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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The genotoxicity of the registration substance was investigated in three in-vitro test system.

Negative results were obtained in Ames test, in mammalian gene mutation test and in micronucleus test.

Overall, no significant genotoxicity can be assigned for the registration substance.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1995
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
E. coli WP2 strain missing; Second experiment performed as a plate incorporation assay, but not as a preincubation assay.
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
S9 mix
Test concentrations with justification for top dose:
Pre-Experiment and Experiment I: 0 (control), 4, 20, 100, 500, 2500, 5000 µg/plate
Experiment II: 0 (control), 4, 20, 100, 500, 2500, 5000 µg/plate
Vehicle / solvent:
ethanol
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
ethanol
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: see below for additional information
Details on test system and experimental conditions:
The assay was performed in two independent experiments:

experiment I: plate incorporation assay with and without induced rat liver S9 mix
experiment II: plate incorporation assay with and without induced rat liver S9 mix

Rat liver S9 mix from Aroclor 1254 induced rats was used.

Top agar was prepared for the Salmonella strains by mixing 100 ml agar (0.6% (w/v) agar, 0.5% (w/v) NaCI) with 10 ml of a 0.5 mM histidine-biotin solution. After mixing, the liquid was poured into a petridish with minimal agar (1.5 % (w/v) agar, Vogel-Bonner E medium with 2 % (wlv) glucose). After incubation for approximately 48 hours at approx. 37 °C in the dark, colonies (hi? revertants) were counted.

DURATION
- Exposure duration: after solidification the plates were incubated upside down for approx. 48 hours at 37°C in the dark

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY: The first experiment was performed with all tester strains using three plates per dose to get information on mutagenicity and toxicity for calculating an appropriate dose range. A reduced rate of spontaneously occuring colonies and visible thinning of the bacterial lawn were used as toxicity indicators. Thinning of the bacterial lawn was evaluated microscopically.
In combination with the second experiment, toxicity testing was performed as follows: 0.1 ml of the different dilutions of the test compound were thoroughly mixed with 0.1 ml of 108 dilution of the overnight culture of TA 100 and plated with histidine and biotin rich top agar (3 plates per dose). The solvent control is compared with the number of colonies per plate in the presence of the test compound. Results are given as a ratio of these values (= surviving fraction).

POSITIVE CONTROL SUBSTANCES:
without metabolic activation: sodium azide (TA 1535, TA 100), 9-aminoacridine (TA 1537), 2-nitrofluorene (TA 98);
with metabolic activation: 2-aminoanthracene (all strains)
Evaluation criteria:
A test article is classified as mutagenic if it has either of the following effects:
a) a test article produces at least a 2-fold increase in the mean number of revertants per plate of at least one of the tester strains over the mean number of revertants per plate of the appropriate vehicle control at complete bacterial background lawn
b) a test article induces a dose-related increase in the mean number of revertants per plate of at least one of the tester strains over the mean number of revertants per plate of the appropriate vehicle control in at least two to three concentrations of the test article at complete bacterial background lawn.
The test results must be reproducible.
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
see below for additional information
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
The test compound was tested at doses of 4 to 5000 microgram/plate and proved to be toxic to most of the bacterial strains at doses of 2500 microgram/plate and above. Thinning of the bacterial lawn and a reduction in the number of colonies were observed at this dose.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Mean mutant number

Exp. I: plate incorporation method without S9 mix

Concentrations given in µg/plate

Strain -- 0 -- 4 -- 20 -- 100 -- 500 -- 2500 -- 5000

TA100 -- 176.7 -- 168.0 -- 201.0 -- 217.0 -- 25.0 -- 1.3 -- 1.0

TA1535 -- 10.7 -- 6.3 -- 8.3 -- 8.0 -- 1.3 -- 1.0 -- 0.3

TA1537 -- 7.0 -- 10.7 -- 9.7 -- 11.3 -- 5.0 -- 0.0 -- 0.0

TA98 -- 27.7 -- 24.0 -- 31.3 -- 23.0 -- 7.3 -- 1.0 -- 0.0

Exp. I: plate incorporation method with rat S9 mix

Concentrations given in µg/plate

Strain -- 0 -- 4 -- 20 -- 100 -- 500 -- 2500 -- 5000

TA100 -- 137.7 -- 207.0 -- 182.7 -- 185.0 -- 165.7 -- 43.0 -- 6.0

TA1535 -- 9.3 -- 11.7 -- 14.7 -- 12.3 -- 10.0 -- 5.0 -- 1.0

TA1537 -- 11.7 -- 8.7 -- 10.0 -- 8.3 -- 6.3 -- 2.3 -- 0.7

TA98 -- 36.3 -- 34.7 -- 36.7 -- 41.7 -- 37.0 -- 15.0 -- 2.0

Exp. II: plate incorporation method without S9 mix

Concentrations given in µg/plate

Strain -- 0 -- 4 -- 20 -- 100 -- 500 -- 2500 -- 5000

TA100 -- 141.7 -- 184.3 -- 146.3 -- 145.7 -- 2.0 -- 1.0 -- 1.0

TA1535 -- 16.0 -- 12.7 -- 7.7 -- 8.7 -- 3.3 -- 0.0 -- 0.0

TA1537 -- 7.7 -- 7.7 -- 8.7 -- 8.3 -- 4.3 -- 0.0 -- 0.0

TA98 -- 26.3 -- 24.3 -- 22.0 -- 31.0 -- 9.3 -- 1.3 -- 0.0

Exp. II:

plate incorporation method with rat liver S9 mix

Concentrations given in µg/plate

Strain -- 0 -- 4 -- 20 -- 100 -- 500 -- 2500 -- 5000

TA100 -- 176.3 -- 172.7 -- 170.3 -- 170.3 -- 123.3 -- 98.7 -- 24.0

TA1535 -- 19.3 -- 13.0 -- 16.7 -- 13.3 -- 15.0 -- 1.3 -- 1.0

TA1537 -- 8.0 -- 7.0 -- 9.7 -- 10.3 -- 6.0 -- 9.3 -- 0.3

TA98 -- 36.7 -- 33.3 -- 36.3 -- 29.3 -- 30.7 -- 8.3 -- 1.3

Conclusions:
The genotoxicity of the registration substance was investigated according to the guideline OECD 471. Negative result was obtained.
Executive summary:

The registration substance was tested for mutagenicity with the strains TA 100, TA 1535, TA 1537 and TA 98 of Salmonella typhimurium.

The registration substance did not increase the number of revertants in any of the bacterial strains with and without metabolic activation. No significant mutagenicity was found.

Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2006-07 - 2007-03
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
mammalian cell gene mutation assay
Target gene:
hypoxanthine-guanine phosphoribosyl transferase
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
- Type and identity of media: minimum essential medium containing 10% fetal calf serum
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes
Metabolic activation:
with and without
Metabolic activation system:
S9-mix (rat liver protein with cofactors)
Test concentrations with justification for top dose:
Doses applied in the gene mutation assay with Behentrimonium chloride (related to active substance) as concentration in μg/mL
Experiment I
without S9 mix 0.1, 0.2, 0.3, 0.6, 1.3, 1.9
with S9 mix 1.3, 2.4, 5.0, 9.9, 19.7, 29.6
Experiment II
without S9 mix 0.6, 1.2, 2.4, 4.7, 6.3, 7.9
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: water
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
water
Positive controls:
yes
Positive control substance:
other: see text below
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium (4h-incubation without serum, 24 h incubations with serum)


DURATION
- Preincubation period: none
- Exposure duration: 4 and 24 h
- Expression time (cells in growth medium): 7 days
- Selection time (if incubation with a selection agent): 8 days


SELECTION AGENT (mutation assays): 6-thioguanine
STAIN (for cytogenetic assays): 10% methylene blue in 0.01% KOH solution


NUMBER OF REPLICATIONS: 5/experiment, 2 experiments plus repetition of the 1st experiment

Positive control substances: ethylmethane sulfonate without metabolic activation and 7,12-Dimethylbenz(a)anthracene with metabolic activation
Metabolic activation: liver S9 was prepared from phenobarbital-treated rats

NUMBER OF CELLS EVALUATED: colonies >50 cells


DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency

Evaluation criteria:
A test item is classified as mutagenic if it reproducibly induces a mutation frequency that is three times above the spontaneous mutation frequency at least at one of the concentrations in the experiment.
The test item is classified as mutagenic if there is a reproducible concentration-related increase of the mutation frequency. Such evaluation may be considered also in the case that a threefold increase of the mutant frequency is not observed.
However, in a case by case evaluation this decision depends on the level of the corresponding negative control data. If there is by chance a low spontaneous mutation rate in the range normally found (0.5 – 31.8 mutants per 106 cells) a concentration-related increase of the mutations within this range has to be discussed. The variability of the mutation rates of negative and solvent controls within all experiments of this study was also taken into consideration.
Statistics:
A linear regression was performed to assess a possible dose dependent increase of mutant frequencies using SYSTAT® statistics software. The number of mutant colonies obtained in the groups treated with the test item was compared to the solvent control groups. A trend is judged as significant whenever the p-value (probability value) is below 0.05. However, both, biological and statistical significance should be considered together.
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
see text below
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: negligible increase at highest dose (pH 7.45 at 78.8 µg/ml vs. 7.37 in solvent control
- Effects of osmolality: negligible: 284 mOsm at highest dose of 78.7 µg/ml vs. 285 in solvent control

Cytotoxicity:

Relevant toxic effects indicated by a relative cloning efficiency I (CE I) below 50% occurred in the first experiment at 1.3 μg/mL and above without metabolic activation and at 9.9 (culture I only) and 19.7 μg/mL and above in the presence of metabolic activation. In the second experiment such toxic effects occurred at 4.7 μg/mL and above. The cultures at 29.6 μg/mL in the first experiment with metabolic activation and at 6.3 and 7.9 μg/mL in the second experiment were not analysable due to exceedingly severe toxic effects. The CE I was zero or well below 10 % at 9.9 (culture I only) and 19.7 μg/mL in the first experiment with and at 1.9 μg/mL without metabolic activation. Still, the data are considered valid since the corresponding cell density of the mass cell cultures used to determine mutagenicity determined at the first sub-cultivation following treatment was above 20 % of the corresponding control value. In the second experiment severe toxicity resulted in CE values of 0 at 4.7 μg/mL and corresponding cell densities of 6.5 % and 9.8 %. Still, the surviving cells recovered and showed cloning efficiency II values of more than 50 % at the time point of selection. The fact, that higher concentrations are tolerated in the second experiment compared to the first experiment without metabolic activation indicates possible protein or lipid binding effects of the test item. The FCS-concentration is 10 % during long term treatment compared to 0 % during 4 h treatment. Test items binding to proteins or lipids of the FCS are less available to the cells and thus, higher concentrations are tolerated even though the treatment period is much longer.

No relevant and reproducible increase of the mutation frequency occurred at any concentration with and without metabolic activation. All mutant frequencies remained well within the historical range of negative and solvent controls. A linear regression analysis (least squares) was performed to assess a possible dose dependent increase of mutant frequencies using SYSTAT®statistics software. No significant dose dependent trend of the mutation frequency indicated by a probability value of <0.05 was calculated in both main experiments.

In both experiments of this study (with and without S9 mix) the range of the negative and solvent controls was from 2.9 up to 10.1 mutant colonies per 106 cells; the range of the groups treated with the test item was from 1.2 up to 12.4 mutant colonies per 106 cells.

EMS (0.300 mg/mL in experiment I and 0.150 mg/mL in experiment II) and DMBA (1.5 μg/mL) were used as positive controls and showed a distinct increase in induced mutant colonies.

Summary of results

Condition -- Conc. (µg/ml) -- Rel. cloning efficiency I (%) -- Cell density (% of control) -- Mutant colonies per 10e6 cells -- Induction factor

Exp. I, 4 hour treatment without S9 mix, Culture 1

Negative control -- -- 100.0 -- 100.0 -- 6.9 --

Solvent control with water -- -- 100.0 -- 100.0 -- 6.4 --1.0

Positive control with EMS -- 300.0 -- 87.6 -- 116.0 -- 119.5 -- 17.2

Test item -- 0.1 -- 92.8 -- culture was not continued

Test item -- 0.2 -- 100.8 -- 103.0 -- 2.0 -- 0.3

Test item -- 0.3 -- 99.5 -- 92.4 -- 5.6 -- 0.9

Test item -- 0.6 -- 98.3 -- 80.6 -- 12.4 -- 2.0

Test item --1.3 -- 22.7 -- 80.6 -- 2.1 -- 0.3

Test item -- 1.9 -- 0.0 -- 34.9 -- 3.4 -- 0.5

Exp. I, 4 hour treatment without S9 mix, Culture 2

Negative control -- -- 100.0 -- 100.0 -- 4.8 --

Solvent control with water -- -- 100.0 -- 100.0 -- 2.9 --1.0

Positive control with EMS -- 300.0 -- 85.3 -- 96.6 -- 94.0 -- 19.6

Test item -- 0.1 -- 85.9 -- culture was not continued

Test item -- 0.2 -- 87.9 -- 111.0 -- 1.5 -- 0.5

Test item -- 0.3 -- 85.8 -- 122.9 -- 3.7 -- 1.3

Test item -- 0.6 -- 76.8 -- 113.4 -- 7.3 -- 2.5

Test item --1.3 -- 21.5 -- 78.1 -- 4.8 -- 1.6

Test item -- 1.9 -- 0.0 -- 30.2 -- 1.5 -- 0.5

Exp. I, 4 hour treatment with S9 mix, Culture 1

Negative control -- -- 100.0 -- 100.0 -- 4.2 --

Solvent control with water -- -- 100.0 -- 100.0 -- 3.6 --1.0

Positive control with DMBA -- 1.5 -- 62.1 -- 67.4 -- 931.2 -- 219.6

Test item -- 1.3 -- 87.2 -- 77.3 -- 1.2 -- 0.3

Test item -- 2.4 -- 82.5 -- 78.4 -- 1.4 -- 0.4

Test item -- 5.0 -- 75.5 -- 58.6 -- 7.9 -- 2.2

Test item -- 9.9 -- 3.6 -- 34.5 -- 4.4 -- 1.2

Test item --19.7 -- 0.0 -- 20.0 -- 3.6 -- 1.0

Test item -- 29.6 -- 0.0 -- culture was not continued

Exp. I, 4 hour treatment with S9 mix, Culture 2

Negative control -- -- 100.0 -- 100.0 -- 7.7 --

Solvent control with water -- -- 100.0 -- 100.0 -- 10.1 --1.0

Positive control with DMBA -- 1.5 -- 21.2 -- 46.4 -- 917.6 -- 119.7

Test item -- 1.3 -- 97.9 -- 96.8 -- 7.8 -- 0.8

Test item -- 2.4 -- 104.6 -- 102.9 -- 7.8 -- 0.8

Test item -- 5.0 -- 93.4 -- 61.6 -- 1.4 -- 0.1

Test item -- 9.9 -- 78.5 -- 58.0 -- 4.6 -- 0.5

Test item -- 19.7 -- 5.1 -- 26.5 -- 3.7 -- 0.4

Test item -- 29.6 -- 0.6 -- culture was not continued

Exp.II, 24 hour treatment without S9 mix, Culture 1

Negative control -- -- 100.0 -- 100.0 -- 3.7 --

Solvent control with water -- -- 100.0 -- 100.0 -- 3.3 --1.0

Positive control with EMS -- 150.0 -- 72.2 -- 101.8 -- 184.8 -- 49.6

Test item -- 0.6 -- 98.1 -- 96.6 -- 7.4 -- 2.3

Test item -- 1.2 -- 91.5 -- 78.0 -- 4.6 -- 1.4

Test item -- 2.4 -- 64.7 -- 30.3 -- 6.9 -- 2.1

Test item -- 4.7 -- 0.0 -- 6.5 -- 2.7 -- 0.8

Test item -- 19.7 -- 0.0 -- culture was not continued

Test item -- 29.6 -- 0.0 -- culture was not continued

Exp.II, 24 hour treatment without S9 mix, Culture 2

Negative control -- -- 100.0 -- 100.0 -- 3.5 --

Solvent control with water -- -- 100.0 -- 100.0 -- 5.4 --1.0

Positive control with EMS -- 150.0 -- 72.9 -- 117.6 -- 210.1 -- 60.5

Test item -- 0.6 -- 104.3 -- 75.3 -- 6.8 -- 1.3

Test item -- 1.2 -- 94.4 -- 73.5 -- 4.2 -- 0.8

Test item -- 2.4 -- 74.3 -- 37.5 -- 8.3 -- 1.6

Test item -- 4.7 -- 0.0 -- 9.8 -- 7.8 -- 1.5

Test item -- 19.7 -- 0.0 -- culture was not continued

Test item -- 29.6 -- 0.0 -- culture was not continued

where indicated low concentration cultures were not continued since a minimum of only four analysable concentrations is required, while high concentration cultures were not continued due to exceedingly strong toxic effects

Conclusions:
The genotoxicity of the registration substance was investigated according to the guideline OECD 476. Negative result was obtained.
Executive summary:

The genotoxic potential of the registration substance was evaluated in a mammalian cell gene mutation assay according to OECD guideline 476.

No increase of the mutation frequency occurred at up to concentrations associated with clear cytotoxicity with and without metabolic activation. No mutagenicity was found.

Endpoint:
in vitro cytogenicity / micronucleus study
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2007
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
other: OECD 487, Draft Version 1
Deviations:
no
Principles of method if other than guideline:
OECD Guideline Draft Proposal for a new Guideline No. 487, Version 1
GLP compliance:
yes (incl. QA statement)
Type of assay:
in vitro mammalian cell micronucleus test
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
Thawed stock cultures were propagated at 37 °C in 80 cm2 plastic flasks (Greiner,
72632 Frickenhausen, Germany). About 5 x 105 cells per flask were seeded in 15 mL of MEM (minimal essential medium; Seromed, 12247 Berlin, Germany) supplemented with 10 % fetal calf serum (FCS; PAA Laboratories GmbH, 35091 Cölbe, Germany). The cells were subcultured twice weekly. The cell cultures were incubated at 37 °C in a humidified atmosphere with 1.5 % carbon dioxide (98.5% air).
Metabolic activation:
with and without
Metabolic activation system:
rat liver S9
Test concentrations with justification for top dose:
Exp. I: with and without S9 mix: 0.8, 1.6, 3.1, 6.3, 12.5, 25.0, 50.0, 100.0, 200.0, and 400.0 µg/mL
Exp. IIA: with and without S9 mix: 0.2, 0.4, 0.8, 1.6, 3.1, 6.3, 12.5, and 25.0 µg/mL
Exp. IIB: without S9 mix: 0.5, 1.0, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 10.0, 12.5, and 15.0 µg/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Water
- Justification for choice of solvent/vehicle: solubility
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
water
Positive controls:
yes
Positive control substance:
mitomycin C
Remarks:
and colcemid and cyclophosphamide
Details on test system and experimental conditions:
Two independent experiments were performed. In Experiment I the exposure period was 4 hours with and without metabolic activation. In Experiment II the exposure period was 20 hours without S9 mix and 4 hours with metabolic activation. The cells were prepared 24 hours (48 hours for Exp. II with S9 mix) after start of treatment with the test item.

METHOD OF APPLICATION: in minimal essential medium

DURATION
- Exposure duration: 4 and 24 hours
- Expression time (cells in growth medium): 24 hours
- Fixation time (start of exposure up to fixation or harvest of cells): 24 hours (48 hours for Exp. II with S9 mix)

SPINDLE INHIBITOR (cytogenetic assays):
STAIN (for cytogenetic assays): May Gruenwald and Giemsa

NUMBER OF REPLICATIONS: 1.5 - 2

NUMBER OF CELLS EVALUATED: 2000

EVALUATION: Evaluation of the cultures was performed manually using NIKON microscopes with 40 x oil immersion objectives. The micronuclei were counted in cells showing a clearly visible cytoplasm area. The criteria for the evaluation of micronuclei are described in the publication of Countryman and Heddle (1976). The micronuclei were stained in the same way as the main nucleus. The area of the micronucleus did not extend the third part of the area of the main nucleus. 2000 cells were scored per test group. The frequency of micronucleated cells was reported as % micronucleated cells.

DETERMINATION OF CYTOTOXICITY
- Method: Proliferation Index

OTHER EXAMINATIONS:


Evaluation criteria:
A test item can be classified as mutagenic if:
- the number of micronucleated cells is not in the range of the historical control data (0.0-2.0% micronucleated cells), and
- either a concentration-related increase in three test groups or a significant increase of micronucleated cells in at least one test group is observed.
A test item can be classified as non-mutagenic if:
- the number of micronucleated cells in all evaluated test groups is in the range of the historical control data (0.0-2.0% micronucleated cells), and/or
- no concentration-related increase in the number of micronucleated cells is observed.
Statistical significance can be confirmed by means of the Chi square test. However, both biological and statistical significance should be considered together. If the criteria above mentioned for the test item are not clearly met, the classification with regard to the historical data and the biological relevance is discussed and/or a confirmatory experiment is performed.
Statistics:
Statistical significance can be confirmed by means of the Chi square test.
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
see textbelow
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
The test item Behentrimonium chloride, suspended (Experiment I) or dissolved (Experiments IIA and llB) in deionised water, was assessed for its potential to induce micronuclei in Chinese hamster V79 cells in vifro in the absence and the presence of metabolic activation by S9 mix.
Three independent experiments were performed. In Experiment I, the exposure period was 4 hours with and without metabolic activation. In Experiment IIA and IIB, the exposure period was 20 hrs without S9 mix and in Experiment 11, the exposure period was 4 hours with metabolic activation. The cells were prepared 24 hours (Exp. I, IIA and llB) and 48 hrs (Exp. IIA) after start of treatment with the test item.
In each experimental group two parallel cultures were set up. Per culture 1000 cells were scored for micronuclei.
Evaluated experimental points after treatment with Behentrimonium chloride:
Exp I: 3.1, 6.3 and 12.5 µg/ml, without S9 mix, 4 hrs exposure, 24 hrs preparation interval
Exp IIA: 3.1, 6.3 and 12.5 µg/ml, without S9 mix, 20 hrs exposure, 24 hrs preparation interval
Exp IIB: 5.0, 6.0, and 7.0 µg/ml, without S9 mix, 20 hrs exposure, 24 hrs preparation interval
Exp I: 6.3, 12.5 and 25.0 µg/ml, with S9 mix, 4 hrs exposure, 24 hrs preparation interval
Exp IIA: 3.1, 6.3 and 12.5 µg/ml, with S9 mix, 4 hrs exposure, 48 hrs preparation interval

In Experiment I, no relevant increase of the pH value or osmolarity was observed (solvent control 271 mOsm, pH 7.3 versus 288 mOsm and pH 7.4 at 400.0 µg/mL). Test item precipitation was observed at a concentration of 50.0 µg/mL and above in the absence of S9 mix and at 25.0 µg/mL and above in the presence of S9 mix. Clear toxic effects indicated by reduced cell numbers were observed after treatment with 12.5 µg/mL (11.3 % of control) and above in the absence of S9 mix. In the presence of S9 mix, concentrations showing clear cytotoxicity were not scorable for cytogenetic damage.
In the absence and presence of S9 mix, no biologically relevant increase in the percentage of micronucleated cells was observed up to the highest scorable concentration. The rates of micronucleated cells after treatment with the test item in the absence of S9 mix (0.95 - 1.55 %) and in the presence of S9 mix (0.85 - 1.55 %) were below the corresponding solvent control values (2.00 and 1.70 %, respectively) and within the range the laboratory's historical control data: 0.0 - 2.0 % micronucleated cells.
In Experiment IIA, test item precipitation was observed at a concentration of 6.3 µg/mL and above in the absence of S9 mix and at 25.0 µg/mL and above in the presence of S9 mix. Clear cytototoxicity (indicated by reduced cell numbers when compared to control values) of about 60% or above were observed after treatment with 12.5 µg/mL (70 % cytotoxicity) and above in the absence of S9 mix, and with 12.5 µg/mL (58 % cytotoxicity) and above in the presence of S9 mix.
In the presence of S9 mix, no biologically relevant increase in the percentage of micronucleated cells was observed up to the highest evaluated concentration of 12.5 µg/mL. The rates of micronucleated cells after treatment with the test item (0.60 - 1.00%) were close to the corresponding solvent control value (0.55 %) and within the range of our historical control data: 0.0 - 2.0 % micronucleated cells. In the absence of S9 mix, dose-related increases in the percentage of micronucleated cells (1 -65 %, 1.75 %, and 2.40 %) were observed at the three evaluated concentrations (3.1, 6.3, and 12.5 µg/mL, respectively). Although all three values were statistically significantly increased compared to the corresponding solvent control value (0.85%), the percentages of micronucleated cells at 3.1 and 6.3 µg/mL (1.65 % and 1.75 %, respectively) were within the laboratorie's historical control data range (0.0 - 2.0 % micronucleated cells), and these increases have to be regarded as biologically irrelevant. At the highest concentration evaluated (12.5µg/mL), given the high cytotoxicity observed (70%), the biological relevance of the increased incidence of micronucleated cells remained ambiguous.
Accordingly, two repeat experiments (Experiment IIB) within a narrow concentration range were performed, in order to verify the observation in Experiment IIA.
The first repeat experiment could not be evaluated, since only two concentrations were available for cytogenetic evaluation, which is not in compliance with the guideline recommendations. In both experiments, concentrations of 7.5 µg/mL and 8.0 µg/mL and above were not scorable for cytogenetic damage due to strong test item induced cytotoxicity.
In the second repeat experiment, test item precipitation was observed at a concentration of 6.0 µglmL and above in the absence of S9 mix. Clear toxic effects indicated by reduced cell numbers were observed after treatment with 7.0 µg/mL (62 % cytotoxicity). No biologically relevant increase in the percentage of micronucleated cells was observed up to the highest scorable concentration. The rates of micronucleated cells after treatment with the test item (0.05 - 1.05 %) were close to the corresponding solvent control values (0.55 %) and within the range of our historical control data: 0.0 - 2.0 % micronucleated cells. Accordingly, the increased incidence of micronucleated cells observed at a concentration associated with high cytotoxicity
(Experiment IIA in the absence of S9 mix) could not be reproduced, and this finding was considered to bear no biological significance.
In Experiment IIB, test item precipitation was observed at a concentration of 6.0 µg/mL and above in the absence of S9 mix. Clear toxic effects indicated by reduced cell numbers were observed after treatment with 7.0 µg/mL.
Colcemid (25 ng/mL to 10 µg/mL), Mitomycin C (0.03 or 0.1 µg/mL) or CPA (2.5 to 25 µg/mL) were evaluated as positive controls and showed a distinct increase in the percentage of micronucleated cells.
In conclusion, it can be stated that under the experimental conditions reported, the test item Behentrimonium chloride did not induce micronuclei in V79 cells (Chinese hamster cell line) in vitro in the absence and the presence of metabolic activation.
Therefore, Behentrimonium chloride has to be considered as non-mutagenic in this in vitro test system, when tested up to cytotoxic test item concentrations (Experiment I, with metabolic activation, Experiment IIA with and without metabolic activation, and Experiment IlB, without metabolic activation) or the highest scorable concentration (Experiment I, with metabolic activation).
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'. Remarks: V79

Dose selection

Dose selection was performed following the current OECD Guideline for in vitro chromosome aberration studies (OECD Guideline no. 473). The highest concentration chosen for the evaluation of genotoxicity should produce clear toxicity with reduced cell growth, determined by the mean of the cell count prior to cell seeding on slides, by > 60 % and/or the occurrence of precipitation. In case of nontoxicity the maximum concentration should be 5 mg/mL, 5 µL/mL or 10 mM, whichever is the lowest, if formulability in an appropriate solvent is possible.

With respect to the molecular weight of the test item, 5000.0 µg/mL of Behentrimonium chloride (approx. 9.7 mM) were applied as top concentration in Experiment I. Test item concentrations between 9.8 and 5000.0 µg/mL (with and without S9 mix) were chosen for the treatment of the cultures. Due to strong toxic effects indicated by reduced cell numbers at test item concentrations of 9.8 µg/mL and above in the absence and presence of metabolic activation, Experiment I was repeated with test item concentrations between 0.8 and 400.0 µg/mL (with and without S9 mix). With regard to the purity of the test item, the corresponding amount of the active substance in the respective test item concentrations was 78.8 %.

In Experiment I, precipitation of the test item in culture medium was observed with 50.0 µg/mL and above in the absence of S9 mix, and with 25.0 µg/mL and above in the presence of S9 mix. Using reduced cell numbers of about 40 % of control or below as an indicator for toxicity in Experiment I, clear toxic effects were observed after 4 hrs treatment with 12.5 µg/mL and above in the absence of S9 mix, and with 50.0 µg/mL and above in the presence of S9 mix. With respect to the results obtained in Experiment I, 25.0 µg/mL was chosen as top concentration for Experiment IIA in the absence and presence of S9 mix. In a confirmatory experiment, designated Experiment llB, concentrations between 2.5 and 20.0 µg/mL, in the absence of S9 mix, were chosen in order to verify the results obtained in Experiment IIA. Due to strong test item induced cytotoxicity at concentrations of 7.5 µg/mL and above, only two concentrations were scorable for cytogenetic damage. Therefore, this experimental part was repeated with test item concentrations between 0.5 and 15.0 µg/mL.

Summary of results of the micronucleus test with Behentrimonium chloride

Exp.

Preparation

Test item

Cell number

Micronucleated

 

interval

concentration

In %

cells

 

 

in µg/mL

of control

in %

Exposure period 4 hrs without S9 mix

I

24 hrs

Negative control

103

0.55

 

 

Solvent control1

100

2.00

 

 

Positive control2

78.9

5.1S

 

 

Positive control3

80.2

3.25S

 

 

Positive control4

78.1

4.70S

 

 

Positive control5

45.8

17.95S

 

 

3.1

88.9

0.95

 

 

6.3

62.7

1.55

 

 

12.5

11.3

1.15

Exposure period 20 hrs without S9 mix

IIA

24 hrs

Negative control

99.5

1.05

 

 

Solvent control1

100

0.85

 

 

Positive control2

51.8

28.3S

 

 

Positive control3

44.4

28.80S

 

 

Positive control4

72.3

9.45S

 

 

Positive control5

61.4

13.85S

 

 

3.1

97.4

1.65S

 

 

6.3 P

57.5

1.75S

 

 

12.5 P

29.9

2.40S

Exposure period 20 hrs without S9 mix

IIB

24 hrs

Negative control

90.0

0.30

 

 

Solvent control1

100

0.55

 

 

Positive control2

40.6

66.35S

 

 

Positive control3

33.8

50.35S

 

 

Positive control4

64.0

6.85S

 

 

Positive control5

69.8

6.00S

 

 

5.0

66.4

1.05

 

 

6.0 P

47.1

0.70

 

 

7.0 P

37.5

0.05

Exposure period 4 hrs with S9 mix

I

24 hrs

Negative control

158.3

0.90

 

 

Solvent control1

100

1.70

 

 

Positive control6

49.0

8.40S

 

 

Positive control7

41.0

5.15S

 

 

6.3

159.4

1.55

 

 

12.5

113.2

1.55

 

 

25.0 P

57.4

0.85

Exposure period 4 hrs with S9 mix

IIA

48 hrs

Negative control

100.8

0.95

 

 

Solvent control1

100

0.55

 

 

Positive control8

67.7

15.55S

 

 

Positive control9

19.9

44.70S

 

 

3.1

82.2

0.70

 

 

6.3

80.9

1.00

 

 

12.5

42.4

0.60

 

P         precipitation occurred

S        number of micronucleated cells statistically significant higher than corresponding control values

1        deioinised water 10%(v/v)

2         colcemid 7.5µg/mL

3         colcemid 10.0 µg/mL

4         mitomycin C 0.03 µg/mL

5         mitomycin C 0.1 µg/mL

6         cyclophosphamide 10 µg/mL

7         cyclophosphamide 25 µg/mL

8         cyclophosphamide 2.5 µg/mL

9         cyclophosphamide 5.0 µg/mL

Conclusions:
The genotoxicity of the registration substance was investigated according to the guideline OECD 487. Negative result was obtained.
Executive summary:

The registration substance was assessed for its clastogenicity according to the guideline OECD 487.

The registration substance did not induce micronuclei in V79 cells (Chinese hamster cell line) in the absence and the presence of metabolic activation at up to the concentration associated with clear cytoxicity. No significant clastogenicity was found.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Due to the deficiency of the available Ames test (E-coli strain missing; no pre-incubation method as the second experiment) a new study was initiated in May 2020. The result will be available in July 2020.

Justification for classification or non-classification

The genotoxicity of the registration substance was investigated in three in-vitro test system.

Negative results were obtained in Ames test, in mammalian gene mutation test and in micronucleus test. No classification is warranted.