Registration Dossier
Registration Dossier
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EC number: 309-912-6 | CAS number: 101357-15-7
Endpoint summary
Administrative data
Key value for chemical safety assessment
Genetic toxicity in vitro
Description of key information
Bacterial Reverse Mutation Assay Key Study: Durward (1993)
The test material was found to be non-mutagenic under the conditions of this test.
Bacterial Reverse Mutation Assay Key Study: Kovács (2018)
Under the conditions of this study, the test material had no mutagenic activity on Escherichia coli WP2 uvrA strain with or without activation.
Bacterial Reverse Mutation Assay Supporting Study: Thompson (1992)
The test material was found to be non-mutagenic under the conditions of this test.
In Vitro Mammalian Chromosome Aberration Test Key Study: Durward (1994)
The material is considered to be non-clastogenic to CHL cells in vitro under the conditions of this test.
Mammalian Cell Gene Mutation Assay Key Study: Morris (2012)
Under the conditions of the test, the test material was found to be negative in the CHO-HPRT mutation assay both in the absence and presence of metabolic activation.
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:
- 09 May 2018 to 18 May 2018
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Deviations:
- yes
- Remarks:
- The experimental design is compatible with the guideline requirements, but only one bacterial strain was used in the study as requested by the Sponsor
- Qualifier:
- according to guideline
- Guideline:
- EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
- Deviations:
- yes
- Remarks:
- The experimental design is compatible with the guideline requirements, but only one bacterial strain was used in the study as requested by the Sponsor
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
- Deviations:
- yes
- Remarks:
- The experimental design is compatible with the guideline requirements, but only one bacterial strain was used in the study as requested by the Sponsor
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- bacterial reverse mutation assay
- Target gene:
- Tryptophan requirement in the Escherichia coli strain.
- Species / strain / cell type:
- E. coli WP2 uvr A
- Details on mammalian cell type (if applicable):
- CELLS USED
- Source of cells: MOLTOX - Molecular Toxicology Inc., Boone, North Carolina, USA
- The strain was stored at -80 ± 10 ºC in the Culture Collection of the Microbiological Laboratory of the testing laboratory. Frozen permanent cultures of the tester strain was prepared from fresh, overnight cultures to which DMSO was added as a cryoprotective agent.
- The phenotype of the tester strain used in the bacterial reverse mutation assays with regard to UV sensitivity (uvrA), as well as spontaneous mutation frequencies are checked regularly according to Ames et al. and Maron and Ames.
- Spontaneous reversion of the test strain to tryptophan independence is measured routinely in mutagenicity experiments and is expressed as the number of spontaneous revertants per plate.
- The day before treatment, the frozen bacterial cultures were thawed at room temperature and 200 μL inoculum were used to inoculate each 50 mL of Nutrient Broth No.2 for the overnight cultures in the assay. The cultures were incubated for 10-14 hours at 37 °C in a Gyrotory water bath shaker.
The viability of each testing culture was determined by plating 0.1 mL of the 10^5, 10^6, 10^7 and 10^8 dilutions prepared by sterile physiological saline on Nutrient Agar plates. The number of viable cell of the cultures was determined by manual counting after approximately 24-hour incubation at 37 °C.
MEDIA USED
- Nutrient Broth No.2 - Metabolic activation:
- with and without
- Metabolic activation system:
- S9 Mix
- Test concentrations with justification for top dose:
- - 5000, 1581, 500, 158.1, 50, 15.81, 5, 1.581 µg/plate
- Based on the results of the preliminary test, 100 mg/mL stock solution was prepared in DMSO, which was diluted by serial dilutions in several steps to obtain the dosing formulations for lower doses. The maximum test concentration was 5000 μg/plate. - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: DMSO
- The solubility of the test material was examined using Distilled water, Dimethyl sulfoxide (DMSO), N,N-Dimethylformamide (DMF) and Acetone. The test material was insoluble in Distilled water at 100 mg/mL concentration. At the same concentration the test material was formed homogeneous suspension with Acetone. The test material was soluble at this concentration using DMSO and DMF (black colour solution was detected in each case). Due to the better biocompatibility, DMSO was selected as vehicle (solvent) for the study. The obtained stock solution (50 μL) with the solution of top agar and phosphate buffer was examined in a test tube without test bacterium suspension to examine the formulation compatibility.
- All dilutions in the main tests of test material were made in the testing laboratory using Dimethyl sulfoxide (DMSO). Test solutions were freshly prepared at the beginning of the experiments in the testing laboratory by diluting the stock solution using the selected solvent and were used within 2 hours after preparation. - Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- methylmethanesulfonate
- other: 2-aminoanthracene with activation (50 µg/plate)
- Details on test system and experimental conditions:
- METHOD OF APPLICATION:
- The study included a Preliminary Compatibility Test, an Initial Mutation Test and a Confirmatory Mutation Test. In the Initial Mutation Test, the plate incorporation method was used. In the Confirmatory Mutation Test, the pre-incubation method was used.
INITIAL MUTATION TEST
- A standard plate incorporation procedure was performed as an Initial Mutation Test. Bacteria (cultured in Nutrient Broth No .2) were exposed to the test material both in the presence and absence of an appropriate metabolic activation system.
- Molten top agar was prepared and kept at 45 °C. The equivalent number of minimal glucose agar plates (three plates per test material concentration and for each control) was properly labelled. The test material and other components were prepared freshly and added to the overlay (45 °C).
- The content of the tubes: top agar 2000 μL, vehicle or test material formulation (or reference controls) 50 μL, overnight culture of test strain 100 μL and phosphate buffer (pH 7.4) or S9 mix 500 μL. This solution was mixed and poured on the surface of minimal agar plates. For activation studies, instead of phosphate buffer, 0.5 mL of the S9 mix was added to each overlay tube. The entire test consisted of non-activated and activated test conditions, with the addition of untreated, negative (solvent) and positive controls. After preparation, the plates were incubated at 37 °C for 48 hours.
CONFIRMATORY MUTATION TEST
- A pre-incubation procedure was performed as a Confirmatory Mutation Test since in the Initial Mutation Test no positive effect was observed.
- For the pre-incubation method, bacteria (cultured in Nutrient Broth No. 2) were exposed to the test material both in the presence and absence of an appropriate metabolic activation system. The equivalent number of minimal glucose agar plates was properly labelled. Molten top agar was prepared and kept at 45 °C.
- Before the overlaying, 50 μL of the test material formulation or its vehicle (or 50 μL of positive reference controls or their solvents), 100 μL of the overnight culture of bacterial cells and 0.5 mL of the S9 mix (activated test conditions) or phosphate buffer pH 7.4 (non-activated test conditions) were added into appropriate tubes to provide direct contact between bacteria and the test material. The tubes (3 tubes per control and 3 tubes for each concentration level) were gently mixed and incubated for 20 minutes at 37 °C in a shaking incubator.
- After the incubation period, 2 mL of molten top agar were added to the tubes, and then the content mixed and poured on the surface of minimal glucose agar plates. The entire test consisted of non-activated and activated test conditions, with the addition of untreated, negative and positive controls. After preparation, the plates were incubated at 37 °C for approximately 48 hours .
EVALUATION OF EXPERIMENTAL DATA
- The colony numbers on the untreated / negative (solvent) / positive control and test material treated plates were determined by manual counting. Visual examination of the plates was also performed; precipitation or signs of growth inhibition (if any) were recorded and reported. The mean number of revertants per plate, the standard deviation and the mutation factor* values were calculated for each concentration level of the test material and for the controls using Microsoft ExcelTM software.
- Mutation factor (MF): mean number of revertants on the test material plate / mean number of revertants on the vehicle control plate.
Criteria for Validity:
The study was considered valid if:
- the number of revertant colonies of the negative (solvent) and positive controls were in the relevant historical control range in the main tests;
- at least five analysable concentrations were presented in the main tests. - Evaluation criteria:
- Criteria for a Positive Response:
A test material was considered mutagenic if:
- a dose–related increase in the number of revertants occurred and/or;
- a reproducible biologically relevant positive response for at least one of the dose groups occurred with or without metabolic activation.
An increase was considered biologically relevant if:
- the number of reversions was more than two times higher than the reversion rate of the negative (solvent) control.
Criteria for a Negative Response:
The test material was considered to have shown no mutagenic activity in this study if it produces neither a dose-related increase in the number of revertants nor a reproducible biologically relevant positive response at any of the dose groups, with or without metabolic activation. - Statistics:
- According to the guidelines, statistical method may be used as an aid in evaluating the test results. However, statistical significance should not be the only determining factor for a positive response.
- Key result
- Species / strain:
- E. coli WP2 uvr A
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity, but tested up to precipitating concentrations
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- INITIAL AND CONFIRMATORY MUTATION TESTS
- In the Initial Mutation Test (using plate incorporation method), the highest revertant rate was observed at 1.581 μg/plate concentration without metabolic activation (the observed mutation factor value was: MF: 1.33). However, there was no dose-response relationship, the observed mutation factor values were below the biologically relevant threshold limit and the number of revertant colonies was within the historical control range.
- In the Confirmatory Mutation Test (using the pre-incubation method), the highest revertant rate was observed at 1.581 μg/plate concentration with metabolic activation (the observed mutation factor value was: MF: 1.16). However, there was no dose-response relationship, the number of revertant colonies did not show any biologically relevant increase compared to the solvent controls and the number of revertant colonies was within the historical control range.
- Higher numbers of revertant colonies compared to the vehicle (solvent) control were detected in the Initial Mutation Test in some other sporadic cases. However, no dose-dependence was observed in those cases and they were below the biologically relevant threshold value. The numbers of revertant colonies were within the historical control range in each case, so they were considered as reflecting the biological variability of the test.
- Precipitate/slight precipitate was observed on the plates in the main tests with and without metabolic activation at the concentrations of 5000, 1581, 500 and/or 158.1μg/plate.
- No inhibitory or toxic effects of the test material were detected in the main tests.
VALIDITY OF THE TESTS
- Untreated, negative (solvent) and positive controls were run concurrently. The mean values of revertant colony numbers of untreated, negative (solvent) and positive control plates were within the historical control range.
- The reference mutagens showed a distinct increase of induced revertant colonies with and without metabolic activation. The viability of the bacterial cells was checked by a plating experiment in each test.
- At least five analysable concentrations were presented of the main tests, the examined concentration range was considered to be adequate.
- The study was therefore, considered to be valid. - Conclusions:
- Under the conditions of this study, the test material had no mutagenic activity on Escherichia coli WP2 uvrA strain with or without activation.
- Executive summary:
The genetic toxicity of the test material was investigated in a study following the design of OECD 471, EU Method B.13/14 and OPPTS 870.5100. Although the experimental design is compatible with the guideline requirements, only one bacterial strain was used in the study as requested by the Sponsor. The testing was performed under GLP conditions.
The experiments were carried out using tryptophan-requiring auxotroph strain of Escherichia coli (Escherichia coli WP2 uvrA) in the presence and absence of a post mitochondrial supernatant (S9 fraction) prepared from the livers of phenobarbital/β-naphthoflavone-induced rats.
The study included a Preliminary Compatibility Test, an Initial Mutation Test (Plate Incorporation Method) and a Confirmatory Mutation Test (Pre-Incubation Method).
Based on the results of the Compatibility Test, the test material was dissolved in Dimethyl sulfoxide (DMSO) at a concentration of 100 mg/mL. The test material concentrations in the Initial Mutation Test and in the Confirmatory Mutation Test were 5000, 1581, 500, 158.1, 50, 15.81, 5 and 1.581 μg/plate.
In the Initial Mutation Test and Confirmatory Mutation Test, the number of revertant colonies did not show any biologically relevant increase compared to the solvent control. There were no dose-related trends and no indication of any treatment-related effect.
Precipitate/slight precipitate was observed on the plates in the main tests with and without metabolic activation at the concentrations of 5000, 1581, 500 and/or 158.1 μg/plate. The precipitation did not adversely affect the colony counting.
No inhibitory or toxic effects of the test material were detected in the study.
The mean values of revertant colonies of the negative (vehicle/solvent) control plates were within the historical control range, the reference mutagens showed the expected increase in the number of revertant colonies, the viability of the bacterial cells was checked by a plating experiment in each test. At least five analysable concentrations were presented of the main tests, the examined concentration range was considered to be adequate. The study was therefore, considered to be valid.
The reported data of this mutagenicity assay show that under the experimental conditions applied the test material did not induce gene mutations by base pair changes in the genome of the strain used.
Under the conditions of this study, the test material had no mutagenic activity on Escherichia coli WP2 uvrA strain with or without activation.
- Endpoint:
- in vitro gene mutation study in bacteria
- Remarks:
- Type of genotoxicity: gene mutation
- Type of information:
- experimental study
- Adequacy of study:
- supporting study
- Study period:
- 19 February 1992 - 3 March 1992
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- other: Study conducted to GLP in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results.
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- bacterial reverse mutation assay
- Target gene:
- Histidine locus
- Species / strain / cell type:
- S. typhimurium, other: TA1535, TA1537, TA1538, TA98 and TA100
- Additional strain / cell type characteristics:
- other: Prior to being used, characterisation checks were carried out to determine the amino-acid requirement, presence of rfa and R factors and the spontaneous reversion rate.
- Metabolic activation:
- with and without
- Metabolic activation system:
- rat liver homogenate metabolising system at 10 % in standard co-factors.
- Test concentrations with justification for top dose:
- Experiment 1: 0, 8, 40, 200, 1000 and 5000 µg/plate
Experiment 2: 0, 312.5, 625, 1250, 2500 and 5000 µg/plate - Vehicle / solvent:
- Dimethyl sulphoxide (DMSO)
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- other: - S9: ENNG 3 µg/plate for TA100 and 5 µg/plate for TA1535; 9AA 80 µg/plate for TA1537; 4NOPD 5 µg/plate for TA1538 and 4NQO 0.2 µg/plate for TA98. + S9: 2AA 2 µg/plate for TA1535 and BP 5 µg/plate for all of the other strains.
- Details on test system and experimental conditions:
- In this assay, overnight sub-cultures of the master slopes were prepared in nutrient broth (Oxoid Limited) and incubated at 37 °C for approximately 10 hours.
Preparation of Test and Control Materials
The test material was accurately weighed and dissolved in dimethyl sulphoxide and appropriate dilutions made on the day of each experiment.
Negative and positive controls were used in parallel with the test material. A solvent treatment group was used as the negative control and the positive control materials were as follows:
-N-Ethyl-N'-nitro-N-nitrosoguanidine (ENNG)
-9-Aminoacridine (9AA)
-4-Nitro-o-phenylenediamine (4NOPD)
-4-Nitroquinoline-1-oxide (4NQO)
-2-Aminoanthracene (2AA)
-Benzo(a)pyrene (BP)
Microsomal Enzyme Fraction
The S9 fraction was prepared from the livers of male Sprague-Dawley rats weighing ~200g. These had each received a single i.p. injection of Aroclor 1254 at 500 mg/kg, 5 days before S9 preparation.
S9 Mix and Top Agar
The S9 Mix was prepared at 4 °C as follows:
S9 5.0 mL
1.65 M KCl/0.4 M MgCl2 1.0 mL
0.1 M Glucose-6-phosphate 2.5 mL
0.1 M NADP 2.0 mL
0.2 M Sodium phosphate buffer (pH 7.4) 25.0 mL
Sterile distilled water 14.5 mL
Top agar was prepared using 0.6 % Difco Bacto agar and 0.5 % sodium chloride. 5 mL of 1.0 mM histidine/1.0 mM biotin solution was added to each 100 mL of top agar.
TEST PROCEDURE
a) Preliminary Toxicity Study
In order to select appropriate dose levels for use in the main study, a preliminary test was carried out to determine the toxicity of the test material to the tester organisms. 0.1 mL of bacterial suspension (TA100), 2 mL of molten, trace histidine supplemented media (histidine/biotin & top agar), 0.1 mL of test solution and 0.5 mL phosphate buffer were over-layed onto sterile plates of Vogel-Bonner agar (minimal agar ~30 mL/plate). Five doses of the test compound and a solvent control (dimethyl sulphoxide) were tested in duplicate. After approximately 48 hours incubation at 37 °C the plates were scored for revertant colonies and examined for a thinning of the background lawn.
b) Mutation Study
EXPERIMENT 1
Five concentrations of the test material were assayed in triplicate against each tester strain, using the direct plate incorporation method in accordance with the standards for mutagenicity tests using micro-organisms.
Test Material and Negative Controls
A 0.1 mL aliquot of one of the bacterial suspensions was placed in sets of sterile test tubes followed by 2.0 mL of molten, trace histidine supplemented, top agar at 45 °C. These sets comprised two test tubes for each bacterial tester strain. 0.1 mL of the appropriately diluted test material or negative control was also added to each of the two test tubes. Into one of the test tubes was placed 0.5 mL of the S9 liver microsome mix; in the other tube 0.5 mL of pH 7.4 buffer was added. This procedure was repeated, in triplicate, for each bacterial strain and for each concentration of test material.
Positive Controls
i) Without Activation
0.1 mL of one of the positive control solutions (ENNG, 9AA, 4NQO or 4NOPD) was added to a test tube containing 2.0 mL of molten, trace histidine supplemented, top agar and 0.1 mL of the appropriate bacterial suspension. Finally 0.5 mL of pH 7.4 buffer was added to the test tube. This procedure was then repeated, in triplicate, for each of the positive controls.
ii) With Activation
0.1 mL of 2AA or BP solution was added to a test tube containing 2.0 mL of molten, trace histidine supplemented, top agar and 0.1 mL of one of the test bacterial suspensions. Finally 0.5 mL of S9 mix was added to the test tube. The procedure was then repeated, in triplicate, for each tester strain.
The contents of each test tube were equally distributed onto the surface of Vogel-Bonner agar plates (one tube per plate). These plates were incubated at 37 °C for approximately 48 hours and the number of revertant colonies counted.
EXPERIMENT 2
The second experiment was performed using fresh bacterial cultures, test material and control solutions in triplicate. - Evaluation criteria:
- Interpretation of Results
For a substance to be considered positive in this test system, it should have induced a dose-related and statistically significant increase in mutation rate in one or more strains of bacteria in the presence and/or absence of the S9 microsomal enzymes in both experiments at sub-toxic dose levels. If the two experiments give conflicting results or equivocal results are obtained then a third experiment may be used to confirm the correct response. - Statistics:
- All data are statistically analysed using the methods recommended by the UKEMS. To be considered negative, the number of induced revertants compared to spontaneous revertants should be less than twofold at each dose level employed, the intervals of which should be between 2 and 5 fold and extend to the limits imposed by toxicity, solubility or up to the maximum recommended dose of 5000 µg/plate. In this case the limiting factor was the maximum recommended dose.
- Species / strain:
- S. typhimurium, other: TA1535, TA1537, TA1538, TA98 and TA100
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Additional information on results:
- Preliminary Toxicity Study.
The dose range used in the preliminary toxicity study was 0, 312.5, 625, 1250, 2500 and 5000 µg/plate. The material was non-toxic in the strain of Salmonella used (TA100).
Mutation Study
The results for the checks on characteristics, viability and spontaneous reversion rate for each tester strain were all found to be satisfactory.
The mean number of revertant colonies obtained for each tester strain following incubation with the test material, with and without metabolic activation, are given in Table 1 for experiment 1 and Table 2 for experiment 2.
No toxicity was exhibited to any of the strains of Salmonella used. A precipitate was observed with doses above 312.5 µg/plate but this did not interfere with the scoring of revertant colonies.
No significant increases in the numbers of revertant colonies of bacteria were recorded for any of the strains of Salmonella used, at any dose level either with or without metabolic activation. - Remarks on result:
- other: all strains/cell types tested
- Conclusions:
- Interpretation of results: Negative
The test material was found to be non-mutagenic under the conditions of this test. - Executive summary:
A test was conducted to assess the gene mutation potential of the test material in an assay carried out in accordance with the OECD Guidelines for the Testing of Chemicals, Protocol No. 471 and also with Method B14 in Commission Directive 84/449/EEC.
Salmonella typhimurium strains TA1535, TA1537, TA1538, TA98 and TA100 were treated with the test material by the Ames plate incorporation method at five dose levels, in triplicate, both with and without metabolic activation. The dose range was determined in a preliminary toxicity assay and was 8 to 5000 µg/plate in the first experiment. The experiment was repeated on a separate day using fresh cultures of the bacterial strains and fresh chemical solutions. In this case the dose range was 312.5 to 5000 µg/plate.
The solvent (dimethyl sulphoxide) control plates gave counts of revertant colonies within the normal range, and all positive control chemicals produced marked increases in the number of revertant colonies, both with and without the metabolising system.
There was no reduction in the growth of the bacterial lawn at any of the dose levels employed in all of the strains of Salmonella used. A precipitate was observed at and above 312.5 µg/plate but this did not interfere with the scoring of revertant colonies.
No significant increase in the numbers of revertant colonies was recorded for any of the bacterial strains with any dose either with or without metabolic activation.
Therefore the test material was found to be non-mutagenic under the conditions of this study.
- Endpoint:
- in vitro gene mutation study in bacteria
- Remarks:
- Type of genotoxicity: gene mutation
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 8 December 1992 - 21 December 1992
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- other: Study conducted to GLP in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results.
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- bacterial reverse mutation assay
- Target gene:
- Histidine locus
- Species / strain / cell type:
- S. typhimurium, other: TA1535, TA1537, TA1538, TA98 and TA100
- Additional strain / cell type characteristics:
- other: Prior to being used, characterisation checks were carried out to determine the amino-acid requirement, presence of rfa and R factors and the spontaneous reversion rate.
- Metabolic activation:
- with and without
- Metabolic activation system:
- rat liver homogenate metabolising system at 10 % in standard co-factors.
- Test concentrations with justification for top dose:
- Experiment 1: 0, 8, 40, 200, 1000 and 5000 µg/plate
Experiment 2: 0, 312.5, 625, 1250, 2500 and 5000 µg/plate - Vehicle / solvent:
- Dimethyl sulphoxide (DMSO)
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- other: - S9: ENNG 3 µg/plate for TA100 and 5 µg/plate for TA1535; 9AA 80 µg/plate for TA1537; 4NOPD 5 µg/plate for TA1538 and 4NQO 0.2 µg/plate for TA98. + S9: 2AA 2 µg/plate for TA1535 and BP 5 µg/plate for all of the other strains.
- Details on test system and experimental conditions:
- In this assay, overnight sub-cultures of the master slopes were prepared in nutrient broth (Oxoid Limited) and incubated at 37 °C for approximately 10 hours.
Preparation of Test and Control Materials
The test material was accurately weighed and dissolved in dimethyl sulphoxide and appropriate dilutions made on the day of each experiment.
Negative and positive controls were used in parallel with the test material. A solvent treatment group was used as the negative control and the positive control materials were as follows:
-N-Ethyl-N'-nitro-N-nitrosoguanidine (ENNG)
-9-Aminoacridine (9AA)
-4-Nitro-o-phenylenediamine (4NOPD)
-4-Nitroquinoline-1-oxide (4NQO)
-2-Aminoanthracene (2AA)
-Benzo(a)pyrene (BP)
Microsomal Enzyme Fraction
The S9 fraction was prepared from the livers of male Sprague-Dawley rats weighing ~200g. These had each received a single i.p. injection of Aroclor 1254 at 500 mg/kg, 5 days before S9 preparation.
S9 Mix and Top Agar
The S9 Mix was prepared at 4 °C as follows:
S9 5.0 mL
1.65 M KCl/0.4 M MgCl2 1.0 mL
0.1 M Glucose-6-phosphate 2.5 mL
0.1 M NADP 2.0 mL
0.2 M Sodium phosphate buffer (pH 7.4) 25.0 mL
Sterile distilled water 14.5 mL
Top agar was prepared using 0.6 % Difco Bacto agar and 0.5 % sodium chloride. 5 mL of 1.0 mM histidine/1.0 mM biotin solution was added to each 100 mL of top agar.
TEST PROCEDURE
a) Preliminary Toxicity Study
In order to select appropriate dose levels for use in the main study, a preliminary test was carried out to determine the toxicity of the test material to the tester organisms. 0.1 mL of bacterial suspension (TA100), 2 mL of molten, trace histidine supplemented media (histidine/biotin & top agar), 0.1 mL of test solution and 0.5 mL phosphate buffer were overlayed onto sterile plates of Vogel-Bonner agar (minimal agar ~30 mL/plate). Five doses of the test compound and a solvent control (dimethyl sulphoxide) were tested in duplicate. After approximately 48 hours incubation at 37 °C the plates were scored for revertant colonies and examined for a thinning of the background lawn.
b) Mutation Study
EXPERIMENT 1
Five concentrations of the test material were assayed in triplicate against each tester strain, using the direct plate incorporation method in accordance with the standards for mutagenicity tests using micro-organisms.
Test Material and Negative Controls
A 0.1 mL aliquot of one of the bacterial suspensions was placed in sets of sterile test tubes followed by 2.0 mL of molten, trace histidine supplemented, top agar at 45 °C. These sets comprised two test tubes for each bacterial tester strain. 0.1 mL of the appropriately diluted test material or negative control was also added to each of the two test tubes. Into one of the test tubes was placed 0.5 mL of the S9 liver microsome mix; in the other tube 0.5 mL of pH 7.4 buffer was added. This procedure was repeated, in triplicate, for each bacterial strain and for each concentration of test material.
Positive Controls
i) Without Activation
0.1 mL of one of the positive control solutions (ENNG, 9AA, 4NQO or 4NOPD) was added to a test tube containing 2.0 mL of molten, trace histidine supplemented, top agar and 0.1 mL of the appropriate bacterial suspension. Finally 0.5 mL of pH 7.4 buffer was added to the test tube. This procedure was then repeated, in triplicate, for each of the positive controls.
ii) With Activation
0.1 mL of 2AA or BP solution was added to a test tube containing 2.0 mL of molten, trace histidine supplemented, top agar and 0.1 mL of one of the test bacterial suspensions. Finally 0.5 mL of S9 mix was added to the test tube. The procedure was then repeated, in triplicate, for each tester strain.
The contents of each test tube were equally distributed onto the surface of Vogel-Bonner agar plates (one tube per plate). These plates were incubated at 37 °C for approximately 48 hours and the number of revertant colonies counted.
EXPERIMENT 2
The second experiment was performed using fresh bacterial cultures, test material and control solutions in triplicate. - Evaluation criteria:
- Interpretation of Results
For a substance to be considered positive in this test system, it should have induced a dose-related and statistically significant increase in mutation rate in one or more strains of bacteria in the presence and/or absence of the S9 microsomal enzymes in both experiments at sub-toxic dose levels. If the two experiments give conflicting results or equivocal results are obtained then a third experiment may be used to confirm the correct response. - Statistics:
- All data are statistically analysed using the methods recommended by the UKEMS. To be considered negative, the number of induced revertants compared to spontaneous revertants should be less than twofold at each dose level employed, the intervals of which should be between 2 and 5 fold and extend to the limits imposed by toxicity, solubility or up to the maximum recommended dose of 5000 µg/plate. In this case the limiting factor was the maximum recommended dose.
- Species / strain:
- S. typhimurium, other: TA1535, TA1537, TA1538, TA98 and TA100
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Additional information on results:
- Preliminary Toxicity Study
The dose range used in the preliminary toxicity study was 0, 312.5, 625, 1250, 2500 and 5000 µg/plate. The test material was non-toxic in the strain of Salmonella used (TA100).
The mean numbers of revertant colonies for the toxicity assay were:
Dose (µg/plate)
0 312.5 2500 625 1250 5000
133.0. 133.5 128.5 132.5P 134.5P 140.5P
P = precipitate observed
Mutation Study
The results for the checks on characteristics, viability and spontaneous reversion rate for each tester strain were all found to be satisfactory.
The mean number of revertant colonies obtained for each tester strain following incubation with the test material, with and without metabolic activation, are given in Table 1 for experiment 1 and Table 2 for experiment 2.
No toxicity was exhibited to any of the strains of Salmonella used. A precipitate was observed at and above 1000 µg/plate; this did not interfere with the scoring of revertant colonies.
No significant increases in the numbers of revertant colonies of bacteria were recorded for any of the strains of Salmonella used, at any dose level either with or without metabolic activation.
The positive control substances all produced marked increases in the number of revertant colonies and the activity of the S9 fraction was found to be satisfactory. - Remarks on result:
- other: all strains/cell types tested
- Conclusions:
- Interpretation of results: Negative
The test material was found to be non-mutagenic under the conditions of this test. - Executive summary:
A test was conducted to assess the gene mutation potential of the test material in an assay carried out in accordance with the OECD Guidelines for the Testing of Chemicals, Protocol No. 471 and also with Method B14 in Commission Directive 84/449/EEC.
Salmonella typhimurium strains TA1535, TA1537, TA1538, TA98 and TA100 were treated with the test material by the Ames plate incorporation method at five dose levels, in triplicate, both with and without metabolic activation. The dose range was determined in a preliminary toxicity assay and was 8 to 5000 µg/plate in the first experiment. The experiment was repeated on a separate day using fresh cultures of the bacterial strains and fresh chemical solutions. In this case the dose range was 312.5 to 5000 µg/plate.
The solvent (dimethyl sulphoxide) control plates gave counts of revertant colonies within the normal range, and all positive control chemicals produced marked increases in the number of revertant colonies, both with and without the metabolising system.
There was no reduction in the growth of the bacterial lawn at any of the dose levels employed in all of the strains of Salmonella used. A precipitate was observed at and above 1000 µg/plate but this did not interfere with the scoring of revertant colonies.
No significant increase in the numbers of revertant colonies was recorded for any of the bacterial strains with any dose either with or without metabolic activation.
Therefore the test material was found to be non-mutagenic under the conditions of this study.
- Endpoint:
- in vitro cytogenicity / chromosome aberration study in mammalian cells
- Remarks:
- Type of genotoxicity: chromosome aberration
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 11 October 1993 - 12 December 1993
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- other: Study conducted to GLP in accordance with generally accepted scientific principles, possibly with incomplete reporting or methodological deficiencies, which do not affect the quality of the relevant results.
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
- Deviations:
- yes
- Remarks:
- The test was conducted before the introduction of the guideline but the method followed is equivalent.
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- in vitro mammalian chromosome aberration test
- Species / strain / cell type:
- other: Chinese hamster lung (CHL)
- Details on mammalian cell type (if applicable):
- The Chinese hamster lung (CHL) cell line, isolated by Koyama et al., (1970) and cloned by Ishidate and Sofuni (1985) was used.
- Metabolic activation:
- with and without
- Metabolic activation system:
- induced rat liver homogenate metabolising system at 50 % in standard co-factors
- Test concentrations with justification for top dose:
- 6 hour treatment: 0, 78.1, 156.25, 312.5 and 625 µg/mL
24 hour treatment: 0, 78.13, 156.25, 312.5 and 625 µg/mL
48 hour treatment: 0, 9.75, 19.5, 39 and 78.13 µg/mL - Vehicle / solvent:
- dimethyl sulphoxide (DMSO)
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- other: Mitomycin C (MMC) 0.075 µg/mL for cultures treated for 24 or 48 hours in the absence of S9 mix; Cyclophosphamide (CP) 10 µg/mL for cultures treated for 6 hours both with and without S9 mix.
- Details on test system and experimental conditions:
- Cell Culture
Cells were grown in Eagle's Minimal Essential medium with Earle's Salts (MEM), supplemented with 10 % foetal bovine serum and antibiotics, at 37 °C with 5 % CO2 in air.
Preparation of Test and Control Materials
The test substance was accurately weighed and dissolved in dimethyl sulphoxide and appropriate dilutions made. Negative and positive controls were used in parallel with the test material.
Preliminary Cytotoxicity Test
A preliminary cytotoxicity test was performed on cell cultures using 24 and 48 hour continuous exposure times without metabolic activation and a 6 hour exposure period both with and without metabolic activation, followed by an 18 hour culture period in treatment-free media. Growth inhibition was estimated by counting the number of cells at the end of the culture period on an electronic cell counter (Coulter) and expressing the cell count as a percentage of the concurrent negative control value. Slides were also prepared from the cells in order to check for the presence of cells in metaphase.
Microsomal Enzyme Fraction
The S9 was prepared from the livers of male Sprague-Dawley rats weighing ~200g. These had received a single i.p. injection of Aroclor 1254 at 500 mg/kg, 5 days before S9 preparation.
Culture Conditions
Cultures were established approximately 24 hours prior to treatment, 0.3 x 10^6 cells and 0.10 x 10^6 cells were seeded per flask for 24 and 48 hour cultures respectively. The cells were exposed to four doses of the test material, negative and positive controls, both with and without metabolic activation. All treatments were performed in duplicate (A + B). Cultures were maintained at 37 °C in a humidified atmosphere of 5 % CO2 in air.
The treatment regimens were as follows:
a) Without Metabolic Activation:
- 6 hours exposure to the test material, a phosphate buffered saline wash and then a further 18 hours culture in treatment-free media prior to cell harvest.
-24 hours continuous exposure to the test material prior to cell harvest.
-48 hours continuous exposure to the test material prior to cell harvest.
b) With Metabolic Activation:
- 6 hours exposure to the test material and S9 mix (0.5 mL per 4.5 mL culture medium, of 50 % S9 in standard co-factors). A phosphate buffered saline wash and then a further 18 hours in treatment-free media prior to cell harvest.
Cell Harvest
Mitosis was arrested by addition of demecolcine (Colcemid 0.1 µg/mL) two hours before the required harvest time. After incubation with demecolcine, the cells were trypsinised to detach them from the tissue culture flask and suspended in 5 mL of culture medium. A sample of the cell suspension from each harvest time was counted to measure growth inhibition at each concentration. The cells were centrifuged (1000 rpm for five minutes), the culture medium drawn off and discarded, and the cells re-suspended in 5 mL 0.075M KCl. After fifteen minutes (including five minutes centrifugation), all but approximately 0.5 mL of hypotonic solution was drawn off and discarded. The cells were re-suspended and then fixed by dropping the KCl suspension into 3 mL fresh methanol/glacial acetic acid (3:1 v/v). The fixative was changed at least twice and the cells stored at 4 °C for at least four hours to ensure complete fixation.
Preparation of Metaphase Spreads
The cells were re-suspended in 3 mL of fresh fixative before centrifugation and re-suspension in 0.5 mL of fixative. Several drops of this suspension were dropped onto clean, wet microscope slides and left to air dry.
Staining
When the slides were dry they were stained in 2 % Gurrs Giemsa R66 for 5 minutes, rinsed, dried and mounted in Depex mounting medium. - Evaluation criteria:
- Scoring of Chromosome Damage
Where possible the first 100 consecutive well-spread metaphases from each culture were counted, and if the cell had 23 to 27 chromosomes, any gaps, breaks or rearrangements were noted according to the simplified system of Savage (1976) recommended in the 1983 UKEMS guidelines for mutagenicity testing.
Aberrations recorded by the slide scorer are checked by a senior cytogeneticist. Cells with greater than 27 chromosomes are scored as aneuploid or polyploid cells and the incidence of polyploid cells % reported. The percentage of cells showing structural chromosome aberrations (gaps, breaks and exchanges) are calculated and reported as both including and excluding those with gaps. - Species / strain:
- other: Chinese hamster lung (CHL)
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Additional information on results:
- Preliminary Cytotoxicity Study
A precipitate was observed at and above 156.25 µg/mL, and formed large clumps at 1250 and 2500 µg/mL. At cell harvest, a precipitate was observed at 2500 µg/mL in the 6 (18) hour cultures and at and above 19.5 µg/mL in the 24 and 48 hour cultures; the precipitate was observed to interfere with the accuracy of the cell counts in some cases. In the 48 hour cultures, no scorable metaphase cells were observed at and above 156.25 µg/mL except at 1250 and 2500 µg/mL. The maximum practical dose level for the 24 hour harvest cultures was selected as 625 µg/mL, and the maximum dose level for the 48 hour cultures was selected as 78.1 µg/mL on the basis of toxicity.
Chromosome Aberration Study
The test material showed the expected absence of toxicity in the cultures harvested at 24 hours. In the 48 hour culture group, a dose-related decrease in the cell count was observed although an abnormally low count for culture A of the vehicle control group gave an overall underestimate of the toxicity of the test material.
The negative control cultures gave values of chromosome aberrations within the expected range. The frequency of aberrations was consistent between the four negative control groups, the highest frequency (1.0 % cells with aberrations including gaps) being seen in the 6 hour treatment group with S9 and in the 48 hour group.
All the positive control cultures, except cyclophosphamide without S9, gave highly significant increases in the frequency of aberrations, indicating that the metabolic activation system was satisfactory and that the test method itself was operating as expected.
The material was seen to induce no significant or dose-related increases in the frequency of aberrations in any of the four treatment cases. It did not induce a significant increase in the numbers of polypolid cells at any dose-level in any of the four treatment cases. - Remarks on result:
- other: all strains/cell types tested
- Conclusions:
- Interpretation of results: Negative
The material is considered to be non-clastogenic to CHL cells in vitro under the conditions of this test. - Executive summary:
A study was conducted to assess the potential of the test material to induce chromosome aberrations. Chinese hamster lung (CHL) cells were treated with the test material at four dose levels, in duplicate, together with negative and positive controls. Four treatment regimens were used: 6 hours exposure both with and without the addition of an induced rat liver homogenate metabolising system at 50 % in standard co-factors followed by an 18-hour expression period; 24 hours continuous exposure and 48 hours continuous exposure.
The dose range was selected on the results of a preliminary toxicity test and was 78.1 to 625 µg/mL for the 6 hour treatment (both with and without S9) and the 24 hour treatment and 9.75 to 78.1 µg/mL for the 48 hour treatment. The upper three dose levels were evaluated for chromosome aberrations in each case.
The negative (solvent) controls gave frequencies of aberrations within the range expected for the CHL cell line.
All the positive control treatments except cyclophosphamide without S9 gave highly significant increases in the frequency of aberrations indicating the satisfactory performance of the test and of the activity of the metabolising system.
The material demonstrated no significant increases in the frequency of cells with aberrations in any of the treatment cases, though it was shown to be toxic to CHL cells in vitro in the 48 hour treatment cases.
The test material was non-clastogenic to CHL cells in vitro under the conditions of this study.
- Endpoint:
- in vitro gene mutation study in mammalian cells
- Remarks:
- Type of genotoxicity: gene mutation
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 28 February 2012 - 23 May 2012
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- other: Study conducted to GLP in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results.
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- other: United Kingdom Environmental Mutagen Society
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- mammalian cell gene mutation assay
- Target gene:
- X-chromosome linked hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus
- Species / strain / cell type:
- Chinese hamster Ovary (CHO)
- Details on mammalian cell type (if applicable):
- - Cell culture: stocks of cells were stored in liquid nitrogen at approximately -196 °C. Cells were routinely cultured in Hams F12 medium, supplemented with 5 % foetal bovine serum and antibiotics (penicillin/streptomycin at 100 units/100 µg per mL) at 37 °C with 5 % CO2 in air.
- Cell cleansing: before the cell stocks were frozen they were cleansed of HPRT-mutants by culturing in HAT medium (Hams F12 medium supplemented with hypoxanthine (13.6 µg/mL, 100 µM), aminopterin (0.0178 µg/mL, 0.4 µM) and thymidine (3.85 µg/mL, 16 µM)) for 4 days. After 4 days in the medium containing HAT, the cells were passaged into HAT-free medium and grown for 4 to 7 days. Bulk frozen stocks of HAT cleansed cells were frozen down, with fresh cultures being recovered from frozen before each experiment. - Additional strain / cell type characteristics:
- not specified
- Metabolic activation:
- with and without
- Metabolic activation system:
- Aroclor 1254-induced rat liver S-9
- Test concentrations with justification for top dose:
- Experiment 1
0, 10, 20, 40, 80, 160, 320 (4-hour exposure without S9)
0, 10, 20, 40, 80, 160, 320, 640 (4-hour exposure with S9, 2 %)
Experiment 2
0, 0.5, 1, 2, 4, 6, 8, 12 (24-hour exposure without S9)
0, 10, 20, 40, 80, 160, 320, 640 (4-hour exposure with S9, 1 %) - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: DMSO
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- Remarks:
- DMSO
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- 7,12-dimethylbenzanthracene
- ethylmethanesulphonate
- Details on test system and experimental conditions:
- For the 4-hour exposure groups of experiment 1 cells were seeded at 1.5 x 10^6/75 cm² flask approximately 48 hours before being exposed to the test or control items. In experiment 2 cells were seeded at 1.5 x 10^6/75 cm² for the 24-hour exposure group and at 3 x 10^6/75 cm² flask for the 4-hour exposure group in the presence of S9 and allowed to attach overnight before being exposed to the test or control items. Duplicate cultures were set up, both in the presence and absence of metabolic activation, with a minimum of five dose levels of test material, vehicle and positive controls. Treatment was for 4 hours in serum free media (Hams F12) or for 24 hours in Hams F12 with 1 % serum in an incubator with a humidified atmosphere of 5 % CO2 in air.
At the end of the treatment period the flasks were washed twice with PBS, trypsinised and the cells suspended in Hams F12 with 5 % FBS. A sample of each dose group cell suspension was counted using a Coulter counter. Cultures were plated out at 2 x 10^6 cells/flask in a 225 cm² flask to allow growth and expression of induced mutants, and in triplicate in 25 cm² flasks at 200 cells/flask for an estimate of cytotoxicity. Cells were grown in growth media and incubated at 37 °C in an incubator with a humidified atmosphere of 5 % CO2 in air.
Cytoxicity flasks were incubated for 6 or 7 days then fixed with methanol and stained with Giemsa. Colonies were manually counted and recorded to estimate cytotoxicity.
During the 7 day expression period the cultures were subcultured and maintained at 2 x 10^6 cells/225 cm² flask on day 3 to maintain logarithmic growth. At the end of the expression period the cell monolayers were trypsinised, cell suspensions counted using a Coulter counter and plated out as follows:
i) In triplicate at 200 cells/25 cm² flask in 5 mL of growth medium to determine cloning efficiency. Flasks were incubated for 6 to 7 days, fixed with methanol and stained with Giemsa. Colonies were manually counted, counts were recorded for each culture and the percentage cloning efficiency for each dose group calculated
ii) At 2 x 10^5 cells/75 cm² flask (5 replicates per group) in Hams F12 growth media (5 % serum), supplemented with 10 µg/mL 6-thioguanine, to determine mutant frequency. The flasks were incubated for 14 days at 37 °C in an incubator with humidified atmosphere of 5 % CO2 in air, then fixed with methanol and stained with Giemsa. Mutant colonies were manually counted and recorded for each flask.
The percentage of viability and mutation frequency per survivor were calculated for each dose group. - Evaluation criteria:
- An assay will be considered valid subject to the following criteria:
- the absolute cloning efficiency of negative controls is between 70 and 115 %
- the background mutant frequency of the vehicle controls are generally in the range of 0 to 25 x 10^-6.
- negative and equivocal responses by the test material must have a positive control mutant frequency that is markedly elevated over the concurrent negative control. Assays without a positive control are only valid if the test material clearly shows mutagenic activity.
- test materials with little or no mutagenic activity should include an acceptable assay where concentrations of the test material have reduced the clonal survival to approximately 10 to 15 % of the average negative controls, reached the maximum recommended dose, or twice the solubility limit of the test material in the culture medium.
- at least five dose levels of test material are required. - Species / strain:
- Chinese hamster Ovary (CHO)
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- in tests without S9
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Additional information on results:
- PRELIMINARY CYTOTOXICITY TEST
A dose range of 9.77 to 2500 µg/mL was used; the maximum dose tested was the maximum achievable dose due to difficulties formulating the test material. A precipitate of the test material was observed in all three exposure groups at the end of the treatment period at all dose levels. The precipitate increased in intensity with increasing dose and became aggregated at and above 312.5 µg/mL in all three exposure groups. Findings from the test show that there was a dose-related reduction in the cloning efficiency in the 4-hour exposure group in the absence of S9 up to 312.5 µg/mL where aggregated precipitate was observed. At 625 µg/mL and above, the cloning efficiency increased demonstrating that the increasing presence of aggregated precipitate reduced exposure of the cells to the test material, indicating that maximum exposure of the test material occurred at dose concentrations before the onset of the aggregated precipitate. The 4-hour exposure group in the presence of S9 showed no dose related reduction in the cloning efficiency and it was considered that the presence of S9 may have been protective against the toxicity of the test material. The 24 hour exposure group demonstrated very marked toxicity and was too toxic for plating at and above 156.25 µg/mL and had a reduction on cloning efficiency of 94 % at 9.77 µg/mL. - Remarks on result:
- other: strain/cell type: HPRT
- Conclusions:
- Interpretation of results: Negative
Under the conditions of the test, the test material was found to be negative in the CHO-HPRT mutation assay both in the absence and presence of metabolic activation. - Executive summary:
The mutagenic potential of the test material was determined in accordance with standardised guidelines OECD 476 and EU Method B.17. During the study, the test material was tested at a minimum of six dose levels, in duplicate, in the CHO-HPRT mutation assay in the absence and presence of metabolic activation with Aroclor-induced rat liver S-9; vehicle and positive controls were also included. Four treatment conditions were used for the test, i.e. in experiment 1, a 4-hour exposure in the presence of S9, at a 2 % final concentration and a 4-hour exposure in the absence of S9. In experiment 2, the 4-hour exposure with addition of S9 was repeated using a 1 % final S9 concentration, whilst in the absence of S9 the exposure time was increased to 24 hours.
Under the conditions of the study the vehicle control gave mutant frequencies within the range expected of CHO cells at the HPRT locus. The positive controls, both in the presence and absence of metabolic activation, gave significant increases in the mutant frequency indicating satisfactory performance of the test and of the metabolising system. The test material demonstrated no significant increases in mutant frequency at any dose level, either with or without metabolic activation, in either the first or second experiment. The test material was, therefore, considered to be non-mutagenic to CHO cells under the conditions of the test.
Referenceopen allclose all
The solubility of the test material in DMSO
Concentration of Test Material in DMSO (mg/mL) |
Solubility in DMSO* |
Solubility in the Top Solution (Test Material Formulation 50 μL + Phosphate Buffer 500 μL + Top Agar 2 mL) |
Test material Concentration in the Test Tube (μg/tube) |
100 |
Solution |
Strong precipitate |
5 000 |
31.62 |
Solution |
Precipitate |
1 581 |
10 |
Solution |
Precipitate |
500 |
3.162 |
Solution |
Slight opalescence |
158.1 |
1 |
Solution |
Solution |
50 |
* The black colour of the test material formulations in the 100 – 0.1 mg/mL concentration range faded in a concentration-related manner.
Summary of the initial mutation test
Concentrations (μg/plate) |
Mean Value of Revertants/ Mutation Factor (MF) |
Escherichia coli WP2 uvrA |
|
-S9 |
+S9 |
||
Untreated control |
Mean |
42.7 |
43.7 |
MF |
1.13 |
0.92 |
|
DMSO control |
Mean |
37.7 |
47.3 |
MF |
1.00 |
1.00 |
|
Distilled water |
Mean |
40.3 |
- |
MF |
1.07 |
- |
|
5 000 |
Mean |
44.3 |
45.7 |
MF |
1.18 |
0.96 |
|
1 581 |
Mean |
37.7 |
52.3 |
MF |
1.00 |
1.11 |
|
500 |
Mean |
42.3 |
50.3 |
MF |
1.12 |
1.06 |
|
158.1 |
Mean |
40.7 |
56.7 |
MF |
1.08 |
1.20 |
|
50 |
Mean |
47.3 |
54.3 |
MF |
1.26 |
1.15 |
|
15.81 |
Mean |
48.7 |
50.0 |
MF |
1.29 |
1.06 |
|
5 |
Mean |
46.0 |
57.3 |
MF |
1.22 |
1.21 |
|
1.581 |
Mean |
50.0 |
49.0 |
MF |
1.33 |
1.04 |
|
2AA (50 μg) |
Mean |
- |
232.7 |
MF |
- |
4.92 |
|
MMS (2 μL) |
Mean |
1049.3 |
- |
MF |
26.02 |
- |
|
Summary table of the confirmatory test
Concentrations (μg/plate) |
Mean Value of Revertants/ Mutation Factor (MF) |
Escherichia coli WP2 uvrA |
|
-S9 |
+S9 |
||
Untreated control |
Mean |
40.0 |
37.3 |
MF |
1.00 |
1.00 |
|
DMSO control |
Mean |
40.0 |
37.3 |
MF |
1.00 |
1.00 |
|
Distilled water |
Mean |
40.0 |
- |
MF |
1.00 |
- |
|
5 000 |
Mean |
35.3 |
37.7 |
MF |
0.88 |
1.01 |
|
1 581 |
Mean |
34.0 |
41.7 |
MF |
0.85 |
1.12 |
|
500 |
Mean |
35.3 |
42.0 |
MF |
0.88 |
1.13 |
|
158.1 |
Mean |
39.7 |
36.3 |
MF |
0.99 |
0.97 |
|
50 |
Mean |
39.7 |
93.0 |
MF |
0.99 |
1.04 |
|
15.81 |
Mean |
37.0 |
38.7 |
MF |
0.93 |
1.04 |
|
5 |
Mean |
42.7 |
40.7 |
MF |
1.07 |
1.09 |
|
1.581 |
Mean |
38.7 |
43.3 |
MF |
0.97 |
1.16 |
|
2AA (50 μg) |
Mean |
- |
234.3 |
MF |
- |
6.28 |
|
MMS (2 μL) |
Mean |
1052.0 |
- |
MF |
26.30 |
- |
|
Initial mutation test (plate incorporation method): Cell count (overnight culture) 2.8 E+09 CFU/mL
Concentration (μg/plate) |
Revertant Colony Number |
||
-S9 |
+S9 |
||
5 000 |
|
46 P |
47 P |
|
40 P |
44 P |
|
|
47 P |
46 P |
|
Mean |
44.3 |
45.7 |
|
SD |
3.79 |
1.43 |
|
MF |
1.18 |
0.96 |
|
1 581 |
|
38 P |
55 P |
|
37 P |
48 P |
|
|
38 P |
54 P |
|
Mean |
37.7 |
52.3 |
|
SD |
0.58 |
3.79 |
|
MF |
1.00 |
1.11 |
|
500 |
|
44 SP |
47 SP |
|
39 SP |
52 SP |
|
|
44 SP |
52 SP |
|
Mean |
42.3 |
50.3 |
|
SD |
2.89 |
2.89 |
|
MF |
1.12 |
1.00 |
|
158.1 |
|
39 |
58 |
|
42 |
55 |
|
|
41 |
57 |
|
Mean |
40.7 |
26.7 |
|
SD |
1.53 |
1.53 |
|
MF |
1.08 |
1.20 |
|
50 |
|
48 |
57 |
|
51 |
48 |
|
|
43 |
58 |
|
Mean |
47.3 |
54.3 |
|
SD |
4.04 |
5.51 |
|
MF |
1.26 |
1.15 |
|
15.81 |
|
46 |
56 |
|
51 |
42 |
|
|
49 |
52 |
|
Mean |
48.7 |
50.0 |
|
SD |
2.52 |
7.71 |
|
MF |
1.29 |
1.06 |
|
5 |
|
46 |
58 |
|
48 |
56 |
|
|
44 |
58 |
|
Mean |
46.0 |
57.3 |
|
SD |
2.52 |
1.15 |
|
MF |
1.29 |
1.21 |
|
1.581 |
|
54 |
49 |
|
42 |
54 |
|
|
54 |
44 |
|
Mean |
50.0 |
49.0 |
|
SD |
6.93 |
5.00 |
|
MF |
1.33 |
1.04 |
|
Untreated control |
|
43 |
47 |
|
43 |
40 |
|
|
42 |
44 |
|
Mean |
42.7 |
43.7 |
|
SD |
0.58 |
3.51 |
|
MF |
1.13 |
0.92 |
|
DMSO control |
|
40 |
47 |
|
37 |
47 |
|
|
36 |
48 |
|
Mean |
37.7 |
47.3 |
|
SD |
2.08 |
0.58 |
|
MF |
1.00 |
1.00 |
|
Distilled water control* |
|
42 |
45 |
|
40 |
48 |
|
|
39 |
48 |
|
Mean |
40.3 |
47.0 |
|
SD |
1053 |
1.73 |
|
MF |
10.7 |
0.99 |
|
Positive control MMS (2 μL) |
|
1080 |
- |
|
1044 |
- |
|
|
1024 |
- |
|
Mean |
1049.3 |
- |
|
SD |
28.38 |
- |
|
MF |
26.02 |
- |
|
Positive control 2AA (50 μL) |
|
- |
228 |
|
- |
238 |
|
|
- |
232 |
|
Mean |
- |
232.7 |
|
SD |
- |
5.03 |
|
MF |
- |
4.92 |
|
* Distilled water was used due to MMS.
P: Precipitate
SP: Slight precipitate
MF: Mutation factor
SD: Standard deviation
+S9: With S9 mix
-S9: Without S9 mix
Mutation factor = mean revertants (test material) / mean revertants (solvent control)
Confirmatory mutation test (Pre-incubation method): Cell count (overnight culture) 2.96 E+09 CFU/mL
Concentration (μg/plate) |
Revertant Colony Number |
||
-S9 |
+S9 |
||
5 000 |
|
34 P |
32 P |
|
37 P |
42 P |
|
|
35 P |
39 P |
|
Mean |
35.3 |
37.7 |
|
SD |
1.53 |
5.13 |
|
MF |
0.8 |
1.01 |
|
1 581 |
|
31 P |
41 P |
|
39 P |
47 P |
|
|
32 P |
37 P |
|
Mean |
34.0 |
41.7 |
|
SD |
4.36 |
5.03 |
|
MF |
0.85 |
1.12 |
|
500 |
|
35 SP |
42 SP |
|
32 SP |
45 SP |
|
|
39 SP |
39 SP |
|
Mean |
35.3 |
42.0 |
|
SD |
3.51 |
3.00 |
|
MF |
0.88 |
1.13 |
|
158.1 |
|
38 SP |
38 SP |
|
42 SP |
39 SP |
|
|
39 SP |
34 SP |
|
Mean |
39.7 |
36.3 |
|
SD |
2.08 |
2.52 |
|
MF |
0.99 |
0.97 |
|
50 |
|
39 |
42 |
|
41 |
37 |
|
|
39 |
38 |
|
Mean |
39.7 |
39.0 |
|
SD |
1.15 |
2.65 |
|
MF |
0.99 |
1.04 |
|
15.81 |
|
39 |
39 |
|
33 |
38 |
|
|
39 |
39 |
|
Mean |
37.0 |
38.7 |
|
SD |
3.46 |
0.58 |
|
MF |
0.93 |
1.04 |
|
5 |
|
44 |
29 |
|
47 |
46 |
|
|
37 |
47 |
|
Mean |
42.7 |
40.7 |
|
SD |
5.13 |
10.12 |
|
MF |
1.07 |
1.09 |
|
1.581 |
|
32 |
45 |
|
45 |
39 |
|
|
39 |
46 |
|
Mean |
38.7 |
43.3 |
|
SD |
6.51 |
3.79 |
|
MF |
0.97 |
1.16 |
|
Untreated control |
|
39 |
37 |
|
42 |
38 |
|
|
39 |
37 |
|
Mean |
10.0 |
37.3 |
|
SD |
1.73 |
0.58 |
|
MF |
1.00 |
1.00 |
|
DMSO control |
|
37 |
38 |
|
44 |
35 |
|
|
39 |
39 |
|
Mean |
40.0 |
37.3 |
|
SD |
3.61 |
2.08 |
|
MF |
1.00 |
1.00 |
|
Distilled water control* |
|
36 |
39 |
|
46 |
38 |
|
|
38 |
39 |
|
Mean |
40.0 |
38.7 |
|
SD |
529 |
0.58 |
|
MF |
1.00 |
1.04 |
|
Positive control MMS (2 μL) |
|
1024 |
- |
|
1088 |
- |
|
|
1044 |
- |
|
Mean |
1052.0 |
- |
|
SD |
32.74 |
- |
|
MF |
26.30 |
- |
|
Positive control 2AA (50 μL) |
|
- |
238 |
|
- |
221 |
|
|
- |
244 |
|
Mean |
- |
234.3 |
|
SD |
- |
11.98 |
|
MF |
- |
6.28 |
|
* Distilled water was used due to MMS.
P: Precipitate
SP: Slight precipitate
MF: Mutation factor
SD: Standard deviation
+S9: With S9 mix
-S9: Without S9 mix
Mutation factor = mean revertants (test material) / mean revertants (solvent control)
Historical control data (period of 2011 – 2017)
Untreated control data |
||||||||||
|
Without metabolic activation (-S9 mix) |
With metabolic activation (+S9 mix) |
||||||||
TA98 |
TA100 |
TA1535 |
TA1537 |
E. coli |
TA98 |
TA100 |
TA1535 |
TA1537 |
E. coli |
|
Mean |
22.5 |
102.3 |
12.0 |
7.7 |
35.2 |
29.0 |
109.7 |
11.5 |
9.4 |
40.4 |
St. dev. |
5.6 |
20.3 |
4.8 |
3.5 |
10.8 |
6.8 |
19.2 |
3.7 |
3.9 |
10.4 |
Range |
9-50 |
54-210 |
1-46 |
1-26 |
11-82 |
10-56 |
65-204 |
1-39 |
1-29 |
16-89 |
n |
1650 |
1636 |
1647 |
1653 |
1662 |
1668 |
1656 |
1667 |
1671 |
1662 |
DMSO Control Data |
||||||||||
|
Without metabolic activation (-S9 mix) |
With metabolic activation (+S9 mix) |
||||||||
TA98 |
TA100 |
TA1535 |
TA1537 |
E. coli |
TA98 |
TA100 |
TA1535 |
TA1537 |
E. coli |
|
Mean |
21.5 |
98.0 |
12.1 |
7.6 |
34.0 |
28.1 |
107.3 |
11.3 |
9.1 |
39.4 |
St. dev. |
5.5 |
19.7 |
4.7 |
3.4 |
10.5 |
6.9 |
20.2 |
3.6 |
3.8 |
10.3 |
Range |
6-55 |
40-217 |
1-43 |
1-27 |
7-81 |
11-67 |
53-229 |
2-33 |
1-29 |
9-85 |
n |
1770 |
1761 |
1770 |
1776 |
1779 |
787 |
1776 |
1790 |
1791 |
1782 |
Distilled Water Control Data |
||||||||||
|
Without metabolic activation (-S9 mix) |
With metabolic activation (+S9 mix) |
||||||||
TA98 |
TA100 |
TA1535 |
TA1537 |
E. coli |
TA98 |
TA100 |
TA1535 |
TA1537 |
E. coli |
|
Mean |
23.3 |
101.7 |
12.1 |
8.5 |
36.2 |
29.7 |
109.7 |
11.3 |
9.9 |
41.5 |
St. dev. |
5.7 |
21.3 |
4.6 |
3.5 |
10.7 |
6.9 |
21.1 |
3.5 |
3.8 |
10.3 |
Range |
11-45 |
45-215 |
2-47 |
2-24 |
12-84 |
10-53 |
64-222 |
3-39 |
1-24 |
13-91 |
n |
351 |
1644 |
1650 |
357 |
1683 |
354 |
1668 |
1677 |
354 |
1677 |
DMF Control Data |
||||||||||
|
Without metabolic activation (-S9 mix) |
With metabolic activation (+S9 mix) |
||||||||
TA98 |
TA100 |
TA1535 |
TA1537 |
E. coli |
TA98 |
TA100 |
TA1535 |
TA1537 |
E. coli |
|
Mean |
20.4 |
90.0 |
11.4 |
7.5 |
36.6 |
27.4 |
98.8 |
11.1 |
8.7 |
39.4 |
St. dev. |
5.3 |
17.0 |
4.4 |
3.4 |
12.8 |
6.9 |
18.4 |
3.4 |
3.5 |
10.6 |
Range |
8-38 |
54-152 |
1-34 |
1-19 |
16-99 |
11-49 |
60-156 |
3-21 |
1-23 |
17-76 |
n |
258 |
258 |
258 |
258 |
249 |
258 |
258 |
258 |
255 |
249 |
Acetone Control Data |
||||||||||
|
Without metabolic activation (-S9 mix) |
With metabolic activation (+S9 mix) |
||||||||
TA98 |
TA100 |
TA1535 |
TA1537 |
E. coli |
TA98 |
TA100 |
TA1535 |
TA1537 |
E. coli |
|
Mean |
22.5 |
98.0 |
12.1 |
7.5 |
35.6 |
28.9 |
107.5 |
11.1 |
8.8 |
40.9 |
St. dev. |
5.1 |
15.1 |
5.8 |
3.0 |
9.7 |
6.7 |
14.5 |
3.4 |
3.4 |
9.2 |
Range |
11-39 |
62-160 |
4-49 |
1-17 |
17-63 |
15-52 |
66-177 |
4-22 |
1-19 |
17-70 |
n |
290 |
291 |
291 |
291 |
288 |
291 |
291 |
294 |
291 |
291 |
Positive Reference Control Data |
||||||||||
|
Without metabolic activation (-S9 mix) |
With metabolic activation (+S9 mix) |
||||||||
TA98 |
TA100 |
TA1535 |
TA1537 |
E. coli |
TA98 |
TA100 |
TA1535 |
TA1537 |
E. coli |
|
Mean |
363.9 |
1216.4 |
1167.3 |
447.3 |
1028.0 |
2409.2 |
2423.8 |
230.9 |
219.7 |
255.1 |
St. dev. |
105.6 |
193.5 |
188.7 |
155.7 |
133.5 |
290.5 |
267.0 |
123.9 |
51.7 |
104.1 |
Range |
152-2336 |
536-2120 |
208-2440 |
149-2104 |
488-1708 |
312-4918 |
1192-5240 |
101-2216 |
117-838 |
125-5212 |
n |
1650 |
1638 |
1647 |
1653 |
1665 |
1668 |
1656 |
1671 |
1671 |
1662 |
TA98: Salmonella typhimurium TA98
TA100: Salmonella typhimurium TA100
TA1535: Salmonella typhimurium TA1535
TA1537: Salmonella typhimurium TA1537
E coli: Escherichia coli WP2uvrA
n: Number of cases
Table 1 Experiment 1
+/- S9 Mix |
Concentration (µg/plate) |
Mean number of colonies/plate |
||||
Base-pair Substitution Type |
Frameshift Type |
|||||
TA100 |
TA1535 |
TA1538 |
TA98 |
TA1537 |
||
- - - - - - |
0 8 40 200 1000 5000 |
154.7 127.3 115.3 119.0 123.7 P 123.5 P |
16.0 15.0 16.0 16.3 16.0 P 12.7 P |
12.7 11.0 8.7 12.0 13.3 P 11.3 P |
16.3 16.3 17.0 17.3 21.7 P 11.0 P |
13.0 10.0 14.3 12.7 11.3 P 10.3 P |
+ + + + + + |
0 8 40 200 1000 5000 |
141.7 144.0 124.3 114.3 146.3 P 129.7 P |
10.0 11.3 12.0 9.0 11.3 P 12.0 P |
16.3 15.0 15.0 15.0 15.0 P 13.7 P |
20.0 18.7 17.3 17.0 20.0 P 14.7 P |
11.7 12.7 14.0 11.7 16.0 P 14.0 P |
Positive Controls |
||||||
- |
Name |
ENNG |
ENNG |
4NOPD |
4NQO |
9AA |
Concentration (µg/plate) |
3.0 |
5.0 |
5.0 |
0.2 |
80 |
|
Mean no. colonies/plate |
654.7 |
325.0 |
16.0.0 |
199.3 |
226.3 |
|
+ |
Name |
BP |
2AA |
BP |
BP |
BP |
Concentration (µg/plate) |
5 |
2 |
5 |
5 |
5 |
|
Mean no. colonies/plate |
288.7 |
157.3 |
166.7 |
156.0 |
95.7 |
|
P = Precipitate
Table 2 Experiment 2
+/- S9 Mix |
Concentration (µg/plate) |
Mean number of colonies/plate |
||||
Base-pair Substitution Type |
Frameshift Type |
|||||
TA100 |
TA1535 |
TA1538 |
TA98 |
TA1537 |
||
- - - - - - |
0 312.5 625 1250 2500 5000 |
163.7 122.7 P 151.0 P 150.3 P 135.0 P 151.0 P |
24.0 20.7 P 19.7 P 19.7 P 20.7 P 17.0 P |
27.0 22.7 P 19.0 P 19.0 P 17.7 P 18.7 P |
30.7 29.3 P 24.7 P 23.0 P 22.7 P 22.3 P |
12.7 13.3 P 11.0 P 11.7 P 11.7 P 8.0 P |
+ + + + + + |
0 312.5 625 1250 2500 5000 |
171.7 149.7 P 167.3 P 162.0 P 158.7 P 154.0 P |
15.3 14.7 P 15.0 P 13.3 P 12.7 P 11.7 P |
21.3 16.7 P 18.0 P 18.7 P 14.7 P 16.0 P |
25.7 23.3 P 21.0 P 21.7 P 19.3 P 18.7 P |
15.0 14.0 P 11.0 P 13.7 P 11.3 P 10.3 P |
Positive Controls |
||||||
- |
Name |
ENNG |
ENNG |
4NOPD |
4NQO |
9AA |
Concentration (µg/plate) |
3.0 |
5.0 |
5.0 |
0.2 |
80 |
|
Mean no. colonies/plate |
576.3 |
317.7 |
220.3 |
148.0 |
531.7 |
|
+ |
Name |
BP |
2AA |
BP |
BP |
BP |
Concentration (µg/plate) |
5 |
2 |
5 |
5 |
5 |
|
Mean no. colonies/plate |
382.3 |
208.0 |
184.3 |
230.3 |
105.0 |
|
P = Precipitate
Table 1 Experiment 1
+/- S9 Mix |
Concentration (µg/plate) |
Mean number of colonies/plate |
||||
Base-pair Substitution Type |
Frameshift Type |
|||||
TA100 |
TA1535 |
TA1538 |
TA98 |
TA1537 |
||
- - - - - - |
0 8 40 200 1000 5000 |
116.0 135.3 122.3 110.0 131.0 P 115.3 P |
17.3 18.7 20.3 12.7 13.3 P 14.7 P |
14.7 17.0 12.7 13.3 16.7 P 12.5 P |
23.3 18.3 14.3 17.3 19.0 P 16.7 P |
10.7 9.3 9.3 10.0 11.7 P 6.7 P |
+ + + + + + |
0 8 40 200 1000 5000 |
116.3 115.7 11.3 120.0 121.0 P 119.7 P |
14.3 12.0 13.0 11.7 13.0 P 13.0 P |
26.0 26.7 17.7 16.3 15.0 P 19.0 P |
26.3 21.7 23.0 24.0 19.7 P 21.7 P |
8.3 10.5 9.7 8.7 8.7 P 9.3 P |
Positive Controls |
||||||
- |
Name |
ENNG |
ENNG |
4NOPD |
4NQO |
9AA |
Concentration (µg/plate) |
3.0 |
5.0 |
5.0 |
0.2 |
80 |
|
Mean no. colonies/plate |
480.7 |
115.0 |
143.0 |
141.3 |
431.0 |
|
+ |
Name |
BP |
2AA |
BP |
BP |
BP |
Concentration (µg/plate) |
5 |
2 |
5 |
5 |
5 |
|
Mean no. colonies/plate |
395.3 |
166.3 |
144.0 |
191.0 |
127.3 |
|
P = Precipitate
Table 2 Experiment 2
+/- S9 Mix |
Concentration (µg/plate) |
Mean number of colonies/plate |
||||
Base-pair Substitution Type |
Frameshift Type |
|||||
TA100 |
TA1535 |
TA1538 |
TA98 |
TA1537 |
||
- - - - - - |
0 312.5 625 1250 2500 5000 |
136.5 129.0 118.7 114.3 P 117.3 P 122.0 P |
21.7 17.3 21.7 19.0 P 18.7 P 22.0 P |
18.7 17.3 18.0 18.0 P 15.3 P 18.3 P |
23.0 19.0 22.7 19. P 21.3 P 21.0 P |
14.0 16.5 13.0 11.7 P 11.3 P 13.0 P |
+ + + + + + |
0 312.5 625 1250 2500 5000 |
127.0 117.0 121.3 113.7 P 131.3 P 122.7 P |
12.7 13.0 12.7 11.7 P 12.7 P 130. P |
21.7 17.0 17.0 15.3 P 18.7 P 18.3 P |
24.0 27.0 19.0 23.7 P 23.0 P 22.7 P |
15.3 13.3 14.0 14.7 P 12.7 P 12.7 P |
Positive Controls |
||||||
- |
Name |
ENNG |
ENNG |
4NOPD |
4NQO |
9AA |
Concentration (µg/plate) |
3.0 |
5.0 |
5.0 |
0.2 |
80 |
|
Mean no. colonies/plate |
499.3 |
188.3 |
231.7 |
165.7 |
309.7 |
|
+ |
Name |
BP |
2AA |
BP |
BP |
BP |
Concentration (µg/plate) |
5 |
2 |
5 |
5 |
5 |
|
Mean no. colonies/plate |
427.7 |
126.3 |
160.3 |
201.3 |
119.7 |
|
P = Precipitate
Table 1 Summary of Chromosome Aberration Assay
6 Hour Treatment Time - No Metabolic Activation
Dose level (µg/mL) |
Replicate Identification |
No. of Cells Scored |
Total Gaps |
Chromatid |
Chromosome |
Total ABS. (+Gaps) |
Total ABS. (-Gaps) |
Cells With ABS. (+Gaps) |
Cells With ABS. (-Gaps) |
||
Breaks |
Exchanges |
Breaks |
Exchanges |
||||||||
Negative Control DMSO |
A B |
100 100 |
0 2 |
0 0 |
0 0 |
0 0 |
0 0 |
0 2 |
0 0 |
0 1 |
0 0 |
Total % |
200 100 |
2 (1.0) |
0 (0.0) |
0 (0.0) |
0 (0.0) |
0 (0.0) |
2 (1.0) |
0 (0.0) |
1 (0.5) |
0 (0.0) |
|
156.25 |
A B |
100 100 |
0 0 |
0 0 |
0 0 |
0 1 |
0 0 |
0 1 |
0 1 |
0 1 |
0 1 |
Total % |
200 100 |
0 (0.0) |
0 (0.0) |
0 (0.0) |
1 (0.5) |
0 (0.0) |
1 (0.5) |
1 (0.5) |
1 (0.5) |
1 (0.5) |
|
312.5 |
A B |
100 100 |
0 0 |
0 1 |
0 0 |
2 0 |
0 0 |
2 1 |
2 1 |
1 1 |
1 1 |
Total % |
200 100 |
0 (0.0) |
1 (0.5) |
0 (0.0) |
2 (1.0) |
0 (0.0) |
3 (1.5) |
3 (1.5) |
2 (1.0) |
2 (1.0) |
|
625 |
A B |
100 100 |
0 0 |
1 0 |
0 0 |
0 2 |
0 0 |
1 2 |
1 2 |
1 1 |
1 1 |
Total % |
200 100 |
0 (0.0) |
1 (0.5) |
0 (0.0) |
2 (1.0) |
0 (0.0) |
3 (1.5) |
3 (1.5) |
2 (1.0) |
2 (1.0) |
|
Figures in brackets = aberrations per 100 cells
Table 2 Summary of Chromosome Aberration Assay
6 Hour Treatment Time - Metabolic Activation
Dose level (µg/mL) |
Replicate Identification |
No. of Cells Scored |
Total Gaps |
Chromatid |
Chromosome |
Total ABS. (+Gaps) |
Total ABS. (-Gaps) |
Cells With ABS. (+Gaps) |
Cells With ABS. (-Gaps) |
||
Breaks |
Exchanges |
Breaks |
Exchanges |
||||||||
Negative Control DMSO |
A B |
100 100 |
0 0 |
0 0 |
0 0 |
0 0 |
2 0 |
2 0 |
2 0 |
2 0 |
2 0 |
Total % |
200 100 |
0 (0.0) |
0 (0.0) |
0 (0.0) |
0 (0.0) |
2 (1.0) |
2 (0.1) |
2 (0.1) |
2 (0.1) |
2 (0.1) |
|
156.25 |
A B |
100 100 |
1 1 |
0 0 |
0 0 |
0 0 |
0 0 |
1 1 |
0 0 |
1 1 |
0 0 |
Total % |
200 100 |
2 (1.0) |
0 (0.0) |
0 (0.0) |
0 (0.0) |
0 (0.0) |
2 (1.0) |
0 (0.0) |
2 (1.0) |
0 (0.0) |
|
312.5 |
A B |
100 100 |
2 1 |
0 2 |
0 0 |
0 0 |
1 1 |
3 4 |
1 3 |
3 4 |
1 3 |
Total % |
200 100 |
3 (1.5) |
2 (1.0) |
0 (0.0) |
0 (0.0) |
2 (1.0) |
7 (3.5) |
4 (2.0) |
7 (3.5) |
4 (2.0) |
|
625 |
A B |
100 100 |
2 3 |
2 1 |
0 0 |
0 0 |
1 1 |
5 5 |
3 2 |
4 5 |
3 2 |
Total % |
200 100 |
5 (2.5) |
3 (1.5) |
0 (0.0) |
0 (0.0) |
2 (1.0) |
10 (5.0) |
5 (2.5) |
9 (4.5) |
5 (2.5) |
|
Figures in brackets = aberrations per 100 cells
Table 3 Summary of Chromosome Aberration Assay
24 Hour Treatment Time - No Metabolic Activation
Dose level (µg/mL) |
Replicate Identification |
No. of Cells Scored |
Total Gaps |
Chromatid |
Chromosome |
Total ABS. (+Gaps) |
Total ABS. (-Gaps) |
Cells With ABS. (+Gaps) |
Cells With ABS. (-Gaps) |
||
Breaks |
Exchanges |
Breaks |
Exchanges |
||||||||
Negative Control DMSO |
A B |
100 100 |
1 0 |
0 0 |
0 0 |
0 0 |
0 0 |
1 0 |
0 0 |
1 0 |
0 0 |
Total % |
200 100 |
1 (0.5) |
0 (0.0) |
0 (0.0) |
0 (0.0) |
0 (0.0) |
1 (0.5) |
0 (0.0) |
1 (0.5) |
0 (0.0) |
|
156.25 |
A B |
100 100 |
3 1 |
0 0 |
0 0 |
1 0 |
0 0 |
4 1 |
1 0 |
3 1 |
1 0 |
Total % |
200 100 |
4 (2.0) |
0 (0.0) |
0 (0.0) |
1 (0.5) |
0 (0.0) |
5 (2.5) |
1 (0.5) |
4 (2.0) |
1 (0.5) |
|
312.5 |
A B |
100 100 |
0 2 |
0 1 |
0 0 |
0 0 |
1 0 |
1 3 |
1 1 |
1 3 |
1 1 |
Total % |
200 100 |
2 (1.0) |
1 (0.5) |
0 (0.0) |
0 (0.0) |
1 (0.5) |
4 (2.0) |
2 (1.0) |
4 (2.0) |
2 (1.0) |
|
625 |
A B |
84 100 |
0 0 |
0 0 |
1 1 |
1 0 |
0 1 |
2 2 |
2 2 |
2 2 |
2 2 |
Total % |
184 100 |
0 (0.0) |
0 (0.0) |
2 (1.1) |
1 (0.5) |
1 (0.5) |
4 (2.2) |
4 (2.2) |
4 (2.2) |
4 (2.2) |
|
Figures in brackets = aberrations per 100 cells
Table 4 Summary of Chromosome Aberration Assay
48 Hour Treatment Time - No Metabolic Activation
Dose level (µg/mL) |
Replicate Identification |
No. of Cells Scored |
Total Gaps |
Chromatid |
Chromosome |
Others |
Total ABS. (+Gaps) |
Total ABS. (-Gaps) |
Cells With ABS. (+Gaps) |
Cells With ABS. (-Gaps) |
||
Breaks |
Exchanges |
Breaks |
Exchanges |
X |
||||||||
Negative Control DMSO |
A B |
100 100 |
1 0 |
0 0 |
0 0 |
0 0 |
1 0 |
0 0 |
2 0 |
1 0 |
2 0 |
1 0 |
Total % |
200 100 |
1 (0.5) |
0 (0.0) |
0 (0.0) |
0 (0.0) |
1 (0.5) |
0 (0.0) |
2 (1.0) |
1 (0.5) |
2 (1.0) |
1 (0.5) |
|
19.5 |
A B |
100 100 |
1 1 |
1 1 |
0 0 |
0 2 |
4 0 |
0 0 |
6 4 |
5 3 |
5 3 |
4 2 |
Total % |
200 100 |
2 (1.0) |
2 (1.0) |
0 (0.0) |
2 (1.0) |
4 (2.0) |
0 (0.0) |
10 (5.0) |
8 (4.0) |
8 (4.0) |
6 (3.0) |
|
39 |
A B |
100 100 |
2 1 |
0 1 |
0 0 |
0 0 |
2 3 |
0 0 |
4 5 |
2 4 |
4 4 |
2 4 |
Total % |
200 100 |
3 (1.5) |
1 (0.5) |
0 (0.0) |
0 (0.0) |
5 (2.5) |
0 (0.0) |
9 (4.5) |
6 (3.0) |
8 (4.0) |
6 (3.0) |
|
78.13 |
A B |
100 100 |
0 1 |
1 0 |
1 0 |
0 0 |
1 0 |
1 0 |
3 1 |
3 0 |
3 1 |
3 0 |
Total % |
200 100 |
1 (0.5) |
1 (0.5) |
1 (0.5) |
0 (0.0) |
1 (0.5) |
1 (0.5) |
4 (2.0) |
3 (1.5) |
4 (2.0) |
3 (1.5) |
|
Figures in brackets = aberrations per 100 cells
X = >10 aberrations per cell (not included in total aberrations)
Table 1: Experiment 1 - 4 hour exposure without S9
| Dose (µg/mL) | Rep. | Day 0 viability | Day 7 viability | Day 7 mutant | |||||||||||||||||
| Colonies/flask (200 cells plated/flask) | % CE | % control | Mean % control | Colonies/flask (200 cells plated/flask) | % CE | % control | Mean % control | Colonies/flask (2x10^5 cells plated/flask) | MF | MFS (10^-6) | Group MFS 10^-6 | ||||||||||
| 0 | A | 146 | 146 | 160 | 75.3 | 100 | 100 | 153 | 135 | 141 | 71.5 | 100 | 100 | 0 | 0 | 1 | 0 | 1 | 2 | 2.8 | 3 |
| B | 101 | 129 | 126 | 59.3 | 100 | 175 | 167 | 136 | 79.7 | 100 | 0 | 0 | 1 | 1 | 0 | 2 | 2.5 | ||||
| 10 P | A | 152 | 139 | 141 | 72 | 95.6 | 116 | 146 | 142 | 136 | 70.7 | 98.8 | 97 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 1 |
| B | 177 | 154 | 156 | 81.2 | 136.8 | 134 | 158 | 161 | 75.5 | 94.8 | 0 | 1 | 1 | 0 | 0 | 2 | 2.6 | ||||
| 20 P | A | 153 | 155 | 154 | 77 | 102.2 | 115 | 149 | 150 | 137 | 72.7 | 101.6 | 97 | 0 | 0 | 0 | 0 | 1 | 1 | 1.4 | 2 |
| B | 151 | 156 | 145 | 75.3 | 127 | 143 | 155 | 146 | 74 | 92.9 | 0 | 1 | 0 | 0 | 1 | 2 | 2.7 | ||||
| 40 P | A | 151 | 151 | 157 | 76.5 | 101.5 | 103 | 129 | 144 | 161 | 72.3 | 101.2 | 91 | 0 | 1 | 1 | 0 | 0 | 2 | 2.8 | 2 |
| B | 128 | 134 | 112 | 62.3 | 105.1 | 116 | 132 | 140 | 64.7 | 81.2 | 0 | 1 | 0 | 0 | 0 | 1 | 1.5 | ||||
| 80 P | A | 110 | 108 | 106 | 54 | 71.7 | 84 | 163 | 156 | 145 | 77.3 | 108.2 | 97 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
| B | 121 | 119 | 102 | 57 | 96.1 | 153 | 119 | 137 | 68.2 | 85.6 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | ||||
| 160 P | A | 53 | 61 | 48 | 27 | 35.8 | 46 | 140 | 132 | 129 | 66.8 | 93.5 | 91 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 6 |
| B | 59 | 80 | 62 | 33.5 | 56.5 | 153 | 133 | 134 | 70 | 87.9 | 3 | 2 | 1 | 3 | 0 | 9 | 12.9 | ||||
| 320 AP | A | 29 | 29 | 36 | 15.7 | 20.8 | 30 | 133 | 153 | 148 | 72.3 | 101.2 | 88 | 0 | 0 | 0 | 2 | 0 | 2 | 2.8 | 2 |
| B | 42 | 48 | 53 | 23.8 | 40.2 | 120 | 133 | 101 | 59 | 74.1 | 1 | 0 | 0 | 0 | 0 | 1 | 1.7 | ||||
| EMS 500 | A | 40 | 33 | 30 | 17.2 | 22.8 | 21 | 105 | 84 | 87 | 46 | 64.3 | 60 | 35 | 38 | 28 | 40 | 29 | 170 | 369.6 | 307 |
| B | 28 | 21 | 20 | 11.5 | 19.4 | 90 | 102 | 74 | 44.3 | 55.6 | 14 | 27 | 27 | 16 | 24 | 108 | 243.6 | ||||
| EMS 750 | A | 16 | 13 | 11 | 6.7 | 8.8 | 9 | 95 | 78 | 71 | 40.7 | 56.9 | 52 | 48 | 49 | 39 | 37 | 30 | 203 | 499.2 | 479 |
| B | 13 | 10 | 9 | 5.3 | 9 | 72 | 77 | 80 | 38.2 | 47.9 | 33 | 42 | 30 | 33 | 37 | 175 | 458.5 | ||||
Table 2: Experiment 1 - 4 hour exposure with S9
| Dose (µg/mL) | Rep. | Day 0 viability | Day 7 viability | Day 7 mutant | |||||||||||||||||
| Colonies/flask (200 cells plated/flask) | % CE | % control | Mean % control | Colonies/flask (200 cells plated/flask) | % CE | % control | Mean % control | Colonies/flask (2x10^5 cells plated/flask) | MF | MFS (10^-6) | Group MFS 10^-6 | ||||||||||
| 0 | A | 171 | 169 | 164 | 84.0 | 100.0 | 100 | 163 | 179 | 161 | 83.8 | 100.0 | 100 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 1 |
| B | 153 | 152 | 148 | 75.5 | 100.0 | 171 | 161 | 159 | 81.8 | 100.0 | 1 | 0 | 0 | 0 | 0 | 1 | 1.2 | ||||
| 10 P | A | 181 | 228 | 193 | 100.3 | 119.4 | 113 | 168 | 56 | 131 | 75.8 | 92.5 | 95 | 0 | 0 | 1 | 0 | 1 | 2 | 2.6 | 1 |
| B | 131 | 151 | 200 | 80.3 | 106.4 | 159 | 167 | 167 | 82.2 | 100.4 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | ||||
| 20 P | A | 173 | 151 | 171 | 82.5 | 98.2 | 108 | 173 | 133 | 160 | 77.7 | 92.6 | 93 | 1 | 0 | 0 | 0 | 0 | 1 | 1.3 | 1 |
| B | 168 | 203 | 164 | 89.2 | 118.1 | 152 | 142 | 162 | 76.0 | 92.9 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | ||||
| 40 P | A | 152 | 167 | 145 | 77.3 | 92.1 | 100 | 144 | 148 | 147 | 73.2 | 87.3 | 93 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 1 |
| B | 179 | 153 | 153 | 80.8 | 107.1 | 151 | 169 | 162 | 80.3 | 98.2 | 0 | 2 | 0 | 0 | 0 | 2 | 2.5 | ||||
| 80 P | A | 130 | 143 | 125 | 66.3 | 79.0 | 89 | 138 | 131 | 145 | 69.0 | 82.3 | 90 | 0 | 0 | 0 | 1 | 0 | 1 | 1.4 | 2 |
| B | 162 | 149 | 138 | 74.8 | 99.1 | 164 | 165 | 152 | 80.2 | 98.0 | 0 | 0 | 0 | 0 | 2 | 2 | 2.5 | ||||
| 160 P | A | 135 | 133 | 144 | 68.7 | 81.7 | 97 | 138 | 158 | 157 | 75.5 | 90.1 | 89 | 0 | 0 | 0 | 3 | 0 | 3 | 4 | 2 |
| B | 172 | 159 | 179 | 85.0 | 112.6 | 158 | 144 | 134 | 72.7 | 88.8 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | ||||
| 320 AP | A | 102 | 78 | 99 | 46.5 | 55.4 | 71 | 151 | 158 | 145 | 75.7 | 90.3 | 93 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 2 |
| B | 134 | 124 | 135 | 65.5 | 86.8 | 155 | 171 | 141 | 77.8 | 95.1 | 2 | 1 | 0 | 0 | 0 | 3 | 3.9 | ||||
| 640 AP | A | 106 | 109 | 111 | 54.3 | 64.7 | 74 | 162 | 168 | 166 | 82.7 | 98.6 | 101 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 1 |
| B | 131 | 116 | 133 | 63.3 | 83.9 | 165 | 169 | 171 | 84.2 | 102.9 | 0 | 0 | 0 | 2 | 0 | 2 | 2.4 | ||||
| DMBA 0.5 | A | 152 | 136 | 136 | 70.7 | 84.1 | 100 | 145 | 131 | 138 | 69.0 | 82.3 | 83 | 15 | 6 | 15 | 15 | 11 | 62 | 89.9 | 172 |
| B | 179 | 177 | 169 | 87.5 | 115.9 | 141 | 129 | 142 | 68.7 | 83.9 | 40 | 26 | 33 | 39 | 36 | 174 | 253.4 | ||||
| DMBA 1 | A | 66 | 69 | 60 | 32.5 | 38.7 | 41 | 102 | 106 | 115 | 53.8 | 64.2 | 63 | 42 | 32 | 40 | 27 | 27 | 168 | 312.1 | 332 |
| B | 60 | 67 | 68 | 32.5 | 43.0 | 93 | 103 | 111 | 51.2 | 62.5 | 28 | 32 | 43 | 46 | 46 | 180 | 351.8 | ||||
Table 3: Experiment 2 - 24 hour exposure without S9
| Dose (µg/mL) | Rep. | Day 0 viability | Day 7 viability | Day 7 mutant | |||||||||||||||||
| Colonies/flask (200 cells plated/flask) | % CE | % control | Mean % control | Colonies/flask (200 cells plated/flask) | % CE | % control | Mean % control | Colonies/flask (2x10^5 cells plated/flask) | MF | MFS (10^-6) | Group MFS 10^-6 | ||||||||||
| 0 | A | 100 | 106 | 111 | 52.8 | 100 | 100 | 151 | 149 | 146 | 74.3 | 100 | 100 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 2 |
| B | 169 | 150 | 162 | 80.2 | 100 | 125 | 133 | 131 | 64.8 | 100 | 1 | 0 | 0 | 1 | 0 | 2 | 3.1 | ||||
| 0.5 | A | 103 | 114 | 97 | 52.3 | 99.1 | 98 | 143 | 138 | 130 | 68.5 | 92.2 | 96 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 2 |
| B | 157 | 149 | 161 | 77.8 | 97.1 | 131 | 139 | 120 | 65 | 100.3 | 0 | 1 | 1 | 0 | 0 | 2 | 3.1 | ||||
| 1 P | A | 94 | 102 | 116 | 52 | 98.4 | 104 | 140 | 152 | 147 | 73.2 | 98.4 | 101 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
| B | 177 | 169 | 178 | 87.3 | 108.9 | 136 | 121 | 142 | 66.5 | 102.6 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | ||||
| 4 P | A | 114 | 119 | 117 | 58.3 | 110.4 | 91 | 116 | 156 | 154 | 71 | 95.5 | 129 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
| B | 124 | 124 | 101 | 58.2 | 72.6 | 209 | 210 | 211 | 105 | 162 | 1 | 0 | 0 | 0 | 0 | 1 | 1 | ||||
| 6 P | A | 60 | 70 | 61 | 31.8 | 60.3 | 43 | 172 | 189 | 171 | 88.7 | 119.3 | 133 | 0 | 0 | 1 | 0 | 0 | 1 | 1.1 | 2 |
| B | 44 | 38 | 38 | 20 | 24.9 | 181 | 199 | 192 | 95.3 | 147 | 0 | 0 | 0 | 2 | 0 | 2 | 2.1 | ||||
| 8 P | A | 21 | 21 | 19 | 10.2 | 19.2 | 18 | 162 | 187 | 161 | 85 | 114.3 | 135 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
| B | 20 | 29 | 28 | 12.8 | 16 | 224 | 188 | 197 | 101.5 | 156.6 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | ||||
| 12 P | A | Too toxic for plating | Too toxic for plating | ||||||||||||||||||
| B | Too toxic for plating | Too toxic for plating | |||||||||||||||||||
| EMS 200 | A | 15 | 12 | 16 | 7.2 | 13.6 | 21 | 103 | 87 | 100 | 48.3 | 65 | 176 | 27 | 33 | 27 | 30 | 28 | 145 | 300 | 239 |
| B | 47 | 42 | 46 | 22.5 | 28.1 | 111 | 101 | 126 | 586.3 | 86.9 | 24 | 24 | 11 | 19 | 22 | 100 | 177.5 | ||||
| EMS 300 | A | 6 | 3 | 6 | 2.5 | 4.7 | 7 | 68 | 70 | 99 | 39.5 | 53.1 | 54 | 95 | 77 | 104 | 87 | 77 | 440 | 1113.9 | 829 |
| B | 14 | 12 | 14 | 6.7 | 8.3 | 122 | 116 | 101 | 45 | 54 | 42 | 56 | 48 | 45 | 54 | 245 | 544.4 | ||||
Table 4: Experiment 2 - 4 hour exposure with S9
| Dose (µg/mL) | Rep. | Day 0 viability | Day 7 viability | Day 7 mutant | |||||||||||||||||
| Colonies/flask (200 cells plated/flask) | % CE | % control | Mean % control | Colonies/flask (200 cells plated/flask) | % CE | % control | Mean % control | Colonies/flask (2x10^5 cells plated/flask) | MF | MFS (10^-6) | Group MFS 10^-6 | ||||||||||
| 0 | A | 166 | 166 | 173 | 84.2 | 100.0 | 100 | 129 | 154 | 140 | 70.5 | 100.0 | 100 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
| B | 152 | 159 | 207 | 86.3 | 100.0 | 140 | 133 | 163 | 72.7 | 100.0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | ||||
| 10 P | A | 144 | 137 | 155 | 72.7 | 86.3 | 98 | 150 | 139 | 140 | 71.5 | 101.4 | 104 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
| B | 177 | 221 | 169 | 94.5 | 109.5 | 153 | 163 | 148 | 77.3 | 106.4 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | ||||
| 20 P | A | 135 | 136 | 145 | 69.3 | 82.4 | 95 | 156 | 161 | 111 | 71.3 | 101.2 | 99 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 1 |
| B | 176 | 184 | 195 | 92.5 | 107.1 | 146 | 137 | 137 | 70.0 | 96.3 | 0 | 0 | 1 | 0 | 0 | 1 | 1.4 | ||||
| 40 P | A | 159 | 161 | 160 | 80.0 | 95.0 | 87 | 128 | 126 | 142 | 66.0 | 93.6 | 97 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
| B | 129 | 146 | 133 | 68.0 | 78.8 | 132 | 158 | 146 | 72.7 | 100.0 | 0 | 2 | 0 | 0 | 0 | 0 | 0 | ||||
| 80 P | A | 163 | 153 | 137 | 75.5 | 89.7 | 88 | 150 | 149 | 153 | 75.3 | 106.9 | 98 | 0 | 0 | 0 | 0 | 0 | 1 | 0 | 1 |
| B | 152 | 169 | 131 | 75.3 | 87.3 | 116 | 138 | 135 | 64.8 | 89.2 | 0 | 1 | 0 | 0 | 0 | 0 | 1.5 | ||||
| 160 P | A | 135 | 129 | 143 | 67.8 | 80.6 | 77 | 141 | 130 | 155 | 71.0 | 100.7 | 98 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
| B | 124 | 133 | 126 | 63.8 | 73.9 | 134 | 141 | 143 | 69.7 | 95.9 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | ||||
| 320 P | A | 136 | 125 | 106 | 61.2 | 72.7 | 73 | 152 | 149 | 186 | 81.2 | 115.1 | 114 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
| B | 131 | 126 | 128 | 64.2 | 74.3 | 172 | 153 | 163 | 81.3 | 111.9 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | ||||
| 640 P | A | 127 | 121 | 115 | 60.5 | 71.9 | 76 | 138 | 119 | 143 | 66.7 | 94.6 | 98 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 1 |
| B | 122 | 150 | 138 | 68.3 | 79.2 | 159 | 130 | 150 | 73.2 | 100.7 | 0 | 0 | 0 | 0 | 1 | 1 | 1.4 | ||||
| DMBA 0.5 | A | 105 | 85 | 91 | 46.8 | 55.6 | 62 | 102 | 77 | 86 | 44.2 | 62.6 | 69 | 11 | 16 | 18 | 12 | 18 | 75 | 169.8 | 185 |
| B | 117 | 135 | 99 | 58.5 | 67.8 | 102 | 119 | 107 | 54.7 | 75.2 | 30 | 22 | 25 | 18 | 14 | 109 | 199.4 | ||||
| DMBA 1 | A | 22 | 23 | 14 | 9.8 | 11.7 | 10 | 63 | 76 | 75 | 35.7 | 50.6 | 50 | 20 | 12 | 21 | 21 | 23 | 97 | 272 | 262 |
| B | 15 | 16 | 10 | 6.8 | 7.9 | 77 | 68 | 69 | 35.7 | 49.1 | 18 | 14 | 20 | 17 | 21 | 90 | 252.3 | ||||
CE = cloning efficiency
MF = mutant frequency
MFS = mutant frequency per survivor
P = precipitate
AP = aggregate precipitate
EMS = ethyl methane sulphonate
DMBA = dimethyl benzanthracene
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 Key Study: Durward (1993)
A test was conducted to assess the gene mutation potential of the test material in an assay carried out in accordance with the OECD Guidelines for the Testing of Chemicals, Protocol No. 471 and also with Method B14 in Commission Directive 84/449/EEC. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).
Salmonella typhimurium strains TA1535, TA1537, TA1538, TA98 and TA100 were treated with the test material by the Ames plate incorporation method at five dose levels, in triplicate, both with and without metabolic activation. The dose range was determined in a preliminary toxicity assay and was 8 to 5000 µg/plate in the first experiment. The experiment was repeated on a separate day using fresh cultures of the bacterial strains and fresh chemical solutions. In this case the dose range was 312.5 to 5 000 µg/plate.
The solvent (dimethyl sulphoxide) control plates gave counts of revertant colonies within the normal range, and all positive control chemicals produced marked increases in the number of revertant colonies, both with and without the metabolising system.
There was no reduction in the growth of the bacterial lawn at any of the dose levels employed in all of the strains of Salmonella used. A precipitate was observed at and above 1 000 µg/plate but this did not interfere with the scoring of revertant colonies.
No significant increase in the numbers of revertant colonies was recorded for any of the bacterial strains with any dose either with or without metabolic activation.
Therefore the test material was found to be non-mutagenic under the conditions of this study.
Bacterial Reverse Mutation Assay Key Study: Kovács (2018)
The genetic toxicity of the test material was investigated in a study following the design of OECD 471, EU Method B.13/14 and OPPTS 870.5100. Although the experimental design is compatible with the guideline requirements, only one bacterial strain was used in the study as requested by the Sponsor. The testing was performed under GLP conditions. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).
The experiments were carried out using tryptophan-requiring auxotroph strain of Escherichia coli (Escherichia coli WP2 uvrA) in the presence and absence of a post mitochondrial supernatant (S9 fraction) prepared from the livers of phenobarbital/β-naphthoflavone-induced rats.
The study included a Preliminary Compatibility Test, an Initial Mutation Test (Plate Incorporation Method) and a Confirmatory Mutation Test (Pre-Incubation Method).
Based on the results of the Compatibility Test, the test material was dissolved in Dimethyl sulfoxide (DMSO) at a concentration of 100 mg/mL. The test material concentrations in the Initial Mutation Test and in the Confirmatory Mutation Test were 5 000, 1 581, 500, 158.1, 50, 15.81, 5 and 1.581 μg/plate.
In the Initial Mutation Test and Confirmatory Mutation Test, the number of revertant colonies did not show any biologically relevant increase compared to the solvent control. There were no dose-related trends and no indication of any treatment-related effect.
Precipitate/slight precipitate was observed on the plates in the main tests with and without metabolic activation at the concentrations of 5 000, 1 581, 500 and/or 158.1 μg/plate. The precipitation did not adversely affect the colony counting.
No inhibitory or toxic effects of the test material were detected in the study.
The mean values of revertant colonies of the negative (vehicle/solvent) control plates were within the historical control range, the reference mutagens showed the expected increase in the number of revertant colonies, the viability of the bacterial cells was checked by a plating experiment in each test. At least five analysable concentrations were presented of the main tests, the examined concentration range was considered to be adequate. The study was therefore, considered to be valid.
The reported data of this mutagenicity assay show that under the experimental conditions applied the test material did not induce gene mutations by base pair changes in the genome of the strain used.
Under the conditions of this study, the test material had no mutagenic activity on Escherichia coli WP2 uvrA strain with or without activation.
Bacterial Reverse Mutation Assay Supporting Study: Thompson (1992)
A test was conducted to assess the gene mutation potential of the test material in an assay carried out in accordance with the OECD Guidelines for the Testing of Chemicals, Protocol No. 471 and also with Method B14 in Commission Directive 84/449/EEC. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).
Salmonella typhimurium strains TA1535, TA1537, TA1538, TA98 and TA100 were treated with the test material by the Ames plate incorporation method at five dose levels, in triplicate, both with and without metabolic activation. The dose range was determined in a preliminary toxicity assay and was 8 to 5000 µg/plate in the first experiment. The experiment was repeated on a separate day using fresh cultures of the bacterial strains and fresh chemical solutions. In this case the dose range was 312.5 to 5 000 µg/plate.
The solvent (dimethyl sulphoxide) control plates gave counts of revertant colonies within the normal range, and all positive control chemicals produced marked increases in the number of revertant colonies, both with and without the metabolising system.
There was no reduction in the growth of the bacterial lawn at any of the dose levels employed in all of the strains of Salmonella used. A precipitate was observed at and above 312.5 µg/plate but this did not interfere with the scoring of revertant colonies.
No significant increase in the numbers of revertant colonies was recorded for any of the bacterial strains with any dose either with or without metabolic activation.
Therefore the test material was found to be non-mutagenic under the conditions of this study.
In Vitro Mammalian Chromosome Aberration test Key Study: Durward (1994)
A study was conducted to assess the potential of the test material to induce chromosome aberrations. The study was awarded a reliability score of 2 in accordance with the criteria set forth by Klimisch et al. (1997).
Chinese hamster lung (CHL) cells were treated with the test material at four dose levels, in duplicate, together with negative and positive controls. Four treatment regimens were used: 6 hours exposure both with and without the addition of an induced rat liver homogenate metabolising system at 50 % in standard co-factors followed by an 18-hour expression period; 24 hours continuous exposure and 48 hours continuous exposure.
The dose range was selected on the results of a preliminary toxicity test and was 78.1 to 625 µg/mL for the 6 hour treatment (both with and without S9) and the 24 hour treatment and 9.75 to 78.1 µg/mL for the 48 hour treatment. The upper three dose levels were evaluated for chromosome aberrations in each case.
The negative (solvent) controls gave frequencies of aberrations within the range expected for the CHL cell line.
All the positive control treatments except cyclophosphamide without S9 gave highly significant increases in the frequency of aberrations indicating the satisfactory performance of the test and of the activity of the metabolising system.
The material demonstrated no significant increases in the frequency of cells with aberrations in any of the treatment cases, though it was shown to be toxic to CHL cells in vitro in the 48 hour treatment cases.
The test material was non-clastogenic to CHL cells in vitro under the conditions of this study.
Mammalian Cell Gene Mutation Assay Key Study: Morris (2012)
The mutagenic potential of the test material was determined in accordance with standardised guidelines OECD 476 and EU Method B.17. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).
During the study, the test material was tested at a minimum of six dose levels, in duplicate, in the CHO-HPRT mutation assay in the absence and presence of metabolic activation with Aroclor-induced rat liver S-9; vehicle and positive controls were also included. Four treatment conditions were used for the test, i.e. in experiment 1, a 4-hour exposure in the presence of S9, at a 2 % final concentration and a 4-hour exposure in the absence of S9. In experiment 2, the 4-hour exposure with addition of S9 was repeated using a 1 % final S9 concentration, whilst in the absence of S9 the exposure time was increased to 24 hours.
Under the conditions of the study the vehicle control gave mutant frequencies within the range expected of CHO cells at the HPRT locus. The positive controls, both in the presence and absence of metabolic activation, gave significant increases in the mutant frequency indicating satisfactory performance of the test and of the metabolising system. The test material demonstrated no significant increases in mutant frequency at any dose level, either with or without metabolic activation, in either the first or second experiment. The test material was, therefore, considered to be non-mutagenic to CHO cells under the conditions of the test.
Justification for classification or non-classification
The registered substance does not meet the criteria for classification for as mutagenic according to the EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008.
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