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Toxicological information

Genetic toxicity: in vitro

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Administrative data

in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of genotoxicity: chromosome aberration
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
From 1989-03-08 to 1989-09-14
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study
Reason / purpose for cross-reference:
reference to other study

Data source

Reference Type:
study report
Report date:

Materials and methods

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

Test material

Constituent 1
Reference substance name:
Quaternary ammonium compounds, bis(hydrogenated tallow alkyl)dimethyl, chlorides
EC Number:
EC Name:
Quaternary ammonium compounds, bis(hydrogenated tallow alkyl)dimethyl, chlorides
Cas Number:


Species / strain
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
The V79 cell line was stored in liquid nitrogen. The thawed stock cultures were propagated at 37°C in 175cm2 plastic flasks. Seeding was done with about 8-10 x 10E5 cells per flask in 30ml of MEM-medium supplemented with 10% fetal calf serum. The cells were subcultured twice weekly.
Metabolic activation:
with and without
Metabolic activation system:
S9 mix (liver post mitochondrial fraction of rats induced with Aroclor 1254)
Test concentrations with justification for top dose:
3 concentrations were used. The highest concentration did not reduce the number of scorable metaphases more than 20% of the negative controls.
Without metabolic activation, the concentration range was 0, 4, 20 and 40 ug/ml.
With metabolic activation, the concentration range was 0, 5, 25 and 50 ug/ml.
Vehicle / solvent:
On the day of the experiment, the test susbtance was dissolved as a solution in aqua.
Controlsopen allclose all
Untreated negative controls:
Untreated cultures
Negative solvent / vehicle controls:
Cultures treated with solvent
Positive controls:
Positive control substance:
Positive control used without metabolic activation.

Migrated to IUCLID6: dissolved in nutrient at the above concentration.
Untreated negative controls:
Untreated cultures
Negative solvent / vehicle controls:
Cultures treated with solvent
Positive controls:
Positive control substance:
Positive control used with metabolic activation.

Migrated to IUCLID6: Endoxan final concentration in nutrient medium at the above concentration.
Details on test system and experimental conditions:


- Exposure duration: 4 hours
- Expression time (cells in growth medium): 24 hours
- Selection time (if incubation with a selection agent): Not documented
- Fixation time (start of exposure up to fixation or harvest of cells): 12 hours

SELECTION AGENT (mutation assays): Not documented
SPINDLE INHIBITOR (cytogenetic assays): Colcemide
STAIN (for cytogenetic assays): The following was the process used for staining:
-staining for 10 minutes in 2% orcein solution.
-rinsing 3 times in distilled water
-rinsing twice in acetone
-brief rinsing in acetone/xylene
-2 minutes in acetone/xylene
-5 minutes in xylene
-10 minutes in xylene
-embedding in Entellan or Eukitt.

NUMBER OF REPLICATIONS: Duplicate - 2 independent cell cultures were used.
NUMBER OF CELLS EVALUATED: 100 metaphases per experimental group were examined.

- Method: mitotic index

- Determination of polyploidy: Not documented
- Determination of endoreplication: Not documented
- Other: Not documented

- Preparation of microscope slides: after treatment with spindle inhibitor each culture recieved a hypotonic treatment (KCl 0.075 M). Afterwards cells were fixed in a methanol/acetic acid mixture (3/1; v/v) and spread on glass slides before staining
- All metaphase analyses were performed blind
- Structural aberrations recorded: gaps, chromatid and chromosome breaks and exchanges, multiple aberrations and pulverizations

Evaluation criteria:
The test substance was classified as mutagenic if it induced a significantly increased aberration rate compared to the negative controls with one of the concentrations tested. The significance was obvious either by an enhancement of the rate clearly exceeding the control range or it was proven by adequate statistics. The test substance was classified as mutagenic if there was a reproducible concentraiton related increase in the aberration rate. The test substance was classified as not mutagenic when it tested negative both with and without metabolic activation.
Binomial statistic with Fischer's exact test to determine mutagenicity using biometry.

Results and discussion

Test results
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
with and without metabolic activation
Cytotoxicity / choice of top concentrations:
18 and 28 hours after treatment at 40 µg/ml without S9 mix
Vehicle controls validity:
not specified
Untreated negative controls validity:
Positive controls validity:
Additional information on results:
In the preliminary cytotoxicity study, it was observed that the test substance was toxic to the V79 cells in the absence of metabolic activation from 50 ug/ml up to the limit of solubility. In the presence of metabolic activation, an indication of toxicity was observed from 100ug/ml up to the limit of solubility. On the basis of these results, the top dose level used in the main experiment was 40 ug/ml with additional dose levels of 4 and 20 ug/ml in the absence of S9 mix. In the presence of S9 mix, 50 ug/ml was selected as the top dose to be used with additional concentrations of 5 and 25 ug/ml also used.

No relevant increase in the number of chromosome aberrations over the range of the negative control was found with any of the concentrations used, neither with or without metabolic activation by S9 mix.
Remarks on result:
other: all strains/cell types tested
Migrated from field 'Test system'.

Applicant's summary and conclusion

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

Under these experimental conditions, no statistically significant increase in the frequency of cells with chromosome aberrations was observed, both with and without S9 mix, using a dose range from 4 to 50 µg/ml (maximal practicable concentration regarding the cytotoxicity of the substance towards V79 cells). The test substance did not show any clastogenic activity in the in vitro mammalian chromosome aberration test with V79 chinese Hamster cells.

The test compound Praepagen WK was not mutagenic in the chromosome aberration test system in vitro with cells of the V79 Chineses hamster cell line. As a result of this, it was not necessary to classify it according to Regulation EC No. 1272/2008.
Executive summary:

Justification for cross reading of Benzyl-dihydrogenated-methyl ammonium chloride (BDHTMAC) to dihydrogenated-dimethyl-ammonium chloride (DHTDMAC) :

An overview of available data existing on common toxicological endpoints for the two substances demonstrates their similar toxicological behaviour: Both substances have a low acute toxicity but strong skin and eye corrosive properties. They are not sensitising and not mutagenic. The 28-day repeated dose toxicity studies performed by oral route in rats conclude to the same NOAEL of 100 mg/kg bw /day. The same biochemical changes (increase in ALAT enzyme) and the same target organ (adrenals) have been identified by these studies. Based on these results, the two substances display a similar toxic profile and it is scientifically appropriate to use cross-reading.

In the Hoechst AG study (1989), the structural analogue 'Quaternary ammonium compounds, bis(hydrogenated tallow alkyl)dimethyl, chlorides (DHTDMAC)

was examined for mutagenic activity in V79 Chinese hamster cells. The induction of chromosome aberrations after in vitro treatment was investigated in the presence and absence of a fraction of liver homogenate for metabolic activation (S9-mix).


A preliminary cytotoxicity experiment was performed in order to select appropriate dose levels for the mutagenicity study. The test substance produced a significant cytotoxic effect (reduction of plating efficiency) without metabolic activation from 50 µg/ml up to a concentration of 200 µg/ml which is the limit of solubility (200 µg/ml) . A cytotoxic effect was also observed with metabolic activation from 100 ug/ml up to the limit of solubility. For mutagenicity testing, two independent cell cultures with and without metabolic activation (S9-mix) were used.


For main experiment dose levels of 4, 20 and 40 ug/ml in the absence of metabolic activation and in the presence of S9-mix metabolic activation dose levels of 5, 25 and 50 ug/ml were used.



The test compound did not induce a significant increase in the number of metaphases with aberrations at any preparation time and dose level of the test substance.

A cytotoxic effect (reduction of mitotic index) of the compound was observed 18 and 28 h after treatment at 40 ug/ml without metabolic activation in the main experiments. Marked increases in the rate of chromosome aberrations were obtained with the positive control substance indicating the sensitivity of the assay.

In conclusion, the test substance does not induce chromosome aberrations in V79 Chinese hamster cells, neither in the presence nor in the absence of a metabolic activation system, under the experimental conditions described.