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Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Negative in all tests conducted:
- Ames test with S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2 uvrA (met. act.: with and without) (OECD TG 471, GLP); tested up to cytotoxic concentrations
- Mammalian cell gene mutation assay (HPRT) in V79 cells (met. act.: with and without) (OECD Guideline 476, GLP); tested up to cytotoxic concentrations
- In vitro mammalian chromosome aberration test with V79 cells (met. act.: with and without) (OECD Guideline 473, GLP); tested up to cytotoxic concentrations

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2004-03-10 to 2004-09-29
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Version / remarks:
1997
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
Version / remarks:
(2000)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
in vitro mammalian chromosome aberration test
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
- Type and identity of media: MEM (Minimal Essential Medium; Seromed)
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
Metabolic activation:
with and without
Metabolic activation system:
Phenobarbital/ß-Naphthoflavone induced rat liver S9 (protein concentration 35.7 mg/mL and 28.9 mg/mL)
Test concentrations with justification for top dose:
Evaluated test concentrations:
Experiment I: without S9 mix/ 4 hrs exposure: 25.0, 50.0, 100.0 µg/mL
with S9 mix/ 4 hrs exposure: 37.5, 75.0, 300.0, 600.0 µg/mL
Experiment II: without S9 mix/ 18 hrs exposure: 6.3, 12.5, 25.0 µg/mL
without S9 mix/ 28 hrs exposure: 18.8 µg/mL
with S9 mix/4 hrs exposure:: 18.8, 37.5, 150.0, 300.0 µg/mL
Experiment III: with S9 mix/ 4 hrs exposure: 300.0, 400.0, 500.0 µg/mL
A table containing all exposure concentrations is given under "Any other informations of materials and methods"
Vehicle / solvent:
deionised water
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
ethylmethanesulphonate
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium (MEM with 10 % FCS)

DURATION
- Exposure duration:
Experiment I: with and without S9 mix 4 hrs
Experiment II: without S9 mix 18 and 28 hrs; with S9 mix 4 hrs
Experiment III: with S9 mix 4 hrs

- Expression time (cells in growth medium): 14 hrs for the 4 hrs exposure groups, no for the 18 and 28 hrs groups.
- Fixation time (start of exposure up to fixation or harvest of cells): 18 and 28 hrs respectively

SPINDLE INHIBITOR (cytogenetic assays): Colcemid (0.2 µg/mL culture medium)
STAIN (for cytogenetic assays): Giemsa

NUMBER OF REPLICATIONS: 3 independent experiments, per group two parallel cultures were set up.

NUMBER OF CELLS EVALUATED: at least 100 methaphase plates were scored per culture. Breaks, fragments, deletions, exchanges, and chromosome disintegrations were recorded as structural chromosome aberrations. Gaps were recorded as well, but not included in the calculation of the aberration rates.

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index

OTHER EXAMINATIONS:
- Determination of polyploidy: number of polyploidy cells in 500 metaphase cells per culture
was determined (% polyploid metaphases)
Evaluation criteria:
A test item is classified as non-clastogenic if:
- the number of induced structural chromosome aberrations in all evaluated dose groups is in the range of laboratories historical control data
(0.0 – 4.0 % aberrant cells, exclusive gaps).
and/or
- no significant increase of the number of structural chromosome aberrations is observed.
A test item is classified as clastogenic if:
- the number of induced structural chromosome aberrations is not in the range of our historical control data
(0.0 – 4.0 % aberrant cells, exclusive gaps).
and
- either a concentration-related or a significant increase of the number of structural chromosome aberrations is observed.
Although the inclusion of the structural chromosome aberrations is the purpose of this study, it is important to include the polyploids and
endoreduplications.
A test item can be classified as aneugenic if:
- the number of induced numerical aberrations is not in the range of laboratories historical control data (0.0 – 8.5 % polyploid cells)
Statistics:
Statistical significance was confirmed by means of the Fisher´s exact test (p < 0.05).
Species / strain:
Chinese hamster lung fibroblasts (V79)
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:
TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: water was chosen as solvent, test substance was suspended in deionised water immediately before treatment.
- Precipitation: was observed, corresponding concentrations were identified in the exposure concentration table under
"any other information on materials and methods".

RANGE-FINDING/SCREENING STUDIES:
In a range finding pre-test on toxicity cell numbers 24 hrs after start of treatment were scored as an indicator for cytotoxicity. Concentrations between 39.1 and 5000µg/mL were applied. Clear toxic effects were observed after 4 hrs treatment with 156.3 µg/mL and above in the absence of S9 mix and with 625µg/mL and above in the presence of S9 mix. In addition, 24 hrs continuous treatment with 78.1 µg/mL and above in the absence of S9 mix induced strong toxic effects.
In the pre-experiment, precipitation of the test item in culture medium was observed after treatment with 39.1 µg/mL and above in the absence and the presence of S9 mix. No relevant influence of the test item on the pH value or osmolarity was observed (solvent control 288 mOsm, pH 7.2 versus 323 mOsm and pH 7.2 at 5000 µg/mL).


ADDITIONAL INFORMATION ON CYTOTOXICITY:
In this study, cytotoxicity was observed in all cytogenetic experiments. In the presence of S9 mix the cell numbers were strongly reduced after treatment with 600 µg/mL (32 % of control) at the 18 hrs preparation interval and with 500µg/mL (43 % if control) at the 28 hrs preparation interval.

MAIN TEST:
In experiment I and II, in the absence and the presence of S9 mix, no biological relevant increase in the number of cells carrying structural chromosome aberrations was observed. The aberration rates of the cells after treatment with the test item (0.0 - 4.8 % abberant cells, exclusive gaps) were close to the range of the solvent control values (0.0 – 1.5 % aberrant cells, exclusive gaps) and close to the range of our historical control data (0.0 – 4.0 % aberrant cells, exclusive gaps).
In the presence of S9 mix two significant (p < 0.05) increases were observed, in experiment I at preparation interval 18 hrs after treatment with 37.5 µg/mL (4 % aberrant cells, exclusive gaps), and in experiment II at preparation interval 28 hrs with 300 µg/mL (4.8 % aberrant cells, exclusive gaps). To prove this slightly increase value exceeding the upper border of our laboratory´s historical control data range an increased sample of 200 metaphase plates per culture was evaluated for cytogenetic damage.
However, the borderline value was confirmed. In addition, a dose related increase in the number of cells carrying structural chromosome aberrations (0.5 %, 2.5 % and 4.8 %) was observed after 4 hrs treatment at 28 hrs preparation interval in the presence of metabolic activation at the upper concentrations evaluated (37.5, 150.0 and 300.0 µg/mL) respectively.

A confirmatory experiment, designated experiment III, was performed to proof these observations. In the repeated experiment in the presence of S9 mix after 4 hrs treatment at prolonged 28 hrs preparation interval no biologically relevant increase in the number of cells carrying structural chromosome aberrations was observed. The aberration rates of the cells after treatment with the test item (1.0 – 3.0 % aberrant cells exclusive gaps) were close to the value of the solvent control (1.5 % aberrant cells, exclusive gaps) and within the range of our historical control data, (0.0 – 4.0 % aberrant cells, exclusive gaps).

Beside the aberration rates were dose related increased (1.0 %, 1.5 % and 3.0 %) in the concentration range evaluated (300 to 500 mg/mL), but the values were clearly within our laboratory´s control data range (0.0 – 4.0 % aberrant cells, exclusive gaps). Finally, the observations of experiment II in the presence of S9 mix (statistical significance, dose dependency, and borderline value) were not confirmed in experiment III and therefore they have to be regarded as biologically irrelevant.

In all experiments, no biologically relevant increase in the rate of polyploid metaphases was found after treatment with the test item (0.9 – 2.5 %) as compared to the rates of the solvent controls (1.9 – 3.0 %).

EMS and CPA were used as positive controls and showed distinct increase in cells with structural chromosome aberrations.
Conclusions:
It can be stated that under the experimental conditions reported, the test item did not induce structural chromosome aberrations as determined by the chromosome aberration test in V79 cells (Chinese hamster cell line) in vitro. The test item is considered to be non-clastogenic in this chromosome aberration test with and without S9 mix when tested up to cytotoxic test item concentrations.
Executive summary:

In a mammalian cell cytogenetics assay according to OECD Guideline 473, 1997 and EU Method B.10, 2000, V79 cell cultures were exposed to partially unsaturated TEA-Esterquat at concentration ranges of 3.1 – 200 µg/mL without metabolic activation and 4.7 – 600 µg/mL in the presence of mammalian metabolic activation. The following concentrations were selected for microscopic analysis:

Experiment I:           without S9 mix/ 4 hrs exposure:       25.0, 50.0, 100.0 µg/mL

                                  with S9 mix/ 4 hrs exposure:            37.5, 75.0, 300.0, 600.0 µg/mL

Experiment II:          without S9 mix/ 18 hrs exposure:     6.3, 12.5, 25.0 µg/mL

                                  without S9 mix/ 28 hrs exposure:     18.8 µg/mL

                                 with S9 mix/4 hrs exposure::            18.8, 37.5, 150.0, 300.0 µg/mL

Experiment III:        with S9 mix/ 4 hrs exposure:            300.0, 400.0, 500.0 µg/mL

In experiment I and II, in the absence and the presence of S9 mix, no biological relevant increase in the number of cells carrying structural chromosome aberrations was observed. The aberration rates of the cells after treatment with the test item (0.0 - 4.8 % abberant cells, exclusive gaps) were close to the range of the solvent control values (0.0 – 1.5 % aberrant cells, exclusive gaps) and close to the range of our historical control data (0.0 – 4.0 % aberrant cells, exclusive gaps).

 

In the presence of S9 mix two significant (p < 0.05) increases were observed, in experiment I at preparation interval 18 hrs after treatment with 37.5 µg/mL (4 % aberrant cells, exclusive gaps), and in experiment II at preparation interval 28 hrs with 300 µg/mL (4.8 % aberrant cells, exclusive gaps). To prove this slightly increase value exceeding the upper border of our laboratory´s historical control data range an increased sample of 200 metaphase plates per culture was evaluated for cytogenetic damage.

However, the borderline value was confirmed. In addition, a dose related increase in the number of cells carrying structural chromosome aberrations (0.5 %, 2.5 % and 4.8 %) was observed after 4 hrs treatment at 28 hrs preparation interval in the presence of metabolic activation at the upper concentrations evaluated (37.5, 150.0 and 300.0 µg/mL) respectively.

A confirmatory experiment III was performed to proof these observations. In the repeated experiment in the presence of S9 mix after 4 hrs treatment at prolonged 28 hrs preparation interval no biologically relevant increase in the number of cells carrying structural chromosome aberrations was observed. The aberration rates of the cells after treatment with the test item (1.0 – 3.0 % aberrant cells exclusive gaps) were close to the value of the solvent control (1.5 % aberrant cells, exclusive gaps) and within the range of our historical control data, (0.0 – 4.0 % aberrant cells, exclusive gaps).

Beside the aberration rates were dose related increased (1.0 %, 1.5 % and 3.0 %) in the concentration range evaluated (300 to 500 mg/mL), but the values were clearly within our laboratory´s control data range (0.0 – 4.0 % aberrant cells, exclusive gaps). Finally, the observations of experiment II in the presence of S9 mix (statistical significance, dose dependency, and borderline value) were not confirmed in experiment III and therefore they have to be regarded as biologically irrelevant.

In all experiments, no biologically relevant increase in the rate of polyploid metaphases was found after treatment with the test item (0.9 – 2.5 %) as compared to the rates of the solvent controls (1.9 – 3.0 %).

Positive controls induced the appropriate response. EMS and CPA were used as positive controls and showed distinct increase in cells with structural chromosome aberrations. 

In conclusion it can be stated that under the experimental conditions reported, the test item did not induce structural chromosome aberrations as determined by the chromosome aberration test in V79 cells (Chinese hamster cell line) in vitro. The test item is considered to be non-clastogenic in this chromosome aberration test with and without S9 mix when tested up to cytotoxic test item concentrations.

There was no evidence of chromosome aberration induced over background.

 

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2009-07-07 to 2009-09-11
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
Qualifier:
according to guideline
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Principles of method if other than guideline:
first experiment 4 hours treatment with and without metabolic activation
second experiment 24 hours treatment without metabolic activation and 4 hours treatment with metabolic activation
GLP compliance:
yes (incl. QA statement)
Type of assay:
mammalian cell gene mutation assay
Target gene:
HPRT
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
- Type and identity of media: MEM (Seromed)
- 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:
Rat liver S9
Test concentrations with justification for top dose:
Experiment I:
without S9 mix: (0.625); 1.25; 2.5; 5.0; 10.0; 20.0; (200); (2000) µg/mL
with S9 mix: 23.4; 46.9; 93.8; 187.5; 375; (1500) µg/mL
Experiment II:
without S9 mix: 2.8; 5.6; 11.3; 22.5; 45; (90); (135); 180 µg/mL
with S9 mix: 12.5; 25; 50; (100); 150; 200 µg/mL

At the concentrations in brackets, the cultures were not analysed. In the first main experiment the cultures at the lowest concentration without metabolic activation (0.625 µg/mL) were not continued since a minimum of only four analysable concentrations is required by the guidelines. The cultures at the concentrations of 200 and 2000 µg/mL without metabolic activation and 1500 µg/mL with metabolic activation were not continued due to exceedingly severe toxic effects. In experiment II the cultures at 90 and 135 µg/mL without metabolic activation and 100 µg/mL with metabolic activation were not continued to avoid analysis of too many precipitating concentrations.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: THF
- Justification for choice of solvent/vehicle: solubility properties
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
with metabolic activation
True negative controls:
no
Positive controls:
yes
Remarks:
with metabolic activation
Positive control substance:
7,12-dimethylbenzanthracene
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
without metabolic activation
True negative controls:
no
Positive controls:
yes
Remarks:
without metabolic activation
Positive control substance:
ethylmethanesulphonate
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: experiment I: 4 hours with and without metabolic activation;
experiment II: 24 hours without and 4 hours with metabolic activation
- Expression time (cells in growth medium): 72 hours
- Selection time (if incubation with a selection agent): 8 days

SELECTION AGENT (mutation assays): Thioguanine

NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED: >1.5x10exp. 6

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency


Evaluation criteria:
A test item producing neither a concentration-related increase of the mutant frequency nor a reproducible positive response at any of the test points is considered to be non-mutagenic in this system.
A mutagenic response is described as follows:
The test item is classified as mutagenic if it induces reproducibly with one of the concentrations a mutation frequency that is three times higher than the spontaneous mutation frequency 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.
In a case by case evaluation this decision depends on the level of the corresponding solvent control data.
Statistics:
Linear regression analysis (least squares) .
Species / strain:
Chinese hamster lung fibroblasts (V79)
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:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: not effected
- Effects of osmolality: not increased
- Precipitation:
Main experiments:
Precipitation of the test item visible to the unaided eye was noted at 20 µg/mL and above in the first experiment without metabolic activation and at 93.8 µg/mL and above in the first experiment with metabolic activation. In the second experiment precipitation as described above occurred at 45.0 µg/mL and above without metabolic activation and at 50.0 µg/mL and above with metabolic activation.


RANGE-FINDING/SCREENING STUDIES:
The highest concentration used in the pre-test was 3000 µg/mL limited by the solubility of the test item in THF. Test item concentrations between 23.4 µg/mL and 3000 µg/mL were used to evaluate toxicity in the presence (4 h treatment) and absence (4 h and 24 h treatment) of metabolic activation. Relevant cytotoxic effects indicated by a relative clon-ing efficiency below 50 % were noted at 23.4 µg/mL and above without metabolic activa-tion and 375 µg/mL and above with metabolic activation following 4 h treatment. Following 24 hours treatment without metabolic activation cytotoxic effects as described above were noted at 187.5 µg/mL and above.

The test medium of the pre-experiment was checked for precipitation at the end of each treatment period (4 or 24 hours) just prior to removal of the test item. Precipitation was noted at 187.5 µg/mL and above after 4 and 24 hours treatment without metabolic activa-tion and at 375 µg/mL and above after 4 hours treatment with metabolic activation.

There was no relevant shift of pH or osmolarity of the medium even at the maximum con-centration of the test item.
Based on the results of the pre-experiment, the individual concentrations of the main experiments were selected. A series of concentrations spaced by a factor of 2 was placed into the lower range of the first experiment up into the precipitating concentration range. An additional larger step was used to include the maximum possible concentration level limited by the solubility properties of the test item. The toxic gradient did not follow a smooth and dose dependent course in the pre-experiment following 4h treatment without metabolic activation. Therefore, the experimental part 4h treatment without metabolic activation of the first main experiment was started with 8 concentrations in order to obtain at least 4 analysable concentrations. In experiment II a narrower spacing was used at high concentrations to cover the precipitating or toxic range more closely.


COMPARISON WITH HISTORICAL CONTROL DATA: Controls within the range of historical data


ADDITIONAL INFORMATION ON CYTOTOXICITY:
Relevant cytotoxic effects as indicated by a relative cloning efficiency I of less than 50 % in both parallel cultures occurred at 10 µg/mL and above in experiment I without metabolic activation and at 187.5 µg/mL and above with metabolic activation. In the second experiment toxic effects as described above occurred at 180 µg/mL without metabolic activation. The cell density at the first subcultivation after treatment showed less severe cytotoxicity in the precipitating concentration range especially without metabolic activation. Even though the relative cloning efficiency 1 was zero or close to zero at 20 µg/mL in the first experiment and at 180 µg/mL in the second experiment without metabolic activation the corresponding values of the cell density remained within the acceptable range. Such a large difference in the onset of cytotoxicity is usually based on binding of the test item to proteins or lipids of cell membranes, serum albumin, or S9. The low-density cell cultures used to determine the cloning efficiency 1 are in such cases more sensitive towards toxic effects than the mass cell cultures used to determine mutagenicity. In the experimental parts with metabolic activation proteins and lipids of the S9 level out the influence of different cell densities. Protein binding effects are also the reason that higher concentrations are tolerated in the second experiment without metabolic activation in spite of the 24h treatment period. However, 10% foetal calf serum have to be added during 24h treatment compared to no foetal calf serum during 4h treatment. Protein binding test items are therefore, better tolerated during long term exposure.
Remarks on result:
mutagenic potential (based on QSAR/QSPR prediction)
Summary Table
    relative cell density mutant   relative cell density mutant  
  conc. S9 cloning % colonies/ induction cloning % colonies/ induction
  µg/mL mix efficiency 1 of control 106cells factor efficiency 1 of control 106cells factor
    % %   % %  
column 1 2 3 4 5 6 7 8 9 10
Experiment I / 4h treatment   culture I          culture II
Solvent control  - 100.0 100.0  6.1 1.0 100.0 100.0  12.3 1.0
Pos. contr. with EMS 150.0 - 116.7  86.4  82.0 13.5  93.8 121.6  69.5 5.6
Test item  0.6 -  97.3 culture was not continued#  81.6 culture was not continued#
Test item  1.3 - 104.3  69.9  9.8 1.6  63.4 103.5  5.1 0.4
Test item  2.5 -  99.0  62.6  10.2 1.7  72.8  78.2  5.0 0.4
Test item  5.0 -  57.5  58.6  13.3 2.2  53.1 106.9  12.9 1.0
Test item  10.0 -  2.3  39.2  15.9 2.6  19.3  95.4  7.8 0.6
Test item 20.0 (p) -  0.0  13.9  25.1 4.2  0.0  24.2  5.4 0.4
Test item 200.0 (p) -  0.0 culture was not continued##  0.0 culture was not continued##
Test item 2000.0 (p) -  0.0 culture was not continued##  0.0 culture was not continued##
       
Solvent control + 100.0 100.0  14.2 1.0 100.0 100.0  12.3 1.0
Pos. contr. with DMBA  1.1 +  58.3  73.5 1336.4 94.0  69.9  84.8 840.9 68.5
Test item  23.4 +  97.3  97.9  19.0 1.3  95.3 119.2  19.7 1.6
Test item  46.9 +  99.3 106.7  19.7 1.4 111.0  79.8  10.1 0.8
Test item 93.8 (p) +  51.3  66.8  27.2 1.9  73.3 108.2  14.4 1.2
Test item 187.5 (p) +  27.9  26.6  31.4 2.2  26.4  46.3  12.3 1.0
Test item 375.0 (p) +  2.8  5.1  25.5 1.8  0.5  2.9  9.0 0.7
Test item 1500.0 (p) +  0.0 culture was not continued##  0.2 culture was not continued##
Experiment II / 24h treatment culture I          culture II
Solvent control  - 100.0 100.0  18.5 1.0 100.0 100.0  15.6  1.0
Pos. contr. with EMS 75.0 - 111.9 127.4 174.3 9.4  84.3  92.9 113.6  7.3
Test item 2.8 -  94.6 114.3  16.7 0.9  79.8  80.3  21.1  1.4
Test item 5.6 - 101.2 116.9  27.8 1.5  78.8  86.6  22.9  1.5
Test item 11.3 -  76.3  77.3  9.9 0.5  84.8 107.3  13.5  0.9
Test item 22.5 -  89.4 123.2  12.5 0.7  52.0 119.0  17.4  1.1
Test item 45.0 (p) -  50.4 107.2  14.6 0.8  56.9 112.8  10.1  0.6
Test item 90.0 (p) -  25.4 culture was not continued###  21.7 culture was not continued###
Test item 135.0 (p) -  9.4 culture was not continued###  4.1 culture was not continued###
Test item 180.0 (p) -  0.2  45.0  21.2 1.1  0.0  62.4  20.6  1.3
Experiment II / 4h treatment      
Solvent control  + 100.0 100.0  11.7 1.0 100.0 100.0  9.2  1.0
Pos. contr. with DMBA 1.1 +  26.2  49.1 1004.6 85.7  23.7  37.0 774.4  84.6
Test item 12.5 +  96.4 145.6  29.1 2.5  99.2  87.7  12.2  1.3
Test item 25.0 + 101.8  85.5  25.6 2.2 107.6  88.2  16.1  1.8
Test item 50.0 (p) + 103.9  90.2  22.3 1.9  97.0  93.4  8.8  1.0
Test item 100.0 (p) +  92.0 culture was not continued### 100.5 culture was not continued###
Test item 150.0 (p) +  69.7 112.5  41.9 3.6  72.3 118.7  12.8  1.4
Test item 200.0 (p) +  72.2  67.3  22.8 1.9  68.3  73.9  16.0  1.8

#    culture was not continued since a minimum of only four analysable concentrations is required
##
   culture was not continued due to exceedingly severe toxic effects
###
 culture was not continued to avoid analysis of too many precipitating concentrations
p
    precipitation visible to the unaided eye

Conclusions:
In conclusion it can be stated that under the experimental conditions reported the test item did not induce gene mutations at the HPRT locus in V79 cells. Therefore, partially unsaturated TEA-Esterquat is considered to be non-mutagenic in this HPRT assay.
Executive summary:

In a mammalian cell gene mutation assay at the HPRT locus according to OECD guideline 476, chinese hamster lung fibroblasts (V79) cells cultured in vitro were exposed to partially unsaturated TEA-Esterquat (100 % a.i. of UVCB description) at the following concentrations in the presence and absence of mammalian metabolic activation (rat liver S9). 

 

Experiment I:

without S9 mix:         0.625;1.25; 2.5; 5.0; 10.0; 20.0; 200; 2000 µg/mL

with S9 mix:              23.4; 46.9; 93.8; 187.5; 375; 1500 µg/mL

Experiment II:

without S9 mix:         2.8; 5.6; 11.3; 22.5; 45; 90; 135;180µg/mL

with S9 mix:              12.5; 25; 50; 100;150; 200µg/mL

 

Concentrations in bold were selected for analysis. THF (Tetrahydrofuran) was selected as solvent. The concentration range of the main experiments was limited by the solubility properties of the test item in aqueous medium and by cytotoxic effects. The assay was performed in two independent experiments, using two parallel cultures each. A treatment period of 4 hours was used for all experiments, with exception of the second experiment with metabolic activation, were a 24 hour treatment period was selected for the cultures.

No substantial and reproducible dose dependent increase of the mutation frequency was observed in both main experiments. Appropriate reference mutagens, used as positive controls, induced a distinct increase in mutant colonies and, thus, showed the sensitivity of the test system and the activity of the metabolic activation system.

The results of this HPRT assay indicate, that partially unsaturated TEA-Esterquat did not cause a positive response in the non-activated and S9-activated systems and was concluded to be negative under the conditions of this study.

This study is classified asacceptable.  This study satisfies the requirement for Test Guideline OECD 476 for in-vitro mutagenicity (mammalian forward gene mutation) data.

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2003-04-15 to 2003-06-20
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
21. July 1997
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
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:
other: Histidin auxotroph
Species / strain / cell type:
E. coli WP2 uvr A pKM 101
Metabolic activation:
with and without
Metabolic activation system:
S9-mix (fraction of Spraque Dawley rat liver incuded with Aroclor 1254). Obtained by Molecular Toxicology, Inc., 157 Industrial Park Dr. Boone, NC 28607, USA.
Test concentrations with justification for top dose:
First plate incorporation test serving as range finding test: with metabolic activation (10 % S9 liver homogenate): 50, 160, 500, 1600, 5000 µg/plate
without metabolic activation: 50, 160, 500, 1600, 5000 µg/plate
Second plate incorporation test: with metabolic activation (10 % S9 rat liver homogenate): 1.6, 5, 16, 50, 160, 500 µg/plate (TA 1537), and 5, 16, 50, 160, 500, 1600 µg/plate (TA 100, TA 1535, TA 98, WP2uvrA),
without metabolic activation: 0.5, 1.6, 5, 16, 50, 160 µg/plate (TA 100, TA 1537), 1.6, 5, 16, 50, 160, 500 µg/plate (TA 1535, TA 98) and 16, 50, 160, 500, 1600, 5000 µg/plate (WP2uvrA)
Third plate incorporation test: with metabolic activation (30 % S9 rat liver homogenate): 1.6, 5, 16, 50, 160, 500 µg/plate (TA 1537), and 5, 16, 50, 160, 500, 1600 µg/plate (TA 100, TA 1535, TA 98, WP2uvrA)
without metabolic activation: 0.5, 1.6, 5, 16, 50, 160 µg/plate (TA 100, TA 1537), and 1.6, 5, 16, 50, 160, 500 µg/plate (TA 1535, TA 98), and 5, 16, 50, 160, 500, 1600 µg/plate (WP2uvrA)
Repeat of third plate incorporation test: with metabolic activation (30 % S9 rat liver homogenat): 50, 160, 500, 1600, 5000 µg/plate (TA 100, WP2uvrA)
without metabolic activation: 50, 160, 500, 1600, 5000 µg/plate (WP2uvrA)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: ethanol
- Justification for choice of solvent/vehicle: Other common vehicles (deionizede water, DMSO) were not appropriate due to insolubility of the test item. Stability and homogeneity (to be reached by stirring) of the test item in the vehicle is guaranteed by the sponsor (Report Ulrike Reploeg, dated 31-Mar-2003) for 4 hours.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
2-nitrofluorene
sodium azide
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene for all strains; with metabolic activation
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation) with 10 and 30 % S9-mix in the third assay.

DURATION
- Preincubation period: not done
- Exposure duration: 48 h

NUMBER OF PLATES EVALUATED: three per dose

NUMBER OF REPLICATIONS: Three independent experiments were performed

DETERMINATION OF CYTOTOXICITY
- Method: toxicity to bacteria was assessed by thinning of the bacterial lawns and/or reduction in the number of colonies.



Evaluation criteria:
Criteria for a valid assay
The assay is considered valid if the following criteria are met:
- the solvent control data are within the laboratory's normal control range for the spontaneous mutant frequency
- the positive controls induce increases in the mutation frequency which are significant and within the laboratory's normal range
Criteria for a positive response
A test compound is classified as mutagenic if it has either of the following effects:
a) it 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) it 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 compound at complete bacterial background lawn. If the test substance does not achieve either of the above criteria, it is considered to show no evidence of mutagenic activity in this system.
Statistics:
No
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A pKM 101
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Solubility: The test item was dissolved in ethanol and a stock solution of 50 mg/ml was prepared for the highest concentration, which provided a final concentration of 5000 µg/plate. Further dilutions of 1600, 500, 160, 50, 16, 5, 1.6 and 0.5 µg/plate were used in the different experiments
- Precipitation: visible precipitation of Praepagen TQ on the plates was observed at 1600 µg/plate and above.

RANGE-FINDING/SCREENING STUDIES:
In the first plate incorporation test with 10 % S9-mix bacteria were treated with doses of 50, 160, 500, 1600 and 5000 µg/plate. Howeever the test item was toxic to all bacteria strains so that evaluation according to the guidelines (5 doses) was not possible. This test served therefore only as range finding test.

COMPARISON WITH HISTORICAL CONTROL DATA:
Values were within the laboratory´s historical control range. Only the number of revertant colonies of the solvent controls in strains TA 1537 and WP2 were slightly out of the historical control data range with ethanol in some experiments. However, the existing control data pool with ethanol is limited as this solvent is used rarely. The historical control data pool for DMSO indicating that all values are in normal range and do not influence the validity of the study.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
Doses in the second plate incorporation test with 10 % S9-mix based on the toxicity to bacteria observed in the first test. In this test toxicity to bacteria in form of incomplete bacterial lawn was observed without metabolic activation at dose levels of 160 µg/plate and above with the strains TA 100, TA 1535, TA 1537 and TA 98 and at dose levels of 500 µg/plate and above with the strain WP2uvrA. In the presence of metabolic activation the test compound proved to be toxic to bacterial strains TA 100, TA 1537 and TA 98 at concentrations of 500 µg/plate and above and at a concentration of 1600 µg/plate to bacterial strains TA 1535 and WP2uvrA.
The top dose level for the third plate incorporation test (30 % S9-mix) was selected on the basis of toxicity to bacteria observed in previous tests. In the third plate incorporation test (with 30% rat liver homogenate) toxicity was observed without metabolic activation at dose levels of 160 µg/plate andabove with the tester strains TA 100, TA 1535, TA 1537 and TA 98. In the presence of metabolic activation the test compound proved to be toxic to the tester strain TA 1537 at a concentration of 500 µg/plate and at a concentration of 1600 µg/plate to bacterial strains TA 1535 and TA 98.

30 % S9-mix reduced toxicity to E. coli WP2 and TA 100 in the third test with the result that the maximum dose showed no toxicity to bacteria as required by the guideline. Therefore, the test with 30 % 89 had to be repeated with this strains (repeat plate incorporation test) using doses up to 5000 µg/plate, In the repeat test with 30 % rat liver homogenate toxicity to bacteria in form of incomplete bacteria lawn was observed with strain TA 100 (+ S9-mix) at a dose level of 1600 µg/plate and with E. coli WP2 at a dose level of 1600 µg/plate and above (-S9-mix).
Slight differences in toxicity to bacteria in the experiments with 30 % S9-mix (WP2 -S9-mix) are probably caused by biological variability.

Sterility control:

The plates for sterility control of test item and S9-mix showed no growth.

Conclusions:
There was no evidence of induced mutant colonies over background in any of the tester strains in the presence or absence of mammalian metabolic activation in this study.
Executive summary:

In a reverse gene mutation assay in bacteria according to OECD guideline 471, 1997, strains TA1535, TA 1537, TA 98 and TA 100 of S. typhimurium and E. coli WP2 were exposed to partially unsaturated TEA-Esterquat. Three independent experiments were performed up to the limit concentration of 5000 µg/plate with and without mammalian metabolic activation (rat liver S9-mix 10 and 30 %).

Significant bacteriotoxic effects of varying severeness were observed, depending on the test strain and the presence of metabolic activation. Generally, bacteriotoxicity was less pronounced in the presence of metabolic activation, especially at concentrations of 30 % S9-mix. Precipitation was observed at 1600 µg/plate and above. There was no evidence of induced mutant colonies over background in any of the tester strains in the presence or absence of mammalian metabolic activation.

The positive controls induced the appropriate responses in the corresponding strains and activity of metabolizing system was confirmed.

There was no evidence of induced mutant colonies over background.

 

 

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

Genetic toxicity in vivo

Description of key information

- in vivo bone marrow micronucleus assay in CFW 1 mouse (OECD guideline 474, GLP); tested up to 5000 mg/kg bw, oral: gavage; no toxic effects (slight reduction in the ratio of polychromatic to normochromatic erythrocytes was determined in female mice 24 and 48 h after administration, indicating possibly a weak toxic effect to the bone marrow)

Link to relevant study records
Reference
Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1989-10-17 to 1990-01-30
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
May 26, 1983
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
Sept. 19, 1984
GLP compliance:
yes
Type of assay:
micronucleus assay
Species:
mouse
Strain:
other: outbred albino mouse, strain CFW 1
Sex:
male/female
Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: bidest. water

Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
- The test item was dispersed in preheated warm bidest. water (50 °C and applicated at a temperature of 35 °C) at an application volume of 20 ml/kg. The test item concentrations were prepared immediately before use. Homogencity was maintained during application using a magnetic stirrer.

The test substance and negative control was administered by oral intubation. the positive control substance was administrated intraperitoneal.

Duration of treatment / exposure:
The animals received the test item once.
Sampling of the bone marrow was carried out on animals 24, 48 and 72 h after treatment.
Frequency of treatment:
single exposure
Post exposure period:
The animals were sacrificed 24, 48 and 72 hours after treatment.
Dose / conc.:
5 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
Dose range finding study: 2 males, 2 females per group (3 groups)
Main study: 6 males, 6 females per group (5 groups)
Control animals:
yes, concurrent vehicle
Positive control(s):
Cyclophosphamide (CP, CAS-No. 50-18-0, Endoxan, Asta Werke, Germany)
- Route of administration: intraperitoneal
- Doses / concentrations: 10 mg/kg dissolved in bidest. water
- Application volume: 10 ml/kg
- Sampling time: 24 h
Tissues and cell types examined:
bone marrow cells
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:
1. Dose range finding study:
LD50, rat determined to be > 5000 mg/kg bw. therefore the maximum tolerated doses: 3000, 4000, and 5000 mg/kg bw were chosen.
2. Main study:
5000 mg/kg bw, chosen because of the results of dose range finding study (it is the maximum tolerated dose for the test).

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):
The animals were sacrificed 24, 48 or 72 hours after treatment under saturated atmosphere of carbondioxide. For each animal both femurs were removed and freed of blood and muscles. The proximal part of the femur were opend and extracted with aid of a syringe in fetal calf serum. The suspension was collected in a siliconised centrifuge tube, filled with 5 ml fetal calf serum. The cell suspensions were centrifuged at 1000 minE-1 (approx 100 g) for 5 minutes, the supernatant was removed with a Pasteur pipette. A drop of serum was left on the cell pellet. The cells of the sediment were carefully mixed.

DETAILS OF SLIDE PREPARATION:
A drop of the cell suspension was placed on a clean and marked slide, previously degreased with alcohol, and immediately spread on the slide. Three slides were prepared per animal.
The slides were air-dried at least overnight and then stained with Giemsa according to modification of Gollapudi and Kamra (Mutation Research 1979, 64, p. 45-46). The slides were fixed in 100 % methanol for 5 minutes, then rinsed twice in bidistilled water and thereafter stained in Giemsa solution (1 vol Giemsa Solution, Merck Art. No. 9204 plus 1 vol glycerol, dissolved 1:6 in bidistilled water).
After air-drying the back side was cleaned, if necessary, with ethanol and then dipped for 3 minutes in xylol.

METHOD OF ANALYSIS:
From the three slides prepared per animal, one slide was chosen and randomly coded. The slides of 5 male and 5 female animals per treatment group were scored microscopically at a mignification of 1000. The number of micronucleated cells was counted in 1000 polychromatic erythrocytes per animal. The ratio of polychromatic to normochromatic erythrocytes was determined by counting and differentiating the first 1000 erythrocytes at the same time.
Averages and standard deviations were calculated after deconding the complete scoring results.
Evaluation criteria:
The micronucleus test is considered acceptable if it meets the following criteria:
- the positive control substance induced a statistically significant increase in the frequency of micronucleated polychromatic erythrocytes.
- the incidence of micronuclei should reasonably fall within the historical control data range of the laboratory.

Test substance is considered positive:
if it induced a biologically as well as a statistically significant increase (p < 0.05) in the frequency of micronuclei at any dose or at any sampling either in the male or in the female groups.
Test substance is considered negative:
if none of the tested doses or sampling times showed a statistically significant (p < 0.05) increase in the incidence of micronuclei, neither in male nor in female groups.
Statistics:
Statistical analysis of data was performed by calculating the statistical significance versus negative controls with the aid of the tables of Kastenbaum and Bowman (Mutation Research, 1970, 9, p. 527-549).
Sex:
male/female
Genotoxicity:
negative
Toxicity:
no effects
Remarks:
A slight reduction in the ration of polychromatic to normochromatic erythrocytes was determined in female mice 24 and 48 h after administration, indicating possibly a weak toxic effect to the bone marrow
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Dose range: 3000, 4000, 5000 mg/kg bw
- Solubility: in bidest. water
- Application volume: 20 ml/kg bw
- No animal up to the highest dose died within the first three days or showed signs of severe morbidity. Slight piloerection was observed at all animals and all doses up to 24 hours after administration.
- Based on the results of this range-finding study 5000 mg/kg bw was selected as appropriate dose for the test.

RESULTS OF DEFINITIVE STUDY
- 5000 mg/kg bw was tested as the maximum dose in the main experiment at 24, 48 and 72 h. The volume administered was 20 ml/kg bw.
- Toxicity: No mortality was was noted after administration of the test item in any of the animals evaluated. A slight reduction in the ratio of polychromatic to normochromatic erythrocytes was determined in female mice 24 and 48 h after administration, indicating possibly a weak toxic effect to the bone marrow.
- Signs of clinical examination: slight piloerection in all animals including positive and negative control.

RESULTS OF NEGATIVE CONTROL GROUP
- no mortality within 24 h
- was in the range of historical control data

RESULTS OF POSITIV CONTROL GROUP
- No mortality within 24 h
- Cyclophoshamide induced a statistically significant increase in the number of micronucleated cells in both sexes.
Conclusions:
It can be stated that under the experimental conditions reported, the test item “partially unsaturated TEA-Esterquat” did not induce structural and/or numerical chromosomal damage in the immature erythrocytes of the mouse.
Therefore, the test item “partially unsaturated TEA-Esterquat” is considered to be negative in the Mammalian Erythrocyte Micronucleus Test.
Executive summary:

In a mouse bone marrow micronucleus assay according to OECD guideline No. 474, 1983, 6 male and 6 female albino mice (CFW1) per group were treated by oral intubation with partially unsaturated TEA-Esterquat at a dose of 5000 mg/kg bw. Bone marrow cells were harvested at 24, 48 and 72 hours post-treatment.

There were no signs of toxicity as indicated by an enhanced mortality rate. A slight reduction in the ratio of polychromatic to normochromatic erythrocytes were determined in female mice 24 and 48 h after administration, indicating possibly a weak toxic effect to the bone marrow.

Partially unsaturated TEA-Esterquat was tested at an adequate dose, based on the results of the range-finding test. The positive control induced the appropriate response.

There was no significant increase in the frequency of micronucleated polychromatic erythrocytes in bone marrow after any treatment time.

This study is classified as acceptable. This study satisfies the requirements of Test Guideline OECD 474 for in-vivo cytogenetic mutagenicity data.

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

Additional information

Reliable and relevant data are available for partially unsaturated TEA-Esterquat. The full set of in vitro tests required by REACH Regulation Annexes VII and VIII is covered with the studies. There was no evidence of mutagenic or genotoxic intrinsic properties in any of the performed studies. Additional data from an in vivo mouse micronucleus Test are available, likewise showing no evidence to cause any chromosomal damage in the bone marrow of mice.


 


In vitro data


A reverse bacterial gene mutation assay (Ames-Test, plate incorporation assay) according to OECD Guideline 471(1997) with partially unsaturated TEA-Esterquat was negative up to the limit concentration of 5000 µg/plate with and without mammalian metabolic activation (rat liver S9-mix 10 and 30 %) in S. typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2 uvrA (pKM101).


Significant bacteriotoxic effects of varying severity were observed, depending on the test strain and the presence of metabolic activation. Generally, bacteriotoxicity was less pronounced in the presence of metabolic activation, especially at concentrations of 30 % S9-mix. Precipitation was observed at 1600 µg/plate and above. The positive controls induced the appropriate responses in the corresponding strains. There was no evidence of induced mutant colonies over background in any of the tester strains in the presence or absence of mammalian metabolic activation.


 


In a mammalian cell gene mutation assay (HPRT locus) according to OECD guideline 476, Chinese hamster lung fibroblasts (V79) cells cultured in vitro were exposed to partially unsaturated TEA-Esterquat (100 % a.i. of UVCB description) at concentrations up to 200 µg/mL in the absence and up to 1500 µg/mL in the presence of mammalian metabolic activation (rat liver S9). 


The concentration range of the main experiments was limited by the solubility of the test item in aqueous medium and by cytotoxic effects. The assay was performed in two independent experiments, using two parallel cultures each. A treatment period of 4 hours was used for all experiments, with the exception of the second experiment with metabolic activation, were a 24 hour treatment period was selected for the cultures. No substantial and reproducible dose dependent increase of the mutation frequency was observed in both experiments. Appropriate reference mutagens, used as positive controls, induced a distinct increase of mutant colonies and, thus, showed the sensitivity of the test system and the activity of the metabolic activation system.


The results of this HPRT assay indicate, that partially unsaturated TEA-Esterquat did not cause a positive response in the non-activated and S9-activated systems and was assessed to be negative under the conditions of this study.


 


In a mammalian cell cytogenicity assay according to OECD Guideline 473 1997, V79 cell cultures were exposed to partially unsaturated TEA-Esterquat at concentration ranges of 3.1 – 200 µg/mL without metabolic activation and 4.7 – 600 µg/mL in the presence of mammalian metabolic activation.


In experiment I and II, in the absence and the presence of S9 mix, no biological relevant increase in the number of cells carrying structural chromosome aberrations was observed. However, in the presence of S9 mix two significant (p < 0.05) increases were observed, in experiment I at preparation interval 18 hrs after treatment with 37.5 µg/mL (4 % aberrant cells, exclusive gaps), and in experiment II at preparation interval 28 hrs with 300 µg/mL (4.8 % aberrant cells, exclusive gaps). In addition, a dose related increase in the number of cells carrying structural chromosome aberrations was observed in experiment II with metabolic activation.


A confirmatory experiment III was performed to verify these observations. In the repeated experiment in the presence of S9 mix after


4 hrs treatment at a prolonged 28 hrs preparation interval no biologically relevant increase in the number of cells carrying structural chromosome aberrations was observed. Although the aberration rates showed a dose related increase, the values were clearly within the historical control data range of the testing laboratory. Finally, the observations of experiment II in the presence of S9 mix were not confirmed in experiment III and therefore they have to be regarded as biologically insignificant.


In all experiments, no biologically relevant increase in the rate of polyploid metaphases was found.


In conclusion it can be stated that under the experimental conditions reported, the test item did not induce structural or numeric chromosome aberrations as determined by the chromosome aberration test in V79 cells (Chinese hamster cell line) in vitro. The test item is considered to be non-clastogenic in this chromosome aberration test with and without S9 mix when tested up to cytotoxic test item concentrations.


 


In vivo data


In a mouse bone marrow micronucleus assay according to OECD guideline No. 474, 1983, 6 male and 6 female albino mice (CFW1) per group were treated by oral intubation with partially unsaturated TEA-Esterquat at a dose of 5000 mg/kg bw. Bone marrow cells were harvested at 24, 48 and 72 hours post-treatment.


There were no signs of toxicity as indicated by an enhanced mortality rate. A slight reduction in the ratio of polychromatic to normochromatic erythrocytes were determined in female mice 24 and 48 h after administration, indicating possibly a weak toxic effect to the bone marrow. The partially unsaturated TEA-Esterquat was tested at an adequate dose, based on the results of the range-finding test. The positive control induced the appropriate response.


There was no significant increase in the frequency of micronucleated polychromatic erythrocytes in bone marrow after any treatment time.


 


There are no data gaps for the endpoint genetic toxicity. No human data are available. However, there is no reason to believe that these results from rat and rabbits would not be applicable to humans.


 


 


Similar results were obtained with the source substance MDEA-Esterquat C16-18 and C18 unsatd.: the substance did not show any genotoxic intrinsic properties in the Ames test, mouse lymphoma assay, chromosome aberration study and in vivo bone marrow micronucleus assay and is therefore considered to be nongenotoxic. These data are included into the dossier to demonstrate, that both substances have a similar toxicological profile.

Justification for classification or non-classification

Based on the available data, the substance does not need to be classified for mutagenicity according to regulation (EC) 1272/2008. Thus, no labelling is required.