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Diss Factsheets

Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
March 1991
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1991
Report date:
1991

Materials and methods

Test guideline
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
26 May 1983
Deviations:
no
Principles of method if other than guideline:
Direct plate incorporation procedure was performed. No E. coli WP2 or S. typhimurium TA102 strain tested; no preincubation test performed. These requirements were first formulated in the adoption of the guideline in 1997 and thus the study was conducted prior to implementation of these requirements. The current OECD TG 471 requires at least 5 test strains and the use of E. coli WP2 strains or Salmonella typhimurium TA 102 to detect certain oxidizing mutagens, cross-linking agents and hydrazines. However, the substance is not a highly reactive agent and is therefore not expected to be a cross-linking agent, has no oxidizing properties and is no hydrazine. Thus, a GLP test according to former versions of OECD TG 471 without E. coli WP2 strains or Salmonella typhimurium TA 102 is considered as sufficient to evaluate the mutagenic activity of the substance in this bacterial test system.
GLP compliance:
yes
Remarks:
- but a QA check was not performed
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
9beta, 11beta-Epoxy-6alpha-fluoro-16alpha-methyl-21-valeryloxy-1,4-pregnadiene-3,20-dione
Cas Number:
65535-29-7
Molecular formula:
C27 H35 F O5
IUPAC Name:
9beta, 11beta-Epoxy-6alpha-fluoro-16alpha-methyl-21-valeryloxy-1,4-pregnadiene-3,20-dione
Test material form:
solid

Method

Target gene:
Histidine gene locus
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1538
Additional strain / cell type characteristics:
not applicable
Species / strain / cell type:
S. typhimurium TA 1537
Additional strain / cell type characteristics:
not applicable
Species / strain / cell type:
S. typhimurium TA 1535
Additional strain / cell type characteristics:
not applicable
Species / strain / cell type:
S. typhimurium TA 100
Additional strain / cell type characteristics:
not applicable
Species / strain / cell type:
S. typhimurium TA 98
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254 induced male rat liver S9 mix
Test concentrations with justification for top dose:
0.1, 0.25, 0.5, 1.0, 2.5, 5.0 mg/plate
Vehicle / solvent:
DMSO
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
2-nitrofluorene
sodium azide
benzo(a)pyrene
cyclophosphamide
other: with metabolic activation: 2-AA
Details on test system and experimental conditions:
NUMBER OF REPLICATIONS:
- Number of cultures per concentration: triplicate
- Number of independent experiments: one

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): Between 13.3 and 24 xE+08 cells/mL
- Test substance added in agar (plate incorporation)

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: background growth inhibition


Evaluation criteria:
The plates were scored for the number of mutant colonies with an automated colony counter (Artek M 982B, Artek Systems Corporation, Farmingdale, NY, USA). The arithmetic means of the number of mutant colonies of the 3 parallel plates in the negative control groups were compared with those of the compound groups. A positive response was considered if at least 5 mg/plate or up to a toxic dose had been tested (or the compound formed precipitates in the agar) and if the number of induced revertants compared to the number of spontaneous ones was reproducibly higher than 2-fold. A dose-dependent increase in the number of revertants was also considered to indicate a mutagenic effect.

Results and discussion

Test resultsopen allclose all
Species / strain:
S. typhimurium TA 102
Remarks:
or E.coli WP2 uvr A
Metabolic activation:
not applicable
Genotoxicity:
not determined
Cytotoxicity / choice of top concentrations:
not determined
Vehicle controls validity:
not examined
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
not examined
Remarks:
The study was conducted prior to implementation of the fifth tester strain into OECD guideline 471. However, based on the additionally performed QSAR analysis, the substance showed no alert for mutagenicity. Please refer to the respective robust study sum
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Remarks:
Precipitates in the agar were found starting at 1 mg/plate without S9 mix and at 2.5 mg/plate with activation.
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Remarks:
Precipitates in the agar were found starting at 1 mg/plate without S9 mix and at 2.5 mg/plate with activation.
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Remarks:
Precipitates in the agar were found starting at 1 mg/plate without S9 mix and at 2.5 mg/plate with activation.
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Remarks:
Precipitates in the agar were found starting at 1 mg/plate without S9 mix and at 2.5 mg/plate with activation.
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH: no data
- Data on osmolality: not data
- Precipitation and time of the determination: Precipitates in the agar were found starting at 1 mg/plate without S9 mix and at 2.5 mg/plate with activation.

STUDY RESULTS
- Concurrent vehicle negative and positive control data:
0.05 ml of the solvents (0.1 M sodium phosphate buffer or DMSO) were plated as negative controls. In order to check the activity of the metabolizing system and the mutability of the bacteria at least two reference mutagens were tested for each strain. Their mutagenic effect occurred either directly {9-AA, 2-NF, NaN3} or after metabolic activation (2-AA, BP, CP). Sterility controls were performed additionally.
According to the evaluation criteria the vehicle controls and the positive control were valid.
Please refer to the tables under any other information on results incl. tables.


Ames test:
- Signs of toxicity: no
- Individual plate counts:
TA1535: 226, 259, 236
TA1537: 126, 127, 146
TA1538: 188, 167, 176
TA100: 193, 198, 178
TA98: 206, 242, 243
- Mean number of revertant colonies per plate and standard deviation:
Please refer to table 2 under any other information on results incl. tables.

Any other information on results incl. tables

None of the five tester strains showed increased reversion to prototrophy with the test item at the concentrations tested, either in the absence or presence of S9 mix.


 


Growth inhibition of the background lawn was not observed. Precipitates in the agar were found starting at 1 mg/plate without S9 mix and at 2.5 mg/plate with metabolic activation.


 


Negative controls and positive controls with known mutagens (9-acridinamine hydrochloride, anthracen-2-amine, benzo(a)pyrene, cyclophosphamide, 2-nitro-9H-fluorene, sodium azide) produced the expected numbers of revertant colonies.


Table 1: Negative and positive control data:



































































































































 



TA1535



TA100



TA1537



TA1538



TA98



 



-S9



+S9



-S9



+S9



-S9



+S9



-S9



+S9



-S9



+S9



DMSO



20±5



14±4



106±11



88±14



9±4



12±1



17±6



41±4



29±6



40±9



phosphate buffer



19±4



12±3



107±4



88±5



8±4



15±4



19±1



41±5



36±2



44±3



2-AA



16±2



102±19



131±13



802±84



10±1



234±13



19±4



1501±62



38±7



1610±19



CP



23±3



89±3



 



 



 



 



 



 



 



 



BaP



 



 



107±8



622±36



10±3



59±7



23±7



135±27



27±3



283±26



2-NF



 



 



 



 



 



 



1046±63



470±32



937±93



420±33



NaN3



630±57



88±9



699±45



145±11



 



 



 



 



 



 



9-AA



 



 



 



 



89±18



37±9



 



 



 



 



mean values ± SD


Table 2:Mean number of revertant colonies per plate and standard deviation



































































































































 



TA1535



TA100



TA1537



TA1538



TA98



 



-S9



+S9



-S9



+S9



-S9



+S9



-S9



+S9



-S9



+S9



DMSO



20±5



14±4



106±11



88±14



9±4



12±1



17±6



41±4



29±6



40±9



phosphate buffer



19±4



12±3



107±4



88±5



8±4



15±4



19±1



41±5



36±2



44±3



0.10



17±4



14±5



106±11



89±3



7±4



15±2



18±6



32±6



33±11



38±8



0.25



21±4



13±1



102±7



87±3



9±4



15±6



16±1



35±11



31±4



45±12



0.50



23±4



15±3



99±7



105±10



8±1



12±1



18±7



37±5



33±4



44±4



1.00



24±2



16±5



123±14



92±8



10±2



11±9



18±4



47±5



30±3



39±5



2.50



19±4



12±1



116±9



112±5



8±3



14±4



19±2



42±12



34±12



47±3



5.00



23±12



14±2



121±8



112±4



6±2



13±3



23±4



36±4



32±6



40±5



 


 


 


 

Applicant's summary and conclusion

Conclusions:
The test item was tested for mutagenic activity effects in five histidine-dependent strains of S. typhimurium (TA 98, TA 100, TA 1535, TA 1537 and TA 1538 ) using the direct plate incorporation procedure equivalent to OECD TG 471. The study was performed with and without metabolic activation, employed a range of the test item concentrations from 0.1 to 5.0 mg per plate. No increased reversion to prototrophy was seen neither without nor with metabolic activation. Growth inhibition of the background lawn was not observed. Precipitation was found starting at 1 mg/plate without S9 mix and at 2.5 mg/plate with metabolic activation. Therefore, the test item was considered to be non-mutagenic in the Salmonella typhimurium reverse mutation assay.
Executive summary:

In a reverse gene mutation assay in bacteria according to OECD TG 471 (adopted 26 May 1983), strains TA 98, TA 100, TA 1535, TA 1537 and TA 1538 of S. typhimurium were exposed to Epoxide-Valerate in DMSO at concentrations of 100, 250, 500, 1000, 2500, and 5000 µg/plate in the presence and absence of mammalian metabolic activation using the plate incorporation method.


The test item was tested limit concentration 5000 µg/plate. None of the five tester strains showed increased reversion to prototrophy at any of the concentrations tested between 100 and 5000 µg/plate, either in the absence or presence of S9 mix. The positive controls induced the appropriate responses in the corresponding strains. Growth inhibition of the background lawn was not observed. Precipitates in the agar were found starting at 1000 µg/plate without S9 mix and at 2500 µg/plate with metabolic activation.


This study is classified as acceptable. This study satisfies the requirement for Test OECD 471 for in vitro mutagenicity (bacterial reverse gene mutation) data.


The test material is considered non-mutagenic under the conditions of the test.