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Genetic toxicity in vitro

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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:
June 1992
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Species / strain / cell type:
S. typhimurium, other: Strains TA 1535, TA 1537, TA 1538, TA 100 and TA 98
Metabolic activation:
with and without
Metabolic activation system:
S9 mix, derived from the livers of male adult rats induced with Aroclor 1254
Test concentrations with justification for top dose:
Concentration range in the main test (with metabolic activation): 1, 10, 100, 1000 and 10000 µg/plate
Concentration range in the main test (without metabolic activation): 1, 10, 100, 1000 and 10000 µg/plate
Vehicle / solvent:
- Solvent: DMSO
- Justification for choice of solvent/vehicle: no data
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: without S9 mix: 9-Aminoacridine (40 µg/plate) with strain TA1537, Sodium azide (5 µg/plate) with strains TA 1535 and TA 100, 2-Nitrofluorene (10 µg/plate) with strains TA 1538 and TA 98; with S9 mix: 2-Aminoanthracene (1 µg/plate) with all tester strains
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Preincubation period: not applicable
- Exposure duration: 48 - 72 h

NUMBER OF REPLICATIONS: triplicates plates for the test material and negative control, two for positive controls

DETERMINATION OF CYTOTOXICITY
- Method: determination of toxicity by semiquantitative evaluation of the background lawn and the number of spontaneous revertant colonies

OTHER EXAMINATIONS:
Sterility test
Evaluation criteria:
- at the end of the assay, a sterility check on S9 mix must show less than two viable colonies per 0.5 mL
- at the end of the test, a sterility check of the test material must show less than two viable colonies per plate
- the positive conrols must produce at least a threefold increase in the number of revertant colonies with regard to the mean value for the respective negative control
- the mean number of spontaneous revertant colonies in the negative controls must correspond to the following values:
strain TA 1535: 20 ± 15; strain TA 1537: 20 ± 15; strain TA 1538: 15 ± 10; strain TA 98: 40 ± 25; strain TA 100: 150 ± 90
Statistics:
no statistics performed
Key result
Species / strain:
other: Salmonella typhimurium: strains TA 1535, TA 1537, TA 1538, TA 98
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
without
Genotoxicity:
other: result could not be used for judgement because of failure of positive control
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
not valid
Key result
Species / strain:
other: Salmonella typhimurium: strains TA 1535, TA 1537, TA 1538, TA 98 and TA 100
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
other: vehicle controls valid with the exception of strain TA 98
Untreated negative controls validity:
not examined
Positive controls validity:
other: positive controls were judged to be valid, even when the increase of revertant colonies was less than 3-fold, but being 2-fold at least
Additional information on results:
- Preliminary test: The genetic characteristics of the bacterial strains were found to be unaltered.
- Test material toxicity: The test material did not induce toxic effects at a concentration of 10 mg/plate both in the absence and presence of the metabolic activation system.
- Negative control: The number of revertant colonies on the negative control plates fell within the normal range with the exception of strain TA 98 without metabolic activation. This negative control (12 ± 2) was below the cited normal range of 40 ± 25 revertant colonies.
- Positive control: The positive controls induced an at least threefold increase in the number of colonies with the exception of the positive controls with strain TA 100 both with and without S9 mix and with strain TA 1535 with S9 mix. In these cases the increase of revertant colonies was not threefold: TA 100 without S9 mix: no increase of number of revertant colonies at all (neg. control mean number of revertants: 150, pos. control: 152), with S9 mix: 2.0-fold; TA 1535 with S9 mix: 2.4-fold.
- Sterility test: The sterility test performed on the test material and on S9 mix did not show any bacterial contamination.
Table #1: Salmonella typhimurium reverse mutation assay without metabolic activation
Concentration
[µg/plate]
Revertant colonies / plate
mean ± SD
Strain TA 1535 Strain TA 1537 Strain TA 1538 Strain TA 98 Strain TA 100
Negative control 22 ± 4 9 ± 3 10 ± 2 12 ± 2 150 ± 16
1 25 ± 5 11 ± 1 12 ± 3 18 ± 2 147 ± 17
10 19 ± 3 11 ± 3 12 ± 3 18 ± 3 160 ± 22
100 20 ± 3 14 ± 2 12 ± 4 14 ± 2 162 ± 19
1000 30 ± 4 11 ± 2 12 ± 4 15 ± 0 170 ± 7
10000 21 ± 4 13 ± 4 12 ± 3 18 ± 8 138 ± 10
Positivecontrol 433 ± 47 96 ± 3 264 ± 31 385 ± 18 152 ± 37
Table #2: Salmonella typhimurium reverse mutation assay with metabolic activation
Concentration
[µg/plate]
Revertant colonies / plate
mean ± SD
Strain TA 1535 Strain TA 1537 Strain TA 1538 Strain TA 98 Strain TA 100
Negative control 28 ± 3 18 ± 3 12 ± 3 18 ± 1 167 ± 9
1 19 ± 7 20 ± 2 16 ± 4 21 ± 3 130 ± 17
10 24 ± 4 12 ± 4 15 ± 2 20 ± 4 101 ± 19
100 28 ± 7 20 ± 3 16 ± 3 24 ± 6 121 ± 20
1000 17 ± 1 19 ± 3 21 ± 2 19 ± 4 113 ± 12
10000 23 ± 3 24 ± 1 16 ± 1 18 ± 2 163 ± 16
Positivecontrol 66 ± 3 480 ± 23 206 ± 14 216 ± 5 335 ± 21

SD = Standard Deviation

Conclusions:
Interpretation of results:
negative without metabolic activation valid for tester strains TA 1535, TA 1537, TA 1538 and TA 98
other: no evaluation of result possible for tester strain TA 100 because of failure of positive control
negative with metabolic activation valid for all strains tested

The test material did not induce an increase in the number of revertant colonies compared to the negative controls and was considered as non-mutagenic in this test-system.
Executive summary:

A test was performed to verify the mutagenic potential of the test material 1,2,3-Propanetricarboxylic acid, 2-hydroxy-, tri-C14-15-alkyl esters on Salmonella typhimurium according to Ames B.N., McCann J. and Yamasaki E. (1975).

The test was carried out on 5 strains of Salmonella typhimurium (TA 1535, TA 1537, TA1538, TA 98, TA 100). The mutagenic activity of the material was assessed by comparing the number of revertant colonies induced with the positive control. This activity was tested in the presence and absence of a metabolizing system and done directly in a petri dish. The test material was tested at concentrations of 100000, 10000, 1000, 100 and 10 µg/mL corresponding to doses of 10000, 1000, 100, 10 and 1 µg/petri dish. The results of the study can be summarized as follows: The test material at maximum concentration 10 mg/petri dish produced no increase in number of revertant colonies in comparison to the negative control. The result of the experiment with tester strain TA 100 without metabolic activation could not be evaluated because of a failure of the respective positive control. Nonetheless the test material 1,2,3-Propanetricarboxylic acid, 2-hydroxy-, tri-C14-15-alkyl esters was judged to be unable to induce mutations under these conditions at least in Salmonella typhimurium tester strains TA 1535, TA 1537, TA 1538 and TA 98.

Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
Please see Analogue Justification
Reason / purpose:
read-across source
Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
but of low order in the maximum practicable concentration
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
Please see Analogue Approach
Reason / purpose:
read-across source
Details on test system and experimental conditions:



DURATION
- Exposure duration:
Pre-experiment and Experiment I: 4 hours with and without S9 mix
Experiment II: 4 hours with S9 mix; 20 and 28 hours without S9 mix
- Fixation time (start of exposure up to fixation or harvest of cells):
Pre-experiment and Experiment I: 20 hours
Experiment II: 20 and 28 hours


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


NUMBER OF REPLICATIONS: 2


NUMBER OF CELLS EVALUATED: 200 well spread metaphases per concentration and control (100 per culture)


DETERMINATION OF CYTOTOXICITY
- Method:
Pre-experiment: The vitality of the cells was measured with the BCA reagent kit which contained the watersoluble and stable BCA (Bicinchonic acid) and an alkaline Cu2+ - solution (Pierce, USA). Cytotoxicity leaded to a reduction of the proliferation rate of the cells. This leaded to a reduction in the protein content of the cell culture as compared to the control cultures and was detected colourometrically (Absorbance at a wave length of 550 nm) after staining by BCA solution.
Experiments I and II: The mitotic index was determined by counting the number of cells in mitosis in 1000 cells to describe a cytotoxic effect. As an additional parameter for cytotoxic effects of the test item the relative cell density was calculated as the mean of twenty cell counts per test group ( cells within the visual field at a 400-fold magnification).

OTHER EXAMINATIONS:
- Determination of polyploidy: yes
- Determination of endoreplication: no
Key result
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
see details below
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

The target substance 1,2,3 -Propanetricarboxylic acid, 2 -hydroxy-, tris(C14 -15 -alkyl) ester

did not cause mutagenic effects towards bacteria in a standard Ames test. Also the source substance Tri (hexyl, octyl, decyl) citrate and the structurally related substance 1,2,3-Propanetricarboxylic acid, 2-hydroxy-, tris(C12-13-branched-alkyl) ester did not cause mutagenicity with and without metabolic activation in Ames tests. The structurally related source substance Tri-C18 -22 (even numbered)-alkyl 2-hydroxypropane-1,2,3-tricarboxylate, is not mutagenic in mouse lymphoma L5178Y cells, in either the absence or the presence of S9 mix, when tested to the maximum practicable concentration of 1000 µg/mL. The structurally related substance Tri (hexyl, octyl, decyl) citrate did not induce structural chromosomal aberrations in the V79 Chinese hamster cell line. Therefore it is concluded that the test substance has no mutagenic properties.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

No classification for mutagenicity is indicated according to the general classification and labeling requirements for dangerous substances and preparations (Directive 67-548-EEC) or the classification, labeling and packaging (CLP) regulation (EC) No 1272/2008.