Cyclododecane

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:

key study

Study period:

06-1993 to 08-1993

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1993

Report date:

1993

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Test guideline 2

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

Test material information

Constituent 1

Reference substance name:

Cyclododecane

EC Number:

206-033-9

EC Name:

Cyclododecane

Cas Number:

294-62-2

Molecular formula:

C12H24

IUPAC Name:

cyclododecane

Details on test material:

Test substance: cyclododecane of Hüls AG, produced 19 May 1993, purity 99.8% (GC-FID area); ID No. 0637/81524, PR No. 1452/931213

Method

Target gene:

mammalian cell system (V79 Chinese hamster lung fibroblasts)

Species / strain

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

modal chromosome numer of 22, cell cycle length of 18 hours
obtained from Dr. Engelhardt of BASF AG

Additional strain / cell type characteristics:

other:

Metabolic activation:

with and without

Metabolic activation system:

S9 mix based on S9 fraction from Aroclor  1254 induced male Wistar rat liver

Test concentrations with justification for top dose:

50; 200; 600 mg/l (+ S9)
10; 20; 100 mg/l (- S9)

Vehicle / solvent:

MEM cell culture medium, containing 1 % acetone

Controls

Untreated negative controls:

yes

Remarks:

solvent control is considered to be the negative control

Negative solvent / vehicle controls:

yes

Remarks:

MEM medium containing 1 % acetone

True negative controls:

no

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

positive control with metabolic activation: cyclophosphamide

Migrated to IUCLID6: without metabolic activation

Details on test system and experimental conditions:

SYSTEM OF TESTING
- Species/cell type: Cloned V79 cells from Dr. Engelhardt of BASF AG,  Germany. Modal chromosome number 22, cell cycle length approx. 18 hours.
- Metabolic activation system: S9 mix based on S9 fraction from Aroclor  1254 induced male Wistar rat liver (Cytotest Cell Research, Rossdorf,  
Germany)
- No. of metaphases analyzed: 100 / culture = 200 / experimental point;  >= 1500 cells / slide scored
ADMINISTRATION: 
- Dosing:   
(1) Initial toxicity test:   10; 50; 100; 200; 400; 600; 1200; 2400; 3600; 5000 mg/l (+/- metabolic  activation)   
without S9 experiment repeated with 0; 5; 10; 20; 50; 100 mg/l due to  very low cell counts at >= 200 mg/l   
(2) Selected for evaluation:   10; 20; 100 mg/l (- metabolic activation)   50; 200; 600 mg/l (+ metabolic activation)
- Number of replicates: 2 (4 in with S9 negative control of experiment 1)
- Application:   Cells were cultured on microscopic slides for approx. 24 hours prior to  treatment.   Medium with S9 mix was replaced by medium 
without S9 mix after 3 hours  exposure in order to limit cytotoxic effects of S9 mix.   
2 hour treatment with 0.2 mg colcemid/l at 18-20 hours exposure (both  experiments) and at 41-43 hours (experiment 2)   Addition of 
50 mM KCl (37 °C) at 20 hours (both experiments) and at 43  hours (experiment 2)   Fixation in methanol / glacial acetic acid (3:1; 4 °C) after further  20  minutes
- Positive and negative control groups and treatment:    
positive, without metabolic activation: 0.02 mg mitomycin C (MMC)/l  
positive, with metabolic activation: 2 or 4 mg cyclophosphamide (CP)/l   
negative: MEM5 Medium containing 1 % acetone (-S9) / MEM0 Medium  containing 1 % acetone +S9 mix

Evaluation criteria:

CRITERIA FOR EVALUATING RESULTS:    Classification of aberrations according to Scott et al. (1990).  Clastogenic if   
(1) Statistically significant (p < 0.05) increase in chromosomal  aberrations (excl. gaps) at one or more concentrations,    
(2) Induced proportion of aberrant cells increase above normal (i.e. >5 %), and   
(3) Positive results must be reproduced in an independent experiment.

Statistics:

The frequency of cells with aberrations (both including and excluding gaps) and the frequency of polyploid cells was compared with the concurrent
vehicle control value using the Chi-square test for the hypothesis of equal means.

Results and discussion

Test results

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

yes; cytotoxicity observed at: 1200 mg/l (+ S9) / 200 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 controls revealed low chromosomal aberration frequencies  (excluding gaps: 0 to 1.0 %; normal: 0 to 5 %).
PRECIPITATION CONCENTRATION: < 10 mg/l. Precipitate mainly disappeared  during the experiment at all concentrations.

Remarks on result:

other: other: V79 Chinese hamster lung cells

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

CONTROLS:
  The positive controls were functional.
  The negative controls revealed low chromosomal aberration frequencies  

excluding gaps: 0 to 1.0 %; normal: 0 to 5 %).
PRECIPITATION CONCENTRATION: < 10 mg/l. Precipitate mainly disappeared  during 

the experiment at all concentrations.
-------------------------------------------
MITOTIC INDEX AND CHROMOSOMAL ABERRATIONS: 
-------------------------------------------
Concentration        % M.I.         % C.A.
-------------------------------------------
- Experiment # 1, without S9 (20 hours)
  neg. control        7.8            1.0
   10 mg/l            8.1            0.0
   20 mg/l            5.2            0.5
  100 mg/l            5.1            0.0
  0.02 mg/l MMC       5.5           14.0 *
-------------------------------------------
- Experiment # 1, with S9 (20 hours)
  neg. control        6.8            1.0
   50 mg/l            5.2            1.0
  200 mg/l            6.4            2.0
  600 mg/l            5.2            1.5
 1200 mg/l            1.6
    2 mg/l CP         5.8           13.0 *
-------------------------------------------
- Experiment # 2, without S9, 20 hours
  neg. control       10.3            1.0
   10 mg/l            9.4            0.0
   20 mg/l            7.9            0.5
  100 mg/l            9.2            0.0
  0.02 mg/l MMC       6.5           12.5 *
-------------------------------------------
  Experiment # 2, without S9, 43 hours
  neg. control        9.2            0.0
  100 mg/l            6.7            0.0
  0.02 mg/l MMC       7.5           12.0 *
-------------------------------------------
- Experiment # 2, with S9, 20 hours
  neg. control        5.9            0.0
   50 mg/l            6.3            2.5
  200 mg/l            6.4            0.0
  600 mg/l            5.6            1.0
    2 mg/l CP         6.8           11.0 *
-------------------------------------------
- Experiment # 2, with S9, 43 hours
  neg. control        5.3            0.0
   600 mg/l           5.6            0.0
     4 mg/l CP        5.5           14.5 *
-------------------------------------------
* indicates significance

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

Under the conditions of this in vitro study cyclododecane did not induce chromosomal aberrations and is therefore considered to be
non-clastogenic.

Executive summary:

In the described in vitro cytogenetic assay with V79 Chinese hamster lung cells, cyclododecane reproducibly did not induce any significant increase in the chromosome aberration frequency, either in the presence or absence of metabolic activation by Arochlor-1254 induced rat liver S9-mix. Cyclododecane is therefore considered to be non-clastogenic to V79 cells under the conditions of this in vitro study.