Registration Dossier

Toxicological information

Genetic toxicity: in vitro

Currently viewing:

Administrative data

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1991
Report Date:
1991

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:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
Principles of method if other than guideline:
Dimethylaminopropylamine (DMAPA) was tested in an in vitro cytogenetics assay using duplicate human lymphocyte cultures both in the absence and presence of metabolic activation (S9 mix), according to the OECD n° 473 Guideline and EC 92/69/EEC B.10 guidelines in compliance with the Principles of Good Laboratory Practice.
With metabolic activation, lymphocytes cultures were exposed for 3 hours to solvent vehicle or DMAPA at 0, 350.5, 500.8, 715.4 µg/ml and then incubated for a further 17 or 41 hours.
Without metabolic activation, lymphocytes cultures were exposed for 20 or 44 hours to solvent vehicle or DMAPA at 0, 120.2, 171.8, 245.4 µg/ml.
The proportion of cells with structural aberrations in negative controls cultures fell within historical solvent control ranges. Positive controls induced statistically significant increases in the proportion of cells with structural aberrations.
Chromosome aberrations were analyzed in cells sampled 20 hours after the start of treatment at 3 consecutive dose levels. The highest concentrations chosen for analysis at this time, 245.4 and 715.4 11µg/ml, induced approximately 51% and 54% mitotic inhibition in the absence and presence of S-9 respectively. The effects of single concentrations only, 245.4 µg/ml without and 715.4 µg/ml with S-9 were investigated at the delayed harvest at which time 0% and 34% mitotic inhibition was induced.
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid

Method

Target gene:
Chromosome defects
Species / strain
Species / strain / cell type:
lymphocytes: human
Details on mammalian cell type (if applicable):
- Type and identity of media: A single female donor was used in this study. The volunteer was not suspected of any virus infection nor had been exposed to high levels of radiation or hazardous chemicals. An appropriate volume of whole blood was drawn from the peripheral circulation on the day of culture. Whole blood cultures were established in sterile disposable centrifuge tubes by placing 0.4 ml heparinised blood into 9.0 ml Hepes-buffered RPMI medium containing 20% (v/v) foetal calf serum and 50 ug/ml gentamycin. Phytohaemagglutinin (PHA) was included at a concentration of 37.5 µl per ml of culture to stimulate the lymphocytes to divide. Cultures were rocked continuously during incubation.
- Properly maintained: yes
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
mammalian liver post-mitochondrial fraction (S-9) prepared from male Sprague Dawley rats induced with Aroclor 1254 (obtained from Molecular Toxicology Incorporated, Annapolis, Maryland, USA.)
Test concentrations with justification for top dose:
Without metabolic activation: 120.2, 171.8, 245.4 µg/ml for 20 hours sampling time and 245.4 µg/ml for 44 hours sampling time.
With metabolic activation: 350.5, 500.8, 715.4 µg/ml for 20 hours sampling time and 715.4 µg/ml for 44 hours sampling time.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: water
- Justification for choice of solvent/vehicle: solubility
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: -S9: Methylmethansulfonate (MMS) 50.0 µg/ml. +S9: Cyclophosphamide (CPA) 25.0 µg/ml
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: 48 hours at 37°C
- Exposure duration: 20 and 44 hours for cultures without metabolic activation and 3 hours for cultures with metabolic activation (followed by 17 and 41 hours of additional sampling time before harvesting)
- Expression time (cells in growth medium): 20 or 44 hours
- Selection time (if incubation with a selection agent): colchicine was added 1.5 hour prior to harvest.

SPINDLE INHIBITOR (cytogenetic assays): colchicine
STAIN (for cytogenetic assays): Giemsa

NUMBER OF REPLICATIONS: 2 (for cultures treated with DMAPA and positive controls) and 4 (for cultures treated with negative controls)

NUMBER OF CELLS EVALUATED: One hundred metaphases from each culture were analysed for chromosome aberrations.

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; percentage of cell in mitosis

OTHER EXAMINATIONS:
- Determination of polyploidy: yes
- Determination of endoreplication: yes
- Other: hyperploidy
Evaluation criteria:
The test chemical was to be considered as clearly positive if:
- statistically significant increases in the proportion of structurally aberrant cells (without gaps) occurred at one or more concentrations
- the proportion of aberrant cells at such data points exceeded the normal range
Statistics:
The proportion of aberrant cells in each replicate were used to establish acceptable heterogeneity between replicates by means of a binomial dispersion test.
The proportion of cells in category 2 for each test treatment condition were compared with the proportion in negative controls by using Fisher's exact test.

Results and discussion

Test results
Key result
Species / strain:
lymphocytes: human
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid

Any other information on results incl. tables

Table 1: Selection of doses

Mitotic Index (%)
Treatment (µm/ml) 20 hours 44 hours
  - S-9 + S-9 - S-9 + S-9
  A B A B A B A B
Solvent 5.8 4.0 5.3 4.1 4.5 3.3 3.5 3.8
20.21 NM NM NM NM NM NM NM NM
28.87 NM NM NM NM NM NM NM NM
41.24 NM NM NM NM NM NM NM NM
58.92 NS NS NM NM NM NM NM NM
84.17 NS NS NM NM NM NM NM NM
120.2 5.8 4.8 NS NS NS NS NM NM
171.8 4.9 4.6 NS NS NS NS NS NS
245.4 1.6 3.2 NS NS 4.2 4.3 NS NS
350.5 0.3 0.4 3.8 3.5 0.7 0.2 NS NS
500.8 0 0 2.9 3.5 0 0 NS NS
715.4 0 0 2.2 2.1 0 0 3.2 1.6
1022 0 0 0 0 0 0 0.6 0.4
NS = not scored NM = not made

Table 2: Cells with structural aberrations

20 hour sampling time, - S-9

Treatment (µg/ml) Replicate Cells scored Cells with aberrations including gaps Cells with aberrations excluding gaps Significance § Mitotic index (mean)
Solvent A 100 1 0   5.8
B 100 1 0   4.0
Totals 200 2 0    (4.9)
120.2 A 100 0  0   5.8
B 100 5 0   4.8
Totals 200 5 0 NS (5.3)
171.8 A 100 5 1   4.9
B 100 1 0   4.6
Totals 200 6 1 NS (4.8)
245.4 A 100 3 2   1.6
B 100 1 0   3.2
Totals 200 4 2 NS (2.4)
MMS, 50 A 25 14 13    
B 25 18 17    
Totals 50 32 30 p <0.001  
§ Statistical significance
NS = not significant

Table 3: Cells with structural aberrations

20 hour sampling time, + S-9

Treatment (µg/ml) Replicate Cells scored Cells with aberrations including gaps Cells with aberrations excluding gaps Significance § Mitotic index (mean)
Solvent A 100 5 2   5.3
B 100 1 0   4.1
Totals 200 6 2   (4.7)
350,5 A 100 3 1   3.8
B 100 5 1   3.5
Totals 200 8 2 NS (3.7)
500,8 A 100 4 2   2.9
B 100 5 4   3.5
Totals 200 9 6 NS (3.2)
715,4 A 100 8 5   2.2
B 100 5 3   2.1
Totals 200 13 8 p<0.05 (2.2)
CPA, 25 A 25 11 7    
B 25 8 6    
Totals 50 19 13 p <0.001  
§ Statistical significance
NS = not significant

Table 4: Cells with structural aberrations

44 hour sampling time, - S-9

Treatment (µg/ml) Replicate Cells scored Cells with aberrations including gaps Cells with aberrations excluding gaps Significance § Mitotic index (mean)
Solvent A 100 3 2   4.5
B 100 4 4   3.3
Totals 200 7 6   (3.9)
245.4 A 100 6 4   4.2
B 100 2 0   4.3
Totals 200 8 4 NS (4.3)

Table 5: Cells with structural aberrations

44 hour sampling time, + S-9

Treatment (µg/ml) Replicate Cells scored Cells with aberrations including gaps Cells with aberrations excluding gaps Significance § Mitotic index (mean)
Solvent A 100 1 1   3.5
B 100 3 1   3.8
Totals 200 4 2   (3.7)
715,4 A 100 5 2   3.2
B 100 2 1   1.6
Totals 200 7 3 NS (2.4)

Cultures treated with DMAPA in the absence and presence of metabolic activation resulted in frequencies of cells with structural aberrations, which were similar to those seen in concurrent negative controls..

Under these experimental conditions, DMAPA did not induce any noteworthy increase in the number of cells with structural chromosome aberration, both with and without metabolic activation at any harvest time.

Applicant's summary and conclusion