2,2,4(or 2,4,4)-trimethylhexanedinitrile

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:

1998-03-03 to 1998-05-15

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1998

Report date:

1998

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

(1983)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

(1992)

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Test material

Test material information

Constituent 1

Reference substance name:

2,2,4(or 2,4,4)-trimethylhexanedinitrile

EC Number:

283-810-9

EC Name:

2,2,4(or 2,4,4)-trimethylhexanedinitrile

Cas Number:

84713-17-7

Molecular formula:

C9H14N2

IUPAC Name:

2,2,4-trimethylhexanedinitrile; 2,4,4-trimethylhexanedinitrile

Details on test material:

2,2,4(or 2,4,4)-Trimethylhexanedinitrile of Creanova Spezialchemie GmbH. Purity 96.5 % (GC-FID area), ID 0637/81824, produced January 1997.

Method

Target gene:

Chromosomes of the mammalian cell system( V79 Chinese hamster lung cells)

Species / strain

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

Cloned V79 cells with a modal chromosome number of 22 and a cell cycle length of approx. 14 h were used. They were originally derived form the lung tissue of a male Chinese hamster and subsequently obtained from BASF AG. The stock culture was maintained under liquid nitrogen.

Metabolic activation:

with and without

Metabolic activation system:

S9 mix based on S9 fraction from  phenobarbital / beta-naphthoflavone co-induced male Wistar rat liver  (Cytotest Cell Research, Rossdorf, Germany, lot 050996)

Test concentrations with justification for top dose:

0; 600; 3000; 4000; 5000 mg/l (+ S9) / 0; 200; 1000; 1800 mg/l (- S9)

Vehicle / solvent:

Cell culture medium (MEM)

Controls

Untreated negative controls:

yes

Remarks:

Cell culture medium (MEM); Negative control=solvent/vehicle control

Negative solvent / vehicle controls:

yes

Remarks:

Cell culture medium (MEM)

True negative controls:

no

Positive controls:

yes

Remarks:

with and without metabolic activation

Positive control substance:

mitomycin C

Remarks:

cyclophosphamide with metabolic activation

Migrated to IUCLID6: without metabolic activation

Details on test system and experimental conditions:

SYSTEM OF TESTING
- Metabolic activation system: S9 mix based on S9 fraction from  phenobarbital / beta-naphthoflavone co-induced male Wistar rat liver  
(Cytotest Cell Research, Rossdorf, Germany, lot 050996)
- No. of metaphases analyzed: 100 / culture = 200 / experimental point;  >= 1000 cells / slide scored
ADMINISTRATION:
- Dosing:
(1) Preliminary toxicity test:   
25; 50; 100; 200; 400; 600; 1000; 1800; 3000; 5000 mg/l (+/- metabolic  activation)
(2) Selected for evaluation:
0; 200; 1000; 1800 mg/l (- metabolic activation)
0; 600; 3000; 5000 mg/l (+ metabolic activation, experiment 1)
0; 600; 3000; 4000; 5000 mg/l (+ metabolic activation, experiment 2)
- Number of replicates: 2
- Application:  Cells were grown in tissue culture dishes for 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 16-18 hours exposure (both experiments) and at 26-28 hours (experiment 2)
Centrifugation and addition of 34 mM sodium citrate (37 °C) at 18 hours (both experiments) and at 28 hours (experiment 2)
Fixation in methanol / glacial acetic acid (3:1; 4 °C) after further 8  minutes and 5 minutes centrifugation
- Positive and negative control groups and treatment:
positive, without metabolic activation: 0.02 and 0.03 mg mitomycin C (MMC)/l
positive, with metabolic activation: 2 mg cyclophosphamide (CP)/l
negative: MEM4 medium (-S9) / MEM0 medium +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) Increase above normal range (0 - 5 %), and
(3) Positive results verified in an independent experiment.

Statistics:

The proportion of cells that was treated with the test substance and harboured structural aberrations (excl. gaps) was compared with the corresponding proportion of the negative controls in the Chi-square test. Probability values of p<0.05 were accepted as statistically significant.

Results and discussion

Test results

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

other: ca. 5000 mg/l (+ S9) / 3000 mg/l (- S9)

Vehicle controls validity:

valid

Untreated negative controls validity:

other: negative control = solvent/vehicle control

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 2.5 %; normal: 0 to 5 %.
PRECIPITATION CONCENTRATION: > 5000 mg/l
MITOTIC INDEX AND CHROMOSOMAL ABERRATIONS: 
A statistically and  biologically significant increase in chromosomal aberrations was observed  at the highest doses with (experiments 1 and 2) and
without (experiment 2) metabolic activation. Osmolality and pH were excluded as reasons for  this finding. A dose-response relationship could not 
be established.

Remarks on result:

other: other: V79 Chinese hamster lung cells

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Concentration        % M.I.         % C.A.
-------------------------------------------
- Experiment # 1, without S9 (18 hours)
  neg. control         7.5            0.0
   200 mg/l            7.5            0.5
  1000 mg/l            4.0            2.0
  1800 mg/l            4.7            0.5
  0.02 mg/l MMC        4.0           15.5 *
  0.03 mg/l MMC        7.0           19.0 *
-------------------------------------------
- Experiment # 1, with S9 (18 hours)
  neg. control         7.3            0.5
   600 mg/l            8.6            1.5
  3000 mg/l            8.9            1.5
  5000 mg/l            5.6           14.5 *
     2 mg/l CP         3.5           20.5 *
-------------------------------------------
- Experiment # 2, without S9, 18 hours
  neg. control         7.3            1.0
   200 mg/l            8.0            2.5
  1000 mg/l            3.5            1.0
  1800 mg/l            2.0            7.0 *
  0.03 mg/l MMC        3.7           25.5 *
-------------------------------------------
- Experiment # 2, with S9, 18 hours
  neg. control         6.7            2.5
   600 mg/l            8.7            1.0
  3000 mg/l            6.0            4.0
  4000 mg/l            3.3           24.2 *
  5000 mg/l            scoring impossible
     2 mg/l CP         5.0           24.5 *
-------------------------------------------
- Experiment # 2, with and without S9, 28 hours: not evaluated
-------------------------------------------
* indicates significance

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
positive

From the experiments performed, it is concluded that under the conditions of this in vitro test system 2,2,4(or 2,4,4)-trimethylhexanedinitrile is an in vitro clastogenic agent.

Executive summary:

The substance 2,2,4(or 2,4,4)-trimethylhexanedinitrile was tested for its ability to induce structural chromosome aberrations in an in vitro mammalian cell system( V79 Chinese hamster lung cells) according to OECD Guideline 473 (1983) and EU method B.10 (1992). Two independent experiments were carried out with and without the addition of phenobarbital-induced rat liver S9 mix. On the basis from the results of the present study, the test substance did induce biologically significant increases in the chromosomal aberration frequency of V79 hamster cells both in the absence and in the presence of metabolic activation and is therefore judged to be an in vitro clastogenic agent.