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Toxicological information

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:
key study
Study period:
February 1994
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1994
Report Date:
1994

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
GLP compliance:
yes (incl. certificate)
Type of assay:
in vitro mammalian chromosome aberration test

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Product name: Amgard V6
Appearance: clear to very pale straw coloured viscous liquid

Method

Species / strain
Species / strain / cell type:
lymphocytes: human, primary culture
Metabolic activation:
with and without
Metabolic activation system:
liver S9, prepared from rats (Sprague-Dawley) treated with Aroclor 1254
Test concentrations with justification for top dose:
- without metabolic activation: 39, 78.1, 156.25 micrograms/ml
- with metabolic activation: 78.1, 156.25, 312.5, 625 micrograms/ml
Vehicle / solvent:
Dimethylsulphoxide (DMSO)
Controls
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
cyclophosphamide for cultures with metabolic activation (25 microgr/mL) Migrated to IUCLID6: cultures without metabolic activation (500 microg/mL)
Details on test system and experimental conditions:
Human lymphocytes were evaluated for chromosome aberrations at three dose levels in the absence of S9 and four levels in the presence of S9, together with positive and negative (solvent) controls.

DURATION
- Preincubation period: 48 hours
- Exposure duration: 4 hours (in presence of S9)
- Expression time (cells in growth medium): 16 hours (in presence of S9)
- Exposure duration: 20 h continous in the absence of S9
- Fixation time: cells stored for at least 4 hours (4degree C) to ensure complete fixation

Spindel inhibitor: Demeclocine (0.1 micrg/ml) added two hours before the harvest time
FIXATION: KCl suspension in methanol/glacial acetic acid (3:1 v/v)
STAIN: Gurrs Giemsa R66 (5%)

NUMBER OF CELLS EVALUATED: 100 well-spread metaphases from each culture

DETERMINATION OF CYTOTOXICITY
- Method: 2000 lymphocytes counted and the number of cells in metaphase recorded and expressed as the mitotic index and as percentage of solvent control value

OTHER EXAMINATIONS:
- Determination of polyploidy, endoreduplication
Evaluation criteria:
A positive response was recorded if the % cells with aberrations markedly exceeded that in the concurrent controls either with or without a clear dose-response relationship. For modest increases in aberration frequency a dose-response relationship is generally required and appropriate statistical tests may be applied in order to record a positive response. Historical control ranges for human primary lymphocytes: 0 to 4% with strucutral aberrations includign gaps and 0 to 2% with structural aberrations excluding gaps and 0 to 1% polyploidy.
Statistics:
Frequency of cells with abberations (incl. and excl. gaps) and frequency of polyploid cells are compared to vehicle control using Fisher's Exact test

Results and discussion

Test results
Species / strain:
lymphocytes: human lymphocytes from donors
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
- The test substance induced a dose-related decrease in the mitotic index both with and without S9 (maximum dose level with scorable metaphase chromosomes: 156.25 microgr/mL without S9 and 625 microgr/mL with S9).
- In the absence of metabolic activation the total number of gaps was slightly increased (in a dose dependent manner), but the increase did not reach statistical significance.
- Test substance induced a statistically significant inrease in the frequency of cells with chromosome aberrations in one of two replictes at a concentration of 312.5 microgr/mL with metabolic activation only when including gaps. The increase was however not significant when gap type aberrations were excluded, but exceeded the historical maximum of the laboratory. No increase was observed at all other concentrations up to 625 microg/ml also when including gaps. As the effect was not reproducible and not dose related it was concluded to be of no toxicological significance.
Three cells contained chromatic exchange. Additionally a further set of slides prepared from the original cell cultures and evaluated for aberrations did not show these type of aberrations. As this effects was non-reproducible and not dose related, so considered to be of no toxicological significance.
- The test substance did not induce a significant increase in polyploid cells at any dose level in any of the treatments.

Applicant's summary and conclusion

Conclusions:
No toxicologically significant increases in the frequency of cells with chromosome aberrations including gaps, either in the presence or absence of a liver enzyme metabolising system (S9) was observed. The test susbstance is therefore considered to be non-clastogenic to human lymphocytes in vitro.
Executive summary:

In a mammalian cell cytogenetics assay, human lymphocytes were evaluated for chromosome aberrations after exposure to 2,2-bis(chloromethyl)trimethylene bis(bis(2-chloroethyl)phosphate at concentrations of 0 (solvent control), 39, 78.1 and 156.25 microgr/mL without metabolic activation and at 0, 78.1, 156.25, 312.5 and 625 microgr/mL with metabolic activation.

Ethyl methanesulphonate (EMS) and cyclophosphamide were used as positive controls, both induced the appropriate response. There was no dose-related positive response of chromosome aberrations after exposure to the test substance compared to the solvent control. At the mid dose (312.5 microgr/mL) with metabolic activation an significant increase in the frequency of cells with chromosome aberrations was observed when including gaps, but not when excluding gaps (but still higher than the historical maximum of the institute). This was not reproducible in a second set of slides at the same concentration and no dose response was observed. Therefore the effect was not considered toxicologically relevant and it can be concluded from this study that the test substance was not clastogenic in this study.

 

This GLP study is conducted according to OECD Guideline No.473 and is classified as acceptable.