Registration Dossier
Registration Dossier
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EC number: 611-173-2 | CAS number: 54605-02-6
Endpoint summary
Administrative data
Key value for chemical safety assessment
Genetic toxicity in vitro
Description of key information
Bromhydrin-valerate is not mutagenic based on bacterial reverse mutation assays with the read-across substance diflucortolone-21-valerate (negative +/- S9 mix in S. typhimurium strains TA 98, TA 100, TA 1535, TA 1537 and TA 1538 (Lang and Sprenger-Semik, 1993; Lang and Schmitt, 1984).
Additionally, QSAR predictions have been performed with both Difluocortolone-valerate and Bromhydrin-valerate. The results for both substances were obtained from predictions made with Leadscope and DEREK. All predictions were negative, thus, substantiating the experimental results obtained with the source substance.
Link to relevant study records
- Endpoint:
- in vitro gene mutation study in mammalian cells
- Type of information:
- experimental study
- Adequacy of study:
- supporting study
- Study period:
- 1992
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
- Version / remarks:
- 4 April 1984
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- in vitro mammalian cell gene mutation test using the Hprt and xprt genes
- Target gene:
- HPRT gene
- Species / strain / cell type:
- Chinese hamster lung fibroblasts (V79)
- Additional strain / cell type characteristics:
- not applicable
- Metabolic activation:
- with and without
- Metabolic activation system:
- Type and composition of metabolic activation system:
- source of S9: The 59 liver microsomal fraction was obtained from the liver of 8 - 12 weeks old male rats, strain Wistar/WU
- method of preparation of S9 mix: The 59 liver microsomal fraction was obtained from the liver of 8 - 12 weeks old male rats, strain Wistar/WU (SAVO, med. Versuchstierzuchten GmbH, 0-7964 Kissleggi weight approximately 150 - 200 g) which received a single i.p. injection of 500 mg/kg b.w. Aroclor 1254 (Antechnika, 0-7500 Karlsruhe,F.R.G.) in olive oil 5 days previously. After cervical dis1ocation, the livers of the animals were removed, washed in 150 rnM KCl and homogenized. The homogenate was diluted 1:4 in KCI and centrifuged twice at 9,000 x g at 4 °C for 10 minutes. A stock of the supernatant containing the microsomes was frozen in ampoules of 2 or 5 ml and stored at -70°C. Small
numbers of the ampoules were kept at -20°C for only several weeks be fore use. The .protein content was determined using the
analysis kit of Bio-Rad Laboratories, 0-8000 München: Bio-Rad protein assay, Catalogue 500 000 6.
- concentration or volume of S9 mix and S9 in the final culture medium : The protein concentration in the 59 preparation was 37.8 mg/mI. Final protein concentration of 0.75 mg/ml in the cultures. - Test concentrations with justification for top dose:
- Aecording to the reeommendations in several guidelines the highest concentration should be 10 mM but not higher than 5 mg/mI unless limited by solubility or cytotoxicity of the test article. In ease of toxicity the plating efficiency may be reduced to less than 50 % and/or eulture growth at subcultivation should be at least 20 % of the corresponding control.
In the pre-test for toxicity (colony forming ability) the plating efficiency of the V79 cells was slightly reduced after treatment with 50.0 µg/ml and 100.0 µg/ml without metabolie activation. Therefore, the main experiments were performed with six concentrations ranging from 5.0 µg/ml to 100.0 µg/ml without and with metabolic activation. During the course of the main experiments four (experiment II without metabolic activation: five concentrations were selected to be evaluated at the end of the experiments concerning concentration range and toxicity.Precipitation of the test article was observed at concentrations higher than 20.0 µg/ml. - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: DMSO
- Justification for percentage of solvent in the final culture medium: 1% - Untreated negative controls:
- yes
- Remarks:
- not further specified
- Negative solvent / vehicle controls:
- yes
- Remarks:
- DMSO
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- 7,12-dimethylbenzanthracene
- ethylmethanesulphonate
- Details on test system and experimental conditions:
- NUMBER OF REPLICATIONS:
- Number of cultures per concentration : a) About 500 cells in 5 ml medium/25 cm²-plastic-flask for
plating efficiency in duplicate per experimental point
b) 2 x E+06 cells in 30 ml medium/175 cm²-plastic-flask for the mutagenicity test, 1 flask per experimental
point
- Number of independent experiments: two
METHOD OF TREATMENT/ EXPOSURE:
- Test substance added in medium
TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable:
- Exposure duration/duration of treatment: 4 h
- Harvest time after the end of treatment (sampling/recovery times): 7 days
FOR GENE MUTATION:
- Expression time (cells in growth medium between treatment and selection): 7 days
- Selection time (if incubation with a selective agent): 7 days
- Fixation time (start of exposure up to fixation or harvest of cells): 15 days
- If a selective agent is used (e.g., 6-thioguanine or trifluorothymidine), indicate its identity, its concentration and, duration and period of cell exposure. 11 µg/ml thioguanine (6TG, SIGMA GmbH, D-8024 Deisenhofen, F.R.G.)
- Number of cells seeded and method to enumerate numbers of viable and mutants cells: About 500 cells in 5 ml medium/25 cm²-plastic-flask for plating efficiency in duplicate per experimental point.
METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method, e.g.: Apre-test was performed in order to determine the concentration range for the mutagenicity experiments. The general culturing and experimental conditions in this pre-test were the same as described below for the mutagenicity experiment. In this pre-test the colony forming ability of approximately 500 single cells
(dulicate cultures per concentration level) after treatment with the test article was observed and compared to the controls. Toxicity of the test artiele was evidenced by a reduction in plating efficiency (PE). - Rationale for test conditions:
- As described in the respective OECD test guideline
- Evaluation criteria:
- A test article is classified as positive if it induces either a significant concentration-related increase in the mutant frequency or a reproducible and significant positive response for at least one of the test points.
A test article producing neither a significant concentration· related increase in the mutant frequency nor a significant and reproducible positive response at any one of the test points is considered non-mutagenic in this system.
A significant response is described as follows:
The test article is classified as mutagenic if it induces reproducibly with at least one of the concentrations a mutation frequency that is three times higher than the spontaneous mutation frequency in the experiment.
The test article is classified as mutagenic if there is a reproducible concentration - related increase in the mutation frequency. Such evaluation may be considered independently of an enhancement factor for induced mutants.
However, in a case by case evaluation this decision depends on the level of the corresponding negative control data. If there is by chance a low spontaneous mutation rate as compared to the range normally found (5 - 45 mutants per 1 x E+06 cells) a seemingly concentration-related increase of the mutations within this range will be regarded to be irrelevant. - Statistics:
- Since the distribution of mutant cells does not follow known statistical models, an adequate statistical method is not available.
- Key result
- Species / strain:
- Chinese hamster lung fibroblasts (V79)
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- 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: not reported
- Precipitation and time of the determination: precipitation occurred from 20.0 µg onwards
RANGE-FINDING/SCREENING STUDIES (if applicable): PE was over 70.=% either with or without metabolic activation, please refer also to any other information on results incl. tables.
STUDY RESULTS
- Concurrent vehicle negative and positive control data: please refer also to any other information on results incl. tables.
Gene mutation tests in mammalian cells:
- Results from cytotoxicity measurements:
o Relative total growth (RTG) or relative survival (RS) and cloning efficiency: PE decreased in both experiments wthout metabolic activation beginning at a concentration of 20 µg, please refer also to any other information on results incl. tables.
- Genotoxicity results:
o Number of cells treated and sub-cultures for each cultures: Approximately 2 x E+06 and 5 x E+02 cells, respectively, were seeded in each flask and treated after 24h of growth for 4 h, subsequently 500 cells were subcultured for plating efficiency and 2 x E+06 cells were cultured for mutagenicity.
o Number of cells plated in selective and non-selective medium: 3 x E+05 cells/flask in selection mediumand 500 cells/falsk for plating efficiency.
HISTORICAL CONTROL DATA (with ranges, means and standard deviation, and 95% control limits for the distribution as well as the number of data)
- Positive historical control data: please refer also to any other information on results incl. tables.
- Negative (solvent/vehicle) historical control data: please refer also to any other information on results incl. tables. - Conclusions:
- The test article ZK 22.612 was assessed for its potential to induce gene mutations at the HGPRT locus using V79 cells of the Chinese hamster in vitro. In both experiments without metabolic activation after treatment with concentrations higher than 20.0 µg/ml the plating efficiency of the cells was clearly reduced. At higher concentrations (80.0 µg/ml and higher), the plating efficiency increased again which may be due to precipitation and less available test article in the medium (see Table I and IV, PE% relative). With metabolic activation in both experiments, up to the highest concentration, no reduction of the plating efficiency of the cells was observed. A similar intermediate toxic effect without metabolic activation was found in the cell densities at the first subcultivation. Reduced cell counts were observed at 40.0µg/ml in experiment I and II and at 80.0 µg/ml in experiment II (see Table I and IV, cell density). Taking into account the mutation rates found in the groups treated with the test article compared to the negative and solvent controls it can be concluded that no relevant increase of gene mutations was observed. The test article did not induce a reproducible concentration-related increase in mutant colony numbers. The mutant values of the groups treated with the test article were in the range of the negative controls.
- Executive summary:
In a mammalian cell gene mutation assay according to OECD test guideline 476 (1984), V79 hamster fibroblast cells cultured in vitro were exposed to Difluocortolone-valerate, (100% a.i.), in DMSO at concentrations of 5.0;10.0; 20.0; 40.0; 60.0 and 80.0 µg/mL (Exp. I) and at 5.0; 20.0; 40.0; 60.0; 80.0 and 100.0 µg/mL (Exp. II) in the presence and absence of mammalian metabolic activation.
Difluocortolone was tested up to cytotoxic concentrations (i.e. 100.0 µg/mL. The positive controls did induce the appropriate response. There was no evidence of induced mutant colonies over background.
- Endpoint:
- in vitro cytogenicity / chromosome aberration study in mammalian cells
- Type of information:
- experimental study
- Adequacy of study:
- supporting study
- Study period:
- 1993
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
- Version / remarks:
- 26 May 1983
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- in vitro mammalian chromosome aberration test
- Species / strain / cell type:
- lymphocytes:
- Details on mammalian cell type (if applicable):
- CELLS USED
- Type and source of cells: human peripheral blood lymphocytes - Metabolic activation:
- with and without
- Metabolic activation system:
- Type and composition of metabolic activation system:
- source of S9: Preparation by CCR
- method of preparation of S9 mix: The 59 liver microsomal fraction was obtained from the livers of 8 - 12 weeks old male rats, strain Wistar/WU (SAVO, med. Versuchstierzuchten GmbH, 0-7964 Kisslegg, F.R.G.; weight approx. 150 - 200 g) which received a single i.p. injection of 500 mg/kg b.w. Aroclor 1254 (Antechnika, 0-7500 Karlsruhe, F.R.G.) in olive
oil 5 days previously. After cervical dislocation, the livers of the animals were removed , washed in 150 rnM KCl and homogenized. The homogenate, diluted 1+3 with KCI was centrifuged twice at 9,000 g for 10 minutes (4 °C). A stock of the supernatant containing the microsomes was frozen in ampoules of 2 or 3 ml and stored at -70 °C .
Small numbers of the ampoules were kept at -20 0 C for only several weeks before use. The protein content was determined using the analysis kit of Bio-Rad Laboratories, 0-8000 München: Bio-Rad protein assay, Catalogue No. 500 000 6.
The protein concentration in the S9 preparation is usually between 20 and 45 mg/mI. The protein concentration of the S9 used in both experiments was 37.8 mg/mI . - Test concentrations with justification for top dose:
- In a pre-test phase (cytotoxicity determination) the concentrations which should be chosen for cytogenetic scoring were determined. In this pre-test phase cytotoxicity of 8 concentrations of the test substance (inclusive negative and positive controls) was deterrnined. All cell cultures were set up at least in duplicate. Exposure times were 4 h (with S9 mix), 22 and 46 h (without S9 mix). The fixation intervals were 22 and 46 hours after start of the exposure. At the 22 h fixation interval, additional cultures (without S9 mix) were used .(solvent controls and four treatment groups) in the presence of BrdU (5-bromodeoxyuridine; 6 µg/ml) to assure that the majority of the cells prepared were in the first metaphase stage after treatment.
Treatment in the pre-test phase was performed with the following concentrations:
Experiment I
without S9 mix (exposure period 22 and 46 h):
22h: 0.3; 1.0; 3.0; 10.0; 30.0; 60.0; 80.0; 100.0 µg/ml
46h: 3.0; 10.0; 30.0; 60.0; 80.0; 100.0 µg/ml
with S9 mix (exposure period 4 h):
22h: 0.3; 1.0; 3.0; 10.0; 30.0; 60.0; 80.0; 100.0 µg/ml
46h: 3.0; 10.0; 30.0; 60.0; 80.0; 100.0 µg/ml
The results of the pre-test phase indicate that both in the absence and presence of S9 mix cytotoxic effects were observed starting at approx. 30.0 µg/ml and increasing up to 100.0 µg/ml. - Vehicle / solvent:
- DMSO
- Justification for percentage of solvent in the final culture medium: 1% - Untreated negative controls:
- yes
- Remarks:
- medium
- Negative solvent / vehicle controls:
- yes
- Remarks:
- DMSO
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- cyclophosphamide
- ethylmethanesulphonate
- Details on test system and experimental conditions:
- NUMBER OF REPLICATIONS:
- Number of cultures per concentration: duplicate
- Number of independent experiments: two
METHOD OF TREATMENT/ EXPOSURE:
- Test substance added in medium
TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: 4, 22, 46 h
FOR CHROMOSOME ABERRATION AND MICRONUCLEUS:
- Spindle inhibitor (cytogenetic assays): indicate the identity of mitotic spindle inhibitor used (e.g., colchicine), its concentration and, duration and period of cell exposure: Colcemide 0.2 µg/mL, 3h before harvest
- Methods of slide preparation and staining technique used including the stain used (for cytogenetic assays): The cultures were harvested by centrifugation 22 h and 46 h after beginning of treatment. The supernatant was discarded and the cells were resuspended in approximately 5 ml hypotonic solution (0.0375 M KCl). The cell suspension was then allowed to stand at 37°C for 20 minutes. After removal of the hypotonic solution by centrifugation the cells were fixed with 3 + 1 absolute methanol + glacial acetic acid. Per experiment slides were prepared by dropping the cell suspension on to a clean microscope slide. The cells were stained with Giemsa (MERCK, 0-6100 Oarmstadt) and according the Fluorescent plus Giemsa technique (6), respectively.
- Number of cells spread and analysed per concentration (number of replicate cultures and total number of cells scored): At least 100 weIl spread metaphases per culture were scored for cytogenetic damage on coded slides except for the positive control cultures where 25 metaphases were scored. Only metaphases with 46 centromer regions were included in the analysis.
- Determination of polyploidy: yes
- Determination of endoreplication: not reported
METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method, e.g.: Cytotoxicity was characterized by the percentages of mitotic suppression in comparison with the controls by counting 1000 cells per culture and duplicate. - Evaluation criteria:
- A test article is classified as mutagenic if it induces reproducibly either a concentration-related increase in the number of structural chromosomal aberrations or a significant and reproducible positive response for at least one of the test points. A test article producing reproducibly neither a concentration-related increase in the number of structural chromosomal aberrations nor a significant and reproducibly positive response at any
one of the test points is considered non-mutagenic in this system.
This can be confirmed by means of the chi-square test. However, both biological and statistical significance should be considered together . - Statistics:
- Statistical significance at the five per cent level (p < 0.05) was evaluated by means of the chi-square test. Evaluation was perforrned only for cells carrying aberrations exclusive gaps.
- Key result
- Species / strain:
- lymphocytes: human
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- 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
- Precipitation and time of the determination: precipitation occurred at the highest concentrations, please refer to the any other information on results incl. tables section.
RANGE-FINDING/SCREENING STUDIES (if applicable): please refer to the any other information on results incl. tables section.
STUDY RESULTS
- Concurrent vehicle negative and positive control data, yes, please refer to the any other information on results incl. tables section.
Chromosome aberration test (CA) in mammalian cells:
- Results from cytotoxicity measurements:
o For lymphocytres in primary cultures: mitotic index (MI), please refer to the any other information on results incl. tables section.
- Genotoxicity results (for both cell lines and lymphocytes)
o Number of cells scored for each culture and concentration, number of cells with chromosomal aberrations and type given separately for each treated and control culture, including and excludling gaps: please refer to the any other information on results incl. tables section.
o Changes in ploidy (polyploidy cells and cells with endoreduplicated chromosomes) if seen
HISTORICAL CONTROL DATA (with ranges, means and standard deviation, and 95% control limits for the distribution as well as the number of data)
- Positive historical control data: not provided
- Negative (solvent/vehicle) historical control data: not provided - Conclusions:
- The test article was assessed for its potential to induce chromosomal aberrations in human lymphocytes in vitro in the absence and presence of an extrinsic metabolizing system (S9 mix) .
Two independent experiments were performed. In each experimental group two parallel cultures were used. 100 metaphases per culture were scored for structural chromosome aberrations except for the positive control cultures where 25 metaphases were scored. Preparation of chromosomes was done 22 h (low, medium and high concentration range) and 46 h (high concentration range) after start of the treatment with the test article which was dissolved in DMSO. The exposure period was 22 hand 46 h without S9 mix and 4 h with S9 mix. In both experiments, the test item was tested up to concentration levels which were in the range were precipitation could be observed and cytotoxic effects did occur as indicated by an obvious reduction of the mitotic index.
In some test groups (especially in exp. I with and without S9 mix at interval 46 h) the evaluation of the mitotic index revealed some variations and did not follow consequently a concentration dependence (see pages 23-24). However, these variations (maybe due to preparation procedure) did not influence the validation of the study.
In both experiments, with test article no biologically relevant increase in the structural chromosomal aberration rate could be found when compared with the range of aberrations in the corresponding negative and solvent controls. These results were obtained at each fixation interval both in the absence and presence of 89 mix. In this study the aberration rates after treatment with the test article in the absence and the presence of S9 mix (exp. I: 0.00 % - 1.50 %; exp. 11: 0.00 % - 1.00 %) were in the range of the control data (exp.I: 0.00 % - 2.00 %; exp.II: 0.00 % - 0.50 %). The occurrence of polyploid metaphases was also reported. In both experiments no biologically relevant deviations from the control da ta (0.0 % - 1.0 %) was found after treatment with the test article (0.0 % - 1.5 %). EMS (0.33 mg/mI) and CPA (30.0 µg/ml) were used as positive controls and showed distinct increases in cells with structural chromosome aberrations.
In conclusion, it can be stated that in the described study and under the experimental conditions reported, the test article did not induce structural chromosome aberrations in human lymphocytes in vitro when tested up to the solubi1ity limit and cytotoxic concentrations. - Executive summary:
In a mammalian cell cytogenetics assay [Chromosome aberration] according to OECD test guideline 473, primary lymphocyte cultures were exposed to Difluocortolone in DMSO at concentrations of
Experiment I
without S9 mix (exposure period 22 hand 46 h):
22 h: 60.0; 80.0; 100.0 µg/ml
46 h: 60.0; 80.0 µg/ml
with S9 mix (exposure period 4 h):
22 h: 10.0; 80.0; 100.0 µg/ml
46 h: 80.0; 100.0 µg/ml
Experiment II
without S9 mix (exposure period 22 hand 46 h):
22 h: 30.0; 60.0; 80.0; 100.0 µg/ml
46 h: 60.0; 100.0 µg/ml
with S9 mix (exposure period 4 h):
22 h: 30.0; 60.0i 80.0i 100.0 µg/ml
46 h: 60.0; 100.0 µg/ml.
Difluocortolone was tested up to cytotoxic or precipitating concentrations.
In both experiments, with test article no biologically relevant increase in the structural chromosomal aberration rate could be found when compared with the range of aberrations in the corresponding negative and solvent controls. These results were obtained at each fixation interval both in the absence and presence of 89 mix. In this study the aberration rates after treatment with the test article in the absence and the presence of S9 mix (exp. I: 0.00 % - 1.50 %; exp. 11: 0.00 % - 1.00 %) were in the range of the control data (exp.I: 0.00 % - 2.00 %; exp.II: 0.00 % - 0.50 %). The occurrence of polyploid metaphases was also reported. In both experiments no biologically relevant deviations from the control data (0.0 % - 1.0 %) was found after treatment with the test article (0.0 % - 1.5 %).
Positive controls induced the appropriate response. There was no evidence of Chromosome aberration induced over background.
- Endpoint:
- in vitro gene mutation study in bacteria
- Type of information:
- (Q)SAR
- Adequacy of study:
- supporting study
- Study period:
- 2021
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- results derived from a valid (Q)SAR model and falling into its applicability domain, with adequate and reliable documentation / justification
- Justification for type of information:
- 1. SOFTWARE
Leadscope model applier (v3.0.2)
2. MODEL (incl. version number)
Leadscope model applier (v3.0.2)
3. SMILES OR OTHER IDENTIFIERS USED AS INPUT FOR THE MODEL
CAS: 59198-70-8; Chemical name: Di-fluocortolone; SMILES: CCCCC(=O)OCC(=O)C3C(C)CC4C2CC(F)C1=CC(=O)C=CC1(C)C2(F)C(O)CC34C
4. SCIENTIFIC VALIDITY OF THE (Q)SAR MODEL
- Defined endpoint: QMRF 4.10. Mutagenicity OECD 471 Bacterial Reverse Mutation Test
- Unambiguous algorithm:
A new ICH M7 compliant expert alert system to predict the mutagenic potential of impurities (white paper) http://www.leadscope.com/white_papers/ICHM7-WhitePaper-0314.pdf
The logic for matching alerts is detailed in "A new ICH M7 compliant expert alert system to predict the mutagenic potential of impurities" (white paper): http://www.leadscope.com/white_papers/ICHM7-WhitePaper-0314.pdf
- Defined domain of applicability: The applicability domain is defined as having at least one chemical in a reference set with at least 30% global similarity to the test structure (using the Leadscope 27,000 chemical fragments as descriptors and the Tanimoto similarity score).
- Appropriate measures of goodness-of-fit and robustness and predictivity: Chemicals/descriptor ratio: 241 alerts for 11,528 reference chemicals (ratio = 48); Alerts are run within the Leadscope model applier that provides the capability to specify one or more compounds (using SMILES, Mol files, SD files, or copying from the clipboard), select and run the alerts, assess the applicability domain, and view the results including an explanation for any prediction (such as a full description of any matched alerts). The performance was assessed using the Hansen dataset comprised of 3,700 chemicals (47% positive).
Concordance = 83%, Sensitivity = 92%, Specificity = 70%, Positive
Predictivity = 81%, Negative Predictivity = 86% , coverage = 95% were
obtained.
- Mechanistic interpretation: Accompanying any positive prediction, any alert(s) that match the test compounds are described including a description of the mechanistic basis from the literature reference that cites the alert.
5. APPLICABILITY DOMAIN
- Descriptor domain: The applicability domain is defined as having at least one chemical in a reference set with at least 30% global similarity to the test structure (using the Leadscope 27,000 chemical fragments as descriptors and the Tanimoto similarity score).
- Structural domain: Leadscope Predictive Data Miner is a software program for systematic sub‐structural analysis of a chemical using predefined structural features stored in a template library, training set‐dependent generated structural features (scaffolds) and calculated molecular descriptors. The feature library contains approximately 27,000 pre‐defined structural features and the structural features chosen for the library are motivated by those typically found in small molecules: aromatics, heterocycles, spacer groups, simple substituents. Leadscope allows for the generation of training set‐dependent structural features (scaffold generation), and these features can be added to the pre‐defined structural features from the library and be included in the descriptor selection process.
- Mechanistic domain: The global model identifies structural features and molecular descriptors which in the model development was found to be statistically significant associated with effect. Many predictions may indicate modes of action that are obvious for persons with expert knowledge for the endpoint
- Similarity with analogues in the training set: The original data set from Kazius et al. (2005) consisted of 4337 molecular structures with corresponding Ames test data.
The structural similarity of the test compound with respect to the training set compounds was analysed and quantified in terms of Tanimoto distance, which provides a quantitative measure of structural relatedness between the test compound and each training set compound. The 25 training set compounds found to be mostly similar to the test compound.
6. ADEQUACY OF THE RESULT
As can be seen from Annex A and B of the QPRF the result is considered adequate due to the presence of almost all structural features of the parent compound which can also be found in the training/validation dataset. Furthermore the prediction substantiate the experimental result for the substance of interest. - Qualifier:
- no guideline followed
- Principles of method if other than guideline:
- - Software tool(s) used including version: Leadscope model applier (v3.0.2)
- Model(s) used: Leadscope model applier (v3.0.2)
- Model description: see field 'Attached justification'
- Justification of QSAR prediction: see field 'Attached justification' - GLP compliance:
- no
- Type of assay:
- bacterial reverse mutation assay
- Species / strain / cell type:
- bacteria, other: Combination of results from the S. typhimurium histidine reversion gene mutation test using tester strains TA97, TA97a, TA1537, TA98, TA100, TA1535, TA102, E.coli (any variant)
- Additional strain / cell type characteristics:
- not applicable
- Remarks:
- The QSAR prediction is based on results from all tester strains recommended by the OECD Test guideline
- Evaluation criteria:
- The model used was the Leadscope Applier which is a statistical model using structural fragments to set an alert. Only descrete organic compounds can be predicted. The model searches for structural fragments and combines them with eight molecular descriptors. Thus, a probability of either a negative or positive result is calculated. If experimental data are available the prediction of the statistical model may be overruled.
- Key result
- Species / strain:
- other: not applicable for in silico study
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Remarks:
- The test item showed no alerts for mutagenicity. Therefore the test item is not considered to be mutagenic.
- Cytotoxicity / choice of top concentrations:
- other: not applicable
- Vehicle controls validity:
- not applicable
- Untreated negative controls validity:
- not applicable
- True negative controls validity:
- not applicable
- Positive controls validity:
- not applicable
- Conclusions:
- Based on the predictions performed with the statistical QSAR model Leadscope Applier Difluocortolone is not mutagenic in a bacterial reverse mutation assay.
- Executive summary:
In a QSAR prediction using Leadscope Model Applier (v3.0.2) the potential of Difluocortolone to induce mutagenicity was assessed. Leadscope uses two parameters to guide the applicability of model domain: 1) having at least one structural feature defined in the model in addition to all the property descriptors; 2) having at least one chemical in a training neighbourhood with at least 30% global similarity to the test structure. In this case the prediction is within the applicability domain, since 28 training compounds were identified in the model training set being structurally similar to the test compound.
The query structure does not match structural alerts or examples for (bacterial in vitro) mutagenicity in Leadscope.
Based on these results Difluocortolone is not considered mutagenic as predicted by Leadscope.
This study is classified as acceptable for assessment based on methodology and documentation. This study satisfies the requirement for Test Guideline OECD 471 for in vitro mutagenicity (bacterial reverse gene mutation) and the data is part of an overall assessment.
- Endpoint:
- in vitro gene mutation study in bacteria
- Type of information:
- (Q)SAR
- Adequacy of study:
- supporting study
- Study period:
- 2021
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- results derived from a valid (Q)SAR model and falling into its applicability domain, with adequate and reliable documentation / justification
- Justification for type of information:
- 1. SOFTWARE
DEREK Nexus 6.1
2. MODEL (incl. version number)
DEREK Nexus 6.1
3. SMILES OR OTHER IDENTIFIERS USED AS INPUT FOR THE MODEL
Di-fluocortolone
4. SCIENTIFIC VALIDITY OF THE (Q)SAR MODEL
- Defined endpoint: TOX 7.6.1. Genetic toxicity in vitro
- Unambiguous algorithm: logic of argumentation. Derek Nexus makes qualitative predictions for and against toxicity through reasoning. For the endpoint of mutagenicity, predictions for toxicity decrease in confidence in the following order: certain>probable>plausible>equivocal. Predictions against toxicity increase in confidence in the following order: inactive (with unclassified and/or misclassified features) inactive
- Defined domain of applicability: The scopes of the structure-activity relationships describing the mutagenicity endpoint are defined by the developer to be the applicability domain for the model. Therefore, if a chemical activates an alert describing a structure-activity for mutagenicity it can be considered to be within the applicability domain. If a compound does not activate an alert or reasoning rule then Derek makes a negative prediction. The applicability of the negative prediction to the query compounds can be determined by an expert, if required, by investigating the presence (or absence) of misclassified and/or unclassified features. The applicability domain of each alert is defined by the alert developer on the basis of the training set data and expert judgement on the chemical and biological factors which affect the mechanism of action for each alert. For non-alerting compounds, users should determine the applicability of negative predictions by evaluating the information supplied by Derek (i.e. the presence or absence of misclassified and/or unclassified features).
- Appropriate measures of goodness-of-fit and robustness and predictivity: n/a
- Mechanistic interpretation: All alerts describing structure-activity relationships for the mutagenicity endpoint have a mechanistic basis wherever possible.
Mechanistic information is detailed in the comments associated with an alert and can include information on both the mechanism of action and biological target. The mechanistic basis of the model was developed a priori by examining the toxicological and mechanistic evidence before developing the structure-activity relationship.
5. APPLICABILITY DOMAIN
- Descriptor domain:
[1]Markush structures encoding activating and deactivating features (known as patterns in the Derek Nexus knowledge base)
[2]count of non-hydrogen atoms
[3]ClogP
[4]2D structural fragments
There is an a priori assumption that patterns and associated reasoning will be used to model toxicity within Derek Nexus. Further, experts identified that misclassified and unclassified features were useful descriptors for determining the reliability of negative predictions for non-alerting compounds.
- Similarity with analogues in the training set: Non-proprietary elements of the training set are available through the references, and illustrated by the examples, within Derek Nexus. The illustrative examples are not available, due to the proprietary nature of Derek Nexus.
6. ADEQUACY OF THE RESULT
Based on the common structure of the substance, the result is considered reliable. - Qualifier:
- no guideline followed
- Principles of method if other than guideline:
- - Software tool(s) used including version: DEREK Nexus 6.1
- Model(s) used: DEREK Nexus 6.1
- Model description: see field 'Attached justification'
- Justification of QSAR prediction: see field 'Attached justification' - GLP compliance:
- no
- Type of assay:
- bacterial reverse mutation assay
- Species / strain / cell type:
- bacteria, other: Predictions are made for the domain of bacteria and can be broken down into species (e.g. Salmonella typhimurium and Escherichia coli)
- Additional strain / cell type characteristics:
- other: The prediction is based on results from all tester strains recommended by the OECD Test Guideline
- Evaluation criteria:
- Two types of models were used to predict the mutagenic potential of the test item.
The DEREK Nexus model was used as a rule-based model which is based on the training set data and expert judgement on the chemical and biological factors which affect the mechanism of action for each alert. The second model used was the Leadscope Applier which is a statistical model using structural fragments to set an alert. If experimental data are available the prediction of the statistical model may be overruled. - Key result
- Species / strain:
- other: Not applicable for in silico study
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- other: not applicable
- Vehicle controls validity:
- not applicable
- Untreated negative controls validity:
- not applicable
- True negative controls validity:
- not applicable
- Positive controls validity:
- not applicable
- Conclusions:
- Based on the predictions performed with the statistical QSAR model Leadscope Applier and the rule-based model DEREK Nexus Diflucortolone is not mutagenic in a bacterial reverse mutation assay.
- Executive summary:
In a QSAR prediction using DEREK Nexus v6.1 the potential of Diflucortolone to induce mutagenicity was assessed. Derek Nexus makes qualitative predictions for and against toxicity through reasoning. For the endpoint of mutagenicity, predictions for toxicity decrease in confidence in the following order: certain>probable>plausible>equivocal. Predictions against toxicity increase in confidence in the following order: inactive (with unclassified and/or misclassified features)<inactive<improbable. Likelihood levels have been shown to correlate with predictivity [Judson et al, 2013]. Multiple data sources (e.g. toxicity data from multiple assays and mechanistic evidence) are synthesised into the structure-activity relationships that underpins Derek Nexus predictions. An appreciation of the assay units applied by alert writers when building the alert training set. However, predictions are not quantitative and, as a result, do not include units.
The query structure does not match a structural alert or examples for (bacterial in vitro) mutagenicity in Derek.
Based on these results Diflucortolone is not considered mutagenic as predicted by DEREK Nexus.
This study is classified as acceptable for assessment based on methodology and documentation. This study satisfy the requirement for Test Guideline OECD 471 for in vitro mutagenicity (bacterial reverse gene mutation) and the data is part of an overall assessment.
- Endpoint:
- in vitro gene mutation study in bacteria
- Type of information:
- (Q)SAR
- Adequacy of study:
- supporting study
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- results derived from a valid (Q)SAR model and falling into its applicability domain, with adequate and reliable documentation / justification
- Justification for type of information:
- 1. SOFTWARE
Leadscope model applier (v3.0.2)
2. MODEL (incl. version number)
Leadscope model applier (v3.0.2)
3. SMILES OR OTHER IDENTIFIERS USED AS INPUT FOR THE MODEL
CAS: 54605-02-6 ; Chemical name: Bromhydrin-valerate ; SMILES: C(C([C@H]3C(C[C@H]4[C@@H]2CC(C1=CC(C=C[C@]1(C)C@@]2(C(C[C@]34C)O)Br)=O)F)C)=O)OCCCCC
4. SCIENTIFIC VALIDITY OF THE (Q)SAR MODEL
- Defined endpoint: QMRF 4.10. Mutagenicity OECD 471 Bacterial Reverse Mutation Test
- Unambiguous algorithm:
A new ICH M7 compliant expert alert system to predict the mutagenic potential of impurities (white paper) http://www.leadscope.com/white_papers/ICHM7-WhitePaper-0314.pdf
The logic for matching alerts is detailed in "A new ICH M7 compliant expert alert system to predict the mutagenic potential of impurities" (white paper): http://www.leadscope.com/white_papers/ICHM7-WhitePaper-0314.pdf
- Defined domain of applicability: The applicability domain is defined as having at least one chemical in a reference set with at least 30% global similarity to the test structure (using the Leadscope 27,000 chemical fragments as descriptors and the Tanimoto similarity score).
- Appropriate measures of goodness-of-fit and robustness and predictivity: Chemicals/descriptor ratio: 241 alerts for 11,528 reference chemicals (ratio = 48); Alerts are run within the Leadscope model applier that provides the capability to specify one or more compounds (using SMILES, Mol files, SD files, or copying from the clipboard), select and run the alerts, assess the applicability domain, and view the results including an explanation for any prediction (such as a full description of any matched alerts). The performance was assessed using the Hansen dataset comprised of 3,700 chemicals (47% positive).
Concordance = 83%, Sensitivity = 92%, Specificity = 70%, Positive
Predictivity = 81%, Negative Predictivity = 86% , coverage = 95% were
obtained.
- Mechanistic interpretation: Accompanying any positive prediction, any alert(s) that match the test compounds are described including a description of the mechanistic basis from the literature reference that cites the alert.
5. APPLICABILITY DOMAIN
- Descriptor domain: The applicability domain is defined as having at least one chemical in a reference set with at least 30% global similarity to the test structure (using the Leadscope 27,000 chemical fragments as descriptors and the Tanimoto similarity score).
- Structural domain: Leadscope Predictive Data Miner is a software program for systematic sub‐structural analysis of a chemical using predefined structural features stored in a template library, training set‐dependent generated structural features (scaffolds) and calculated molecular descriptors. The feature library contains approximately 27,000 pre‐defined structural features and the structural features chosen for the library are motivated by those typically found in small molecules: aromatics, heterocycles, spacer groups, simple substituents. Leadscope allows for the generation of training set‐dependent structural features (scaffold generation), and these features can be added to the pre‐defined structural features from the library and be included in the descriptor selection process.
- Mechanistic domain: The global model identifies structural features and molecular descriptors which in the model development was found to be statistically significant associated with effect. Many predictions may indicate modes of action that are obvious for persons with expert knowledge for the endpoint
- Similarity with analogues in the training set: The original data set from Kazius et al. (2005) consisted of 4337 molecular structures with corresponding Ames test data.
The structural similarity of the test compound with respect to the training set compounds was analysed and quantified in terms of Tanimoto distance, which provides a quantitative measure of structural relatedness between the test compound and each training set compound. The 25 training set compounds found to be mostly similar to the test compound.
6. ADEQUACY OF THE RESULT
As can be seen from Annex A and B of the QPRF the result is considered adequate due to the presence of almost all structural features of the parent compound which can also be found in the training/validation dataset. Furthermore the prediction substantiate the experimental result for the substance of interest. - Qualifier:
- no guideline followed
- Principles of method if other than guideline:
- - Software tool(s) used including version: Leadscope model applier (v3.0.2)
- Model(s) used: Leadscope model applier (v3.0.2)
- Model description: see field 'Attached justification'
- Justification of QSAR prediction: see field 'Attached justification' - GLP compliance:
- no
- Type of assay:
- bacterial reverse mutation assay
- Species / strain / cell type:
- other: Combination of results from the S. typhimurium histidine reversion gene mutation test using tester strains TA97, TA97a, TA1537, TA98, TA100, TA1535, TA102, E.coli (any variant)
- Additional strain / cell type characteristics:
- other: The QSAR prediction is based on results from all tester strains recommended by the OECD Test guideline
- Evaluation criteria:
- The model used was the Leadscope Applier which is a statistical model using structural fragments to set an alert. Only descrete organic compounds can be predicted. The model searches for structural fragments and combines them with eight molecular descriptors. Thus, a probability of either a negative or positive result is calculated. If experimental data are available the prediction of the statistical model may be overruled.
- Key result
- Species / strain:
- other: Not applicable for in silico study
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- other: not applicable
- Vehicle controls validity:
- not applicable
- Untreated negative controls validity:
- not applicable
- True negative controls validity:
- not applicable
- Positive controls validity:
- not applicable
- Conclusions:
- Based on the predictions performed with the statistical QSAR model Leadscope Applier Bromhydrin-valerate is not mutagenic in a bacterial reverse mutation assay.
- Executive summary:
In a QSAR prediction using Leadscope Model Applier (v3.0.2) the potential of Bromhydrin-valerate to induce mutagenicity was assessed. Leadscope uses two parameters to guide the applicability of model domain: 1) having at least one structural feature defined in the model in addition to all the property descriptors; 2) having at least one chemical in a training neighbourhood with at least 30% global similarity to the test structure. In this case the prediction is within the applicability domain, since 37 training compounds were identified in the model training set being structurally similar to the test compound.
The query structure does not match structural alerts or examples for (bacterial in vitro) mutagenicity in Leadscope.
Based on these results Bromhydrin-valerate is considered not mutagenic as predicted by Leadscope.
This study is classified as acceptable for assessment based on methodology and documentation. This study satisfy the requirement for Test Guideline OECD 471 for in vitro mutagenicity (bacterial reverse gene mutation) and the data is part of an overall assessment.
- Endpoint:
- in vitro gene mutation study in bacteria
- Type of information:
- (Q)SAR
- Adequacy of study:
- supporting study
- Study period:
- 2021
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- results derived from a valid (Q)SAR model and falling into its applicability domain, with adequate and reliable documentation / justification
- Justification for type of information:
- 1. SOFTWARE
DEREK Nexus 6.1
2. MODEL (incl. version number)
DEREK Nexus 6.1
3. SMILES OR OTHER IDENTIFIERS USED AS INPUT FOR THE MODEL
CAS 54605-02-6
4. SCIENTIFIC VALIDITY OF THE (Q)SAR MODEL
- Defined endpoint: TOX 7.6.1. Genetic toxicity in vitro
- Unambiguous algorithm: logic of argumentation. Derek Nexus makes qualitative predictions for and against toxicity through reasoning. For the endpoint of mutagenicity, predictions for toxicity decrease in confidence in the following order: certain>probable>plausible>equivocal. Predictions against toxicity increase in confidence in the following order: inactive (with unclassified and/or misclassified features) inactive
- Defined domain of applicability: The scopes of the structure-activity relationships describing the mutagenicity endpoint are defined by the developer to be the applicability domain for the model. Therefore, if a chemical activates an alert describing a structure-activity for mutagenicity it can be considered to be within the applicability domain. If a compound does not activate an alert or reasoning rule then Derek makes a negative prediction. The applicability of the negative prediction to the query compounds can be determined by an expert, if required, by investigating the presence (or absence) of misclassified and/or unclassified features. The applicability domain of each alert is defined by the alert developer on the basis of the training set data and expert judgement on the chemical and biological factors which affect the mechanism of action for each alert. For non-alerting compounds, users should determine the applicability of negative predictions by evaluating the information supplied by Derek (i.e. the presence or absence of misclassified and/or unclassified features).
- Appropriate measures of goodness-of-fit and robustness and predictivity: n/a
- Mechanistic interpretation: All alerts describing structure-activity relationships for the mutagenicity endpoint have a mechanistic basis wherever possible.
Mechanistic information is detailed in the comments associated with an alert and can include information on both the mechanism of action and biological target. The mechanistic basis of the model was developed a priori by examining the toxicological and mechanistic evidence before developing the structure-activity relationship.
5. APPLICABILITY DOMAIN
- Descriptor domain:
[1]Markush structures encoding activating and deactivating features (known as patterns in the Derek Nexus knowledge base)
[2]count of non-hydrogen atoms
[3]ClogP
[4]2D structural fragments
There is an a priori assumption that patterns and associated reasoning will be used to model toxicity within Derek Nexus. Further, experts identified that misclassified and unclassified features were useful descriptors for determining the reliability of negative predictions for non-alerting compounds.
- Similarity with analogues in the training set: Non-proprietary elements of the training set are available through the references, and illustrated by the examples, within Derek Nexus. The illustrative examples are not available, due to the proprietary nature of Derek Nexus.
6. ADEQUACY OF THE RESULT
Based on the common structure of the substance, the result is considered reliable. - Qualifier:
- no guideline followed
- Principles of method if other than guideline:
- - Software tool(s) used including version: DEREK Nexus 6.1
- Model(s) used: DEREK Nexus 6.1
- Model description: see field 'Attached justification'
- Justification of QSAR prediction: see field 'Attached justification' - GLP compliance:
- no
- Type of assay:
- bacterial reverse mutation assay
- Species / strain / cell type:
- bacteria, other: Predictions are made for the domain of bacteria and can be broken down into species (e.g. Salmonella typhimurium and Escherichia coli)
- Additional strain / cell type characteristics:
- other: The prediction is based on results from all tester strains recommended by the OECD Test Guideline
- Evaluation criteria:
- Two types of models were used to predict the mutagenic potential of the test item.
The DEREK Nexus model was used as a rule-based model which is based on the training set data and expert judgement on the chemical and biological factors which affect the mechanism of action for each alert. The second model used was the Leadscope Applier which is a statistical model using structural fragments to set an alert. If experimental data are available the prediction of the statistical model may be overruled. - Key result
- Species / strain:
- bacteria, other: Not applicable for in silico study
- Metabolic activation:
- not applicable
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- other: not applicable
- Vehicle controls validity:
- not applicable
- Untreated negative controls validity:
- not applicable
- True negative controls validity:
- not applicable
- Positive controls validity:
- not applicable
- Conclusions:
- Based on the predictions performed with the statistical QSAR model Leadscope Applier and the rule-based model DEREK Nexus Bromhydrin-valerate is not mutagenic in a bacterial reverse mutation assay.
- Executive summary:
In a QSAR prediction using DEREK Nexus v6.1 the potential of Bromhydrin-valerate to induce mutagenicity was assessed. Derek Nexus makes qualitative predictions for and against toxicity through reasoning. For the endpoint of mutagenicity, predictions for toxicity decrease in confidence in the following order: certain>probable>plausible>equivocal. Predictions against toxicity increase in confidence in the following order: inactive (with unclassified and/or misclassified features)<inactive<improbable. Likelihood levels have been shown to correlate with predictivity [Judson et al, 2013]. Multiple data sources (e.g. toxicity data from multiple assays and mechanistic evidence) are synthesised into the structure-activity relationships that underpins Derek Nexus predictions. An appreciation of the assay units applied by alert writers when building the alert training set. However, predictions are not quantitative and, as a result, do not include units.
The query structure does not match a structural alert or examples for (bacterial in vitro) mutagenicity in Derek.
Based on these results Bromhydrin-valerate is not considered mutagenic as predicted by DEREK Nexus.
This study is classified as acceptable for assessment based on methodology and documentation. This study satisfy the requirement for Test Guideline OECD 471 for in vitro mutagenicity (bacterial reverse gene mutation) and the data is part of an overall assessment.
- Endpoint:
- in vitro gene mutation study in bacteria
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- June to July 1992
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- comparable to guideline study with acceptable restrictions
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Version / remarks:
- 26 May 1983
- Deviations:
- yes
- Remarks:
- no plate incorporation procedure performed; E. coli WP2uvrA and/or Salmonell typhimurium TA 102 were not tested
- Principles of method if other than guideline:
- Preincubation modification was performed
- GLP compliance:
- yes
- Type of assay:
- bacterial reverse mutation assay
- Target gene:
- Histidine gene locus
- Species / strain / cell type:
- S. typhimurium TA 98
- 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 1535
- 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 1538
- Additional strain / cell type characteristics:
- not applicable
- Metabolic activation:
- with and without
- Metabolic activation system:
- Aroclor 1254 induced male rat liver S9 mix
Type and composition of metabolic activation system:
- source of S9
- method of preparation of S9 mix
- concentration or volume of S9 mix and S9 in the final culture medium
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability) - Test concentrations with justification for top dose:
- All strains: 0.05, 0.1, 0.25, 0.5, 1.0, 2.0 mg/plate
- Vehicle / solvent:
- DMSO
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- other: without metabolic activation: 9-AA, 2-NF, NaN3; with metabolic activation: 2-AA, BP, CP, DMNA.
- Details on test system and experimental conditions:
- NUMBER OF REPLICATIONS:
- Number of cultures per concentration: triplicate
- Number of independent experiments: for TA 100 two, the remaining strains: one
METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): 1E-06 dilution
- Test substance added in agar (plate incorporation) and for TA 100 as preincubation
METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method, e.g.: background growth inhibition
METHODS FOR MEASUREMENTS OF GENOTOXICITY: The plates were scored for the number of mutant colonies with an automated colony counter (Biotran II, Model C 111, New Brunswiek Scientific Co., Edison, N.J.), except for the plates in the preincubation test without S9 mix which were scored manually because of precipitates in the agar. 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 spontaneous was higher than 2-fold. Also a dose dependent increase in the number of revertants was considered to indicate a mutagenic effect. - Rationale for test conditions:
- as recommended by the test guideline
- Evaluation criteria:
- 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 spontaneous was higher than 2-fold. Also a dose dependent increase in the number of revertants was considered to indicate a mutagenic effect.
- Key result
- Species / strain:
- S. typhimurium TA 98
- 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
- Key result
- Species / strain:
- S. typhimurium 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
- Key result
- 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:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 1537
- 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
- Key result
- Species / strain:
- S. typhimurium TA 1538
- 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
- Conclusions:
- The purpose of the Ames test was to investigate whether diflucortolone-21-valerate can induce point mutations using the preincubation modification. The five histidine-dependent strains of S. typhimurium TA1535, TA100, TA1537, TA1538 and TA98 were tested. The study was performed with and without metabolic activation, employed a range of diflucortolone-21-valerate concentrations from 0.05 to 2.0 mg per plate. No increased reversion to prototrophy were seen neither without nor with metabolic activation. Precipitates in the agar were found starting at 0.25 or 0.50 mg diflucortolone-21-valerate/plate without S9 mix and 2 mg diflucortolone-21-valerate /plate with S9 mix. Growth inhibition of the background lawn was not observed.
- Executive summary:
In a reverse gene mutation assay in bacteria according to OECD test guideline 471 (1983), strains TA 98, TA 100, TA 1535, TA 1537 and TA 1538 of S. typhimurium were exposed to Difluocortolone valerate, (100% a.i.), in DMSO at concentrations of 0.05, 0.1, 0.25, 0.5, 1.0, 2.0 mg/plate in the presence and absence of mammalian metabolic activation using the pre-incubation method.
Difluocortolone valerate was tested up to insoluble concentrations starting at 2.5 mg with S9 mix and at 0.1 mg/plate without S9 mix. The positive controls induced the appropriate responses in the corresponding strains. There was no evidence of induced mutant colonies over background.
This study satisfies the requirement for Test Guideline OECD 471 for in vitro mutagenicity (bacterial reverse gene mutation) data.
- Endpoint:
- in vitro gene mutation study in bacteria
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- June to Aug 1984
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- comparable to guideline study with acceptable restrictions
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Version / remarks:
- 26 May 1983
- Deviations:
- yes
- Remarks:
- preincubation modification performed only with strain TA 100, no E.coli WP2uvrA or TA102 was tested
- GLP compliance:
- yes
- Type of assay:
- bacterial reverse mutation assay
- Target gene:
- Histidine gene locus
- Species / strain / cell type:
- S. typhimurium TA 98
- 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 1535
- 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 1538
- Additional strain / cell type characteristics:
- not applicable
- Metabolic activation:
- with and without
- Metabolic activation system:
- Aroclor 1254 induced male rat liver S9 mix
Type and composition of metabolic activation system: S9-liver mix (rat)
- source of S9: S9, derived from male Sprague-Dawley rats pretreated with Aroclor 1254, was obtained from Organon Teknika Co., Durham, NC, USA, [S9 batch no. 33499; protein content 39.5 mg/mL; activity (37°C; pmoles/min/mg S9 protein) 7-hydroxyresorufin, 8286].
- concentration or volume of S9 mix and S9 in the final culture medium: The components of the standard S9 mix were 8 mM MgCI2, 33 mM KCI, 5 mM glucose-6-phosphate, 4 mM NADP, 100 mM sodium phosphate, pH 7.4 and S9 at a concentration of 0.3 mL per mL of mix. 0.5 mL S9 mix was added to 0.1 mL bacterial suspension and 0.05 mL of the test item. - Test concentrations with justification for top dose:
- without preincubation: 0.1, 0.25, 0.5, 1.0, 2.5, 5.0 mg/plate
with preincubation: 0.1, 0.25, 0.5, 0.75, 1.0, 2.0, 4.0, 6.0 mg/plate (only S. typhymurium TA100) - Vehicle / solvent:
- DMSO
- 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, additionally DMNA was used as positive control
- 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): E+06 dilution
- Test substance added in preincubation
METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: background growth inhibition
METHODS FOR MEASUREMENTS OF GENOTOXICITY: The plates were scored for the number of mutant colonies with an automated colony counter (Artek M 9828, 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 indueed revertants compared to the number of spontaneous ones was higher than 2-fold. A dose-dependent increase in the number of revertants was also considered to indieate a mutagenic effect. - Evaluation criteria:
- 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 indueed revertants compared to the number of spontaneous ones was higher than 2-fold. A dose-dependent increase in the number of revertants was also considered to indieate a mutagenic effect.
- Key result
- Species / strain:
- S. typhimurium TA 98
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity, but tested up to precipitating concentrations
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- 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, but tested up to precipitating concentrations
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- 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, but tested up to precipitating concentrations
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 1537
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity, but tested up to precipitating concentrations
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 1538
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity, but tested up to precipitating concentrations
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Conclusions:
- ZK 22612 (diflucortolone-21-valerate) was examined for mutagenic activity in five histidine-dependent strains of S. typhimurium (TA1535, TA100, TA1537, TA1538, TA98) using the direct plate incorporation procedure. The study was performed with and without metabolic activation, employed a range of concentrations from 0.1 to 5.0 mg per plate. No increased reversion to prototrophy were seen neither without nor with metabolic activation. Also an additionally performed test with strain TA 100 using the preincubation procedure did not show a mutagenic effect of the test item. Precipitates in the agar were found generally in all concentrations without S9 mix, at 2.5 and 5 mg per plate with S9 mix and in the preincubation test starting at 1 mg per plate with S9 mix. Growth inhibition of the background lawn was not observed.
- Executive summary:
In a reverse gene mutation assay in bacteria according to OECD test guideline 471 (1983), strains TA 98, TA 100, TA 1535, TA 1537 and TA 1538 of S. typhimurium were exposed to Difluocortolone valerate, (100% a.i.), in DMSO at concentrations of 0.1, 0.25, 0.5, 1,0, 2.5, and 5.0 mg/plate in the presence and absence of mammalian metabolic activation using the pre-incubation method. Strain TA 100 was additionally exposed to the test item in DMSO at concentrations of 0.1, 0.25, 0.5, 0.75, 1.0, 2.0, 4.0, and 6.0 mg/plate using the plate incorporation method.
Difluocortolone valerate was tested up to insoluble concentrations starting at 2.5 mg with S9 mix and at 0.1 mg/plate without S9 mix. The positive controls induced the appropriate responses in the corresponding strains. There was no evidence of induced mutant colonies over background.
This study satisfies the requirement for Test Guideline OECD 471 for in vitro mutagenicity (bacterial reverse gene mutation) data.
Referenceopen allclose all
PRE-TEST FOR TOXICITY
Plating Efficiency Assay without metabolic activation 504 single cells were seeded into each flask. | ||||
conc. per mL | Colonies counted | mean | PE% relative | |
| Flask 1 | Flask 2 |
|
|
Negative control | 257 | 307 | 282.0 | 100.0 |
Solvent control (DMSO) | 267 | 287 | 277.0 | 100.0 |
0.10 µg | 282 | 272 | 277.0 | 100.0 |
1.00 µg | 299 | 277 | 288.0 | 104.0 |
3.00 µg | 278 | 275 | 276.0 | 99.8 |
10.00 µg | 296 | 284 | 290.0 | 104.7 |
20.00 µg | 229 | 242 | 235.5 | 85.0 |
50.00 µg* | 196 | 213 | 204.5 | 73.8 |
100.00 µg* | 189 | 204 | 196.5 | 70.9 |
200.00 µg* | 266 | 234 | 250.0 | 90.3 |
| ||||
Plating Efficiency Assay with metabolic activation 504 single cells were seeded into each flask. | ||||
Negative control | 284 | 273 | 278.5 | 100.0 |
Solvent control (DMSO) | 222 | 237 | 229.5 | 100.0 |
0.10 µg | 281 | 301 | 291.0 | 126.8 |
1.00 µg | 267 | 272 | 269.5 | 117.4 |
3.00 µg | 281 | 268 | 274.5 | 119.6 |
10.00 µg | 288 | 304 | 296.0 | 129.0 |
20.00 µg | 247 | 275 | 261.0 | 113.7 |
50.00 µg* | 274 | 244 | 259.0 | 112.9 |
100.00 µg* | 257 | 265 | 261.0 | 113.7 |
200.00 µg* | 267 | 257 | 262.0 | 114.2 |
* Precipitation | ||||
Table I: Toxicity data; experiment I | ||||||||||
| no. of cells /flask* | PE%** absolute | PE%*** relative | cells/mL at 1st subcultivation | cell density; % of control | |||||
| conc. µg/mL | S9 mix | seeded I/II | found I | found II | mean |
|
|
|
|
Column | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 |
negative control | 0.00 | - | 504 | 274 | 301 | 287.5 | 57.0 | 100.0 | 3346667 |
|
Solvent control (DMSO) | 0.00 | - | 504 | 285 | 310 | 297.5 | 59.0 | 100.0 | 3353333 |
|
Positive control EMS | 600 | - | 504 | 304 | 226 | 265.0 | 52.6 | 92.2 | 2846667 |
|
Test item | 5.00 | - | 504 | 231 | 295 | 263.0 | 52.2 | 88.4 | 3133333 |
|
| 10.00 | - | 504 | 234 | 256 | 245.0 | 48.6 | 82.4 | not performed |
|
| 20.00° | - | 504 | 241 | 229 | 235.0 | 46.6 | 79.0 | 3433333 |
|
| 40.00° | - | 504 | 0 | 0 | 0.0 | 0.0 | 0.0 | 2333333 |
|
| 60.00° | - | 504 | 0 | 0 | 0.0 | 0.0 | 0.0 | not performed |
|
| 80.00° | - | 504 | 82 | 104 | 93.0 | 18.5 | 31.3 | 3280000 |
|
negative control | 0.00 | + | 504 | 353 | 310 | 331.5 | 65.8 | 100.0 | 4926667 |
|
Solvent control (DMSO) | 0.00 | + | 504 | 286 | 322084 | 304.0 | 60.3 | 100.0 | 5473333 |
|
Positive control DMBA | 600 | + | 504 | 81 | 304 | 82.5 | 16.4 | 27.1 | 2586667 |
|
Test item | 5.00 | + | 504 | 337 | 296 | 320.5 | 63.6 | 105.4 | 5620000 |
|
| 10.00 | + | 504 | 317 | 315 | 306.5 | 60.8 | 100.8 | not performed |
|
| 20.00° | + | 504 | 304 | 340 | 309.5 | 61.4 | 101.8 | 4986667 |
|
| 40.00° | + | 504 | 346 | 316 | 343.0 | 68.1 | 112.8 | 4946667 |
|
| 60.00° | + | 504 | 303 | 321 | 309.5 | 61.4 | 101.8 | not performed |
|
| 80.00° | + | 504 | 328 |
| 324.5 | 64.4 | 106.7 | 5153333 |
|
* only colonles wlth more than 50 cells 7 days after seeding were scored ** PE absolute (value column 6 / value column 3 x 100) *** PE relative (value column 6 / value column 6 of corresponding control x 100) ° Precipitation | ||||||||||
|
|
|
|
|
|
|
|
|
|
|
Table II: Mutagenicity data; experiment I (part 1: cell survival) | ||||||||||
|
|
| no. of cells /flask* | factor calculated** | cells seeded | cells survived*** | ||||
| conc. µg/mL | S9 mix | seeded I/II | found I | found II | mean |
|
|
|
|
Column | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 |
|
negative control | 0.00 | - | 547 | 441 | 449 | 445.0 | 0.81 | 441000 | 357210 |
|
Solvent control (DMSO) | 0.00 | - | 500 | 320 | 381 | 350.5 | 0.70 | 465000 | 325500 |
|
Positive control EMS | 600 | - | 558 | 385 | 368 | 376.5 | 0.67 | 450000 | 301500 |
|
Test item | 5.00 | - | 510 | 408 | 389 | 398.5 | 0.78 | 450000 | 351000 |
|
| 10.00 | - | culture was not continued |
|
|
|
| |||
| 20.00° | - | 565 | 333 | 327 | 330.0 | 0.58 | 444000 | 257520 |
|
| 40.00° | - | 513 | 408 | 375 | 391.5 | 0.76 | 495000 | 376200 |
|
| 60.00° | - | culture was not continued |
|
|
|
| |||
| 80.00° | - | 500 | 286 | 313 | 299.5 | 0.60 | 450000 | 270000 |
|
negative control | 0.00 | + | 506 | 368 | 366 | 367.0 | 0.73 | 495000 | 361350 |
|
Solvent control (DMSO) | 0.00 | + | 503 | 439 | 407 | 423.0 | 0.84 | 441000 | 370440 |
|
Positive control DMBA | 600 | + | 582 | 348 | 310 | 329.0 | 0.57 | 477000 | 271890 |
|
Test item | 5.00 | + | 525 | 402 | 439 | 420.5 | 0.80 | 507000 | 405600 |
|
| 10.00 | + | culture was not continued |
|
|
|
| |||
| 20.00° | + | 591 | 508 | 496 | 502.0 | 0.85 | 456000 | 387600 |
|
| 40.00° | + | 512 | 494 | 463 | 478.5 | 0.93 | 483000 | 449190 |
|
| 60.00° | + | culture was n457ot continued |
|
|
|
| |||
| 80.00° | + | 561 |
| 513 | 485.0 | 0.86 | 444000 | 381840 |
|
* only colonies with more than 50 cells 7 days after seeding in normal medium were scored **. factor calculated (value column 6 I value column 3) *** cells survived after plating in 1G containing medium (value column 8 x value column 7) ° Precipitation . | ||||||||||
|
|
|
|
|
|
|
|
|
|
|
Table III: Mutagenicity data; experiment I (part 2: mutation rates) | ||||||||||
| number of mutant colonies per flask* found after plating in TG medium | mean | standard deviation | ** mutant colonies /E+06 cells | ||||||
| conc. µg/mL | S9 mix | I | II | III | IV | V | |||
Column | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 |
negative control | 0.00 | - | 3 | 1 | 2 | 4 | 6 | 3.2 | 1.9 | 9.0 |
Solvent control (DMSO) | 0.00 | - | 5 | 8 | 5 | 6 | 4 | 5.6 | 1.5 | 17.2 |
Positive control EMS | 600 | - | 66 | 73 | 85 | 83 | 75 | 76.4 | 7.7 | 253.4 |
Test item | 5.00 | - | 7 | 6 | 9 | 15 | 11 | 9.6 | 3.6 | 27.4 |
| 10.00 | - |
| |||||||
| 20.00° | - | 5 | 5 | 7 | 4 | 6 | 5.4 | 1.1 | 21.0 |
| 40.00° | - | 8 | 9 | 6 | 7 | 6 | 7.2 | 1.3 | 19.1 |
| 60.00° | - |
| |||||||
| 80.00° | - | 0 | 3 | 3 | 4 | 6 | 3.2 | 2.2 | 11.9 |
negative control | 0.00 | + | 6 | 6 | 8 | 10 | 10 | 8.0 | 2.0 | 22.1 |
Solvent control (DMSO) | 0.00 | + | 8 | 9 | 10 | 8 | 3 | 7.6 | 2.7 | 20.5 |
Positive control DMBA | 600 | + | 163 | 169 | 187 | 170 | 197 | 177.2 | 14.2 | 651.7 |
Test item | 5.00 | + | 15 | 5 | 15 | 14 | 6 | 11.0 | 5.0 | 27.1 |
| 10.00 | + |
|
|
|
|
|
|
|
|
| 20.00° | + | 8 | 10 | 11 | 7 | 18 | 10.8 | 4.3 | 27.9 |
| 40.00° | + | 9 | 9 | 6 | 10 | 7 | 8.2 | 1.6 | 18.3 |
| 60.00° | + |
|
|
|
|
|
|
|
|
| 80.00° | + | 9 | 9 | 6 | 10 | 11 | 9.0 | 1.9 | 23.6 |
* only colonies with more than 50 cells 8 days after seeding in TG medium were scored ** value column 8 (this page) x E+06/ value column 9 of table II . ° Preclpitation | ||||||||||
| ||||||||||
Table IV: Toxicity data; experiment II | ||||||||||
|
|
| no. of cells /flask* | PE%** absolute | PE%*** relative | cells/mL at 1st subcultivation | cell density; % of control | |||
|
|
| seeded I/II | found I | found II | mean | ||||
| conc. µg/mL | S9 mix |
|
|
|
|
|
|
|
|
Column | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 |
negative control | 0.00 | - | 530 | 306 | 265 | 285.5 | 53.9 | 100.0 | 3426667 | 100.0 |
Solvent control (DMSO) | 0.00 | - | 530 | 328 | 267 | 297.5 | 56.1 | 100.0 | 4493333 | 100.0 |
Positive control EMS | 600 | - | 530 | 71 | 75 | 73.0 | 13.8 | 25.6 | 2166667 | 58.1 |
Test item | 5.00 | - | 530 | 291 | 286 | 288.5 | 54.4 | 97.0 | 4660000 | 103.7 |
| 20.00° | - | 530 | 277 | 247 | 262.0 | 49.4 | 88.1 | not performed | |
| 40.00° | - | 530 | 232 | 212 | 222.0 | 41.9 | 74.6 | 3853333 | 85.8 |
| 60.00° | - | 530 | 111 | 115 | 113.0 | 21.3 | 38.0 | 1993333 | 44.4 |
| 80.00° | - | 530 | 147 | 203 | 175.0 | 33.0 | 58.8 | 2173333 | 48.4 |
| 100.00° | - | 530 | 168 | 144 | 156.0 | 29.4 | 52.4 | 3753333 | 83.5 |
negative control | 0.00 | + | 530 | 276 | 261 | 268.5 | 50.7 | 100.0 | 5513333 | 100.0 |
Solvent control (DMSO) | 0.00 | + | 530 | 298 | 283 | 290.5 | 54.8 | 100.0 | 5426667 | 100.0 |
Positive control DMBA | 600 | + | 530 | 33 | 44 | 38.5 | 7.3 | 13.3 | 2913333 | 53.7 |
Test item | 5.00 | + | 530 | 304 | 293 | 298.5 | 56.3 | 102.8 | 6020000 | 110.9 |
| 20.00° | + | 530 | 276 | 304 | 290.0 | 54.7 | 99.8 | not performed | |
| 40.00° | + | 530 | 296 | 285 | 290.5 | 54.8 | 100.0 | 6013333 | 110.8 |
| 60.00° | + | 530 | 281 | 300 | 290.5 | 54.8 | 100.0 | not performed | |
| 80.00° | + | 530 | 305 | 314 | 309.5 | 58.4 | 106.5 | 5846667 | 107.7 |
| 100.00° | + | 530 | 291 | 276 | 283.5 | 53.5 | 97.6 | 5693333 | 104.9 |
* only colonles with more than 50 cells 7 days after seeding were scored ** PE absolute (value column 6 / value column 3 x 100) *** PE, relative (value column 6 / value colunln 6 of corresponding control x 100) ° Precipitation | ||||||||||
Table V: Mutagenicity data; experiment II (part 1: cell survival) | ||||||||||
|
|
| no. of cells /flask* | factor calculated** | cells seeded | cells survived*** |
| |||
|
|
| seeded I/II | found I | found II | mean |
| |||
| conc. µg/mL | S9 mix |
|
|
|
|
|
|
|
|
Column | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 |
|
negative control | 0.00 | - | 526 | 304 | 313 | 308.5 | 0.59 | 442500 | 261075 |
|
Solvent control (DMSO) | 0.00 | - | 538 | 260 | 254 | 257.0 | 0.48 | 439500 | 210960 |
|
Positive control EMS | 600 | - | 518 | 137 | 158 | 147.5 | 0.28 | 475500 | 133140 |
|
Test item | 5.00 | - | 546 | 448 | 528 | 488.0 | 089 | 387000 | 344430 |
|
| 20.00° | - |
| |||||||
| 40.00° | - | 554 | 358 | 381 | 369.5 | 0.67 | 480000 | 321600 |
|
| 60.00° | - | 546 | 352 | 395 | 373.5 | 0.65 | 465000 | 302250 |
|
| 80.00° | - | 554 | 386 | 307 | 346.5 | 0.63 | 460500 | 290115 |
|
| 100.00° | - | 558 | 380 | 380 | 380.0 | 0.68 | 439500 | 298860 |
|
negative control | 0.00 | + | 516 | 335 | 331 | 333.0 | 0.65 | 390000 | 253500 |
|
Solvent control (DMSO) | 0.00 | + | 573 | 334 | 306 | 320.0 | 0.56 | 450000 | 252000 |
|
Positive control DMBA | 600 | + | 539 | 254 | 249 | 251.5 | 0.47 | 405000 | 190350 |
|
Test item | 5.00 | + | 502 | 264 | 411 | 337.5 | 0.67 | 435000 | 291450 |
|
| 20.00° | + |
| |||||||
| 40.00° | + | 501 | 322 | 317 | 319.5 | 0.64 | 375000 | 240000 |
|
| 60.00° | + |
| |||||||
| 80.00° | + | 573 | 421 | 441 | 431.0 | 0.75 | 450000 | 337500 |
|
| 100.00° | + | 501 | 349 | 345 | 347.0 | 0.69 | 442500 | 305325 |
|
* only colonies with more than 50 cells 7 days after seeding in normal medium were scored . ** factor calcuJated (value coJumn 6 / value column 3) , ***. cells survlved after plating in TG containing medium (value column 8 x value column 7) ° Precipitation | ||||||||||
Tabie VI: Mutagenicity data; experiment II (part 2: mutation rates) | ||||||||||
| conc. µg/mL | S9 mix | number of mutant colonies per flask* found after plating in TG medium | mean | standard deviation | ** mutant colonies /E+06 cells | ||||
I | II | III | IV | V |
|
|
| |||
Column | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 |
negative control | 0.00 | - | 3 | 3 | 5 | 3 | 4 | 3.6 | 0.9 | 13.8 |
Solvent control (DMSO) | 0.00 | - | 5 | 5 | 2 | 4 | 3 | 3.8 | 1.3 | 1.80 |
Positive control EMS | 600 | - | 139 | 166 | 147 | 157 | 152 | 152.2 | 10.2 | 1143.2 |
Test item | 5.00 | - | 5 | 4 | 3 | 5 | 3 | 4.0 | 1.0 | 11.6 |
| 20.00° | - |
| |||||||
| 40.00° | - | 3 | 5 | 7 | 7 | 4 | 5.2 | 1.8 | 16.2 |
| 60.00° | - | 1 | 4 | 2 | 1 | 1 | 1.8 | 1.3 | 6.0 |
| 80.00° | - | 3 | 2 | 3 | 2 | 3 | 2.6 | 0.5 | 9.0 |
| 100.00° | - | 3 | 6 | 2 | 5 | 2 | 3.6 | 1.8 | 12.0 |
negative control | 0.00 | + | 8 | 2 | 7 | 4 | 9 | 6.0 | 2.9 | 23.7 |
Solvent control (DMSO) | 0.00 | + | 4 | 7 | 11 | 6 | 5 | 6.6 | 2.7 | 26.2 |
Positive control DMBA | 600 | + | 122 | 120 | 112 | 116 | 118 | 117.6 | 3.8 | 617.8 |
Test item | 5.00 | + | 7 | 4 | 4 | 4 | 7 | 5.2 | 1.6 | 17.8 |
| 20.00° | + |
| |||||||
| 40.00° | + | 2 | 1 | 2 | 2 | 1 | 1.6 | 0.5 | 6.7 |
| 60.00° | + |
| |||||||
| 80.00° | + | 2 | 5 | 2 | 1 | 1 | 2.2 | 1.6 | 6.5 |
| 100.00° | + | 5 | 3 | 7 | 7 | 5 | 5.4 | 1.7 | 17.7 |
* only colonies with more than 50 cells 8 days after seeding in TG medium were scored ** value column 8 (this page) x E+06 / value column 9 of table V . ° Precitpitation | ||||||||||
Experiment I; mitotic index (exposure period 22 hand 46 h) | |||||||
Test group | conc/ mL | S9 mix | fixation interval (h) | mitotic index [%]* Vessel | abs. | rel. | |
1 | 2 | ||||||
Negative control (medium) |
| - | 22 | 16.6 | 12.1 | 14.4 | 100.0 |
Solvent control (DMSO) | 1% | - | 22 | 9.5 | 10.8 | 10.2 | 100.0 |
Positive control (EMS) | 0.33 mg | - | 22 | 8.9 | 6.9 | 7.9 | 55.1 |
Test item | 0.3 µg | - | 22 | 10.8 | 8.9 | 9.9 | 97.0 |
| 1.0 µg | - | 22 | 13.8 | 8.2 | 11.0 | 108.4 |
| 3.0 µg | - | 22 | 9.0 | 12.2 | 10.6 | 104.4 |
| 10.0 µg | - | 22 | 11.4 | 8.8 | 10.1 | 99.5 |
| 30.0 µg | - | 22 | 11.4 | 7.8 | 9.6 | 94.6 |
| 60.0 µg | - | 22 | 7.5 | 13.8 | 10.7 | 104.9 |
| 80.0 µg | - | 22 | 11.5 | 10.4 | 11.0 | 107.9 |
| 100.0 µg | - | 22 | 5.2 | 6.1 | 5.7 | 55.7 |
|
|
|
|
|
|
|
|
Solvent control (DMSO) | 1% | - | 46 | 12.9 | 14.8 | 13.9 | 100.0 |
Test item | 3.0 µg | - | 46 | 4.9 | 8.1 | 6.5 | 46.9 |
| 10.0 µg | - | 46 | 3.4 | 2.8 | 3.1 | 22.4 |
| 30.0 µg | - | 46 | 5.8 | 4.8 | 5.3 | 38.3 |
| 60.0 µg | - | 46 | 5.5 | 3.8 | 4.7 | 33.6 |
| 80.0 µg | - | 46 | 4.2 | 1.5 | 2.9 | 20.6 |
| 100.0 µg | - | 46 | 6.4 | 4.2 | 5.3 | 38.3 |
* The mitotic index was determined in 1000 cells from each of the two vessels per test group | |||||||
Experiment I; mitotic index (exposure period 4 h) | |||||||
Test group | conc/ mL | S9 mix | fixation interval (h) | mitotic index [%]* Vessel | abs. | rel. | |
1 | 2 | ||||||
Negative control (medium) |
| + | 22 | 14.1 | 14.8 | 14.5 | 100.0 |
Solvent control (DMSO) | 1% | + | 22 | 10.4 | 15.8 | 13.1 | 100.0 |
Positive control (CPA) | 30.0 µg | + | 22 | 3.8 | 10.4 | 7.1 | 49.1 |
Test item | 0.3 µg | + | 22 | 8.3 | 9.1 | 8.7 | 66.4 |
| 1.0 µg | + | 22 | 12.5 | 8.8 | 10.7 | 81.3 |
| 3.0 µg | + | 22 | 12.4 | 11.8 | 12.1 | 92.4 |
| 10.0 µg | + | 22 | 8.2 | 12.0 | 10.1 | 77.1 |
| 30.0 µg | + | 22 | 6.1 | 8.9 | 7.5 | 57.3 |
| 60.0 µg | + | 22 | 8.4 | 8.2 | 8.3 | 63.4 |
| 80.0 µg | + | 22 | 9.8 | 7.1 | 8.5 | 64.5 |
| 100.0 µg | + | 22 | 6.1 | 2.3 | 4.2 | 32.1 |
|
|
|
|
|
|
|
|
Solvent control (DMSO) | 1% | + | 46 | 16.6 | 13.5 | 15.1 | 100.0 |
Test item | 3.0 µg | + | 46 | 9.8 | 7.6 | 8.7 | 57.8 |
| 10.0 µg | + | 46 | 8.9 | 10.6 | 9.8 | 64.8 |
| 30.0 µg | + | 46 | 9.8 | 6.4 | 8.1 | 53.8 |
| 60.0 µg | + | 46 | 11.0 | 8.4 | 9.7 | 64.5 |
| 80.0 µg | + | 46 | 11.8 | 9.6 | 10.7 | 71.1 |
| 100.0 µg | + | 46 | 8.9 | 8.3 | 8.6 | 57.1 |
*The mitotic index was determined in 1000 cells from each of the two vessels per test group. | |||||||
Experiment II; mitotic index (exposure period 22 hand 46 h) | |||||||
Test group | conc/ mL | S9 mix | fixation interval (h) | mitotic index [%]* Vessel | abs. | rel. | |
1 | 2 | ||||||
Negative control (medium) |
| - | 22 | 18.3 | 21.0 | 19.7 | 100.0 |
Solvent control (DMSO) | 1% | - | 22 | 12.0 | 15.4 | 13.7 | 100.0 |
Positive control (EMS) | 0.33 mg | - | 22 | 13.1 | 11.7 | 12.4 | 63.1 |
Test item | 3.0 µg | - | 22 | 13.3 | 10.7 | 12.0 | 87.6 |
| 10.0 µg | - | 22 | 11.5 | 11.3 | 11.4 | 83.2 |
| 30.0 µg | - | 22 | 10.9 | 11.6 | 11.3 | 82.1 |
| 60.0 µg | - | 22 | 10.5 | 10.0 | 10.3 | 74.8 |
| 80.0 µg | - | 22 | 10.5 | 12.4 | 11.5 | 83.6 |
| 100.0 µg | - | 22 | 7.9 | 5.7 | 6.8 | 49.6 |
Solvent control (DMSO) | 1% | - | 46 | 6.9 | 7.9 | 7.4 | 100.0 |
Test item | 30.0 µg | - | 46 | 3.5 | 4.5 | 4.0 | 54.1 |
| 60.0 µg | - | 46 | 3.3 | 5.3 | 4.3 | 58.1 |
| 80.0 µg | - | 46 | 2.6 | 3.9 | 3.3 | 43.9 |
| 100.0 µg | - | 46 | 4.0 | 3.5 | 3.8 | 50.7 |
* The mitotic index was determined in 1000 cells from each of the two vessels per test group | |||||||
Experiment II; mitotic index (exposure period 4 h) | |||||||
Test group | conc/ mL | S9 mix | fixation interval (h) | mitotic index [%]* Vessel | abs. | rel. | |
1 | 2 | ||||||
Negative control (medium) |
| + | 22 | 15.8 | 14.1 | 15.0 | 100.0 |
Solvent control (DMSO) | 1% | + | 22 | 13.2 | 11.0 | 12.1 | 100.0 |
Positive control (CPA) | 30.0 µg | + | 22 | 3.5 | 8.3 | 5.9 | 39.5 |
Test item | 3.0 µg | + | 22 | 12.7 | 15.4 | 14.1 | 116.1 |
| 10.0 µg | + | 22 | 12.3 | 13.5 | 12.9 | 106.6 |
| 30.0 µg | + | 22 | 9.1 | 9.5 | 9.3 | 76.9 |
| 60.0 µg | + | 22 | 7.6 | 9.9 | 8.8 | 72.3 |
| 80.0 µg | + | 22 | 8.8 | 7.8 | 8.3 | 68.6 |
| 100.0 µg | + | 22 | 9.2 | 6.7 | 8.0 | 65.7 |
Solvent control (DMSO) | 1% | + | 46 | 12.6 | 12.8 | 12.7 | 100.0 |
Test item | 30.0 µg | + | 46 | 7.5 | 8.0 | 7.8 | 61.0 |
| 60.0 µg | + | 46 | 9.5 | 8.8 | 9.2 | 72.0 |
| 80.0 µg | + | 46 | 6.9 | 5.1 | 6.0 | 47.2 |
| 100.0 µg | + | 46 | 4.0 | 8.4 | 6.2 | 48.8 |
*The mitotic index was determined in 1000 cells from each of the two vessels per test group. | |||||||
Summary of results:
Experiment I: mutagenicity data (exposure period 22 h) fixation interval: 22 h after start of the treatment | ||||||
Test group | no. of cells analysed | Conc./mL | S9 mix | % aberrant cells | ||
incl. gaps | excl. gaps | exchanges | ||||
Negative control | 200 |
| - | 1.00 | 0.00 | 0.00 |
Solvent control - DMSO | 200 | 1.0% | - | 3.00 | 2.00 | 0.00 |
Positive control - EMS | 50 | 330 µg | - | 16.00 | 16.00 | 2.00 |
Test item | 200 | 60.0 µg | - | 0.50 | 0.00 | 0.00 |
| 200 | 80.0 µg | - | 3.00 | 1.50 | 0.00 |
| 200 | 100.0 µg | - | 0.50 | 0.00 | 0.00 |
Experiment I: mutagenicity data (exposure period 46 h) fixation interval: 46 h after start of the treatment | ||||||
Test group | no. of cells analysed | Conc./mL | S9 mix | % aberrant cells | ||
incl. gaps | excl. gaps | exchanges | ||||
Solvent control - DMSO | 200 | 1.0% | - | 1.00 | 0.00 | 0.00 |
Test item | 200 | 60.0 µg | - | 0.00 | 0.00 | 0.00 |
| 200 | 80.0 µg | - | 1.00 | 1.00 | 0.50 |
Experiment II: mutagenicity data (exposure period 22 h) fixation interval: 22 h after start of the treatment | ||||||
Test group | no. of cells analysed | Conc./mL | S9 mix | % aberrant cells | ||
incl. gaps | excl. gaps | exchanges | ||||
Negative control | 200 |
| - | 0.50 | 0.00 | 0.00 |
Solvent control - DMSO | 200 | 1.0% | - | 0.00 | 0.00 | 0.00 |
Positive control - EMS | 50 | 330 µg | - | 14.00 | 14.00 | 0.00 |
Test item | 200 | 30.0 µg |
| 0.00 | 0.00 | 0.00 |
| 200 | 60.0 µg | - | 0.50 | 0.50 | 0.00 |
| 200 | 80.0 µg | - | 1.00 | 0.50 | 0.00 |
| 200 | 100.0 µg | - | 0.50 | 0.50 | 0.00 |
Experiment II: mutagenicity data (exposure period 46 h) fixation interval: 46 h after start of the treatment | ||||||
Test group | no. of cells analysed | Conc./mL | S9 mix | % aberrant cells | ||
incl. gaps | excl. gaps | exchanges | ||||
Solvent control - DMSO | 200 | 1.0% | - | 0.50 | 0.00 | 0.00 |
Test item | 200 | 60.0 µg | - | 0.00 | 0.00 | 0.00 |
| 200 | 100.0 µg | - | 1.00 | 1.00 | 0.00 |
Experiment I: mutagenicity data (exposure period 4 h) fixation interval: 22 h after start of the treatment | ||||||
Test group | no. of cells analysed | Conc./mL | S9 mix | % aberrant cells | ||
incl. gaps | excl. gaps | exchanges | ||||
Negative control | 200 |
| + | 0.50 | 0.50 | 0.00 |
Solvent control - DMSO | 200 | 1.0% | + | 3.00 | 1.00 | 0.00 |
Positive control - CPA | 50 | 30.0 µg | + | 12.00 | 10.00 | 4.00 |
Test item | 200 | 10.0 µg | + | 1.50 | 1.00 | 0.00 |
| 200 | 80.0 µg | + | 3.50 | 1.50 | 0.50 |
| 200 | 100.0 µg | + | 2.00 | 1.50 | 0.50 |
Experiment I: mutagenicity data (exposure period 4 h) fixation interval: 46 h after start of the treatment | ||||||
Test group | no. of cells analysed | Conc./mL | S9 mix | % aberrant cells | ||
incl. gaps | excl. gaps | exchanges | ||||
Solvent control - DMSO | 200 | 1.0% | + | 2.00 | 1.00 | 0.00 |
Test item | 200 | 80.0 µg | + | 0.00 | 0.00 | 0.00 |
| 200 | 100.0 µg | + | 1.50 | 1.00 | 0.00 |
Experiment II: mutagenicity data (exposure period 4 h) fixation interval: 22 h after start of the treatment | ||||||
Test group | no. of cells analysed | Conc./mL | S9 mix | % aberrant cells | ||
incl. gaps | excl. gaps | exchanges | ||||
Negative control | 200 |
| + | 0.00 | 0.00 | 0.00 |
Solvent control - DMSO | 200 | 1.0% | + | 0.00 | 0.00 | 0.00 |
Positive control - CPA | 50 | 30.0 µg | + | 10.00 | 10.00 | 2.00 |
Test item | 200 | 30.0 µg | + | 0.00 | 0.00 | 0.00 |
| 200 | 60.0 µg | + | 0.00 | 0.00 | 0.00 |
| 200 | 80.0 µg | + | 1.50 | 1.00 | 0.00 |
| 200 | 100.0 µg | + | 0.00 | 0.00 | 0.00 |
Experiment II: mutagenicity data (exposure period 4 h) fixation interval: 46 h after start of the treatment | ||||||
Test group | no. of cells analysed | Conc./mL | S9 mix | % aberrant cells | ||
incl. gaps | excl. gaps | exchanges | ||||
Solvent control - DMSO | 200 | 1.0% | + | 0.50 | 0.50 | 0.00 |
Test item | 200 | 60.0 µg | + | 0.50 | 0.00 | 0.00 |
| 200 | 100.0 µg | + | 0.00 | 0.00 | 0.00 |
None of the five tester strains showed increased reversion to prototrophy with diflucortolone-21-valerate at the concentrations tested, either in the absence or presence of S9 mix.
Precipitates in the agar were found starting at 0.25 or 0.50 mg diflucortolone-21-valerate/plate without S9 mix and 2 mg diflucortolone-21-valerate /plate with S9 mix.
Growth inhibition of the background lawn was not observed.
Negative controls and positive controls with known mutagens (9-acridinamine, hydrochloride; anthracen-2-amine; benzo[a]pyrene; cyclophosphamide; 2-nitro-9H-fluorene; sodium azide; N-nitrosodimethylamine) produced the expected numbers of revertant colonies.
TA 1535 | Revertants per plate
| Quotien | ||||||||||||
Dose/Plate | -S9 | M | SD | +S9 | M | SD | -S9 | +S9 | ||||||
DMSO | 50µL | 18 | 16 | 4 | 10 | 12 | 3 | 1.0 | 1.0 | |||||
|
| 12 |
|
| 12 |
|
|
|
| |||||
|
| 19 |
|
| 15 |
|
|
|
| |||||
Phosphate Buffer | 50µL | 20 | 17 | 4 | 13 | 14 | 2 | 1.0 | 1.2 | |||||
|
| 13 |
|
| 16 |
|
|
|
| |||||
|
| 17 |
|
| 14 |
|
|
|
| |||||
Test item | 0.05 | 23 | 18 | 5 | 16 | 19 | 6 | 1.1 | 1.5 | |||||
|
| 14 |
|
| 25 |
|
|
|
| |||||
|
| 17 |
|
| 15 |
|
|
|
| |||||
| 0.10 | 13 | 17 | 4 | 9 | 15 | 6 | 1.1 | 1.2 | |||||
|
| 19 |
|
| 21 |
|
|
|
| |||||
|
| 20 |
|
| 15 |
|
|
|
| |||||
| 0.25 | 10 | 18 | 9 | 17 | 15 | 6 | 1.1 | 1.2 | |||||
|
| 15P |
|
| 8 |
|
|
|
| |||||
|
| 28 |
|
| 19 |
|
|
|
| |||||
| 0.5 | 15 | 19 | 4 | 12 | 13 | 4 | 1.1 | 1.1 | |||||
|
| 19P |
|
| 10 |
|
|
|
| |||||
|
| 22 |
|
| 17 |
|
|
|
| |||||
| 1.0 | 16 | 19 | 3 | 13 | 13 | 5 | 1.2 | 1.1 | |||||
|
| 22P |
|
| 9 |
|
|
|
| |||||
|
| 20 |
|
| 18 |
|
|
|
| |||||
| 2.0 | 15 | 15 | 4 | 17 | 12 | 4 | 0.9 | 1.0 | |||||
|
| 18P |
|
| 10P |
|
|
|
| |||||
|
| 11 |
|
| 10 |
|
|
|
| |||||
2-AA | 5.0 µg | 21 | 18 | 3 | 228 | 214 | 12 | 1.1 | 17.4 | |||||
|
| 17 |
|
| 210 |
|
|
|
| |||||
|
| 16 |
|
| 205 |
|
|
|
| |||||
CP | 400 µg | 32 | 29 | 4 | 707 | 685 | 23 | 1.8 | 55.5 | |||||
|
| 30 |
|
| 686 |
|
|
|
| |||||
|
| 25 |
|
| 661 |
|
|
|
| |||||
NaN3 | 5.0 µg | 860 | 851 | 25 | 29 | 21 | 8 | 52.1 | 1.7 | |||||
|
| 871 |
|
| 22 |
|
|
|
| |||||
|
| 823 |
|
| 13 |
|
|
|
| |||||
Titer: Dilution: E+06 Values: 99/110/101 Titer/mL: 10.3E+08 | ||||||||||||||
P = Precipitation M= Mean SD= Standard-Deviation | ||||||||||||||
| ||||||||||||||
| ||||||||||||||
TA 100 | Revertants per plate | Quotien | ||||||||||||
Dose/Plate | -S9 | M | SD | +S9 | M | SD | -S9 | +S9 | ||||||
DMSO | 50µL | 108 | 101 | 10 | 113 | 110 | 3 | 1.0 | 1.0 | |||||
|
| 89 |
|
| 107 |
|
|
|
| |||||
|
| 105 |
|
| 111 |
|
|
|
| |||||
Phosphate Buffer | 50µL | 120 | 106 | 18 | 109 | 104 | 17 | 1.1 | 0.9 | |||||
|
| 112 |
|
| 119 |
|
|
|
| |||||
|
| 86 |
|
| 85 |
|
|
|
| |||||
Test item | 0.05 | 121 | 114 | 6 | 128 | 123 | 5 | 1.1 | 1.1 | |||||
|
| 109 |
|
| 119 |
|
|
|
| |||||
|
| 111 |
|
| 121 |
|
|
|
| |||||
| 0.10 | 87 | 92 | 5 | 115 | 110 | 5 | 0.9 | 1.0 | |||||
|
| 94 |
|
| 106 |
|
|
|
| |||||
|
| 96 |
|
| 108 |
|
|
|
| |||||
| 0.25 | 114 | 98 | 15 | 103 | 108 | 4 | 1.0 | 1.0 | |||||
|
| 95P |
|
| 111 |
|
|
|
| |||||
|
| 84 |
|
| 110 |
|
|
|
| |||||
| 0.5 | 99 | 105 | 10 | 113 | 107 | 5 | 1.0 | 1.0 | |||||
|
| 116P |
|
| 105 |
|
|
|
| |||||
|
| 100 |
|
| 103 |
|
|
|
| |||||
| 1.0 | 106 | 100 | 5 | 117 | 115 | 6 | 1.0 | 1.0 | |||||
|
| 96P |
|
| 108 |
|
|
|
| |||||
|
| 99 |
|
| 119 |
|
|
|
| |||||
| 2.0 | 122 | 111 | 11 | 117 | 118 | 8 | 1.1 | 1.1 | |||||
|
| 100P |
|
| 126P |
|
|
|
| |||||
|
| 110 |
|
| 110 |
|
|
|
| |||||
2-AA | 5.0 µg | 133 | 126 | 8 | 513 | 519 | 7 | 1.2 | 4.7 | |||||
|
| 127 |
|
| 517 |
|
|
|
| |||||
|
| 117 |
|
| 527 |
|
|
|
| |||||
DMNA | 5.0 µL | 122 | 121 | 10 | 1084 | 1124 | 302 | 1.2 | 10.2 | |||||
|
| 130 |
|
| 1444 |
|
|
|
| |||||
|
| 110 |
|
| 843 |
|
|
|
| |||||
NaN3 | 5.0 µg | 677 | 647 | 26 | 155 | 144 | 16 | 6.4 | 1.3 | |||||
|
| 629 |
|
| 151 |
|
|
|
| |||||
|
| 636 |
|
| 125 |
|
|
|
| |||||
Titer: Dilution: E+06 Values: 80/61/71 Titer/mL: 7.1E+08 | ||||||||||||||
P = Precipitation M= Mean SD= Standard-Deviation | ||||||||||||||
TA 1537 |
| Revertants per plate | Quotient | |||||||||||
Dose/Plate | -S9 | M | SD | +S9 | M | SD | -S9 | +S9 | ||||||
DMSO | 50µL | 7 | 8 | 3 | 17 | 19 | 2 | 1.0 | 1.0 | |||||
|
| 6 |
|
| 19 |
|
|
|
| |||||
|
| 11 |
|
| 20 |
|
|
|
| |||||
Phosphate Buffer | 50µL | 7 | 8 | 1 | 20 | 18 | 2 | 1.0 | 1.0 | |||||
|
| 8 |
|
| 19 |
|
|
|
| |||||
|
| 8 |
|
| 16 |
|
|
|
| |||||
Test item | 0.05 | 8 | 8 | 4 | 17 | 16 | 1 | 1.0 | 0.9 | |||||
|
| 4 |
|
| 15 |
|
|
|
| |||||
|
| 11 |
|
| 17 |
|
|
|
| |||||
| 0.10 | 9 | 12 | 3 | 23 | 18 | 5 | 1.5 | 1.0 | |||||
|
| 12 |
|
| 17 |
|
|
|
| |||||
|
| 14 |
|
| 14 |
|
|
|
| |||||
| 0.25 | 12 | 9 | 4 | 15 | 18 | 5 | 1.1 | 1.0 | |||||
|
| 9 |
|
| 24 |
|
|
|
| |||||
|
| 5 |
|
| 15 |
|
|
|
| |||||
| 0.5 | 9 | 10 | 2 | 15 | 15 | 7 | 1.2 | 0.8 | |||||
|
| 8P |
|
| 9 |
|
|
|
| |||||
|
| 12 |
|
| 22 |
|
|
|
| |||||
| 1.0 | 7 | 8 | 1 | 10 | 13 | 4 | 1.0 | 0.7 | |||||
|
| 8P |
|
| 12 |
|
|
|
| |||||
|
| 9 |
|
| 17 |
|
|
|
| |||||
| 2.0 | 5 | 7 | 2 | 17 | 12 | 6 | 0.8 | 0.6 | |||||
|
| 9P |
|
| 6P |
|
|
|
| |||||
|
| 6 |
|
| 13 |
|
|
|
| |||||
2-AA | 5.0 µg | 8 | 12 | 4 | 67 | 69 | 8 | 1.5 | 3.7 | |||||
|
| 14 |
|
| 62 |
|
|
|
| |||||
|
| 15 |
|
| 77 |
|
|
|
| |||||
B[a]P | 10.0 µg | 10 | 10 | 1 | 120 | 132 | 11 | 1.2 | 7.1 | |||||
|
| 10 |
|
| 133 |
|
|
|
| |||||
|
| 9 |
|
| 142 |
|
|
|
| |||||
9-AA | 80.0 µg | 259 | 265 | 10 | 378 | 386 | 27 | 33.1 | 20.7 | |||||
|
| 259 |
|
| 416 |
|
|
|
| |||||
|
| 276 |
|
| 363 |
|
|
|
| |||||
Titer: Dilution: E+06 Values: 61/68/69 Titer/mL: 6.6E+08 | ||||||||||||||
P = Precipitation M= Mean SD= Standard-Deviation | ||||||||||||||
TA 1538 | Revertants per plate | Quotient | ||||||||||||
Dose/Plate | -S9 | M | SD | +S9 | M | SD | -S9 | +S9 | ||||||
DMSO | 50µL | 21 | 20 | 2 | 23 | 27 | 3 | 1.0 | 1.0 | |||||
|
| 18 |
|
| 28 |
|
|
|
| |||||
|
| 22 |
|
| 29 |
|
|
|
| |||||
Phosphate Buffer | 50µL | 25 | 26 | 6 | 26 | 29 | 3 | 1.3 | 1.1 | |||||
|
| 21 |
|
| 28 |
|
|
|
| |||||
|
| 32 |
|
| 32 |
|
|
|
| |||||
Test item | 0.05 | 20 | 15 | 6 | 26 | 28 | 2 | 0.7 | 1.1 | |||||
|
| 16 |
|
| 29 |
|
|
|
| |||||
|
| 9 |
|
| 29 |
|
|
|
| |||||
| 0.10 | 21 | 21 | 7 | 26 | 26 | 1 | 1.0 | 1.0 | |||||
|
| 28 |
|
| 25 |
|
|
|
| |||||
|
| 15 |
|
| 27 |
|
|
|
| |||||
| 0.25 | 20 | 20 | 4 | 31 | 28 | 3 | 1.0 | 1.0 | |||||
|
| 17 |
|
| 27 |
|
|
|
| |||||
|
| 24 |
|
| 25 |
|
|
|
| |||||
| 0.5 | 20 | 22 | 4 | 27 | 29 | 4 | 1.1 | 1.1 | |||||
|
| 26P |
|
| 26 |
|
|
|
| |||||
|
| 19 |
|
| 33 |
|
|
|
| |||||
| 1.0 | 27 | 24 | 4 | 26 | 29 | 3 | 1.2 | 1.1 | |||||
|
| 19P |
|
| 31 |
|
|
|
| |||||
|
| 26 |
|
| 31 |
|
|
|
| |||||
| 2.0 | 26 | 19 | 7 | 17 | 19 | 3 | 0.9 | 0.7 | |||||
|
| 19P |
|
| 18P |
|
|
|
| |||||
|
| 12 |
|
| 23 |
|
|
|
| |||||
2-AA | 5.0 µg | 20 | 19 | 2 | 577 | 546 | 36 | 0.9 | 20.5 | |||||
|
| 20 |
|
| 554 |
|
|
|
| |||||
|
| 17 |
|
| 506 |
|
|
|
| |||||
B[a]P | 10.0 µg | 18 | 18 | 5 | 395 | 416 | 26 | 0.9 | 15.6 | |||||
|
| 13 |
|
| 445 |
|
|
|
| |||||
|
| 22 |
|
| 409 |
|
|
|
| |||||
2-NF | 10.0 µg | 987 | 973 | 35 | 215 | 233 | 22 | 47.9 | 8.7 | |||||
|
| 999 |
|
| 257 |
|
|
|
| |||||
|
| 934 |
|
| 227 |
|
|
|
| |||||
Titer: Dilution: E+06 Values: 59/66/56 Titer/mL: 6.0E+08 | ||||||||||||||
P = Precipitation M= Mean SD= Standard-Deviation | ||||||||||||||
TA 98 | Revertants per plate | Quotient | ||||||||||||
Dose/Plate | -S9 | M | SD | +S9 | M | SD | -S9 | +S9 | ||||||
DMSO | 50µL | 28 | 27 | 3 | 35 | 38 | 4 | 1.0 | 1.0 | |||||
|
| 29 |
|
| 37 |
|
|
|
| |||||
|
| 24 |
|
| 43 |
|
|
|
| |||||
Phosphate Buffer | 50µL | 30 | 30 | 1 | 42 | 39 | 5 | 1.1 | 1.0 | |||||
|
| 29 |
|
| 33 |
|
|
|
| |||||
|
| 30 |
|
| 41 |
|
|
|
| |||||
Test item | 0.05 | 32 | 25 | 7 | 35 | 35 | 7 | 0.9 | 0.9 | |||||
|
| 25 |
|
| 29 |
|
|
|
| |||||
|
| 19 |
|
| 42 |
|
|
|
| |||||
| 0.10 | 29 | 27 | 4 | 31 | 32 | 2 | 1.0 | 0.8 | |||||
|
| 30 |
|
| 32 |
|
|
|
| |||||
|
| 23 |
|
| 34 |
|
|
|
| |||||
| 0.25 | 29 | 34 | 9 | 32 | 32 | 2 | 1.3 | 0.8 | |||||
|
| 29P |
|
| 34 |
|
|
|
| |||||
|
| 44 |
|
| 31 |
|
|
|
| |||||
| 0.5 | 33 | 35 | 2 | 39 | 36 | 3 | 1.3 | 0.9 | |||||
|
| 36P |
|
| 33 |
|
|
|
| |||||
|
| 37 |
|
| 36 |
|
|
|
| |||||
| 1.0 | 30 | 26 | 4 | 37 | 37 | 1 | 1.0 | 1.0 | |||||
|
| 25P |
|
| 38 |
|
|
|
| |||||
|
| 23 |
|
| 37 |
|
|
|
| |||||
| 2.0 | 24 | 22 | 3 | 48 | 45 | 6 | 0.8 | 1.2 | |||||
|
| 19P |
|
| 48P |
|
|
|
| |||||
|
| 24 |
|
| 38 |
|
|
|
| |||||
2-AA | 5.0 µg | 14 | 15 | 2 | 460 | 509 | 42 | 0.6 | 13.3 | |||||
|
| 15 |
|
| 532 |
|
|
|
| |||||
|
| 17 |
|
| 534 |
|
|
|
| |||||
B[a]P | 10.0 µg | 39 | 31 | 8 | 398 | 359 | 50 | 1.1 | 9.4 | |||||
|
| 24 |
|
| 375 |
|
|
|
| |||||
|
| 30 |
|
| 303 |
|
|
|
| |||||
2-NF | 10.0 µg | 1252 | 1232 | 82 | 204 | 217 | 15 | 45.6 | 5.7 | |||||
|
| 1142 |
|
| 233 |
|
|
|
| |||||
|
| 1302 |
|
| 215 |
|
|
|
| |||||
Titer: Dilution: E+06 Values: 107/110/101 Titer/mL: 10.6E+08 | ||||||||||||||
P = Precipitation M= Mean SD= Standard-Deviation | ||||||||||||||
None of the five tester strains showed increased reversion to prototrophy with ZK 22612 (diflucortolone-21-valerate) at the concentrations tested, either in the presence or absence of S9 mix. Also in an additionally performed test with TA 100 using the preincubation procedure ZK 22612 did not show a mutagenic effect.
Growth inhibition of the background lawn was not observed.
Precipitates in the agar were found generally in all concentrations without S9 mix, at 2.5 and 5 mg per plate with S9 mix and in the preincubation test starting at 1 mg per plate with S9 mix.
Negative controls and positive controls with known mutagens (9-aminoacridine, anthracene-2-amine, benzo(a)pyrene, cyclophosphamide, 2-nitrofluorene, sodium azide) produced the expected numbers of revertant colonies.
Number of revertant colonies per plate, arithmetic means and standard deviations obtained with Salmonella typhimurium TA 1535 following exposure to the test item (dissolved in DMSO) and known mutagens. | |||||||
Addition | Amount/plate | with S9 mix | without S9 mix | ||||
Individual plate count | x̅ | SD | Individual plate count | x̅ | SD | ||
DMSO | 0.1 mL | 17 | 15 | 2.0 | 24 | 22 | 4.9 |
|
| 13 |
|
| 16 |
|
|
|
| 15 |
|
| 25 |
|
|
phosphate buffer | 0.1 mL | 15 | 16 | 4.2 | 26 | 25 | 3.2 |
|
| 13 |
|
| 27 |
|
|
|
| 21 |
|
| 21 |
|
|
2-AA | 2.0 µg | 225 | 203 | 19.1 | 23 | 18 | 4.0 |
|
| 191 |
|
| 16 |
|
|
|
| 193 |
|
| 16 |
|
|
CP | 400.0 µg | 289 | 345 | 49.4 | 37 | 45 | 8.6 |
|
| 381 |
|
| 43 |
|
|
|
| 366 |
|
| 54 |
|
|
test item | 0.1 mg | 8 | 12 | 4.0 | 28 | 25P | 4.9 |
|
| 12 |
|
| 27 |
|
|
|
| 16 |
|
| 19 |
|
|
| 0.25 mg | 24 | 18 | 5.3 | 31 | 23P | 8.0 |
|
| 14 |
|
| 15 |
|
|
|
| 16 |
|
| 22 |
|
|
| 0.5 mg | 22 | 16 | 5.5 | 29 | 32P | 4.6 |
|
| 13 |
|
| 29 |
|
|
|
| 12 |
|
| 37 |
|
|
| 1.0 mg | 17 | 16 | 2.3 | 29 | 22P | 6.7 |
|
| 17 |
|
| 20 |
|
|
|
| 13 |
|
| 16 |
|
|
| 2.5 mg | 8 | 11P | 4.2 | 16 | 20P | 3.8 |
|
| 16 |
|
| 23 |
|
|
|
| 10 |
|
| 22 |
|
|
| 5.0 mg | 5 | 10P | 4.7 | 21 | 21P | 2.5 |
|
| 14 |
|
| 19 |
|
|
|
| 12 |
|
| 24 |
|
|
cell population: 2E+08 per plate p= precipitates | |||||||
| |||||||
Number of revertant colonies per plate, arithmetic means and standard deviations obtained with Salmonella typhimurium TA 100 following exposure to the test item (dissolved in OMSO) and known mutagens. | |||||||
Addition | Amount/plate | with S9 mix | without S9 mix | ||||
Individual plate count | x̅ | SD | Individual plate count | x̅ | SD | ||
DMSO | 0.1 mL | 106 | 97 | 8.1 | 128 | 104 | 21.8 |
|
| 93 |
|
| 86 |
|
|
|
| 91 |
|
| 97 |
|
|
phosphate buffer | 0.1 mL | 112 | 101 | 14.4 | 148 | 127 | 30.4 |
|
| 85 |
|
| 105 |
|
|
|
| 107 |
|
| - |
|
|
2-AA | 2.0 µg | 1107 | 1160 | 75.7 | 90 | 98 | 6.8 |
|
| 1127 |
|
| 100 |
|
|
|
| 1247 |
|
| 103 |
|
|
B[a]P | 5.0 µg | 273 | 267 | 29.5 | 86 | 91 | 14.6 |
|
| 235 |
|
| 107 |
|
|
|
| 293 |
|
| 79 |
|
|
NaN3 | 2.0 µg | 478 | 476 | 1.5 | 718 | 711 | 6.4 |
|
| 476 |
|
| 706 |
|
|
|
| 475 |
|
| 708 |
|
|
test item | 0.1 mg | 102 | 98 | 3.8 | 112 | 108P | 15.9 |
|
| 96 |
|
| 121 |
|
|
|
| 95 |
|
| 90 |
|
|
| 0.25 mg | 93 | 97 | 6.4 | 111 | 107P | 8.7 |
|
| 104 |
|
| 113 |
|
|
|
| 93 |
|
| 97 |
|
|
| 0.5 mg | 102 | 102 | 3.5 | 103 | 106P | 12.3 |
|
| 105 |
|
| 120 |
|
|
|
| 98 |
|
| 96 |
|
|
| 1.0 mg | 114 | 103 | 9.8 | 112 | 108P | 4.7 |
|
| 97 |
|
| 110 |
|
|
|
| 97 |
|
| 103 |
|
|
| 2.5 mg | 112 | 98P | 12.2 | 102 | 106P | 6.4 |
|
| 90 |
|
| 113 |
|
|
|
| 92 |
|
| 102 |
|
|
| 5.0 mg | 91 | 91P | 6.5 | 116 | 110P | 7.2 |
|
| 97 |
|
| 112 |
|
|
|
| 84 |
|
| 102 |
|
|
cell population: 3xE+08 per plate; - not plated; P precipitates | |||||||
| |||||||
Number of revertant colonies per plate, arithmetic means and standard deviations obtained with Salmonella typhimurium TA 1537 following exposure to the test item (dissolved in DMSO) and known mutagens. | |||||||
Addition | Amount/plate | with S9 mix | without S9 mix | ||||
Individual plate count | x̅ | SD | Individual plate count | x̅ | SD | ||
DMSO | 0.1 mL | 18 | 13 | 4.2 | 12 | 10 | 2.5 |
|
| 12 |
|
| 7 |
|
|
|
| 10 |
|
| 10 |
|
|
2-AA | 5.0 µg | 152 | 188 | 33.4 | 8 | 7 | 1.5 |
|
| 194 |
|
| 5 |
|
|
|
| 218 |
|
| 7 |
|
|
B[a]P | 10.0 µg | 33 | 35 | 6.8 | 6 | 7 | 2.6 |
|
| 43 |
|
| 10 |
|
|
|
| 30 |
|
| 5 |
|
|
2-NF | 10.0 µg | 80 | 87 | 13.6 | 53 | 57 | 10.0 |
|
| 79 |
|
| 49 |
|
|
|
| 103 |
|
| 68 |
|
|
9-AA | 100.0 µg | - | - | - | 601 | 561 | 36.3 |
|
| - |
|
| 530 |
|
|
|
| - |
|
| 552 |
|
|
Test item | 0.1 mg | 8 | 11 | 4.6 | 5 | 6P | 1.5 |
|
| 16 |
|
| 6 |
|
|
|
| 8 |
|
| 8 |
|
|
| 0.25 mg | 13 | 10 | 2.6 | 8 | 9P | 4.0 |
|
| 8 |
|
| 5 |
|
|
|
| 9 |
|
| 13 |
|
|
| 0.5mg | 10 | 11 | 3.2 | 10 | 7P | 2.5 |
|
| 9 |
|
| 7 |
|
|
|
| 15 |
|
| 5 |
|
|
| 1.0 mg | 14 | 12 | 2.0 | 5 | 8P | 3.1 |
|
| 12 |
|
| 11 |
|
|
|
| 10 |
|
| 7 |
|
|
| 2.5 mg | 5 | 8P | 3.1 | 11 | 11P | 0.6 |
|
| 9 |
|
| 12 |
|
|
|
| 11 |
|
| 11 |
|
|
| 5.0 mg | 7 | 7P | 1.0 | 8 | 6P | 2.1 |
|
| 8 |
|
| 5 |
|
|
|
| 6 |
|
| 4 |
|
|
cell population: 8E+07per plate; - not tested; p precipitates | |||||||
| |||||||
Nurnber of revertant colonies per plate, arithrnetic means and standard deviations obtained with Salmonella typhimurium TA 1538 following exposure to the test item (dissolved in DMSO) and known mutagens. | |||||||
Addition | Amount/plate | with S9 mix | without S9 mix | ||||
Individual plate count | x̅ | SD | Individual plate count | x̅ | SD | ||
DMSO | 0.1 mL | 26 | 32 | 6.6 | 11 | 17 | 6.0 |
|
| 31 |
|
| 23 |
|
|
|
| 39 |
|
| 18 |
|
|
2-AA | 5.0 µg | 1839 | 1630 | 184.0 | 34 | 30 | 6.4 |
|
| 1561 |
|
| 23 |
|
|
|
| 1491 |
|
| 34 |
|
|
B[a]P | 10.0 µg | 99 | 99 | 10.5 | 19 | 17 | 4.4 |
|
| 109 |
|
| 20 |
|
|
|
| 88 |
|
| 12 |
|
|
2-NF | 10.0 µg | 448 | 434 | 13.5 | 1166 | 1239 | 70.2 |
|
| 421 |
|
| 1305 |
|
|
|
| 434 |
|
| 1245 |
|
|
Test item | 0.1 mg | 26 | 31 | 5.0 | 20 | 18P | 4.4 |
|
| 36 |
|
| 21 |
|
|
|
| 30 |
|
| 13 |
|
|
| 0.25 mg | 29 | 37 | 8.0 | 11 | 12P | 2.3 |
|
| 45 |
|
| 15 |
|
|
|
| 36 |
|
| 11 |
|
|
| 0.5mg | 26 | 33 | 6.6 | 12 | 11P | 1.7 |
|
| 34 |
|
| 9 |
|
|
|
| 39 |
|
| 12 |
|
|
| 1.0 mg | 34 | 32 | 2.5 | 18 | 17P | 1.7 |
|
| 29 |
|
| 15 |
|
|
|
| 32 |
|
| 18 |
|
|
| 2.5 mg | 46 | 35P | 9.8 | 12 | 12P | 3.5 |
|
| 29 |
|
| 9 |
|
|
|
| 29 |
|
| 16 |
|
|
| 5.0 mg | 26 | 27P | 2.1 | 10 | 12P | 2.0 |
|
| 25 |
|
| 12 |
|
|
|
| 29 |
|
| 14 |
|
|
cell population: 8 x E+07 per plate p precipitates | |||||||
| |||||||
Number of revertant colonies per plate, arithmetic means and standard deviations obtained with Salmonella typhimurium TA 98 following exposure to the test item (dissolved in DMSO) and known mutagens. | |||||||
Addition | Amount/plate | with S9 mix | without S9 mix | ||||
Individual plate count | x̅ | SD | Individual plate count | x̅ | SD | ||
DMSO | 0.1 mL | 44 | 38 | 5.2 | 31 | 29 | 9.7 |
|
| 35 |
|
| 18 |
|
|
|
| 35 |
|
| 37 |
|
|
2-AA | 5.0 µg | 1007 | 956 | 53.7 | 21 | 27 | 6.0 |
|
| 900 |
|
| 33 |
|
|
|
| 962 |
|
| 27 |
|
|
B[a]P | 10.0 µg | 129 | 124 | 4.0 | 22 | 28 | 6.7 |
|
| 122 |
|
| 26 |
|
|
|
| 122 |
|
| 35 |
|
|
2-NF | 10.0 µg | 99 | 100 | 11.5 | 180 | 196 | 13.7 |
|
| 112 |
|
| 205 |
|
|
|
| 89 |
|
| 202 |
|
|
Test item | 0.1 mg | 33 | 37 | 3.2 | 30 | 26P | 3.5 |
|
| 39 |
|
| 24 |
|
|
|
| 38 |
|
| 24 |
|
|
| 0.25 mg | 35 | 36 | 1.2 | 24 | 22P | 2.6 |
|
| 35 |
|
| 23 |
|
|
|
| 37 |
|
| 19 |
|
|
| 0.5mg | 43 | 38 | 4.2 | 29 | 27P | 2.0 |
|
| 37 |
|
| 27 |
|
|
|
| 35 |
|
| 25 |
|
|
| 1.0 mg | 48 | 39 | 8.1 | 27 | 28P | 3.1 |
|
| 33 |
|
| 31 |
|
|
|
| 35 |
|
| 25 |
|
|
| 2.5 mg | 33 | 27P | 5.9 | 22 | 25P | 3.6 |
|
| 35 |
|
| 24 |
|
|
|
| 44 |
|
| 29 |
|
|
| 5.0 mg | 26 | 34P | 7.5 | 37 | 33P | 9.1 |
|
| 41 |
|
| 40 |
|
|
|
| 35 |
|
| 23 |
|
|
cell population:2 xE+08 per plate - not tested; P precipitates | |||||||
Number of revertant colonies per plate. arithmetic means and standard deviations obtained with Salmonella typhimurium TA 100 following exposure to the test item (dissolved in DMSO) and known mutagens without S9 mix or in the presence of various concentrations of S9, using the preincubation procedure. | ||||||||||
Addition | Amount | with 10% S9 mix | with 30% S9 mix | without S9 mix | ||||||
Individual plate count | x̅ | SD | Individual plate count | x̅ | SD | Individual plate count | x̅ | SD | ||
DMSO | 0.1 mL | 108 | 104 | 4.0 | 101 | 94 | 16.3 | 105 | 100 | 14.6 |
|
| 100 |
|
| 105 |
|
| 84 |
|
|
|
| 105 |
|
| 75 |
|
| 112 |
|
|
phosphate buffer | 0.1 mL | 114 | 120 | 6.6 | 125 | 112 | 14.0 | 105 | 124 | 19.0 |
|
| 119 |
|
| 113 |
|
| 124 |
|
|
|
| 127 |
|
| 97 |
|
| 143 |
|
|
2-AA | 2.0 µg | 833 | 839 | 75.7 | 411 | 399 | 17.7 | 117 | 126 | 9.0 |
|
| 917 |
|
| 386 |
|
| 135 |
|
|
|
| 766 |
|
| - |
|
| 127 |
|
|
NaN3 | 2.0 µg | 411 | 389 | 31.8 | 204 | 196 | 7.6 | 564 | 581 | 15.1 |
|
| 366 |
|
| 189 |
|
| 588 |
|
|
|
| - |
|
| 194 |
|
| 592 |
|
|
N-nitroso-dimethyl-amine | 5:0 µL | 144 | 133 | 12.9 | 1006 | 889 | 102.0 | 143 | 125 | 15.7 |
|
| 119 |
|
| 819 |
|
| 120 |
|
|
|
| 137 |
|
| 841 |
|
| 113 |
|
|
test item | 0.1 mg | 105 | 104 | 7.5 | 77 | 88 | 9.8 | 90 | 89P | 9.1 |
|
| 111 |
|
| 96 |
|
| 79 |
|
|
|
| 96 |
|
| 91 |
|
| 97 |
|
|
| 0.25 mg | 112 | 101 | 9.6 | 96 | 80 | 13.8 | 112 | 123P | 9.2 |
|
| 97 |
|
| 70 |
|
| 128 |
|
|
|
| 94 |
|
| 75 |
|
| 128 |
|
|
| 0.5 mg | 106 | 100 | 11.8 | 106 | 101 | 9.2 | 121 | 118P | 11.2 |
|
| 107 |
|
| 106 |
|
| 128 |
|
|
|
| 86 |
|
| 90 |
|
| 106 |
|
|
| 0.75 mg | 98 | 114 | 15.0 | 90 | 100 | 10.0 | 98 | 109P | 14.4 |
|
| 115 |
|
| 110 |
|
| 125 |
|
|
|
| 128 |
|
| 99 |
|
| 103 |
|
|
| 1.0 mg | 110 | 103 | 5.8 | 102 | 108P | 12.5 | 110 | 103P | 9.1 |
|
| 100 |
|
| 122 |
|
| 93 |
|
|
|
| 100 |
|
| 99 |
|
| 107 |
|
|
| 2.0 mg | 136 | 122 | 13.1 | 88 | 87P | 0.6 | 123 | 107P | 16.0 |
|
| 121 |
|
| 87 |
|
| 107 |
|
|
|
| 110 |
|
| 87 |
|
| 91 |
|
|
| 4.0 mg | 109 | 120P | 17.1 | 92 | 82P | 10.0 | 111 | 105P | 9.5 |
|
| 112 |
|
| 82 |
|
| 94 |
|
|
|
| 140 |
|
| 72 |
|
| 110 |
|
|
| 6.0 mg | 112 | 112P | 4.5 | 56 | 58P | 4.4 | 112 | 113P | 8.5 |
|
| 107 |
|
| 63 |
|
| 105 |
|
|
|
| 116 |
|
| 55 |
|
| 122 |
|
|
cell population: 1xE+08 per plate; - plate contaminated; P precipitates | ||||||||||
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Genetic toxicity in vivo
Description of key information
Reimann, 1993, study conducted with Difluocortolone according to OECD test guideline 474, result: negative
Link to relevant study records
- Endpoint:
- in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
- Type of information:
- experimental study
- Adequacy of study:
- supporting study
- Study period:
- 1993
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
- Version / remarks:
- 26 May 1983
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- mammalian erythrocyte micronucleus test
- Species:
- mouse
- Strain:
- NMRI
- Details on species / strain selection:
- The mouse is an animal which has been used for many years as suitable experimental animal in cytogenetic investigations. There are many data available from such investigations which may be helpful in the interpretation of results from the micronucleus test. In addition, the mouse is an experimental animal in many physiological, pharmacological and toxicological studies. Data from such experiments also may be useful for the design and the performance of the micronucleus test.
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: Charles River Wiga GmbH Sandhofer Weg 7, D-8741 Sulzfeld 1
- Age at study initiation: minimum 10 weeks
- Weight at study initiation: 23.0 g (minimum) - 38.5 g (maximum)
- Housing: individually in MakroIon Type I, with wire mesh top
- Diet (e.g. ad libitum): pelleted standard diet, ad libitum (ALTROMIN 1324, 0-4937 Lage/Lippe)
- Water (e.g. ad libitum): tap water, ad libitum (Gemeindewerke D-6101 Roßdorf)
- Acclimation period: minimum 5 days
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21± 3
- Humidity (%): 30-70
- Photoperiod (hrs dark / hrs light): 12/12 - Route of administration:
- intraperitoneal
- Vehicle:
- NaCl/Myrj 53
- 20mL/kg bw
- Lot/batch no. (if required): G/M 93-3 - Details on exposure:
- At the beginning of the treatment the animals were weighed and the individual volume to be administered was adjusted to the animals body weight. The animals received the test article intraperitoneally, once. Twelve animals, six males and six females, were treated. per dose group. Collection of the bone marrow was done 24 hand 48 h after treatment.
- Duration of treatment / exposure:
- The animals received the test article intraperitoneally, once. Twelve animals, six males and six females, were treated. per dose group. Collection of the bone marrow was done 24 hand 48 h after treatment.
- Frequency of treatment:
- once
- Post exposure period:
- 24 and 48 h
- Dose / conc.:
- 2 000 mg/kg bw (total dose)
- Dose / conc.:
- 700 mg/kg bw (total dose)
- Dose / conc.:
- 200 mg/kg bw (total dose)
- No. of animals per sex per dose:
- 6
- Control animals:
- yes, concurrent vehicle
- Positive control(s):
- cyclophosphamide
- Justification for choice of positive control(s): At 25°C only 3.5 % of its potency is lost after 24 hours
- Route of administration: i.p.
- Doses / concentrations: 30 mg/kg b.w. 10 ml/kg b.w. - Tissues and cell types examined:
- micronuclei in PCEs
1000 polychromatic erythrocytes (PCE) were analysed per animal for micronuclei. To describe a cytotoxic effect the ratio between polychromatic and normochromatic erythrocytes was determined in the same sampIe and expressed as normochromatic erythrocytes per 1000 PCEs. The analysis was performed with coded slides. Five animals per sex and group were evaluated as described. The remaining animalof each test group was evaluated in case an animal had died in its test group. - Details of tissue and slide preparation:
- CRITERIA FOR DOSE SELECTION: A preliminary study on acute toxicity was performed with the same strain and under identical conditions as in the mutagenicity study. The following three doses were applied in the main experiment:
200, 700, and 2000 mg/kg body weight.
TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields): Intraperitoneal injection and collection of bone marrow after 24 and 48 h.
DETAILS OF SLIDE PREPARATION: The animals were sacrificed by cervical dislocation. The femora were removed, the epiphyses were cut off and the marrow was flushed out with fetal calf serum, using a 5 ml syringe. The cell suspension was centrifuged at 1,500 rpm for 10 minutes and the supernatant was discarded. A small drop of the resuspended cell pellet was spread on a slide. The smear was air-dried and then stained with May-Grünwald (MERCK, 0-6100 Darmstadt) /Giemsa (Gurr, BDH Limited Poole, Great Britain). Cover slips were mounted with EUKITT (KINDLER, 0-7800 Freiburg). At least one slide was made from each bone marrow sampIe. 1000 polychromatic erythrocytes (PCE) were analysed per animal for micronuclei. - Evaluation criteria:
- A test article is classified as mutagenic if it induces a statistically significant increase in the number of micronucleated polychromatic erythrocytes for at least one of the test points.
A test article producing no statistically significant increase in the number of micronucleated polychromatic erythrocytes at any of the test points is considered non-mutagenic in this system.
This can be confirmed by means of the nonparametrie Mann-Whitney test.
However, both biological and statistical significance are considered together. - Key result
- Sex:
- male/female
- Genotoxicity:
- negative
- Toxicity:
- yes
- Vehicle controls validity:
- valid
- Negative controls validity:
- not examined
- Positive controls validity:
- valid
- Additional information on results:
- RESULTS OF RANGE-FINDING STUDY
- Dose range: Dose of 2000 mg/kg bw i.p.
- Clinical signs of toxicity in test animals: 96 hours after treatment 1 male and 1 female died. Therefore, 2000 mg / kg b.w. were estimated to be close to the maximum tolerated dose within the experimental period.
- Evidence of cytotoxicity in tissue analysed: not reported
RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei (for Micronucleus assay): Only after 48 h post-treatment at 2000 mg/kg bw.
- Ratio of PCE/NCE (for Micronucleus assay): please refer to any other information on results incl. tables
- Conclusions:
- The test article was assessed in the micronucleus assay for its potential to induce micronuclei in polychromatic erythrocytes (PCE) in the bone marrow of the mouse. The test article was suspended in NaCl/Myrj 53. This suspending agent was used as negative control. The volume administered intraperitoneally was 20 ml/kg body weight (b.w.). 24 h and 48 h after a single application of the test article the bone marrow cells were collected for micronuclei analysis. Ten animals (5 males, 5 females) per test group were evaluated for the occurrence of micronuclei in PCEs. Per animal 1000 PCEs were scored for micronuclei.
In comparison to-the corresponding negative controls there was no significant enhancement in the frequency of the detected micronuclei at any preparation interval or dose level of the test articIe. The mean values of micronuclei observed after treatment were in the same range as compared to the negative control groups.
In conclusion, it can be stated that during the study described and under the experimental conditions reported, the test article did not induce micronuclei as determined by the micronucleus test with bone marrow cells of the mouse. - Executive summary:
In a NMRI mouse bone marrow micronucleus assay according to OECD test guideline 474, 5 mice/sex/dose were treated intraperitoneally with Difluocortolone (100 % a.i.) at doses of 0, 200, 700, 2000 mg/kg bw. Bone marrow cells were harvested at 24h and 48h post-treatment. The vehicle was NaCl/Myrj 53.
There were signs of toxicity at 2000 mg/kg bw after 48h post-treatment period. The animals expressed toxic reactions. The ratio of normochromatic to polychromatic erythrocytes was affected by the treatment, indicating that the test article had cytotoxic properties. However, Difluocortolone was tested at an adequate dose based on the preliminary toxicity test. The positive control induced the appropriate response. There was not a significant increase in the frequency of micronucleated polychromatic erythrocytes in bone marrow after any treatment time.
Reference
Preliminary toxicity test:
toxic reactions | hours post-treatment male/female | ||||
1h | 6h | 24h | 48h | 72h | |
reduction of spontaneous activity | 2/2 | 2/2 | 2/2 | 1/0 | 1/1 |
eyelid closure | 2/2 | 2/2 | 2/0 | 1/0 | 1/0 |
apathy | 2/2 | 0/0 | 0/0 | 0/0 | 1/1 |
abdominal position | 2/2 | 0/0 | 0/0 | 0/0 | 0/0 |
Summary of results
test group | dose mg/kg bw | sampling time (h) | PCEs with micronuclei(%) | range | PCE/NCE |
vehicle | 0 | 24 | 0.11 | 0-4 | 1000/752 |
test item | 200 | 24 | 0.12 | 0-3 | 1000/827 |
| 700 | 24 | 0.18 | 0-4 | 1000/850 |
| 2000 | 24 | 0.17 | 0-4 | 1000/937 |
CPA | 30 | 24 | 2.06 | 14-38 | 1000/849 |
vehicle | 0 | 48 | 0.11 | 0-3 | 1000/824 |
| 200 | 48 | 0.16 | 0-4 | 1000/913 |
| 700 | 48 | 0.16 | 0-3 | 1000/955 |
| 2000 | 48 | 0.14 | 0-3 | 1000/1366 |
Additional information
For bromhydrin-valerate (CAS No. 54605-02-6) no genetic toxicity data are available. Therefore, genetic toxicity in vitro data of diflucortolone-21-valerate (CAS No. 59198-70-8) were used since these data are regarded as representative. In diflucortolone-21-valerate the bromine atom in position 9 of the target molecule (bromhydrin-valerate) is replaced by a fluorine atom. No other changes in the molecule occured. No relevant toxicological effects are expected by the change of a halogen atom (Br) versus another (F). A search for structure-analogue substances using the QSAR OECD Toolbox 3.4 recommended diflucortolone-21-valerate as one out of 8 category substances for a read-across approach (for additional information see QSAR OECD Toolbox Report on Bromhydrin-valerat in "Attached justification"). A justification for read-across is attached in chapter 13 of this IUCLID file.
For bromhydrin-valerate (CAS No. 54605-02-6) no genetic toxicity data are available. Therefore, genetic toxicity in vitro data of diflucortolone-21-valerate (CAS No. 59198-70-8) were used since these data are regarded as representative. In diflucortolone-21-valerate the bromine atom in position 9 of the target molecule (bromhydrin-valerate) is replaced by a fluorine atom. No other changes in the molecule occured. No relevant toxicological effects are expected by the change of a halogen atom (Br) versus another (F). A search for structure-analogue substances using the QSAR OECD Toolbox 3.4 recommended diflucortolone-21-valerate as one out of 8 category substances for a read-across approach (for additional information see QSAR OECD Toolbox Report on Bromhydrin-valerat in "Attached justification"). A justification for read-across is attached in chapter 13 of this IUCLID file.
For Difluocortolone the following experimental data are available:
In a reverse gene mutation assay in bacteria according to OECD test guideline 471 (1983), strains TA 98, TA 100, TA 1535, TA 1537 and TA 1538 of S. typhimurium were exposed to Difluocortolone valerate, (100% a.i.), in DMSO at concentrations of 0.05, 0.1, 0.25, 0.5, 1.0, 2.0 mg/plate in the presence and absence of mammalian metabolic activation using the pre-incubation method.
Difluocortolone valerate was tested up to insoluble concentrations starting at 2.5 mg with S9 mix and at 0.1 mg/plate without S9 mix. The positive controls induced the appropriate responses in the corresponding strains. There was no evidence of induced mutant colonies over background.
In a second reverse gene mutation assay in bacteria according to OECD test guideline 471 (1983), strains TA 98, TA 100, TA 1535, TA 1537 and TA 1538 of S. typhimurium were exposed to Difluocortolone valerate, (100% a.i.), in DMSO at concentrations of 0.1, 0.25, 0.5, 1,0, 2.5, and 5.0 mg/plate in the presence and absence of mammalian metabolic activation using the pre-incubation method. Strain TA 100 was additionally exposed to the test item in DMSO at concentrations of 0.1, 0.25, 0.5, 0.75, 1.0, 2.0, 4.0, and 6.0 mg/plate using the plate incorporation method.
Difluocortolone valerate was tested up to insoluble concentrations starting at 2.5 mg with S9 mix and at 0.1 mg/plate without S9 mix. The positive controls induced the appropriate responses in the corresponding strains. There was no evidence of induced mutant colonies over background.
In a mammalian cell gene mutation assay according to OECD test guideline 476 (1984), V79 hamster fibroblast cells cultured in vitro were exposed to Difluocortolone-valerate, (100% a.i.), in DMSO at concentrations of 5.0;10.0; 20.0; 40.0; 60.0 and 80.0 µg/mL (Exp. I) and at 5.0; 20.0; 40.0; 60.0; 80.0 and 100.0 µg/mL (Exp. II) in the presence and absence of mammalian metabolic activation.
Difluocortolone was tested up to cytotoxic concentrations (i.e. 100.0 µg/mL. The positive controls did induce the appropriate response. There was no evidence of induced mutant colonies over background.
In a mammalian cell cytogenetics assay [Chromosome aberration] according to OECD test guideline 473, primary lymphocyte cultures were exposed to Difluocortolone in DMSO at concentrations of
Experiment I
without S9 mix (exposure period 22 hand 46 h):
22 h: 60.0; 80.0; 100.0 µg/ml
46 h: 60.0; 80.0 µg/ml
with S9 mix (exposure period 4 h):
22 h: 10.0; 80.0; 100.0 µg/ml
46 h: 80.0; 100.0 µg/ml
Experiment II
without S9 mix (exposure period 22 hand 46 h):
22 h: 30.0; 60.0; 80.0; 100.0 µg/ml
46 h: 60.0; 100.0 µg/ml
with S9 mix (exposure period 4 h):
22 h: 30.0; 60.0i 80.0i 100.0 µg/ml
46 h: 60.0; 100.0 µg/ml.
Difluocortolone was tested up to cytotoxic or precipitating concentrations.
In both experiments, with test article no biologically relevant increase in the structural chromosomal aberration rate could be found when compared with the range of aberrations in the corresponding negative and solvent controls. These results were obtained at each fixation interval both in the absence and presence of 89 mix. In this study the aberration rates after treatment with the test article in the absence and the presence of S9 mix (exp. I: 0.00 % - 1.50 %; exp. 11: 0.00 % - 1.00 %) were in the range of the control data (exp.I: 0.00 % - 2.00 %; exp.II: 0.00 % - 0.50 %). The occurrence of polyploid metaphases was also reported. In both experiments no biologically relevant deviations from the control data (0.0 % - 1.0 %) was found after treatment with the test article (0.0 % - 1.5 %).
Positive controls induced the appropriate response. There was no evidence of Chromosome aberration induced over background.
In a NMRI mouse bone marrow micronucleus assay according to OECD test guideline 474, 5 mice/sex/dose were treated intraperitoneally with Difluocortolone (100 % a.i.) at doses of 0, 200, 700, 2000 mg/kg bw. Bone marrow cells were harvested at 24h and 48h post-treatment. The vehicle was NaCl/Myrj 53.
There were signs of toxicity at 2000 mg/kg bw after 48h post-treatment period. The animals expressed toxic reactions. The ratio of normochromatic to polychromatic erythrocytes was affected by the treatment, indicating that the test article had cytotoxic properties. However, Difluocortolone was tested at an adequate dose based on the preliminary toxicity test. The positive control induced the appropriate response. There was not a significant increase in the frequency of micronucleated polychromatic erythrocytes in bone marrow after any treatment time.
Furthermore, the potential to induce mutagenicity was assessed by QSAR predictions performed with Leadscope and DEREK for both substances
In QSAR predictions using Leadscope Model Applier (v3.0.2) the potential of Bromhydrin-valerate and Difluocortolone to induce mutagenicity was assessed. Leadscope uses two parameters to guide the applicability of model domain: 1) having at least one structural feature defined in the model in addition to all the property descriptors; 2) having at least one chemical in a training neighbourhood with at least 30% global similarity to the test structure. In the case of Bromhydrin-valerate the prediction is within the applicability domain, since 37 training compounds were identified in the model training set being structurally similar to the test compound. In the case of Difluocortolone the prediction is within the applicability domain, since 28 training compounds were identified in the model training set being structurally similar to the test compound.
The query structures (Bromhydrin-valerate and Difluocortolone) do not match structural alerts or examples for (bacterial in vitro) mutagenicity in Leadscope. Based on these results Bromhydrin-valerate and Difluocortolone are considered not mutagenic as predicted by Leadscope.
In QSAR predictions using DEREK Nexus v6.1 the potential of Bromhydrin-valerate and Difluocortolone to induce mutagenicity was assessed. Derek Nexus makes qualitative predictions for and against toxicity through reasoning. For the endpoint of mutagenicity, predictions for toxicity decrease in confidence in the following order: certain>probable>plausible>equivocal. Predictions against toxicity increase in confidence in the following order: inactive (with unclassified and/or misclassified features)<inactive<improbable. Likelihood levels have been shown to correlate with predictivity [Judson et al, 2013]. Multiple data sources (e.g. toxicity data from multiple assays and mechanistic evidence) are synthesised into the structure-activity relationships that underpins Derek Nexus predictions. An appreciation of the assay units applied by alert writers when building the alert training set. However, predictions are not quantitative and, as a result, do not include units.
The query structures (bromhydrin-valerate and Difluocortolone) do not match a structural alert or examples for (bacterial in vitro) mutagenicity in Derek.
Based on these results both substances are not considered mutagenic as predicted by DEREK Nexus.
This studies are classified as acceptable for assessment based on methodology and documentation. This studies satisfy the requirement for Test Guideline OECD 471 for in vitro mutagenicity (bacterial reverse gene mutation) and the data is part of an overall assessment.
Justification for classification or non-classification
Based on the study results of genetic toxicity in vitro tests with the read-across substance diflucortolone-21-valerate and the performed QSAR predictions classification of bromhydrin-valerate is not required according to Regulation (EC) No. 1272/2008 (CLP).
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