytotoxic concentrations. A statistically significant increase to 3-30% cells with aberrations (vs. controls) was reported only at the top concentration with metabolic activation. At this concentration a marked decrease in the mitotic index was observed. Positive controls induced the appropriate response. 

There was evidence of Chromosome aberration induced over background.

This study is classified as acceptable.  This study satisfies the requirement for Test Guideline OECD 473 for in vitro cytogenetic mutagenicity data. 

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