Registration Dossier

Diss Factsheets

Administrative data

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:
1998-01-27 to 1998-02-19
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted in compliance with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results. The study report was conclusive and done to a valid guideline.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1998
Report date:
1998

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
Ethyl (3aR,7R,7aR)-2,2-dimethyl-7-[(methylsulfonyl)oxy]-3a,6,7,7a-tetrahydro-1,3-benzodioxole-5-carboxylate
EC Number:
606-548-2
Cas Number:
204254-84-2
Molecular formula:
C13 H20 O7 S
IUPAC Name:
Ethyl (3aR,7R,7aR)-2,2-dimethyl-7-[(methylsulfonyl)oxy]-3a,6,7,7a-tetrahydro-1,3-benzodioxole-5-carboxylate
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
White powder
Purity 98.5%
Retest date: 17 October 1998

Method

Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535
Species / strain / cell type:
S. typhimurium TA 97
Species / strain / cell type:
S. typhimurium TA 98
Species / strain / cell type:
S. typhimurium TA 100
Species / strain / cell type:
S. typhimurium TA 102
Metabolic activation:
with and without
Metabolic activation system:
S9
Test concentrations with justification for top dose:
Preliminary test with TA100 (+/- S9): 0, 1.6, 5, 16, 50, 166, 500, 1666, 3333 and 5000 ug per plate.
Main experiment: 50 - 5000 ug/plate.
Vehicle / solvent:
Dimethylsulfoxide
Controls
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
sodium azide
other: ICR 191
Details on test system and experimental conditions:
Nutrient broth cultures of each strain, supplemented with 9 % DMSO were stored in liquid nitrogen. Subcultures with a normal spontaneous frequency were stored in NB + 9 % DMSO at -75°C + 20°C. The strain identities and characteristics were periodically checked by the recommended procedures (De Serres and Shelby, 1979; Zeiger et al., 1981; Maron and Ames, 1983).
For use in tests, cultures of the strains were grown overnight at 37°C in a shaking water bath in a NB liquid medium. The strain TA102 was incubated with 0.3 pg tetracycline per ml NB medium in order to ensure the presence of an adequate number of plasmids (Albertini and Gocke, 1988). The growth of the overnight cultures was controlled by measuring the optical density on a photometer (Lumetron Colorimeter) at 650 nm. Each
bacterial strain was diluted 10’6 in 0.85 % sodium chloride, and 100 pI of the last dilution step was plated on a NB complete medium. Two replicate plates were incubated at 37°C, upside down, for two days. The number of colonies was registered and the number of cells plated on VB medium was calculated.
The sensitivity of the Salmonella typhimurium strains was verified using the following positive controls: sodium azide with strains TA1535 and TA100, ICR 191 with strain TA97, 2-nitrofluorene with strain TA98, and MMC with strain TA102. Moreover, 2-aminoanthracene was used with all strains with and without metabolic activation to examine the activity of the S9 mix.
Evaluation criteria:
A positive result was defined as a reproducible, dose-related increase in the number of his+ revertants. The increase should reach at least a doubling of the number of spontaneous revertants for Salmonella typhimurium strains TA1535 and TA98. For strains TA97, TA100 and TA102 a 1.5 - fold increase over control values might be indicative of a mutagenic effect provided the negative control values fall within the historical control data.

Results and discussion

Test results
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Table of Revertants per plate for the reverse mutation assay (mean values and standard deviations)

 Strain  TA1535  TA1535  TA97  TA97  TA98  TA98  TA100  TA100  TA102  TA102
 Activation  -S9  +S9  -S9  +S9  -S9  +S9  -S9  +S9  -S9  +S9
 Concentration ug/plate                    
 0  17±5  7 ±2  150 ±1  187 ±38  21 ±9  25  ±6 206  ±  210  ±21  371  ±40 465  ± 13 
 50 19  ± 10  ± 165  ± 178  ±  25  ±3  28  ±8  201  ±11  209  ±8  380  ±10  492  ±34
 166  18  ±3  6  ±3  147  ±5  184  ±7  21  ±3  26  ±2  185  ±7  198  ±1  408  ±49  466  ±14
 500  17  ±4  7  ±2  171  ±9  175  ±13  22  ±4  25  ±8  200  ±22  212  ±32  385  ±17  456  ±7
 1666  15  ±2  7  ±1  148  ±19  168  ±13  37  ±28  26  ±2  203  ±13  196  ±16  378  ±33  419  ±36
 5000  20  ±4  6  ±1  155  ±19  201  ±27  20  ±5  28  ±3  198  ±14  194  ±9  306  ±12  418  ±17

Table of Revertants per plate after pre-incubation for the reverse mutation assay (mean values and standard deviations)

 Strain  TA1535  TA1535  TA97  TA97  TA98  TA98  TA100  TA100  TA102  TA102
 Activation  -S9  +S9  -S9  +S9  -S9  +S9  -S9  +S9  -S9  +S9
 Concentration ug/plate                    
 0  12±6  5 ±3  184±19  236 ±23  19 ±5  15  ±5 173±12  191±14  322 ±13 428 ±26 
 50 18 ±4  ±1  211 ± 227 ±15   16 ±2  20 ±3  194 ±8  186 ±14  325 ±11  456 ±23
 166  16 ±1  6  ±3  210 ±7  251  ±7  14 ±7  17 ±1  163 ±18  166 ±11  319  ±9  431 ±32
 500  20 ±4  9 ±4  217 ±3  238 ±6  21 ±8  20 ±6  174 ±1  171 ±27  335 ±16  461 ±25
 1666  16 ±4  10 ±4  216 ±16  230 ±12  14 ±3  17 ±3  117 ±8  175 ±9  258 ±31  475 ±12
 5000  19 ±3  8 ±3  229 ±47  226 ±9  17 ±2  21 ±3  183 ±23  177 ±12  241 ±19  427 ±60

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative both with and without S9 activation

Neither the test item or its metabolites formed under the experimental conditions were mutagenic in the Ames test.
Executive summary:

Introduction: The test item was evaluated in the Ames test. For determination of the top dose a preliminary toxicity experiment was performed using strain TA100 (+S9). The compound, a white powder, was dissolved in dimethylsulfoxide (DMSO). No toxic effects or increase in revertants were noted up to 5,000 ug/plate. Precipitation in the aqueous medium was noted at 3333 ug/plate.

Results: Based upon the preliminary test the concentration range of 50 -5,000 ug/plate was selected for the main experiment. Upon addition to the aqueous medium in the standard plate incorporation assay a milky suspension was observed at 5’000 pg/plate, which disappeared after shaking. Using the preincubation assay increasingly milky suspensions were formed starting at 1’666 pg/plate. For all strains without S9 the plates at the highest test concentration of 5’000 pg/plate had to be counted manually due to strong precipitation of the compound (preincubation assay).

The mutant frequencies of the controls were in the range of the historical controls and published data. The positive controls induced significant increases in the mutant frequencies verifying the sensitivity of the strains used.

For TA100 the spontaneous rate using the standard assay was at the upper range. Nevertheless the experiment was considered acceptable, since the positive controls verified the responsiveness of the culture used. Neither in the standard plate incorporation nor in the preincubation assay any increase in the mutant frequency was observed for all five tester strains (TA1535, TA97, TA98,

TA100, and TA102) after treatment with the test item.

Conclusion: Neither the test item or its metabolites formed under the experimental conditions were mutagenic in the Ames test.