Registration Dossier
Registration Dossier
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EC number: 211-889-1 | CAS number: 705-86-2
Endpoint summary
Administrative data
Key value for chemical safety assessment
Genetic toxicity in vitro
Description of key information
Bacterial reverse mutation test:
The registered substance, Decan-5-olide (CAS No. 705-86-2) tested non-mutagenic (negative) in Salmonella Typhimurium 1535, TA 1537, TA 98, TA 100 and TA 102 tester strains in the presence and absence of S9 metabolic activation system. The test was performed according to OECD TG 471 and in compliance with GLP.
In vitro mammalian chromosome aberration study:
The registered substance, Decan-5-olide (CAS No. 705-86-2) was tested non-clastogenic (negative) as it did not induce structural chromosomal aberration in human peripheral blood lymphocyte either in the presence (1% and 2%) or absence of S9 metabolic activation system. The test was performed according to OECD TG 473 and in compliance with GLP.
In vitro mammalian cell gene mutation assay:
The test substance, Decan-5-olide (CAS No. 705-86-2) was tested non-mutagenic (negative) in cultured Chinese Hamster Ovary (CHO) cells in the presence and absence of S9 metabolic activation when CHO cells were exposed at ≤ 5.0 mM for 3 hrs or more. The test was performed according to OECD TG 476 and in compliance with GLP.
Link to relevant study records
- Endpoint:
- in vitro gene mutation study in bacteria
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 21 May 2020 to 29 June 2020
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Justification for type of information:
- The study contains experimental data with the registered substance.
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Version / remarks:
- Adopted: July 21st, 1997
- GLP compliance:
- yes
- Type of assay:
- bacterial reverse mutation assay
- Specific details on test material used for the study:
- Appearance: Colorless clear liquid
Purity (GC): 98.4% - Target gene:
- Histidine Operon
- Species / strain / cell type:
- S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
- Details on mammalian cell type (if applicable):
- Not applicable
- Metabolic activation:
- with and without
- Metabolic activation system:
- Type and composition of metabolic activation system:
- source of S9: Aroclor 1254 induced S9 was procured from Defence Research and Development Establishment, Nagpur (India)
- method of preparation of S9 mix : Appropriate quantity of S9 supernatant was mixed with S9 cofactor solution, which contains D-glucose-6-phosphate 0.8 g, β-NADP 1.75 g, MgCl2 1.0 g, KCl 1.35 g, Na2HPO4 6.4 g, NaH2PO4.H2O 1.4 g in 500 ml of distilled water
- concentration or volume of S9 mix and S9 in the final culture medium
: 10% v/v
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability): Metabolic capability of S9 is certified. - Test concentrations with justification for top dose:
- Test concentrations:
0,0 (NC), 0.0 (VC) 0.050, 0.158, 0.501, 1.582, and 5.000 mg/plate
Justification:
Test concentrations were selected based on a preliminary cytotoxicity experiment. This pre-experiment was performed with strains TA 98 and TA 100. Eight concentrations (0.001, 0.005, 0.015, 0.050, 0.158, 0.501, 1.582 and 5.000 mg/plate) were tested for toxicity and mutation induction with 3 plates each (triplicates). The cytotoxicity was detected as a reduction in the number of spontaneous revertants or inhibition of the bacterial background lawn growth. - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The test chemical was solulble in DMSO at 50 mg/ml. - Untreated negative controls:
- yes
- Remarks:
- Distilled water
- Negative solvent / vehicle controls:
- yes
- Remarks:
- Dimethyl sulfoxide
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- sodium azide
- methylmethanesulfonate
- other: 4-Nitro-o-phenylenediamine for strains TA1537, TA98 (without metabolic activation) 2-Aminoanthracene for strains TA 1535, TA1537, TA98, TA 100 and TA102
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in agar (plate incorporation- Trial I); preincubation (Trial II)
DURATION
- Preincubation period: 60 min
- Exposure duration: 48 hrs
- Expression time (cells in growth medium): 48 hrs
- Selection time (if incubation with a selection agent): NA
- Fixation time (start of exposure up to fixation or harvest of cells): NA
SELECTION AGENT (mutation assays): NA
SPINDLE INHIBITOR (cytogenetic assays): NA
STAIN (for cytogenetic assays): NA
NUMBER OF REPLICATIONS: Each concentration, including the negative, vehicle and positive controls was tested in triplicates in two independent experiments (Trial I-II).
METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: NA
DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other: Cytotoxicity was determined as a reduction in revertant counts and /or inhibition of the background lawn growth. - Evaluation criteria:
- The substance was considered as a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA98, TA100, and TA102) or thrice (strains TA1535 and TA1537) the colony count of the corresponding vehicle/(solvent) control was observed. A dose-dependent increase was considered biologically relevant if the threshold is exceeded at more than one concentration. An increase exceeding the threshold at only one concentration was judged as biologically relevant if reproduced in an independent second experiment. A dose-dependent increase in the number of revertant colonies below the threshold was regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remained within the historical range of negative control and vehicle control, such an increase was not considered biologically relevant.
- Statistics:
- The mean values of the plates for each concentration together with standard deviation were compared with the spontaneous reversion rates of solvent treated cultures. Microsoft Office Excel-based calculation was used for descriptive statistical analysis.
- Key result
- Species / strain:
- S. typhimurium, other: TA1535, TA1537, TA98, TA100, TA102
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- Reduction in the number of revertant counts was seen at 5 mg/plate with and without S9 mix in Trial I and Trial II.
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- True negative controls validity:
- not examined
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH: No data available
- Data on osmolality: No data available
- Possibility of evaporation from medium: No data available
- Water solubility: Insoluble in water, but soluble in DMSO at 50 mg/ml.
- Precipitation: No precipitation was observed in 5 mg/plate concentration.
RANGE-FINDING/SCREENING STUDIES (if applicable):
To evaluate the cytotoxicity of the substance, a pre-experiment was performed with strains TA 98 and TA 100 according to the plate incorporation methods. Bacterial cells were exposed to the concentrations of 0.0 (NC), 0.0 (VC), 0.001, 0.005, 0.015, 0.050, 0.158, 0.501, 1.582 and 5 mg/plate for 48 hours using triplicates.
At concentrations of 0.001-0.158 mg/plate, no reduction in colony count or clearing of the background lawn was observed. At 0.501 mg/plate, there was no decrease in revertant counts but a slight inhibition of the background lawn growth. At 1.582 mg/plate, moderate inhibition of the background lawn and no substantial decrease in the number of revertant colonies were seen. At 5 mg/plate marked reduction in the number of revertant counts and moderate inhibition of the background lawn was observed. Hence, the mutagenicity test was performed with the following test concentrations: 0.050, 0.158, 0.501, 1.582, and 5.000 mg/plate.
STUDY RESULTS
- Positive control data : Positive controls induced an unequivocal increase in revertant counts in all the five tester strains compared to respective controls used.
HISTORICAL CONTROL DATA (with ranges, means and standard deviation, and 95% control limits for the distribution as well as the number of data)
- Positive historical control data: Please refer to the table.
- Negative (solvent/vehicle) historical control data: Please refer to the table. - Remarks on result:
- other: No mutagenic potential
- Conclusions:
- The registered substance, Decan-5-olide (CAS No. 705-86-2) tested non-mutagenic (negative) in Salmonella Typhimurium 1535, TA 1537, TA 98, TA 100 and TA 102 tester strains in the presence and absence of S9 metabolic activation system. The test was performed according to OECD TG 471 and in compliance with GLP.
- Executive summary:
The potential of Decan-5-olide (CAS No. 705-86-2) to induce point mutations or frameshifts with the histidine operon was tested in Salmonella Typhimurium 1535, TA 1537, TA 98, TA 100 and TA 102 tester strains in the presence and absence of a metabolic activation system. Cofactor-supplemented liver S9 microsomal fraction was used as an exogenous metabolic activation system. Dimethyl sulfoxide was selected as a vehicle of the test substance. Test concentrations were selected based on a preliminary cytotoxicity test. This pre-experiment was performed with strains TA 98 and TA 100 according to the plate incorporation methods. Bacterial cells were exposed to the substance at concentrations of 0.0 (NC), 0.0 (VC), 0.001, 0.005, 0.015, 0.050, 0.158, 0.501, 1.582 and 5 mg/plate for 48 hours using triplicates. At concentrations of 0.001-0.158 mg/plate, no reduction in colony count or clearing of the background lawn was observed. At 0.501 mg/plate, there was no decrease in revertant counts but a slight inhibition of the background lawn growth. At 1.582 mg/plate, moderate inhibition of the background lawn and no substantial decrease in the number of revertant colonies were seen. At 5 mg/plate marked reduction in the number of revertant counts and moderate inhibition of the background lawn was observed. Hence, the mutagenicity test was performed with the following test concentrations: 0.050, 0.158, 0.501, 1.582, and 5.000 mg/plate with and without S9 metabolic activation. The main test consisted of two trials.
Trial I was performed according to the plate incorporation method using five test concentrations along with the negative, vehicle, and concurrent positive controls with the remaining three strains, i.e., TA1537, TA1535, and TA102. For TA98 and TA 100 the revertant colony counts were directly incorporated in the Trial-I from the pre-experiment up to the required five concentrations (from 0.050 mg/plate to 5.000 mg/plate). Trial-II was performed independently with all the five tester strains and the negative, vehicle, and positive controls by preincubation method to confirm the results of Trial-I. Results: No substantial increase in the number of revertant colonies compared to the vehicle control was observed at concentrations tested in any of the five tester strains, either in the presence or absence of S9 metabolic activation in both trials. Cytotoxicity was detected at 5 mg/plate as a reduction in the number of revertant colonies. The positive controls induced unequivocal increases in revertant counts in all the five tester strains compared to vehicle controls in Trial I and Trial II. Conclusion: The test chemical did not induce gene mutations by base pair changes or frameshifts in the genome of the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and TA 102 in the presence and absence of S9 metabolic activation system. The test was performed according to OECD TG 471.
- Endpoint:
- in vitro cytogenicity / chromosome aberration study in mammalian cells
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 30 December 2019 to 10 March 2020
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Justification for type of information:
- The study contains experimental results with the registered substance.
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 473 (In Vitro Mammalian Chromosomal Aberration Test)
- Version / remarks:
- Adopted on 29th July 2016
- GLP compliance:
- yes
- Type of assay:
- in vitro mammalian chromosome aberration test
- Specific details on test material used for the study:
- Purity: 99.10%
- Species / strain / cell type:
- lymphocytes: Human peripheral blood lymphocytes
- Details on mammalian cell type (if applicable):
- CELLS USED
- Type and source of cells: Human blood
For lymphocytes:
- Sex, age and number of blood donors: 24 to 33 years of age range
- Whether whole blood or separated lymphocytes were used: Whole blood
- Whether blood from different donors were pooled or not: No data
MEDIA USED
- Type and composition of media, CO2 concentration, humidity level, temperature, if applicable: RPMI - 1640
- Properly maintained: Yes
- Periodically checked for Mycoplasma contamination: Yes
- Periodically checked for karyotype stability: Yes
- Periodically 'cleansed' against high spontaneous background: NA - Metabolic activation:
- with and without
- Metabolic activation system:
- Type and composition of metabolic activation system:
- source of S9 : Aroclor 1254-induced S9 was procured from Defence Research and Development Establishment, Nagpur
- method of preparation of S9 mix : Appropriate quantity of S9 supernatant was mixed with S9 cofactor solution, which contains 0.80 g of D-glucose-6-phosphate, 1.00 g of MgCl2, 1.35 g of KCl, 6.40 g of Na2HPO4, 1.40 g of NaH2PO4.H2O, 1.75 g of β-NADP in 500 mL of RO water. During the experiment, S9 mix was freshly prepared.
- concentration or volume of S9 mix and S9 in the final culture medium: 1 % v/v for Phase I of experiment and 2 % v/v for Phase II of experiment.
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability): No data - Test concentrations with justification for top dose:
- Test concentrations:0.0 (C), 0.0 (VC), 0.250, 0.5 and 1.0 mg/mL.
Justification:
The test concentrations were selected based on a preliminary cytotoxicity test. The cytotoxicity was assessed at test concentrations of 0.0 (NC), 0.0 (VC), 0.5 (T1), 1.0 (T2) and 2.0 (T3) mg/mL both in the presence and absence of S9 metabolic activation. Cytotoxicity was determined by a reduction in the mitotic index at each test concentration compared to vehicle control data. The results showed extensive cytotoxicity at 2 mg/ml; thus, 1.0 mg/ml was selected as the highest test concentration for the CA test. - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The test chemical was soluble in DMSO at 200 mg/ml. - Untreated negative controls:
- yes
- Remarks:
- Distilled water
- Negative solvent / vehicle controls:
- yes
- Remarks:
- DMSO
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- ethylmethanesulphonate
- other: Cyclophosphamide monohydrate, 30 µg/ml, with S9 mix
- Details on test system and experimental conditions:
- Number of cultures used: Duplicate cultures were used for each test concentration and control.
Number of independent experiments: Two (Phase 1 and Phase 2)
FOR CHROMOSOME ABERRATION AND MICRONUCLEUS:
- Spindle inhibitor (cytogenetic assays): indicate the identity of mitotic spindle inhibitor used (e.g., colchicine), its concentration and, duration and period of cell exposure.: Colcemide (0.3 µg/mL) was added 3 hour prior to harvesting and kept under incubation at 37 ± 2 °C
- Methods of slide preparation and staining technique used including the stain used (for cytogenetic assays): The slides were prepared by dropping the cell suspension onto a clean ice-chilled microscope slide. The slides were dried on the slide warmer and labelled. Two slides were made from each sample. The cells were stained with 5 % fresh Giemsa stain in phosphate buffer and mounted using DPX. All slides, including those of positive, vehicle and negative controls, were independently coded before microscopic analysis.
- Number of cells spread and analysed per concentration (number of replicate cultures and total number of cells scored): 1000 cells per slides
- Criteria for scoring chromosome aberrations (selection of analysable cells and aberration identification): Chromosomal and chromatid breaks, acentric fragments, deletions, exchanges, pulverisation, polyploidy (including endoreduplication) and disintegrations were recorded as structural chromosomal aberrations. Gaps were recorded as well, but they were not included in the calculation of the aberration rates. Only metaphases with 46 2 centromere regions were included in the analysis. To describe a cytotoxic effect the mitotic index (% cells in mitosis) were determined.
- Determination of polyploidy: Yes
- Determination of endoreplication: Yes
METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method, e.g.: background growth inhibition; mitotic index (MI); relative population doubling (RPD); relative increase in cell count (RICC); replication index; cytokinesis-block proliferation index; cloning efficiency; relative total growth (RTG); relative survival (RS); other: Mitotic Index - Evaluation criteria:
- A test item was classified as clastogenic if:
- At least one of the test concentrations exhibits a statistically significant increase compared with the concurrent vehicle control
- If the increase is dose-related
- Any of the results are outside the historical vehicle control range.
A test item was not classified as non - clastogenic if:
- None of the test concentrations exhibits a statistically significant increase compared with the concurrent vehicle control
- If there is no dose-related increase
- All results are inside the historical vehicle control range.
Statistical significance was confirmed using the non-parametric Mann-Whitney Test. However, both biological and statistical significance were considered together.
If the above-mentioned criteria for the test item were not clearly met, the classification with regard to the historical data and the biological relevance was discussed and/or a confirmatory experiment was performed. - Statistics:
- Statistical significance at the p < 0.05 was evaluated by means of the non-parametric Mann-Whitney test.
- Key result
- Species / strain:
- lymphocytes: Human peripheral lymphocytes
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- At 1.0 mg/ml, the cytotoxicity was 56.08% (-S9), 58.54% (+S9) and 57.57% (-S9), 55.85% (+S9) in Phase 1 and Phase 2, respectively.
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- True negative controls validity:
- not examined
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH: The pH of the test item in the culture medium was assessed at 0 h and 4 hrs after incubation at 37 ± 2 °C. No significant change in pH was observed at 0 h and 4 hrs when compared with negative controls.
- Water solubility: Insoluble in water but soluble in DMSO at 200 mg/ml.
- Precipitation and time of the determination: Precipitation was not observed at 2 mg/ml it was selected as the highest test concentration in the preliminary cytotoxicity assay.
RANGE-FINDING/SCREENING STUDIES (if applicable):
To evaluate the cytotoxicity of the test item, a preliminary cytotoxicity test was performed both in the presence and in absence of the S9 metabolic activation system (1%). Cells were exposed to test concentrations of 0.0 (NC), 0.0 (VC), 0.5, 1.0 and 2.0 mg/ml and the cytotoxicity was determined by calculation of the Mitotic index (MI) at each test concentration and negative, vehicle and positive controls. The concentration which yielded 55±5% cytotoxicity (i.e., reduction in MI to 45±5% of the vehicle control) was selected as the highest test concentration. At 1.0 mg/ml, 54.94% and 57.80% of cytotoxicity were observed in the presence and absence of S9 metabolic activation, respectively. Hence, 1.0 mg/ml was selected as the highest concentration for the main study both in the presence and in the absence of metabolic activation.
Chromosome aberration test (CA) in mammalian cells:
- Results from cytotoxicity measurements:
o For lymphocytres in primary cultures: mitotic index (MI): In the absence of S9 mix, the mean mitotic index observed was 10.05 (NC), 9.93 (VC), 5.43, (T1), 419 (T2), 3.58 (T3) and 8.20 (PC). In the presence of S9 mix, the mean mitotic index observed was 10.195 (NC), 9.92 (VC), 5.49 (T1), 4.47 (T2), 3.03 (T3) and 8.49 (PC).
HISTORICAL CONTROL DATA (with ranges, means and standard deviation, and 95% control limits for the distribution as well as the number of data)
- Positive historical control data: Please refer to tabular data in the section “Any other information on results inc tables”.
- Negative (solvent/vehicle) historical control data: Please refer to tabular the section “Any other information on results inc tables”.
- Remarks on result:
- other: No clastogenic potential
- Conclusions:
- The registered substance, Decan-5-olide (CAS No. 705-86-2) was tested non-clastogenic (negative) as it did not induce structural chromosomal aberration in human peripheral blood lymphocyte either in the presence (1% and 2%) or absence of S9 metabolic activation system. The test was performed according to OECD TG 473 and in compliance with GLP.
- Executive summary:
The clastogenic potential of Decan-5-olide (CAS No. 705-86-2) was tested according to OECD TG 473 in human peripheral blood lymphocytes and in the presence and absence of S9 metabolic activation. Dimethyl sulfoxide (DMSO) was selected as a vehicle of the test substance. The pH of the test item in the culture medium was assessed at 0 hours and 4 hours after incubation at 37°C. No significant change in pH was observed at times tested when compared with negative control. Hence, 2.0 mg/mL was selected as the highest concentration for a preliminary cytotoxicity test. Cytotoxicity was assessed at the concentrations of 0.0 (NC), 0.0 (VC), 0.5, 1.0 and 2.0 mg/ml along with concurrent positive control substances in the presence and absence of S9 metabolic activation. The cytotoxicity was determined by the calculation of the Mitotic index (MI) at each test concentration and negative, vehicle and positive controls. The concentration which yielded 55±5% cytotoxicity (i.e., reduction in MI to 45±5% of the vehicle control) was selected as the highest test concentration. At 1.0 mg/ml, 54.94% and 57.80% of cytotoxicity were observed in the presence and absence of S9 metabolic activation, respectively. Hence, 1.0 mg/ml was selected as the highest concentration for the main study both in the presence and in the absence of metabolic activation.
The main study was performed in two independent phases: In Phase I experiment, the duplicates cultures were exposed to the test chemical for 4 hours both in the absence and in the presence of S9 metabolic activation system (1%).The mean percentage of aberrant cells was 0.333 (NC), 0.333 (VC), 0.333 (at 0.250 mg/ml), 0.333 (at 0.5 mg/ml), 0.667 (at 1.0 mg/ml) and 10.667 (PC) in the absence of metabolic activation and 0.333 (NC), 0.333 (VC), 0.333 (0.250 mg/ml), 0.333 (0.5 mg/ml), 0.667 (1.0 mg/ml) and 10.333 (PC) in the presence of metabolic activation. Treatment with Ethyl methanesulfonate at the concentration of 600 µg/ml in the absence of metabolic activation and Cyclophosphamide monohydrate at the concentration of 30 µg/mL in the presence of metabolic activation (1%) caused significant increases in percent aberrant cells (≥10.33% aberrant cells). Even though the analysis did not reveal any statistical significance, the increases were biologically significant. Cytotoxicity was observed at 1.0 mg/ml in the presence and absence of S9 metabolic activation. The observed mean mitotic index in the absence of metabolic activation were 10.03, 9.79, 6.64, 5.11, 4.30 and 8.73 and in the presence of metabolic activation were 10.18, 9.89, 6.80, 5.59, 4.10 and 8.69 for 0.0 (NC), 0.0 (VC) 0.250 (T1), 0.5 (T2) and 1.0 (T3) mg/mL and 30 µg/mL(+S9), 600 ug/ml (-S9) of PC concentrations, respectively. The Phase II experiment was performed to confirm the negative results obtained in the absence and in the presence of metabolic activation in Phase I. In the absence of metabolic activation, test items concentrations used were 0.0 (NC), 0.0 (VC) 0.250, 0.5 and 1.0 mg/mL and 600 µg/mL (PC; ethylmethanesulfonate). In the presence of metabolic activation (2%), test items concentrations used were 0.0 (NC), 0.0 (VC) 0.250, 0.5 and 1.0 mg/mL and 30 µg/mL (PC; cyclophosphamide monohydrate).The duration of exposure to the test item in presence of a metabolic activation system was 4 hours and in absence of metabolic activation was 24 hours. The mean percent aberrant cells were 0.333 (NC), 0.333 (VC) 0.333, 0.667, 0.667 and 10.333 (PC) in the absence of metabolic activation and 0.333 (NC),0.667 (VC),0.333, 0.333, 0.667 and 10.000 (PC) in the presence of metabolic activation at the concentrations of 0.0 (NC), 0.0 (VC) 0.250, 0.5 and 1.0 mg/ml and positive control. Treatment with Ethyl methanesulfonate at the concentration of 600 µg/mL in the absence of metabolic activation and Cyclophosphamide monohydrate at the concentration of 30 µg/mL in the presence of metabolic activation (2%) caused significant increases in percent aberrant cells (≥10.00% aberrant cells). Though the analysis did not reveal any statistical significance, the increases were biologically significant. The increased frequency of aberrations observed in the concurrent positive control groups (Phase I and II) validated the sensitivity of the test system, the suitability of the methods and conditions employed in the experiment. Treatment with test item both in the absence and presence of S9 mix induced slight to moderate reductions of the mitotic index at low (0.250 mg/mL) and middle (0.5 mg/mL) doses. The observed mean mitotic index in the absence of metabolic activation were 10.13, 9.90,6.40,5.05,4.20and 8.29 and in the presence of metabolic activation were 10.03, 9.90,6.14,5.35,4.37and 8.68 for 0.0 (NC), 0.0 (VC) 0.250, 0.5 and 1.0 and 30 µg/mL(+S9), 600 ug/ml(-S9) of PC concentrations, respectively. Conclusion: The test chemical was found to be non-clastogenic (negative) as it did not induce structural chromosomal aberration in human peripheral blood lymphocytes both in the presence (1% and 2%) and in the absence of S9 metabolic activation.
- Endpoint:
- in vitro gene mutation study in mammalian cells
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Justification for type of information:
- Data is from study report
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
- GLP compliance:
- yes
- Type of assay:
- in vitro mammalian cell gene mutation test using the Hprt and xprt genes
- Target gene:
- Cells deficient in hypoxanthine-guanine phosphoribosyl transferase (HPRT) due to the mutation HPRT+/- to HPRT-/- are resistant to cytotoxic effects of 6-thioguanine (TG). HPRT proficient cells are sensitive to TG (which causes inhibition of cellular metabolism and halts further cell division since HPRT enzyme activity is important for DNA synthesis), so mutant cells can proliferate in the presence of TG, while normal cells, containing hypoxanthine-guanine phosphoribosyl transferase cannot. This in vitro test is an assay for the detection of forward gene mutations at the in hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus on the X chromosomes of hypodiploid, modal No. 20, CHO cells. Gene and chromosome mutations are considered as an initial step in the carcinogenic process.The hypodiploid CHO cells are exposed to the test item with and without exogenous metabolic activation. Following an expression time the descendants of the treated cell population are monitored for the loss of functional HPRT enzyme.HPRT catalyses the transformation of the purine analogue 6-thioguanine (TG) and thus rendering the analogue cytotoxic to normal cells. Hence, cells with mutations in the HPRT gene cannot phosphoribosylate the analogue and survive treatment with TG.Therefore, mutated cells are able to proliferate in the presence of TG whereas the non-mutated cells die. However, the mutant phenotype requires a certain period of time before it is completely expressed. The phenotypic expression is achieved by allowing exponential growth of the cells for 7 days.
- Species / strain / cell type:
- Chinese hamster Ovary (CHO)
- Details on mammalian cell type (if applicable):
- Cell line used: Chinese Hamster Ovary (CHO) cells
- Type and identity of media: Ham’s F12K (Kaighn’s) Medium including 2 mM Glutamine supplemented with 10% Foetal Bovine Serum (FBS; qualified, EU-approved, South America origin), penicillin (50 U/ml) and streptomycin (50 µg/ml).
- Properly maintained: Yes
- Periodically checked for Mycoplasma contamination: No detection for mycoplasma was performed
- Periodically checked for karyotype stability: No detection for karyotype stability was performed - Additional strain / cell type characteristics:
- other: Hypodiploid, modal No. 20
- Cytokinesis block (if used):
- Not specified
- Metabolic activation:
- with and without
- Metabolic activation system:
- S9 liver microsomal fraction obtained from Arcolor 1254-induced male Sprague-Dawley rats (Supplier: Molecular Toxicology Inc. via Trinova Biochem GmbH, Giessen, Germany)
- Test concentrations with justification for top dose:
- 0, 0.5, 1.0, 2.5 or 5.0 mM
- Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: Ethanol
- Justification for choice of solvent/vehicle:Phenethyl phenylacetate was easily dissolved in ethanol. - Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- Remarks:
- Ethanol
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- 7,12-dimethylbenzanthracene
- other: N-ethyl-N-nitrosourea (ENU)
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: In medium with pre-incubation
DURATION- Preincubation period:One week involving 3 days of incubation with Hypoxanthine-aminopterin-thymidine (HAT) in medium as a mutant cleansing stage, followed by overnight incubation with hypoxanthine-thymidine (HT) in medium prior to a 3-4 days incubation in regular cell medium. After seeding and prior to treatment, the mutant-free cells were incubated for an additional of 24 hours.
- Exposure duration:3 hours
- Expression time (cells in growth medium): 7 days
- Selection time (if incubation with a selection agent): 14 days
- Fixation time (start of exposure up to fixation or harvest of cells): 7 days (harvest of cells)
SELECTION AGENT (mutation assays): 6-thioguanine (TG)
SPINDLE INHIBITOR (cytogenetic assays): Not applicable
STAIN (for cytogenetic assays): Crystal violet
NUMBER OF REPLICATIONS: A minimum of 2 replicates per dose concentration including negative and positive control.
NUMBER OF CELLS EVALUATED: 5 x 10 E5 cells were plated 7 days after treatment and whatever cells left, after 14 days of incubation with the selection medium, were evaluated.
DETERMINATION OF CYTOTOXICITY- Method: mitotic index; cloning efficiency; relative total growth; other: After being exposed to the test chemical for 3 hours, in the absence or presence of S9, cells were trypsinized and 0.5 x 10 E5 cells per well was seeded in duplicates from two parallel duplicate cultures into 6-well plates in fresh medium. The relative total growth and cytotoxicity was evaluated 24 and 48 hours after seeding.
- OTHER: - Rationale for test conditions:
- No data
- Evaluation criteria:
- The plates were scored for total number of colonies by manual counting. As a result, the mutation frequency could be calculated
- Statistics:
- Mean were observed.
- Key result
- Species / strain:
- Chinese hamster Ovary (CHO)
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- No data
- Remarks on result:
- other: No mutagenic effect were observed.
- Conclusions:
- The test substance, Decan-5-olide (CAS No. 705-86-2) was tested non-mutagenic (negative) in cultured Chinese Hamster Ovary (CHO) cells in the presence and absence of S9 metabolic activation when CHO cells were exposed at ≤ 5.0 mM for 3 hrs or more. The test was performed according to OECD TG 476 and in compliance with GLP.
- Executive summary:
An in vitro mammalian cell gene mutation study was designed and conducted to determine the genotoxicity profile of the given test chemical as per OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test) when administered to Chinese Hamster Ovary (CHO) cells.
A preliminary dose-finding study was conducted prior to the main study. A range of different test chemical concentrations were tested in 96-well plates and analyzed by two commonly used assays, i.e. the colorimetric assay of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and the bicinchoninic acid (BCA) assay to assess cell viability and protein concentration, respectively.
From the basis of the results from the MTT and BCA assays, test concentrations of the test chemical was chosen to be included in the gene toxicity test.
In the genotoxicity test, the given test chemical was administered to CHO cells for 3 hrs at the dose levels of 0, 0.5, 1.0, 2.5 or 5.0 mM and in the absence or presence of exogenous metabolic activation. CHO cells representing the negative controls were exposed to the vehicle. Positive controls, such as N-ethyl-N-nitrosourea (ENU) experiments without metabolic activation and 7,12-dimethylbenz(a) anthracene in experiments with metabolic activation, were also included in each test.
The results showed indication of gene mutations occurring only in the positive control ENU while no other treatment gave rise to gene toxicity. No cytotoxic effects was observed in CHO cells when exposed towards test chemical for 3 hrs in the absence or presence of S9 liver microsomal fraction.
Based on the results of the current study, it can be concluded that the given test chemical does not give rise to gene mutations when exposed at ≤ 5.0 mM for 3 hrs or more, and it does not give rise to cytotoxicity in CHO cells at concentrations of ≤ 5.0 mM.
Referenceopen allclose all
TABLE 1 : REVERTANT COUNT FOR PRE-EXPERIMENT
Dose (mg/plate) |
R |
Absence (-S9) of Metabolic Activation |
Presence (+S9) of Metabolic Activation |
||
TA100 |
TA 98 |
TA100 |
TA 98 |
||
NC (0.00) |
R1 |
94 |
14 |
104 |
15 |
R2 |
95 |
19 |
108 |
19 |
|
R3 |
92 |
15 |
103 |
21 |
|
VC (0.00) |
R1 |
125 |
26 |
142 |
31 |
R2 |
126 |
28 |
137 |
28 |
|
R3 |
129 |
31 |
141 |
33 |
|
T1 (0.001) |
R1 |
103 |
23 |
136 |
19 |
R2 |
108 |
26 |
130 |
25 |
|
R3 |
104 |
21 |
129 |
21 |
|
T2 (0.005) |
R1 |
114 |
18 |
125 |
19 |
R2 |
116 |
24 |
129 |
19 |
|
R3 |
111 |
20 |
124 |
23 |
|
T3 (0.015) |
R1 |
100 |
25 |
119 |
27 |
R2 |
105 |
26 |
123 |
29 |
|
R3 |
103 |
21 |
126 |
30 |
|
T4 (0.050) |
R1 |
96 |
29 |
116 |
32 |
R2 |
100 |
24 |
118 |
28 |
|
R3 |
102 |
26 |
120 |
25 |
|
T5 (0.158) |
R1 |
113 |
19 |
120 |
29 |
R2 |
118 |
26 |
124 |
27 |
|
R3 |
114 |
23 |
126 |
24 |
|
T6 (0.501) |
R1 |
120 ++++ |
20 ++++ |
119 ++++ |
20 ++++ |
R2 |
117 ++++ |
22 ++++ |
122 ++++ |
25 ++++ |
|
R3 |
123 ++++ |
24 ++++ |
115 ++++ |
21 ++++ |
|
T7 (1.582) |
R1 |
114 +++ |
26 +++ |
125 +++ |
18 +++ |
R2 |
109 +++ |
25 +++ |
120 +++ |
25 +++ |
|
R3 |
111 +++ |
23 +++ |
122 +++ |
21 +++ |
|
T8 (5.000) |
R1 |
81 +++ |
15 +++ |
93 +++ |
15 +++ |
R2 |
87 +++ |
12 +++ |
97 +++ |
19 +++ |
|
R3 |
85 +++ |
16 +++ |
100 +++ |
13 +++ |
|
PC |
R1 |
908 |
516 |
1136 |
824 |
R2 |
1012 |
492 |
1056 |
752 |
|
R3 |
972 |
524 |
1128 |
800 |
|
NC = Negative control, VC = Vehicle Control, PC = Positive control, R = Replicate, ++++=Slight inhibition, +++ = Moderate inhibition.
T = Test concentration (T8: Highest, T1: Lowest)
4-Nitro-o-phenylenediamine [10μg/plate]: TA 98
Sodium azide [10μg/plate]: TA 100,
2-Aminoanthracene [2.5μg/plate]: TA98, TA100
TABLE 2 - REVERTANT COUNT IN PLATE INCORPORATION METHOD (TRIAL- I)
Dose (mg/plate) |
R |
In the Presence (+S9) of Metabolic Activation |
||||
TA 1537 |
TA 1535 |
TA 98 |
TA 100 |
TA 102 |
||
NC (0.00) |
R1 |
4 |
11 |
15 |
104 |
240 |
R2 |
5 |
8 |
19 |
108 |
228 |
|
R3 |
5 |
10 |
21 |
103 |
234 |
|
VC (0.00) |
R1 |
7 |
14 |
31 |
142 |
296 |
R2 |
6 |
16 |
28 |
137 |
285 |
|
R3 |
8 |
13 |
33 |
141 |
291 |
|
T1 (0.050) |
R1 |
5 |
14 |
32 |
116 |
281 |
R2 |
5 |
14 |
28 |
118 |
295 |
|
R3 |
7 |
12 |
25 |
120 |
269 |
|
T2 (0.158) |
R1 |
7 |
12 |
29 |
120 |
268 |
R2 |
6 |
16 |
27 |
124 |
257 |
|
R3 |
5 |
13 |
24 |
126 |
238 |
|
T3 (0.501) |
R1 |
6 |
10 |
20 |
119 |
235 |
R2 |
8 |
10 |
25 |
122 |
248 |
|
R3 |
5 |
12 |
21 |
115 |
252 |
|
T4 (1.582) |
R1 |
5 |
14 |
18 |
125 |
267 |
R2 |
5 |
11 |
25 |
120 |
284 |
|
R3 |
6 |
12 |
21 |
122 |
278 |
|
T5 (5.000) |
R1 |
4 |
9 |
15 |
93 |
251 |
R2 |
4 |
8 |
19 |
97 |
246 |
|
R3 |
5 |
11 |
13 |
100 |
239 |
|
PC |
R1 |
152 |
320 |
824 |
1136 |
1784 |
R2 |
180 |
424 |
752 |
1056 |
1680 |
|
R3 |
168 |
408 |
800 |
1128 |
1624 |
|
Dose (mg/plate) |
R |
In the Absence (-S9) of Metabolic Activation |
||||
TA 1537 |
TA 1535 |
TA 98 |
TA 100 |
TA 102 |
||
NC (0.00) |
R1 |
3 |
9 |
14 |
94 |
226 |
R2 |
3 |
10 |
19 |
95 |
235 |
|
R3 |
4 |
8 |
15 |
92 |
240 |
|
VC (0.00) |
R1 |
5 |
15 |
26 |
125 |
286 |
R2 |
6 |
17 |
28 |
126 |
306 |
|
R3 |
6 |
14 |
31 |
129 |
291 |
|
T1 (0.050) |
R1 |
5 |
14 |
29 |
96 |
261 |
R2 |
4 |
12 |
24 |
100 |
255 |
|
R3 |
5 |
12 |
26 |
102 |
269 |
|
T2 (0.158) |
R1 |
5 |
12 |
19 |
113 |
251 |
R2 |
6 |
11 |
26 |
118 |
229 |
|
R3 |
4 |
14 |
23 |
114 |
243 |
|
T3 (0.501) |
R1 |
5 |
14 |
20 |
120 |
262 |
R2 |
5 |
15 |
22 |
117 |
248 |
|
R3 |
6 |
12 |
24 |
123 |
269 |
|
T4 (1.582) |
R1 |
5 |
10 |
26 |
114 |
224 |
R2 |
4 |
11 |
25 |
109 |
251 |
|
R3 |
4 |
13 |
23 |
111 |
238 |
|
T5 (5.000) |
R1 |
3 |
8 |
15 |
81 |
238 |
R2 |
5 |
8 |
12 |
87 |
252 |
|
R3 |
3 |
10 |
16 |
85 |
230 |
|
PC |
R1 |
112 |
1080 |
516 |
908 |
1608 |
R2 |
124 |
1032 |
492 |
1012 |
1584 |
|
R3 |
136 |
1008 |
524 |
972 |
1704 |
|
TABLE 3 - REVERTANT COUNT IN PRE-INCUBATION METHOD (TRIAL- II)
Dose (mg/plate) |
R |
In the Presence (+S9) of Metabolic Activation |
||||
TA 1537 |
TA 1535 |
TA 98 |
TA 100 |
TA 102 |
||
NC (0.00) |
R1 |
4 |
10 |
20 |
109 |
266 |
R2 |
5 |
9 |
24 |
113 |
281 |
|
R3 |
5 |
11 |
23 |
110 |
260 |
|
VC (0.00) |
R1 |
7 |
15 |
27 |
126 |
282 |
R2 |
7 |
18 |
30 |
120 |
295 |
|
R3 |
6 |
16 |
29 |
124 |
290 |
|
T1 (0.050) |
R1 |
5 |
12 |
24 |
120 |
267 |
R2 |
8 |
11 |
27 |
116 |
272 |
|
R3 |
5 |
14 |
25 |
119 |
256 |
|
T2 (0.158) |
R1 |
7 |
13 |
23 |
119 |
286 |
R2 |
6 |
11 |
22 |
116 |
294 |
|
R3 |
4 |
11 |
25 |
114 |
275 |
|
T3 (0.501) |
R1 |
6 |
14 |
23 |
120 |
283 |
R2 |
8 |
13 |
25 |
123 |
272 |
|
R3 |
5 |
11 |
26 |
119 |
263 |
|
T4 (1.582) |
R1 |
4 |
15 |
25 |
117 |
278 |
R2 |
5 |
13 |
21 |
115 |
283 |
|
R3 |
6 |
14 |
23 |
114 |
291 |
|
T5 (5.000) |
R1 |
6 |
9 |
19 |
120 |
255 |
R2 |
6 |
12 |
24 |
123 |
268 |
|
R3 |
4 |
10 |
23 |
121 |
261 |
|
PC |
R1 |
140 |
296 |
820 |
1624 |
1344 |
R2 |
136 |
256 |
936 |
1528 |
1440 |
|
R3 |
128 |
240 |
884 |
1496 |
1392 |
|
Dose (mg/plate) |
R |
In the Absence (-S9) of Metabolic Activation |
||||
TA 1537 |
TA 1535 |
TA 98 |
TA 100 |
TA 102 |
||
NC (0.00) |
R1 |
5 |
8 |
16 |
108 |
256 |
R2 |
5 |
8 |
18 |
105 |
260 |
|
R3 |
4 |
11 |
21 |
109 |
271 |
|
VC (0.00) |
R1 |
8 |
16 |
26 |
126 |
292 |
R2 |
6 |
17 |
30 |
125 |
286 |
|
R3 |
6 |
14 |
32 |
128 |
280 |
|
T1 (0.050) |
R1 |
7 |
15 |
24 |
123 |
278 |
R2 |
6 |
16 |
27 |
125 |
264 |
|
R3 |
5 |
12 |
21 |
121 |
260 |
|
T2 (0.158) |
R1 |
6 |
10 |
23 |
114 |
272 |
R2 |
5 |
10 |
25 |
118 |
283 |
|
R3 |
5 |
13 |
22 |
113 |
268 |
|
T3 (0.501) |
R1 |
5 |
14 |
26 |
119 |
264 |
R2 |
8 |
13 |
23 |
121 |
270 |
|
R3 |
6 |
14 |
28 |
123 |
282 |
|
T4 (1.582) |
R1 |
7 |
11 |
20 |
117 |
275 |
R2 |
5 |
10 |
20 |
120 |
280 |
|
R3 |
5 |
9 |
24 |
122 |
286 |
|
T5 (5.000) |
R1 |
5 |
14 |
19 |
118 |
276 |
R2 |
4 |
12 |
20 |
115 |
283 |
|
R3 |
4 |
11 |
22 |
116 |
272 |
|
PC |
R1 |
148 |
960 |
528 |
1112 |
1352 |
R2 |
152 |
1016 |
620 |
1000 |
1424 |
|
R3 |
160 |
928 |
592 |
1080 |
1480 |
|
TABLE 4: MEAN REVERTANT COUNT IN PLATE INCORPORATION METHOD (TRIAL-I)
Dose (mg/plate) |
In the Presence (+S9) of Metabolic Activation |
|||||||||
TA 1537 |
TA 1535 |
TA 98 |
TA 100 |
TA 102 |
||||||
MEAN |
SD |
MEAN |
SD |
MEAN |
SD |
MEAN |
SD |
MEAN |
SD |
|
NC (0.00) |
4.67 |
0.58 |
9.67 |
1.53 |
18.33 |
3.06 |
105.00 |
2.65 |
234.00 |
6.00 |
VC (0.00) |
7.00 |
1.00 |
14.33 |
1.53 |
30.67 |
2.52 |
140.00 |
2.65 |
290.67 |
5.51 |
T1 (0.050) |
5.67 |
1.15 |
13.33 |
1.15 |
28.33 |
3.51 |
118.00 |
2.00 |
281.67 |
13.01 |
T2 (0.158) |
6.00 |
1.00 |
13.67 |
2.08 |
26.67 |
2.52 |
123.33 |
3.06 |
254.33 |
15.18 |
T3 (0.501) |
6.33 |
1.53 |
10.67 |
1.15 |
22.00 |
2.65 |
118.67 |
3.51 |
245.00 |
8.89 |
T4 (1.582) |
5.33 |
0.58 |
12.33 |
1.53 |
21.33 |
3.51 |
122.33 |
2.52 |
276.33 |
8.62 |
T5 (5.000) |
4.33 |
0.58 |
9.33 |
1.53 |
15.67 |
3.06 |
96.67 |
3.51 |
245.33 |
6.03 |
PC |
166.67 |
14.05 |
384.00 |
56.00 |
792.00 |
36.66 |
1106.67 |
44.06 |
1696.00 |
81.19 |
Dose (mg/plate) |
In the Absence (-S9) of Metabolic Activation |
|||||||||
TA 1537 |
TA 1535 |
TA 98 |
TA 100 |
TA 102 |
||||||
MEAN |
SD |
MEAN |
SD |
MEAN |
SD |
MEAN |
SD |
MEAN |
SD |
|
NC (0.00) |
3.33 |
0.58 |
9.00 |
1.00 |
16.00 |
2.65 |
93.67 |
1.53 |
233.67 |
7.09 |
VC (0.00) |
5.67 |
0.58 |
15.33 |
1.53 |
28.33 |
2.52 |
126.67 |
2.08 |
294.33 |
10.41 |
T1 (0.050) |
4.67 |
0.58 |
12.67 |
1.15 |
26.33 |
2.52 |
99.33 |
3.06 |
261.67 |
7.02 |
T2 (0.158) |
5.00 |
1.00 |
12.33 |
1.53 |
22.67 |
3.51 |
115.00 |
2.65 |
241.00 |
11.14 |
T3 (0.501) |
5.33 |
0.58 |
13.67 |
1.53 |
22.00 |
2.00 |
120.00 |
3.00 |
259.67 |
10.69 |
T4 (1.582) |
4.33 |
0.58 |
11.33 |
1.53 |
24.67 |
1.53 |
111.33 |
2.52 |
237.67 |
13.50 |
T5 (5.000) |
3.67 |
1.15 |
8.67 |
1.15 |
14.33 |
2.08 |
84.33 |
3.06 |
240.00 |
11.14 |
PC |
124.00 |
12.00 |
1040.00 |
36.66 |
510.67 |
16.65 |
964.00 |
52.46 |
1632.00 |
63.50 |
Key:-
NC= Negative Control,VC= Vehicle Control,T =Test concentration (T5: Highest, T1: Lowest),R= Replicate
PC= Positive control
2-Aminoanthracene [2.5μg/plate]: TA
1537, TA1535, TA 98, TA 100
2- Aminoanthracene [10μg/plate]: TA
102
Sodium azide [10μg/plate]: TA 1535, TA 100
4-Nitro-o-phenylenediamine: TA 1537 [50μg/plate], TA 98 [10μg/plate]
Methyl methanesulfonate [4μl/plate]: TA 102
TABLE 5 : MEAN REVERTANT COUNT IN PRE-INCUBATION METHOD (TRIAL-II)
Dose (mg/plate) |
In the Presence (+S9) of Metabolic Activation |
|||||||||
TA 1537 |
TA 1535 |
TA 98 |
TA 100 |
TA 102 |
||||||
MEAN |
SD |
MEAN |
SD |
MEAN |
SD |
MEAN |
SD |
MEAN |
SD |
|
NC (0.00) |
4.67 |
0.58 |
10.00 |
1.00 |
22.33 |
2.08 |
110.67 |
2.08 |
269.00 |
10.82 |
VC (0.00) |
6.67 |
0.58 |
16.33 |
1.53 |
28.67 |
1.53 |
123.33 |
3.06 |
289.00 |
6.56 |
T1 (0.050) |
6.00 |
1.73 |
12.33 |
1.53 |
25.33 |
1.53 |
118.33 |
2.08 |
265.00 |
8.19 |
T2 (0.158) |
5.67 |
1.53 |
11.67 |
1.15 |
23.33 |
1.53 |
116.33 |
2.52 |
285.00 |
9.54 |
T3 (0.501) |
6.33 |
1.53 |
12.67 |
1.53 |
24.67 |
1.53 |
120.67 |
2.08 |
272.67 |
10.02 |
T4 (1.582) |
5.00 |
1.00 |
14.00 |
1.00 |
23.00 |
2.00 |
115.33 |
1.53 |
284.00 |
6.56 |
T5 (5.000) |
5.33 |
1.15 |
10.33 |
1.53 |
22.00 |
2.65 |
121.33 |
1.53 |
261.33 |
6.51 |
PC |
134.67 |
6.11 |
264.00 |
28.84 |
880.00 |
58.10 |
1549.33 |
66.61 |
1392.00 |
48.00 |
Dose (mg/plate) |
In the Absence (-S9) of Metabolic Activation |
|||||||||
TA 1537 |
TA 1535 |
TA 98 |
TA 100 |
TA 102 |
||||||
MEAN |
SD |
MEAN |
SD |
MEAN |
SD |
MEAN |
SD |
MEAN |
SD |
|
NC (0.00) |
4.67 |
0.58 |
9.00 |
1.73 |
18.33 |
2.52 |
107.33 |
2.08 |
262.33 |
7.77 |
VC (0.00) |
6.67 |
1.15 |
15.67 |
1.53 |
29.33 |
3.06 |
126.33 |
1.53 |
286.00 |
6.00 |
T1 (0.050) |
6.00 |
1.00 |
14.33 |
2.08 |
24.00 |
3.00 |
123.00 |
2.00 |
267.33 |
9.45 |
T2 (0.158) |
5.33 |
0.58 |
11.00 |
1.73 |
23.33 |
1.53 |
115.00 |
2.65 |
274.33 |
7.77 |
T3 (0.501) |
6.33 |
1.53 |
13.67 |
0.58 |
25.67 |
2.52 |
121.00 |
2.00 |
272.00 |
9.17 |
T4 (1.582) |
5.67 |
1.15 |
10.00 |
1.00 |
21.33 |
2.31 |
119.67 |
2.52 |
280.33 |
5.51 |
T5 (5.000) |
4.33 |
0.58 |
12.33 |
1.53 |
20.33 |
1.53 |
116.33 |
1.53 |
277.00 |
5.57 |
PC |
153.33 |
6.11 |
968.00 |
44.54 |
580.00 |
47.16 |
1064.00 |
57.69 |
1418.67 |
64.17 |
Key:-NC= Negative Control,VC= Vehicle Control,T =Test concentration (T5: Highest, T1: Lowest),R= Replicate
PC= Positive control
2-Aminoanthracene [2.5μg/plate]: TA
1537, TA1535, TA 98, TA 100
2- Aminoanthracene [10μg/plate]:TA
102
Sodium azide [10μg/plate]: TA 1535, TA 100
4-Nitro-o-phenylenediamine: TA 1537 [50μg/plate], TA 98 [10μg/plate]
Methyl methanesulfonate [4μl/plate]: TA 102
HISTORICAL CONTROL DATA
These data represent the laboratory's historical control data.
Trial I (Plate Incorporation Method) |
||||||
Strains |
Metabolic Activation |
Treatment |
Mean |
SD |
Max |
Min |
TA 1537 |
S9 + |
Negative control |
6 |
2 |
10 |
2 |
S9 - |
6 |
2 |
10 |
2 |
||
S9 + |
Solvent control |
6 |
2 |
10 |
2 |
|
S9 - |
6 |
2 |
10 |
2 |
||
S9 + |
Positive control |
168 |
38 |
245 |
92 |
|
S9 - |
175 |
43 |
261 |
89 |
||
TA 1535 |
S9 + |
Negative control |
12 |
3 |
18 |
7 |
S9 - |
12 |
3 |
18 |
7 |
||
S9 + |
Solvent control |
13 |
3 |
18 |
7 |
|
S9 - |
13 |
3 |
18 |
7 |
||
S9 + |
Positive control |
336 |
211 |
757 |
86 |
|
S9 - |
1200 |
263 |
1726 |
674 |
||
TA 98 |
S9 + |
Negative control |
24 |
6 |
36 |
11 |
S9 - |
23 |
6 |
35 |
11 |
||
S9 + |
Solvent control |
25 |
6 |
37 |
13 |
|
S9 - |
23 |
5 |
33 |
13 |
||
S9 + |
Positive control |
1099 |
312 |
1722 |
476 |
|
S9 - |
815 |
284 |
1383 |
248 |
||
TA 100 |
S9 + |
Negative control |
117 |
28 |
173 |
61 |
S9 - |
114 |
26 |
166 |
62 |
||
S9 + |
Solvent control |
116 |
28 |
172 |
60 |
|
S9 - |
113 |
26 |
165 |
61 |
||
S9 + |
Positive control |
1488 |
390 |
2268 |
709 |
|
S9 - |
1311 |
298 |
1906 |
715 |
||
TA 102 |
S9 + |
Negative control |
274 |
42 |
358 |
190 |
S9 - |
271 |
55 |
382 |
161 |
||
S9 + |
Solvent control |
279 |
65 |
409 |
150 |
|
S9 - |
277 |
82 |
442 |
112 |
||
S9 + |
Positive control |
1648 |
305 |
2258 |
1037 |
|
S9 - |
1896 |
364 |
2624 |
1168 |
||
Mean = mean value of revertants/plate, SD = standard deviation, Min = -2SD, Max = +2SD
8 HISTORICAL CONTROL DATA (Contd.)
Trial II (Pre-Incubation Method) |
||||||
Strains |
Metabolic Activation |
Treatment |
Mean |
SD |
Max |
Min |
TA 1537 |
S9 + |
Negative control |
6 |
2 |
10 |
2 |
S9 - |
6 |
2 |
10 |
2 |
||
S9 + |
Solvent control |
6 |
2 |
10 |
3 |
|
S9 - |
6 |
2 |
10 |
2 |
||
S9 + |
Positive control |
170 |
39 |
249 |
91 |
|
S9 - |
182 |
43 |
268 |
96 |
||
TA 1535 |
S9 + |
Negative control |
13 |
3 |
18 |
7 |
S9 - |
12 |
3 |
18 |
7 |
||
S9 + |
Solvent control |
13 |
3 |
18 |
8 |
|
S9 - |
13 |
3 |
18 |
7 |
||
S9 + |
Positive control |
299 |
197 |
694 |
145 |
|
S9 - |
1244 |
260 |
1765 |
724 |
||
TA 98 |
S9 + |
Negative control |
24 |
6 |
35 |
13 |
S9 - |
23 |
5 |
33 |
13 |
||
S9 + |
Solvent control |
24 |
5 |
35 |
14 |
|
S9 - |
23 |
5 |
32 |
14 |
||
S9 + |
Positive control |
1269 |
275 |
1819 |
719 |
|
S9 - |
740 |
210 |
1160 |
320 |
||
TA 100 |
S9 + |
Negative control |
117 |
25 |
166 |
67 |
S9 - |
113 |
23 |
159 |
66 |
||
S9 + |
Solvent control |
116 |
22 |
159 |
73 |
|
S9 - |
112 |
20 |
151 |
73 |
||
S9 + |
Positive control |
1469 |
347 |
2163 |
775 |
|
S9 - |
1352 |
263 |
1878 |
827 |
||
TA 102 |
S9 + |
Negative control |
281 |
32 |
345 |
218 |
S9 - |
276 |
28 |
331 |
220 |
||
S9 + |
Solvent control |
281 |
34 |
350 |
212 |
|
S9 - |
276 |
34 |
344 |
207 |
||
S9 + |
Positive control |
1595 |
287 |
2168 |
1022 |
|
S9 - |
1753 |
248 |
2248 |
1258 |
||
Mean = mean value of revertants/plate, SD = standard deviation, Min = -2SD, Max = +2SD
MITOTIC INDEX - CYTOTOXICITY TEST
Treatment |
R |
Mitotic Index (%) |
|||||||
In the Absence of Metabolic Activation (-S9) |
In the Presence of Metabolic Activation (1% S9) |
||||||||
Mitotic Index |
Mean |
SD |
Percent Reduction |
Mitotic Index |
Mean |
SD |
Percent Reduction |
||
NC |
R1 |
10.10 |
10.05 |
0.08 |
- |
10.09 |
10.19 |
0.15 |
- |
R2 |
9.99 |
10.30 |
|||||||
VC |
R1 |
9.87 |
9.93 |
0.08 |
- |
9.86 |
9.92 |
0.08 |
- |
R2 |
9.98 |
9.98 |
|||||||
T1 |
R1 |
4.99 |
5.43 |
0.63 |
45.25 |
5.80 |
5.49 |
0.43 |
44.61 |
R2 |
5.88 |
5.19 |
|||||||
T2 |
R1 |
4.40 |
4.19 |
0.30 |
57.80 |
4.31 |
4.47 |
0.22 |
54.94 |
R2 |
3.98 |
4.63 |
|||||||
T3 |
R1 |
3.65 |
3.58 |
0.11 |
63.98 |
2.46 |
3.03 |
0.80 |
69.43 |
R2 |
3.50 |
3.60 |
|||||||
PC |
R1 |
8.29 |
8.20 |
0.14 |
17.42 |
8.39 |
8.49 |
0.14 |
14.45 |
R2 |
8.10 |
8.58 |
|||||||
Key:R = Replicate,NC = Negative control, VC = Vehicle control,PC = Positive control,SD = Standard Deviation
SUMMARY OF MITOTIC INDEX - CA TEST
Treatment |
Mitotic Index (%) |
|||
Phase I |
||||
In the Absence of Metabolic Activation (-S9) |
In the Presence of Metabolic Activation (1% S9) |
|||
Mean |
SD |
Mean |
SD |
|
NC |
10.03 |
0.06 |
10.18 |
0.15 |
VC |
9.79 |
0.14 |
9.89 |
0.15 |
T1 (0.250 mg/mL) |
6.64 |
0.36 |
6.80 |
2.26 |
T2 (0.5 mg/mL) |
5.11 |
0.34 |
5.59 |
0.27 |
T3 (1.0 mg/mL) |
4.30 |
0.15 |
4.10 |
0.14 |
PC |
8.73 |
0.65 |
8.69 |
0.44 |
Treatment |
Mitotic Index (%) |
|||
Phase II |
||||
In the Absence of Metabolic Activation (-S9) |
In the Presence of Metabolic Activation (2% S9) |
|||
Mean |
SD |
Mean |
SD |
|
NC |
10.13 |
0.20 |
10.03 |
0.06 |
VC |
9.90 |
0.13 |
9.90 |
0.15 |
T1 (0.250 mg/mL) |
6.40 |
0.85 |
6.14 |
0.20 |
T2 (0.5 mg/mL) |
5.05 |
0.35 |
5.35 |
0.07 |
T3 (1.0 mg/mL) |
4.20 |
0.43 |
4.37 |
0.20 |
PC |
8.29 |
0.16 |
8.68 |
0.17 |
Key: NC = Negative control, VC = Vehicle controlPC = Positive control, MI = Mitotic Index, -S9 = In the absence of metabolic activation, +S9 = In the presence of metabolic activation
SUMMARY OF PERCENT ABERRANT CELLS
Treatment |
Percent Aberrant Cells |
|||
Phase I |
||||
In the Absence of Metabolic Activation (-S9) |
In the Presence of Metabolic Activation (1% S9) |
|||
Mean |
SD |
Mean |
SD |
|
NC |
0.333 |
0.471 |
0.333 |
0.471 |
VC |
0.333 |
0.471 |
0.333 |
0.471 |
T1 (0.250 mg/mL) |
0.333 |
0.471 |
0.333 |
0.471 |
T2 (0.5 mg/mL) |
0.333 |
0.471 |
0.333 |
0.471 |
T3 (1.0 mg/mL) |
0.667 |
0.000 |
0.667 |
0.000 |
PC |
10.333 |
0.471 |
10.333 |
0.471 |
Treatment |
Percent Aberrant Cells |
|||
Phase II |
||||
In the Absence of Metabolic Activation (-S9) |
In the Presence of Metabolic Activation (2% S9) |
|||
Mean |
SD |
Mean |
SD |
|
NC |
0.333 |
0.471 |
0.333 |
0.471 |
VC |
0.333 |
0.471 |
0.667 |
0.000 |
T1 (0.250 mg/mL) |
0.333 |
0.471 |
0.333 |
0.471 |
T2 (0.5 mg/mL) |
0.667 |
0.000 |
0.333 |
0.471 |
T3 (1.0 mg/mL) |
0.667 |
0.000 |
0.667 |
0.000 |
PC |
10.333 |
0.471 |
10.000 |
0.943 |
Key: NC = Negative Control, VC = Vehicle control, SD = Standard Deviation, PC = Positive Control
INDIVIDUAL OBSERVATION OF SLIDES FOR MITOTIC INDEX AND CHROMOSOME ABERRATIONS
Phase I [In the Absence of Metabolic Activation, (-S9)]
Treatment |
Culture No. |
Mitotic Index |
Frequencies of Aberration |
Total No. of Aberration |
Percentage of Aberrated Cells |
|
NC |
R1 |
10.07 |
1csb |
1 |
0.67 | |
R2 |
9.99 |
- |
0 |
0.00 | ||
VC |
R1 |
9.69 |
- |
0 |
0.00 | |
R2 |
9.89 |
1 fragment |
1 |
0.67 | ||
T1 (0.250 mg/mL) |
R1 |
6.39 |
- |
0 |
0.00 | |
R2 |
6.90 |
1 ctb |
1 |
0.67 | ||
T2 (0.5 mg/mL) |
R1 |
4.86 |
1 fragment |
1 |
0.67 | |
R2 |
5.35 |
- |
0 |
0.00 | ||
T3 (1.0 mg/mL) |
R1 |
4.40 |
1 ctb |
1 |
0.67 | |
R2 |
4.19 |
1 fragment |
1 |
0.67 | ||
PC |
R1 |
9.19 |
4 ctb, 2 cte, 2 ctg, 3 csb, 1 cse, 2 csg, 2 DC, 7 fragments |
23 |
11.33 | |
R2 |
8.28 |
3 ctb, 1 cte, 2 ctg, 2 csb, 1 cse, 2 csg, 1 DC, 1 AC, 9 fragments |
22 |
10.00 |
Phase I [In the Presence of Metabolic Activation (1% S9)]
Treatment |
Culture No. |
Mitotic Index |
Frequencies of Aberration |
Total No. of Aberration |
Percentage of Aberrated Cells |
NC |
R1 |
10.08 |
- |
0 |
0.00 |
R2 |
10.29 |
1 fragmnet |
1 |
0.67 |
|
VC |
R1 |
9.99 |
- |
0 |
0.00 |
R2 |
9.78 |
1 ctb |
1 |
0.67 |
|
T1 (0.250 mg/mL) |
R1 |
8.39 |
1 fragment |
1 |
0.67 |
R2 |
5.20 |
- |
0 |
0.00 |
|
T2 (0.5 mg/mL) |
R1 |
5.40 |
- |
0 |
0.00 |
R2 |
5.78 |
1 csb |
1 |
0.67 |
|
T3 (1.0 mg/mL) |
R1 |
4.20 |
1 fragment |
1 |
0.67 |
R2 |
4.00 |
1 ctg, 1 fragment |
2 |
0.67 |
|
PC |
R1 |
9.00 |
4 ctb, 1 cte, 2 ctg, 2csb,2 cse, 1 csg, 1 DC, 2 AC, 7 fragments |
22 |
10.67 |
R2 |
8.38 |
3 ctb, 2 cte, 2 ctg, 1 csb, 3 cse, 3 csg, 1 DC, 9 fragments |
24 |
10.00 |
Key: MI = Mitotic Index, ctg = Chromatid gap, ctb = Chromatid break, csg = Chromosomal gap, csb = Chromosomal break, DC = Dicentric, cte = Chromatid exchange, cse = Chromosome exchange, NC = Negative Control, VC = Vehicle Control, PC = Positive Control
INDIVIDUAL OBSERVATION OF SLIDES FOR MITOTIC INDEX AND CHROMOSOME ABERRATIONS (Contd.)
Phase II [In the Absence of Metabolic Activation (-S9)]
Treatment |
Culture No. |
Mitotic Index |
Frequencies of Aberration |
Total No. of Aberration |
Percentage of Aberrated Cells |
NC |
R1 |
10.27 |
- |
0 |
0.00 |
R2 |
9.99 |
1 fragment |
1 |
0.67 |
|
VC |
R1 |
9.80 |
1 ctb |
1 |
0.67 |
R2 |
9.99 |
- |
0 |
0.00 |
|
T1 (0.250 mg/mL) |
R1 |
5.80 |
1 cse |
1 |
0.67 |
R2 |
7.00 |
- |
0 |
0.00 |
|
T2 (0.5 mg/mL) |
R1 |
4.80 |
1 ctb |
1 |
0.67 |
R2 |
5.29 |
1 DC |
1 |
0.67 |
|
T3 (1.0 mg/mL) |
R1 |
3.90 |
1 ctb, 1 ctg |
2 |
0.67 |
R2 |
4.50 |
1 fragment |
1 |
0.67 |
|
PC |
R1 |
8.40 |
4 ctb, 1 cte, 3 ctg, 3 csb, 2 csg, 1 DC, 1 AC, 9 fragments |
25 |
10.00 |
R2 |
8.18 |
4 ctb, 2 cte, 5 ctg, 2 csb, 1 cse, 3 csg, 1DC, 1AC, 9 fragments |
28 |
10.67 |
Phase II [In the Presence of Metabolic Activation (2% S9)]
Treatment |
Culture No. |
Mitotic Index |
Frequencies of Aberration |
Total No. of Aberration |
Percentage of Aberrated Cells |
|
NC |
R1 |
9.99 |
1 fragment |
1 |
0.67 |
|
R2 |
10.07 |
- |
0 |
0.00 |
||
VC |
R1 |
9.79 |
1 ctb |
1 |
0.67 |
|
R2 |
10.00 |
1 fragment |
1 |
0.67 |
||
T1 (0.250 mg/mL) |
R1 |
6.00 |
- |
0 |
0.00 |
|
R2 |
6.29 |
1 csb |
1 |
0.67 |
||
T2 (0.5 mg/mL) |
R1 |
5.39 |
- |
0 |
0.00 |
|
R2 |
5.30 |
1 fragment |
1 |
0.67 |
||
T3 (1.0 mg/mL) |
R1 |
4.23 |
1 ctb |
1 |
0.67 |
|
R2 |
4.51 |
1 ctg, 1 fragment |
2 |
0.67 |
||
PC |
R1 |
8.80 |
3 ctb, 1 cte, 3 ctg, 3 csb, 1 cse, 2 csg, 1 DC, 1 AC, 11 fragments |
26 |
10.67 |
|
R2 |
8.57 |
2 ctb, 2 cte, 2 ctg, 1 csb, 2 cse, 2 csg, 1DC, 10 fragments |
22 |
9.33 |
Key: MI = Mitotic Index, ctg = Chromatid gap, ctb = Chromatid break, csg = Chromosomal gap, csb = Chromosomal break, DC = Dicentric, cte = Chromatid exchange, cse = Chromosome exchange, NC = Negative Control, VC = Vehicle Control, PC = Positive Control.
HISTORICAL DATA
HISTORICAL DATA FOR IN VITRO MAMMALIAN CHROMOSOMAL ABERRATION TEST (CA-H) |
||||||||||
S.No. |
Study No. |
Vehicle |
Phase I |
Phase II |
||||||
Absence of S9 |
Presence of S9 |
Absence of S9 |
Presence of S9 |
|||||||
Vehicle Control |
Positive Control |
Vehicle Control |
Positive Control |
Vehicle Control |
Positive Control |
Vehicle Control |
Positive Control |
|||
1 |
1151 |
DMSO |
0.000 |
9.000 |
0.000 |
10.500 |
0.000 |
9.000 |
0.000 |
8.000 |
2 |
1333 |
DMSO |
0.000 |
8.000 |
0.000 |
7.500 |
0.500 |
8.500 |
0.500 |
9.000 |
3 |
2060 |
DMSO |
0.500 |
8.000 |
0.000 |
7.000 |
1.500 |
6.500 |
0.000 |
9.000 |
4 |
2450 |
DMSO |
0.000 |
10.000 |
0.000 |
10.500 |
0.000 |
11.500 |
0.000 |
12.000 |
5 |
2452 |
DMSO |
0.000 |
10.000 |
0.000 |
8.500 |
0.000 |
9.500 |
0.000 |
8.500 |
6 |
3000 |
PBS |
0.000 |
7.500 |
0.000 |
8.500 |
0.000 |
11.000 |
0.000 |
10.000 |
7 |
3313 |
DMSO |
0.000 |
8.000 |
0.000 |
10.500 |
0.500 |
9.500 |
0.000 |
9.500 |
8 |
3422 |
DMSO |
0.000 |
9.000 |
0.500 |
10.000 |
1.000 |
9.500 |
1.000 |
8.500 |
9 |
3665 |
RPMI |
0.500 |
8.500 |
0.000 |
7.500 |
0.000 |
8.500 |
0.500 |
8.000 |
10 |
3801 |
Sodium Phosphate Buffer |
1.500 |
9.500 |
1.000 |
9.000 |
1.000 |
9.500 |
0.500 |
9.500 |
11 |
3862 |
DMSO |
1.500 |
9.500 |
1.000 |
9.000 |
1.000 |
9.500 |
0.500 |
9.500 |
12 |
4792 |
PBS |
0.500 |
7.500 |
0.500 |
8.500 |
0.500 |
8.500 |
0.000 |
8.000 |
13 |
4938 |
DMSO |
0.500 |
8.500 |
1.000 |
8.500 |
0.500 |
8.000 |
1.000 |
8.000 |
14 |
5123 |
DMSO |
0.333 |
9.000 |
0.667 |
8.667 |
0.333 |
9.667 |
0.333 |
9.000 |
15 |
5739 |
Ethyl alcohol |
0.333 |
10.333 |
0.333 |
10.000 |
0.333 |
9.667 |
0.333 |
10.667 |
16 |
5824 |
PBS |
0.333 |
10.000 |
0.333 |
11.000 |
0.333 |
9.333 |
0.333 |
10.000 |
17 |
6461 |
PBS |
0.333 |
10.000 |
0.333 |
9.667 |
0.333 |
9.000 |
0.333 |
10.000 |
18 |
6196 |
RPMI |
0.333 |
11.000 |
0.333 |
10.000 |
0.333 |
10.667 |
0.333 |
10.000 |
19 |
6121 |
DMSO |
0.667 |
8.667 |
0.667 |
9.667 |
0.667 |
9.667 |
0.667 |
9.333 |
6 HISTORICAL DATA (Contd.)
HISTORICAL DATA FOR IN VITRO MAMMALIAN CHROMOSOMAL ABERRATION TEST (CA-H) |
||||||||||
S.No. |
Study No. |
Vehicle |
Phase I |
Phase II |
||||||
Absence of S9 |
Presence of S9 |
Absence of S9 |
Presence of S9 |
|||||||
Vehicle Control |
Positive Control |
Vehicle Control |
Positive Control |
Vehicle Control |
Positive Control |
Vehicle Control |
Positive Control |
|||
20 |
6678 |
DMSO |
0.333 |
10.333 |
0.333 |
10.000 |
0.333 |
9.667 |
0.333 |
10.667 |
21 |
6687 |
DMSO |
0.333 |
11.333 |
0.333 |
11.333 |
0.333 |
12.333 |
0.333 |
12.000 |
22 |
6221 |
DMSO |
0.333 |
9.667 |
0.333 |
10.667 |
0.333 |
9.667 |
0.333 |
10.333 |
23 |
6834 |
DMSO |
0.333 |
10.333 |
0.333 |
11.333 |
0.333 |
11.333 |
0.333 |
10.667 |
24 |
6759 |
PBS |
0.667 |
10.667 |
0.000 |
10.000 |
0.333 |
12.000 |
0.333 |
11.333 |
25 |
6430 |
DMSO |
0.333 |
9.000 |
0.333 |
10.000 |
0.667 |
9.667 |
0.667 |
9.667 |
26 |
7703 |
DMSO |
0.333 |
10.000 |
0.333 |
10 |
0.333 |
10.333 |
0.333 |
10.333 |
27 |
7576 |
RPMI |
0.333 |
10.000 |
0.333 |
10.667 |
0.333 |
10.333 |
0.333 |
10.333 |
28 |
7572 |
DMSO |
0.667 |
10.333 |
0.667 |
10 |
0.667 |
9.667 |
0.333 |
10 |
29 |
7574 |
Ethyl alcohol |
0.333 |
10.333 |
0.333 |
10.333 |
0.333 |
10.000 |
0.333 |
10.333 |
30 |
7434 |
DMSO |
0.667 |
10.000 |
0.333 |
10.667 |
0.333 |
9.667 |
0.333 |
11.000 |
31 |
7708 |
DMSO |
0.333 |
9.667 |
0.333 |
10.333 |
0.333 |
9.333 |
0.333 |
9.667 |
32 |
7263 |
DMSO |
0.333 |
10.667 |
0.333 |
10.333 |
0.667 |
10.667 |
0.667 |
9.667 |
33 |
8072 |
DMSO |
0.333 |
10.333 |
0.333 |
10.000 |
0.333 |
10.333 |
0.333 |
10.333 |
34 |
4825 |
DMSO |
0.667 |
9.000 |
0.667 |
9.333 |
0.333 |
10.000 |
0.667 |
9.000 |
35 |
8112 |
DMSO |
0.333 |
9.667 |
0.333 |
10.000 |
0.333 |
10.667 |
0.333 |
10.333 |
36 |
8142 |
DMSO |
0.333 |
10.000 |
0.333 |
10.333 |
0.333 |
10.000 |
0.333 |
10.333 |
37 |
8091 |
DMSO |
0.333 |
10.333 |
0.333 |
10.333 |
0.333 |
10.000 |
0.333 |
10.000 |
38 |
8174 |
RPMI |
0.333 |
11.000 |
0.333 |
10.000 |
0.333 |
9.667 |
0.333 |
10.667 |
39 |
7657 |
DMSO |
0.333 |
10.333 |
0.333 |
11.333 |
0.333 |
10.000 |
0.333 |
11.000 |
6 HISTORICAL DATA (Contd.)
HISTORICAL DATA FOR IN VITRO MAMMALIAN CHROMOSOMAL ABERRATION TEST (CA-H) |
||||||||||
S.No. |
Study No. |
Vehicle |
Phase I |
Phase II |
||||||
Absence of S9 |
Presence of S9 |
Absence of S9 |
Presence of S9 |
|||||||
Vehicle Control |
Positive Control |
Vehicle Control |
Positive Control |
Vehicle Control |
Positive Control |
Vehicle Control |
Positive Control |
|||
40 |
8176 |
DMSO |
0.333 |
11.333 |
0.333 |
8.667 |
0.333 |
10.667 |
0.333 |
10.000 |
41 |
8541 |
DMSO |
0.667 |
9.667 |
0.333 |
10.000 |
0.667 |
9.667 |
0.333 |
10.000 |
42 |
8064 |
DMSO |
0.333 |
10.667 |
0.667 |
9.667 |
0.333 |
10.333 |
0.333 |
10.000 |
43 |
8486 |
DMSO |
0.333 |
10.000 |
0.333 |
10.333 |
0.333 |
10.333 |
0.667 |
11.333 |
44 |
8660 |
RPMI |
0.333 |
11.000 |
0.333 |
10.000 |
0.333 |
10.333 |
0.333 |
10.000 |
45 |
8722 |
DMSO |
0.667 |
10.000 |
0.667 |
10.667 |
0.667 |
9.333 |
0.667 |
10.666 |
46 |
8670 |
DMSO |
0.333 |
10.000 |
0.333 |
11.000 |
0.333 |
10.667 |
0.667 |
10.000 |
47 |
8680 |
DMSO |
0.333 |
10.333 |
0.667 |
10.333 |
0.333 |
10.000 |
0.333 |
10.000 |
48 |
9845 |
DMSO |
0.333 |
10.000 |
0.333 |
11.000 |
0.333 |
10.669 |
0.333 |
10.667 |
Mean |
|
0.392 |
9.750 |
0.374 |
9.858 |
0.422 |
9.882 |
0.374 |
9.934 |
|
SD |
|
0.308 |
0.952 |
0.259 |
1.005 |
0.279 |
0.988 |
0.225 |
0.954 |
|
Mean + 2SD |
|
1.009 |
11.654 |
0.892 |
11.868 |
0.980 |
11.857 |
0.824 |
11.843 |
|
Mean - 2SD |
|
-0.224 |
7.846 |
-0.144 |
7.847 |
-0.136 |
7.907 |
-0.077 |
8.025 |
|
GENE MUTATION TEST
Table 1A.Effect of 5-Decanolide exposure on gene toxicity in CHO cells. After being exposed to the test chemical for 3 hrs, cells was washed with sterile PBS and then incubated for 7 days at 37°C, 5% CO2. After 7 days, cells were re-seeded in new 6-well plates in the absence or presence of 10mM TG as a selection agent and returned to the incubator for 14 days at 37°C, 5% CO2. On day 15, all 6-well plates were stained with crystal violet and the number of colonies were counted manually. The results are presented as the total number of colonies found in the number of independent wells analyzed (e.g. 0 colonies in 4 wells will give 0/4) (n = 2 samples from 2 independent cultures).
|
With S9 |
Without S9 |
||
|
with TG |
without TG |
with TG |
without TG |
Neg. control |
0/4 |
773/4 |
0/4 |
856/4 |
Pos. control |
0/4 |
788/4 |
36/4 |
769/4 |
0.5 mM |
0/4 |
773/4 |
0/4 |
720/4 |
1.0 mM |
0/4 |
603/4 |
0/4 |
747/4 |
2.5 mM |
0/4 |
666/4 |
0/4 |
725/4 |
5.0 mM |
0/4 |
611/4 |
0/4 |
403/4 |
Table 1B.Mutation frequency in CHO cells after 3 hrs of exposure to 5-Decanolide in the absence or presence of 4% S9 liver microsomal fraction. N/A, no colonies present in the samples selected with TG, i.e. no mutation frequency could be determined.
|
With S9 |
Without S9 |
Neg. control |
N/A |
N/A |
Pos. control |
N/A |
4.18x10-4 |
0.5 mM |
N/A |
N/A |
1.0 mM |
N/A |
N/A |
2.5 mM |
N/A |
N/A |
5.0 mM |
N/A |
N/A |
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Genetic toxicity in vivo
Endpoint conclusion
- Endpoint conclusion:
- no study available
Additional information
Bacterial reverse mutation test:
The potential of Decan-5-olide (CAS No. 705-86-2) to induce point mutations or frameshifts with the histidine operon was tested in Salmonella Typhimurium 1535, TA 1537, TA 98, TA 100 and TA 102 tester strains in the presence and absence of a metabolic activation system. Cofactor-supplemented liver S9 microsomal fraction was used as an exogenous metabolic activation system. Dimethyl sulfoxide was selected as a vehicle of the test substance. Test concentrations were selected based on a preliminary cytotoxicity test. This pre-experiment was performed with strains TA 98 and TA 100 according to the plate incorporation methods. Bacterial cells were exposed to the substance at concentrations of 0.0 (NC), 0.0 (VC), 0.001, 0.005, 0.015, 0.050, 0.158, 0.501, 1.582 and 5 mg/plate for 48 hours using triplicates. At concentrations of 0.001-0.158 mg/plate, no reduction in colony count or clearing of the background lawn was observed. At 0.501 mg/plate, there was no decrease in revertant counts but a slight inhibition of the background lawn growth. At 1.582 mg/plate, moderate inhibition of the background lawn and no substantial decrease in the number of revertant colonies were seen. At 5 mg/plate marked reduction in the number of revertant counts and moderate inhibition of the background lawn was observed. Hence, the mutagenicity test was performed with the following test concentrations: 0.050, 0.158, 0.501, 1.582, and 5.000 mg/plate with and without S9 metabolic activation. The main test consisted of two trials.
Trial I was performed according to the plate incorporation method using five test concentrations along with the negative, vehicle, and concurrent positive controls with the remaining three strains, i.e., TA1537, TA1535, and TA102. For TA98 and TA 100 the revertant colony counts were directly incorporated in the Trial-I from the pre-experiment up to the required five concentrations (from 0.050 mg/plate to 5.000 mg/plate). Trial-II was performed independently with all the five tester strains and the negative, vehicle, and positive controls by preincubation method to confirm the results of Trial-I. Results: No substantial increase in the number of revertant colonies compared to the vehicle control was observed at concentrations tested in any of the five tester strains, either in the presence or absence of S9 metabolic activation in both trials. Cytotoxicity was detected at 5 mg/plate as a reduction in the number of revertant colonies. The positive controls induced unequivocal increases in revertant counts in all the five tester strains compared to vehicle controls in Trial I and Trial II. Conclusion: The test chemical did not induce gene mutations by base pair changes or frameshifts in the genome of the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and TA 102 in the presence and absence of S9 metabolic activation system. The test was performed according to OECD TG 471.
In vitro mammalian chromosome aberration test:
The clastogenic potential of Decan-5-olide (CAS No. 705-86-2) was tested according to OECD TG 473 in human peripheral blood lymphocytes and in the presence and absence of S9 metabolic activation. Dimethyl sulfoxide (DMSO) was selected as a vehicle of the test substance. The pH of the test item in the culture medium was assessed at 0 hours and 4 hours after incubation at 37°C. No significant change in pH was observed at times tested when compared with negative control. Hence, 2.0 mg/mL was selected as the highest concentration for a preliminary cytotoxicity test. Cytotoxicity was assessed at the concentrations of 0.0 (NC), 0.0 (VC), 0.5, 1.0 and 2.0 mg/ml along with concurrent positive control substances in the presence and absence of S9 metabolic activation. The cytotoxicity was determined by the calculation of the Mitotic index (MI) at each test concentration and negative, vehicle and positive controls. The concentration which yielded 55±5% cytotoxicity (i.e., reduction in MI to 45±5% of the vehicle control) was selected as the highest test concentration. At 1.0 mg/ml, 54.94% and 57.80% of cytotoxicity were observed in the presence and absence of S9 metabolic activation, respectively. Hence, 1.0 mg/ml was selected as the highest concentration for the main study both in the presence and in the absence of metabolic activation.
The main study was performed in two independent phases: In Phase I experiment, the duplicates cultures were exposed to the test chemical for 4 hours both in the absence and in the presence of S9 metabolic activation system (1%).The mean percentage of aberrant cells was 0.333 (NC), 0.333 (VC), 0.333 (at 0.250 mg/ml), 0.333 (at 0.5 mg/ml), 0.667 (at 1.0 mg/ml) and 10.667 (PC) in the absence of metabolic activation and 0.333 (NC), 0.333 (VC), 0.333 (0.250 mg/ml), 0.333 (0.5 mg/ml), 0.667 (1.0 mg/ml) and 10.333 (PC) in the presence of metabolic activation. Treatment with Ethyl methanesulfonate at the concentration of 600 µg/ml in the absence of metabolic activation and Cyclophosphamide monohydrate at the concentration of 30 µg/mL in the presence of metabolic activation (1%) caused significant increases in percent aberrant cells (≥10.33% aberrant cells). Even though the analysis did not reveal any statistical significance, the increases were biologically significant. Cytotoxicity was observed at 1.0 mg/ml in the presence and absence of S9 metabolic activation. The observed mean mitotic index in the absence of metabolic activation were 10.03, 9.79, 6.64, 5.11, 4.30 and 8.73 and in the presence of metabolic activation were 10.18, 9.89, 6.80, 5.59, 4.10 and 8.69 for 0.0 (NC), 0.0 (VC) 0.250 (T1), 0.5 (T2) and 1.0 (T3) mg/mL and 30 µg/mL(+S9), 600 ug/ml (-S9) of PC concentrations, respectively. The Phase II experiment was performed to confirm the negative results obtained in the absence and in the presence of metabolic activation in Phase I. In the absence of metabolic activation, test items concentrations used were 0.0 (NC), 0.0 (VC) 0.250, 0.5 and 1.0 mg/mL and 600 µg/mL (PC; ethylmethanesulfonate). In the presence of metabolic activation (2%), test items concentrations used were 0.0 (NC), 0.0 (VC) 0.250, 0.5 and 1.0 mg/mL and 30 µg/mL (PC; cyclophosphamide monohydrate).The duration of exposure to the test item in presence of a metabolic activation system was 4 hours and in absence of metabolic activation was 24 hours. The mean percent aberrant cells were 0.333 (NC), 0.333 (VC) 0.333, 0.667, 0.667 and 10.333 (PC) in the absence of metabolic activation and 0.333 (NC),0.667 (VC),0.333, 0.333, 0.667 and 10.000 (PC) in the presence of metabolic activation at the concentrations of 0.0 (NC), 0.0 (VC) 0.250, 0.5 and 1.0 mg/ml and positive control. Treatment with Ethyl methanesulfonate at the concentration of 600 µg/mL in the absence of metabolic activation and Cyclophosphamide monohydrate at the concentration of 30 µg/mL in the presence of metabolic activation (2%) caused significant increases in percent aberrant cells (≥10.00% aberrant cells). Though the analysis did not reveal any statistical significance, the increases were biologically significant. The increased frequency of aberrations observed in the concurrent positive control groups (Phase I and II) validated the sensitivity of the test system, the suitability of the methods and conditions employed in the experiment. Treatment with test item both in the absence and presence of S9 mix induced slight to moderate reductions of the mitotic index at low (0.250 mg/mL) and middle (0.5 mg/mL) doses. The observed mean mitotic index in the absence of metabolic activation were 10.13, 9.90,6.40,5.05,4.20and 8.29 and in the presence of metabolic activation were 10.03, 9.90,6.14,5.35,4.37and 8.68 for 0.0 (NC), 0.0 (VC) 0.250, 0.5 and 1.0 and 30 µg/mL(+S9), 600 ug/ml(-S9) of PC concentrations, respectively. Conclusion: The test chemical was found to be non-clastogenic (negative) as it did not induce structural chromosomal aberration in human peripheral blood lymphocytes both in the presence (1% and 2%) and in the absence of S9 metabolic activation.
In vitro gene mutation test in mammalian cells:
An in vitro mammalian cell gene mutation study was designed and conducted to determine the genotoxicity profile of the given test chemical as per OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test) when administered to Chinese Hamster Ovary (CHO) cells. A preliminary dose-finding study was conducted prior to the main study. A range of different test chemical concentrations was tested in 96-well plates and analyzed by two commonly used assays, i.e. the colourimetric assay of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and the bicinchoninic acid (BCA) assay to assess cell viability and protein concentration, respectively. From the basis of the results from the MTT and BCA assays, test concentrations of the test chemical were chosen to be included in the gene toxicity test. In the genotoxicity test, the given test chemical was administered to CHO cells for 3 hrs at the dose levels of 0, 0.5, 1.0, 2.5 or 5.0 mM and in the absence or presence of exogenous metabolic activation. CHO cells representing the negative controls were exposed to the vehicle. Positive controls, such as N-ethyl-N-nitrosourea (ENU) experiments without metabolic activation and 7,12-dimethylbenz(a) anthracene in experiments with metabolic activation, were also included in each test. The results showed an indication of gene mutations occurring only in the positive control ENU while no other treatment gave rise to gene toxicity. No cytotoxic effects were observed in CHO cells when exposed to test chemical for 3 hrs in the absence or presence of S9 liver microsomal fraction. Based on the results of the current study, it can be concluded that the given test chemical does not give rise to gene mutations when exposed at ≤ 5.0 mM for 3 hrs or more, and it does not give rise to cytotoxicity in CHO cells at concentrations of ≤ 5.0 mM.
Justification for classification or non-classification
The registered substance, Decan-5-olide (CAS No. 705-86-2) did not induce gene mutation in bacterial and mammalian cells and induced no structural chromosomal aberration in mammalian cells in the presence and absence of an exogenous metabolic activation system. The in vitro genetic toxicity tests, presented in this dossier were performed according to concurrent OECD test guidelines and in compliance with GLP. Hence, Decan-5-olide (CAS No. 705-86-2) classified as Non-Classified for germ cell mutagenicity according to Regulation (EC) No 1272/2008 on the classification, labelling and packaging of substances and mixtures (CLP Regulation).
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