Registration Dossier

Toxicological information

Genetic toxicity: in vitro

Currently viewing:

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:
2002-07-02 to 2003-02-13
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2003
Report Date:
2003

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
Qualifier:
according to
Guideline:
other: EEC Council Directive 2000/32, Annex 4A
Deviations:
no
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Isophorone diisocyanate of Degussa AG, Batch No. 1103211, purity > 99.5 %

Method

Target gene:
not applicable
Species / strain
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):
CHO cells from Dr. A.T. Natarajan (State University of Leiden, Netherlands)
Additional strain / cell type characteristics:
other: This cell line derives from the CHO isolate originally described by Kao and Puck (1968)
Metabolic activation:
with and without
Metabolic activation system:
from Phenobarbital and betanaphthoflavone induced Sprague-Dawley rat liver (S9 homogenate)
Test concentrations with justification for top dose:
0; 10.0; 20.0; 40.0 mg/l (+/- S9); additionally 5 mg/l (- S9)
Vehicle / solvent:
DMSO (dimethyl sulfoxide) 1% (v/v)
Controls
Untreated negative controls:
yes
Remarks:
details see below
Negative solvent / vehicle controls:
yes
Remarks:
1% (v/v)
True negative controls:
no
Positive controls:
yes
Positive control substance:
mitomycin C
Remarks:
positive control with metabolic activation: Cyclophoshamide

Migrated to IUCLID6: without metabolic activation
Details on test system and experimental conditions:
SYSTEM OF TESTING
- Species/cell type: CHO cells as described by Kao and Puck (1968),  obtained from Dr. A.T. Natarajan (State University of Leiden, Netherlands)
- Metabolic activation system:   S9 homogenate prepared from young male Sprague-Dawley rat livers, co-induced  with phenobarbital and 
betanaphthoflavone. Batches No. 2002/9 and 2002/14
- No. of metaphases analyzed:    100 / culture except 50 for positive controls with chromosomal  aberration rates > 50 % (excl. gaps)
ADMINISTRATION: 
- Dosing:    0.625; 1.25; 2.50; 5.0; 10.0; 20.0; 40.0; 80.0 mg/l (+/- S9)   
Doses selected for scoring:   10; 20; 40 mg/l (Experiment 1 +/- S9; Experiment 2 + S9)     5; 10; 20 mg/l (Experiment 2 - S9)
- Number of replicates: 2
- Application:    Approx. 300,000 cells each seeded in 25 cm2 flasks approx. 20 hours  before treatment   
Treatment time 3 hours, 
harvest time 20 hours (approx. 1.5 cell cycles)   
Addition of 0.2 mg/l colcemid for last 3 hours (Spindel inhibitor)
- Positive and negative control groups and treatment:    
negative: untreated
negative: DMSO (dimethyl sulfoxide, CAS RN 67-68-5)   
positive -S9: 0.30 or 0.45 mg mitomycin C/l   
positive +S9: 15 and 23 mg cyclophosphamide/l
- Harvesting/Stain
cells brought into suspension by trypsinization, centrifuged cell pellet is resuspendes and fixed, washed in freshly prepated metanol: acetic acid
cell suspension are dropped onto glass slides and air-dried. At least 3 slides each dose are stained in 3% Giemsa in tap water, slides made
permanent with Eukitt
Evaluation criteria:
CRITERIA FOR EVALUATING RESULTS:
(i) statistically significant increases in the incidence of cells bearing  aberrations at any dose-level over the concurrent control, AND
(ii) the increases must exceed the historical control values, AND
(iii) the increases are reproduced in both replicate cultures
Statistics:
Fisher's Exact Test

Results and discussion

Test results
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
the following doses used for scoring of aberrations revealed cytotoxicity: 40 mg/l (cell viability reduced to 26% of the control); 20 mg/l (viability reduced to 55% of the control); other doeses: no relevant cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
GENOTOXIC EFFECTS: 
- With metabolic activation: Dose related increase in chromosomal  aberrations
- Without metabolic activation: Dose related increase in chromosomal  aberrations
OTHER OBSERVATIONS: 
pH and osmolality of the treatment media were not  obviously affected by the test substance.
Remarks on result:
other:
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

CHROMOSOMAL ABERRATIONS (excluding gaps): 
-----------------------------------------------
Concentration      % Chromosomal aberrations
-----------------------------------------------
- Experiment # 1     with S9       without S9
  untreated              0.0             0.5
  Solvent                 0.5             0.5
  10 mg/l IPDI         0.0             1.5
  20 mg/l IPDI          5.5 *           2.5
  40 mg/l IPDI          8.5 ***     10.5 ***
  0.3 mg/l MMC          -           57.0 ***
  15 mg/l CPA         32.0 ***       -
-----------------------------------------------
- Experiment # 2     with S9       without S9
  untreated           0.5             0.0
  Solvent                1.5             1.0
   5 mg/l IPDI            -              0.5
  10 mg/l IPDI          3.5            3.5
  20 mg/l IPDI          4.0          12.0 ***
  40 mg/l IPDI        19.5 ***         -
  0.3 mg/l MMC         -            27.0 ***
  15 mg/l CPA         42.0 ***         -
-----------------------------------------------
IPDI = isophorone diisocyanate (test substance)
MMC = mitomycin-C (positive control)
CPA = cyclophosphamide (positive control)
Significance: * p<0.05; ** p<0.01; *** p<0.001
-----------------------------------------------
PRECIPITATION CONCENTRATION: Solubility in DMSO at 500 g/l. Precipitation  at > 62.5 g/l upon addition to Ham's F10.
Cell growth results:: 
-----------------------------------------------
Concentration        % relative cell growth
-----------------------------------------------
- Experiment # 1     with S9       without S9
  untreated             122           106
  Solvent              100           100
  10 mg/l IPDI          97             85
  20 mg/l IPDI           80             55
  40 mg/l IPDI           76             26
  0.3 mg/l MMC        -             71
  15 mg/l CPA           53             -
-----------------------------------------------
- Experiment # 2     with S9       without S9
  untreated             107              99
  Solvent                100            100
   5 mg/l IPDI            -             86
  10 mg/l IPDI          71              71
  20 mg/l IPDI           73              65
  40 mg/l IPDI           21               -
  0.3 mg/l MMC          -             66
  15 mg/l CPA          66               -
-----------------------------------------------

Applicant's summary and conclusion

Conclusions:
On the basis of the result of this study it is concluded that the test substance Isophorone Diisocyanate (IPDI) induces chromosomal aberrations in Chines hamster ovary cells after in vitro treatment under the reported experimental conditions.
Executive summary:

The test item Isophorone Diisocyanate (IPDI) was assayed for the ability to cause chromosomal damage in Chinese hamster ovary cells, following in vitro treatment in the absence and presence of metabolic activation with S9 homogenate. Two independend assays for chromosomal damage were performed. For both experiments the cells were treated with the test item in concentrations which are described in the study for 3 hours in the presence and absence of S9 metabolism. Cells were harvested after 20 hours, corresponding to approximately 1.5 cell cycle. For both experiments, following treatment with IPDI statistically significant increases in the incidence of cells bearing aberrations, including and excluding gaps, were observed in the absence and presence of S9 metabolism. It is concluded that IPDI induces chromosomal aberrations in Chinese hamster ovary cells after in vitro treatment under the reported experimental conditions.