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Genetic toxicity in vitro

Description of key information

Negative resuls obtained on bacterial mutagenicity study performed on potassoum sodium tartrate (Ames test) with and without metabolic activation. Negative results obtained also in in vitro chromosomal

aberration test performed on chinese hamster lung fibroblasts, employing analogue substance potassium hydrogen tartrate, as part of a mutagenicity screening programme sponsored by the Japanese

Ministry of Health ands Welfare.

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:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
supporting study
Study period:
1982
Reliability:
2 (reliable with restrictions)
Qualifier:
equivalent or similar to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
yes
Remarks:
No metabolic activation employed
GLP compliance:
not specified
Type of assay:
bacterial reverse mutation assay
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
S. typhimurium, other: TA 92 and TA 94
Metabolic activation:
with and without
Metabolic activation system:
S9 fraction prepared from livers of pretreated rats with PCB compund Kanechlor KC-400
Test concentrations with justification for top dose:
Reported 6 concentrations. Maximum concentration tested is 0.5 mg/plate.
Vehicle / solvent:
Diluted in a solution of 0.1 N sodium hydroxyde.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
not specified
Remarks:
Details nos providad in paper, althogh numerous positive substances are reported.
Details on test system and experimental conditions:

Cells cultured overnight were pre-incubated with both the test sample and the S-9 mix for 20 min at 37°C before plating. Duplicate plates were used for each of six different concentrations of the sample.
The number of revertant (his +) colonies was scored after incubation at 37°C for 2 days.
Evaluation criteria:
The result was considered positive if the number of colonies found was twice the number in the control (exposed to the appropriate solvent or untreated).
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:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
not specified
Species / strain:
S. typhimurium, other: TA 92 and TA 94
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
not specified
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Conclusions:
Interpretation of results (migrated information):
negative

The analogue substance potassium bitartrate (potassium hydrogen tartrate) was tested up to a maximum concentration of 0.5 mg/ml on Salmonella strains TA 1535, TA 1537, TA 98, TA 92, TA 94 and TA 100, with and without metabolic activation as part of a programme sponsored by the Japanese Ministry of Health and Welfare to test common food additives. Results obtained on all strains tested at the maximum concentrations tested were negative.
Executive summary:

The analogue substance potassium bitartrate (potassium hydrogen tartrate) was tested up to a maximum concentration of 0.5 mg/ml on Salmonella strains TA 1535, TA 1537, TA 98, TA 92, TA 94 and TA 100, with and without metabolic activation as part of a programme sponsored by the Japanese Ministry of Health and Welfare to test common food additives. Results obtained on all strains tested at the maximum concentrations tested were negative. These results are considered valid to support conclusions of the analogue reference substance Potassoim Sodium Tartrate.

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:
1979
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Well performed, well described study, commissioned in 1979 by USFDA and performed by reputed laboratory. Raw data available and method essentially as described in EU test method B13/14. Lack of evidence of GLP is only reason for not asigning Klimisch 1.
Qualifier:
equivalent or similar to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
yes
Remarks:
Duplicate instead of triplicate plates. However tests were performed twice.
GLP compliance:
not specified
Type of assay:
bacterial reverse mutation assay
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
Arochlor-1254 stimulated rat-liver homogenate (S9 mix) according to Ames et al.
Test concentrations with justification for top dose:
A range-finding assay was performes on strain TA 100 at concentrations of 0.3 - 10.000 µg/plate.
Sunsequently two assays were performed using the following test concentrations: 0.3, 3.3, 33.3, 100.0, 333.3, 1000.0, 3333.3 and 10000 µg/plate. In the second assay only the 6 highest concentrations were used.
Vehicle / solvent:
0.067 Potassium Phosphate buffer is used as vehicle / solvent (ph 7.0).
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
sodium azide
Untreated negative controls:
yes
Positive controls:
yes
Positive control substance:
other: 2-anthramine
Untreated negative controls:
yes
Positive controls:
yes
Positive control substance:
9-aminoacridine
Untreated negative controls:
yes
Positive controls:
yes
Positive control substance:
2-nitrofluorene
Untreated negative controls:
yes
Positive controls:
yes
Positive control substance:
2-acetylaminofluorene
Details on test system and experimental conditions:
Test performed according to test developed by B. Ames. Briefly, innoculum from stck cultures is grown overnight a 37°C in Oxoid nutrient broth. After stationary overnight growth cultures are shaken for 3 - 4 h for optimal exponential growth.The standard plata-incorporation method is employed and test substance and controls are plated, with and without metabolic activation. Plates are incubated for 48 h at 37 °C and revertants are counted and recorded. Metabolic activation mixture contains 10% S9 fraction.
Evaluation criteria:
Not recordad, as no positive response was found. Presumably the modified two-fold rule is employed as stated in source publications and reference test method.
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:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Report states that in the second experiment performed abnormally high numver of revertabts were seen with strains TA 1535 and TA100 (in samples and negative controls). This was subsequently attributed to the presence of traces of ethylene oxide in the plates used.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

TABLE 1. SALMONELLA TYPHIMURIUM STRAIN TA1535. FDA COMPOUND F76-019 (Potassium Sodium Tartrate)

Compound  Metabolic  Micrograms of compound  Histidine revertants per plate  
  Activation added per plate   Experiment 1*     Experiment 2*  
        14 March 1978     7 July 1978  
          Average     Average
Negative control −   31 36 41 19 14 15
  −   37 60   14 14  
  +   48 35 41 4 4 5
  +   37 45   7 4  
Positive controls                
Sodium azide − 0,5 264     344 256 300
2-Anthramine − 1 37     7    
  + 1 119     76    
F76-019 − 0,3       14 13 14
  − 3,3       15 18 17
  − 33,3 30 67 49 13 14 14
  − 100 44 41 43 13 9 11
  − 333,3 37 39 38 13 14 14
  − 1000 51 45 48 14 7 11
  − 3333,3 42 52 47 15 17 16
  − 10000 24 50 37 16 8 12
  + 0,3       9 9 9
  + 3,3       8 6 7
  + 33,3 54 84 69 4 9 7
  + 100 43 113 78 5 6 6
  + 333,3 63 70 67 7 4 6
  + 1000 54 63 59 4 5 5
  + 3333,3 56 67 62 4 8 6
  + 10000 51 53 52 11 2 7
                 

TABLE 2. SALMONELLA TYPHIMURIUM STRAIN TA1537. FDA COMPOUND F76-019 (Potassium Sodium Tartrate)
Compound  Metabolic  Micrograms of compound                                                   Histidine revertants per plate  
  Activation added per plate   Experiment 1     Experiment 2  
        28 February 1978     14 March 1978  
          Average     Average
Negative control −   17 24 16 6 3 5
  −   9 14   4 5  
  +   12 5 11 11 3 7
  +   15 13   10 4  
Positive controls                
9-Aminoacridine − 50 150 219 185 154    
2-Anthramine − 1 NT     3    
  + 1 NT     41    
F76-019 − 0,3 12 13 13      
  − 3,3 10 19 15      
  − 33,3 15 20 18 5 4 5
  − 100 17 7 12 6 2 4
  − 333,3 13 15 14 5 5 5
  − 1000 9 12 11 1 8 5
  − 3333,3 14 23 19 1 5 3
  − 10000 18 9 14 5 4 5
  + 0,3 14 14 14      
  + 3,3 16 19 18      
  + 33,3 15 16 16 6 12 9
  + 100 18 18 18 8 9 9
  + 333,3 17 10 14 7 8 8
  + 1000 13 11 12 11 4 8
  + 3333,3 14 12 13 6 3 5
  + 10000 13 24 19 8 4 6

TABLE 3. SALMONELLA TYPHIMURIUM STRAIN TA1538. FDA COMPOUND F76-019 (Potassium Sodium Tartrate)
Compound  Metabolic  Micrograms of compound                                                   Histidine revertants per plate  
  Activation added per plate   Experiment 1     Experiment 2  
        28 February 1978     14 March 1978  
          Average     Average
Negative control −   13 13 13 9 14 11
  −   12 13   9 10  
  +   21 16 22 24 19 21
  +   28 23   18 21  
Positive controls                
2-Nitrofluorene − 5 597 787 692 591    
2-Anthramine − 1 NT     19    
  + 1 NT     89    
F76-019 − 0,3 13 17 15      
  − 3,3 14 11 13      
  − 33,3 21 17 19 20 8 14
  − 100 5 14 10 14 4 9
  − 333,3 29 21 25 19 17 18
  − 1000 14 8 11 15 7 11
  − 3333,3 18 10 14 14 16 15
  − 10000 13 19 16 20 14 17
  + 0,3 9 32 21      
  + 3,3 14 19 17      
  + 33,3 26 37 32 29 23 26
  + 100 30 34 32 27 22 25
  + 333,3 31 23 27 28 21 25
  + 1000 29 20 25 25 22 24
  + 3333,3 21 27 24 25 25 25
  + 10000 44 24 34 27 15 21

TABLE 4. SALMONELLA TYPHIMURIUM STRAIN TA98. FDA COMPOUND F76-019 (Potassium Sodium Tartrate)
Compound  Metabolic  Micrograms of compound                                                   Histidine revertants per plate  
  Activation added per plate   Experiment 1     Experiment 2  
        28 February 1978     14 March 1978  
          Average     Average
Negative control −   28 21 24 19 18 15
  −   20 27   11 12  
  +   37 38 36 18 37 27
  +   41 29   27 27  
Positive controls                
2-Nitrofluorene − 5 345 373 359 359    
2-Anthramine − 2,5 39     18    
  + 2,5 965     104    
F76-019 − 0,3 26 17 22      
  − 3,3 25 21 23      
  − 33,3 23 19 21 31 11 21
  − 100 25 22 24 19 21 20
  − 333,3 26 18 22 15 17 16
  − 1000 20 23 22 15 27 21
  − 3333,3 22 33 28 19 14 17
  − 10000 12 15 14 17 13 15
  + 0,3 51 42 47      
  + 3,3 30 37 34      
  + 33,3 32 48 40 39 48 44
  + 100 27 35 31 36 35 36
  + 333,3 37 38 38 18 36 27
  + 1000 42 47 45 18 37 28
  + 3333,3 38 30 34 21 36 29
  + 10000 32 37 35 29 24 27

TABLE 6. ESCHERICHIA COLI WP2. FDA COMPOUND F76-019 (Potassium Sodium Tartrate)
Compound  Metabolic  Micrograms of compound                                                   Tryptophan Revertants per plate  
  Activation added per plate   Experiment 1     Experiment 2  
        28 February 1978     14 March 1978  
          Average     Average
Negative control −   65 45 50 33 36 33
  −   60 31   31 30  
  +   55 53 57 57 45 40
  +   65 56   24 34  
Positive controls                
AF2 − 0,1 104 102 103 1581    
2-Anthramine − 10 72     NT*    
  + 10 166          
F76-019 − 0,3 43 54 49      
  − 3,3 53 48 51      
  − 33,3 39 55 47 42 28 35
  − 100 45 37 41 32 39 36
  − 333,3 51 39 45 39 43 41
  − 1000 42 30 36 39 29 34
  − 3333,3 44 52 48 23 48 36
  − 10000 57 48 53 29 49 39
  + 0,3 57 37 47      
  + 3,3 52 53 53      
  + 33,3 48 48 48 43 44 44
  + 100 53 48 51 50 66 58
  + 333,3 53 50 52 50 36 43
  + 1000 63 55 59 46 57 52
  + 3333,3 48 50 49 50 48 49
  + 10000 52 56 54 53 60 57

Conclusions:
Interpretation of results (migrated information):
negative

Potassium sodium tartrate tested in the Ames test with and without metabolic activation on Salmonella strains TA 1535, TA 1537, TA98, TA 100 and E.coli strain WP2 did not reveal any significant increase in revertants attributable to the test concentrations tested, neither signs of toxicity (as seen by background lawn observation) up to a maximum concentration of 10 mg/plate. Thereby the sample is considered to be negative in the bacterial mutagenicity test performed.
Executive summary:

Potassium sodium tartrate tested in the Ames test with and without metabolic activation on Salmonella strains TA 1535, TA 1537, TA98, TA 100 and E.coli strain WP2 did not reveal any significant increase in revertants attributable to the test concentrations tested, neither signs of toxicity (as seen by background lawn observation) up to a maximum concentration of 10 mg/plate. Thereby the sample is considered to be negative in the bacterial mutagenicity test performed.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
1982
Reliability:
2 (reliable with restrictions)
Qualifier:
equivalent or similar to guideline
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
Deviations:
yes
Remarks:
No metabolic activation employed
GLP compliance:
not specified
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):
Established originaly from lung of newborn female at the Cancer Research Institute, Tokyo (1970). Maintained by 4-day passages in Minnimum Essential Medoum (MEM) of GIBCO. The modal chromosomal number is 25 and the doubling time was approximately 15h.
Metabolic activation:
without
Test concentrations with justification for top dose:
Three concentrations were used, although the report only states maximum concentration employed which is 2 mg/ml. This concentration is stated to be that producing 50% growth inhibition, as determined in a prelimnary study.
Vehicle / solvent:
Test medium
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
not specified
Details on test system and experimental conditions:
The cells were exposed to each sample at three different doses for 24 and 48 hr. No metabolic activation systems were applied. The maximum dose of each sample was selected by a preliminary test in which the dose needed for 50% cell-growth inhibition was estimated using a cell densitometer (Monocellater, Olympus Co., Ltd).
Previous studies indicated that the osmotic pressure of the medium generally rose with sample concentrations of more than 10 raM, SO that the maximum dose for some samples was limited to around this level, at which cytotoxic effects were not necessarily observed.

Chromosome preparations were made as follows. Colcemid (final concn 0.2/~g/ml) was added to the culture 2 hr before cell harvesting. The cells were then trypsinized and suspended in a hypotonic KCI solution (0.075 M) for 13 min at room temperature. After centrifugation the cells were fixed with acetic acid-methanol (1:3, v/v) and spread on clean glass slides. After air-drying, the slides were stained with Giemsa solution (1.5%, at pH 6.8; E. Merck) for 12-15 min. A hundred well-spread metaphases were observed under the microscope (x 600 with a nocover objective lens). The incidence of polyploid cells as well as of cells with structural chromosomal aberrations such as chromatid or chromosome gaps, breaks, exchanges, ring formations, fragmentations and others, was recorded on each culture plate.
.
Evaluation criteria:
The results were considered to be negative if the incidence was less than 4.9%, equivocal if it was between 5.0 and 9.9%, and positive if it was more than 10.0 %. Untreated cells and solvent-treated cells served as negative controls, in which the incidence of aberrations was usually less than 3.0%
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
other: Reported as valid although not verifiable in report avaiable
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Conclusions:
Interpretation of results (migrated information):
negative without metabolic activation

The analogue substance potassium bitartrate (potassium hydrogen tartrate) was tested up to a maximum concentration of 2 mg/ml on a Chinse Hamster lung established cell line and tested without metabolic activation. Results obtained at maximum non severly toxic concentrations after 48 hours of exposure do not indicate any significant amounts of structural aberrations or polyploidy (1%). Thereby the results point to the tested substancxe as being negative for clastogenesis in this mammalian cell line.
Executive summary:

The analogue substance potassium bitartrate (potassium hydrogen tartrate) was tested up to a maximum concentration of 2 mg/ml on a Chinse Hamster lung established cell line and tested without metabolic activation. Results obtained at maximum non severly toxic concentrations after 48 hours of exposure do not indicate any significant amounts of structural aberrations or polyploidy (1%). Thereby the results point to the tested substancxe as being negative for clastogenesis in this mammalian cell line.

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

Additional information

Justification for classification or non-classification

Potassium sodium tartrate tested in the Ames test with and without metabolic activation on Salmonella strains TA 1535, TA 1537, TA98,

TA 100 and E.coli strain WP2 did not reveal any significant increase in revertants attributable to the test concentrations tested, neither

signs of toxicity (as seen by background lawn observation) up to a maximum concentration of 10 mg/plate. Thereby the sample is

considered to be negative in the bacterial mutagenicity test performed.

The analogue substance potassium bitartrate (potassium hydrogen tartrate) was tested up to a maximum concentration of 2 mg/ml on a

Chinse Hamster lung established cell line and tested without metabolic activation. Results obtained at maximum non severly toxic

concentrations after 48 hours of exposure do not indicate any significant amounts of structural aberrations or polyploidy (1%). Thereby

the results point to the tested substancxe as being negative for clastogenesis in this mammalian cell line.

In the same set of experiments, potassium bitartrate was tested up to a maximum concentration of 0.5 mg/ml on Salmonella strains TA

1535, TA 1537, TA 98, TA 92, TA 94 and TA 100, with and without metabolic activation as part of a programme sponsored by the

Japanese Ministry of Health and Welfare to test common food additives. Results obtained on all strains tested at the maximum

concentrations tested were negative. These results are considered valid to support conclusions of the analogue reference substance

Potassoim Sodium Tartrate.