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Diss Factsheets

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
May - June 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report date:
2016

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
Trimethylacetic anhydride
EC Number:
216-263-1
EC Name:
Trimethylacetic anhydride
Cas Number:
1538-75-6
Molecular formula:
C10H18O3
IUPAC Name:
2,2-dimethylpropanoic anhydride
Test material form:
liquid

Method

Target gene:
histidine gene locus
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
Phenobarbital/β-naphthoflavone induced rat liver S9
Test concentrations with justification for top dose:
Experiment 1 (Plate incorporation test): 3; 10; 33; 100; 333; 1000; 2500 and 5000 μg/plate. Since only moderate toxic effects were observed 5000 μg/plate were chosen as maximal concentration.

Experiment 2 (pre-incubation test):
TA 98: 10; 33; 100; 333; 1000; 2500 and 5000 μg/plate
TA 100: 3; 10; 33; 100; 333; 1000; 2500 and 5000 μg/plate
Remaining strains: 33; 100; 333; 1000; 2500 and 5000 μg/plate
Vehicle / solvent:
DMSO
The solvent was chosen because of its solubility properties and its relative nontoxicity to the bacteria.
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
methylmethanesulfonate
other: 2-aminoanthracene, 4-nitro-o-phenylene-diamine
Details on test system and experimental conditions:
METHOD OF APPLICATION:

Experiment 1: in agar (plate incorporation);
Experiment 2: preincubation;

DURATION
- Preincubation period: 60 minutes
- Exposure duration: at least 48 hours

Each concentration and the controls were tested in triplicate.
Evaluation criteria:
A test item is considered as a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA 98, TA 100 and 102) or thrice (strains TA 1535 and TA 1537) the colony count of the corresponding solvent control is observed.
A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration.
An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment.
A dose dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative and solvent controls such an increase is not considered biologically relevant.
Statistics:
According to the OECD guideline 471, a statistical analysis of the data is not mandatory.

Results and discussion

Test results
Key result
Species / strain:
other: TA 1535, TA 1537, TA 98, TA 100, TA 102
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:
The test item precipitated in the overlay agar in the test tubes from 1000 to 5000 μg/plate in the absence of S9 mix and at 5000 in the presence of S9 mix. Precipitation of the test item in the overlay agar on the incubated agar plates was observed in Experiment II in the presence of S9 mix at 5000 μg/plate. The undissolved particles had no influence on the data recording.

Reduced background growth or a reduction in the number of revertants were observed at the following concentrations:
TA1535 and TA 102 - none
TA 1537 - from 1000 µg/plate on without S9 mix in Experiment I and from 2500 µg/plate without S9 mix in experiment II; none with S9 mix
TA 98 - from 1000 µg/plate on without S9 mix in Experiment I and II; none with S9 mix
TA 100 - from 333 µg/plate on wihout S9 mix in Experiment I and II; with S9 mix only at 5000 µg/plate in experiment II

Any other information on results incl. tables

Table 1: Summary of results from the salmonella typhimurium reverse mutation assay of experiment 1 (plate incorporation assay) with the test substance (mean +/- SD of revertants per plate).

 Substance  Dose (µg/plate)  Without metabolic activation
     TA 1535 TA 1537  TA 98  TA 100  TA 102 
 DMSO  

11 ± 1

11 ± 3

 28 ± 8  174 ± 8 527 ± 21 
 Untreated   12 ± 4  11 ± 5  35 ± 4  174 ± 28  561 ± 70 
 Test substance  12 ±3 10 ± 1  27 ± 7  176 ± 14  531 ± 18 
  10  12 ± 4  9 ± 3  27 ± 4  171 ± 12  564 ± 28 
   33 13 ± 5  11 ± 5  30 ± 10  172 ± 15  564 ± 27 
   100 11 ± 4  10 ± 5  37 ± 8  149 ± 14  537 ± 14 
   333 13 ± 1  11 ± 4  23 ± 9   73 ± 9 552 ± 28 
   1000  9 ± 2 9 ± 2  33 ± 15  53 ± 8   520 ± 17
   2500 14 ± 5  10 ± 1  28 ± 3  31 ± 6  526 ± 38 
   5000 10 ± 1  9 ± 6  5 ± 2  6 ± 3  542 ± 17 
 sodium azide 10  1305 ± 124       2215 ± 128  
 4 -nitro-o-phenylene-diamine 10       392 ± 17    
 4 -nitro-o-pneylene-diamine 50     85 ± 3      
 methyl methane sulfonate  2.0 µl          6183 ± 412
 Substance Dose (µg/plate)               With metabolic activation
     TA 1535  TA 1537  TA 98  TA 100  TA 102
 DMSO    11 ± 4 15 ± 3  45 ± 0   169 ± 3  638 ± 7
 Untreated    11 ± 5  17 ± 6 40 ± 8   176 ± 30 665 ± 13 
 Test substance  3 10 ± 1   10 ± 4 49 ± 17  173 ± 18   732 ± 9
   10  14 ± 3 10 ± 5   37 ± 2 177 ± 9  735 ± 20 
   33 14 ± 3   13 ± 4 33 ± 12   182 ± 18  768 ± 12
   100  12 ± 2 12 ± 1  41 ± 3   161 ± 23 764 ± 7 
   333 14 ± 3  10 ± 3  41 ± 4  163 ± 3  750 ± 10 
   1000  14 ± 5 10 ± 4  42 ± 7  187 ± 7   772 ± 7
   2500 10 ± 5   12 ± 3 29 ± 3  171 ± 13  726 ± 58 
   5000  9 ± 5 15 ± 4  30 ± 6  113 ± 15  698 ± 53 
 2 -aminoanthracene 2.5   383 ± 34 235 ± 24   5046 ± 72 4825 ± 97   
 2 -aminoanthracene  10.0          1501 ± 13

Table 2: Summary of results from the salmonella typhimurium reverse mutation assay of experiment 2 (pre-incubation test) with the test substance (mean +/- SD of revertants per plate).

 Substance Dose (µg/plate)   Without metabolic activation
     TA 1535 TA 1537   TA 98 TA 100   TA 102
 DMSO    10 ± 4  8 ± 2  30 ± 6  147 ± 9  500 ± 47
 Untreated    11 ± 2  9 ± 2  29 ± 7 193 ± 6   457 ± 32
 test substance  3        145 ± 16  
   10      29 ± 3  149 ± 8  
   33  9 ± 2 9 ± 2   29 ± 3 142 ± 8   479 ± 17
   100  8 ± 2 8 ± 2   30 ± 9  68 ± 13  495 ± 4
   333  9 ± 3 8 ± 3   19 ± 4  51 ± 8  473 ± 20
   1000  7 ± 2  10 ± 2 15 ± 6   34 ± 9  405 ± 9
   2500  9 ± 2 8 ± 2   1 ± 1 1 ± 1   333 ± 28
   5000  8 ± 3 1 ± 1   1 ± 1  0 ± 1  297 ± 13
 Sodium azide  10  1051 ± 93      1958 ± 135  
 4 -nitro-o-phenylene-diamine  10      352 ± 32    
 4 -nitro-o-phenylene-diamine  50    85 ± 19      
 methyl methane sulfonate  2.0 µL        4348 ± 421
 Substance  Dose (µg/plate)              With metabolic activation
     TA 1535  TA 1537  TA 98  TA 100  TA 102
 DMSO    15 ± 1 16 ± 5  45 ± 6   132 ± 24  633 ± 5
 Untreated    12 ± 2  18 ± 6  38 ± 1 197 ± 24   667 ± 11
 Test substance  3        121 ± 11  
   10      44 ± 7  145 ± 4  
   33  13 ± 5 16 ± 5  37 ± 11  139 ± 14  605 ± 27 
   100  12 ± 5 13 ± 5  34 ± 11   151 ± 14 659 ± 67 
   333  12 ± 6  15 ± 5  36 ± 5  107 ± 8  616 ± 5
   1000  14 ± 3 16 ± 5   40 ± 16  133 ± 16  646 ± 67
   2500  12 ± 4  12 ± 3  40 ± 2  89 ± 2  583 ± 72
   5000  11 ± 5  14 ± 2 38 ± 2   36 ± 8  534 ± 49
 2 -aminoanthracene  2.5  385 ± 12  178 ± 28  3323 ± 333 3923 ± 461  
 2 -aminoanthracene            1575 ± 177

No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with Trimethylessigsäureanhydrid at any concentration level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance. Appropriate reference mutagens were used as positive controls. They showed a distinct in-crease in induced revertant colonies.

Applicant's summary and conclusion

Executive summary:

This study was performed to investigate the potential of the test substance to induce gene mutations (according to OECD guideline 471) according to the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100, and TA 102. The assay was performed in two independent experiments both with and without liver microsomal activation in concentrations up to 5000 µg/plate. It can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used. Therefore, the test substance is considered to be non-mutagenic in this Salmonella typhimurium reverse mutation assay.