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EC number: 500-241-6 | CAS number: 69011-36-5 1 - 2.5 moles ethoxylated
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Genetic toxicity in vitro
Description of key information
Bacterial reverse mutation assay (Ames test, OECD 471): negative with and without metabolic activation
In vitro mammalian cell micronucleus test (cultured peripheral human lymphocytes, OECD 487): negative with and without metabolic activation
In vitro mammalian cell gene mutation test using the Hprt gene (Chinese hamster lung fibroblasts (V79), OECD 476): negative with and without metabolic activation
Conclusions based on data obtained with isotridecanol, ethoxylated, < 2.5 EO (CAS No. 69011-36-5, EC No. 500-241-6).
Link to relevant study records
- Endpoint:
- in vitro cytogenicity / micronucleus study
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 27 May - 10 Oct 2019
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)
- Version / remarks:
- adopted in 2016
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- in vitro mammalian cell micronucleus test
- Target gene:
- Not applicable
- Species / strain / cell type:
- lymphocytes: cultured peripheral human lymphocytes
- Details on mammalian cell type (if applicable):
- CELLS USED
For lymphocytes:
- Sex, age and number of blood donors: Female, 35 and 33 years, two (Main Assay I); female, 27 and 28 years, two (Main Assay II); male, 30 years, one (Main Assay III)
- Whether whole blood or separated lymphocytes were used: whole blood
- Whether blood from different donors were pooled or not: pooled (Main Assays I and II), not pooled (Main Assay III)
- Mitogen used for lymphocytes: phytohaemagglutinin (PHA)
MEDIA USED
- Type and composition of media:
Culture medium:
RPMI 1640 1x (Dutch modification): 500 mL
Foetal Calf Serum (FCS): 100 mL
L-Glutamine (200 mM): 6.25 mL
Antibiotic solution (not further specified): 1.25 mL
Treatment medium (3 h exposure):
+S9:
Test item solution or solvent/vehicle: 0.05 mL
S9 mix: 1.00 mL
Culture medium (without PHA): 3.95 mL
-S9:
Test item solution or solvent/vehicle: 0.05 mL
Culture medium (without PHA): 4.95 mL
Treatment medium (continous exposure):
Test item as supplied, test item solution or solvent/vehicle: 0.025 mL
Culture medium (without PHA): 4.975 mL - Cytokinesis block (if used):
- Cytochalasin B (6 μg/mL)
- Metabolic activation:
- with and without
- Metabolic activation system:
- Type and composition of metabolic activation system:
- source of S9: cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with phenobarbital and 5,6-benzoflavone.
- Species: rat
- Strain: Sprague Dawley
- Sex: male
- Age: 5 - 6 weeks
- Weight: 175 - 199 g
- Tissue: liver
- Inducing Agents: phenobarbital and 5,6-benzoflavone
- Producer: TRINOVA BIOCHEM GmbH, Giessen, Germany
- Batch Number: 3971
- Quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability): yes - Test concentrations with justification for top dose:
- Main Assay I:
3 h exposure (with and without metabolic activation): 0.195, 0.293, 0.439, 0.658, 0.988, 1.48, 2.22, 3.33, 5.00 μL/mL
31 h exposure (without metabolic activation): 0.130, 0.195, 0.293, 0.439, 0.658, 0.988, 1.48, 2.22, 3.33, 5.00 μL/mL
Haemolysis was observed at all doses in all experiments.
Main Assay II:
3 h exposure (with and without metabolic activation): 0.00780, 0.0117, 0.0176, 0.0263, 0.0395, 0.0593, 0.0889, 0.133, 0.200 µL/mL
31 h exposure (without metabolic activation): 0.000520, 0.000780, 0.00117, 0.00176, 0.00263, 0.00395, 0.00593, 0.00889, 0.0133, 0.0200 µL/mL
Cytotoxicity was observed in the 3 h experiments in the presence of metabolic activation.
Main Assay III:
3 h exposure (with metabolic activation): 0.0654, 0.0752, 0.0865, 0.0994, 0.114, 0.132, 0.151, 0.174, 0.200 µL/mL - Vehicle / solvent:
- - Vehicle/solvent used: dimethylsulfoxide (DMSO)
- Justification for choice of solvent/vehicle: Due to the limited solubility of the test substance in water, DMSO was selected as the vehicle. This vehicle was selected since it is compatible with the survival of cells and the S9 metabolic activation system. - Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- colchicine
- cyclophosphamide
- Details on test system and experimental conditions:
- NUMBER OF REPLICATIONS:
- Number of cultures per concentration: duplicate
- Number of independent experiments : three
METHOD OF TREATMENT/ EXPOSURE:
- Test material added in medium
TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable: 48 h
- Exposure duration/duration of treatment: 3 h (3 h exposure experiments) and 31 h (continous exposure experiments)
- Harvest time after the end of treatment (sampling/recovery times): 32 h in 3 h exposure experiments and 31 h in continous exposure experiments
FOR CHROMOSOME ABERRATION AND MICRONUCLEUS:
- If cytokinesis blocked method was used for micronucleus assay: cytB 6 µL/mL medium, added 3 h after start of treatment
- Methods of slide preparation and staining technique used including the stain used: The lymphocyte cultures were centrifuged and the supernatant was removed. The cells were resuspended in hypotonic solution and fresh methanol/acetic acid fixative was added. The fixative was changed several times by centrifugation and resuspension. A few drops of the cell suspension were transfered to glass slides which were allowed to air dry before staining with acridine Orange in phosphate buffered saline (PBS).
- Number of cells spread and analysed per concentration (number of replicate cultures and total number of cells scored): 2000 binucleated cells per cell culture were scored
- Criteria for scoring micronucleated cells (selection of analysable cells and micronucleus identification):
1. The micronucleus diameter was less than 1/3 of the nucleus diameter
2. The micronucleus diameter was greater than 1/16 of the nucleus diameter
3. No overlapping with the nucleus was observed
4. The aspect was the same as the chromatin
METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: cytokinesis-block proliferation index (CBPI), 1000 cells counted per concentration - Evaluation criteria:
- EVALUATION CRITERIA
A test item is considered clearly positive if the following criteria are met:
1. Significant increases in the proportion of micronucleated cells over the concurrent controls occur at one or more concentrations.
2. The proportion of micronucleated cells at such data points exceeds the normal range based on historical control values (95% control limits).
3. There is a significant dose-effect relationship.
A item is considered clearly negative if the following criteria are met:
1. None of the dose levels shows a statistically significant increase in the incidence of micronucleated cells.
2. There is no concentration-related increase when evaluated with the Cochran-Armitage trend test.
3. All the results are inside the distribution of the historical control data (95% control limits).
ACCEPTANCE CRITERIA
The assay is considered valid if the following criteria are met:
1. The incidence of micronucleated cells of the negative control is within the distribution range of our historical control values.
2. Concurrent positive controls induce responses that are compatible with those generated in our historical positive control database and produce a statistically significant increase compared with the concurrent negative control.
3. Adequate cell proliferation is observed in solvent control cultures.
4. The appropriate number of doses and cells is analysed. - Statistics:
- A modified χ2 test was used to compare the number of cells with micronuclei in control and treated cultures.
Cochran-Armitage Trend Test (one-sided) was performed for the assessment of the concentration-response relationship. - Key result
- Species / strain:
- lymphocytes: cultured peripheral human lymphocytes
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- Strong cytotoxicity was observed at and above 0.130 μL/mL in the absence and presence of metabolic activation.
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- True negative controls validity:
- not examined
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH: Treatment with the test item did not change the pH at any dose level in any experiment.
- Data on osmolality: Treatment with the test item did not increases the osmolality at any dose level in any experiment.
- Precipitation and time of the determination: Following treatment with the test item, no opacity or precipitation of the medium was observed at the beginning or by the end of treatment in any experiment.
STUDY RESULTS
- Concurrent vehicle negative and positive control data are reported in tabular form under 'Any other information on results incl. tables'.
HISTORICAL CONTROL DATA
- The incidence of micronucleated cells of the negative controls was within the distribution range of our historical control values, data are presented in tabular form under 'Any other information on results incl. tables'. - Conclusions:
- Interpretation of results: negative
- Endpoint:
- in vitro gene mutation study in mammalian cells
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 05 June - 11 Oct 2019
- 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 using the Hprt and xprt genes)
- Version / remarks:
- adopted in 2016
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- in vitro mammalian cell gene mutation test using the Hprt and xprt genes
- Target gene:
- HPRT locus
- Species / strain / cell type:
- Chinese hamster lung fibroblasts (V79)
- Details on mammalian cell type (if applicable):
- CELLS USED
- Source of cells: Dr. J. Thacker, MRC Radiobiology Unit, Harwell, UK.
- Suitability of cells: Cell type selected is listed as one of the recommended cell types in OECD guideline 476.
For cell lines:
- Absence of Mycoplasma contamination: checked periodically
- Periodically checked for karyotype stability: yes
- Periodically ‘cleansed’ of spontaneous mutants: yes
MEDIA USED
- Type and composition of media, CO2 concentration, humidity level, temperature:
Eagle’s Minimal Essential Medium (EMEM) Minimal medium:
EMEM (10X): 58.7 mL
L-glutamine (200 mM): 5.9 mL
Sodium bicarbonate (7.5%): 15.7 mL
Non-essential amino acids (100X): 5.9 mL
Streptomycin sulphate (50000 IU/mL) + Penicillin G (50000 IU/mL): 1.2 mL
Sterile distilled water: 500 mL
EMEM Complete medium:
EMEM Minimal medium: 900 mL
Foetal Calf Serum (FCS): 100 mL
- Incubation conditions: 37 °C in a 5% CO2 atmosphere (100% nominal relative humidity) - Metabolic activation:
- with and without
- Metabolic activation system:
- Type and composition of metabolic activation system:
- source of S9: cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with phenobarbital and 5,6-benzoflavone.
- Species: rat
- Strain: Sprague Dawley
- Sex: male
- Age: 5 - 6 weeks
- Weight: 175 - 199 g
- Tissue: liver
- Inducing Agents: phenobarbital and 5,6-benzoflavone
- Producer: TRINOVA BIOCHEM GmbH, Giessen, Germany
- Batch Number: 3971
- Quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability): yes - Test concentrations with justification for top dose:
- Cytotoxicity test (+/-S9): 0.00328, 0.00819, 0.0205, 0.0512, 0.128, 0.320, 0.800, 2.00, 5.00 µL/mL
No cells survived at and above 0.0512 µL/mL in the absence of metabolic activation and at and above 0.320 µL/mL in the presence of metabolic activation.
Mutation assay (-S9): 0.00205, 0.00410, 0.00819, 0.0102, 0.0128, 0.0160, 0.0200 µL/mL
Mutation assay (+S9): 0.0307, 0.0614, 0.123, 0.154, 0.192, 0.240, 0.300 µL/mL
The selection of the concentrations used in the main experiments was based on data from the cytotoxicity test. - Vehicle / solvent:
- - Vehicle/solvent used: dimethylsulfoxide (DMSO)
- Justification for choice of solvent/vehicle: Due to the limited solubility of the test substance in water, DMSO was selected as the vehicle. This vehicle was selected since it is compatible with the survival of cells and the S9 metabolic activation system. - Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- 7,12-dimethylbenzanthracene
- ethylmethanesulphonate
- Details on test system and experimental conditions:
- NUMBER OF REPLICATIONS:
- Number of cultures per concentration: duplicate
- Number of independent experiments : single
METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding: 2 x 10E6
- Test material added in medium
TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: 3 h
FOR GENE MUTATION:
- Expression time (cells in growth medium between treatment and selection): 8 days
- Selection time (if incubation with a selective agent): 10 - 15 days
- Selective agent used: 6-thioguanine, 7.5 µg/mL
- Number of cells seeded and method to enumerate numbers of viable and mutants cells: 1 x 10E5
METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: Relative survival (RS)
METHODS FOR MEASUREMENTS OF GENOTOXICIY
- Method: Mutant frequency (MF) per million surviving cells - Evaluation criteria:
- EVALUATION CRITERIA
A test item is considered to be clearly positive if:
– At least one of the test concentrations exhibits a statistically significant increase, compared with the concurrent solvent/vehicle control.
– The increase is concentration-related.
– Any of the results are outside the distribution of the historical negative control data (95% confidence limits).
A test item is considered to be clearly negative if:
– None of the test concentrations exhibits a statistically significant increase, compared with the concurrent solvent/vehicle control.
– There is no concentration-related increase.
– All results are inside the distribution of the historical negative control data (95% confidence limits).
ACCEPTANCE CRITERIA
The assay was considered valid if the following criteria were met:
1. The mutant frequency of the solvent/vehicle control is within the 95% control limits of the distribution of the laboratory’s historical control database.
2. The positive controls induce responses that are compatible with those generated in the historical control database and produce a statistically significant increase in mutant frequency, compared with the concurrent solvent/vehicle control.
3. Two experimental conditions are tested (i.e. with and without metabolic activation), unless one gives positive results.
4. Adequate number of cells (i.e. at least 20 x 10E6 at treatment time and at least 2 x 10E6 during the expression period) and concentrations (at least 4 with appropriate cytotoxicity) are analysable.
5. The selection of dose levels is consistent with those indicated in § 4.3 of the Study Protocol. - Statistics:
- Individual mutation frequencies were transformed to induce homogeneous variance and normal distribution. The mutant frequency in the solvent control and treated cultures was compared using the Dunnett’s test (one-tailed). For each experimental point, the corrected sum of squares of transformed mutation frequencies was calculated.For each experimental point, the t value was calculated. For each comparison of treatment with control, the calculated t value was compared with tabulated critical values for the one tailed Dunnett’s test. The results of the experiment were subjected to an Analysis of Variance in which the effect of replicate culture and dose level in explaining the observed variation were examined.
- Key result
- Species / strain:
- Chinese hamster lung fibroblasts (V79)
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- Strong cytotoxicity was observed at and above a test substance concentration of 0.0205 µL/mL in cytotoxicity test (RS < 10%); concentrations in main assays were selected accordingly.
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- True negative controls validity:
- not examined
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH: Treatment with the test item did not change the pH at any dose level in any experiment.
- Data on osmolality: Treatment with the test item did not increases the osmolality at any dose level in any experiment.
- Precipitation and time of the determination: No precipitation of the test item was noted at any dose level.
RANGE-FINDING/SCREENING STUDIES
A preliminary cytotoxicity assay was performed using a single culture at each test point both in the presence and absence of metabolic activation. No positive controls were included. The test item was assayed at concentrations of 0.00328, 0.00819, 0.0205, 0.0512, 0.128, 0.320, 0.800, 2.00 and 5.00 µL/mL. In the absence of S9 mix, no cell survived treatment at and above concetrations of 0.0512 µL/mL while in the presence of S9 mix, no cell survived treatment at and above 0.320 μL/mL. Based on these findings the test item concentrations in the main assay were selected.
STUDY RESULTS
- Treatment with the positive control items gave marked responses that were compatible with those generated in the historical control database and produced a statistically significant increase in mutant frequency, compared with the concurrent solvent/vehicle control, indicating the correct functioning of the test system. An adequate number of cells and concentrations was analysed; concurrent vehicle negative and positive control data are reported in tabular form under 'Any other information on results incl. tables'.
HISTORICAL CONTROL DATA
- The mutant frequencies in the negative control cultures fell within the 95% control limits of the distribution of the laboratory’s historical control data; data are presented in tabular form under 'Any other information on results incl. tables'. - Conclusions:
- Interpretation of results: negative
- Endpoint:
- in vitro gene mutation study in bacteria
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 17 May - 24 June 2019
- 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:
- adopted in 1997
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
- Version / remarks:
- adopted in 2008
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- bacterial reverse mutation assay
- Target gene:
- his operon (S. typhimurium strains) and trp operon (E. coli strain)
- Species / strain / cell type:
- S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
- Species / strain / cell type:
- E. coli WP2 uvr A
- Metabolic activation:
- with and without
- Metabolic activation system:
- Type and composition of metabolic activation system:
- source of S9: cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with phenobarbital and 5,6-benzoflavone.
- Species: rat
- Strain: Sprague Dawley
- Sex: male
- Age: 5 - 6 weeks
- Weight: 175 - 199 g
- Tissue: liver
- Inducing Agents: phenobarbital and 5,6-benzoflavone
- Producer: TRINOVA BIOCHEM GmbH, Giessen, Germany
- Batch Number: 4009
- Quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability): yes - Test concentrations with justification for top dose:
- Main assay I (plate incorporation) and main assay II (pre-incubation):
TA1535, WP2 uvrA, TA98 (+/-S9): 0.313, 0.625, 1.25, 2.50, 5.00 µL/plate
TA1537 (+/-S9) and TA100 (+S9): 0.00977, 0.0195, 0.0391, 0.0781, 0.156, 0.313 µL/plate
TA100 (-S9): 0.00244, 0.00488, 0.00977, 0.0195, 0.0391, 0.0781 µL/plate
Main assay III (pre-incubation):
TA1535 (-S9): 0.00977, 0.0195, 0.0391, 0.0781, 0.156, 0.313 µL/plate
TA1537 (-S9): 0.000610, 0.00122, 0.00244, 0.00488, 0.00977, 0.0195 µL/plate
WP2 uvrA (-S9): 0.0391, 0.0781, 0.156, 0.313, 0.625, 1.25 µL/plate
TA100 (-S9): 0.000305, 0.000610, 0.00122, 0.0024, 0.00488, 0.00977 µL/plate
The test concentrations were selected based on the results of a toxicity test with all 5 strains in which no precipitation occured. However, severe cytotoxicity at most of the concentrations tested with TA1537 and TA100 strains (both in the absence and presence of S9) was observed up to and including the highest concentration of 5 µL/plate. - Vehicle / solvent:
- - Vehicle used: dimethylsulfoxide (DMSO)
- Justification for choice of solvent/vehicle: DMSO was selected based on the solubility of the test substance and its compatibility with the survival of the bacteria and the S9 metabolic activity. The test item was found to be miscible at 100 μL/mL. This allowed a maximum concentration of 5.00 μL/plate. - 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
- methylmethanesulfonate
- other: 2-Aminoanthracene (2-AA), +S9
- Details on test system and experimental conditions:
- NUMBER OF REPLICATIONS:
- Number of cultures per concentration: triplicate
- Number of independent experiments : three
METHOD OF TREATMENT/ EXPOSURE:
- Test substance added in agar (plate incorporation, main assay I) and preincubation (main assays II and III)
TREATMENT AND HARVEST SCHEDULE:
- Preincubation period: 30 min at 37 °C (main assays II and III)
- Exposure duration/duration of treatment: 72 h at 37 °C
METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: decline in the number of spontaneous revertants, a thinning of the background lawn or a microcolony formation - Evaluation criteria:
- The test material may be considered positive in this test system if the following criteria are met:
For the test item to be considered mutagenic, two-fold (or more) increases in mean revertant numbers must be observed at two consecutive dose levels or at the highest practicable dose level only. In addition, there must be evidence of a dose-response relationship showing increasing numbers of mutant colonies with increasing dose levels. All cultures must demonstrate the characteristic mean number of spontaneous revertants in the vehicle controls. - Statistics:
- Mean values and standard deviation were calculated.
- 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: no cytotoxicity +/- S9; preincubation method: at and above 0.156 µL/plate +S9, no cytotoxicity -S9.
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- True negative controls validity:
- not examined
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 1537
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- Plate incorporation method: at and above 0.156 µL/plate +/- S9; preincubation method: at and above 0.00977 µL/plate -S9, at and above 0.156 µL/plate +S9.
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- True negative controls validity:
- not examined
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 98
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- True negative controls validity:
- not examined
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 100
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- Plate incorporation method: at and above 0.313 µL/plate +S9 and 0.0781 µg/plate -S9; preincubation method: at and above 0.313 µL/plate +S9 and 0.00488 µL/plate -S9.
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- True negative controls validity:
- not examined
- 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:
- cytotoxicity
- Remarks:
- Plate incorporation method: no cytotoxicity +/s S9; preincubation method: at and above 0.625 µL/plate -S9, no cytotoxicity +S9.
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- True negative controls validity:
- not examined
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation and time of the determination: No precipitation of the test item was observed at the end of the incubation period at any concentration in any experiment.
RANGE-FINDING/SCREENING STUDY:
Concentrations of 0.0500, 0.158, 0.500, 1.58 and 5.00 μL/plate of the test item in the presence and absence of S9 mix in S. typhimurium TA1935, TA1937, TA98, TA100 and E. coli WP2 uvr A were tested in an plate incorporation experiment. Severe toxicity, as indicated by thinning of the background lawn and/or reduction in revertant numbers, was observed at all or almost all concentrations with TA1537 and TA100 tester strains both in the absence and presence of S9 mix. The highest concentration analysed was selected based on the solubility of the test substance.
STUDY RESULTS
- Concurrent vehicle negative and positive control data are presented in tabular form under 'Any other information on results incl. tables'.
Ames test:
- Individual plate counts are presented in tabular form under 'Any other information on results incl. tables'.
- Mean number of revertant colonies per plate and standard deviation are presented in tabular form under 'Any other information on results incl. tables'.
HISTORICAL CONTROL DATA:
- Mean plate counts for untreated and positive control plates fell within the normal range based on historical control data. Data are presented in tabular form under 'Any other information on results incl. tables'. - Conclusions:
- Interpretation of results: negative
Referenceopen allclose all
Table 1: Proliferation index
Main Assay I | ||||||||
Treatment (µL/mL) | Culture No. | MonoN | BiN | PoliN | Total cells | CBPI | Mean Value | Cytotoxicity (%) |
Treatment: 3 h, - S9 | ||||||||
Test Item* | 19 | Only mononucleated cells | ||||||
0.195 | 20 | |||||||
Treatment: 3 h, +S9 | ||||||||
Solvent* | 21 | 124 | 219 | 157 | 500 | 2.066 | 2.052 | 0 |
1% | 22 | 123 | 235 | 142 | 500 | 2.038 | ||
Test Item* | 39 | 378 | 122 | 0 | 500 | 1.244 | 1.235 | 78 |
0.195 | 40 | 388 | 111 | 1 | 500 | 1.226 | ||
Test Item* | 37 | Only mononucleated cells | ||||||
0.293 | 38 | |||||||
Test Item* | 35 | Only mononucleated cells | ||||||
0.439 | 36 | |||||||
Treatment: 31 h, - S9 | ||||||||
Test Item* | 65 | Only mononucleated cells | ||||||
0.130 | 66 | |||||||
Main Assay II | ||||||||
Treatment (µL/mL) | Culture No. | MonoN | BiN | PoliN | Total cells | CBPI | Mean Value | Cytotoxicity (%) |
Treatment: 3 h, -S9 | ||||||||
Solvent* | 71 | 122 | 275 | 103 | 500 | 1.962 | 1.953 | 0 |
1% | 72 | 138 | 252 | 110 | 500 | 1.944 | ||
Test Item* | 85 | 121 | 256 | 123 | 500 | 2.004 | 1.983 | -3 |
0.0176 | 86 | 143 | 233 | 124 | 500 | 1.962 | ||
Test Item* | 83 | 123 | 274 | 103 | 500 | 1.960 | 1.925 | 3 |
0.0263 | 84 | 155 | 245 | 100 | 500 | 1.890 | ||
Test Item* | 81 | 154 | 248 | 98 | 500 | 1.888 | 1.897 | 6 |
0.0395 | 82 | 139 | 269 | 92 | 500 | 1.906 | ||
Test Item* | 79 | 120 | 294 | 86 | 500 | 1.932 | 1.878 | 8 |
0.0593 | 80 | 154 | 280 | 66 | 500 | 1.824 | ||
Test Item* | 77 | 305 | 177 | 18 | 500 | 1.426 | 1.445 | 53 |
0.0889 | 78 | 289 | 190 | 21 | 500 | 1.464 | ||
Test Item* | 75 | Very few mononucleated cells recovered onto slides | ||||||
0.133 | 76 | |||||||
Test Item* | 73 | No cells recovered onto slides | ||||||
0.200 | 74 | |||||||
Treatment: 3 h, +S9 | ||||||||
Solvent* | 91 | 144 | 217 | 139 | 500 | 1.990 | 1.802 | 0 |
1% | 92 | 243 | 207 | 50 | 500 | 1.614 | ||
Test Item* | 103 | 165 | 211 | 124 | 500 | 1.918 | 1.911 | -14 |
0.0263 | 104 | 166 | 216 | 118 | 500 | 1.904 | ||
Test Item* | 101 | 162 | 249 | 89 | 500 | 1.854 | 1.857 | -7 |
0.0395 | 102 | 169 | 232 | 99 | 500 | 1.860 | ||
Test Item* | 99 | 154 | 242 | 104 | 500 | 1.900 | 1.832 | -4 |
0.0593 | 100 | 204 | 210 | 86 | 500 | 1.764 | ||
Test Item* | 97 | 179 | 237 | 84 | 500 | 1.810 | 1.861 | -7 |
0.0889 | 98 | 134 | 276 | 90 | 500 | 1.912 | ||
Test Item* | 95 | 276 | 209 | 15 | 500 | 1.478 | 1.469 | 42 |
0.133 | 96 | 290 | 190 | 20 | 500 | 1.460 | ||
Test Item* | 93 | Very few mononucleated cells recovered onto slides | ||||||
0.200 | 94 | |||||||
Cyclophosphamide | 111 | 310 | 182 | 8 | 500 | 1.396 | 1.394 | 51 |
20.0 µg/mL | 112 | 311 | 182 | 7 | 500 | 1.392 | ||
Cyclophosphamide | 113 | 316 | 176 | 8 | 500 | 1.384 | 1.392 | 51 |
15.0 µg/mL | 114 | 306 | 188 | 6 | 500 | 1.400 | ||
Treatment: 31 h, - S9 | ||||||||
Solvent* | 115 | 118 | 273 | 109 | 500 | 1.982 | 1.951 | 0 |
0.5% | 116 | 140 | 260 | 100 | 500 | 1.920 | ||
Test Item* | 129 | 172 | 266 | 62 | 500 | 1.780 | 1.796 | 16 |
0.00176 | 130 | 164 | 266 | 70 | 500 | 1.812 | ||
Test Item* | 127 | 147 | 293 | 60 | 500 | 1.826 | 1.782 | 18 |
0.00263 | 128 | 188 | 255 | 57 | 500 | 1.738 | ||
Test Item* | 125 | 168 | 298 | 34 | 500 | 1.732 | 1.733 | 23 |
0.00395 | 126 | 170 | 293 | 37 | 500 | 1.734 | ||
Test Item* | 123 | 187 | 289 | 24 | 500 | 1.674 | 1.656 | 31 |
0.00593 | 124 | 206 | 269 | 25 | 500 | 1.638 | ||
Test Item* | 121 | 228 | 265 | 7 | 500 | 1.558 | 1.535 | 44 |
0.00889 | 122 | 251 | 242 | 7 | 500 | 1.512 | ||
Test Item* | 119 | 316 | 183 | 1 | 500 | 1.370 | 1.393 | 59 |
0.0133 | 120 | 293 | 206 | 1 | 500 | 1.416 | ||
Test Item* | 117 | 391 | 109 | 0 | 500 | 1.218 | 1.225 | 76 |
0.0200 | 118 | 384 | 116 | 0 | 500 | 1.232 | ||
Colchicine | 137 | 495 | 5 | 0 | 500 | 1.010 | 1.007 | 99 |
0.0800 µg/mL | 138 | 498 | 2 | 0 | 500 | 1.004 | ||
Colchicine | 139 | 487 | 13 | 0 | 500 | 1.026 | 1.028 | 97 |
0.0400 µg/mL | 140 | 485 | 15 | 0 | 500 | 1.030 | ||
Main Assay III | ||||||||
Treatment (µL/mL) | Culture No. | MonoN | BiN | PoliN | Total cells | CBPI | Mean Value | Cytotoxicity (%) |
Treatment: 3 h, -S9 | ||||||||
Solvent* | 141 | 297 | 164 | 39 | 500 | 1.484 | 1.482 | 0 |
1% | 142 | 308 | 144 | 48 | 500 | 1.480 | ||
Test Item* | 159 | 344 | 110 | 46 | 500 | 1.404 | 1.484 | 0 |
0.0654 | 160 | 290 | 138 | 72 | 500 | 1.564 | ||
Test Item* | 157 | 324 | 128 | 48 | 500 | 1.448 | 1.436 | 10 |
0.0752 | 158 | 325 | 138 | 37 | 500 | 1.424 | ||
Test Item* | 155 | 319 | 140 | 41 | 500 | 1.444 | 1.441 | 9 |
0.0865 | 156 | 321 | 139 | 40 | 500 | 1.438 | ||
Test Item* | 153 | 333 | 138 | 29 | 500 | 1.392 | 1.389 | 19 |
0.0994 | 154 | 335 | 137 | 28 | 500 | 1.386 | ||
Test Item* | 151 | 380 | 103 | 17 | 500 | 1.274 | 1.266 | 45 |
0.114 | 152 | 379 | 113 | 8 | 500 | 1.258 | ||
Test Item* | 149 | 805 | 190 | 5 | 1000 | 1.200 | 1.213 | 56 |
0.132 | 150 | 783 | 208 | 9 | 1000 | 1.226 | ||
Test Item* | 147 | 428 | 72 | 0 | 500 | 1.144 | 1.134 | 72 |
0.151 | 148 | 438 | 62 | 0 | 500 | 1.124 | ||
Test item* | 145 | Few cells recovered onto slides | ||||||
0.174 | 146 | |||||||
Test Item* | 143 | 396 | 82 | 22 | 500 | 1.252 | ||
0.200 | 144 | Few cells recovered onto slides | ||||||
Cyclophosphamide | 161 | 356 | 142 | 2 | 500 | 1.292 | 1.280 | 42 |
20.0 µg/mL | 162 | 366 | 134 | 0 | 500 | 1.268 | ||
Cyclophosphamide | 163 | 343 | 150 | 7 | 500 | 1.328 | 1.310 | 36 |
15.0 µg/mL | 164 | 358 | 138 | 4 | 500 | 1.292 | ||
*: 2 replicates | ||||||||
MonoN: | Mononucleated cells | |||||||
BiN: | Binucleated cells | |||||||
PoliN: | Polinucleated cells | |||||||
CBPI: | Cytokinesis block proliferation index | |||||||
% Cytotoxicity: | Relative to vehicle/solvent control cultures | |||||||
Mn: | Micronucleus/micronuclei |
Table 2: Scoring of micronuclei
Main Assay II | ||||||
Dose level (μL/mL) | Culture No. | Cells scored (BiN cells) |
BiN cells with 1 Mn |
BiN cells with 2 Mn |
BiN cells with >2 Mn |
BiN cells with Mn |
Treatment: 3 h, -S9 | ||||||
Solvent 1% | 71 | 1000 | 1 | 0 | 0 | 1 |
72 | 1000 | 3 | 0 | 0 | 3 | |
0.0395 | 81 | 1000 | 3 | 0 | 0 | 3 |
82 | 1000 | 2 | 0 | 0 | 2 | |
0.0593 | 79 | 1000 | 3 | 0 | 0 | 3 |
80 | 1000 | 2 | 0 | 0 | 2 | |
0.0889 | 77 | 1000 | 4 | 0 | 0 | 4 |
78 | 1000 | 1 | 0 | 0 | 1 | |
Cyclophosphamide 15.0 µg/mL | 113 | 1000 | 20 | 0 | 0 | 20 |
114 | 1000 | 45 | 1 | 0 | 46 | |
Treatment: 31 h, -S9 | ||||||
Solvent 0.5% | 115 | 1000 | 7 | 0 | 0 | 7 |
116 | 1000 | 2 | 0 | 0 | 2 | |
0.00263 | 127 | 1000 | 3 | 0 | 0 | 3 |
128 | 1000 | 4 | 0 | 0 | 4 | |
0.00593 | 123 | 1000 | 7 | 0 | 0 | 7 |
124 | 1000 | 1 | 0 | 0 | 1 | |
0.0133 | 119 | 1000 | 3 | 0 | 0 | 3 |
120 | 1000 | 4 | 0 | 0 | 4 | |
Colchicine 0.0400 µg/mL |
139 | 1000 | 21 | 5 | 0 | 26 |
140 | 1000 | 16 | 6 | 0 | 22 | |
Main Assay III | ||||||
Dose level (μL/mL) | Culture No. | Cells scored (BiN cells) |
BiN cells with 1 Mn |
BiN cells with 2 Mn |
BiN cells with >2 Mn |
BiN cells with Mn |
Treatment: 3 h, +S9 | ||||||
Solvent 1% | 141 | 1000 | 9 | 1 | 0 | 10 |
142 | 1000 | 7 | 3 | 0 | 10 | |
0.0752 | 157 | 1000 | 9 | 0 | 0 | 9 |
158 | 1000 | 9 | 0 | 0 | 9 | |
0.0994 | 153 | 1000 | 5 | 0 | 0 | 5 |
154 | 1000 | 7 | 0 | 0 | 7 | |
0.132 | 149 | 1000 | 8 | 2 | 0 | 10 |
150 | 1000 | 7 | 0 | 0 | 7 | |
Cyclophosphamide 20.0 µg/mL | 161 | 1000 | 28 | 4 | 0 | 32 |
162 | 1000 | 35 | 4 | 0 | 39 | |
MonoN: | Mononucleated cells | |||||
BiN: | Binucleated cells | |||||
PoliN: | Polinucleated cells | |||||
CBPI: | Cytokinesis block proliferation index | |||||
% Cytotoxicity: | Relative to vehicle/solvent control cultures | |||||
Mn: | Micronucleus/micronuclei |
Table 3: Historical control data
UNTREATED AND SOLVENT CONTROLS | |||
-S9 | +S9 | ||
Short treatment time (3 h) | Long treatment time (31-32 h) | Short treatment time (3 hours) | |
Mean | 0.33 | 0.39 | 0.36 |
SD | 0.21 | 0.30 | 0.24 |
n | 64 | 61 | 64 |
UCL | 0.75 | 0.96 | 0.85 |
LCL | 0.00 | 0.00 | 0.00 |
POSITIVE CONTROL | ||
+S9 | -S9 | |
Short treatment time (3 h) | Long treatment time (31-32 h) | |
Cyclophosphamide | Colchicine | |
Mean | 2.97 | 3.10 |
SD | 1.40 | 1.30 |
n | 51 | 46 |
Minimum | 0.95 | 1.10 |
Maximum | 7.65 | 8.50 |
SD: | Standard deviation |
UCL: | Upper Confidence Limit (Mean value + 2SD) |
LCL: | Lower Confidence Limit (Mean value - 2SD) |
Table 1: Summary | |||||||
Without metabolic activation | With metabolic activation | ||||||
Dose level (µL/mL) | %RS | MF | Sig | Dose level (µL/mL) | %RS | MF | Sig |
0.00 | 100 | 4.26 | 0.00 | 100 | 14.5 | ||
0.00205 | 82 | 15.3 | * | 0.0307 | 88 | 9.04 | NS |
0.00410 | 99 | 11.3 | NS | 0.0614 | 105 | 6.02 | NS |
0.00819 | 65 | 15.0 | * | 0.123 | 63 | 16.8 | NS |
0.0102 | 62 | 9.40 | NS | 0.154 | 91 | 13.9 | NS |
0.0128 | 23 | 9.66 | NS | 0.192 | 64 | 6.05 | NS |
0.0160 | £ | £ | 0.240 | 29 | 14.7 | NS | |
0.0200 | £ | £ | 0.300 | £ | £ | ||
EMS 10.0 mM |
57 | 2681 | ** | DMBA 10.0 µg/mL |
51 | 241 | ** |
Table 2: Survival after treatment | |||||||||||
SURVIVAL AFTER TREATMENT WITHOUT METABOLIC ACTIVATION | |||||||||||
Treatment | Dose Level (µL/mL) | 106 cells post treatment | Plate counts | Mean | CE | Adjusted CE | Mean | RS (%) | |||
Sovent control | A | 0.00 | 20.2 | 116 | 108 | 115 | 113 | 0.57 | 0.47 | 0.55 | 100 |
B | 20.4 | 158 | 144 | 152 | 151 | 0.76 | 0.63 | ||||
Test Item | A | 0.00205 | 19.6 | 89 | 93 | 93 | 92 | 0.46 | 0.37 | 0.45 | 82 |
B | 16.2 | 164 | 160 | 157 | 160 | 0.80 | 0.53 | ||||
Test Item | A | 0.00410 | 17.4 | 104 | 104 | 107 | 105 | 0.53 | 0.37 | 0.54 | 99 |
B | 17.6 | 199 | 203 | 191 | 198 | 0.99 | 0.71 | ||||
Test Item | A | 0.00819 | 11.4 | 131 | 138 | 137 | 135 | 0.68 | 0.32 | 0.36 | 65 |
B | 12.1 | 155 | 160 | 163 | 159 | 0.80 | 0.40 | ||||
Test Item | A | 0.0102 | 12.6 | 133 | 130 | 137 | 133 | 0.67 | 0.34 | 0.34 | 62 |
B | 11.9 | 131 | 135 | 145 | 137 | 0.69 | 0.33 | ||||
Test Item | A | 0.0128 | 7.4 | 89 | 89 | 90 | 89 | 0.45 | 0.14 | 0.13 | 23 |
B | 7.7 | 76 | 76 | 78 | 77 | 0.38 | 0.12 | ||||
Test Item | A | 0.0160 | 1.3 | ||||||||
B | 1.3 | £ | £ | £ | |||||||
Test item | A | 0.0200 | 1.3 | ||||||||
B | 1.7 | £ | £ | £ | |||||||
EMS | 10.0 mM | 17 | 88 | 90 | 90 | 89 | 0.45 | 0.31 | 0.31 | 57 | |
Baseline count | 24.4 | ||||||||||
SURVIVAL AFTER TREATMENT WITH METABOLIC ACTIVATION | |||||||||||
Treatment | Dose Level (µL/mL) | 106 cells post treatment | Plate counts | Mean | CE | Adjusted CE | Mean | RS (%) | |||
Sovent control | A | 0.00 | 30 | 144 | 152 | 152 | 149 | 0.75 | 1.07 | 1.00 | 100 |
B | 30.3 | 129 | 131 | 124 | 128 | 0.64 | 0.93 | ||||
Test Item | A | 0.0307 | 24.9 | 160 | 150 | 152 | 154 | 0.77 | 0.92 | 0.88 | 88 |
B | 26.7 | 136 | 136 | 125 | 132 | 0.66 | 0.85 | ||||
Test Item | A | 0.0614 | 26.4 | 170 | 167 | 165 | 167 | 0.84 | 1.06 | 1.05 | 105 |
B | 29.7 | 147 | 146 | 144 | 146 | 0.73 | 1.04 | ||||
Test Item | A | 0.123 | 21 | 125 | 118 | 112 | 118 | 0.59 | 0.60 | 0.63 | 63 |
B | 21.8 | 120 | 131 | 127 | 126 | 0.63 | 0.66 | ||||
Test Item | A | 0.154 | 21.6 | 154 | 152 | 154 | 153 | 0.77 | 0.79 | 0.91 | 91 |
B | 25.2 | 172 | 174 | 165 | 170 | 0.85 | 1.03 | ||||
Test Item | A | 0.192 | 23.6 | 125 | 138 | 139 | 134 | 0.67 | 0.76 | 0.64 | 64 |
B | 21.9 | 100 | 100 | 102 | 101 | 0.50 | 0.53 | ||||
Test Item | A | 0.240 | 11 | 116 | 113 | 118 | 116 | 0.58 | 0.31 | 0.29 | 29 |
B | 10 | 114 | 119 | 114 | 116 | 0.58 | 0.28 | ||||
Test item | A | 0.300 | 0.5 | ||||||||
B | 0.4 | £ | £ | £ | |||||||
DMBA | 10.0 µg/mL | 25.2 | 80 | 82 | 90 | 84 | 0.42 | 0.51 | 0.51 | 51 | |
Baseline count | 20.9 |
Table 3: Cloning efficiency | |||||||
CLONING EFFICIENCY WITHOUT METABOLIC ACTIVATION | |||||||
Treatment | Dose Level (µL/mL ) | Plate counts | Mean | %CE | |||
Solvent control | A | 0.00 | 134 | 131 | 140 | 118 | 59 |
B | 99 | 96 | 105 | ||||
Test Item | A | 0.00205 | 86 | 80 | 86 | 88 | 44 |
B | 92 | 95 | 90 | ||||
Test Item | A | 0.00410 | 160 | 160 | 160 | 185 | 93 |
B | 211 | 211 | 209 | ||||
Test Item | A | 0.00819 | 140 | 142 | 140 | 120 | 60 |
B | 103 | 100 | 97 | ||||
Test Item | A | 0.0102 | 151 | 150 | 159 | 149 | 75 |
B | 145 | 144 | 145 | ||||
Test Item | A | 0.0128 | 197 | 200 | 200 | 166 | 83 |
B | 131 | 134 | 132 | ||||
EMS | 10.0 mM | 70 | 70 | 79 | 73 | 37 | |
CLONING EFFICIENCY WITH METABOLIC ACTIVATION | |||||||
Treatment | Dose Level (µL/mL) | Plate counts | Mean | %CE | |||
Solvent control | A | 0.00 | 108 | 112 | 101 | 111 | 55 |
B | 110 | 122 | 110 | ||||
Test Item | A | 0.0307 | 130 | 128 | 123 | 144 | 72 |
B | 159 | 156 | 167 | ||||
Test Item | A | 0.0614 | 113 | 111 | 108 | 108 | 54 |
B | 101 | 106 | 109 | ||||
Test Item | A | 0.123 | 104 | 101 | 93 | 98 | 49 |
B | 96 | 97 | 99 | ||||
Test Item | A | 0.154 | 104 | 96 | 97 | 101 | 50 |
B | 105 | 104 | 97 | ||||
Test Item | A | 0.192 | 98 | 98 | 102 | 108 | 54 |
B | 112 | 113 | 122 | ||||
Test Item | A | 0.240 | 122 | 120 | 112 | 136 | 68 |
B | 151 | 146 | 165 | ||||
DMBA | 10.0 µg/mL | 131 | 155 | 148 | 145 | 72 |
Table 4: Mutation frequency | |||||||||||||||||||
MUTATION FREQUENCY WITHOUT METABOLIC ACTIVATION | |||||||||||||||||||
Treatment | Dose Level (µL/mL) | Plate counts | Tot. | Mean | SD | MF | MF Pooled cultures | t | Sig. | ||||||||||
Solvent control | A | 0.00 | 1 | 0 | 1 | 0 | 0 | 0 | 0 | 0 | 1 | 0 | 7 | 0.4 | 0.67 | 5.19 | 4.26 | ||
0 | 0 | 0 | 2 | 0 | 0 | 0 | 0 | 0 | 2 | ||||||||||
B | 0 | 0 | 0 | 0 | 0 | 1 | 0 | 0 | 0 | 0 | 3 | 0.2 | 0.37 | 3.00 | |||||
0 | 1 | 1 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | ||||||||||
Test item | A | 0.00205 | 0 | 0 | 0 | 2 | 0 | 0 | 1 | 0 | 0 | 1 | 11 | 0.6 | 0.76 | 13.10 | 15.31 | 3.060 | * |
0 | 0 | 1 | 2 | 1 | 2 | 1 | 0 | 0 | 0 | ||||||||||
B | 1 | 1 | 1 | 0 | 3 | 0 | 0 | 0 | 3 | 0 | 16 | 0.8 | 1.01 | 17.33 | |||||
0 | 0 | 1 | 0 | 0 | 2 | 1 | 0 | 2 | 1 | ||||||||||
Test item | A | 0.00410 | 4 | 0 | 2 | 1 | 0 | 1 | 2 | 1 | 0 | 1 | 21 | 1.05 | 1.00 | 13.13 | 11.34 | 2.335 | NS |
1 | 0 | 2 | 0 | 2 | 1 | 0 | 1 | 1 | 1 | ||||||||||
B | 0 | 2 | 2 | 1 | 0 | 2 | 0 | 2 | 1 | 0 | 21 | 1.05 | 0.89 | 9.98 | |||||
3 | 0 | 1 | 1 | 1 | 1 | 1 | 2 | 1 | 0 | ||||||||||
Test item | A | 0.00819 | 1 | 0 | 0 | 1 | 1 | 1 | 0 | 0 | 1 | 0 | 11 | 0.55 | 0.69 | 7.82 | 14.96 | 2.976 | * |
0 | 0 | 1 | 1 | 0 | 0 | 0 | 2 | 0 | 2 | ||||||||||
B | 2 | 2 | 0 | 2 | 0 | 2 | 1 | 1 | 2 | 1 | 25 | 1.3 | 0.97 | 25.00 | |||||
1 | 1 | 4 | 0 | 1 | 1 | 2 | 1 | 1 | 0 | ||||||||||
Test item | A | 0.0102 | 2 | 1 | 0 | 3 | 0 | 2 | 0 | 0 | 0 | 1 | 14 | 0.7 | 1.03 | 9.13 | 9.40 | 1.841 | NS |
3 | 1 | 0 | 0 | 0 | 0 | 0 | 1 | 0 | 0 | ||||||||||
B | 2 | 0 | 2 | 0 | 1 | 0 | 1 | 0 | 1 | 0 | 14 | 0.7 | 0.73 | 9.68 | |||||
1 | 1 | 1 | 1 | 2 | 0 | 0 | 0 | 1 | 0 | ||||||||||
Test item | A | 0.0128 | 2 | 1 | 0 | 0 | 1 | 2 | 1 | 2 | 3 | 0 | 19 | 1.0 | 1.05 | 9.55 | 9.66 | 1.913 | NS |
2 | 1 | 0 | 0 | 0 | 3 | 1 | 0 | 0 | 0 | ||||||||||
B | 0 | 2 | 0 | 0 | 1 | 0 | 0 | 2 | 2 | 0 | 13 | 0.7 | 0.99 | 9.82 | |||||
0 | 0 | 0 | 0 | 0 | 0 | 0 | 3 | 1 | 2 | ||||||||||
EMS | 10.0 mM | 88 | 79 | 98 | 94 | 96 | 103 | 134 | 123 | 121 | 128 | 1957 | 97.9 | 18.39 | 2681 | 28.190 | ** | ||
120 | 82 | 85 | 85 | 104 | 86 | 83 | 79 | 71 | 98 | ||||||||||
MUTATION FREQUENCY WITH METABOLIC ACTIVATION | |||||||||||||||||||
Treatment | Dose Level (µL/mL) | Plate counts | Tot. | Mean | SD | MF | MF Pooled cultures | t | Sig. | ||||||||||
Solvent control | A | 0.00 | 1 | 0 | 1 | 0 | 1 | 1 | 0 | 0 | 1 | 0 | 11 | 0.6 | 0.51 | 10.28 | 14.48 | ||
0 | 1 | 1 | 0 | 1 | 1 | 1 | 0 | 1 | 0 | ||||||||||
B | 1 | 1 | 3 | 0 | 0 | 0 | 1 | 0 | 3 | 0 | 21 | 1.1 | 1.10 | 18.42 | |||||
1 | 0 | 2 | 3 | 1 | 1 | 0 | 2 | 2 | 0 | ||||||||||
Test item | A | 0.0307 | 0 | 2 | 0 | 1 | 0 | 1 | 1 | 1 | 0 | 1 | 9 | 0.5 | 0.60 | 7.09 | 9.04 | -1.360 | NS |
0 | 0 | 1 | 0 | 0 | 0 | 1 | 0 | 0 | 0 | ||||||||||
B | 1 | 2 | 1 | 1 | 2 | 0 | 0 | 1 | 1 | 0 | 17 | 0.9 | 0.75 | 10.58 | |||||
2 | 1 | 0 | 1 | 0 | 0 | 1 | 1 | 0 | 2 | ||||||||||
Test item | A | 0.0614 | 1 | 0 | 1 | 0 | 1 | 0 | 0 | 1 | 0 | 1 | 8 | 0.4 | 0.50 | 7.23 | 6.02 | -2.365 | NS |
0 | 0 | 1 | 0 | 0 | 0 | 0 | 0 | 1 | 1 | ||||||||||
B | 0 | 1 | 0 | 0 | 0 | 1 | 0 | 0 | 0 | 1 | 5 | 0.25 | 0.44 | 4.75 | |||||
0 | 0 | 1 | 0 | 0 | 1 | 0 | 0 | 0 | 0 | ||||||||||
Test item | A | 0.123 | 0 | 0 | 1 | 1 | 2 | 1 | 0 | 0 | 0 | 3 | 15 | 0.75 | 0.79 | 15.10 | 16.78 | 0.583 | NS |
1 | 1 | 1 | 0 | 1 | 1 | 1 | 1 | 0 | 0 | ||||||||||
B | 1 | 3 | 1 | 0 | 0 | 0 | 1 | 0 | 3 | 1 | 18 | 0.9 | 0.97 | 18.49 | |||||
1 | 0 | 0 | 1 | 2 | 2 | 1 | 1 | 0 | 0 | ||||||||||
Test item | A | 0.154 | 2 | 1 | 2 | 1 | 1 | 0 | 2 | 1 | 0 | 1 | 19 | 1.0 | 0.89 | 19.19 | 13.93 | -0.144 | NS |
1 | 0 | 1 | 0 | 2 | 0 | 3 | 1 | 0 | 0 | ||||||||||
B | 0 | 0 | 0 | 0 | 0 | 1 | 1 | 0 | 1 | 1 | 9 | 0.45 | 0.51 | 8.82 | |||||
0 | 1 | 1 | 0 | 0 | 0 | 0 | 1 | 1 | 1 | ||||||||||
Test item | A | 0.192 | 1 | 0 | 0 | 0 | 0 | 0 | 0 | 1 | 0 | 0 | 4 | 0.2 | 0.41 | 4.03 | 6.05 | -2.454 | NS |
0 | 1 | 0 | 0 | 1 | 0 | 0 | 0 | 0 | 0 | ||||||||||
B | 0 | 2 | 1 | 1 | 0 | 0 | 1 | 0 | 0 | 0 | 9 | 0.5 | 0.60 | 7.78 | |||||
0 | 0 | 0 | 1 | 1 | 1 | 0 | 1 | 0 | 0 | ||||||||||
Test item | A | 0.240 | 2 | 1 | 0 | 0 | 0 | 0 | 1 | 1 | 0 | 0 | 17 | 0.9 | 0.93 | 14.41 | 14.71 | 0.162 | NS |
3 | 1 | 2 | 1 | 2 | 0 | 0 | 1 | 0 | 2 | ||||||||||
B | 3 | 1 | 1 | 0 | 0 | 3 | 2 | 0 | 0 | 2 | 23 | 1.2 | 1.09 | 14.94 | |||||
3 | 2 | 1 | 1 | 2 | 0 | 1 | 0 | 1 | 0 | ||||||||||
DMBA | 10.0 µg/mL | 15 | 17 | 16 | 19 | 16 | 15 | 20 | 16 | 17 | 15 | 348 | 17.4 | 2.01 | 240.6 | 10.909 | ** | ||
20 | 12 | 16 | 20 | 18 | 18 | 16 | 20 | 21 | 18 |
CE = Cloning efficiency
RS = Relative survival
SD = Standard deviation
Tot = Total number of mutant colonies
MF = Mutant frequency per million survival cells
£ = Insufficient number of cells recovered after treatment period
Sig = Significance level
NS = Not statistically significant
* = Statistically significant at p < 0.05
** = Statistically significant at p < 0.01
*** = Statistically significant at p > 0.001
Table 5: Historical control data | |
HISTORICAL DATA OF NEGATIVE/SOLVENT CONTROL mutation frequencies per million surviving cells (up to May 2019) |
|
WITHOUT METABOLIC ACTIVATION | WITH METABOLIC ACTIVATION |
Day 8 / Day 9 | Day 8 / Day 9 |
Mean value | |
9.49 | 11.19 |
Standard deviation | |
8.65 | 8.82 |
Number of experiments | |
72 | 73 |
Upper confidence limit (5%) | |
26.8 | 28.8 |
Observed range | |
1.45 - 65.14 | 1.48 - 59.72 |
HISTORICAL DATA OF POSITIVE CONTROL mutation frequencies per million surviving cells (up to November 2018) |
|
WITHOUT METABOLIC ACTIVATION | WITH METABOLIC ACTIVATION |
Day 8 / Day 9 | Day 8 / Day 9 |
Mean value | |
1147.38 | 503.30 |
Standard deviation | |
401.15 | 282.27 |
Number of experiments | |
72 | 73 |
Observed range | |
391 - 2631 | 109.86 - 1670.18 |
Table 1: Detailed results of Main Assay I | ||||||||||
MAIN ASSAY I | Plate incorporation method | |||||||||
Strain: TA1535 | ||||||||||
Dose-level [µL/plate] | Without metabolic activation | Mean | S. E. | With metabolic activation | Mean | S. E. | ||||
Plate counts | Plate counts | |||||||||
Untreated | 14 | 20 | 14 | 16 | 2.0 | 16 | 14 | 18 | 16 | 1.2 |
0.00 | 15 | 17 | 14 | 15 | 0.9 | 18 | 20 | 17 | 18 | 0.9 |
0.313 | 14 | 16 | 19 | 16 | 1.5 | 18 | 17 | 18 | 18 | 0.3 |
0.625 | 17 | 14 | 15 | 15 | 0.9 | 17 | 20 | 17 | 18 | 1.0 |
1.25 | 17 | 17 | 16 | 17 | 0.3 | 18 | 14 | 13 | 15 | 1.5 |
2.50 | 17 | 16 | 21 | 18 | 1.5 | 14 | 14 | 16 | 15 | 0.7 |
5.00 | 15 | 20 | 19 | 18 | 1.5 | 15 | 16 | 14 | 15 | 0.6 |
Controls | S9 | Plate counts | Mean | S. E. | ||||||
Sodium Azide | 1 µg/plate | - | 532 | 489 | 507 | 509 | 12.5 | |||
DMSO | 100 µL/plate | + | 18 | 20 | 17 | 18 | 0.9 | |||
2-Aminoanthracene | 1 µg/plate | + | 161 | 172 | 154 | 162 | 5.2 | |||
Strain: TA1537 | ||||||||||
Dose-level [µL/plate] | Without metabolic activation | Mean | S. E. | With metabolic activation | Mean | S. E. | ||||
Plate counts | Plate counts | |||||||||
Untreated | 17 | 17 | 18 | 17 | 0.3 | 24 | 20 | 17 | 20 | 2.0 |
0.00 | 19 | 22 | 16 | 19 | 1.7 | 20 | 20 | 21 | 20 | 0.3 |
0.00977 | 16 | 17 | 22 | 18 | 1.9 | 21 | 24 | 24 | 23 | 1.0 |
0.0195 | 17 | 22 | 18 | 19 | 1.5 | 23 | 25 | 20 | 23 | 1.5 |
0.0391 | 15 | 19 | 14 | 16 | 1.5 | 19 | 23 | 21 | 21 | 1.2 |
0.0781 | 12 | 14 | 16 | 14 | 1.2 | 25 | 23 | 22 | 23 | 0.9 |
0.156 | 14 | 16 | 16 | 15* | 0.7 | 22 | 18 | 16 | 19* | 1.8 |
0.313 | 12 | 12 | 10 | 11** | 0.7 | 20 | 24 | 21 | 22** | 1.2 |
Controls | S9 | Plate counts | Mean | S. E. | ||||||
DMSO | 100 µL/plate | - | 19 | 22 | 16 | 19 | 1.7 | |||
9-Aminoacridine | 50 µL/plate | - | 207 | 163 | 182 | 184 | 12.7 | |||
DMSO | 100 µL/plate | + | 20 | 20 | 21 | 20 | 0.3 | |||
2-Aminoanthracene | 1 µL/plate | + | 117 | 121 | 129 | 122 | 3.5 | |||
* : Slight thinning of the background lawn | ||||||||||
**: Moderate thinning of the background lawn | ||||||||||
Strain: WP2 uvrA | ||||||||||
Dose-level [µL/plate] | Without metabolic activation | Mean | S. E. | With metabolic activation | Mean | S. E. | ||||
Plate counts | Plate counts | |||||||||
Untreated | 27 | 31 | 25 | 28 | 1.8 | 37 | 31 | 42 | 37 | 3.2 |
0.00 | 26 | 31 | 34 | 30 | 2.3 | 33 | 28 | 34 | 32 | 1.9 |
0.313 | 29 | 27 | 24 | 27 | 1.5 | 31 | 36 | 32 | 33 | 1.5 |
0.625 | 29 | 28 | 31 | 29 | 0.9 | 32 | 35 | 35 | 34 | 1.0 |
1.25 | 26 | 28 | 34 | 29 | 2.4 | 38 | 36 | 35 | 36 | 0.9 |
2.50 | 26 | 26 | 29 | 27 | 1.0 | 24 | 29 | 32 | 28 | 2.3 |
5.00 | 27 | 24 | 24 | 25 | 1.0 | 25 | 29 | 30 | 28 | 1.5 |
Controls | S9 | Plate counts | Mean | S. E. | ||||||
MMS | 500 µg/pl | - | 144 | 152 | 153 | 150 | 2.8 | |||
DMSO | 100 µL/pl | + | 33 | 28 | 34 | 32 | 1.9 | |||
2-Aminoanthracene | 10 µg/pl | + | 130 | 127 | 131 | 129 | 1.2 | |||
Strain: TA98 | ||||||||||
Dose-level [µL/plate] | Without metabolic activation | Mean | S. E. | With metabolic activation | Mean | S. E. | ||||
Plate counts | Plate counts | |||||||||
Untreated | 28 | 30 | 26 | 28 | 1.2 | 39 | 32 | 39 | 37 | 2.3 |
0.00 | 27 | 30 | 26 | 28 | 1.2 | 40 | 37 | 41 | 39 | 1.2 |
0.313 | 24 | 27 | 21 | 24 | 1.7 | 34 | 43 | 37 | 38 | 2.6 |
0.625 | 24 | 21 | 28 | 24 | 2.0 | 38 | 33 | 39 | 37 | 1.9 |
1.25 | 26 | 31 | 25 | 27 | 1.9 | 30 | 32 | 33 | 32 | 0.9 |
2.50 | 26 | 27 | 25 | 26 | 0.6 | 38 | 34 | 37 | 36 | 1.2 |
5.00 | 28 | 26 | 26 | 27 | 0.7 | 38 | 37 | 37 | 37 | 0.3 |
Controls | S9 | Plate counts | Mean | S. E. | ||||||
DMSO | 100 µL/plate | - | 27 | 30 | 26 | 28 | 1.2 | |||
2-Nitrofluorene | 2 µL/plate | - | 140 | 166 | 152 | 153 | 7.5 | |||
DMSO | 100 µL/plate | + | 40 | 37 | 41 | 39 | 1.2 | |||
2-Aminoanthracene | 1 µL/plate | + | 317 | 356 | 406 | 360 | 25.8 | |||
Strain: TA100 | ||||||||||
Dose-level [µL/plate] | Without metabolic activation | Mean | S. E. | With metabolic activation | Mean | S. E. | ||||
Plate counts | Plate counts | |||||||||
Untreated | 117 | 113 | 113 | 114 | 1.3 | 114 | 127 | 127 | 123 | 4.3 |
0.00 | 126 | 127 | 121 | 125 | 1.9 | 115 | 108 | 102 | 108 | 3.8 |
0.00244 | 140 | 141 | 136 | 139 | 1.5 | NT | ||||
0.00488 | 140 | 145 | 133 | 139 | 3.5 | NT | ||||
0.00977 | 136 | 140 | 140 | 139 | 1.3 | 108 | 109 | 110 | 109 | 0.6 |
0.0195 | 133 | 133 | 120 | 129 | 4.3 | 102 | 106 | 113 | 107 | 3.2 |
0.0391 | 126 | 127 | 122 | 125 | 1.5 | 107 | 106 | 116 | 110 | 3.2 |
0.0781 | 90 | 91 | 83 | 88* | 2.5 | 129 | 121 | 124 | 125 | 2.3 |
0.156 | NT | 109 | 109 | 108 | 109 | 0.3 | ||||
0.313 | NT | 98 | 97 | 86 | 94* | 3.8 | ||||
Controls | S9 | Plate counts | Mean | S. E. | ||||||
Sodium Azide | 1 µg/plate | - | 621 | 655 | 724 | 667 | 30.3 | |||
DMSO | 100 µL/plate | + | 115 | 108 | 102 | 108 | 3.8 | |||
2-Aminoanthracene | 1 µg/plate | + | 918 | 1020 | 1083 | 1007 | 48.1 | |||
* : Slight thinning of the background lawn | ||||||||||
NT: Not tested |
Table 2: Detailed results of Main Assay II | ||||||||||
MAIN ASSAY II | Pre-incubation method | |||||||||
Strain: TA1535 | ||||||||||
Dose-level [µL/plate] | Without metabolic activation | Mean | S. E. | With metabolic activation | Mean | S. E. | ||||
Plate counts | Plate counts | |||||||||
Untreated | 21 | 20 | 17 | 19 | 1.2 | 13 | 14 | 14 | 14 | 0.3 |
0.00 | 18 | 18 | 21 | 19 | 1.0 | 14 | 16 | 13 | 14 | 0.9 |
0.313 | 13 | 15 | 17 | 15** | 1.2 | 17 | 13 | 15 | 15 | 1.2 |
0.625 | 13 | 15 | 16 | 15** | 0.9 | 15 | 14 | 14 | 14 | 0.3 |
1.25 | 14 | 16 | 15 | 15** | 0.6 | 16 | 15 | 15 | 15 | 0.3 |
2.50 | 17 | 15 | 19 | 17** | 1.2 | 12 | 15 | 16 | 14 | 1.2 |
5.00 | 12 | 12 | 11 | 12** | 0.3 | 12 | 11 | 12 | 12 | 0.3 |
Controls | S9 | Plate counts | Mean | S. E. | ||||||
Sodium Azide | 1 µg/plate | - | 382 | 416 | 384 | 28.1 | 11.0 | |||
DMSO | 50 µg/plate | + | 14 | 16 | 13 | 14 | 0.9 | |||
2-Aminoanthracene | 1 µg/plate | + | 113 | 113 | 110 | 21.4 | 1.0 | |||
**: Moderate thinning of the background lawn | ||||||||||
Strain: TA1537 | ||||||||||
Dose-level [µL/plate] | Without metabolic activation | Mean | S. E. | With metabolic activation | Mean | S. E. | ||||
Plate counts | Plate counts | |||||||||
Untreated | 20 | 16 | 15 | 17 | 1.5 | 23 | 22 | 21 | 22 | 0.6 |
0.00 | 18 | 18 | 21 | 19 | 1.0 | 21 | 20 | 20 | 20 | 0.3 |
0.00977 | 15 | 14 | 12 | 14* | 0.9 | 25 | 20 | 23 | 23 | 1.5 |
0.0195 | 10 | 11 | 12 | 11** | 0.6 | 23 | 20 | 20 | 21 | 1.0 |
0.0391 | 8 | 8 | 7 | 8** | 0.3 | 18 | 23 | 15 | 19 | 2.3 |
0.0781 | 4 | 5 | 7 | 5** | 0.9 | 17 | 18 | 21 | 19 | 1.2 |
0.156 | 8 | 6 | 9 | 8*** | 0.9 | 17 | 17 | 15 | 16* | 0.7 |
0.313 | 5 | 7 | 5 | 6*** | 0.7 | 14 | 12 | 18 | 15** | 1.8 |
Controls | S9 | Plate counts | Mean | S. E. | ||||||
DMSO | 50 µL/plate | - | 18 | 18 | 21 | 19.1 | 1.0 | |||
9-Aminoacridine | 50 µL/plate | - | 92 | 86 | 94 | 31.3 | 2.4 | |||
DMSO | 50 µL/plate | + | 21 | 20 | 20 | 20.0 | 0.3 | |||
2-Aminoanthracene | 1 µL/plate | + | 78 | 78 | 85 | 20.3 | 2.3 | |||
*: Slight thinning of the background lawn | ||||||||||
** : Moderate thinning of the background lawn | ||||||||||
***: Severe thinning of the background lawn | ||||||||||
Strain: WP2 uvrA | ||||||||||
Dose-level [µL/plate] | Without metabolic activation | Mean | S. E. | With metabolic activation | Mean | S. E. | ||||
Plate counts | Plate counts | |||||||||
Untreated | 30 | 31 | 32 | 31 | 0.6 | 37 | 35 | 38 | 37 | 0.9 |
0.00 | 29 | 29 | 29 | 29 | 0.0 | 35 | 33 | 39 | 36 | 1.8 |
0.313 | 30 | 30 | 30 | 30 | 0.0 | 32 | 41 | 41 | 38 | 3.0 |
0.625 | 28 | 28 | 26 | 27* | 0.7 | 33 | 39 | 40 | 37 | 2.2 |
1.25 | 29 | 22 | 22 | 24** | 2.3 | 39 | 35 | 32 | 35 | 2.0 |
2.50 | 20 | 25 | 24 | 23*** | 1.5 | 35 | 32 | 37 | 35 | 1.5 |
5.00 | 25 | 29 | 25 | 26*** | 1.3 | 28 | 26 | 26 | 27 | 0.7 |
Controls | S9 | Plate counts | Mean | S. E. | ||||||
MMS | 500 µg/plate | - | 112 | 114 | 117 | 23.4 | 1.5 | |||
DMSO | 50 µg/plate | + | 35 | 33 | 39 | 5.2 | 1.8 | |||
2-Aminoanthracene | 20 µg/plate | + | 135 | 150 | 167 | 30.5 | 9.2 | |||
*: Slight thinning of the background lawn | ||||||||||
** : Moderate thinning of the background lawn | ||||||||||
***: Severe thinning of the background lawn | ||||||||||
Strain: TA98 | ||||||||||
Dose-level [µL/plate] | Without metabolic activation | Mean | S. E. | With metabolic activation | Mean | S. E. | ||||
Plate counts | Plate counts | |||||||||
Untreated | 27 | 31 | 32 | 30 | 1.5 | 35 | 35 | 36 | 35 | 0.3 |
0.00 | 33 | 34 | 29 | 32 | 1.5 | 34 | 39 | 39 | 37 | 1.7 |
0.313 | 26 | 32 | 25 | 28 | 2.2 | 29 | 31 | 30 | 30 | 0.6 |
0.625 | 32 | 31 | 25 | 29 | 2.2 | 28 | 30 | 34 | 31 | 1.8 |
1.25 | 29 | 29 | 29 | 29 | 0.0 | 30 | 30 | 29 | 30 | 0.3 |
2.50 | 24 | 25 | 25 | 25 | 0.3 | 27 | 25 | 28 | 27 | 0.9 |
5.00 | 27 | 25 | 29 | 27 | 1.2 | 30 | 35 | 28 | 31 | 2.1 |
Controls | S9 | Plate counts | Mean | S. E. | ||||||
DMSO | 50 µl/plate | - | 33 | 34 | 29 | 1.2 | 1.5 | |||
2-Nitrofluorene | 2 µl/plate | - | 125 | 123 | 127 | 4.5 | 1.2 | |||
DMSO | 50 µl/plate | + | 34 | 39 | 39 | 6.2 | 1.7 | |||
2-Aminoanthracene | 2 µl/plate | + | 507 | 681 | 643 | 1.9 | 52.8 | |||
Strain: TA100 | ||||||||||
Dose-level [µL/plate] | Without metabolic activation | Mean | S. E. | With metabolic activation | Mean | S. E. | ||||
Plate counts | Plate counts | |||||||||
Untreated | 125 | 118 | 128 | 124 | 3.0 | 156 | 158 | 147 | 154 | 3.4 |
0.00 | 124 | 132 | 122 | 126 | 3.1 | 147 | 148 | 159 | 151 | 3.8 |
0.00244 | 128 | 123 | 132 | 128 | 2.6 | NT | ||||
0.00488 | 120 | 122 | 120 | 121 | 0.7 | NT | ||||
0.00977 | 78 | 79 | 78 | 78* | 0.3 | 123 | 120 | 128 | 124 | 2.3 |
0.0195 | 98 | 97 | 91 | 95** | 2.2 | 121 | 116 | 118 | 118 | 1.5 |
0.0391 | 89 | 86 | 85 | 87*** | 1.2 | 115 | 128 | 124 | 122 | 3.8 |
0.0781 | 82 | 89 | 80 | 84*** | 2.7 | 129 | 127 | 121 | 126 | 2.4 |
0.156 | NT | 137 | 134 | 126 | 132 | 3.3 | ||||
0.313 | NT | 78 | 77 | 68 | 74* | 3.2 | ||||
Controls | S9 | Plate counts | Mean | S. E. | ||||||
Sodium Azide | 1 µg/pl | - | 680 | 542 | 623 | 615 | 40.0 | |||
DMSO | 50 µL/pl | + | 147 | 148 | 159 | 151 | 3.8 | |||
2-Aminoanthracene | 2 µg/pl | + | 1316 | 1086 | 1218 | 1207 | 66.6 | |||
*: Slight thinning of the background lawn | ||||||||||
** : Moderate thinning of the background lawn | ||||||||||
***: Severe thinning of the background lawn | ||||||||||
NT: Not tested |
Table 3: Detailed results of Main Assay III | |||||||
MAIN ASSAY III | Pre-incubation method | ||||||
Strain: TA1535 | |||||||
Dose-level [µL/plate] | Without metabolic activation | Mean | S. E. | ||||
Plate counts | |||||||
Untreated | 14 | 14 | 14 | 14 | 0.0 | ||
0.00 | 15 | 15 | 14 | 15 | 0.3 | ||
0.00977 | 18 | 21 | 21 | 20 | 1.0 | ||
0.0195 | 15 | 14 | 15 | 15 | 0.3 | ||
0.0391 | 15 | 17 | 15 | 16 | 0.7 | ||
0.0781 | 16 | 13 | 13 | 14 | 1.0 | ||
0.156 | 12 | 12 | 13 | 12* | 0.3 | ||
0.313 | 13 | 13 | 12 | 13** | 0.3 | ||
Controls | S9 | Plate counts | Mean | S. E. | |||
Sodium Azide | 1 µg/plate | - | 412 | 463 | 394 | 423 | 20.7 |
* : Slight thinning of the background lawn | |||||||
**: Moderate thinning of the background lawn | |||||||
Strain: TA1537 | |||||||
Dose-level [µL/plate] | Without metabolic activation | Mean | S. E. | ||||
Plate counts | |||||||
Untreated | 13 | 15 | 14 | 14 | 0.6 | ||
0.00 | 14 | 17 | 16 | 16 | 0.9 | ||
0.000610 | 19 | 20 | 16 | 18 | 1.2 | ||
0.00122 | 15 | 16 | 13 | 15 | 0.9 | ||
0.00244 | 18 | 19 | 20 | 19 | 0.6 | ||
0.00488 | 17 | 15 | 16 | 16 | 0.6 | ||
0.00977 | 10 | 10 | 12 | 11** | 0.7 | ||
Controls | S9 | Plate counts | Mean | S. E. | |||
DMSO | 50 µL/pl | - | 14 | 17 | 16 | 16 | 0.9 |
9-Aminoacridine | 50 µg/pl | - | 89 | 103 | 127 | 106 | 11.1 |
**: Moderate thinning of the background lawn | |||||||
Strain: WP2 uvrA | |||||||
Dose-level [µL/plate] | Without metabolic activation | Mean | S. E. | ||||
Plate counts | |||||||
Untreated | 30 | 26 | 27 | 28 | 1.2 | ||
0.00 | 34 | 31 | 27 | 31 | 2.0 | ||
0.0391 | 29 | 26 | 32 | 29 | 1.7 | ||
0.0781 | 25 | 30 | 29 | 28 | 1.5 | ||
0.156 | 27 | 26 | 34 | 29 | 2.5 | ||
0.313 | 27 | 29 | 30 | 29 | 0.9 | ||
0.625 | 26 | 22 | 22 | 23* | 1.3 | ||
1.25 | 21 | 23 | 24 | 23** | 0.9 | ||
Controls | S9 | Plate counts | Mean | S. E. | |||
MMS | 500 µg/pl | - | 156 | 161 | 179 | 165 | 7.0 |
*: Slight thinning of the background lawn | |||||||
**: Moderate thinning of the background lawn | |||||||
Strain: TA100 | |||||||
Dose-level [µL/plate] | Without metabolic activation | Mean | S. E. | ||||
Plate counts | |||||||
Untreated | 154 | 142 | 141 | 146 | 4.2 | ||
0.00 | 120 | 136 | 131 | 129 | 4.7 | ||
0.000305 | 131 | 134 | 139 | 135 | 2.3 | ||
0.000610 | 141 | 121 | 121 | 128 | 5.7 | ||
0.00122 | 156 | 138 | 159 | 151 | 6.6 | ||
0.00244 | 121 | 129 | 128 | 126 | 2.5 | ||
0.00488 | 92 | 100 | 90 | 94* | 3.1 | ||
0.00977 | 88 | 76 | 78 | 81** | 3.7 | ||
Controls | S9 | Plate counts | Mean | S. E. | |||
Sodium Azide | 1 µg/plate | - | 715 | 883 | 779 | 792 | 49.0 |
* : Slight thinning of the background lawn | |||||||
**: Moderate thinning of the background lawn |
Table 4: Historical control data | |||||||||
TA 1535 Plate incorporation | TA 1535 Pre-incubation | ||||||||
Negative Control | Positive control | Negative Control | Positive control | ||||||
-S9 | +S9 | -S9 | +S9 | -S9 | +S9 | -S9 | +S9 | ||
Mean | 18 | 17 | 518 | 136 | Mean | 18 | 17 | 528 | 98 |
SD | 2.6 | 2.2 | 81.7 | 27.9 | SD | 2.5 | 2.2 | 78.4 | 14.7 |
UCL | 23.2 | 21.3 | 681.3 | 191.6 | UCL | 23.0 | 21.5 | 684.5 | 127.2 |
LCL | 12.8 | 12.7 | 354.6 | 79.9 | LCL | 12.8 | 12.7 | 370.7 | 68.5 |
n | 491 | 492 | 491 | 492 | n | 393 | 379 | 393 | 378 |
min | 13 | 12 | 299 | 81 | min | 12 | 12 | 374 | 54 |
max | 16 | 22 | 750 | 241 | max | 25 | 23 | 774 | 179 |
TA 1537 Plate incorporation | TA 1537 Pre-incubation | ||||||||
Negative Control | Positive control | Negative Control | Positive control | ||||||
-S9 | +S9 | -S9 | +S9 | -S9 | +S9 | -S9 | +S9 | ||
Mean | 17 | 21 | 177 | 114 | Mean | 18 | 21 | 169 | 98 |
SD | 2.3 | 2.9 | 40 | 21 | SD | 2.3 | 2.4 | 46.4 | 12.4 |
UCL | 21.7 | 26.7 | 257.2 | 156.7 | UCL | 22.1 | 25.7 | 261.8 | 122.3 |
LCL | 12.6 | 15.2 | 97.0 | 72 | LCL | 13.0 | 16.1 | 76.3 | 72.8 |
n | 495 | 494 | 495 | 494 | n | 386 | 371 | 386 | 370 |
min | 11 | 15 | 92 | 75 | min | 12 | 15 | 72 | 69 |
max | 27 | 33 | 407 | 181 | max | 27 | 27 | 412 | 142 |
TA 98 Plate incorporation | TA 98 Pre-incubation | ||||||||
Negative Control | Positive control | Negative Control | Positive control | ||||||
-S9 | +S9 | -S9 | +S9 | -S9 | +S9 | -S9 | +S9 | ||
Mean | 31 | 38 | 162 | 508 | Mean | 31 | 39 | 163 | 628 |
SD | 3.3 | 3.9 | 29.3 | 157.8 | SD | 3.1 | 3.2 | 26.7 | 180 |
UCL | 37.7 | 46.0 | 220.3 | 823.5 | UCL | 37.4 | 45.8 | 216.5 | 987.7 |
LCL | 24.3 | 30.5 | 103.0 | 192.2 | LCL | 25.1 | 33.1 | 109.8 | 267.6 |
n | 497 | 497 | 497 | 497 | n | 377 | 353 | 377 | 353 |
min | 23 | 29 | 109 | 203 | min | 24 | 33 | 115 | 188 |
max | 43 | 53 | 259 | 994 | max | 43 | 50 | 256 | 1377 |
TA 100 Plate incorporation | TA 100 Pre-incubation | ||||||||
Negative Control | Positive control | Negative Control | Positive control | ||||||
-S9 | +S9 | -S9 | +S9 | -S9 | +S9 | -S9 | +S9 | ||
Mean | 141 | 151 | 633 | 1192 | Mean | 143 | 150 | 676 | 1118 |
SD | 17.7 | 18.3 | 157.0 | 230.2 | SD | 16.7 | 15.5 | 154.3 | 201.8 |
UCL | 176.1 | 188.0 | 947.3 | 1652.1 | UCL | 176.6 | 181.2 | 984.8 | 1521.5 |
LCL | 105.4 | 114.3 | 319.3 | 731.4 | LCL | 109.7 | 119.2 | 367.5 | 714.2 |
n | 499 | 498 | 499 | 498 | n | 386 | 366 | 386 | 365 |
min | 109 | 86 | 144 | 607 | min | 112 | 119 | 325 | 745 |
max | 182 | 196 | 1081 | 2257 | max | 192 | 197 | 1081 | 1701 |
WP2 uvrA Plate incorporation | WP2 uvrA Pre-incubation | ||||||||
Negative Control | Positive control | Negative Control | Positive control | ||||||
-S9 | +S9 | -S9 | +S9 | -S9 | +S9 | -S9 | +S9 | ||
Mean | 28 | 34 | 178 | 194 | Mean | 28 | 33 | 189 | 198 |
SD | 2.9 | 3.5 | 20.1 | 36.9 | SD | 3.0 | 3.2 | 41.6 | 33.8 |
UCL | 34.0 | 40.5 | 218.7 | 268,3 | UCL | 33.7 | 39.5 | 272.0 | 265.5 |
LCL | 22.4 | 26.7 | 138.1 | 120,6 | LCL | 21.7 | 26.5 | 105.5 | 130.3 |
n | 430 | 430 | 430 | 430 | n | 315 | 302 | 315 | 301 |
min | 21 | 26 | 127 | 117 | min | 20 | 24 | 113 | 129 |
max | 38 | 47 | 240 | 333 | max | 38 | 44 | 351 | 322 |
SD: Standard Deviation | |||||||||
UCL: Upper Confidence Limit (mean value + 2 SD) | |||||||||
LCL: Lower Confidence Limit (mean value - 2 SD) | |||||||||
n: number of experiments |
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 assay
Mutagenicity in bacteria was assessed in a study performed with the registered substance isotridecanol, ethoxylated, < 2.5 EO (CAS No. 69011-36-5, EC No. 500-241-6) according to OECD guideline 471 (adopted 1997) under GLP conditions (Sasol/BASF, 2019a). Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and E. coli strain WP2 uvrA were treated using the plate incorporation method and the preincubation method, both with and without the addition of a metabolic activation system (rat liver S9 mix). The dose levels selected for the different strains in the plate incorporation experiment (first assay) were the following: TA1535, WP2 uvrA, TA98 (±S9): 0.313, 0.625, 1.25, 2.50 and 5.00 µg/plate; TA1537 (±S9) and TA100 (+S9): 0.00977, 0.0195, 0.0391, 0.0781, 0.156 and 0.313 µg/plate; TA100 (-S9): 0.00244, 0.00488, 0.00977, 0.0195, 0.0391 and 0.0781 µg/plate. Cytotoxicity was observed with TA1537 and TA100 tester strains at the highest dose levels, both in the absence and presence of S9 mix. No cytotoxicity was observed with the remaining tester strains and, therefore, the same dose levels were applied in the pre-incubation assay (second assay). However, in this assay, severe cytotoxicity was observed with most tester strains in the absence of metabolic activation and a third experiment (pre-incubation method) was necessary in order to have a sufficiently high number of analysable concentrations. In this third assay the following dose levels were used: TA1535 (-S9): 0.00977, 0.0195, 0.0391, 0.0781, 0.156 and 0.313 µg/plate; TA1537 (-S9): 0.000610, 0.00122, 0.00244, 0.00488, 0.00977 and 0.0195 µg/plate; WP2 uvrA (-S9): 0.0391, 0.0781, 0.156, 0.313, 0.625 and 1.25 µg/plate; TA100 (-S9): 0.000305, 0.000610, 0.00122, 0.00244, 0.00488 and 0.00977 µg/plate. All tests were carried out in triplicates. The vehicle (dimethyl sulfoxide, DMSO) control and untreated control produced counts of revertant colonies within an acceptable range. All positive controls used induced significant increases in the frequency of revertant colonies, both with and without the metabolising system. No mutagenic activity of the test substance to any of the tester strains was observed with and without metabolic activation at any dose level. Thus, under the conditions of this test, the test substance can be regarded as not mutagenic in bacteria.
In vitro mammalian cell micronucleus test
The registered substance isotridecanol, ethoxylated, < 2.5 EO (CAS No. 69011-36-5, EC No. 500-241-6) was assayed for its ability to induce micronuclei in human lymphocytes, following in vitro treatment in the presence and absence of an S9 metabolic activation system (rat liver S9 mix). Experiments were conducted according to OECD guideline 487 (adopted 2016) and observing GLP conditions (Sasol/BASF, 2020). A total of three experiments were performed: a short-term treatment, in which cells were treated for 3 h both in the absence and presence of S9 mix and harvested at 32 h, and a long-term (continuous) treatment where cells were treated in the absence of S9 mix, until harvest at 31 h. The dose levels applied were 0.195, 0.293, 0.439, 0.658, 0.988, 1.48, 2.22, 3.33 and 5.00 µL/mL. An additional dose of 0.130 µL/mL was included for the continuous treatment. Dimethyl sulfoxide (DMSO) served as vehicle. Since severe cytotoxicity was observed at all dose levels in all experiments, a second assay needed to be performed. The dose levels applied in the second assay were: ±S9 (3 h treatment, 31 h harvest): 0.00780, 0.0117, 0.0176, 0.0263, 0.0395, 0.0593, 0.0889, 0.133 and 0.200 µL/mL; -S9 (32 h treatment, 32 h harvest): 0.000520, 0.000780, 0.00117, 0.00176, 0.00263, 0.00395, 0.00593, 0.00889, 0.0133 and 0.0200 µL/mL. In the presence of S9 metabolic activation an adequate level of cytotoxicity to select dose levels for scoring was not achieved. Therefore, this treatment series was repeated by using a very narrowly spaced concentration interval. The following dose levels were selected: 0.0654, 0.0752, 0.0865, 0.0994, 0.114, 0.132, 0.151, 0.174 and 0.200 µL/mL. Each experiment included appropriate negative and positive controls. Two replicate cell cultures were prepared at each test point. The actin polymerisation inhibitor Cytochalasin B was added after 3 h treatment to allow the selective analysis of micronucleus frequency in binucleated cells. The cytokinesis block proliferation index (CBPI) was calculated in order to evaluate cytotoxicity. 2000 binucleated cells per culture were scored to assess the frequency of micronucleated cells as well as the number of micronuclei per cell, while 1000 cells in total per concentration were assessed for cytotoxicity. Adequate cell proliferation was observed in negative control cultures and an appropriate number of doses and cells was analysed. The negative and positive control treatments yielded the expected results. No statistically significant increase in the incidence of micronucleated cells over the concurrent solvent control value was observed at any dose level at any treatment condition for the test substance. It can therefore be concluded that the test substance does not induce micronuclei in human lymphocytes after in vitro treatment under the reported experimental conditions.
In vitro mammalian cell gene mutation test
The mutagenic potential of the registered substance isotridecanol, ethoxylated, < 2.5 EO (CAS No. 69011-36-5, EC No. 500-241-6) in mammalian cells was assessed in a HPRT-assay according to OECD guideline 476 (adopted 2016) under GLP conditions (Sasol/BASF, 2019b). Following pre-tests with concentrations ranging from 0.00328 - 5.00 µL/mL, Chinese hamster V79 cells were exposed for 3 h to test substance concentrations of 0.00205, 0.00410, 0.00819, 0.0102, 0.0128, 0.0160 and 0.0200 µg/mL in the absence of metabolic activation, and at concentrations of 0.0307, 0.0614, 0.123, 0.154, 0.192, 0.240 and 0.300 µL/mL in the presence of metabolic activation by rat liver S9 mix. In both treatment series cytotoxic effects were observed at one or several concentrations. No relevant increase in mutant numbers or mutant frequency was observed following treatment with the test substance at any dose level, in the absence or presence of S9 mix. Negative and positive control treatments were included in each mutation experiment. Significant increases in mutant numbers or mutant frequency were obtained with the positive control treatments, indicating the correct functioning of the assay system. It is concluded that the test substance does not induce gene mutation in Chinese hamster V79 cells after in vitro treatment in the absence or presence of S9 metabolic activation under the reported experimental conditions.
Justification for classification or non-classification
The available data on genetic toxicity obtained with isotridecanol, ethoxylated, < 2.5 EO (CAS No. 69011-36-5, EC No. 500-241-6) do not meet the criteria for classification according to the CLP Regulation (EC) No. 1272/2008 and are therefore conclusive but not sufficient for classification.
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