Registration Dossier

Toxicological information

Genetic toxicity: in vitro

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Administrative data

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:
weight of evidence
Study period:
2000-05-12 - 2000-11-06
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: GLP-study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2000
Report Date:
2000

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
Ninth Addendum (adopted 1997-07-21)
Deviations:
no
Qualifier:
according to
Guideline:
other: EEC Directive 92/69, L 383 A, Annex V, B14, dated December 29, 1992
Deviations:
no
Qualifier:
according to
Guideline:
EPA OTS 798.5265 (The Salmonella typhimurium Bacterial Reverse Mutation Test)
Deviations:
no
Qualifier:
according to
Guideline:
other: EPA7J2-C-96-219, June 1996 (Public Draft)
Deviations:
no
Principles of method if other than guideline:
None
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: liquid
Details on test material:
- Name of test material (as cited in study report): ATEHC
- Physical state: liquid
- Expiration date of the lot/batch: November 01, 2000
- Stability under test conditions: pure at RT: min. 6 month
- Storage condition of test material: room temperature, dry, closed containers
- Other: Chemical Name: Acetyl-tri-(2-ethylhexyl)-citrat
- Other: Safety precautions: Routine hygienic procedures were sufficient to assure personnel health and safety.

Method

Target gene:
Histidine
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Details on mammalian cell type (if applicable):
TA 1537: his C 3076; rfa; uvrB: frame shift mutations
TA 98: his D 3052; rfa"; uvrB"; R-factor: frame shift mutations
TA 1535: his G 46; rfa"; uvrB": base-pair substitutions
TA 100: his G46; rfa"; uvrB"; R-factor: base-pair substitutions
TA102: his G 428 (pAQl); rfa"; R-factor: base-pair substitutions
Additional strain / cell type characteristics:
other: The strains are constructed to differentiate between base pair (TA 1535, TA 100, TA 102) and frameshift (TA 1537, TA 98) mutations.
Metabolic activation:
with and without
Metabolic activation system:
mammalian microsome enzyme activation mixture
Test concentrations with justification for top dose:
100, 316.2, 1000, 2000, 3000 and 4000 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The solvent was compatible with the survival of the bacteria and the S9 activity
Controls
Untreated negative controls:
yes
Remarks:
water
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
Positive controls:
yes
Positive control substance:
other: NaN3 (sodium azide), 4-NOPD (4-nitro-o-phenylene-diamine), MMS (methyl methane sulfonate) and 2-AA (2-aminoanthracene)
Details on test system and experimental conditions:
For the plate incorporation method the following materials were mixed in a test tube and poured over the surface of a minimal agar plate:
100 µL Test solution at each dose level, solvent or negative control or reference mutagen solution (positive control),
500 µL S9 mix (for the test with metabolic activation) or S9 mix substitution buffer (for test without metabolic activation),
100 µL Bacteria suspension (cf. Preparation of Bacteria, preculture of the strain),
2000 µL Overlay agar.
For the pre-incubation method 100 µL of the test item solution was preincubated with the tester strains (100 µL) and sterile buffer or the metabolic activation system (500 µL) for 60 minutes at 37 °C prior to adding the overlay agar (2000 µL) and pouring onto the surface of a minimal agar plate.
For each strain and dose level, including the controls, three plates were used.
After solidification the plates were inverted and incubated at 37 °C for at least 48 h in the dark.
Evaluation criteria:
The Mutation Factor is calculated by dividing the mean value of the revertant counts through the mean values of the solvent control (the exact and not the rounded values are used for calculation).
A test is considered acceptable if for each strain:
- the bacteria demonstrate their typical responses to crystal violet and ampicillin
- the control plates without S9 mix are within the following ranges (range of spontaneous reversion frequencies, mean of three replicates):
TA 1535: 5-30
TA 1537: 4-32
TA98: 18-63
TA 100: 79-197
TA 102: 173-396
- corresponding background growth on both negative control and test plates is observed.
- the positive controls show a distinct enhancement over the control plate.

A test item is considered as mutagenic if:
- a dose-related increase in the number of revertants occurs and/or
- a reproducible biologically relevant positive response for at least one of the test points occurs in at least one strain with or without metabolic activation.
According to the new OECD guidelines, the biological relevance of the results will be the criterion for the interpretation of results, a statistical evaluation of the results is not regarded as necessary.

A test item producing neither a dose related increase in the number of revertants nor a reproducible biologically relevant positive response at any of the test points is considered non-mutagenic in this system.
A biologically relevant increase is described as follows:
- if in strain TA 100 and TA 102 the number of reversions is at least twice as high
- if in strains TA 1535, TA 1537 and TA 98 the number of reversions is at least three times higher as compared to the spontaneous reversion rate.
Statistics:
According to the new OECD guidelines, the biological relevance of the results will be the criterion for the interpretation of results, a statistical evaluation of the results is not regarded as necessary.

Results and discussion

Test results
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not determined
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Slight toxic effects of the test item (indicated by a reduction of the spontaneous rate) were observed at the highest investigated dose without metabolic activation in the tester strains TA 100 (2nd experiment) and TA 1537 (1st experiment).
No substantial increases in revertant colony numbers of any of the five tester strains were observed following treatment with ATEHC at any concentration level, either in the presence or absence of metabolic activation (S9-mix) in experiment I and II. There was also no tendency of higher mutation rates with increasing concentrations in the range beyond the generally acknowledged border of biological relevance in both experiments independently performed.
The reference mutagens showed a distinct increase of induced revertant colonies.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Experiment I (Plate-incorporation Test)

Test Strain : TA 1535

 Dose Group     Revertants per plate                 Mutation Factor   
   dose/plate  -S9  M  +/-SD  +S9  M  +/-SD  -S9  +S9
 Water    15  15  4  9  9  2  1.8 0.8 
    12       7        
     19      10        
 DMSO    12  9  3  10  11  2  1.0 1.0 
     6      13        
     8      10        
 Test item  100.0 µg/plate  9  11  2  13  10  4  1.2  0.9
     12            
     11      11        
 Test item  316.2 µg/plate  14  13  2  11  8  4  1.5 0.8 
     11      10        
     13      4        
 Test item  1000 µg/plate  9 P  9  4  7  9  2  1.0 0.9 
     5 P      11        
     13 P      10        
 Test item  2000 µg/plate  10 P  9  3  14  13  2  1.1 1.2 
     12 P      11        
     6 P      14        
 Test item  3000 µg/plate  8 P  9  1  6  9  4  1.0 0.9 
     9 P      14        
     10 P      8        
 Test item  4000 µg/plate  12 P  11  2  5  9  4  1.3  0.8
     12 P      10        
     9 P      12        
 Na-azide  10 µg/plate  1161  1118  94  *  /  /  128.9
     1182              
     1010              
 2 -AA  2.5 µg/plate  * /    /  195  227  28  / 20.6 
           238        
           248        

M: Mean

+/-SD: Standard-deviation

B : Background lawn reduced

N : No background lawn

C: Contamination

P: Precipitation

+S9: With S9 mix

-S9: Without S9 mix

*: Not tested

Mutation factor = mean revertants (test item) / mean revertants (solvent control)

Test Strain : TA 1537

 Dose Group     Revertants per plate                 Mutation Factor   
   dose/plate  -S9  M  +/-SD  +S9  M  +/-SD  -S9  +S9
 Water    11  7  4  14  13  2  1.0  1.4
     4      14        
     7      11        
 DMSO    8  8  2  7  9  3  1.0  1.0
     6      12        
     9      9        
 Test item  100.0 µg/plate  13  9  4  15  10  4  1.1  1.1
     8      8        
     5      7        
 Test item  316.2 µg/plate  10  8  3  9  11  3  1.0  1.2
     4      14        
     9      11        
 Test item  1000 µg/plate  13 P  9  4  7  6  1  1.1  0.7
     5 P      5        
     8 P      7        
 Test item  2000 µg/plate  12 P 11   2  8  7  1  1.4  0.8
     9 P      7        
     12 P      6        
 Test item  3000 µg/plate  8 P 10   3  11  7  4  1.4  0.8
     14 P      7        
     9 P      4        
 Test item  4000 µg/plate  6 P  5  1  11  15  4  0.6  1.6
     4      17        
     4      18        
 4 -NOPD  40 µg/plate  368 334   30  *  /  /  43.6  /
     313              
     321              
 2 -AA  2.5 µg/plate  *  /  /  308  328  18  /  35.2
           339        
           338        

Test Strain : TA 102

 Dose Group     Revertants per plate                 Mutation Factor   
   dose/plate  -S9  M  +/-SD  +S9  M  +/-SD  -S9  +S9
 Water    283  295  17  306  315  9  1.1 1.0 
     315      323        
     287      316        
 DMSO    273  263  10  316  330  15  1.0  1.0
     253      329        
     264      346        
 Test item  100.0 µg/plate  263  256  7  287  301  27  1.0  0.9
     250      332        
     254      285        
 Test item  316.2 µg/plate  284  281  6  312  322  17  1.1  1.0
     274      342        
     285      312        
 Test item  1000 µg/plate  282 P  282  39  335  334  4  1.1  1.0
     321 P      337        
     243 P      330        
 Test item  2000 µg/plate  238 P  241  31  348  328  39  0.9  1.0
    273 P      352        
     212 P      283        
 Test item  3000 µ/plate  237 P  252  30  371 P  350  26  1.0  1.1
     233 P      358 P        
     287 P      321 P        
 Test item  4000 µg/plate  235 P  246  11  294 P  333  36  0.9  1.0
     246 P      339 P        
     256 P      365 P        
 MMS  1 µg/plate  2009  2031  149  *  /  /  7.7  /
     1895              
     2190              
 2 -AA  10 µg/plate  *  /  /  507  531  36  /  1.6
           513        
           573        

Test Strain : TA 98

 Dose Group     Revertants per plate                 Mutation Factor   
   dose/plate  -S9  M  +/-SD  +S9  M  +/-SD  -S9  +S9
 Water    20  23  3  36  39  3  1.1  1.0
     25      41        
     24      40        
 DMSO    18  21  3  34  38  4  1.0  1.0
     23      41        
     21      40        
 Test item  100.0 µg/plate  20  24  6  43  41  3  1.2  1.1
     22      38        
    31       41        
 Test item  316.2 µg/plate  21  26  4  44  39  8  1.2  1.0
     28      29        
     28      43        
 Test item  1000 µg/plate  22  27  6  40  38  2  1.3  1.0
     34      36        
     25      37        
 Test item  2000 µg/plate  26  24  2  35  40  7  1.2  1.0
    23       37        
     24      48        
 Test item  3000 µ/plate  22  27  5  27  35  7  1.3  0.9
     28      41        
     31      38        
 Test item  4000 µg/plate  30  28  4  37  35  5  1.4  0.9
     24      30        
     31      39        
 4 -NOPD  10 µ/plate  1558  1695  125  *  /  /  82.0  /
    1804               
     1724              
 2 -AA  2.5 µg/plate  /  /  827  918  222  /  24.0
           1171        
           757        

Test Strain : TA 100

 Dose Group     Revertants per plate                 Mutation Factor   
   dose/plate  -S9  M  +/-SD  +S9  M  +/-SD  -S9  +S9
 Water    117  107  8  93  104  10  1.2  1.0
     102      107        
     103      112        
 DMSO    92  89  6  105  103  10  1.0  1.0
     83      92        
     93      112        
 Test item  100.0 µg/plate  100  94  9  105  92  13  1.1  0.9
     84      92        
     97      79        
 Test item  316.2 µg/plate  75  86  10  67  83  16  1.0  0.8
     92      99        
     92      83        
 Test item  1000 µg/plate  112  104  17  84  88  10  1.2  0.9
     84     99         
     115      80        
 Test item  2000 µg/plate  107  105  5  85  96  10  1.2  0.9
     108      105        
     99      98        
 Test item  3000 µg/plate  100  97  12  91  96  5  1.1  0.9
     84      101        
     107      95        
 Test item  4000 µg/plate  77  91  12  89  86  3  1.0  0.8
     97      85        
     99      83        
 Na-azide  10 µg/plate  968  1012  48  *  /  /  11.3  /
     1063              
     1006              
 2 -AA  2.5 µg/plate  *  /  /  2100  2030  98  /  19.7
           2071        
           1918        

Experiment II (Pre-incubation Test)

Test Strain : TA 1535

 Dose Group     Revertants per plate                 Mutation Factor   
   dose/plate  -S9  M  +/-SD  +S9  M  +/-SD  -S9  +S9
 Water    12  11  3  10  14  3  0.8  1.0
     13      15        
     8      16        
 DMSO    12  14  3  9  14  6  1.0  1.0
     18      20        
     13      13        
 Test item  100.0 µg/plate  13  12  2  10  11  4  0.8  0.8
     10      8        
     12      15        
 Test item  316.2 µg/plate  9  11  3  14  13  4  0.7  1.0
     9      17        
     14      9        
 Test item  1000 µg/plate  13  13  1  16  17  2  0.9  1.2
     14      16        
     12      19        
 Test item  2000 µg/plate 15   14  2  20  16  4  1.0  1.1
     15      15        
     11      13        
 Test item  3000 µ/plate  4  10  6  9  13  4  0.7  0.8
     16      13        
     10      16        
 Test item  4000 µg/plate  12 P  10  3  9 P  11  2  0.7  0.8
     7 P      11 P        
     12 P      13 P        
 Na-azide  10 µ/plate  1391  1307  77  *  /  /  91.2  /
     1240              
     1289              
 2 -AA  2.5 µg/plate  *  /  /  193  167  31  /  12.0
           133        
           176        

M: Mean

+/-SD: Standard-deviation

B : Background lawn reduced

N : No background lawn

C: Contamination

P: Precipitation

+S9: With S9 mix

-S9: Without S9 mix

*: Not tested

Mutation factor = mean revertants (test item) / mean revertants (solvent control)

Test Strain : TA 1537

 Dose Group     Revertants per plate                 Mutation Factor   
   dose/plate  -S9  M  +/-SD  +S9  M  +/-SD  -S9  +S9
 Water    8  7  1  16  16  2  0.6  1.5
     6      14        
     7      18        
 DMSO    8 11   4  13  11  3  1.0  1.0
     15      12        
     10      8        
 Test item  100.0 µg/plate  9  10  1  13  10  3 0.9   0.9
     10      8        
     11      10        
 Test item  316.2 µg/plate  14  12  3  6  10  5  1.1  0.9
     14      9        
     8      15        
 Test item  1000 µg/plate  11  9  2  6  10  4 0.9   0.9
     10     10         
     7      14        
 Test item  2000 µg/plate  13  14  5  13 10   3  1.3  0.9
     10      7        
     19      10        
 Test item  3000 µg/plate  9  12  4  10  9  2  1.1  0.9
     11      7        
     16      11        
 Test item  4000 µg/plate  11 P  11  3  14 P  10  4  1.0  0.9
     8 P      6 P        
     14 P      9 P        
 4 -NOPD  40 µg/plate  179  181  5  *  /  /  16.5  /
    178               
     187              
 2 -AA  2.5 µg/plate  *  /  /  445  417  26  /  37.9
           411        
           394        

Test Strain : TA 102

 Dose Group     Revertants per plate                 Mutation Factor   
   dose/plate  -S9  M  +/-SD  +S9  M  +/-SD  -S9  +S9
 Water    285  299  12  478  461  15  1.0  1.2
     306      453        
     305      451        
 DMSO    304  307  32  377  389  40  1.0  1.0
     277      434        
     340      355        
 Test item  100.0 µg/plate  316  307  11  270  291  25  1.0  0.8
     294      285        
     310      319        
 Test item  316.2 µg/plate  343  322  18  333  344  13  1.1  0.9
     309      358        
     315      340        
 Test item  1000 µg/plate  321  289  29  340  361  19  0.9  0.9
     264      376        
     283      366        
 Test item  2000 µg/plate  301  293  8  353  339  29  1.0  0.9
     292      358        
     286      306        
 Test item  3000 µg/plate  316  278  33  355  351  12  0.9  0.9
     261      337        
     256      361        
 Test item  4000 µg/plate  283 P  289  9  325 P  351  23  0.9  0.9
     286 P      358 P        
     299 P      370 P        
 MMS  1 µg/plate  1459  1378  78  *  /  /  4.5  /
     1371              
     1303              
 2 -AA  10 µg/plate  *  /  /  937  960  20  /  2.5
           974        
           969        

Test Strain : TA 98

 Dose Group     Revertants per plate                 Mutation Factor   
   dose/plate  -S9  M  +/-SD  +S9  M  +/-SD  -S9  +S9
 Water    30  35  5  49  47  4  1.6  1.2
     39      42        
     36      50        
 DMSO    19  21  2  42  41  5  1.0  1.0
     23      36        
     22      45        
 Test item  100.0 µg/plate  12  16  11  51  50  1  0.8  1.2
     28      49        
     8      50        
 Test item  316.2 µg/plate  19  19  4  50  49  4  0.9  1.2
     23      53        
     16      45        
 Test item  1000 µg/plate  17  20  4  44  42  4  1.0  1.0
     24      45        
     20      37        
 Test item  2000 µg/plate  17  22  5  34  41  10  1.0  1.0
     27      52        
     21      37        
 Test item  3000 µ/plate  17  24  8  45  47  2  1.1  1.1
     24      47        
     32      48        
 Test item  4000 µg/plate  19 P  19  2  40 P  44  8  0.9  1.1
     21 P      38 P        
     17 P      53 P        
 4 -NOPD  10 µg/plate  2639  2743  92  *  /  /  128.6  /
     2812              
     2778              
 2 -AA  2.5 µg/plate  *  /  2338  2555  207  /  62.3
           2576        
          2751         

Test Strain : TA 100

 Dose Group     Revertants per plate                 Mutation Factor   
   dose/plate  -S9  M  +/-SD  +S9  M  +/-SD  -S9  +S9
 Water    108  113  15  116  125  11  1.1  1.0
     102      121        
     130      137        
 DMSO    103  101  2  121  122  12  1.0  1.0
     101      111        
     100      134        
 Test item  100.0 µg/plate  101  95  9  102  109  6  0.9  0.9
     99      113        
     85      113        
 Test item  316.2 µg/plate  135  124  11  103  109  7  1.2  0.9
     124      116        
     114      109        
 Test item  1000 µg/plate  132  112  17  117  117  7  1.1  1.0
     100      124        
     105      110        
 Test item  2000 µg/plate  115  103  12  103  112  8  1.0  0.9
     92      118        
     102      115        
 Test item  3000 µ/plate 84   89  14  107  125  16  0.9  1.0
     79      131        
    105       136        
 Test item  4000 µg/plate  93  52  42  127 P  112  15  0.5  0.9
     53 P     97 P         
     9 P      111 P        
 Na-azide  10 µg/plate  772  741  28  *  /  /  7.3  /
     734              
    718               
 2 -AA  2.5 µg/plate  *  /  /  2340  2224  324  /  18.2
           2474        
           1858        

Applicant's summary and conclusion

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

The test item did not cause gene mutations by base pair changes or frameshifts in the genome of the tester strains used during the described mutagenicity test and under the experimental conditions reported.
Executive summary:

In order to investigate the potential of the test substance for its ability to induce gene mutations the plate incorporation test (experiment I) and the preincubation test (experiment II) were performed with the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and TA 102 (2000, GLP study according to OECD 471 and EU method B.14). The test item was tested in two independent experiments at several concentrations. Each assay was conducted with and without metabolic activation (S9 mix). The concentrations, including the controls, were tested in triplicate. The following concentrations of the test item were prepared and used in the experiments: 100.0, 316.2, 1000, 2000, 3000 and 4000 µg/plate. Slight toxic effects of the test item (indicated by a reduction of the spontaneous rate) were observed at the highest investigated dose without metabolic activation in the tester strains TA 100 (2nd experiment) and TA 1537 (1st experiment). No substantial increases in the revertant colony numbers of any of the five test strains were detected at any dose level of the test item either with or without metabolic activation in both independently performed experiments. Therefore, the test item is considered to be non-mutagenic in this bacterial reverse mutation assay. As positive controls reference mutagens were tested in parallel to the test item. They showed a distinct increase of induced revertant colonies.

It can be assumed that the same applies to tributyl citrate (CAS 77-94-1) as it is a near analogue to the test substance acetyltri-2 -ethylhexylcitrat (ATEHC).