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EC number: 613-915-0 | CAS number: 66233-43-0
- Life Cycle description
- Uses advised against
- Endpoint summary
- Appearance / physical state / colour
- Melting point / freezing point
- Boiling point
- Density
- Particle size distribution (Granulometry)
- Vapour pressure
- Partition coefficient
- Water solubility
- Solubility in organic solvents / fat solubility
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- Endpoint summary
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- Additional information on environmental fate and behaviour
- Ecotoxicological Summary
- Aquatic toxicity
- Endpoint summary
- Short-term toxicity to fish
- Long-term toxicity to fish
- Short-term toxicity to aquatic invertebrates
- Long-term toxicity to aquatic invertebrates
- Toxicity to aquatic algae and cyanobacteria
- Toxicity to aquatic plants other than algae
- Toxicity to microorganisms
- Endocrine disrupter testing in aquatic vertebrates – in vivo
- Toxicity to other aquatic organisms
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- Additional ecotoxological information
- Toxicological Summary
- Toxicokinetics, metabolism and distribution
- Acute Toxicity
- Irritation / corrosion
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- Carcinogenicity
- Toxicity to reproduction
- Specific investigations
- Exposure related observations in humans
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- Additional toxicological data
Endpoint summary
Administrative data
Key value for chemical safety assessment
Genetic toxicity in vitro
Description of key information
Delta-9(11)-fluocortolone-valerate is not mutagenic based on bacterial reverse mutation assays with the read-across substance fluocortolone (negative +/- S9 mix in S. typhimurium strains TA 98, TA 100, TA 1535, TA 1537 and TA 1538 (Reimann and Jarzombek, 2005; Reimann and Görke, 1997; Lang and Schmitt, 1984) - negative +/- S9 mix inE. coli strain WP2 uvrA (Reimann and Jarzombek, 2005)). Additionally, two QSAR predictions have been performed (Leadscope and DEREK, 2021). Both predictions revealed negative results.
Link to relevant study records
- 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: 66233-43-0 ; Chemical name: DELTA-9(11)-Fluocortolone-Valerate
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:
- bacteria, other: Not applicable for in silico study
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- other: Not applicable for in silico study
- 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 DELTA-9(11)-Fluocortolone-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 DELTA-9(11)-Fluocortolone-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 DELTA-9(11)-Fluocortolone-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 66233-43-0
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:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- other: Not applicable for in silico study
- 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 DELTA-9(11)-Fluocortolone-Valerate is not mutagenic in a bacterial reverse mutation assay.
- Executive summary:
In a QSAR prediction using DEREK Nexus v6.1 the potential of DELTA-9(11)-Fluocortolone-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 DELTA-9(11)-Fluocortolone-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 cytogenicity / chromosome aberration study in mammalian cells
- Type of information:
- experimental study
- Adequacy of study:
- other information
- Study period:
- Sep 1996
- 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:
- 21 July 1997
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- in vitro mammalian chromosome aberration test
- Target gene:
- not specified
- Species / strain / cell type:
- lymphocytes: human primary cells
- Details on mammalian cell type (if applicable):
- - Type and identity of media: MCCoy's 5a medium
- Properly maintained: yes - Metabolic activation:
- with and without
- Metabolic activation system:
- S9 rat liver homogenate
Type and composition of metabolic activation system:
- source of S9: S9 (batch no. 050296), derived from male Wistar rats pretreated with Aroclor 1254, protein content 29.5 mg/mL was prepared by CCR (Cytotest Cell Research, Roßdorf, Germany)
- concentration or volume of S9 mix and S9 in the final culture medium: The components of the standard S9 mix were 12.2% (v/v) S9, 4 mmol/L NADP, 5 mmol/L glucose-6-phosphate, 8 mmol/L MgCI2, 34.3 mmol/L KCI and 50 mmol/L sodium phosphate butter, pH 7.4. One mL freshly prepared S9 mix was added to a 5 mL blood culture to give a final S9 concentration of 2% (v/v). - Test concentrations with justification for top dose:
- Assay without S9 mix:
1st harvesting: 25, 50, 100, 125, 150, 175, 200, 225 and 250 µg/ml ; 2nd harvsting: 50, 100, 150 and 200 µg/ml
Assay with S9 mix:
1st harvesting: 100, 250, 500, 550, 600, 650, 700, 750, 800, 850 and 900 µg/ml ; 2nd harvsting: 500, 600, 700 and 800 µg/ml - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: DMSO
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- cyclophosphamide
- other: -S9 mix: triaziquone
- Details on test system and experimental conditions:
- NUMBER OF REPLICATIONS:
- Number of cultures per concentration: duplicate
- Number of independent experiments: One
METHOD OF TREATMENT/ EXPOSURE:
- Test substance added in medium
TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable: 48 h and additional 4 h for culture with metabolic activation
- Exposure duration/duration of treatment: 21 h or 44 h without S9 mix and 23 h and 44 h with metabolic activation
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. To arrest the cells in the metaphase, colcemide (final concentration 0.08 µg/mL) was added ca. 3.5 hours before the cells were harvested.
- If cytokinesis blocked method was used for micronucleus assay: indicate the identity of cytokinesis blocking substance (e.g. cytoB), its concentration, and duration and period of cell exposure.
- Methods of slide preparation and staining technique used including the stain used (for cytogenetic assays): Drops of concentrated cell suspension were placed on slides, which were allowed to air-dry before being stained with 10% (v/v) Giemsa and mounted with Eukitt on the following day.
For assessment of cell cycle kinetics, the slides were stained using a modified fluorescence-plus-Giemsa technique (FPG-technique). The slides were stained for 15 minutes with Hoechst 33258 (final concentration 4.5 µg/mL), rinsed and covered with phosphate buffer (pH 6.9) for irradiation with ultra-violet light (e.g. Heraeus Sterisol®, 30 watt). After 90 min incubation in 12 x SSC at 60°C the slides were stained with 7% (v/v) Giemsa for ca. 10 min, then rinsed with water and air-dried. After drying, the slides were mounted with Eukitt on the following day. On the basis of a "BUdR-control-culture", which was harvested after 2 days
incubation with BUdR, the success of the differential staining was demonstrated in each instance.
- Number of cells spread and analysed per concentration (number of replicate cultures and total number of cells scored): For the analysis of chromosomal aberrations, 100 cells per replicate culture were scored if possible, i.e. 200 cells per concentration level, except for the positive control in which only 100 cells were scored to prove the positive response.
The number of chromosomes per metaphase was determined on the television monitor. Only metaphases with 2 n = 46 chromosomes were included in the analysis. The coordinates of all metaphase spreads with structural aberrations were recorded using the vernier scale at the microscope stage.
All structural chromosome aberrations were assigned artificial lesion (or break) scores as folIows:
- chromatid and chromosome (isochromatid) breaks, acentric fragments and deletions were scored as one lesion each
- exchanges, rings and dicentrics were designated 2 lesions each.
- Criteria for scoring chromosome aberrations (selection of analysable cells and aberration identification): All metaphase spreads were examined for both chromatid and chromosome aberrations, notably achromatic lesions (gaps), breaks, acentric fragments, deletions and exchange with the following exception: an achromatic lesion (AL) may show a non-staining region greater than the diameter of the chromatid if there is no dislocation of the apparently
detached part. The clastogenic response of the compound was evaluated by calculating the percentage of aberrant cells excluding ALs. The aberration frequency (% cells showing breaks excluding cells with ALs) in untreated or solvent-treated human peripherallymphocyte cultures should be in the range of 0 - 3%. A break incidence of up to 3% is therefore classified as a negative response, especially if a dose-dependent increase does not exist.
- Determination of polyploidy: The incidence of cells with numerical alterations in chromosome number (excl. aneuploidy) such as endoreduplication and polyploidy was also recorded.
- Determination of endoreplication: Endoreduplicated cells (endopolyploidy) were classified as quasitetraploid metaphases eXhibiting chromosome pairing, whereas a polyploid cell is any metaphase containing multiple copies of the haploid number (n) of the chromosome complement, e.g. 3 n, 4 n etc.
METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method, e.g.: mitotic index (MI); other: cell cycle progression
- Any supplementary information relevant to cytotoxicity: Detectable levels of chromosomal aberrations are usually found only at doses of clastogens
wh ich induce some evidence of cytotoxicity.
The highest dose chosen for evaluation should be one which causes a reduction in the mitotic index of approximately 50% or corresponds to the substance's sOlubility limit but does not exceed 10-2 mol/L or 5 mg/mL. On the basis of these considerations 3 increasing concentrations were selected for the first harvesting time and 1 concentration for the second harvesting time in order to evaluate chromosomal aberrations. In this study the highest test substance concentrations induced marked cytotoxicity as indicated by a clear reduction (≥ 50%) of the mitotic index. Mitotic indices (percentage of cells in mitosis) were compiled from counts of 1000 stimulated cells per parallel culture. For determination of the cell cycle progression, 100 metaphase spreads per culture, treated with BUdR and stained with the FPG-technique, were analyzed for the occurrence of first, second and third mitosis in order to demonstrate that the first harvesting time chosen corresponds to approximately one to one and a half cell cycles after start of treatment. - Evaluation criteria:
- please refer to any other information on materials and methods.
- Statistics:
- The positive control group and the dose groups were compared to the appropriate negative control using Fisher's exact test, each at the level of significance a = 0.05.
- Key result
- Species / strain:
- lymphocytes: human primary cells
- Metabolic activation:
- without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- Positive controls validity:
- valid
- Key result
- Species / strain:
- lymphocytes: human primary cells
- Metabolic activation:
- with
- Genotoxicity:
- ambiguous
- Remarks:
- positive at 1st harvesting time point at precipitating concentration
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- Positive controls validity:
- valid
- Conclusions:
Fluocortolone did not show biological relevant clastogenic effects under the conditions of the test. Classification is not required.
In the chromosomal aberration test in primary human lymphocytes fluocortolone (ZK 10445) was tested up 900 µg/ml. Cytotoxicity and precipitation was the dose limiting factors. No clastogenic effects could be observed when tested without extrinsic metabolising system. Fluocortolone showed clastogenic effect in human lymphocytes when tested with extrinsic metabolising system at first harvesting time point at precipitating concentrations, which is assumed to be of no biological relevance.- Executive summary:
In a mammalian cell cytogenetics assay [Chromosome aberration] according to OECD test guideline 473, primary lymphocyte cultures were exposed to [Fluocortolone, (100% a.i.), in DMSO at concentrations of 25, 50, 100, 125, 150, 175, 200, 225 and 250 µg/mL (first harvesting time) and 50, 100, 150 and 200 µg/mL (second harvesting time) without metabolic activation and at 100, 250, 500, 550, 600, 650, 700, 750, 800, 850 and 900 µg/mL (first harvesting time) and 500, 600, 700 and 800 µg/mL (second harvesting time) with metabolic activation.
Fluocortolon was tested up to cytotoxic and precipitating concentrations. No clastogenic effects could be observed when tested without extrinsic metabolising system. Fluocortolone showed a clastogenic effect in human lymphocytes when tested with extrinsic metabolising system at first harvesting time point at precipitating concentrations, which is assumed to be of no biological relevance. Positive controls induced the appropriate response.
- Endpoint:
- in vitro cytogenicity / chromosome aberration study in mammalian cells
- Type of information:
- experimental study
- Adequacy of study:
- other information
- Study period:
- Jul to Aug 1996
- 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:
- 21 July 1997
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- in vitro mammalian chromosome aberration test
- Target gene:
- not specified
- Species / strain / cell type:
- lymphocytes: human primary cells
- Details on mammalian cell type (if applicable):
- - Type and identity of media: MCCoy's 5a medium
- Properly maintained: yes - Additional strain / cell type characteristics:
- not applicable
- Metabolic activation:
- with and without
- Metabolic activation system:
- S9 rat liver homogenate
Type and composition of metabolic activation system:
- source of S9: S9 (batch no. 050296), derived from male Wistar rats pretreated with Aroclor 1254, protein content 29.5 mg/mL was prepared by CCR (Cytotest Cell Research, Roßdorf, Germany).
- concentration or volume of S9 mix and S9 in the final culture medium: The
components of the standard S9 mix were 12% (v/v) 89, 4 mmol/L NADP, 5 mmol/L glucose-6-phosphate, 8 mmol/L MgCI2, 34.3 mmol/L KCI and 50 mmol/L sodium phosphate buffer, pH 7.4. Immediately after addition of the test compounds, 1 mL freshly prepared S9 mix was added to a 5 mL blood culture to give a final S9 concentration of 2% (v/v). - Test concentrations with justification for top dose:
- Assay without S9 mix:
1st harvesting: 10, 25, 50, 75, 100, 125, 150, 175, 200, 225, 250, 275 and 300 µg/ml ; 2nd harvsting: 10, 50, 100, 150 and 200 µg/ml
Assay with S9 mix:
1st harvesting: 10, 50, 100, 200, 400, 600, 800 and 1000 µg/ml ; 2nd harvsting: 200, 400, 600 and 1000 µg/ml - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The solubility of fluocortolone was tested in DMSO, yielding a solubility of > 100 mg/mL. But when a stock solution was added at 1 % to tissue culture medium containing 15% (v/v) FCS precipitations of the test compound were visible starting at 600 µg/mL. - Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- cyclophosphamide
- other: -S9 mix: triaziquone
- Details on test system and experimental conditions:
- NUMBER OF REPLICATIONS:
- Number of cultures per concentration: duplicate
- Number of independent experiments: One
METHOD OF TREATMENT/ EXPOSURE:
- Test substance added in medium
TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable: 48 h
- Exposure duration/duration of treatment: without S9 mix: 21h first harvesting, 44 h second harvesting; with S9 mix: 23 h first harvesting, 44h second harvesting
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. To arrest the cells in the metaphase, colcemide (final concentration 0.08 µg/mL) was added
ca. 3.5 h before the cells were harvested.
- Methods of slide preparation and staining technique used including the stain used (for cytogenetic assays): The cells were collected by centrifugation, exposed to 1 % (w/v) tri-sodiumcitrate-2-hydrate hypotonie solution (for swelling) and fixed in glacial acetic acid/methanol, 1 +3. Drops of concentrated cell suspension were placed on slides, which were allowed to air-dry before being stained with 10% (v/v) Giemsa and mounted with Eukitt. For assessment of cell cycle kinetics, the slides were stained using a modified fluorescence-plus-Giemsa technique (FPG-technique). The slides were stained for 15 minutes with Hoechst 33258 (final concentration 4.5 µg/mL, rinsed and covered with phosphate butter (pH 6.9) during irradiation with ultra-violet light (e.g. Heraeus Sterisol®, 30 watt). After 90 min incubation in 12 x SSC at 60°C the slides were stained with 7% (v/v) Giemsa for ca. 10min, then rinsed with water and air-dried. After drying, the slides were mounted with Eukitt. On the basis of a "BUdR-control-culture", which was harvested after 2 days incubation with BUdR, the success of the differential staining was demonstrated in each instance.
- Number of cells spread and analysed per concentration (number of replicate cultures and total number of cells scored): For the analysis of chromosomal aberrations, 100 cells per replicate culture were scored if possible, i.e. 200 cells per concentration level, except for the positive control in which only 100 cells were scored to prove the positive response. The number of chromosomes per metaphase was determined on the
television monitor. Only metaphases with 2 n = 46 chromosomes were included in the analysis.
- Criteria for scoring chromosome aberrations (selection of analysable cells and aberration identification): Using the vernier scale at the microscope stage, the coordinates of all metaphase spreads with structural aberrations were recorded.
All metaphase spreads were examined for both chromatid and chromosome aberrations, notably achromatic lesions (gaps), breaks, acentric fragments, deletions and exchange figures, with the following exception: an achromatic lesion (AL) may show a non-staining region greater than the diameter of the chromatid if there is no dislocation of the apparently detached part.
The incidence of cells with numerical alterations in chromosome number (excl. aneuploidy) such as endoreduplication and polyploidy was also recorded.
Endoreduplicated cells (endopolyploidy) were classified as quasitetraploid metaphases
exhibiting chromosome pairing, whereas a polyploid cell is any metaphase containing multiple copies of the haploid number (n) of the chromosome complement, e.g. 3 n, 4 n etc.
All structural chromosome aberrations were assigned artificial lesion (or break) scores as folIows:
chromatid and chromosome (isochromatid) breaks, acentric fragments and deletions were scored as one lesion each
exchanges, ringsand dicentrics were designated 2 lesions each.
The clastogenic potential of the compound was evaluated by calculating the breakage rate and the percentage of aberrant cells excluding ALs. The aberration frequency (% cells showing breaks excluding cells with ALs) in untreated or solvent-treated human peripheral Iymphocyte cultures should be in the range of 0 - 3%. A break incidence of up to 3% is therefore classified as a negative response, especially if a dose-dependent increase does not exist.
METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method, e.g.: mitotic index (MI); other: cell cycle progression
- Any supplementary information relevant to cytotoxicity: For determination of the cell cycle progression, 100 metaphase spreads per culture, treated with BUdR and stained with the FPG-technique, were analyzed for the occurrence of first, second and third mitosis in order to demonstrate that the first harvesting time chosen corresponds to approximately one to one and a half cell cycles after start of treatment. - Evaluation criteria:
- please refer to any other information on materials and methods incl. tables
- Statistics:
- The positive control group and the dose groups were compared to the appropriate negative control using Fisher's exact test, each at the level of significance α = 0.05.
- Key result
- Species / strain:
- lymphocytes: human primary cells
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- Positive controls validity:
- valid
- Conclusions:
- Fluocortolone did not show clastogenic effects under the conditions of the test. Classification is not required.
In the chromosomal aberration test in primary human lymphocytes fluocortolone (ZK 10445) was tested up 1000 µg/ml reaching the cytotoxic and precipitating concentrations. No clastogenic effects of fluocortolone tested with and without extrinsic metabolising system could be observed. - Executive summary:
In a mammalian cell cytogenetics assay [Chromosome aberration] according to OECD test guideline 473, primary 10, 25, 50, 75, 100, 125, 150, 175,200, 225, 250, 275 and 300 µg/mL (first harvesting time) and 10, 50, 100, 150 and 200 µg/mL (second harvesting time).without metabolic activation and at 10, 50, 100, 200, 400, 600, 800 and 1000 µg/mL (first harvesting time) and 200, 400, 600 and 1000 µg/mL (second harvesting time) with metabolic activation.
Fluocortolon was tested up to cytotoxic and precipitating concentrations. No clastogenic effects could be observed when tested without or with extrinsic metabolising system. Positive controls induced the appropriate response, thus the test is considered valid. Fluocortolon exhibited no clastogenic effect under the conditions of the test.
- Endpoint:
- in vitro DNA damage and/or repair study
- Type of information:
- experimental study
- Adequacy of study:
- other information
- Study period:
- Mar to Apr 1998
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 482 (Genetic Toxicology: DNA Damage and Repair, Unscheduled DNA Synthesis in Mammalian Cells In Vitro)
- Version / remarks:
- 23 October 1986
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- DNA damage and repair assay, unscheduled DNA synthesis in mammalian cells in vitro
- Target gene:
- not applicable
- Species / strain / cell type:
- hepatocytes: primary cells, female rat
- Details on mammalian cell type (if applicable):
- - Type and identity of media: Williams E
- Properly maintained: yes - Additional strain / cell type characteristics:
- not applicable
- Test concentrations with justification for top dose:
- 10, 20, 40, 60, 80, 100, 125, 150 and 200 µg/mL
- Vehicle / solvent:
- - Vehicle(s)/solvent(s) used:DMSO
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- Positive controls:
- yes
- Positive control substance:
- 2-acetylaminofluorene
- Details on test system and experimental conditions:
- NUMBER OF REPLICATIONS:
- Number of cultures per concentration: Sextuplicate cultures were established for fluocortolone, 2-AAF (positive control), DMSO (solvent control) and medium (negative control) treatment.
- Number of independent experiments: One
METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): 2E+05 cell/mL
- Test substance added in medium
TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable: 2 h for attachment
- Exposure duration/duration of treatment: 18 h
METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method, e.g.: Neutral red assay - Evaluation criteria:
- For quantification of UDS three slides per concentration and control and 50 cells per slide, i.e. 150 cells per treatment, were evaluated, if possible.
The following criteria were used for cell counting:
- only mono-nucleated cells were selected for analysis
- only cells with normal morphology were analysed
- isolated nuclei with no surrounding cytoplasma were disregarded
- cells with unusual staining artefacts were not analysed
- heavily labelled S-phase cells were excluded from counting
- all other normal cells, up to 50 per slide, were analysed.
According to recommendations for in vitro genotoxicity studies, as a rule the highest dose chosen for evalution should be one which causes a reduction of the parameter for cytotoxicity by approximately 50% or corresponds to the substance's solubility limit, i.e. a concentration which causes visible precipitates, but should not exceed E-02 mol/L or 5 mg/mL. On the basis of this consideration the highest and at least four consecutive concentrations were selected for the evaluation of UDS, whereby the highest concentration chosen was clearly cytotoxic.
A test chemical of a particular concentration response relationship level is considered clearly positive if:
- the test chemical yields NNG > 5, and at least 20% of cells are found to be in repair
- any induction of UDS is repraduced in an independent experiment.
However, only increased net nuclear grain values which are based on enhanced nuclear grain counts are considered relevant. Increased net nuclear grain values which are mainly due to decreased cytoplasmic grains are considered to be non-relevant. Thus, for the etablishment of a positive UDS response, nuclear and net nuclear grain counts should be taken into consideration together.
Since, however, no apprapriate statistical methods are established for evaluation of UDS-tests, only the biological significance is taken into account for the assessment of the data. - Statistics:
- The following were calculated for each slide and for each treatment:
- the average and standard deviation (SD) of the nuclear (NG) and cytoplasm grain (CG) counts as well as the net nuclear grain (NNG) values (nuclear grains minus mean cytoplasm grains counted in three cytoplasm areas equivalent to the nucleus size).
- the percentage of cells responding or in repair (i.e. NNG > 5 ). - Key result
- Species / strain:
- hepatocytes: primary cells, female rat
- Metabolic activation:
- not applicable
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Conclusions:
- Fluocortolone did not increase the DNA repair synthesis and is therefore not considered to be genotoxic under the conditions of this test. Classification is not required.
- Executive summary:
In an unscheduled DNA synthesis assay according to OECD guideline 482 (1986), primary rat hepatocyte cultures from female Wistar rats were exposed to Fluocortolone in DMSO and at concentrations of 0, 10, 20, 40, 60, 80, 100, 125, 150 and 200 µg/mL for 18 h.
The viability of the hepatocytes was reduced by 34% at a fluocortolone concentration of 150 µg/mL and by 67% at 200 µg/mL. Therefore, 150 µg/mL and five consecutive concentrations (10, 20, 40, 80 and 125 µg/mL) were scored for UDS in this study. There was no increase of unscheduled DNA synthesis at any non-toxic or toxic concentration. The positive control (2-AAF, 1E-06 mol/L) induced the appropriate response. There was no evidence that unscheduled DNA synthesis, as determined by nuclear silver grain counts was induced.
This study is classified as acceptable. This study satisfies the requirement for Test Guideline OECD 482 for other genotoxic mutagenicity data. Therefore, it can be concluded that in the study described and under the experimental
conditions reported, fluocortolone did not induce repairable DNA damage leading to increased DNA repair synthesis (UDS) when tested up to clearly cytotoxic concentrations in cultured male rat hepatocytes.
- Endpoint:
- in vitro DNA damage and/or repair study
- Type of information:
- experimental study
- Adequacy of study:
- other information
- Study period:
- Apr 1997
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 482 (Genetic Toxicology: DNA Damage and Repair, Unscheduled DNA Synthesis in Mammalian Cells In Vitro)
- Version / remarks:
- 23 October 1986
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- DNA damage and repair assay, unscheduled DNA synthesis in mammalian cells in vitro
- Target gene:
- not applicable
- Species / strain / cell type:
- hepatocytes: primary cells, male
- Details on mammalian cell type (if applicable):
- - Type and identity of media: Williams E
- Properly maintained: yes - Additional strain / cell type characteristics:
- not applicable
- Metabolic activation:
- not applicable
- Test concentrations with justification for top dose:
- 10, 20, 40, 60, 80, 100, 125, 150 and 200 µg/ml
- Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: DMSO
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- Positive controls:
- yes
- Positive control substance:
- 2-acetylaminofluorene
- Details on test system and experimental conditions:
- NUMBER OF REPLICATIONS:
- Number of cultures per concentration: Sextuplicate cultures were established for fluocortolone, 2-AAF (positive control), DMSO (solvent control) and medium (negative control) treatment.
- Number of independent experiments: One
METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): 2E+05 cell/mL
- Test substance added in medium
TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable: 2 h for attachment
- Exposure duration/duration of treatment: 18 h
METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method, e.g.: Neutral red assay - Evaluation criteria:
- For quantification of UDS three slides per concentration and control and 50 cells per slide, i.e. 150 cells per treatment, were evaluated, if possible.
The following criteria were used for cell counting:
- only mono-nucleated cells were selected for analysis
- only cells with normal morphology were analysed
- isolated nuclei with no surrounding cytoplasma were disregarded
- cells with unusual staining artefacts were not analysed
- heavily labelled S-phase cells were excluded from counting
- all other normal cells, up to 50 per slide, were analysed.
According to recommendations for in vitro genotoxicity studies, as a rule the highest dose chosen for evalution should be one which causes a reduction of the parameter for cytotoxicity by approximately 50% or corresponds to the substance's solubility limit, i.e. a concentration which causes visible precipitates, but should not exceed E-02 mol/L or 5 mg/mL. On the basis of this consideration the highest and at least four consecutive concentrations were selected for the evaluation of UDS, whereby the highest concentration chosen was clearly cytotoxic.
A test chemical of a particular concentration response relationship level is considered clearly positive if:
- the test chemical yields NNG > 5, and at least 20% of cells are found to be in repair
- any induction of UDS is repraduced in an independent experiment.
However, only increased net nuclear grain values which are based on enhanced nuclear grain counts are considered relevant. Increased net nuclear grain values which are mainly due to decreased cytoplasmic grains are considered to be non-relevant. Thus, for the etablishment of a positive UDS response, nuclear and net nuclear grain counts should be taken into consideration together.
Since, however, no apprapriate statistical methods are established for evaluation of UDS-tests, only the biological significance is taken into account for the assessment of the data. - Statistics:
- The following were calculated for each slide and for each treatment:
- the average and standard deviation (SD) of the nuclear (NG) and cytoplasm grain (CG) counts as well as the net nuclear grain (NNG) values (nuclear grains minus mean cytoplasm grains counted in three cytoplasm areas equivalent to the nucleus size).
- the percentage of cells responding or in repair (i.e. NNG > 5 ). - Key result
- Species / strain:
- hepatocytes: primary male rat cells
- Metabolic activation:
- not applicable
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Conclusions:
- Fluocortolone did not induce repairable DNA damage leading to increased DNA repair synthesis (UDS)
- Executive summary:
In an unscheduled DNA synthesis assay according to OECD guideline 482 (1986), primary rat hepatocyte cultures from male Wistar rats were exposed to Fluocortolone in DMSO and at concentrations of 0, 10, 20, 40, 60, 80, 100, 125, 150 and 200 µg/mL for 18 h.
The viability of the hepatocytes was reduced by 48% at a fluocortolone concentration of 150 µg/mL. Therefore, 150 µg/mLand five consecutive concentrations (10, 20, 40, 80 and 125 µg/mL) were scored for UDS in this study. There was no increase of unscheduled DNA synthesis at any non-toxic or toxic concentration. The positive control (2-AAF, 1E-06 mol/L) induced the appropriate response. There was no evidence that unscheduled DNA synthesis, as determined by nuclear silver grain counts was induced.
This study is classified as acceptable. This study satisfies the requirement for Test Guideline OECD 482 for other genotoxic mutagenicity data. Therefore, it can be concluded that in the study described and under the experimental
conditions reported, fluocortolone did not induce repairable DNA damage leading to increased DNA repair synthesis (UDS) when tested up to clearly cytotoxic concentrations in cultured male rat hepatocytes.
- Endpoint:
- in vitro gene mutation study in mammalian cells
- Type of information:
- experimental study
- Adequacy of study:
- other information
- Study period:
- Nov 1995 to Apr 1996
- 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:
- 21 July 1997
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- mammalian cell gene mutation assay
- Target gene:
- HPRT gene locus
- Species / strain / cell type:
- Chinese hamster lung fibroblasts (V79)
- Details on mammalian cell type (if applicable):
- - Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes - Additional strain / cell type characteristics:
- not specified
- Metabolic activation:
- with and without
- Metabolic activation system:
- Type and composition of metabolic activation system: rat liver homogenate (S9 mix)
- source of S9: S9, derived from male Sprague-Dawley rats pretreated with Aroclor 1254, was obtained from Organon Teknika Co., Durham, NC, USA, (batch no.39904; protein content 43.7 mg/mL; activity 2008 pmoles 7-hydroxyresorufin formed per min per mg S9 protein).
- concentration or volume of S9 mix and S9 in the final culture medium: The composition of the co-factor solution was concentrated to yield the following concentrations in the S9 mix:
8 mmol/LMgCI2; 33 mmol/L KCI; 5 mmol/L glucose-6-phosphate; 5 mmol/L NADP in 0.1 mol/L sodium phosphate butter, pH 7.4. - Test concentrations with justification for top dose:
- 0.05, 0.1, 0.2, 0.3, 0.4, 0.5 mg/mL
The highest dose chosen for evaluation should be clearly cytotoxic, i.e. it should cause a reduction in the plating efficiency or in the cell number in the mass-culture (cell survival). Nontaxic compounds will be tested up to a range in which precipitates are visible in the medium but not exceeding 1E-02 mol/L or 5 mg/mL, whichever is lower. In a pre-experiment the cytotoxicity of fluocortolone (dissolved in DMSO) was tested up to a maximum of 2.5 mg/mL. The test compound was toxic at 500 µg/mL and formed visible precipitates starting at 1000 µg/mL with and without S9 mix; therefore, 500 µg/mL was chosen as the highest concentration with/without S9 mix in the HPRT-test. - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: solubility - Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- yes
- Positive controls:
- yes
- Positive control substance:
- 7,12-dimethylbenzanthracene
- ethylmethanesulphonate
- other: 1st test: - S9 mix: MNNG
- Details on test system and experimental conditions:
- NUMBER OF REPLICATIONS:
- Number of cultures per concentration: duplicate cultures
- Number of independent experiments: two
METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): 1.5E+06 cells/175 cm² flask
- Test substance added in medium
TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: 4h
- Harvest time after the end of treatment (sampling/recovery times): For mutation expression the cells were grown for seven days with one subculturing (ca 1.5 E+06 cells/175 cm² flask). For subsequent mutant selection two sets of five 75 cm² plastic flasks (ea. 5 E+05 cells/flask) per concentration
were established in selection medium containing 11 µg/mL 6-thioguanine (Sigma, Deisenhofen, Germany). After a selection period of 10 days the colonies were fixated and stained with 10% methylene blue in 0.01 % KOH solution.
FOR GENE MUTATION:
- Expression time (cells in growth medium between treatment and selection): 7 days
- Selection time (if incubation with a selective agent): 10 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: selection medium containing 11 µg/mL 6-thioguanine (Sigma, Deisenhofen, Germany). After a selection period of 10 days the colonies were fixated and stained with 10% methylene blue in 0.01 % KOH solution.
METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method, other: To check the cell survival parallel to the mutant selection a second plating efficiency was performed.
METHODS FOR MEASUREMENTS OF GENOTOXICIY:
The mutant frequency per 106 cells is calculated on the basis of mutant counts, number of cells seeded into the selective (6-thioguanine) medium, and the absolute percentage of the second plating efficiency (i.e., mean mutant count x E+06 + number of cells seeded x percentage of second PE x 0.01 = mutant frequency per E+06 cells).
The calculated individual and mean mutant colonies per E+06 cells are reported as whole figures whereby the values between 0 and 1 are treated as 1. - Evaluation criteria:
- So far no satisfactory mathematical methods are available for statistical analysis of mammalian cell mutagenicity experiments suchas those performed here. Our experience has shown that the following predetermined descriptive criteria are the most useful for interpretation of the results:
An evaluation is made only after a repeat experiment has been carried out.
The evaluation of the results is performed as folIows:
The test substance is classified as mutagenic if it induces with one of the test substance concentrations, reproducibly, a mutation frequency that is three times higher than the spontaneous mutant frequency in this experiment.
The test substance is classified as mutagenic if there is a reproducible concentration-related increase in the mutation frequency. Such an evaluation may be considered independently of the enhancement factor for induced mutants.
However, in a case-by-case evaluation both decisions depend on the level of the
corresponding negative contra I data. If there is, by chance, a very low spontaneous mutation rate as compared to the range found in this laboratory, a seemingly concentration-related increase in the mutations or a factor of three or even more within this range may be regarded as being irrelevant. When considerable variations in the results occur, clarification is sought in additional experiments. - Species / strain:
- Chinese hamster lung fibroblasts (V79)
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- at ≥ 300 µg/mL
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation and time of the determination: Visible precipitates in the cell culture medium, however, could be observed starting at 400 µg/mL only in HPRT-test I whithout metabolic acitivation.
RANGE-FINDING/SCREENING STUDIES (if applicable): In a pre-experiment the cytotoxicity of fluocortolone (dissolved in DMSO) was tested up to a maximum of 2.5 mg/mL. The test eompound was taxie at 500 µg/mL and formed visible
precipitates starting at 1000 mg/mL with and without S9 mix; therefore, 500 µg/mL was chosen as the highest concentration with/without S9 mix in the HPRT-test.
Gene mutation tests in mammalian cells:
- Results from cytotoxicity measurements:
o Relative total growth (RTG) or relative survival (RS) and cloning efficiency
In HPRT-test I fluocortolone showed astrang cytotoxic effect starting at
400 µg/mL. While the cell cultures of the plating efficiencies showed no survival, the population growths were decreased by about 70%. The second test revealed a clear cytotoxicity of fluocortolone even starting at 300 µg/mL. This is indicated by a marked reduction in the plating efficiencies. However, the population growth was reduced first starting at 350 µg/mL. - Conclusions:
- V79 cells were cultured for 4 hours with fluocortolone at concentrations between 50 and 500 µg/mL with and without metabolic activation. No biologically relevant increase in the number of mutant colonies was observed compared to controls. Toxicity was recorded at concentrations ≥ 300 µg/mL.
- Executive summary:
In a mammalian cell gene mutation assay [HPRT assay] according to OECD test guideline 476 (1997), V79 cells cultured in vitro were exposed to Fluocortolone in DMSO at concentrations of 50, 100, 200, 300, 400 and 500 µg/mL and 50, 150, 300, 350, and 400 µg/mL (in a second test) in the presence and absence of mammalian metabolic activation [S9 mix].
Fluocortolone was tested up to cytotoxic concentrations (i.e., 400 µg/mL). In HPRT-test I fluocortolone showed a strong cytotoxic effect starting at 400 µg/mL. The second test revealed a clear cytotoxicity of fluocortolone even starting at 300 µg/mL. This is indicated by a marked reduction in the plating efficiencies. However, the population growth was reduced first starting at 350 µg/mL. Visible precipitates in the cell culture medium, however, could be observed starting at 400 µg/mL only in HPRT-test I without metabolic activation. The positive controls did induce the appropriate response. There was no evidence of induced mutant colonies over background.
- Endpoint:
- in vitro gene mutation study in bacteria
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- June to Sep 1984
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- comparable to guideline study
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Deviations:
- yes
- Remarks:
- preincubation modification performed only with strain TA 100
- 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:
- rat liver homogenate (S9 mix)
- 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:
- other: without metabolic activation: 9-AA, 2-NF, NaN3, with metabolic activation: 2-AA, BP, CP; in test with preincubation additionally DMNA was used as positive control.
- Key result
- Species / strain:
- S. typhimurium TA 1538
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- growth inhibition starting at 0.75 mg/plate (preincubation procedure) or at 1.0 mg/plate (plate incorporation procedure)
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- True negative controls validity:
- not examined
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 1537
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- growth inhibition starting at 0.75 mg/plate (preincubation procedure) or at 1.0 mg/plate (plate incorporation procedure)
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- True negative controls validity:
- not examined
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 1535
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- growth inhibition starting at 0.75 mg/plate (preincubation procedure) or at 1.0 mg/plate (plate incorporation procedure)
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- True negative controls validity:
- not examined
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 100
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- growth inhibition starting at 0.75 mg/plate (preincubation procedure) or at 1.0 mg/plate (plate incorporation procedure)
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- True negative controls validity:
- not examined
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 98
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- growth inhibition starting at 0.75 mg/plate (preincubation procedure) or at 1.0 mg/plate (plate incorporation procedure)
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- True negative controls validity:
- not examined
- Positive controls validity:
- valid
- Conclusions:
- Fluocortolone was tested for mutagenic activity effects 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 fluocortolon concentrations from 0.1 to 6.0 mg per plate. No increased reversion to prototrophy were seen neither without nor with metabolic activation. Growth inhibition of the background lawn was observed at fluocortolone concentrations starting at 1.0 mg/plate. An additionally performed test with strain TA 100 using the preincubation procedure did not show a mutagenic effect of the test item. Precipitates were found partially in the preincubation test at 6.0 mg per plate.
- Executive summary:
In a reverse gene mutation assay in bacteria according to OECD guideline 471 (1983), Salmonella typhimurium strains TA98, TA100, TA1535, TA1537, and TA1538 were exposed to Fluocortolon in DMSO in concentrations of 0.1, 0.25, 0.5, 1.0, 2.5, 5.0 mg/plate in the absence and presence of mammalian metabolic activation (rat liver S9 mix) in the plate incorporation assay and with the preincubation method at concentrations of 0.1, 0.25, 0.5, 0.75, 1.0, 2.0, 4.0, 6.0 mg/plate (only S. typhimurium TA100). Cytotoxicity was observed from 1.0 and 0.75 mg/plate upwards with the plate incorporation method and the preincubation method, respectively.
Precipitation was partially observed at the highest concentration (6.0 mg/plate). The positive controls induced the appropriate responses in the corresponding strains. The mean numbers of revertant colonies in the negative controls were within the ranges of the historical control data.
There was no evidence of an increase in the number of revertant colonies that exceeded twice background in any of the tester strains (TA98, TA 100, TA1538, TA1535, and TA1537) examined at dose levels up to 6.0 mg/plate in the absence and presence of a metabolic activation source (S9). Therefore, test substance was considered to be non-genotoxic (non-mutagenic) under the conditions employed (plate incorporation/preincubation assay).
There was no evidence of induced mutant colonies over background.
Under the conditions of the study, the test substance was negative for mutagenic potential.
- Endpoint:
- in vitro gene mutation study in bacteria
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- June to Aug 2004
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Version / remarks:
- 1997
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- 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
- Species / strain / cell type:
- E. coli WP2 uvr A
- 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: Liver homogenates (S9: 9000 x g fraction), derived from male Sprague-Dawley rats pre-treated with Aroclor 1254, was obtained from ICN/Cappel Pharmaceuticals, Inc., Aurora, Ohio, USA, [S9 batch number:7756F; protein content 35.0 mg/mL; EROD activity: 4593.2 pmoles 7-hydroxyresorufin/min/mg S9 protein].
- concentration or volume of S9 mix and S9 in the final culture medium: The components of the standard S9 mix were 8 mmol/L MgCl;, 33 mmol/L KCl, 5 mmol/L glucose-6-phosphate, 4 mmol/L NADP, 100 mmol/L sodium phosphate, pH 7.4 and S9.at a concentration of0.1 mL per mL of mix (standard plate incorporation and E.coli) and for the preincubation test S9 at a concentration of 0.3 mL per mL of mix (only S.typhimurium). - Test concentrations with justification for top dose:
- Concentrations of test item (fluocortolone containing 5% ZK 344937 as impurity): 0.01, 0.05, 0.1, 0.25, 0.5, 1.0, 2.5, 5.0 mg/plate (all strains)
- Vehicle / solvent:
- DMSO
- Untreated negative controls:
- yes
- Remarks:
- phosphate buffer
- Negative solvent / vehicle controls:
- yes
- Remarks:
- DMSO
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- 2-nitrofluorene
- sodium azide
- N-dimethylnitrosamine
- benzo(a)pyrene
- cyclophosphamide
- ethylmethanesulphonate
- other: anthracene-2-amine (with metabolic activation), N-methyl-N'-nitro-N-nitrosoguanidine (without metabolic activation), 4-nitro-o-phenylenediamine (without metabolic activation)
- Details on test system and experimental conditions:
- NUMBER OF REPLICATIONS:
- Number of cultures per concentration: triplicate
- Number of independent experiments: two
METHOD OF TREATMENT/ EXPOSURE:
- Test substance added in agar (plate incorporation); preincubation
METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method, e.g.: background growth inhibition - Rationale for test conditions:
- As recommended by the OECD test guideline
- Evaluation criteria:
- please refer to 'Statistics'
- Statistics:
- The arithmetic means of the number ofmutant colonies of the 3 parallel plates in the negative control groups were compared with those of the compound groups. However, in the tables the given mean values are rounded to the nearest integer. A positive response was considered if the number of revertants of the compound groups compared to the number of revertants of the negative group was reproducibly higher than 2-fold. A dose-dependent increase in the number of
revertants was also considered to indicate a mutagenic effect. - Key result
- Species / strain:
- S. typhimurium TA 1538
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- plate incorporation method: growth inhibition at 1.0 mg/plate and above (+/- S9 mix) preincubation modification: growth inhibition at 0.1 mg/plate and above (- S9 mix) or at at 1.0 mg/plate and above (+ S9 mix)
- 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:
- cytotoxicity
- Remarks:
- plate incorporation method: growth inhibition at 1.0 mg/plate and above (+/- S9 mix) preincubation modification: growth inhibition at 0.1 mg/plate and above (- S9 mix) or at at 1.0 mg/plate and above (+ S9 mix)
- 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:
- cytotoxicity
- Remarks:
- plate incorporation method: growth inhibition at 1.0 mg/plate and above (+/- S9 mix) preincubation modification: growth inhibition at 0.1 mg/plate and above (- S9 mix) or at at 1.0 mg/plate and above (+ S9 mix)
- 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:
- cytotoxicity
- Remarks:
- plate incorporation method: growth inhibition at 1.0 mg/plate and above (+/- S9 mix) preincubation modification: growth inhibition at 0.1 mg/plate and above (- S9 mix) or at at 1.0 mg/plate and above (+ S9 mix)
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 98
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- plate incorporation method: growth inhibition at 1.0 mg/plate and above (+/- S9 mix) preincubation modification: growth inhibition at 0.1 mg/plate and above (- S9 mix) or at at 1.0 mg/plate and above (+ S9 mix)
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- E. coli WP2 uvr A
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Conclusions:
- The mutagenic potential of the test item (fluocortolone batch containing 5% of ZK 344937 as impurity) was evaluated in a Salmonella/microsome test with the S. typhimurium strains TA 98, TA 100, TA 1535, TA 1537 and TA 1538 as well as the E. coli strain WP2 uvrA in the presence and absence of S9 mix according to OECD TG 471. Evidence of mutagenic activity was not seen up to the maximum recommended dose level of 5.0 mg/plate. No substantial increases in revertant colony numbers of any of the six tester strains were observed at any dose level in the presence and absence of metabolic activation. Precipitation was observed at the maximum dose level of 5.0 mg/plate. Therefore, the test item was considered to be non-mutagenic in the Salmonella typhimurium reverse mutation assay (plate incorporation method and preincubation modification).
- Executive summary:
In a reverse gene mutation assay in bacteria according to OECD guideline 471 (1997), Salmonella typhimurium strains TA98, TA100, TA1535, TA1537, TA1538 and E.coli strain WP2uvrA were exposed to Fluocortolon in DMSO in concentrations of 0.01, 0.05, 0.1, 0.25, 0.5, 1.0, 2.5, 5.0 mg/plate in the absence and presence of mammalian metabolic activation (rat liver S9 mix) in the plate incorporation assay and with the preincubation method. Cytotoxicity was observed from 1.0 mg/plate upwards (in the refinement only with S9 mix).Without metabolic activation cytotoxicity appeared at 0.1 mg/plate in the refinement.
Precipitation was observed at the highest concentration (5.0 mg/plate). The positive controls induced the appropriate responses in the corresponding strains. The mean numbers of revertant colonies in the negative controls were within the ranges of the historical control data.
There was no evidence of an increase in the number of revertant colonies that exceeded twice background in any of the tester strains (TA98, TA 100, TA1538, TA1535, TA1537 or E.coli WP2uvrA) examined at dose levels up to 5.0 mg/plate in the absence and presence of a metabolic activation source (S9). Therefore, test substance was considered to be non-genotoxic (non-mutagenic) under the conditions employed (preincubation assay).
There was no evidence of induced mutant colonies over background.
Under the conditions of the study, the test substance was negative for mutagenic potential.
- Endpoint:
- in vitro gene mutation study in bacteria
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- Apr to June 1996
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- comparable to guideline study
- 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
- Principles of method if other than guideline:
- Preincubation modification was performed
- GLP compliance:
- yes (incl. QA statement)
- 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:
- rat liver homogenate (S9 mix)
- Test concentrations with justification for top dose:
- All strains: 0.1, 0.25, 0.5, 1.0, 2.5, 5.0 mg/plate
TA 1538 without S9 mix: 0.05, 0.1, 0.25, 0.5, 1.0, 2.5, 5.0 mg/plate - Vehicle / solvent:
- DMSO
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- 9-aminoacridine
- 2-nitrofluorene
- sodium azide
- N-dimethylnitrosamine
- benzo(a)pyrene
- cyclophosphamide
- other: with metabolic activation: 2-AA
- Details on test system and experimental conditions:
- NUMBER OF REPLICATIONS:
- Number of cultures per concentration: triplicate
- Number of independent experiments: one
METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): E-06 dilution
- Test substance added: preincubation
METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method, e.g.: background growth inhibition - Evaluation criteria:
- The plates were scored for the number of mutant colonies with an automated colony counter (Artek M 982B, Artek Systems Corporation, Farmingdale, NY, USA). In exceptional cases where reliable automatic counting is not possible, e. g. due to distinct precipitates of the test compounds,the colonies scored manually. The arithmetic means of the number of mutant colonies of the 3 parallel plates in the negative contral graups 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 (ar the compound formed precipitates in the agar) and if the number of induced revertants compared to the number of spontaneaus ones was higher than 2-fold. A dose-dependent increase in the number of revertants was also considered to indicate a mutagenic effect.
A toxic effect of the substance on the background lawn of non-revertant bacteria and precipitates in the agar were examined stereomicrascopically. - Key result
- Species / strain:
- S. typhimurium TA 1538
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- growth inhibition at 1.0 mg/plate and above
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- True negative controls validity:
- not examined
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 1537
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- growth inhibition at 1.0 mg/plate and above
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- True negative controls validity:
- not examined
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 1535
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- growth inhibition at 1.0 mg/plate and above
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- True negative controls validity:
- not examined
- Positive controls validity:
- not examined
- Key result
- Species / strain:
- S. typhimurium TA 100
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- growth inhibition at 1.0 mg/plate and above
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- True negative controls validity:
- not examined
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 98
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- growth inhibition at 1.0 mg/plate and above
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- True negative controls validity:
- not examined
- Positive controls validity:
- valid
- Conclusions:
- The purpose of the Ames test was to investigate whether fluocortolone 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 fluocortolon concentrations from 0.1 to 5.0 mg per plate and 0.05 to 5.0 mg/plate (TA1538 without S9 mix). No increased reversion to prototrophy were seen neither without nor with metabolic activation. Growth inhibition of the background lawn was observed at fluocortolone concentrations of 1.0 mg/plate and above.
- Executive summary:
In a reverse gene mutation assay in bacteria according to OECD guideline 471 (1983), Salmonella typhimurium strains TA98, TA100, TA1535,TA 1538 and TA1537 were exposed to Fluocotrolon in in DMSO in concentrations of All strains: 0.1, 0.25, 0.5, 1.0, 2.5, 5.0 mg/plate in the absence and presence of mammalian metabolic activation (rat liver S9 mix) and TA 1538 without S9 mix: 0.05, 0.1, 0.25, 0.5, 1.0, 2.5, 5.0 mg/plate in the preincubation assay. Cytotoxicity was observed from 1.0 mg/plate upwards.
Precipitation was observed at the highest concentration (5.0 mg/plate). The positive controls induced the appropriate responses in the corresponding strains. The mean numbers of revertant colonies in the negative controls were within the ranges of the historical control data.
There was no evidence of an increase in the number of revertant colonies that exceeded twice background in any of the five tester strains (TA98, TA 100, TA1538, TA1535, or TA1537) examined at dose levels up to 5.0 mg/plate in the absence and presence of a metabolic activation source (S9). Therefore, test substance was considered to be non-genotoxic (non-mutagenic) in Salmonella tester strains TA98, TA100, TA1538, TA1535, and TA 1537 under the conditions employed (preincubation assay).
There was no evidence of induced mutant colonies over background.
Under the conditions of the study, the test substance was negative for mutagenic potential.
Referenceopen allclose all
No clastogenic effects could be observed when tested without extrinsic metabolising system. The concentration of 100 µg/ml induced as sign of cytotoxicity >50% reduction of mitotic index.
Fluocortolone tested with S9 mix showed clastogenic effect at first harvesting time point at the precipitating concentrations of 600 µg/ml (5.5% abarrant cells) and 650 µg/ml (9% aberrant cells). No clastogenic effects could be observed at the second time point.
Chromosomal aberrations in human Iymphocytes (whole blood culture); test without S9 mix - first harvesting time, after 21-hour treatment | |||||
Test substance | Concentration [µg/mL] | Number of Metaphases analyzed | % aberrant metaphases (excl. AL) | Mitotic index 1) | |
Absolute | relative2) | ||||
DMSO (solvent control) | 1% (v/v) | 200 | 1.5 | 11.4 | 100 |
Test item | 25 | 200 | 0 | 9.0 | 79 |
| 50 | not analyzed |
| not scored |
|
| 100 | not analyzed |
| not scored |
|
| 125 | 200 | 1.0 | 6.0 | 53 |
| 150 | not analyzed |
| not scored |
|
| 175 | 200 | 2.0 | 4.1 | 36 |
| 200 | not analyzed |
| not scored |
|
| 225 | not analyzed |
| not scored |
|
| 250 | not analyzed |
| 2.8 | 25 |
Triaziquone (positive control) | 7.5E-08 mol/L | 100 | 14* | 6.4 | 56 |
DMSO = dimethyl sulfoxide 1) mean values 2) The values are related to the solvent control. * Fisher's exact test: p < 0.05, compared to the concurrent solvent control | |||||
|
|
|
|
|
|
Chromosomal aberrations in human Iymphocytes (whole blood culture); test without S9 mix - second harvesting time, after 44-hour treatment - | |||||
Test substance | |||||
Absolute | relative2) | ||||
DMSO (solvent control) | 1% (v/v) | 200 | 1.0 | 15.5 | 100 |
Test item | 50 | not analyzed |
| not scored |
|
| 100 | 200 | 1.5 | 6.6 | 43 |
| 150 | not analyzed |
| not scored |
|
| 200 | not analyzed |
| not scored |
|
DMSO = dimethyl sulfoxide 1) mean values 2) The values are related to the solvent control | |||||
| |||||
Chromosomal aberrations in human Iymphocytes (whole blood culture); test with S9 mix - first harvesting time, 19 hours after 4-hour treatment - | |||||
Test substance | Concentration [µg/mL] | Number of Metaphases analyzed | % aberrant metaphases (excl. AL) | Mitotic index 1) | |
Absolute | relative2) | ||||
DMSO (solvent control) | 1% (v/v) | 200 | 0.5 | 13.7 | 100 |
Test item | 100 | 200 | 0.5 | 9.8 | 72 |
| 250 | not analyzed |
| not scored |
|
| 500 | not analyzed |
| 10.5 | 77 |
| 550 | not analyzed |
| not scored |
|
| 600 | 200 | 5.5* | 12.0 | 88 |
| 650 | 200 | 9.0* | 6.9 | 50 |
| 700 | not analyzed |
| 2.2 | 16 |
| 750p) | not harvested |
|
|
|
| 800 p) | not harvested |
|
|
|
| 850 p) | not harvested |
|
|
|
| 900 p) | not harvested |
|
|
|
Cyclophosphamide (positive control) | 1.0E-05 mol/L | 100 | 18* | 8.5 | 62 |
DMSO = dimethyl sulfoxide 1) mean values 2) The values are related to the solvent control. p) = precipitates *= Fisher's exact test: p < 0.05, compared to the concurrent solvent control | |||||
| |||||
Chromosomal aberrations in human Iymphocytes (whole blood culture); test with S9 mix - second harvesting time, 40 hours after 4-hour treatment | |||||
Test substance | Concentration [µg/mL] | Number of Metaphases analyzed | % aberrant metaphases (excl. AL) | Mitotic index 1) | |
Absolute | relative2) | ||||
DMSO (solvent control) | 1% (v/v) | 200 | 1.0 | 17.5 | 100 |
test item | 500 | not analyzed |
| not scored |
|
| 600 | not analyzed |
| not scored |
|
| 700p) | 200 | 3.0 | 13.9 | 79 |
| 800p) | not scorable3) |
| not scored |
|
DMSO = dimethyl sulfoxide 1) mean values 2) The values are related to the solvent control. 3) due to less test material caused by cytotoxicity of the test compound p) = precipitates | |||||
| |||||
Proliferation kinetics of human Iymphocytes (whole blood culture) treated with fluocortolone; test without S9 mix - first harvesting time after 21-hour treatment | |||||
Test substance | Concentration [µg/mL] | Number of Metaphases scored | % cells1) | ||
M1 | M2 | M3 | |||
DMSO (solvent control) | 1% (v/v) | 100 | 78 | 22 | 0 |
test item | 125 | 100 | 99 | 1 | 0 |
DMSO = dimethyl sulfoxide 1) % cells which have completed one (M1), two (M2) or three (M3) cell cycles in BUdR at the harvesting time for chromosomal aberrations | |||||
| |||||
Proliferation kinetics of human Iymphocytes (whole blood culture) treated with fluocortolone; test with S9 mix - first harvesting time, 19 hours after 4-hour treatment | |||||
Test substance | Concentration [µg/mL] | Number of Metaphases scored | % cells1) |
|
|
|
|
| M1 | M2 | M3 |
DMSO (solvent control) | 1% (v/v) | 100 | 82 | 18 | 0 |
test item | 500 | 100 | 97 | 3 | 0 |
DMSO = dimethyl sulfoxide 1) % cells which have completed one (M1), two (M2) or three (M3) cell cycles in BUdR at the harvesting time for chromosomal aberrations |
In the assay without S9 mix the concentration of 175 µg/ml induced as sign of cytotoxicity >50% reduction of mitotic index. In the assay +S9 mix fluocortolone was tested up to the concentration of 600 µg/ml at which precipitation of the test compound was observed.
Chromosomal aberrations in human Iymphocytes (whole blood culture); test without S9 mix - first harvesting time, after 21-hour treatment | |||||
Test substance | Concentration [µg/mL] | Number of Metaphases analyzed | % aberrant metaphases (excl. AL) | BR1) | Mitotic index [%] |
DMSO (solvent control) | 1% (v/v) | 200 | 0.5 | 0.5 | 12.4 |
Test item | 10 | not analyzed |
| not scored |
|
| 25 | 200 | 0 | 0 | 10.2 |
| 50 | not analyzed |
| not scored |
|
| 75 | not analyzed |
|
| 8.7 |
| 100 | not analyzed |
| not scored |
|
| 125 | 200 | 1.0 | 1.0 | 9.7 |
| 150 | not analyzed |
| not scored |
|
| 175 | 200 | 2.0 | 3.0 | 6.0 |
| 200 | not analyzed |
|
| 5.7 |
| 225 | not scorable |
|
|
|
| 250 | not scorable |
|
|
|
| 275 | not harvested |
|
|
|
| 300 | not harvested |
|
|
|
Triaziquone (positive control) | 7.5E-08 mol/L | 100 | 28* | 41 | 9.2 |
1) BR = Breakage rate per 100 metaphases, calculation from the following breakage events: B', B" = 1 RB', DIC, RING = 2 (i. e. the estimated number of breaks involved in the production of the different exchange configurations) MA and DIS are included in the percentage of aberrant metaphases but excluded from the breakage rate. *-Fisher's exact test: p < 0.05, compared to the concurrent solvent control | |||||
|
|
|
|
|
|
Chromosomal aberrations in human Iymphocytes (whole blood culture); test without S9 mix - second harvesting time, after 44-hour treatment - | |||||
Test substance | Concentration [µg/mL] | Number of Metaphases analyzed | % aberrant metaphases (excl. AL) | BR1) | Mitotic index [%] |
DMSO (solvent control) | 1% (v/v) | 200 | 1.0 | 1.0 | 15.3 |
Test item | 10 | not analyzed |
|
| not scored |
| 50 | not analyzed |
|
| not scored |
| 100 | not analyzed |
|
| not scored |
| 150 | 200 | 4.5* | 4.5 | 5.9 |
| 200 | not analyzed |
|
|
|
1) BR = Breakage rate per 100 metaphases, calculation from the following breakage events: B', B" = 1 RB', DIC, RING = 2 (i. e. the estimated number of breaks involved in the production of the different exchange configurations) MA and DIS are included in the percentage of aberrant metaphases but excluded from the breakage rate. *-Fisher's exact test: p < 0.05, compared to the concurrent solvent control | |||||
| |||||
Chromosomal aberrations in human Iymphocytes (whole blood culture); test with S9 mix - first harvesting time, 19 hours after 4-hour treatment - | |||||
Test substance | Concentration [µg/mL] | Number of Metaphases analyzed | % aberrant metaphases (excl. AL) | BR1) | Mitotic index [%] |
DMSO (solvent control) | 1% (v/v) | 200 | 0 | 0 | 9.5 |
Test item | 10 | not analyzed |
|
| not scored |
| 50 | 200 | 1.5 | 1.5 | 10.1 |
| 100 | not analyzed |
|
| not scored |
| 200 | 200 | 1.5 | 1.5 | 9.6 |
| 400 | not analyzed |
|
| 9.5 |
| 600 p) | 200 | 2.0 | 4.0 | 11.1 |
| 800 p) | not scorable2) |
|
|
|
| 1000p) | not harvested |
|
|
|
Cyclophosphamide (positive control) | 1.0E-05 mol/L | 100 | 15* | 20 | 6.9 |
1) BR = Breakage rate per 100 metaphases, calculation from the following breakage events: B', B" = 1 RB', DIC, RING = 2 (i. e. the estimated number of breaks involved in the production of the different exchange configurations) MA and DIS are included in the percentage of aberrant metaphases but excluded from the breakage rate. 2) since there were not enough cells/metaphases due to the cytotoxicity of the test compound P = precipitates *Fisher's exact test: p < 0.05, compared to the concurrent solvent control | |||||
| |||||
Chromosomal aberrations in human Iymphocytes (whole blood culture); test with S9 mix - second harvesting time, 40 hours after 4-hour treatment | |||||
Test substance | Concentration [µg/mL] | Number of Metaphases analyzed | % aberrant metaphases (excl. AL) | BR1) | Mitotic index [%] |
DMSO (solvent control) | 1% (v/v) | 200 | 0.5 | 0.5 | 14.9 |
test item | 200 | not analyzed |
|
| not scored |
| 400 | not analyzed |
|
| not scored |
| 600p) | 200 | 0 | 0 | 17.0 |
| 1000p) | not scorable2) |
|
|
|
1) BR = Breakage rate per 100 metaphases, calculation from the following breakage events: B', B" = 1 RB', DIC, RING = 2 (i. e. the estimated number of breaks involved in the production of the different exchange configurations) MA and DIS are included in the percentage of aberrant metaphases but excluded from the breakage rate. 2) due to less test material caused by the cytotoxicity of the test compound P = precipitates | |||||
| |||||
Proliferation kinetics of human Iymphocytes (whole blood culture) treated with fluocortolone; test without S9 mix - first harvesting time after 21-hour treatment | |||||
Test substance | Concentration [µg/mL] | Number of Metaphases scored | % cells1) | ||
M1 | M2 | M3 | |||
DMSO (solvent control) | 1% (v/v) | 200 | 74 | 26 | 0 |
test item | 100 | 200 | 91 | 9 | 0 |
DMSO = dimethyl sulfoxide 1) % cells which have completed one (M1), two (M2) or three (M3) cell cycles in BUdR at the harvesting time for chromosomal aberrations | |||||
| |||||
Proliferation kinetics of human Iymphocytes (whole blood culture) treated with fluocortolone; test with S9 mix - first harvesting time, 19 hours after 4-hour treatment | |||||
Test substance | Concentration [µg/mL] | Number of Metaphases scored | % cells1) |
|
|
|
|
| M1 | M2 | M3 |
DMSO (solvent control) | 1% (v/v) | 200 | 90 | 10 | 0 |
test item | 100 | 200 | 90 | 10 | 0 |
DMSO = dimethyl sulfoxide 1) % cells which have completed one (M1), two (M2) or three (M3) cell cycles in BUdR at the harvesting time for chromosomal aberrations |
The viability of the hepatocytes was reduced by 34% at a concentration of 150µg/ml test substance and by 67% at a concentration of 200 µg/mL.
Cytotoxicity test with fluocortolone
Concentration | Neutral red absorption at 540 nm1) | Percentage of viable cells relative to the control (%)1) |
Medium | 0.389 | 129.7 |
1%DMSO | 0.300 | 100.0 |
10 µg/ml | 0.241 | 80.3 |
20 µg/ml | 0.241 | 80.3 |
40 µg/ml | 0.216 | 72.0 |
60 µg/ml | 0.340 | 113.3 |
80 µg/ml | 0.225 | 75.0 |
100 µg/ml | 0.192 | 64.0 |
125 µg/ml | 0.213 | 71.0 |
150 µg/ml | 0.198 | 66.0 |
200 µg/ml | 0.099 | 33.0 |
2-AAF (1 x 10-6 mol/1) | 0.251 | 83.7 |
1) mean values of 2 cultures
UDS test with fluocortolone on cultured female rat hepatocytes:
summarized data (mean nuclear and cytoplasmic grain counts, mean net nuclear grain values)
Test substance | Concentration in culture | Number of cells scored | Mean NG ±SD | Mean CG ±SD | Mean NNG ±SD | Cells (%) in repair (NNG > 5) |
Medium | -- | 150 | 10.0 ± 4.2 | 16.2 ± 5.6 | -6.2 ±4.4 | 0.7 |
DMSO | 1% | 150 | 8.5 ± 3.4 | 14.9 ± 3.9 | -6.4 ±3.5 | 0.0 |
FIuocortolone | 10 µg/mL | 150 | 8.6±3.9 | 14.7 ± 3.8 | -6.2 ±4.5 | 0.7 |
20 µg/mL | 150 | 8.1 ± 3.8 | 11.8 ± 3.1 | -3.7 ±3.9 | 2.Ö | |
40 µg/mL | 150 | 9.5 ± 4.1 | 13.8 ± 3.8 | -4.2 ±5.0 | 2.7 | |
80 µg/mL | 150 | 8.7±4.0 | 11.4 ± 3.4 | -2.7 ±4.1 | 4.7 | |
125 µg/mL | 150 | 8.2 ±4.0 | 10.8 ± 2.9 | -2.7 ± 4.1 | 3.3 | |
150 µg/mL | 150 | 7.4 ± 3.9 | 7.9 ± 3.1 | -0.5±3.7 | 8.7 | |
2-AAF | 1 x 1o-6 mol/L | 150 | 42.2 ± 18.7 | 16.0 ± 6.1 | 26.2 ± 16.0 | 97.3 |
NG: nuclear grain counts
CG: cytoplasmic grain counts
NNG: net nuclear grain counts (mean NG minus mean CG)
SD: standard deviation
The viability of the hepatocytes was reduced by 48% at a concentration of 150µg/ml test substance.
Cytotoxicity test with fluocortolone (ZK 10445)
Concentration | Neutral red absorption at 540 nm1l | Percentage of viable cells relative to the control (%) 1l |
Medium | 0.324 | 153.6 |
1% DMSO | 0.211 | 100.0 |
2-AAF (1 x 1o-6 mol/I) | 0.225 | 106.6 |
200 µg /ml ., | 0.020 | 9.5 |
150 µg /ml | 0.110 | 52.1 |
125 µg /ml | 0.128 | 60.7 |
100 µg /ml | 0.135 | 64.0 |
80 µg /ml | 0.118 | 56.0 |
60 µg /ml | 0.128 | 60.7 |
40 µg /ml | 0.165 | 78.2 |
20 µg /ml | 0.227 | 107.6 |
10 µg /ml | 0.256 | 121.3 |
1) mean values of 2 cultures
UDS test with f!uocortolone (ZK 10445) on cultured male rat hepatocytes: Summarized data (mean nuclear and cytoplasmic grain counts, mean net nuclear grain values)
Test substance | Concentration in culture | Number of cells scored | Mean NG ±SD | Mean CG ±SD | Mean NNG ±SD | Cells (%) in repair (NNG > 5) |
DMSO | 1% | 150 | 1.0 ± 1.1 | 4.1 ± 1.6 | -3.2 ± 1.7 | 0.0 |
2-AAF | 1 x 1o-6 mol/I | 150 | 20.2 ± 7.7 | 4.9 ± 1.9 | 15.2 ± 6.9 | 94.7 |
Fluocortolone | 150 µg/ml | 150 | 2.4±2.1 | 3.3 ± 1.5 | -0.9±2.1 | 0.0 |
Fluocortolone | 125 µg/ml | 150 | 3.6 ± 2.7 | 4.5 ± 1.8 | -0.9 ±2.6 | 2.7 |
Fluocortolone | 80 µg/ml | 150 | 4.4 ± 3.7 | 5.0 ± 3.1 | -0.6 ± 3.1 | 3.3 |
Fluocortolone | 40 µg/ml | 150 | 2.4 ± 1.8 | 4.1 ± 1.5 | -1.7±2.0 | 0.0 |
Fluocortolone | 20 µg/ml | 150 | 2.9 ± 2.3 | 6.4 ± 2.5 | -3.5 ±2.7 | 0.7 |
Fluocortolone | 10 µg/ml | 150 | 2.5 ± 2.1 | 6.8 ± 2.4 | -4.3 ± 2.4 | 0.0 |
NG: nuclear grain counts
CG: cytoplasmic grain counts
NNG: net nuclear grain counts (mean NG minus mean CG)
SD: standard deviation
Cytotoxicity was observed with fluorocortolone at concentrations ¿ 300 µg/mL resulting in the clear reduction in the plating efficancies and in a moderate effect in the population growths.
Table 1: Toxicity data; HPRT-test I without S9 mix
Column |
1 |
2 |
3 |
4 |
5 | 6② | 7③ | 9④ |
9⑤ |
Test compound |
Concentration |
Number of cells per flask ① |
PE%
absolute |
PE%
relative |
Population growth absolute |
Population growth % relative | |||
seeded 1/11 |
found 1 II |
mean | |||||||
DMSO |
1% |
500 |
310 | 448 |
379 |
76 |
100 |
3868020 |
100 |
(solvent |
| 500 | 542 | 550 | 546 | 109 | 100 | 4008960 | 100 |
control) |
|
|
|
|
|
|
|
|
|
Fluo- |
50 µg/mL |
500 |
541 |
417 |
479 |
96 |
126 |
5042520 |
130 |
cortolone |
| 500 | 537 | 506 | 522 | 104 | 96 | 4259520 | 106 |
| 100 µg/mL | 500 | 525 | 510 | 518 | 104 | 137 | 4494420 | 116 |
|
| 500 | 477 | 465 | 471 | 94 | 86 | 3664440 | 91 |
| 200 µg/mL | 500 | 520 | 514 | 517 | 103 | 136 | 3836700 | 99 |
|
| 500 | 506 | 527 | 517 | 103 | 95 | 3554820 | 89 |
| 300 µg/mL | 500 | 453 | 435 | 444 | 89 | 117 | 4118580 | 106 |
|
| 500 | 350 | 392 | 371 | 74 | 68 | 4165560 | 104 |
| 400 µg/miPl | 500 | 1 | 0 | 1 | 0 | 0 | 1064880 | 28 |
|
| 500 | 0 | 0 | 0 | 0 | 0 | 1393740 | 35 |
| 500 µg/mL Pl | 500 | 0 | 0 | 0 | 0 | 0 | t | -- |
|
| 500 | 0 | 0 | 0 | 0 | 0 | t | -- |
MNNG |
0.15 µg/mL |
500 |
140 |
106 |
123 |
25 |
32 |
2302020 |
60 |
(positive |
| 500 | 143 | 140 | 142 | 28 | 26 | 2176740 | 54 |
control) |
|
|
|
|
|
|
|
|
|
(1) only colonies with more than 50 cells 7 days after seeding were scored
(2) PE absolute (value column 5/value column 2 x 100)
(3) PE relative (value column 5/value column 5 of corresponding control x 100)
(4) no. of cells per mL at subculture (3 days after treatment)
(5) % of solvent control (three days after treatment)
p precipitates
t toxic
Table 2: Mutagenicity data (part 1); HPRT-test I without S9 mix
Column | 1 | 2 | 3 | 4 | 5 | 6 ② | 7③ | 8④ |
Test compound | Concentration | Number of cells per flask ① | Factor calculated | Cells seeded | Cells surviving | |||
seeded I/II | found I II | mean | ||||||
DMSO | 1% | 500 | 427 | 477 |
452 | 0.90 | 554529 | 499076 |
(solvent |
| 500 | 452 | 440 | 446 | 0.89 | 555830 | 494689 |
control) |
|
|
|
|
|
|
|
|
Fluo- | 50 µg/mL | 500 | 381 | 360 | 371 | 0.74 | 555899 | 411365 |
cortolone |
| 500 | 415 | 364 | 390 | 0.78 | 556862 | 434352 |
| 100 µg/mL | 500 | 460 | 445 | 453 | 0.91 | 555830 | 505805 |
|
| 500 | 403 | 419 | 411 | 0.82 | 556036 | 455950 |
| 200 µg/mL | 500 | 383 | 369 | 376 | 0.75 | 556724 | 417543 |
|
| 500 | 397 | 403 | 400 | 0.80 | 556036 | 444829 |
| 300 µg/mL | 500 | 413 | 400 | 407 | 0.81 | 556105 | 450445 |
|
| 500 | 393 | 390 | 392 | 0.78 | 555487 | 433280 |
| 400 µg/mLP) | 500 | 309 | 303 | 309 | 0.61 | 556655 | 339560 |
|
| 500 | 356 | 335 | 346 | 0.69 | 556449 | 383950 |
| 500 µg/miP> | t t | -- -- | -- -- | -- -- | -- -- | -- . -- | -- -- |
MNNG | 0.15 µg/mL | 500 | 204 | 183 | 194 |
0.39 | 530723 | 206982 |
(positive |
| 500 | 173 | 177 | 175 | 0.35 | 536289 | 187701 |
control) |
|
|
|
|
|
|
|
|
①only colonies with more than 50 cells 6 days after seeding in normal medium were scored
②actor calculated (value column 5/value column 2)
③ cells seeded in TG medium (selective medium)
④ cells surviving after plating in TG medium (value column 7 x value column 6)
p) precipitates
t) toxic
Table 3: Mutagenicity data (part 2); HPRT-test I without S9 mix
Column | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | |
Test compound | Concentration | Number of mutant cells per flask ① found after plating in TG medium | Standard deviation | Mutant colonies② per 106 cells | ||||||
I | II | III | IV | V | mean | individual | mean | |||
DMSO (solvent control) | 1% | 15 14 | 19 16 | 17 17 | 16 14 | 21 12 | 17.6 14.6 | 2.4 1.9 | 35 30 |
33 |
Fluo- | 50 µg/mL | 10 | 10 | 5 | 10 | 9 | 8.8 | 2.2 | 21 |
|
cortolone |
| 10 | 9 | 9 | 6 | 7 | 8.2 | 1.6 | 19 | 20 |
| 100 µg/mL | 10 | 8 | 6 | 2 | 2 | 5.6 | 3.6 | 11 |
|
|
| 17 | 12 | 6 | 11 | 8 | 10.8 | 4.2 | 24 | 18 |
| 200 µg/mL | 11 | 10 | 12 | 12 | 11 | 11.2 | 0.8 | 27 |
|
|
| 18 | 12 | 16 | 20 | 19 | 17.0 | 3.2 | 38 | 33 |
| 300 µg/mL | 2 | 6 | 7 | 3 | 4 | 4.4 | 2.1 | 10 |
|
|
| 7 | 11 | 12 | 10 | 17 | 11.4 | 3.6 | 26 | 18 |
| 400 µg/mL P) | 12 | 9 | 7 | 6 | 4 | 7.6 | 3.0 | 22 |
|
|
| 7 | 10 | 9 | 3 | 14 | 8.6 | 4.0 | 22 | 22 |
| 500 µg/mLP) | t t | -- -- | -- -· | -- - | -- -- | -- -- | -- -· | -· |
-- |
MNNG (positive control) | 0.15 µg/mL | 146 171 | 138 171 | 132 162 | 157 202 | 158 191 | 146.2 179.4 | 11.5 16.5 | 706 956 |
831 |
① only colonies with more than 50 cells 10 days after seeding in TG medium were scored
② value column 7 (Table 3) x 106/value column 8 (Table 2)
p) precipitates
t) toxic
Table 4: Toxicity data; HPRT-test I with S9 mix
Column | 1 | 2 | 3 | 4 | 5 | 6 ② | 7③ | 8④ | 9⑤ |
Test compound |
Concentration | Number of cells per flask① |
PE%
absolute | PE%
relative |
Population growth absolute |
Population growth % relative | |||
seeded I/II | found I II | mean | |||||||
DMSO | 1% | 500 | 453 | 457 | 455 |
91 | 100 | 3100680 |
100 |
(solvent |
| 500 | 482 | 453 | 468 | 94 | 100 | 3366900 | 100 |
control) |
|
|
|
|
|
|
|
|
|
Fluo- | 50 µg/mL | 500 | 498 | 527 | 513 | 103 | 113 | 3868020 | 125 |
cortolone |
| 500 | 527 | 481 | 504 | 101 | 108 | 4384800 | 130 |
| 100 µg/mL | 500 | 482 | 481 | 482 | 96 | 106 | 3116340 | 101 |
|
| 500 | 499 | 465 | 482 | 96 | 103 | 3257280 | 97 |
| 200 µg/mL | 500 | 456 | 489 | 473 | 95 | 104 | 3774060 | 122 |
|
| 500 | 449 | 439 | 444 | 89 | 95 | 3695760 | 110 |
| 300 µg/mL | 500 | 157 | 200 | 179 | 36 | 39 | 2411640 | 78 |
|
| 500 | 206 | 221 | 214 | 43 | 46 | 2286360 | 68 |
| 400 µg/mL | 500 | 0 | 0 | 0 | 0 | 0 | t | -- |
|
| 500 | 0 | 0 | 0 | 0 | 0 | t | -- |
| 500 µg/mL | 500 | 0 | 0 | 0 | 0 | 0 | .t | -- |
|
| 500 | 0 | 0 | 0 | 0 | 0 | t | -- |
DMBA |
15 µg/mL |
500 | 140 | 173 | 157 |
31 |
35 |
1910520 | 62 |
(positive |
| 500 | 152 | 157 | 155 | 31 | 33 | 2302020 | 68 |
control) |
|
|
|
|
|
|
|
|
|
(1) only colonies with more than 50 cells 7 days after seeding were scored
(2) PE absolute (value column 5/value column 2 x 100)
(3) PE relative (value column 5/value column 5 of corresponding control x 100)
(4) no. of cells per mL at subculture (3 days after treatment)
(5) % of solvent control {three days after treatment)
t) toxic
Table 5: Mutagenicity data (part 1); HPRT-test I with S9 mix
Column | 1 | 2 | 3 | 4 | 5 | 6 ② | 7③ | 8④ |
Test compound | Concentration | Number of cells per flask ① | Factor calculated | Calls seeded | Calls surviving | |||
seeded I/II | found I II | mean | ||||||
DMSO | 1% | 500 | 569 | 588 | 579 | 1.16 | 554324 | 643016 |
(solvent |
| 500 | 383 | 457 | 420 | 0.84 | 555761 | 466839 |
control) |
|
|
|
|
|
|
|
|
Fluo- | 50 µg/mL | 500 | 402 | 417 443
570 441
453 383
383 386 -- -- -- -- | 410 | 0.82 0.89
1.17 0.90
0.94 0.79
0.81 0.78 -- -- -- -- | 556586 555830
555213 557483
555624 556449
556655 557069 -- -- -- -- | 456401 494689
649599 501735
522287 439595
450891 434514 -- -- -- -- |
cortolone |
| 500 | 449 | 446 | ||||
| 100 µg/mL | 500 | 600 | 585 | ||||
|
| 500 | 461 | 451 | ||||
| 200 µg/mL | 500 | 482 | 468 | ||||
|
| 500 | 406 | 395 | ||||
| 300 µg/mL | 500 | 431 | 407 | ||||
|
| 500 | 394 | 390 | ||||
| 400 µg/mL | t | -- | -- | ||||
|
| t | -- | -- | ||||
| 500 µg/mL | t | -- | -- | ||||
|
| t | -- | -- | ||||
DMBA |
15 µg/mL |
500 | 469 |
406 | 438 |
0.88 |
553710 |
487265 |
(positive |
| 500 | 369 | 405 | 387 | 0.77 | 555556 | 427778 |
control) |
|
|
|
|
|
|
|
|
(1) only colonies with more than 50 cells 6 days after seeding in normal medium were scored
(2) factor calculated (value column 5/value column 2)
(3) cells seeded in TG medium (selective medium)
(4) cells surviving after plating in TG medium (value column 7 x value column 6)
t) toxic
Table 6: Mutagenicity data (part 2); HPRT-test I with S9 mix
Column | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | |
Test compound | Concentration | Number of mutant cells per flask ① found after plating in TG medium | Standard deviation | Mutant colonies② per 106 cells | ||||||
I | II | III | IV | V | mean | individual | mean | |||
DMSO (solvent control) | 1% | 6 4 | 3 5 | 5 5 | 1 7 | 7 3 | 4.4 4.8 | 2.4 1.5 | 7 10 |
9 |
Fluo- | 50 µg/mL | 5 | 1 2 5 10
4 6
8 3 -- -- -- -- |
2 2 8 9
3 9
6 9 -- -- -- -- | 1 1 4 14
3 11
4 5 -- -- -- -- | 0 7
8 13
3 8
3 5 -- -- -- -- | 1.8 3.4
6.6 10.4
3.0 8.6
4.8 6.2 -- -- -- -- | 1.9 2.5 1.9 3.2
0.7 1.8
2.2 2.7 -- -- -- -- | 4 7 10 21
6 20
11 14 -- -- -- -- |
|
cortolone |
| 5 | 6 | |||||||
| 100 µg/mL | 8 |
| |||||||
|
| 6 | 16 | |||||||
| 200 µg/mL | 2 |
| |||||||
|
| 9 | 13 | |||||||
| 300 µg/mL | 3 |
| |||||||
|
| 9 | 13 | |||||||
| 400 µg/mL | t |
| |||||||
| t | -- | ||||||||
| 500 µg/mL | t t | -- | |||||||
DMBA (positive control) | 15 µg/mL | 93 82 | 115 98 | 96 84 | 109 85 | 103 103 | 103.2 90.4 | 9.1 9.4 | 212 211 |
212 |
①only colonies with more than 50 cells 10 days after seeding in TG medium were scored
②value column 7 (Table 6) x 106/value column 8 (Table 5)
t) toxic
Table 7: Toxicity data; HPRT-test II without S9 mix
Column |
1 |
2 | 3 |
4 |
5 | 6 ② | 7③ | 8④ | 9⑤ |
Test compound |
Concentration |
Number of cells per flask ① |
PE%
absolute |
PE%
relative |
Population growth absolute |
Population growth % relative | |||
seeded found I/II I II |
mean | ||||||||
Medium |
|
500 |
531 |
487 |
509 |
102 |
100 |
2677860 |
100 |
control |
| 500 | 477 | 504 | 491 | 98 | 100 | 2442960 | 100 |
DMSO | 1% | 500 | 541 | 485 | 513 | 103 | 100 | 2850120 | 100 |
(solvent |
| 500 | 510 | 531 | 521 | 104 | 100. | 2724840 | 100 |
control) |
|
|
|
|
|
|
|
|
|
Fluo- |
50 µg/mL |
500 |
521 |
491 |
506 |
101 |
99 |
3398220 |
119 |
cortolone |
| 500 | 517 | 489 | 503 | 101 | 97 | 3319920 | 122 |
| 150 µg/mL | 500 | 544 | 452 | 498 | 100 | 97 | 2724840 | 96 |
|
| 500 | 557 | 532 | 545 | 109 | 105 | 3633120 | 133 |
| 300 µg/mL | 500 | 115 | 83 | 99 | 20 | 19 | 2912760 | 102 |
|
| 500 | 107 | 136 | 122 | 24 | 23 | 3288600 | 121 |
| 350 µg/mL | 500 | 0 | 0 | 0 | 0 | 0 | 1331100 | 47 |
|
| 500 | 1 | 1 | 1 | 1 | 1 | 1237140 | 45 |
| 400 µg/mL | 500 | 0 | 0 | 0 | 0 | 0 | t | -- |
| 500 | 0 | 0 | 0 | 0 | 0 | t | -- | |
EMS | 0.7 µg/mL | 500 | 263 | 227 | 245 |
49 | 48 | 1863540 |
70 |
(positive |
| 500 | 232 | 219 | 226 | 45 | 46 | 2004480 | 82 |
control) |
|
|
|
|
|
|
|
|
|
(1) only colonies with more than 50 cells 7 days after seeding were scored
(2) PE absolute (value column 5/value column 2 x 100)
(3) PE relative (value column 5/value column 5 of corresponding control x 100)
(4) no. of cells per mL at subculture (3 days after treatment)
(5) % of solvent control (three days after treatment)
t) toxic
Table 8:Mutagenicity data (part 1); HPRT-test II without S9 mix
Column | 1 | 2 | 3 | 4 | 5 | 6② | 7③ | 8④ |
Test compound | Concentration | Number of cells per flask ① | Factor calculated | Cells seeded | Cells surviving | |||
seeded I/II | found I II | mean | ||||||
Medium |
| 500 | 430 | 443 | 437 | 0.87 | 555213 | 483035 |
control |
| 500 | 385 | 405 | 395 | 0.79 | 555693 | 438997 |
DMSO | 1% | 500 | 470 | 514 | 492 | 0.98 | 555899 | 544781 |
(solvent |
| 500 | 423 | 422 | 423 | 0.85 | 555144 | 471872 |
control) |
|
|
|
|
|
|
|
|
Fluo- | 50 µg/mL | 500 | 393 | 400 | 397 | 0.79 | 552215 | 436250 |
cortolone |
| 500 | 417 | 444 | 431 | 0.86 | 553029 | 475605 |
| 150 µg/mL | 500 | 439 | 411 | 425 | 0.85 | 553574 | 470538 |
|
| 500 | 384 | 392 | 388 | 0.78 | 553914 | 432053 |
| 300 µg/mL | 500 | 419 | 397 | 408 | 0.82 | 552758 | 453262 |
|
| 500 | 384 | 434 | 409 | 0.82 | 553846 | 454154 |
| 350 µg/mL | 500 | 359 | 396 | 378 | 0.76 | 552826 | 420148 |
|
| 500 | 292 | 320 | 306 | 0.61 | 555830 | · 339056 |
| 400 µg/mL | t | -- | -- | -- | -- | -- | -- |
|
| t | -- | -- | -- | -- | -- | -- |
EMS (positive | 0.7 µg/mL | 500 C | 394 -- | 406 -- | 400 -- | 0.80 -- | 548981 -- | 439185 -- |
control) |
|
|
|
|
|
|
|
|
(1) only colonies with more than 50 cells 6 days after seeding in normal medium were scored
(2) factor calculated (value column 5/value column 2)
(3) cells seeded in TG medium (selective medium)
(4) cells surviving after plating in TG medium (value column 7 x value column 6)
t) toxic
C) contaminated
Table 9:Mutagenicity data (part 2); HPRT-test II without S9 mix
Column | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | |
Test compound | Concentration | Number of mutant cells per flask ① found after plating in TG medium | Standard deviation | Mutant colonies② per 106 cells | ||||||
I | II | III | IV | V | mean | individual | mean | |||
Medium control |
|
1 0 | 3 1 | 3 0 |
2 1 |
2 4 |
2.2 1.2 |
0.8 1.6 |
5 3 |
4 |
DMSO (solvent | 1% | 1 3 | 0 1 | 2 2 | 2 1 | 1 3 | 1.2 2.0 | 0.8 1.0 | 2 4 |
3 |
control) |
|
|
|
|
|
|
|
|
|
|
Fluo- | 50 µg/mL | 2 | 1 | 0 | 2 | 5 | 2.0 | 1.9 | 5 |
|
cortolone |
| 1 | 2 | 6 | 0 | 2 | 2.2 | 2.3 | 5 | 5 |
| 150 µg/mL | 1 | 2 | 2 | 0 | 5 | 2.0 | 1.9 | 4 |
|
|
| 1 | 2 | 2 | 1 | 3 | 1.8 | 0.8 | 4 | 4 |
| 300 µg/mL | 3 | 3 | 4 | 2 | 2 | 2.8 | 0.8 | 6 |
|
|
| 4 | 2 | 2 | 3 | 1 | 2.4 | 1.1 | 5 | 6 |
| 350 µg/mL | 3 | 2 | 5 | 5 | 4 | 3.8 | 1.3 | 9 |
|
|
| 4 | 2 | 2 | 1 | 0 | 1.8 | 1.5 | 5 | 7 |
| 400 µg/mL | t t | -- -- | -- -- | -- -- | -- -- | -- -- | -- -- | -- -- |
-- |
EMS (positive | 0.7 µg/mL | 197 C | 182 -- | 167 -- | 197 -- | 201 -- | 188.8 -- |
14.2 -- | 430 -- |
430 |
control) |
|
|
|
|
|
|
|
|
|
|
① only colonies with more than 50 cells 10 days after seeding in TG medium were scored
② value column 7 (Table 9) x 106/value column 8 (Table 8)
t) toxic
C) contaminated
Table 10: Toxicity data; HPRT-test II with S9 mix
Column |
1 |
2 |
3 |
4 |
5 | 6 ② | 7③ | 8④ | 9⑤ |
Test compound |
Concentration | Number of cells per flask ① | PE%
absolute | PE%
relative | Population growth absolute | Population growth % relative | |||
seeded I/II | found I II | mean | |||||||
DMSO |
1% |
500 | 490 | 451 | 471 | 94 |
100 | 2771820 | 100 |
{solvent |
| 500 | 438 | 472 | 455 | 91 | 100 | 3163320 | 100 |
control) |
|
|
|
|
|
|
|
|
|
Fluo- | 50 µg/mL | 500 | 553 | 457 | 505 | 101 | 107 | 3633120 | 131 |
cortolone |
| 500 | 481 | 449 | 465 | 93 | 102 | 3789720 | 120 |
| 150 µg/mL | 500 | 457 | 426 | 442 | 88 | 94 | 3022380 | 109 |
|
| 500 | 544 | 443 | 494 | 99 | 109 | 3695760 | 117 |
| 250 µg/mL | 500 | 414 | 473 | 444 | 89 | 94 | 2834460 | 102 |
|
| 500 | 423 | 438 | 431 | 86 | 95 | 3413880 | 108 |
| 300 µg/mL | 500 | 117 | 126 | 122 | 24 | 26 | 2599560 | 94 |
|
| 500 | 130 | 144 | 137 | 27 | 30 | 2176740 | 69 |
| 350 µg/mL | 500 | 1 | 1 | 1 | 0 | 0 | 704700 | 25 |
|
| 500 | 0 | 0 | 0 | 0 | 0 | 767340 | 24 |
| 400 µg/mL | 500 | 0 | 0 | 0 | 0 | 0 | . t | -- |
|
| 500 | 0 | 0 | 0 | 0 | 0 | t | -- |
DMBA |
15 µg/mL | 500 | 42 | 31 | 37 | 7 | 8 | 1988820 | 72 |
(positive |
| 500 | 30 | 33 | 32 | 6 | 7 | 1910520 | 60 |
control) |
|
|
|
|
|
|
|
|
|
(1) only colonies with more than 50 cells 7 days after seeding were scored
(2) PE absolute (value column 5/value column 2 x 100)
(3) PE relative (value column 5/value column 5 of corresponding control x 100)
(4) no. of cells per mL at subculture (3 days after treatment)
(5) % of solvent control (three days after treatment)
t) toxic
Table 11: Mutagenicity data (part 1); HPRT-test II with S9 mix
Column | 1 | 2 | 3 | 4 | 5 | 6② | 7③ | 8④ |
Test compound | Concentration | Number of cells per flask① | Factor calculated | Cells seeded | Cells surviving | |||
seeded I/II | found I II | mean | ||||||
DMSO | 1% | 500 | 410 | 393 | 402 | 0.80 |
555487 |
444390 |
(solvent |
| 500 | 451 | 457 | 454 | 0.91 | 554187 | 504310 |
control) |
|
|
|
|
|
|
|
|
Fluo- |
50 µg/mL |
500 | 406 |
356 |
381 |
0.76 |
554939 |
421754 |
cortolone |
| 500 | 421 | 418 | 420 | 0.84 | 555487 | 466609 |
| 150 µg/mL |
C | -- | -- | -- | -- | -- | -- |
|
| 500 | 448 | 427 | 438 | 0.88 | 554734 | 488166 |
| 250 µg/mL |
C | -- | -- | -- | -- | -- | -- |
|
| 500 | 460 | 452 | 456 | 0.91 | 555418 | 505430 |
| 300 µg/mL | 500 | 407 | 372 | 390 | 0.78 | 554734 | 432693 |
|
| 500 | 507 | 508 | 508 | 1.02 | 552690 | 563744 |
| 350 µg/mL | 500 | 375 | 363 | 369 | 0.74 | 552079 | 408538 |
|
| 500 | 398 | 421 | 410 | 0.82 | 553369 | · 453763 |
| 400 µg/mL | t t | -- -- | -- -- | -- -- | -- -- | -- -- | -- -- |
DMBA (positive | 15 µg/mL |
C 500 | -- 409 | -- 379 | -- 394 | -- 0.79 | -- 552961 | -- 436839 |
control) |
|
|
|
|
|
|
|
|
(1) only colonies with more than 50 cells 6 days after seeding in normal medium were scored
(2) factor calculated (value column 5/value column 2)
(3) cells seeded in TG medium (selective medium)
(4)cells surviving after plating in TG medium (value column 7 x value column 6)
t) toxic
C) contaminated
Table 12: Mutagenicity data (part 2); HPRT-test II with S9 mix
Column | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | |
Test compound | Concentration | Number of mutant cells per flask ①found after plating in TG medium | Standard deviation | Mutant colonies② per 106 cells | ||||||
I | II | III | IV | V | mean | individual | mean | |||
DMSO (solvent control) | 1% | 1 0 | 1 1 | 0 1 | 2 0 | 1 1 | 1.0 0.6 | 0.7 0.5 | 2 1 |
2 |
Fluo- |
50 µg/mL |
6 |
2 |
1 |
6 |
3 |
3.6 |
2.3 |
9 |
|
cortolone |
| 3 | 0 | 0 | 3 | 1 | 1.4 | 1.5 | 3 | 6 |
| 150 µg/mL |
C | -- | -- | - | -- | -- | -- | -- |
|
|
| 1 | 1 | 4 | 3 | 0 | 1.8 | 1.6 | 4 | 4 |
| 250 µg/mL |
C | -- | -- | - | -- | -- | -- | -- |
|
|
| 0 | 1 | 1 | 3 | 0 | 1.0 | 1.2 | 2 | 2 |
| 300 µg/mL | 1 | 1 | 0 | 1 | 1 | 0.8 | 0.4 | 2 |
|
|
| 4 | 3 | 1 | 0 | 3 | 2.2 | 1.6 | 4 | 3 |
| 350 µg/mL | 0 | 0 | 0 | 0 | 0 | 0.0 | 0.0 | 0 |
|
|
| 2 | 1 | 1 | 1 | 0 | 1.0 | 0.7 | 2 | 1 |
| 400 µg/mL | t t | -- -- | -- -- | - - | -- -- | -- -- | -- -- | -- -- |
-- |
DMBA (positive | 15 µg/mL |
C 77 | -- 80 | -- 65 | - 67 | -- 67 | -- 71.2 | -- 6.8 | -- 163 |
163 |
control) |
|
|
|
|
|
|
|
|
|
|
(1) only colonies with more than 50 cells 10 days after seeding in TG medium were scored
(2) value column 7 (Table 12) x 106/value column 8 (Table 11)
t) toxic
C) contaminated
None of the five tester strains showed increased reversion to prototrophy with fluocortolone (ZK 10.445) 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 10.445 did not show a mutagenic effect.
Growth inhibition of the background lawn could be observed on TA 1535, starting at 1 mg/plate, on TA 1537 and TA 1538 starting at 2.5 mg/plate and in the preincubation procedure on TA 100 starting at 0.75 mg/plate.
Precipitates in the ag ar were found partially in the preincubation test at 6.0 mg/plate.
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.
TA 1535 plate incorporation | |||||||
Addition | Amount/plate | with S9 mix | without S9 mix | ||||
individual plate count | mean | SD | individual plate count | mean | SD | ||
DMSO | 0.1 mL | 17 |
|
| 24 |
|
|
|
| 13 | 15 | 2.0 | 16 | 22 | 4.9 |
|
| 15 |
|
| 25 |
|
|
phosphate buffer |
| 15 |
|
| 26 |
|
|
|
| 12 | 16 | 4.2 | 27 | 25 | 3.2 |
|
| 21 |
|
| 21 |
|
|
2-AA | 2.0 µg | 225 |
|
| 23 |
|
|
|
| 191 | 203 | 19.1 | 16 | 18 | 4.0 |
|
| 193 |
|
| 16 |
|
|
CP | 400.0µg | 289 |
|
| 37 |
|
|
|
| 381 | 345 | 49.4 | 43 | 45 | 8.6 |
|
| 366 |
|
| 54 |
|
|
test item | 0.1 mg | 21 |
|
| 14 |
|
|
|
| 18 | 16 | 6.8 | 19 | 22 | 9.3 |
|
| 8 |
|
| 32 |
|
|
| 0.25 mg | 13 |
|
| 26 |
|
|
|
| 11 | 13 | 2.0 | 32 | 29 | 3.1 |
|
| 15 |
|
| 28 |
|
|
| 0.5 mg | 13 |
|
| 26 |
|
|
|
| 21 | 14 | 7.0 | 25 | 25 | 1.5 |
|
| 7 |
|
| 23 |
|
|
| 1.0 mg | 11 |
|
| 24 |
|
|
|
| 12 | 12 | 1.0 | 23 | 221 | 3.2 |
|
| 13 |
|
| 18 |
|
|
| 2.5 mg | 11 |
|
| 7 |
|
|
|
| 11 | 11 | 0.0 | 14 | 91 | 4.4 |
|
| 11 |
|
| 6 |
|
|
| 5.0 mg | 2 |
|
| 1 |
|
|
|
| 4 | 31 | 1.2 | 1 | 11 | 0.6 |
|
| 4 |
|
| 0 |
|
|
cell population: 2E+08per plate; 1 background lawn reduced
| |||||||
TA 100 plate incorporation | |||||||
Addition | Amount/plate | with S9 mix | without S9 mix | ||||
individual plate count | mean | SD | individual plate count | mean | SD | ||
DMSO | 0.1 mL | 106 |
|
| 128 |
|
|
|
| 93 | 97 | 8.1 | 86 | 104 | 21.8 |
|
| 91 |
|
| 97 |
|
|
phosphate buffer | 0.1 mL | 112 |
|
| 148 |
|
|
|
| 85 | 101 | 14.4 | 105 | 127 | 30.4 |
|
| 107 |
|
| - |
|
|
2-AA | 2.0 µg | 1107 |
|
| 90 |
|
|
|
| 1127 | 1160 | 75.7 | 100 | 98 | 6.8 |
|
| 1247 |
|
| 103 |
|
|
BP | 5.0µg | 273 |
|
| 86 |
|
|
|
| 235 | 267 | 29.5 | 107 | 91 | 14.6 |
|
| 293 |
|
| 79 |
|
|
NaN3 | 2.0 µg | 478 |
|
| 718 |
|
|
|
| 476 | 476 | 1.5 | 706 | 711 | 6.4 |
|
| 475 |
|
| 708 |
|
|
test item | 0.25 mg | 85 |
|
| 101 |
|
|
|
| 88 | 85 | 3.0 | 96 | 100 | 4.0 |
|
| 82 |
|
| 104 |
|
|
| 0.5 mg | 103 |
|
| 95 |
|
|
|
| 73 | 88 | 21.2 | 90 | 93 | 1.5 |
|
| - |
|
| - |
|
|
| 1.0 mg | 88 |
|
| 111 |
|
|
|
| 97 | 97 | 8.5 | 102 | 101 | 10.5 |
|
| 105 |
|
| 90 |
|
|
| 2.5 mg | 117 |
|
| 111 |
|
|
|
| 81 | 96 | 18.7 | 115 | 107 | 10.6 |
|
| 90 |
|
| 95 |
|
|
| 5.0 mg | 96 |
|
| 91 |
|
|
|
| 76 | 92 | 14.8 | 77 | 76 | 15.5 |
|
| 105 |
|
| 60 |
|
|
cell population: 3 E+08 per plate; - not tested
| |||||||
TA 100 (b) plate incorporation | |||||||
Addition | Amount/plate | with S9 mix | without S9 mix | ||||
|
| individual plate count | mean | SD | individual plate count | mean | SD |
DMSO | 0.1 mL | 88 |
|
| 89 | 87 | 11.7 |
|
| 80 | 81 | 7.0 | 97 |
|
|
|
| 74 |
|
| 74 |
|
|
phosphate buffer | 0.1 mL | 101 |
|
| 107 | 111 | 3.5 |
|
| 87 | 89 | 10.7 | 111 |
|
|
|
| 80 |
|
| 114 |
|
|
2-AA | 2.0 µg | 1081 |
|
| 87 | 96 | 7.8 |
|
| 1010 | 908 | 240.0 | 100 |
|
|
|
| 634 |
|
| 101 |
|
|
BP | 5.0µg | 298 |
|
| 120 | 110 | 12.3 |
|
| 324 | 297 | 27.5 | 113 |
|
|
|
| 269 |
|
| 96 |
|
|
NaN3 | 2.0 µg | 297 |
|
| 509 | 501 | 31.2 |
|
| 320 | 312 | 12.7 | 528 |
|
|
|
| 318 |
|
| 467 |
|
|
test item | 0.1 mg | 88 |
|
| 110 | 113 | 3.8 |
|
| 96 | 93 | 4.4 | 117 |
|
|
|
| 95 |
|
| 111 |
|
|
| 0.25 mg | 85 |
|
| 96 | 104 | 9.2 |
|
| 78 | 85 | 7.5 | 114 |
|
|
|
| 93 |
|
| 102 |
|
|
| 0.5 mg | 93 |
|
| 92 | 108 | 15.5 |
|
| 76 | 85 | 8.6 | 123 |
|
|
|
| 87 |
|
| 108 |
|
|
| 1.0 mg | 77 |
|
| 133 | 123 | 12.3 |
|
| 90 | 86 | 7.8 | 109 |
|
|
|
| 91 |
|
| 126 |
|
|
| 2.5 mg | 94 |
|
| 134 | 127 | 17.0 |
|
| 104 | 105 | 11.0 | 140 |
|
|
|
| 116 |
|
| 108 |
|
|
| 5.0 mg | 80 |
|
| 100 | 107 | 12.4 |
|
| 63 | 79 | 16.0 | 99 |
|
|
|
| 95 |
|
| 121 |
|
|
cell population: 1E+08 per plate
| |||||||
TA1537 plate incorporation | |||||||
Addition | Amount/plate | with S9 mix | without S9 mix | ||||
individual plate count | mean | SD | individual plate count | mean | SD | ||
DMSO | 0.1 mL | 18 |
|
| 12 |
|
|
|
| 12 | 13 | 4.2 | 7 | 10 | 2.5 |
|
| 10 |
|
| 10 |
|
|
2-AA | 5.0 µg | 152 |
|
| 8 |
|
|
|
| 194 | 188 | 33.4 | 5 | 7 | 1.5 |
|
| 218 |
|
| 7 |
|
|
BP | 10.0 µg | 33 |
|
| 6 |
|
|
|
| 43 | 35 | 6.8 | 10 | 7 | 2.6 |
|
| 30 |
|
| 5 |
|
|
2-NF | 10.0 µg | 80 |
|
| 53 |
|
|
|
| 79 | 87 | 13.6 | 49 | 57 | 10.0 |
|
| 103 |
|
| 68 |
|
|
9-AA | 100.0 µg | - |
|
| 601 |
|
|
|
| - | - | - | 530 | 561 | 36.3 |
|
| - |
|
| 552 |
|
|
test item | 0.1 mg | 11 |
|
| 8 |
|
|
|
| 10 | 12 | 2.1 | 13 | 9 | 3.6 |
|
| 14 |
|
| 6 |
|
|
| 0.25 mg | 13 |
|
| 13 |
|
|
|
| 17 | 14 | 2.6 | 9 | 10 | 2.6 |
|
| 12 |
|
| 8 |
|
|
| 0.5 mg | 15 |
|
| 9 |
|
|
|
| 9 | 14 | 4.2 | 9 | 8 | 2.3 |
|
| 17 |
|
| 5 |
|
|
| 1.0 mg | 12 |
|
| 17 |
|
|
|
| 11 | 13 | 2.1 | 7 | 10 | 6.1 |
|
| 15 |
|
| 6 |
|
|
| 2.5 mg | 9 |
|
| 8 |
|
|
|
| 9 | 9 | 0.6 | 2 | 51 | 3.1 |
|
| 8 |
|
| 4 |
|
|
| 5.0 mg | 1 |
|
| 2 |
|
|
|
| 3 | 3 | 1.5 | 6 | 31 | 2.6 |
|
| 4 |
|
| 1 |
|
|
cell population: 8 E+07 per plate - not tested; 1 background lawn reduced
| |||||||
TA1538 plate incorporation | |||||||
Addition | Amount/plate | with S9 mix | without S9 mix | ||||
individual plate count | mean | SD | individual plate count | mean | SD | ||
DMSO | 0.1 mL | 26 |
|
| 11 |
|
|
|
| 31 | 32 | 6.6 | 23 | 17 | 6.0 |
|
| 39 |
|
| 18 |
|
|
2-AA | 5.0 µg | 1839 |
|
| 34 |
|
|
|
| 1561 | 1630 | 184.0 | 23 | 30 | 6.4 |
|
| 1491 |
|
| 34 |
|
|
BP | 10.0 µg | 99 |
|
| 19 |
|
|
|
| 109 | 99 | 10.5 | 20 | 17 | 4.4 |
|
| 88 |
|
| 12 |
|
|
2-NF | 10.0 µg | 448 |
|
| 1166 |
|
|
|
| 421 | 434 | 13.5 | 1306 | 1239 | 70.2 |
|
| 434 |
|
| 1245 |
|
|
test item | 0.1 mg | 29 |
|
| 17 |
|
|
|
| 30 | 33 | 6.1 | 13 | 15 | 2.1 |
|
| 40 |
|
| 14 |
|
|
| 0.25 mg | 42 |
|
| 18 |
|
|
|
| 21 | 33 | 10.8 | 11 | 15 | 3.5 |
|
| 36 |
|
| 15 |
|
|
| 0.5 mg | 38 |
|
| 11 |
|
|
|
| 35 | 37 | 1.7 | 11 | 11 | 0.6 |
|
| 38 |
|
| 12 |
|
|
| 1.0 mg | 31 |
|
| 14 |
|
|
|
| 29 | 30 | 1.2 | 16 | 16 | 2.0 |
|
| 29 |
|
| 18 |
|
|
| 2.5 mg | 32 |
|
| 8 |
|
|
|
| 33 | 34 | 2.6 | 8 | 81 | 0.6 |
|
| 37 |
|
| 9 |
|
|
| 5.0 mg | 31 |
|
| 7 |
|
|
|
| 16 | 25 | 8.1 | 4 | 61 | 2.1 |
|
| 29 |
|
| 8 |
|
|
cell population: 8E+07 per plate 1 background lawn reduced
| |||||||
TA 98 plate incorporation | |||||||
Addition | Amount/plate | with S9 mix | without S9 mix | ||||
individual plate count | mean | SD | individual plate count | mean | SD | ||
DMSO | 0.1 mL | 44 |
|
| 31 |
|
|
|
| 35 | 38 | 5.2 | 18 | 29 | 9.7 |
|
| 35 |
|
| 37 |
|
|
2-AA | 5.0 µg | 1007 |
|
| 21 |
|
|
|
| 900 | 956 | 53.7 | 33 | 27 | 6.0 |
|
| 962 |
|
| 27 |
|
|
BP | 10.0 µg | 129 |
|
| 22 |
|
|
|
| 122 | 124 | 4.0 | 26 | 28 | 6.7 |
|
| 122 |
|
| 35 |
|
|
2-NF | 10.0 µg | 99 |
|
| 180 |
|
|
|
| 112 | 100 | 11.5 | 205 | 196 | 13.7 |
|
| 89 |
|
| 202 |
|
|
test item | 0.1 mg | 38 |
|
| 33 |
|
|
|
| 37 | 37 | 1.5 | 25 | 28 | 4.2 |
|
| 35 |
|
| 27 |
|
|
| 0.25 mg | 44 |
|
| 24 |
|
|
|
| 41 | 39 | 6.2 | 21 | 23 | 2.1 |
|
| 32 |
|
| 25 |
|
|
| 0.5 mg | 43 |
|
| 20 |
|
|
|
| 33 | 38 | 5.0 | 26 | 22 | 3.2 |
|
| 37 |
|
| 21 |
|
|
| 1.0 mg | 45 |
|
| 24 |
|
|
|
| 32 | 37 | 7.2 | 13 | 21 | 7.4 |
|
| 33 |
|
| 27 |
|
|
| 2.5 mg | 40 |
|
| 24 |
|
|
|
| 39 | 38 | 2.6 | 25 | 24 | 0.6 |
|
| 35 |
|
| 24 |
|
|
| 5.0 mg | 36 |
|
| 37 |
|
|
|
| 46 | 44 | 6.8 | 24 | 27 | 8.5 |
|
| 49 |
|
| 21 |
|
|
cell population: 2 E+08 per plate |
TA 100 preincubation (c) | ||||||||||
Addition | Amount/plate | with 10% S9 mix | With 30% S9 mix | without S9 mix | ||||||
individual plate count | mean | SD | individual plate count | mean | SD | individual plate count | mean | SD | ||
DMSO | 0.1 mL | 76 |
|
| 84 |
|
| 95 |
|
|
|
| 75 | 77 | 3.2 | 79 | 84 | 4.5 | 79 | 84 | 9.9 |
|
| 81 |
|
| 88 |
|
| 77 |
|
|
phosphate buffer | 0.1 mL | 96 |
|
| 102 |
|
| 95 |
|
|
|
| 100 | 97 | 2.6 | 123 | 111 | 11.0 | 89 | 96 | 7.5 |
|
| 95 |
|
| 107 |
|
| 104 |
|
|
2-AA | 2.0 µg | 545 |
|
| 263 |
|
| 106 |
|
|
|
| 522 | 563 | 52.4 | 262 | 268 | 9.5 | 101 | 100 | 6.6 |
|
| 622 |
|
| 279 |
|
| 93 |
|
|
NaN3 | 2.0 µg | 398 |
|
| 183 |
|
| 615 |
|
|
|
| 419 | 409 | 10.6 | 199 | 189 | 8.7 | 572 | 594 | 21.5 |
|
| 411 |
|
| 185 |
|
| 595 |
|
|
N-nitroso-dimethylamine | 5 µL | 167 |
|
| 1215 |
|
| 119 |
|
|
|
| 190 | 174 | 14.2 | 1197 | 1246 | 70.0 | 109 | 106 | 15.3 |
|
| 164 |
|
| 1326 |
|
| 89 |
|
|
test item | 0.1 mg | 120 |
|
| 90 |
|
| 100 |
|
|
|
| 94 | 102 | 15.3 | 76 | 88 | 11.1 | 83 | 87 | 11.2 |
|
| 93 |
|
| 98 |
|
| 79 |
|
|
| 0.25 mg | 95 |
|
| 96 |
|
| 102 |
|
|
|
| 90 | 91 | 3.6 | 77 | 86 | 9.5 | 93 | 97 | 4.6 |
|
| 88 |
|
| 86 |
|
| 96 |
|
|
| 0.5 mg | 84 |
|
| 94 |
|
| 71 |
|
|
|
| 88 | 88 | 4.5 | 98 | 90 | 10.0 | 76 | 83 | 17.1 |
|
| 93 |
|
| 79 |
|
| 103 |
|
|
| 0.75 mg | 99 |
|
| 85 |
|
| 66 |
|
|
|
| 68 | 84 | 15.6 | 94 | 89 | 4.7 | 74 | 651 | 9.5 |
|
| 86 |
|
| 87 |
|
| 55 |
|
|
| 1.0 mg | 76 |
|
| 96 |
|
| 51 |
|
|
|
| 94 | 87 | 9.6 | 101 | 104 | 9.8 | 44 | 481 | 4.9 |
|
| 91 |
|
| 115 |
|
| - |
|
|
| 2.0 mg | 60 |
|
| 103 |
|
|
|
|
|
|
| 95 | 741 | 18.3 | 67 | 881 | 18.7 |
|
|
|
|
| 68 |
|
| 94 |
|
|
|
|
|
| 4.0 mg | 37 |
|
| 58 |
|
|
|
|
|
|
| 50 | 441 | 6.5 | 66 | 631 | 4.2 |
|
|
|
|
| 44 |
|
| 64 |
|
|
|
|
|
| 6.0 mg | 20p |
|
| 21 |
|
|
|
|
|
|
| 26p | 221 | 3.8 | 24 | 341 |
|
|
|
|
|
| 19p |
|
| 56 |
|
|
|
|
|
cell population: 1 E+08 per plate -: plate contaminated 1: background lawn reduced p: precipitates |
None of the six tester strains showed increased reversion to prototrophy with the test item (fluocortolone containing 5% ZK 344937 as impurity) at the doses tested in the two tests (direct plate incorporation procedure and preincubation modification), either in the absence or presence of S9 mix.
Precipitates in the agar were observed at the maximum dose of 5.0 mg per plate.
In the direct plate incorporation assay with Salmonella typhimurium growth inhibition of the background lawn was observed from 1.0 mg per plate onwards without or with metabolic activation. When using the preincubation modification growth inhibition of the background lawn was observed in the experiments with Salmonella typhimurium from 0.1 mg per plate onwards without metabolic activation and from 1.0 mg per plate onwards with metabolic activation.
No growth inhibition ofthe background lawn was observed in the experiments with Escherichia coli WP2uvrA.
Negative controls and positive controls with known mutagens (anthracene-2-amine, benzo[a]pyrene, cyclophosphamide, ethyl methanesulfonate, N-methyl-N' -nitro-N-nitrosoguanidine, 2-nitro-9H-fluorene, 4-nitro-o-phenylenediamine, sodium azide, N-nitrosodimethylamine) produced the expected distinct increase in the number of revertant colonies.
Direct plate incorporation test on Salmonella typhimurium strain TA98 | |||||||||
|
| -S9 | M | +/-SD | +S9 | M | +/-SD | -S9 | +S9 |
DMSO | 50 µL | 17 | 19 | 2 | 33 | 34 | 2 | 1.0 | 1.0 |
|
| 19 |
|
| 36 |
|
|
|
|
|
| 21 |
|
| 32 |
|
|
|
|
Phosphate buffer | 50 µL | 19 | 21 | 2 | 40 | 40 | 7 | 1.1 | 1.2 |
|
| 22 |
|
| 47 |
|
|
|
|
|
| 22 |
|
| 34 |
|
|
|
|
ZK10445/ZK344937 | 0.1 mg | 15 | 13 | 3 | 41 | 33 | 7 | 0.7 | 1.0 |
|
| 10 |
|
| 31 |
|
|
|
|
|
| 15 |
|
| 28 |
|
|
|
|
ZK10445/ZK344937 | 0.25 mg | 17 | 16 |
| 31 | 38 | 6 | 0.8 | 1.1 |
|
| 15 |
|
| 42 |
|
|
|
|
|
| 15 |
|
| 42 |
|
|
|
|
ZK10445/ZK344937 | 0.5 mg | 7 | 9 | 3 | 42 | 39 | 5 | 0.5 | 1.1 |
|
| 9 |
|
| 33 |
|
|
|
|
|
| 12 |
|
| 41 |
|
|
|
|
ZK10445/ZK34493 7 | 1.0 mg | 10 B | 11 | 3 | 29 | 34 | 5 | 0.6 | 1.0 |
|
| 14 B |
|
| 38 |
|
|
|
|
|
| 8 B |
|
| 36 |
|
|
|
|
ZK10445/ZK344937 | 2.5 mg | 7 B | 8 |
| 33 B | 37 | 4 | 0.4 | 1.1 |
|
| 8 B |
|
| 36 B |
|
|
|
|
|
| 8 B |
|
| 41 B |
|
|
|
|
ZK10445/ZK34493 7 | 5.0 mg | 1 mPB |
|
| 45 mPB | 40 | 5 | 0.0 | 1.2 |
|
| 1 mPB |
|
| 38 mPB |
|
|
|
|
|
| 0 mPB |
|
| 36 mPB |
|
|
|
|
Anthracene-2-amine | 2.5 µg | 12 | 12 | 5 | 709 | 778 | 60 | 0.6 | 23.1 |
|
| 8 |
|
| 822 |
|
|
|
|
|
| 17 |
|
| 802 |
|
|
|
|
Benzo[a]pyrene | 2.5 µg | 8 | 9 |
| 243 | 243 | 6 | 0.5 | 7.2 |
|
| 10 |
|
| 238 |
|
|
|
|
|
| 8 |
|
| 249 |
|
|
|
|
2-Nitrofluorene | 10.0 µg | 731 | 721 | 21 | 229 | 244 | 13 | 37.9 | 7.2 |
|
| 697 |
|
| 251 |
|
|
|
|
|
| 735 |
|
| 251 |
|
|
|
|
|
|
|
|
|
|
|
|
|
|
Direct plate incorporation test on Salmonella typhimurium strain TA 100 | |||||||||
|
|
|
|
|
|
|
|
|
|
|
| -S9 | M | +/-SD | +S9 | M | +/-SD | -S9 | +S9 |
|
|
|
|
|
|
|
|
|
|
DMSO | 50 µL | 100 | 103 | 4 | 92 | 94 | 3 | 1.0 | 1.0 |
|
| 101 |
|
| 92 |
|
|
|
|
|
| 108 |
|
| 98 |
|
|
|
|
Phosphate buffer | 50µL | 101 | 107 | 6 | 90 | 95 | 5 | 1.0 | 1.0 |
|
| 109 |
|
| 96 |
|
|
|
|
|
| 112 |
|
| 99 |
|
|
|
|
ZK10445/ZK344937 | 0.1mg | 100 | 87 | 11 | 86 | 89 | 2 | 0.8 | 0.9 |
|
| 81 |
|
| 90 |
|
|
|
|
|
| 80 |
|
| 90 |
|
|
|
|
ZK10445/ZK344937 | 0.25 mg | 82 | 88 | 8 | 88 | 85 | 16 | 0.9 | 0.9 |
|
| 84 |
|
| 100 |
|
|
|
|
|
| 97 |
|
| 68 |
|
|
|
|
ZK10445/ZK344937 | 0.5mg | 100 | 87 | 15 | 87 | 79 | 7 | 0.8 | 0.8 |
|
| 71 |
|
| 76 |
|
|
|
|
|
| 89 |
|
| 73 |
|
|
|
|
ZK10445/ZK344937 | 1.0mg | 82 | 86 | 6 | 98 | 85 | 11 | 0.8 | 0.9 |
|
| 93 |
|
| 82 |
|
|
|
|
|
| 83 |
|
| 76 |
|
|
|
|
ZK10445/ZK344937 | 2.5mg | 80B | 88 | 12 | 106B | 94 | 12 | 0.9 | 1.0 |
|
| 102B |
|
| 94B |
|
|
|
|
|
| 83B |
|
| 83B |
|
|
|
|
ZK10445/ZK344937 | 5.0mg | 28 mPB | 25 | 4 | 47 mB | 46 | 8 | 0.2 | 0.5 |
|
| 21 mPB |
|
| 38 mPB |
|
|
|
|
|
| 25 mPB |
|
| 53 mPB |
|
|
|
|
Anthracene-2-amine | 2.5µg | 112 | 114 | 10 | 939 | 953 | 38 | 1.1 | 10.1 |
|
| 105 |
|
| 996 |
|
|
|
|
|
| 125 |
|
| 924 |
|
|
|
|
Benzo[a]pyrene | 2.5µg | 83 | 83 | 0 | 478 | 445 | 29 | 0.8 | 4.7 |
|
| 83 |
|
| 425 |
|
|
|
|
|
| 83 |
|
| 433 |
|
|
|
|
Sodium azide | 5.0µg | 437 | 453 | 27 | 164 | 158 | 5 | 4.4 | 1.7 |
|
| 485 |
|
| 154 |
|
|
|
|
|
| 438 |
|
| 157 |
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
Direct plate incorporation test on Salmonella typhimurium strain TA1535 | |||||||||
|
| -S9 | M | +/-SD | +S9 | M | +/-SD | -S9 | +S9 |
|
|
|
|
|
|
|
|
|
|
DMSO | 50 µL | 15 | 17 | 3 | 16 | 14 | 3 | 1.0 | 1.0 |
|
| 21 |
|
| 14 |
|
|
|
|
|
| 15 |
|
| 11 |
|
|
|
|
Phosphate buffer | 50 µL | 21 | 19 | 4 | 12 | 16 | 3 | 1.1 | 1.2 |
|
| 21 |
|
| 18 |
|
|
|
|
|
| 14 |
|
| 18 |
|
|
|
|
ZK10445/ZK344937 | 0.1mg | 26 | 22 | 5 | 11 | 12 | 5 | 1.3 | 0.9 |
|
| 24 |
|
| 8 |
|
|
|
|
|
| 16 |
|
| 18 |
|
|
|
|
ZK10445/ZK344937 | 0.25mg | 21 | 19 | 4 | 9 | 10 | 3 | 1.1 | 0.8 |
|
| 15 |
|
| 8 |
|
|
|
|
|
| 22 |
|
| 14 |
|
|
|
|
ZK10445/ZK344937 | 0.5mg | 16 | 20 | 3 | 9 | 11 | 2 | 1.2 | 0.8 |
|
| 22 |
|
| 12 |
|
|
|
|
|
| 22 |
|
| 13 |
|
|
|
|
ZK10445/ZK344937 | 1.0mg | 23 | 22 | 3 | 6 | 11 | 4 | 1.3 | 0.8 |
|
| 25 |
|
| 13 |
|
|
|
|
|
| 19 |
|
| 13 |
|
|
|
|
ZK10445/ZK344937 | 2.5mg | 15 | 15 | 2 | 14 | 12 | 2 | 0.9 | 0.9 |
|
| 17 |
|
| 12 |
|
|
|
|
|
| 13 |
|
| 11 |
|
|
|
|
ZK10445/ZK344937 | 5.0mg | 10 mPB | 13 | 3 | 11 mPB | 10 |
| 0.7 | 0.7 |
|
| 13 mPB |
|
| 9 mPB |
|
|
|
|
|
| 15 mPB |
|
| 9 mPB |
|
|
|
|
Anthracene-2-amine | 2.5µg | 15 | 19 | 4 | 113 | 107 | 8 | 1.1 | 7.8 |
|
| 20 |
|
| 98 |
|
|
|
|
|
| 23 |
|
| 110 |
|
|
|
|
Cyclophosphamide | 400.0 µg | 36 | 35 | 7 | 364 | 365 |
| 2.0 | 26.7 |
|
| 41 |
|
| 366 |
|
|
|
|
|
| 27 |
|
| 364 |
|
|
|
|
Sodium azide | 5.0µg | 505 | 498 | 22 | 114 | 133 | 21 | 29.3 | 9.7 |
|
| 515 |
|
| 156 |
|
|
|
|
|
| 473 |
|
| 129 |
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
Direct plate incorporation test on Salmonella typhimurium strain TA1538 | |||||||||
|
| -S9 | M | +/-SD | +S9 | M | +/-SD | -S9 | +S9 |
|
|
|
|
|
|
|
|
|
|
DMSO | 50 µL | 9 | 12 | 3 | 26 | 23 | 5 | 1.0 | 1.0 |
|
| 12 |
|
| 17 |
|
|
|
|
|
| 14 |
|
| 26 |
|
|
|
|
Phosphate buffer | 50 µL | 8 | 8 |
| 27 | 28 | 2 | 0.7 | 1.2 |
|
| 9 |
|
| 27 |
|
|
|
|
|
| 7 |
|
| 31 |
|
|
|
|
ZK10445/ZK344937 | 0.1mg | 11 | 8 | 3 | 17 | 22 | 7 | 0.7 | 1.0 |
|
| 5 |
|
| 30 |
|
|
|
|
|
| 9 |
|
| 19 |
|
|
|
|
ZK10445/ZK344937 | 0.25mg | 8 | 7 |
| 22 | 18 | 6 | 0.6 | 0.8 |
|
| 7 |
|
| 22 |
|
|
|
|
|
| 7 |
|
| 11 |
|
|
|
|
ZK10445/ZK344937 | 0.5mg | 8 | 7 | 2 | 17 | 18 |
| 0.6 | 0.8 |
|
| 8 |
|
| 19 |
|
|
|
|
|
| 4 |
|
| 18 |
|
|
|
|
ZK10445/ZK344937 | 1.0mg | 8 | 7 | 2 | 25 | 25 |
| 0.6 | 1.1 |
|
| 9 |
|
| 25 |
|
|
|
|
|
| 5 |
|
| 26 |
|
|
|
|
ZK10445/ZK344937 | 2.5mg | 2 mB | 2 |
| 27 | 24 | 3 | 0.2 | 1.0 |
|
| 2 mB |
|
| 21 |
|
|
|
|
|
| 3 mB |
|
| 23 |
|
|
|
|
ZK10445/ZK344937 | 5.0mg | 0 mPB | 0 | 0 | 13 mPB | 16 | 4 | 0.0 | 0.7 |
|
| 0 mPB |
|
| 20 mPB |
|
|
|
|
|
| 0 mPB |
|
| 15 mPB |
|
|
|
|
Anthracene-2-amine | 2.5µg | 4 | 7 | 4 | 615 | 621 | 19 | 0.6 | 27.0 |
|
| 6 |
|
| 606 |
|
|
|
|
|
| 12 |
|
| 643 |
|
|
|
|
2-Nitrofluorene | 10.0µg | 811 | 735 | 101 | 312 | 304 | 8 | 63.0 | 13.2 |
|
| 620 |
|
| 296 |
|
|
|
|
|
| 774 |
|
| 304 |
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
Direct plate incorporation test on Salmonella typhimurium strain TA1537 | |||||||||
|
| -S9 | M | +/-SD | +S9 | M | +/-SD | -S9 | +S9 |
|
|
|
|
|
|
|
|
|
|
DMSO | 50 µL | 6 | 9 | 3 | 10 | 12 | 2 | 1.0 | 1.0 |
|
| 9 |
|
| 14 |
|
|
|
|
|
| 11 |
|
| 12 |
|
|
|
|
Phosphate buffer | 50 µL | 8 | 8 |
| 14 | 11 | 3 | 1.0 | 0.9 |
|
| 9 |
|
| 9 |
|
|
|
|
|
| 8 |
|
| 11 |
|
|
|
|
ZK1044S/ZK344937 | 0.1mg | 6 | 8 | 2 | 10 | 11 | 4 | 0.9 | 0.9 |
|
| 9 |
|
| 8 |
|
|
|
|
|
| 9 |
|
| 15 |
|
|
|
|
ZK10445/ZK344937 | 0.25mg | 8 | 8 | 3 | 7 | 11 | 4 | 0.9 | 0.9 |
|
| 5 |
|
| 15 |
|
|
|
|
|
| 10 |
|
| 12 |
|
|
|
|
ZK! 0445/ZK344937 | 0.5mg | 5 | 6 | 3 | 8 | 8 |
| 0.7 | 0.7 |
|
| 9 |
|
| 9 |
|
|
|
|
|
| 4 |
|
| 7 |
|
|
|
|
ZK! 044S/ZK344937 | 1.0mg | 5B | 5 |
| 8B | 9 | 2 | 0.6 | 0.8 |
|
| 6B |
|
| 8B |
|
|
|
|
|
| 4B |
|
| 11B |
|
|
|
|
ZKI 0445/ZK344937 | 2.5mg | 1 mB | 1 |
| 4 mB | 5 | 1 | 0.1 | 0.4 |
|
| 1 mB |
|
| 5 mB |
|
|
|
|
|
| 0 mB |
|
| 5 mB |
|
|
|
|
ZK10445/ZK344937 | 5.0mg | 0 mPB | 0 | 0 | 5 mPB | 3 | 2 | 0.0 | 0.2 |
|
| 0 mPB |
|
| 1 mPB |
|
|
|
|
|
| 0 mPB |
|
| 2 mPB |
|
|
|
|
Anthracene-2-amine | 2.5mg | 8 | 7 |
| 101 | 98 | 8 | 0.8 | 8.1 |
|
| 7 |
|
| 103 |
|
|
|
|
|
| 7 |
|
| 89 |
|
|
|
|
4-NPDA | 10.0 µg | 63 | 66 | 7 | 17 | 21 | 5 | 7.6 | 1.8 |
|
| 74 |
|
| 26 |
|
|
|
|
|
| 60 |
|
| 20 |
|
|
|
|
|
|
|
|
|
|
|
|
|
|
| |||||||||
Direct plate incorporation assay E.coli WP2uvrA |
| -S9 | M | +/-SD | +S9 | M | +/-SD | -S9 | +S9 |
|
|
|
|
|
|
|
|
|
|
DMSO | 50 µL | 19 | 22 | 3 | 29 | 31 | 3 | 1.0 | 1.0 |
|
| 23 |
|
| 34 |
|
|
|
|
|
| 25 |
|
| 29 |
|
|
|
|
Phosphate buffer | 50 µL | 22 | 25 | 4 | 32 | 33 | 2 | 1.1 | 1.1 |
|
| 30 |
|
| 31 |
|
|
|
|
|
| 23 |
|
| 35 |
|
|
|
|
ZK10445/ZK344937 | 0.1mg | 25 | 21 | 4 | 21 | 28 | 8 | 0.9 | 0.9 |
|
| 20 |
|
| 27 |
|
|
|
|
|
| 18 |
|
| 36 |
|
|
|
|
ZKl 0445/ZK344937 | 0.25 mg | 23 | 25 | 3 | 31 | 29 | 2 | 1.1 | 1.0 |
|
| 29 |
|
| 27 |
|
|
|
|
|
| 24 |
|
| 30 |
|
|
|
|
ZKl 0445/ZK344937 | 0.5mg | 18 | 18 | 5 | 27 | 32 | 6 | 0.8 | 1.0 |
|
| 23 |
|
| 31 |
|
|
|
|
|
| 14 |
|
| 38 |
|
|
|
|
ZKl 0445/ZK344937 | 1.0mg | 29 | 24 | 5 | 20 | 28 | 7 | 1.1 | 0.9 |
|
| 20 |
|
| 29 |
|
|
|
|
|
| 22 |
|
| 34 |
|
|
|
|
ZK l 0445/ZK344937 | 2.5mg | 23 | 20 | 3 | 38 | 33 | 4 | 0.9 | 1.1 |
|
| 21 |
|
| 31 |
|
|
|
|
|
| 17 |
|
| 30 |
|
|
|
|
ZKl 0445/ZK344937 | 5.0mg | 15 mP | 16 | 2 | 26 mP | 25 | 2 | 0.7 | 0.8 |
|
| 15 m P |
|
| 23 mP |
|
|
|
|
|
| 18 mP |
|
| 2S mP |
|
|
|
|
Anthracene-2-amine | 10.0 µg | 25 | 23 | 2 | 155 | 192 | 34 | 1.0 | 6.3 |
|
| 21 |
|
| 200 |
|
|
|
|
|
| 22 |
|
| 221 |
|
|
|
|
EMS | 5.0µL | 612 | 572 | 75 | 490 | 553 | 62 | 25.6 | 18.0 |
|
| 619 |
|
| 614 |
|
|
|
|
|
| 48S |
|
| 555 |
|
|
|
|
MNNG | 5.0µg | 289 | 268 | 33 | 222 | 238 | 23 | 12.0 | 7.8 |
|
| 285 |
|
| 229 |
|
|
|
|
|
| 230 |
|
| 264 |
|
|
|
|
|
|
|
|
|
|
|
|
|
|
Preincubation test on Salmonella typhimurium strain T A98 | |||||||||
|
| -S9 | M | +/-SD | +S9 | M | +/-SD | -S9 | +S9 |
|
|
|
|
|
|
|
|
|
|
DMSO | 50 µL | 14 | 14 | 0 | 47 | 40 | 8 | 1.0 | 1.0 |
|
| 14 |
|
| 41 |
|
|
|
|
|
| 14 |
|
| 31 |
|
|
|
|
Phosphate buffer | 50 µL | 10 | 13 | 3 | 34 | 44 | 10 | 1.0 | 1.1 |
|
| 16 |
|
| 54 |
|
|
|
|
|
| 14 |
|
| 44 |
|
|
|
|
ZK!0445/ZK344937 | 0.1mg | 14 | 12 | 2 | 32 | 36 | 5 | 0.9 | 0.9 |
|
| 12 |
|
| 36 |
|
|
|
|
|
| 11 |
|
| 41 |
|
|
|
|
ZKl 0445/ZK344937 | 0.25mg | 11 | 10 | 2 | 37 | 33 | 4 | 0.7 | 0.8 |
|
| 7 |
|
| 30 |
|
|
|
|
|
| 11 |
|
| 33 |
|
|
|
|
ZK10445/ZK344937 | 0.5mg | 3mB | 5 | 2 | 29 | 35 | 5 | 0.4 | 0.9 |
|
| 7 mB |
|
| 37 |
|
|
|
|
|
| 5 mB |
|
| 39 |
|
|
|
|
ZK10445/ZK344937 | 1.0mg | 1022) B1) | 78 | 28 | 20B | 22 | 2 | 5.6 | 0.5 |
|
| 482) B1) |
|
| 24B |
|
|
|
|
|
| 852) B1) |
|
| 21B |
|
|
|
|
ZK10445/ZK344937 | 2.5mg | 782) B1) | 29 | 43 | 18B | 19 | 3 | 2.0 | 0.5 |
|
| 6 B1) |
|
| 17B |
|
|
|
|
|
| 2 B1) |
|
| 23B |
|
|
|
|
ZK10445/ZK344937 | 5.0mg | 1mPB1) | 1 | 1 | 15mPB | 17 | 9 | 0.0 | 0.4 |
|
| 0 mPB1) |
|
| 27mPB |
|
|
|
|
|
| 1 mPB1) |
|
| 10mPB |
|
|
|
|
Anthracene-2-amine | 2.5µg | 13 | 13 | 4 | 244 | 245 | 13 | 1.0 | 6.2 |
|
| 17 |
|
| 259 |
|
|
|
|
|
| 10 |
|
| 233 |
|
|
|
|
Anthracene-2-amine | 3.5µg | 22 | 23 |
| 326 | 376 | 57 | 1.6 | 9.5 |
|
| 22 |
|
| 438 |
|
|
|
|
|
| 24 |
|
| 365 |
|
|
|
|
Benzo[a]pyrene | 2.5µg | 5 | 9 | 7 | 166 | 153 | 18 | 0.7 | 3.9 |
|
| 6 |
|
| 161 |
|
|
|
|
|
| 17 |
|
| 132 |
|
|
|
|
2-Nitrofluorene | 10.0 µg | 798 | 799 | 25 | 245 | 217 | 30 | 57.0 | 5.5 |
|
| 774 |
|
| 221 |
|
|
|
|
|
| 824 |
|
| 185 |
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
Preincubation test on Salmonella typhimurium strain TA98 (a) | |||||||||
|
| -S9 | M | +/-SD | +S9 | M | +/-SD | -S9 | +S9 |
DMSO | 50 µL | 16 | 18 | 3 |
| / | / | 1.0 | / |
|
| 22 |
|
|
|
|
|
|
|
|
| 16 |
|
|
|
|
|
|
|
Phosphate buffer | 50 µL | 20 | 23 | 3 |
| / | / | 1.3 | / |
|
| 25 |
|
|
|
|
|
|
|
|
| 23 |
|
|
|
|
|
|
|
ZK!0445/ZK344937 | 0.05mg | 23 | 18 | 5 |
| / | / | 1.0 | / |
|
| 14 |
|
|
|
|
|
|
|
|
| 17 |
|
|
|
|
|
|
|
ZKl 0445/ZK344937 | 0.10mg | 10 | 13 | 3 |
| / | / | 0.7 | / |
|
| 15 |
|
|
|
|
|
|
|
|
| 15 |
|
|
|
|
|
|
|
ZK!0445/ZK344937 | 0.25mg | 20 | 15 | 5 |
| / | / | 0.8 | / |
|
| 14 |
|
|
|
|
|
|
|
|
| 11 |
|
|
|
|
|
|
|
ZK10445/ZK344937 | 0.5 mg | 4mB | 2 | 2 |
| / | / | 0.1 | / |
|
| 1mB |
|
|
|
|
|
|
|
|
| 2mB |
|
|
|
|
|
|
|
ZK10445/ZK344937 | 0.75mg | 1mB | 1 | 2 |
| / | / | 0.1 | / |
|
| 0mB |
|
|
|
|
|
|
|
|
| 3mB |
|
|
|
|
|
|
|
ZK10445/ZK344937 | 1.0mg | 472)mB1) | 24 | 20 |
| / | / | 1.3 | / |
|
| 8mB1) |
|
|
|
|
|
|
|
|
| 172)mB1) |
|
|
|
|
|
|
|
ZK10445/ZK344937 | 2.0mg | 0mB1) | 6 | 11 |
| / | / | 0.4 | / |
|
| 0mB1) |
|
|
|
|
|
|
|
|
| 192)mB1) |
|
|
|
|
|
|
|
ZK10445/ZK344937 | 3.0mg | 12mB1) | 9 | 3 |
| / | / | 0.5 | / |
|
| 6mB1) |
|
|
|
|
|
|
|
|
| 10mB1) |
|
|
|
|
|
|
|
2-NF | 10.0 µg | 958 | 874 | 149 |
| / | / | 48.5 | / |
|
| 702 |
|
|
|
|
|
|
|
|
| 961 |
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
Preincubation test on Salmonella typhimurium strain TA100 | |||||||||
|
| -S9 | M | +/-SD | +S9 | M | +/-SD | -S9 | +S9 |
DMSO | 50 µL | 88 | 93 | 4 | 100 | 101 | 8 | 1.0 | 1.0 |
|
| 96 |
|
| 110 |
|
|
|
|
|
| 94 |
|
| 94 |
|
|
|
|
Phosphate buffer | 50 µL | 101 | 101 | 2 | 102 | 109 | 10 | 1.1 | 1.1 |
|
| 100 |
|
| 105 |
|
|
|
|
|
| 103 |
|
| 121 |
|
|
|
|
ZKl0445/ZK344937 | 0.1mg | 77 | 83 | 13 | 95 | 88 | 10 | 0.9 | 0.9 |
|
| 73 |
|
| 77 |
|
|
|
|
|
| 98 |
|
| 93 |
|
|
|
|
ZK10445/ZK344937 | 0.25 mg | 74 | 75 | 2 | 110 | 98 | 12 | 0.8 | 1.0 |
|
| 77 |
|
| 86 |
|
|
|
|
|
| 74 |
|
| 98 |
|
|
|
|
ZK10445/ZK344937 | 0.5mg | 69 | 41 | 24 | 98 | 91 | 7 | 0.4 | 0.9 |
|
| 24 |
|
| 84 |
|
|
|
|
|
| 30 |
|
| 91 |
|
|
|
|
ZK10445/ZK344937 | 1.0mg | 5 mB | 11 | 5 | 90 | 82 | 20 | 0.1 | 0.8 |
|
| 15 mB |
|
| 59 |
|
|
|
|
|
| 12 mB |
|
| 97 |
|
|
|
|
ZK10445/ZK34493 7 | 2.5mg | 25 m B | 23 | 4 | 103B | 86 | 20 | 0.2 | 0.8 |
|
| 26 m B |
|
| 91B |
|
|
|
|
|
| 18 mB |
|
| 64B |
|
|
|
|
ZK10445/ZK344937 | 5.0mg | 18 mPB | 20 | 2 | 42mPB | 53 | 12 | 0.2 | 0.5 |
|
| 22 mPB |
|
| 66mPB |
|
|
|
|
|
| 20 mPB |
|
| 50mPB |
|
|
|
|
Anthracene-2-amine | 2.5µg | 113 | 110 | 14 | 234 | 225 | 9 | 1.2 | 2.2 |
|
| 95 |
|
| 217 |
|
|
|
|
|
| 123 |
|
| 223 |
|
|
|
|
Anthracene-2-amine | 3.5µg | 76 | 88 | 21 | 281 | 322 | 40 | 1.0 | 3.2 |
|
| 77 |
|
| 361 |
|
|
|
|
|
| 112 |
|
| 325 |
|
|
|
|
DMNA | 5.0µL | 88 | 88 | 0 | 1561 | 1471 | 127 | 0.9 | 14.5 |
|
| 88 |
|
| 1526 |
|
|
|
|
|
| 88 |
|
| 1325 |
|
|
|
|
Sodium azide | 5.0µg | 498 | 486 | 12 | 130 | 128 | 3 | 5.2 | 1.3 |
|
| 485 |
|
| 124 |
|
|
|
|
|
| 475 |
|
| 130 |
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
Preincubation test on Salmonella typhimurium strain TA100 | |||||||||
|
| -S9 | M | +/-SD | +S9 | M | +/-SD | -S9 | +S9 |
DMSO | 50µL | 104 | 102 | 3 |
| I | / | 1.0 |
|
|
| 98 |
|
|
|
|
|
|
|
|
| 104 |
|
|
|
|
|
|
|
Phosphate buffer | 50 µL | 90 | 99 | 8 |
| I | / | 1.0 |
|
|
| 102 |
|
|
|
|
|
|
|
|
| 105 |
|
|
|
|
|
|
|
ZK! 0445/ZK344937 | 0.01mg | 89 | 89 | 2 |
| I | / | 0.9 |
|
|
| 88 |
|
|
|
|
|
|
|
|
| 91 |
|
|
|
|
|
|
|
ZK10445/ZK34493 7 | 0.05mg | 86 | 90 | 4 |
| I | / | 0.9 |
|
|
| 90 |
|
|
|
|
|
|
|
|
| 94 |
|
|
|
|
|
|
|
ZK10445/ZK344937 | 0.1mg | 91 | 79 | 16 |
| I | / | 0.8 | / |
|
| 61 |
|
|
|
|
|
|
|
|
| 84 |
|
|
|
|
|
|
|
ZK10445/ZK344937 | 0.25 mg | 52 | 66 | 14 |
| I | / | 0.6 |
|
|
| 65 |
|
|
|
|
|
|
|
|
| 80 |
|
|
|
|
|
|
|
ZK10445/ZK344937 | 0.5mg | 16 mB | 19 | 6 |
| I | / | 0.2 |
|
|
| 26 mB |
|
|
|
|
|
|
|
|
| 15 mB |
|
|
|
|
|
|
|
ZK10445/ZK344937 | 1.0mg | 30 mB | 22 | 8 |
| I | / | 0.2 |
|
|
| 15 mB |
|
|
|
|
|
|
|
|
| 21 mB |
|
|
|
|
|
|
|
Sodium azide | 5.0µg | 726 | 690 | 41 |
| I | / | 6.8 |
|
|
| 699 |
|
|
|
|
|
|
|
|
| 646 |
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
Preincubation test on Salmonella typhimurium strain TA1535 | |||||||||
|
| -S9 | M | +/-SD | +S9 | M | +/-SD | -S9 | +S9 |
DMSO | 50 µL | 17 | 18 |
| 19 | 16 | 4 | 1.0 | 1.0 |
|
| 18 |
|
| 11 |
|
|
|
|
|
| 18 |
|
| 18 |
|
|
|
|
Phosphate buffer | 50 µL | 22 | 24 | 2 | 13 | 13 |
| 1.4 | 0.8 |
|
| 25 |
|
| 13 |
|
|
|
|
|
| 25 |
|
| 12 |
|
|
|
|
ZK10445/ZK344937 | 0.1mg | 12 | 13 |
| 17 | 16 | 5 | 0.7 | 1.0 |
|
| 12 |
|
| 20 |
|
|
|
|
|
| 14 |
|
| II |
|
|
|
|
ZK10445/ZK344937 | 0.25 mg | II | 13 | 2 | 12 | 18 | 8 | 0.7 | 1.1 |
|
| 13 |
|
| 27 |
|
|
|
|
|
| 14 |
|
| 15 |
|
|
|
|
ZK10445/ZK344937 | 0.5mg | 13B | 12 | 2 | 20 | 21 | 6 | 0.7 | 1.3 |
|
| 10B |
|
| 27 |
|
|
|
|
|
| 12B |
|
| 15 |
|
|
|
|
ZK10445/ZK344937 | 1.0mg | 7B | 7 |
| 8B | 11 | 2 | 0.4 | 0.7 |
|
| 8B |
|
| 12B |
|
|
|
|
|
| 7B |
|
| 12B |
|
|
|
|
ZK10445/ZK344937 | 2.5mg | 6B | 8 | 2 | 7B | 7 | 3 | 0.5 | 0.4 |
|
| 9B |
|
| 4B |
|
|
|
|
|
| 9B |
|
| 9B |
|
|
|
|
ZK10445/ZK344937 | 5.0mg | 7 mPB | 5 | 2 | 4 mPB | 3 | 0.3 | 0.2 |
|
|
| 4 mPB |
|
| 4 mPB |
|
|
|
|
|
| 4 mPB |
|
| 2 mPB |
|
|
|
|
Anthracene-2-amine | 2.5µg | 13 | 11 | 2 | 62 | 63 | 10 | 0.6 | 3.9 |
|
| 9 |
|
| 54 |
|
|
|
|
|
| 10 |
|
| 73 |
|
|
|
|
Anthracene-2-amine | 3.5µg | 9 | 10 |
| 69 | 62 | 6 | 0.5 | 3.9 |
|
| 10 |
|
| 59 |
|
|
|
|
|
| 10 |
|
| 57 |
|
|
|
|
Cyclophosphamide | 400.0 µg | 26 | 32 | 6 | 491 | 530 | 38 | 1.8 | 33.1 |
|
| 37 |
|
| 533 |
|
|
|
|
|
| 33 |
|
| 566 |
|
|
|
|
Sodium azide | 5µg | 431 | 423 | 26 | 52 | 39 | 12 | 24.0 | 2.4 |
|
| 445 |
|
| 37 |
|
|
|
|
|
| 394 |
|
| 28 |
|
|
|
|
|
|
|
|
|
|
|
|
|
|
Preincubation test on Salmonella typhimurium strain T A1538 | |||||||||
|
| -S9 | M | +/-SD | +S9 | M | +/-SD | -S9 | +S9 |
DMSO | 50 µL | 10 | 11 | 4 | 20 | 21 | 2 | 1.0 | 1.0 |
|
| 15 |
|
| 23 |
|
|
|
|
|
| 7 |
|
| 21 |
|
|
|
|
Phosphate buffer | 50 µL | 12 | 13 |
| 18 | 18 | 3 | 1.2 | 0.8 |
|
| 14 |
|
| 15 |
|
|
|
|
|
| 13 |
|
| 20 |
|
|
|
|
ZK10445/ZK344937 | 0.1mg | 10 | 9 |
| 10 | 12 | 2 | 0.9 | 0.6 |
|
| 9 |
|
| 14 |
|
|
|
|
|
| 9 |
|
| 13 |
|
|
|
|
ZK10445/ZK344937 | 0.25 mg | II | 10 | 3 | 20 | 18 | 2 | 0.9 | 0.9 |
|
| 12 |
|
| 18 |
|
|
|
|
|
| 7 |
|
| 17 |
|
|
|
|
ZK10445/ZK344937 | 0.5mg | 9 mB | 4 | 5 | 15 | 20 | 5 | 0.3 | 0.9 |
|
| 1 mB |
|
| 25 |
|
|
|
|
|
| 1 mB |
|
| 19 |
|
|
|
|
ZK10445/ZK344937 | 1.0mg | 1 mB | 0 |
| 15 | 12 | 3 | 0.0 | 0.5 |
|
| 0 mB |
|
| 10 |
|
|
|
|
|
| 0 mB |
|
| 10 |
|
|
|
|
ZK10445/ZK344937 | 2.5mg | 0 mB | 0 | 0 | 17B | 14 | 4 | 0.0 | 0.7 |
|
| 0mB |
|
| 16B |
|
|
|
|
|
| 0 mB |
|
| 10B |
|
|
|
|
ZK10445/ZK34493 7 | 5.0mg | 0 mPB | 0 | 0 | 5mPB | 4 |
| 0.0 | 0.2 |
|
| 0 mPB |
|
| 3mPB |
|
|
|
|
|
| 0 mPB |
|
| 3mPB |
|
|
|
|
Anthracene-2-amine | 2.5µg | 6 | 7 | 1 | 198 | 192 | 9 | 0.7 | 9.0 |
|
| 8 |
|
| 182 |
|
|
|
|
|
| 8 |
|
| 197 |
|
|
|
|
Anthracene-2-amine | 3.5µg | 6 | 6 | 1 | 246 | 233 | 24 | 0.6 | 10.9 |
|
| 7 |
|
| 248 |
|
|
|
|
|
| 6 |
|
| 206 |
|
|
|
|
2-Nitrofluorene | 10.0 µg | 794 | 789 | 9 | 195 | 215 | 20 | 73.9 | 10.1 |
|
| 778 |
|
| 214 |
|
|
|
|
|
| 794 |
|
| 235 |
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
Preincubation test on Salmonella typhimurium strain TA1538 (a) | |||||||||
|
| -S9 | M | +/-SD | +S9 | M | +/-SD | -S9 | +S9 |
DMSO | 50 µL | 12 | 13 | 1 |
| I | I | 1.0 | / |
|
| 14 |
|
|
|
|
|
|
|
|
| 13 |
|
|
|
|
|
|
|
Phosphate buffer | 50 µL | 9 | 9 | 1 |
| I | I | 0.7 | I |
|
| 9 |
|
|
|
|
|
|
|
|
| 10 |
|
|
|
|
|
|
|
ZK10445/ZK344937 | 0.01 mg | 11 | 9 | 2 |
| I | I | 0.7 | I |
|
| 7 |
|
|
|
|
|
|
|
|
| 9 |
|
|
|
|
|
|
|
ZK10445/ZK344937 | 0.05 mg | 18 | 12 | 6 |
| / | / | 0.9 | / |
|
| 11 |
|
|
|
|
|
|
|
|
| 6 |
|
|
|
|
|
|
|
ZK10445/ZK344937 | 0.1mg | 5 B | 5 | 1 |
| / | / | 0.4 | / |
|
| 4 mB |
|
|
|
|
|
|
|
|
| 6 mB |
|
|
|
|
|
|
|
ZK10445/ZK344937 | 0.25 mg | 11mB | 7 | 4 | / | I | 0.5 | I |
|
|
| 3mB |
|
|
|
|
|
|
|
|
| 6mB |
|
|
|
|
|
|
|
ZK10445/ZK344937 | 0.5 mg | 19mB | 6 | 11 | / | I | 0.5 | / |
|
|
| 0mB |
|
|
|
|
|
|
|
|
| 0 mB |
|
|
|
|
|
|
|
ZK10445/ZK344937 | 1.0 mg | 0 mB 0mB | 0 | 0 | I | / | 0.0 | / |
|
|
| 0 mB |
|
|
|
|
|
|
|
2-Nitrofluorene | l0.0 µg | 899 | 976 | 70 | I | / | 75.1 | / |
|
|
| 994 |
|
|
|
|
|
|
|
|
| l036 |
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
Preincubation test on Salmonella typhimurium strain TA1537 | |||||||||
|
| -S9 | M | +/-SD | +S9 | M | +/-SD | -S9 | +S9 |
DMSO | 50 µL | 4 | 8 | 3 | 18 | 21 | 4 | 1.0 | 1.0 |
|
| 10 |
|
| 26 |
|
|
|
|
|
| 9 |
|
| 19 |
|
|
|
|
Phosphate buffer | 50 µL | 5 | 5 |
| 10 | 11 | 2 | 0.7 | 0.5 |
|
| 5 |
|
| 11 |
|
|
|
|
|
| 6 |
|
| 13 |
|
|
|
|
ZK1 0445/ZK344937 | 0.1mg | 5 | 7 | 2 | 22 | 19 | 4 | 0.9 | 0.9 |
|
| 7 |
|
| 14 |
|
|
|
|
|
| 8 |
|
| 21 |
|
|
|
|
ZK10445/ZK344937 | 0.25 mg | 2 mB | 3 | 2 | 14 | 13 | 2 | 0.4 | 0.6 |
|
| 3 mB |
|
| 11 |
|
|
|
|
|
| 5 mB |
|
| 13 |
|
|
|
|
ZK10445/ZK344937 | 0.5mg | 1 mB | 10 | 13 | 23 | 14 | 8 | 1.3 | 0.7 |
|
| 25mB |
|
| 9 |
|
|
|
|
|
| 3 mB |
|
| 10 |
|
|
|
|
ZK10445/ZK344937 | 1.0mg | 1 m B | 0 |
| 13 | 12 | 2 | 0.0 | 0.6 |
|
| 0 mB |
|
| 13 |
|
|
|
|
|
| 0 mB |
|
| 10 |
|
|
|
|
ZK10445/ZK344937 | 2.5mg | 0 mB | 0 |
| 6B | 9 | 3 | 0.0 | 0.4 |
|
| l mB |
|
| 8B |
|
|
|
|
|
| 0 mB |
|
| 12B |
|
|
|
|
ZK10445/ZK344937 | 5.0mg | 2 mPB | 1 |
| 0mPB | 2 | 2 | 0.1 | 0.1 |
|
| 0 mPB |
|
| 2mPB |
|
|
|
|
|
| 0 mPB |
|
| 4mPB |
|
|
|
|
Anthracene-2-amine | 5.0µg | 9 | 9 | 0 | 58 | 59 | 4 | 1.2 | 2.8 |
|
| 9 |
|
| 56 |
|
|
|
|
|
| 9 |
|
| 63 |
|
|
|
|
4-NPDA | 10.0 µg | 67 | 57 | 12 | 24 | 26 | 2 | 7.4 | 1.2 |
|
| 43 |
|
| 28 |
|
|
|
|
|
| 60 |
|
| 26 |
|
|
|
|
|
|
|
|
|
|
|
|
|
|
Preincubation test on Salmonella typhimurium strain TA1537 (a) | |||||||||
|
| -S9 | M | +/-SD | +S9 | M | +/-SD | -S9 | +S9 |
DMSO | 50 µL | 11 | 11 | 1 |
| / | / | 1.0 | / |
|
| 11 |
|
|
|
|
|
|
|
|
| 10 |
|
|
|
|
|
|
|
Phosphate buffer | 50µL | 10 | 9 | 1 |
| / | / | 0.9 | / |
|
| 8 |
|
|
|
|
|
|
|
|
| 10 |
|
|
|
|
|
|
|
ZK10445/ZK344937 | 0.01 mg | 9 | 10 | 1 |
| / | / | 0.9 | / |
|
| 10 |
|
|
|
|
|
|
|
|
| 10 |
|
|
|
|
|
|
|
ZK10445/ZK344937 | 0.05 mg | 5 | 5 | 1 |
| / | / | 0.4 | / |
|
| 5 |
|
|
|
|
|
|
|
|
| 4 |
|
|
|
|
|
|
|
ZK10445/ZK344937 | 0.1mg | 5 | 5 | 1 |
| / | / | 0.4 | / |
|
| 5 |
|
|
|
|
|
|
|
|
| 4 |
|
|
|
|
|
|
|
ZK10445/ZK344937 | 0.25 mg | 4 mB | 51 | 75 |
| / | / | 4.8 | / |
|
| 1382)B1) |
|
|
|
|
|
|
|
|
| 11B1) |
|
|
|
|
|
|
|
ZK10445/ZK344937 | 0.5 mg | 1mB | 53 | 50 |
| / | / | 5.0 | / |
|
| 1012)B1) |
|
|
|
|
|
|
|
|
| 582)B1) |
|
|
|
|
|
|
|
ZK10445/ZK344937 | 1.0 mg | 0mB1) | 24 | 42 |
| / | / | 2.3 | / |
|
| 0mB1) |
|
|
|
|
|
|
|
|
| 732)B1) |
|
|
|
|
|
|
|
4-NPDA | 10.0µg | 65 | 70 | 8 |
| / | / | 6.6 | / |
|
| 79 |
|
|
|
|
|
|
|
|
| 67 |
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
Preincubation test on Salmonella typhimurium strain TA1537 (d) | |||||||||
|
| -S9 | M | +/-SD | +S9 | M | +/-SD | -S9 | +S9 |
|
|
|
|
|
|
|
|
|
|
DMSO | 50 µL | 8 | 7 | 1 |
| / | / | 1.0 | / |
|
| 8 |
|
|
|
|
|
|
|
|
| 6 |
|
|
|
|
|
|
|
Phosphate buffer | 50 µL | 5 | 8 | 4 |
| / | / | 1.1 | / |
|
| 13 |
|
|
|
|
|
|
|
|
| 7 |
|
|
|
|
|
|
|
ZK10445 | 2.5µg | 7 | 9 | 2 |
| / | / | 1.2 | / |
|
| 9 |
|
|
|
|
|
|
|
|
| 10 |
|
|
|
|
|
|
|
ZK10445 | 5µg | 5 | 6 | 1 |
| / | / | 0.8 | / |
|
| 5 |
|
|
|
|
|
|
|
|
| 7 |
|
|
|
|
|
|
|
ZK10445 | 10µg | 9 | 10 | 1 |
| / | / | 1.3 | / |
|
| 10 |
|
|
|
|
|
|
|
|
| 10 |
|
|
|
|
|
|
|
ZK10445 | 25µg | 5 | 8 | 4 |
| / | / | 1.1 | / |
|
| 13 |
|
|
|
|
|
|
|
|
| 6 |
|
|
|
|
|
|
|
ZK10445 | 50µg | 7B | 4 | 3 |
| / | / | 0.5 | / |
|
| 1B |
|
|
|
|
|
|
|
|
| 3B |
|
|
|
|
|
|
|
ZK10445 | 100µg | 4B | 6 | 2 |
| / | / | 0.8 | / |
|
| 7B |
|
|
|
|
|
|
|
|
| 7B |
|
|
|
|
|
|
|
ZK10445 | 250µg | 612)B1) | 27 | 30 |
| / | / | 3.6 | / |
|
| 9 B1) |
|
|
|
|
|
|
|
|
| 10 B1) |
|
|
|
|
|
|
|
ZK10445 | 500µg | 0 B1) | 2 | 3 |
| / | / | 0.2 | / |
|
| 0 B1) |
|
|
|
|
|
|
|
|
| 5 B1) |
|
|
|
|
|
|
|
ZK10445 | 1000µg | 0 B1) | 0 | 0 |
| / | / | 0.0 | / |
|
| 0 B1) |
|
|
|
|
|
|
|
|
| 0 B1) |
|
|
|
|
|
|
|
4-NPDA | 10.0µg | 89 | 85 | 11 |
| / | / | 11.6 | / |
|
| 93 |
|
|
|
|
|
|
|
|
| 73 |
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
Preincubation test on Escherichia coli WP2uvrA | |||||||||
|
| -S9 | M | +/-SD | +S9 | M | +/-SD | -S9 | +S9 |
DMSO | 50 µL | 25 | 24 | 3 | 39 | 36 | 3 | 1.0 | 1.0 |
|
| 27 |
|
| 34 |
|
|
|
|
|
| 21 |
|
| 34 |
|
|
|
|
Phosphate buffer | 50 µL | 32 | 31 | 1 | 48 | 47 | 2 | 1.3 | 1.3 |
|
| 31 |
|
| 44 |
|
|
|
|
|
| 31 |
|
| 48 |
|
|
|
|
ZK344937/10445 | 0.1mg | 28 | 24 | 5 | 34 | 40 | 6 | 1.0 | 1.1 |
|
| 18 |
|
| 42 |
|
|
|
|
|
| 26 |
|
| 45 |
|
|
|
|
ZK344937/10445 | 0.25mg | 28 | 26 | 2 | 38 | 35 | 3 | 1.1 | 1.0 |
|
| 25 |
|
| 32 |
|
|
|
|
|
| 25 |
|
| 36 |
|
|
|
|
ZK344937/10445 | 0.5mg | 26 | 29 | 4 | 49 | 43 | 6 | 1.2 | 1.2 |
|
| 33 |
|
| 44 |
|
|
|
|
|
| 29 |
|
| 37 |
|
|
|
|
ZK344937/10445 | 1.0mg | 19 | 21 | 10 | 25 | 24 | 5 | 0.9 | 0.7 |
|
| 32 |
|
| 28 |
|
|
|
|
|
| 13 |
|
| 19 |
|
|
|
|
ZK344937/10445 | 2.5mg | 16 | 17 | 1 | 27 | 27 | 1 | 0.7 | 0.8 |
|
| 18 |
|
| 28 |
|
|
|
|
|
| 16 |
|
| 26 |
|
|
|
|
ZK344937/10445 | 5.0mg | 10mP | 14 | 3 | 25 | 27 | 5 | 0.6 | 0.7 |
|
| 15mP |
|
| 32 |
|
|
|
|
|
| 16mP |
|
| 23 |
|
|
|
|
Anthracene-2-amine | 50.0µg | 70 | 63 | 10 | 151 | 159 | 9 | 2.6 | 4.4 |
|
| 51 |
|
| 169 |
|
|
|
|
|
| 67 |
|
| 156 |
|
|
|
|
EMS | 5.0µL | 1550 | 1475 | 65 | 1682 | 1667 | 73 | 60.6 | 46.7 |
|
| 1437 |
|
| 1588 |
|
|
|
|
|
| 1438 |
|
| 1731 |
|
|
|
|
MNNG | 5.0µg | 489 | 451 | 46 | 89 | 111 | 34 | 18.5 | 3.1 |
|
| 400 |
|
| 151 |
|
|
|
|
|
| 463 |
|
| 94 |
|
|
|
|
M: Mean +/-SD: Standard-deviation p: Precipitation +S9: With S9 mix -S9: Without S9 mix B: Background lawn reduced C: Contamination /: Not tested Quotient= Mean revertants (test substance) Mean revertants (solvent) 1) : Background lawn was extremely or totally suppressed 2) : Counts include, however, small "pinpoint" colonies which could not be unequivocally distinguished from normal colonies |
None of the five tester strains showed increased reversion to prototrophy with fluocortolone at the concentrations tested, either in the absence or presence of S9 mix.
Precipitates in the agar were found at the highest concentration (5.0 mg/plate) tested in all experiments.
Generally, growth inhibition of the background lawn was observed fram 1.0 mg fluocortolone/plate onwards in all tested strains without and with S9 mix.
Negative contrals and positive contrals 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 | Quotient | ||||||
Dose/Plate | -S9 | M | SD | +S9 | M | SD | -S9 | +S9 | |
DMSO | 50µL | 21 | 28 | 8 | 16 | 14 | 2 | 1.0 | 1.0 |
|
| 26 |
|
| 14 |
|
|
|
|
|
| 36 |
|
| 12 |
|
|
|
|
Phosphate Buffer | 50µL | 25 | 24 | 3 | 13 | 18 | 4 | 0.9 | 1.3 |
|
| 21 |
|
| 20 |
|
|
|
|
|
| 26 |
|
| 21 |
|
|
|
|
Test item | 0.10mg | 19 | 20 | 7 | 13 | 14 | 5 | 0.7 | 1.0 |
|
| 28 |
|
| 10 |
|
|
|
|
|
| 14 |
|
| 19 |
|
|
|
|
| 0.25mg | 18 | 28 | 9 | 16 | 16 | 2 | 1.0 | 1.1 |
|
| 36 |
|
| 14 |
|
|
|
|
|
| 29 |
|
| 17 |
|
|
|
|
| 0.50mg | 32 | 28 | 10 | 24 | 20 | 3 | 1.0 | 1.5 |
|
| 17 |
|
| 18 |
|
|
|
|
|
| 36 |
|
| 19 |
|
|
|
|
| 1.0mg | 19 | 19 | 3 | 16B | 19 | 3 | 0.7 | 1.4 |
|
| 16 |
|
| 21 |
|
|
|
|
|
| 22 |
|
| 20 |
|
|
|
|
| 2.5mg | 26 B | 23 | 4 | 16B | 13 | 3 | 0.8 | 0.9 |
|
| 19 |
|
| 11 |
|
|
|
|
|
| 23 |
|
| 12 |
|
|
|
|
| 5.0mg | 24B | 19 | 5 | 7B | 6 | 2 | 0.7 | 0.4 |
|
| 19P |
|
| 6P |
|
|
|
|
|
| 15 |
|
| 4 |
|
|
|
|
2-AA | 5µg | 25 | 24 | 3 | 182 | 151 | 32 | 0.9 | 10.8 |
|
| 21 |
|
| 153 |
|
|
|
|
|
| 27 |
|
| 118 |
|
|
|
|
CP | 400µg | 79 | 78 | 4 | 375 | 361 | 17 | 2.8 | 25.8 |
|
| 73 |
|
| 342 |
|
|
|
|
|
| 81 |
|
| 366 |
|
|
|
|
NaN3 | 5µg | 387 | 416 | 27 |
|
|
| 15.0 |
|
|
| 439 |
|
|
|
|
|
|
|
|
| 423 |
|
|
|
|
|
|
|
| |||||||||
TA 100 |
| Revertants per plate | Quotient | ||||||
Dose/Plate | -S9 | M | SD | +S9 | M | SD | -S9 | +S9 | |
DMSO | 50µL | 119 | 108 | 12 | 114 | 104 | 11 | 1.0 | 1.0 |
|
| 109 |
|
| 106 |
|
|
|
|
|
| 96 |
|
| 93 |
|
|
|
|
Phosphate Buffer | 50µL | 104 | 105 | 10 | 101 | 105 | 6 | 1.0 | 1.0 |
|
| 95 |
|
| 112 |
|
|
|
|
|
| 115 |
|
| 103 |
|
|
|
|
Test item | 0.10mg | 120 | 103 | 19 | 110 | 108 | 3 | 1.0 | 1.0 |
|
| 105 |
|
| 105 |
|
|
|
|
|
| 83 |
|
| 108 |
|
|
|
|
| 0.25mg | 104 | 101 | 16 | 109 | 106 | 5 | 0.9 | 1.0 |
|
| 115 |
|
| 109 |
|
|
|
|
|
| 84 |
|
| 100 |
|
|
|
|
| 0.50mg | 108 | 100 | 12 | 116 | 106 | 12 | 0.9 | 1.0 |
|
| 106 |
|
| 108 |
|
|
|
|
|
| 86 |
|
| 93 |
|
|
|
|
| 1.0mg | 82 | 77 | 44 | 94B | 104 | 12 | 0.7 | 1.0 |
|
| 30 |
|
| 118 |
|
|
|
|
|
| 118 |
|
| 101 |
|
|
|
|
| 2.5mg | 87 | 57 | 27 | 81B | 87 | 6 | 0.5 | 0.8 |
|
| 50 |
|
| 87 |
|
|
|
|
|
| 34 |
|
| 93 |
|
|
|
|
| 5.0mg | 5 | 36 | 28 | 39B | 51 | 10 | 0.3 | 0.5 |
|
| 59 |
|
| 56P |
|
|
|
|
|
| 45 |
|
| 58 |
|
|
|
|
2-AA | 5µg | 128 | 124 | 12 | 585 | 583 | 19 | 1.1 | 5.6 |
|
| 133 |
|
| 601 |
|
|
|
|
|
| 111 |
|
| 563 |
|
|
|
|
DMNA | 5µL | 118 | 115 | 6 | 391 | 337 | 57 | 1.1 | 3.2 |
|
| 119 |
|
| 277 |
|
|
|
|
|
| 108 |
|
| 344 |
|
|
|
|
NaN3 | 5µg | 551 | 603 | 48 |
|
|
| 5.6 |
|
|
| 645 |
|
|
|
|
|
|
|
|
| 614 |
|
|
|
|
|
|
|
| |||||||||
TA 1537 |
| Revertants per plate | Quotient | ||||||
Dose/Plate | -S9 | M | SD | +S9 | M | SD | -S9 | +S9 | |
DMSO | 50µL | 17 | 16 | 4 | 11 | 14 | 3 | 1.0 | 1.0 |
|
| 19 |
|
| 17 |
|
|
|
|
|
| 12 |
|
| 15 |
|
|
|
|
Phosphate Buffer | 50µL | 16 | 17 | 2 | 16 | 19 | 7 | 1.0 | 1.3 |
|
| 19 |
|
| 27 |
|
|
|
|
|
| 15 |
|
| 13 |
|
|
|
|
Test item | 0.10mg | 12 | 14 | 2 | 13 | 16 | 2 | 0.9 | 1.1 |
|
| 15 |
|
| 17 |
|
|
|
|
|
| 14 |
|
| 17 |
|
|
|
|
| 0.25mg | 12 | 10 | 2 | 16 | 19 | 3 | 0.6 | 1.3 |
|
| 9 |
|
| 19 |
|
|
|
|
|
| 8 |
|
| 22 |
|
|
|
|
| 0.50mg | 12 | 11 | 1 | 13 | 18 | 6 | 0.7 | 1.3 |
|
| 11 |
|
| 18 |
|
|
|
|
|
| 10 |
|
| 24 |
|
|
|
|
| 1.0mg | 21 | 16 | 6 | 21B | 17 | 3 | 1.0 | 1.2 |
|
| 18 |
|
| 15 |
|
|
|
|
|
| 10 |
|
| 15 |
|
|
|
|
| 2.5mg | 10B | 9 | 5 | 8B | 9 | 2 | 0.6 | 0.6 |
|
| 4 |
|
| 11 |
|
|
|
|
|
| 14 |
|
| 8 |
|
|
|
|
| 5.0mg | 9B | 7 | 3 | 4B | 3 | 2 | 0.4 | 0.2 |
|
| 8P |
|
| 0P |
|
|
|
|
|
| 4 |
|
| 4 |
|
|
|
|
9-AA | 35µg | 27B | 40 | 13 |
|
|
| 2.5 |
|
|
| 52 |
|
|
|
|
|
|
|
|
| 40 |
|
|
|
|
|
|
|
9-AA | 40µg | 70B | 75 | 18 |
|
|
| 4.7 |
|
|
| 61 |
|
|
|
|
|
|
|
|
| 95 |
|
|
|
|
|
|
|
2-AA | 5µg |
|
|
| 51 | 64 | 13 |
| 4.4 |
|
|
|
|
| 76 |
|
|
|
|
|
|
|
|
| 64 |
|
|
|
|
BaP | 5µg |
|
|
| 60 | 61 | 5 |
| 4.3 |
|
|
|
|
| 66 |
|
|
|
|
|
|
|
|
| 57 |
|
|
|
|
| |||||||||
TA 1538 |
| Revertants per plate | Quotient | ||||||
Dose/Plate | -S9 | M | SD | +S9 | M | SD | -S9 | +S9 | |
DMSO | 50µL | 10 | 8 | 2 | 25 | 28 | 3 | 1.0 | 1.0 |
|
| 6 |
|
| 31 |
|
|
|
|
|
| 7 |
|
| 28 |
|
|
|
|
Phosphate Buffer | 50µL | 14 | 15 | 2 | 31 | 31 | 3 | 2.0 | 1.1 |
|
| 15 |
|
| 28 |
|
|
|
|
|
| 17 |
|
| 33 |
|
|
|
|
Test item | 0.10mg | 13 | 12 | 2 | 36 | 31 | 6 | 1.5 | 1.1 |
|
| 12 |
|
| 24 |
|
|
|
|
|
| 10 |
|
| 34 |
|
|
|
|
| 0.25mg | 7 | 9 | 3 | 25 | 32 | 7 | 1.2 | 1.1 |
|
| 13 |
|
| 39 |
|
|
|
|
|
| 7 |
|
| 32 |
|
|
|
|
| 0.50mg | 7B | 7 | 4 | 21 | 25 | 7 | 1.0 | 0.9 |
|
| 4 |
|
| 33 |
|
|
|
|
|
| 11 |
|
| 21 |
|
|
|
|
| 1.0mg | 0B | 4 | 4 | 20B | 22 | 7 | 0.5 | 0.8 |
|
| 8 |
|
| 16 |
|
|
|
|
|
| 4 |
|
| 29 |
|
|
|
|
| 2.5mg | 3B | 3 | 3 | 24B | 23 | 4 | 0.3 | 0.8 |
|
| 5 |
|
| 26 |
|
|
|
|
|
| 0 |
|
| 19 |
|
|
|
|
| 5.0mg | 10B | 3 | 6 | 16B | 17 | 5 | 0.4 | 0.6 |
|
| 0P |
|
| 23P |
|
|
|
|
|
| 0 |
|
| 13 |
|
|
|
|
2-NF | 10µg | 874 | 881 | 20 |
|
|
| 115.0 |
|
|
| 904 |
|
|
|
|
|
|
|
|
| 866 |
|
|
|
|
|
|
|
2-AA | 5µg |
|
|
| 442 | 467 | 24 |
| 16.7 |
|
|
|
|
| 468 |
|
|
|
|
|
|
|
|
| 490 |
|
|
|
|
BaP | 10µg |
|
|
| 91 | 89 | 2 |
| 3.2 |
|
|
|
|
| 88 |
|
|
|
|
|
|
|
|
| 89 |
|
|
|
|
| |||||||||
TA 1538 |
| Revertants per plate | Quotient | ||||||
Dose/Plate | -S9 | M | SD | +S9 | M | SD | -S9 | +S9 | |
DMSO | 50µL | 18 | 17 | 1 |
|
|
| 1.0 |
|
|
| 17 |
|
|
|
|
|
|
|
|
| 16 |
|
|
|
|
|
|
|
Phosphate Buffer | 50µL | 11 | 11 | 2 |
|
|
| 0.7 |
|
|
| 10 |
|
|
|
|
|
|
|
|
| 13 |
|
|
|
|
|
|
|
Test item | 0.05mg | 10 | 15 | 4 |
|
|
| 0.9 |
|
|
| 18 |
|
|
|
|
|
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| 16 |
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| 0.10mg | 16 | 11 | 5 |
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| 0.6 |
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| 10 |
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| 6 |
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| 0.25mg | 11 | 15 | 5 |
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| 0.9 |
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| 13 |
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| 20 |
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| 0.50mg | 8B | 9 | 1 |
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| 0.5 |
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| 9 |
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| 10 |
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| 1.0mg | 12B | 8 | 4 |
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| 0.5 |
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| 5 |
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| 6 |
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| 2.5mg | 13B | 10 | 3 |
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| 0.6 |
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| 8 |
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| 8 |
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| 5.0mg | 0B | 3 | 4 |
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| 0.2 |
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| 7P |
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| 2 |
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2-NF | 10µg | 965 | 928 | 33 |
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| 54.6 |
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| 903 |
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| 916 |
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TA 98 |
| Revertants per plate | Quotient | ||||||
Dose/Plate | -S9 | M | SD | +S9 | M | SD | -S9 | +S9 | |
DMSO | 50µL | 27 | 30 | 3 |
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| 10. |
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| 30 |
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| 32 |
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Phosphate Buffer | 50µL | 31 | 32 | 6 |
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| 1.1 |
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| 27 |
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| 38 |
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Test item | 0.10mg | 36 | 33 | 4 |
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| 1.1 |
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| 35 |
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| 29 |
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| 0.25mg | 28 | 33 | 6 |
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| 1.1 |
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| 39 |
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| 32 |
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| 0.50mg | 22 | 23 | 1 |
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| 0.8 |
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| 22 |
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| 24 |
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| 1.0mg | 6B | 8 | 3 |
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| 0.3 |
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| 8 |
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| 11 |
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| 2.5mg | 10B | 9 | 5 |
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| 0.3 |
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| 13 |
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| 4 |
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| 5.0mg | 11B | 7 | 3 |
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| 0.2 |
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| 5P |
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| 6 |
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2-AA | 5µg | 38 | 37 | 1 |
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| 1.2 |
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| 36 |
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| 36 |
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BaP | 10µg | 38 | 32 | 6 |
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| 1.1 |
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| 27 |
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| 30 |
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2-NF | 10µg | 1051 | 1160 | 103 |
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| 39.1 |
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| 1173 |
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| 1255 |
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B: Background lawn reduced P: Precipitation M: Mean SD: Standard-Deviation |
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Genetic toxicity in vivo
Description of key information
Delta-9(11)-fluocortolone-valerate is not mutagenic based on micronucleus test according to OECD test guideline 474 with the read-across substance fluocortolone.
Link to relevant study records
- Endpoint:
- in vivo mammalian somatic cell study: cytogenicity / bone marrow chromosome aberration
- Type of information:
- experimental study
- Adequacy of study:
- other information
- Study period:
- Sep 1995
- 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
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- micronucleus assay
- Species:
- mouse
- Strain:
- NMRI
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: Charles River GmbH, Sulzfeld, Germany
- Age at study initiation: ca. 9 to 10 weeks old
- Weight at study initiation: males: ca. 29 - 36 g; females: ca. 25 - 29 g
- Assigned to test groups randomly: yes
- Fasting period before study: ca. 17-21 h
- Housing: individually, in Makrolon® type II cages containing wood-chip bedding
- Diet (e.g. ad libitum): Altromin ® R; Lage; Germany, ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 13 days
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22
- Humidity (%): 60-62
- Photoperiod (hrs dark / hrs light): 12/12
- Route of administration:
- intraperitoneal
- Vehicle:
- - Vehicle(s)/solvent(s) used: 0.9% NaCI; 0.085% MyrjC8i 53 in bidist. water
- Concentration of test material in vehicle: 7.5; 15 or 30 mg fluocortolone/ml (pH 3.4-4.0)
- Amount of vehicle: 10 mL/kg i.p. - Details on exposure:
- TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 10 mL/kg i.p.
- Concentration (if solution): 7.5, 15, 30, 3 mg/mL
- Constant volume or concentration used: yes - Duration of treatment / exposure:
- single
- Frequency of treatment:
- once
- Post exposure period:
- 24 and 48 hours
- Dose / conc.:
- 75 mg/kg bw/day (nominal)
- Dose / conc.:
- 150 mg/kg bw/day (nominal)
- Dose / conc.:
- 300 mg/kg bw/day (nominal)
- No. of animals per sex per dose:
- 10/sex/dose (test substance and negative control)
Positive control animals: 5/sex - Control animals:
- yes, concurrent vehicle
- Positive control(s):
- cyclophosphamide
- Route of administration: gavage
- Doses / concentrations: 30 mg/kg - Tissues and cell types examined:
- bone marrow of femur
- Details of tissue and slide preparation:
- CRITERIA FOR DOSE SELECTION: It was known from acute toxicity studies in male and female mice after single i.p. application that the LD50 of fluocortolone was 395-510 mg/kg. Therefore, in order to achieve toxic effects while avoiding mortality, 300 mg/kg was chosen as highest dose in the micronucleus test.
TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields): All animals were treated once in the morning. The animals of the negative control group and the three dose groups were treated intraperitoneally with the vehicle or the test substance. The positive contral substance cyclophosphamide was administered by gavage. Furthermore, in the highest dose group 3 reserve animals of each sex were also treated in order to replace moribund or dead animals, if necessary. 5 males and 5 females from the negative control and the fluocortolone groups were killed by carbon dioxide asphyxiation at 24 or 48 hours after treatment (the positive control animals were killed 24 hours after treatment) and both femurs were dissected out from each animal.
DETAILS OF SLIDE PREPARATION: The bone marrow was flushed/aspirated into fetal calf serum. The resulting cell suspensions
were centrifuged and smears were prepared from drops of the cell pellets which had been resuspended in a few drops of serum. The slides were air-dried and stained using May-Gruenwald and Giemsa solutions. The slides were coded and analysed "blind" in random order.
METHOD OF ANALYSIS: The stained smears were examined using oi! immersion high power magnification in regions where cells were weil spread and stained. The slides were examined for the incidence of micronucleated cells per 2000 polychromatic (PCE) and 1000 normochromatic (NCE) erythrocytes per anima!. The ratio of polychromatic to normochromatic erythrocytes was calculated on the basis of 1000 NCE scored.
- Statistics:
- The statistical analysis was conducted for each of the following variables:
P1: proportion of micronucleated PCE
P2: proportion of micronucleated NCE
P3: ratio of PCE/NCE
For investigation of treatment differences of the variables P1 and P2 were arc sin √pi transformed. The analyses were conducted separately for the sampie times. Regarding the first sampie time one-sided t-tests were performed to assess the difference between positive and negative controls with pooled values for both sexes; the positive control group was then excluded from further analysis. Thereafter in a two-factorial analysis of variance (factors "sex", "treatment") for each sampie time it was investigated as to whether treatment effect was present. In case of significant interactions, comparisons between the control and each of the treatment groups were conducted separately for each sex. Where no significant interactions
occurred but a global treatment effect, comparisons were performed with values pooled for both sexes. The pair-wise comparisons were performed with one-sided t-tests (increase of P1 and P2, decrease of P3), using the error estimate of the analysis of variance table. The test levels were always α = 0.05 (least significant differences test-LSD). - Key result
- Sex:
- male/female
- Genotoxicity:
- negative
- Toxicity:
- yes
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei (for Micronucleus assay): In male animals 48 hours post application a significant decrease in the ratio of PCE and NCE was observed for all three dose groups as compared to the control, indicating a bone marrow depression induced by the test compound. Regarding micronucleated PCE counts there were neither biologically relevant nor statistically significant differences (p > 0.05) at any sampling time. However, a significant increase in the number of micronucleated NCE was observed for all three dose groups as compared to the negative control at the early sampling time 24 hours post application. Since this statistically significant increase is obviously due to an unusually low NCE rate (0.4‰) , in the vehicle control in comparison to the historical control (0.7-1.1‰, MV 0.89‰ ± 0.17), this effect is considered to be of no biological relevance. This assessment is strongly supported by the fact that micronuclei first appear in PCE at 15 hours after treatment and only the NCE will have micronuclei induced by a recent treatment within 2-3 days of sampling. Consequently, 24 hours after treatment test substance-related micronuclei cannot be expected in the mature normochromatic erythrocytes (NCE). And anyway for the micronucleus assay in the bone marrow, it is essential to base the conclusion primarily upon micronuclei in polychromatic erythrocytes, i.e. the frequency of micronucleated immature erythrocytes is the principal end-point. However, these were not increased by the test substance in this study.
- Ratio of PCE/NCE (for Micronucleus assay): see 'Any other information on results incl. tables'- Conclusions:
Fluocortolone did not show any mutagenic/clastogenic potential under the test conditions. Classification is not reqiured.- Executive summary:
In a NMRI mouse bone marrow micronucleus assay according to OECD test guideline 474 (1983), (10/sex/dose) mice were treated i.p. with Fluocortolone (100% a.i.) at doses of 0, 75, 150, 300 mg/kg bw. Bone marrow cells were harvested at 24 and 48 h post-treatment. The vehicle was 0.9% NaCI; 0.085% MyrjC8i 53 in bidist. water.
Slight to moderate apathy was seen as a sign of systemic toxicity dose dependently in all treated groups. In male animals 48 hours after exposure a significant decrease in the ratio of PCE and NCE was observed for all three dose groups, indicating bone marrow suppression. Fluocortolone was tested at an adequate dose based on preliminary tests. 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.
Fluocortolone did not show any mutagenic/clastogenic potential under the test conditions.
Reference
Slight to moderate apathy was seen as a sign of systemic toxicity dose dependently in all treated groups. In male animals 48 hours after exposure a significant decrease in the ratio of PCE and NCE was observed for all three dose groups, indicating bone marrow suppression.
Summary of results -
Frequency of micronucleated polychromatic erythrocytes (in ‰) from 2000 cells scored per animal and ratio PCE/NCE in mouse bone marrow after a single i.p. administration of the test item
Sampling times, 24 h and 48 h after treatment
Treatment | Dosage (mg/kg) | Number of animals | 24 h post application | |||
Male | Female | Male and Female | ||||
PCE (M) (MV ± SD) | PCE (M) (MV ± SD) | PCE (M) (MV ± SD) | Ratioa) PCE/NCE | |||
Negative control (vehicle) |
| 5M +5 F | 0.70 ± 0.27 | 0.60 ± 0.42 | 0.65 ± 0.34 | 0.99 ± 0.06 |
Test item | 75 | 5M +5 F | 1.10±0.74 | 0.90 ± 0.42 | 1.00 ± 0.58 | 0.97 ± 0.07 |
| 150 | 5M +5 F | 1.00 ± 0.50 | 0.90 ± 0.55 | 0.95 ± 0.50 | 0.98 ± 0.09 |
| 300 | 5M +5 F | 1.50 ± 0.94 | 0.60 ± 0.22 | 1.05 ± 0.80 | 0.93 ± 0.07 |
Cyclophosphamide | 30 | 5M +5 F | 10.20 ± 4.72 | 12.50 ± 1.46 | 11.35*± 3.51 | 0.85*± 0.08 |
| ||||||
Treatment | Dosage (mg/kg) | Number of animals | 48 h post application | |||
Male | Female | Male and Female | ||||
PCE (M) (MV ± SD) | PCE (M) (MV ± SD) | PCE (M) (MV ± SD) | Ratioa) PCE/NCE | |||
Negative control (vehicle) |
| 5M +5 F | 0.60 ± 0.42 | 0.70 ± 0.57 | 0.65 ± 0.47 | 0.94 ± 0.07 |
Test item | 75 | 5M +5 F | 0.50 ± 0.50 | 1.00 ± 0.61 | 0.75 ± 0.59 | 0.90 ± 0.06 |
| 150 | 5M +5 F | 0.90 ± 0.55 | 0.80 ± 0.27 | 0.85 ± 0.41 | 0.89 ± 0.05 |
| 300 | 5M +5 F | 0.70 ± 0.57 | 0.50 ± 0.50 | 0.60 ± 0.52 | 0.88 ± 0.05 |
Cyclophosphamide | 30 | -- | -- | -- | -- | -- |
| ||||||
PCE = polychromatic erythrocytes NCE = normochromatic erythrocytes PCE(M) = micronucleated polychromatic erythrocytes SD = standard deviation MV = mean value a) = calculated on the basis of 1000 NCE's scored per animal * p < 0.05, compared to negative control |
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Additional information
For delta-9(11)-fluocortolone-valerate (CAS No. 66233-43-0) no genetic toxicity data are available. Therefore, genetic toxicity in vitro data of fluocortolone (CAS No. 152-97-6) were used since these data are regarded as representative as most likely ester cleavage of delta-9(11)-fluocortolone-valerate occurs under physiological conditions. A search for structure-analogue substances using the QSAR OECD Toolbox 3.4 recommended fluocortolone as one out of 4 category substances for a read-across approach (for additional information see QSAR OECD Toolbox Report on Delta-9(11)-Fluocortolon-valerat in "Attached justification"). A general justification for read-across is attached to chapter 13 of this IUCLID file.
Fluocortolone was tested for mutagenic activity effects in five histidine-dependent strains of S. typhimurium (TA1535, TA100, TA1537, TA1538, TA98) using the direct plate incorporation procedure (Lang and Schmitt, 1984). The study was performed with and without metabolic activation, employed a range of fluocortolon concentrations from 0.1 to 6.0 mg per plate. No increased reversion to prototrophy were seen neither without nor with metabolic activation. Growth inhibition of the background lawn was observed at fluocortolone concentrations starting at 1.0 mg/plate. An additionally performed test with strain TA 100 using the preincubation procedure did not show a mutagenic effect of the test item. Precipitates were found partially in the preincubation test at 6.0 mg per plate.
The purpose of a further Ames test was to investigate whether fluocortolone can induce point mutations using the preincubation modification (Reimann and Görke, 1997). 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 fluocortolone concentrations from 0.1 to 5.0 mg per plate and 0.05 to 5.0 mg/plate (TA1538 without S9 mix). No increased reversion to prototrophy were seen neither without nor with metabolic activation. Growth inhibition of the background lawn was observed at fluocortolone concentrations of 1.0 mg/plate and above.
In a third study the mutagenic potential of the test item (fluocortolone batch containing 5% of ZK 344937 as impurity) was evaluated in a Salmonella/microsome test with the S. typhimurium strains TA 98, TA 100, TA 1535, TA 1537 and TA 1538 as well as the E. coli strain WP2 uvrA in the presence and absence of S9 mix according to OECD TG 471 (Reimann and Jarzombek, 2005) .Evidence of mutagenic activity was not seen up to the maximum recommended dose level of 5.0 mg/plate. No substantial increases in revertant colony numbers of any of the six tester strains were observed at any dose level in the presence and absence of metabolic activation. Precipitation was observed at the maximum dose levelof 5.0 mg/plate. Therefore, the test item was considered to be non-mutagenic in the Salmonella typhimurium reverse mutation assay (plate incorporation method and preincubation modification).
Further information on in vitro and in vivo genotoxicity of fluocortolone:
In an HPRT test V79 cells were cultured for 4 hours with fluocortolone at concentrations between 50 and 500 µg/mL with and without metabolic activation. No biologically relevant increase in the number of mutant colonies was observed compared to controls. Toxicity was recorded starting at concentrations of 300 µg/mL (Reimann and Naumann, 1998).
Fluocortolone did not show any genotoxic potential in an in vitro UDS test with primary male rat hepatocytes when tested up to the toxic concentration (Reimann and Jarzombek, 1999).
Fluocortolone was tested to induce unscheduled repairable DNA damage in female rat primary hepatocytes with and without metabolic activation. The test substance did not increase the DNA repair synthesis and is therefore not considered to be genotoxic under the conditions of this test (Becker and Jarzombek, 2000).
In the chromosomal aberration test in primary human lymphocytes fluocortolone was tested up 1000 µg/ml reaching the cytotoxic and precipitating concentrations. No clastogenic effects of fluocortolone tested with and without extrinsic metabolising system could be observed (Reimann and Gramlich, 1999).
In the chromosomal aberration test in primary human lymphocytes fluocortolone was tested up 900 µg/ml. Cytotoxicity and precipitation was the dose limiting factors. No clastogenic effects could be observed when tested without extrinsic metabolising system. Fluocortolone showed clastogenic effect in human lymphocytes when tested with extrinsic metabolising system at first harvesting time point at precipitating concentrations, which is assumed to be of no biological relevance (Reimann and Gramlich, 2000).
In a QSAR prediction using Leadscope Model Applier (v3.0.2) the potential of DELTA-9(11)-Fluocortolone-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 DELTA-9(11)-Fluocortolone-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.
In a QSAR prediction using DEREK Nexus v6.1 the potential of DELTA-9(11)-Fluocortolone-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 DELTA-9(11)-Fluocortolone-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.
In a micronucleus test fluocortolone was applied intraperitoneally at dose levels of 0, 75, 150 or 300 mg/kg bw to mice. Bone marrow smears were prepared at 24 and 48 hours after application and 2000 polychromatic erythrocytes were scored for micronuclei per animal. Slight to moderate apathy was seen as a sign of systemic toxicity dose dependently in all treated groups. Fluocortolone did not show any mutagenic/clastogenic potential under the test conditions (Reimann and Bokowsky, 1997).
Justification for classification or non-classification
Based on the study results of genetic toxicity in vitro tests with the read-across substance fluocortolone classification of delta-9(11)-fluocortolone-valerate is not required according to Regulation (EC) No. 1272/2008 (CLP).
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