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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:
2005
Reliability:
1 (reliable without restriction)

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2005
Report Date:
2005

Materials and methods

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

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Batch 19902-20
Expiry 31.3.2007

Method

Target gene:
TK
Species / strain
Species / strain / cell type:
human lymphoblastoid cells (TK6)
Metabolic activation:
with and without
Metabolic activation system:
rat liver S9
Test concentrations with justification for top dose:
Without S9 : From 700 to 1000 micrograms/ml (3h exposure time, 24 h fixation time).
With S9 : From 925 to 1200 microgram/ml (3h exposure time, 24 h fixation time).
Without S9 : From 500 to 1000 micrograms/ml (48h exposure time, 48 h fixation time).
With S9 : From 1000 to 1200 microgram/ml (48 h exposure time, 48 h fixation time).
Controlsopen allclose all
Untreated negative controls:
no
Negative solvent / vehicle controls:
no
True negative controls:
no
Positive controls:
yes
Positive control substance:
mitomycin C
Remarks:
Without metabolic activation

Migrated to IUCLID6: 0.5 µg/ml for a 3 h exposure period, 0.2 µg/ml for a 24 h exposure period and 0.1 µg/ml for a 48 h exposure period.
Untreated negative controls:
no
Negative solvent / vehicle controls:
no
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
With metabolic activation (+S9-mix)

Migrated to IUCLID6: 10 µg/ml for a 3 h exposure period (24 h fixation time)l for a 3 h exposure period (24 h fixation time).
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
RPMI 1640 medium
True negative controls:
no
Positive controls:
no
Details on test system and experimental conditions:
Cultured peripheral human lymphocytes were used as test system. Peripheral human lymphocytes are recommended in international guidelines (e.g.OECD and EEC). Blood was collected from healthy adult, non-smoking male volunteers.
Evaluation criteria:
A test substance was considered positive (clastogenic) in the chromosome aberration test if:
a) It induced a dose-related statistically significant (Chi-square test, P < 0.05) increase in the number of cells with chromosome aberrations.
b) A statistically significant and biologically relevant increase in the frequencies of the number of cells with chromosome aberrations was
observed in the absence of a clear dose-response relationship.
A test substance was considered negative (not clastogenic) in the chromosome aberration test if none of the tested concentrations induced
a statistically significant (Chi-square test, P c 0.05) increase in the number of cells with chromosome aberrations.
The preceding criteria are not absolute and other modifying factors might enter into the final evaluation decision.
The incidence of aberrant cells (cells with one or more chromosome aberrations, inclusive or exclusive gaps) for each exposure group was compared to that of the solvent control using Chi-square statistics.
Statistics:
Chi-square statistics.
Effects were considered staticticaly significant if p ≤ 0.05

Results and discussion

Test results
Species / strain:
human lymphoblastoid cells (TK6)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
The substance did not induce a statistically relevant increase in the number of chromosome aberrations in the absence and presence of S9 mix. The substance increased the number of polyploidy cells and cells with endoreduplication. The substance induced a statistically relevant increase in the number of chromosome aberrations at the highest cytotoxic concentrations only, both when gaps were included and excluded. Since the type of aberrations observed were mainly breaks and gaps the increases were not dose-related and the number of cells with aberrations were within the range of historical controls. The conclusion is that these increases were considered not to be biologically relevant.

In this experiment, it was technically challenging to obtain reproducible results regarding dose ranges associated with the desired level of cytotoxicity (50% decrease in mitotic index). The first experiment required 4 trials in order to obtain the 50% decrease in mitotic index. The second experimen required 6 trials in order to obtain the scorable cells.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative without metabolic activation
negative with metabolic activation

Substance was tested in chromosome aberrations test (OECD 473) using cultured periphesal human lymphocytes. The test is valid and the substance is not clastogenic in human lymphocytes under the experimental conditions, in the presence and absence of S9. Substance may have the potential to disturb mitotic processes and cell cycle progression due to polyploidy and endoreduplication.
Executive summary:

This report describes the effect of the substance on the number of chromosome aberrations in cultured peripheral human lymphocytes in the presence and absence of a metabolic activation system (Phenobarbital and B-naphthoflavone induced rat liver S9-mix).

The possible clastogenicity of N- { 2-[2-(Dimethylamine)Ethoxy]Ethyl ) -N-Methyl- l,3-Propanediamine was tested in two independent experiments.

In the first cytogenetic assay, substance was tested up to 925 µg/ml for a 3 hour exposure time with a 24 hour fixation time in the absence of S9-fraction. In the presence of 1.8% (vlv) S9-fraction, was tested up to 1015 µg/ml for a 3 hour exposure time with a 24 hour fixation time. Appropriate toxicity was reached at these dose levels.

In the second cytogenetic assay, the substance was tested up to 850 µg/rnl for a 24 hour continuous exposure time with a 24 hour fixation time and up to 800 µg/ml for a 48 hour continuous exposure time with a 48 hour fixation time in the absence of S9-mix. In

the presence of S9-mix the substance was tested up to 1080 µg/ml for a 3 hour exposure time with a 48 hour fixation time. Appropriate toxicity was reached at these dose levels.

Positive control chemicals, mitomycin C and cyclophosphamide, both produced a statistically significant increase in the incidence of cells with chromosome aberrations, indicating that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly.

Finally, it is concluded that this test is valid and that N-(2-[2-(Dimethylamine)Ethoxy]Ethyl}-N-Methyl-1,3-Propanediamine is not clastogenic in human lymphocytes under the experimental conditions described in this report. N- (2-[2-(Dimethylamine)Ethoxy]Ethyl ) -N-Methyl- l,3-Propanediamine may have the potential to disturb mitotic processes and cell cycle progression.