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EC number: 287-673-6 | CAS number: 85566-63-8
- Life Cycle description
- Uses advised against
- Endpoint summary
- Appearance / physical state / colour
- Melting point / freezing point
- Boiling point
- Density
- Particle size distribution (Granulometry)
- Vapour pressure
- Partition coefficient
- Water solubility
- Solubility in organic solvents / fat solubility
- Surface tension
- Flash point
- Auto flammability
- Flammability
- Explosiveness
- Oxidising properties
- Oxidation reduction potential
- Stability in organic solvents and identity of relevant degradation products
- Storage stability and reactivity towards container material
- Stability: thermal, sunlight, metals
- pH
- Dissociation constant
- Viscosity
- Additional physico-chemical information
- Additional physico-chemical properties of nanomaterials
- Nanomaterial agglomeration / aggregation
- Nanomaterial crystalline phase
- Nanomaterial crystallite and grain size
- Nanomaterial aspect ratio / shape
- Nanomaterial specific surface area
- Nanomaterial Zeta potential
- Nanomaterial surface chemistry
- Nanomaterial dustiness
- Nanomaterial porosity
- Nanomaterial pour density
- Nanomaterial photocatalytic activity
- Nanomaterial radical formation potential
- Nanomaterial catalytic activity
- Endpoint summary
- Stability
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- Environmental data
- Additional information on environmental fate and behaviour
- Ecotoxicological Summary
- Aquatic toxicity
- Endpoint summary
- Short-term toxicity to fish
- Long-term toxicity to fish
- Short-term toxicity to aquatic invertebrates
- Long-term toxicity to aquatic invertebrates
- Toxicity to aquatic algae and cyanobacteria
- Toxicity to aquatic plants other than algae
- Toxicity to microorganisms
- Endocrine disrupter testing in aquatic vertebrates – in vivo
- Toxicity to other aquatic organisms
- Sediment toxicity
- Terrestrial toxicity
- Biological effects monitoring
- Biotransformation and kinetics
- Additional ecotoxological information
- Toxicological Summary
- Toxicokinetics, metabolism and distribution
- Acute Toxicity
- Irritation / corrosion
- Sensitisation
- Repeated dose toxicity
- Genetic toxicity
- Carcinogenicity
- Toxicity to reproduction
- Specific investigations
- Exposure related observations in humans
- Toxic effects on livestock and pets
- Additional toxicological data
Endpoint summary
Administrative data
Key value for chemical safety assessment
Genetic toxicity in vitro
Description of key information
The key studies were conducted to internationally recognised testing guidelines and with GLP certification.
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:
- 25 October 2017 - 20 December 2017
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Version / remarks:
- 21 July 1997
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- bacterial reverse mutation assay
- Specific details on test material used for the study:
- Bernel Ester DCM (CAS number 85566-63-8), batch number P6978, was a clear liquid. It was received on 07 September 2017 and stored at 15-25°C protected from light. Purity was stated as 92.78% (assumed 100% for testing) and the expiry date was given as 06 September 2018, see Certificate of Analysis. The test article information and certificate of analysis provided by the Sponsor are considered an adequate description of the characterisation, purity and stability of the test article. Determinations of stability and characteristics of the test article were the responsibility of the Sponsor.
Preliminary solubility data indicated that Bernel Ester DCM was soluble in ethanol at concentrations equivalent to at least 100 mg/mL. A maximum concentration of 5000 µg/plate was selected for Experiment 1, in order that initial treatments were performed up to this maximum recommended concentration according to current regulatory guidelines (OECD, 1997). A maximum concentration of 5000 µg/plate was also selected for Experiment 2.
Test article stock solutions were prepared by formulating Bernel Ester DCM under subdued lighting in ethanol with the aid of vortex mixing, to give the maximum required treatment concentration. Subsequent dilutions were made using ethanol. The test article solutions were protected from light and used within approximately 6 hours of initial formulation. - Target gene:
- Histidine locus.
- Species / strain / cell type:
- S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
- Metabolic activation:
- with and without
- Metabolic activation system:
- mammalian liver post-mitochondrial fraction (S-9)
- Test concentrations with justification for top dose:
- Mutation Experiment 1 (S-9+-): 5, 16, 50, 160, 500, 1600, 5000 µg/plate
Mutation Experiment 2 (S-9+-): 160, 320, 625, 1250, 2500, 5000 µg/plate - Vehicle / solvent:
- Ethanol
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- Positive controls:
- yes
- Positive control substance:
- 9-aminoacridine
- 2-nitrofluorene
- sodium azide
- benzo(a)pyrene
- mitomycin C
- other: 2-aminoanthracene
- Details on test system and experimental conditions:
- Test System
The test system was suitably labelled to clearly identify the study number, bacterial strain, test article concentration (where appropriate), positive and vehicle controls, in the absence or presence of S-9 mix.
Mutation Experiments
Bernel Ester DCM was tested for mutation (and toxicity) in five strains of Salmonella typhimurium (TA98, TA100, TA1535, TA1537 and TA102), in two separate experiments, at the concentrations detailed previously, using triplicate plates without and with S-9 for test article, vehicle and positive controls. These platings were achieved by the following sequence of additions to molten agar at 45±1°C:
• 0.1 mL bacterial culture
• 0.1 mL of test article solution/vehicle control or 0.05 mL of positive control
• 0.5 mL 10% S-9 mix or buffer solution
followed by rapid mixing and pouring on to Vogel-Bonner E agar plates. When set, the plates were inverted and incubated at 37±1°C protected from light for 3 days. Following incubation, these plates were examined for evidence of toxicity to the background lawn, and where possible revertant colonies were counted (see Colony Enumeration).
As the results of Experiment 1 were negative, treatments in the presence of S-9 in Experiment 2 included a pre-incubation step. Quantities of test article, vehicle control solution (reduced to 0.05 mL) or positive control, bacteria and S-9 mix detailed above, were mixed together and incubated for 20 minutes at 37±1°C, with shaking, before the addition of 2 mL molten agar at 45±1°C. Plating of these treatments then proceeded as for the normal plate-incorporation procedure. In this way, it was hoped to increase the range of mutagenic chemicals that could be detected in the assay.
Volume additions for the Experiment 2 pre-incubation treatments were reduced to 0.05 mL due to the vehicle (ethanol) employed in this study. This, and some other organic vehicles, are known to be near to toxic levels when added at volumes of 0.1 mL in this assay system when employing the pre-incubation methodology. By reducing the addition volume to 0.05 mL per plate, it was hoped to minimise or eliminate any toxic effects of the vehicle that may have otherwise occurred.
Toxicity Assessment
The background lawns of the plates were examined for signs of toxicity. Other evidence of toxicity may have included a marked reduction in revertants compared to the concurrent vehicle controls and/or a reduction in mutagenic response.
Colony Enumeration
Colonies were counted electronically using a Sorcerer Colony Counter (Perceptive Instruments) or manually where confounding factors such as precipitation, contamination or bubbles or splits in the agar affected the accuracy of the automated counter.
Analysis of Results
Treatment of Data
Individual plate counts were recorded separately and the mean and standard deviation of the plate counts for each treatment were determined. Control counts were compared with the laboratory’s historical control ranges. Data were considered acceptable if the vehicle control counts fell within the calculated historical control ranges and the positive control plate counts were comparable with the historical control ranges.
The presence or otherwise of a concentration response was checked by non-statistical analysis, up to limiting levels (for example toxicity, precipitation or 5000 µg/plate). However, adequate interpretation of biological relevance was of critical importance.
Acceptance Criteria
The assay was to be considered valid if all the following criteria were met:
1. The vehicle control counts fell within the laboratory’s historical control ranges
2. The positive control chemicals induced increases in revertant numbers of ≥1.5 fold (in strain TA102), ≥2-fold (in strains TA98 and TA100) or ≥3-fold (in strains TA1535 and TA1537) the concurrent vehicle control confirming discrimination between different strains, and an active S-9 preparation. - Evaluation criteria:
- For valid data, the test article was considered to be mutagenic if:
1. A concentration related increase in revertant numbers was ≥1.5-fold (in strain TA102), ≥2-fold (in strains TA98 or TA100) or ≥3-fold (in strains TA1535 or TA1537) the concurrent vehicle control values
2. The positive trend/effects described above were reproducible.
The test article was considered positive in this assay if both of the above criteria were met.
The test article was considered negative in this assay if neither of the above criteria were met.
Results which only partially satisfied the above criteria were dealt with on a case-by-case basis. Biological relevance was taken into account, for example consistency of response within and between concentrations and (where applicable) between experiments. - Species / strain:
- S. typhimurium TA 98
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Species / strain:
- S. typhimurium TA 100
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Species / strain:
- S. typhimurium TA 1535
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Species / strain:
- S. typhimurium TA 1537
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Species / strain:
- S. typhimurium TA 102
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- Toxicity, Solubility and Concentration Selection
Experiment 1 treatments of all the tester strains were performed in the absence and in the presence of S-9, using final concentrations of Bernel Ester DCM at 5, 16, 50, 160, 500, 1600 and 5000 µg/plate, plus vehicle and positive controls. Following these treatments evidence of toxicity in the form of a slight thinning of the background bacterial lawn was observed at 5000 µg/plate in strain TA98 in the absence and presence of S-9 and strain TA98 in the presence of S-9 only.
Experiment 2 treatments of all the tester strains were performed in the absence and in the presence of S-9. The maximum test concentration of 5000 µg/plate was retained for all strains. Narrowed concentration intervals were employed covering the range 160-5000 µg/plate, in order to examine more closely those concentrations of Bernel Ester DCM approaching the maximum test concentration and considered therefore most likely to provide evidence of any mutagenic activity. In addition, all treatments in the presence of S-9 were further modified by the inclusion of a pre incubation step. In this way, it was hoped to increase the range of mutagenic chemicals that could be detected using this assay system. Following these treatments no evidence of toxicity was observed.
Precipitation was observed on the test plates at concentrations of 5000 µg/plate.
Data Acceptability and Validity
The individual mutagenicity plate counts were averaged to give mean values, which are presented in Section 8. From the data it can be seen that vehicle control counts fell within the laboratory’s historical ranges. The positive control chemicals all induced increases in revertant numbers of ≥1.5-fold (in strain TA102), ≥2-fold (in strains TA98 and TA100) or ≥3 fold (in strains TA1535 and TA1537) the concurrent vehicle controls confirming discrimination between different strains, and an active S-9 preparation. The study therefore demonstrated correct strain and assay functioning and was accepted as valid.
Mutation
Following Bernel Ester DCM treatments of all the test strains in the absence and presence of S-9, no increases in revertant numbers were observed that were ≥1.5-fold (in strain TA102), ≥2-fold (in strains TA98 and TA100) or ≥3-fold (in strains TA1535 and TA1537) the concurrent vehicle control. This study was considered therefore to have provided no evidence of any Bernel Ester DCM mutagenic activity in this assay system. - Conclusions:
- It was concluded that Bernel Ester DCM did not induce mutation in five histidine-requiring strains (TA98, TA100, TA1535, TA1537 and TA102) of Salmonella typhimurium when tested under the conditions of this study. These conditions included treatments at concentrations up to 5000 µg/plate (the maximum recommended concentration according to current regulatory guidelines) in the absence and in the presence of a rat liver metabolic activation system (S-9).
- Executive summary:
Bernel Ester DCM was assayed for mutation in five histidine-requiring strains (TA98, TA100, TA1535, TA1537 and TA102) of Salmonella typhimurium, both in the absence and in the presence of metabolic activation by an Aroclor 1254-induced rat liver post-mitochondrial fraction (S-9), in two separate experiments.
All Bernel Ester DCM treatments in this study were performed using formulations prepared in ethanol.
Experiment 1 treatments of all the tester strains were performed in the absence and in the presence of S-9, using final concentrations of Bernel Ester DCM at 5, 16, 50, 160, 500, 1600 and 5000 µg/plate, plus vehicle and positive controls. Following these treatments evidence of toxicity was observed at 5000 µg/plate in strain TA98 in the absence and presence of S-9 and strain TA98 in the presence of S-9 only.
Experiment 2 treatments of all the tester strains were performed in the absence and in the presence of S-9. The maximum test concentration of 5000 µg/plate was retained for all strains. Narrowed concentration intervals were employed covering the range 160-5000 µg/plate, in order to examine more closely those concentrations of Bernel Ester DCM approaching the maximum test concentration and considered therefore most likely to provide evidence of any mutagenic activity. In addition, all treatments in the presence of S-9 were further modified by the inclusion of a pre‑incubation step. In this way, it was hoped to increase the range of mutagenic chemicals that could be detected using this assay system. Following these treatments, no evidence of toxicity was observed.
Precipitation was observed on all test plates at a concentration of 5000 µg/plate.
Vehicle and positive control treatments were included for all strains in both experiments. The mean numbers of revertant colonies fell withinacceptable ranges for vehicle control treatments, and were elevated by positive control treatments.
Following Bernel Ester DCM treatments of all the test strains in the absence and presence of S-9, no increases in revertant numbers were observed that were ≥1.5-fold (in strain TA102), ≥2-fold (in strains TA98 or TA100) or ≥3-fold (in strains TA1535 or TA1537) the concurrent vehicle control. This study was considered therefore to have provided no evidence of any Bernel Ester DCM mutagenic activity in this assay system.
It was concluded that Bernel Ester DCM did not induce mutation in five histidine‑requiring strains (TA98, TA100, TA1535, TA1537 and TA102) of Salmonella typhimurium when tested under the conditions of this study. These conditions included treatments at concentrations up to 5000 µg/plate (the maximum recommended concentration according to current regulatory guidelines) in the absence and in the presence of a rat liver metabolic activation system (S-9).
- Endpoint:
- in vitro cytogenicity / micronucleus study
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 19/9/2017 to 13/3/2018
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)
- Version / remarks:
- 23 July 2010
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- in vitro mammalian cell micronucleus test
- Specific details on test material used for the study:
- Bernel Ester DCM (CAS number 85566-63-8), batch number P6978, was a clear liquid, with a molecular weight of 340 g/L. It was received on 07 September 2017 and stored at 15-25°C protected from light. Purity was stated as 92.78% (assumed 100% for testing) and the expiry date was given as 06 September 2018, see Certificate of Analysis. The test article information and certificate of analysis provided by the Sponsor are considered an adequate description of the characterisation, purity and stability of the test article.
- Species / strain / cell type:
- lymphocytes:
- Details on mammalian cell type (if applicable):
- Human lymphocytes prepared form the pooled blood of two female doners
- Additional strain / cell type characteristics:
- not applicable
- Metabolic activation:
- with and without
- Metabolic activation system:
- mammalian liver post-mitochondrial fraction (S-9)
- Test concentrations with justification for top dose:
- Preliminary solubility data indicated that Bernel Ester DCM was soluble in acetone at concentrations up to at least 342.9 mg/mL. The solubility limit in culture medium was in the range of 53.58 to 107.2 µg/mL, as indicated by precipitation at the higher concentration which persisted following 24 hours incubation at 37±1°C. A maximum concentration of 200 µg/mL was selected for the cytotoxicity Range-Finder Experiment, in order that treatments were performed up to a precipitating treatment concentration (OECD, 2016). Concentrations for the Micronucleus Experiment were selected based on the results of this cytotoxicity Range-Finder Experiment.
Test article stock solutions were prepared by formulating Bernel Ester DCM under subdued lighting in acetone, with the aid of vortex mixing, to give the maximum required treatment concentration. Subsequent dilutions were made using acetone. The test article solutions were protected from light and used within approximately
3.5 hours of initial formulation. The following concentration ranges were tested:
Experiment Treatment Concentration Range Final Concentration Range
(mg/mL) (µg/mL)
Range-Finder
3+21, -S-9 0.07256 to 20.00 0.7256 to 200.0
3+21, +S-9 0.07256 to 20.00 0.7256 to 200.0
24+24, -S-9 0.07256 to 20.00 0.7256 to 200.0
Micronucleus Experiment
3+21, -S-9 0.500 to 6.000 5.000 to 60.00
3+21, +S-9 1.000 to 10.000 10.000 to 100.00
24+24, -S-9 0.500 to 6.000 5.000 to 60.00 - Vehicle / solvent:
- Acetone
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- Positive controls:
- yes
- Positive control substance:
- cyclophosphamide
- mitomycin C
- other: vinblastine
- Evaluation criteria:
- Evaluation Criteria
For valid data, the test article was considered to induce clastogenic and/or aneugenic events if:
1. A statistically significant increase in the frequency of MNBN cells at one or more concentrations was observed
2. An incidence of MNBN cells at such a concentration that exceeded the normal range in both replicates was observed
3. A concentration-related increase in the proportion of MNBN cells was observed (positive trend test).
The test article was considered positive in this assay if all of the above criteria were met.
The test article was considered negative in this assay if none of the above criteria were met.
Results which only partially satisfied the above criteria were dealt with on a case-by-case basis. Evidence of a concentration-related effect was considered useful but not essential in the evaluation of a positive result (Scott et al., 1990). Biological relevance was taken into account, for example consistency of response within and between concentrations, or effects occurring only at very toxic concentrations (Thybaud et al., 2007). - Key result
- Species / strain:
- lymphocytes: 24+24 hour treatment
- Metabolic activation:
- with
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- lymphocytes: 24+24 hour treatment
- Metabolic activation:
- without
- Genotoxicity:
- positive
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- lymphocytes: 3+21 hour treatment
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Conclusions:
- It is concluded that Bernel Ester DCM showed evidence of micronucleus induction in cultured human peripheral blood lymphocytes following 24+24 hour treatment in the absence of an Aroclor-induced rat liver metabolic activation system (S-9). In the same test system, Bernel Ester DCM did not induce biologically relevant increases in micronuclei following 3+21 hour treatment in the absence and presence of S-9. Sporadic increases were noted following 3+21 hour treatment in the absence of S-9 but there was poor reproducibility between replicates and therefore this observation was considered of questionable biological relevance. Maximum concentrations analysed were limited by toxicity under all treatment conditions (in line with current regulatory test guidelines for the in vitro micronucleus assay).
- Executive summary:
Bernel Ester DCM was tested in an in vitro micronucleus assay using duplicate human lymphocyte cultures prepared from the pooled blood of two female donors in a single experiment. Treatments covering a broad range of concentrations, separated by narrow intervals, were performed both in the absence and presence of metabolic activation (S-9) from Aroclor 1254-induced rats. The test article was formulated in acetone and the highest concentrations tested in the Micronucleus Experiment, (limited by toxicity) were determined following a preliminary cytotoxicity Range-Finder Experiment.
Treatments were conducted (as detailed in the following summary table) 48 hours following mitogen stimulation by phytohaemagglutinin (PHA). The test article concentrations for micronucleus analysis were selected by evaluating the effect of Bernel Ester DCM on the replication index (RI). Micronuclei were analysed at three or four concentrations and a summary of the data is presented in the following table:
Treatment
Concentration (mg/mL)
Cytotoxicity (%)$
Mean MNBN Cell Frequency (%)
Historical Control Range (%)#
Statistical Significance
3+21 hour -S-9
Vehiclea
-
0.65
0.20 – 1.00
-
UTC
-
0.85
-
5.000
0
0.55
NS
25.00
18
0.85
NS
30.00
45
0.80
NS
32.50
50
1.03
p≤0.05
*MMC, 0.30
32
5.00
p≤0.001
3+21 hour +S-9
Vehiclea
-
0.83
0.20 – 1.07
-
UTC
-
0.60
-
10.00
12
0.98
NS
45.00
29
0.68
NS
65.00
57
0.70
NS
*CPA, 3.00
20
2.20
p≤0.001
24+24 hour -S-9
Vehiclea
-
0.65
0.10 – 0.90
-
15.00
2
0.55
NS
22.50
22
0.45
NS
27.50
48
1.20
p≤0.05
*VIN, 0.04
21
3.10
p≤0.001
Appropriate negative (vehicle and untreated) control cultures were included in the test system under each treatment condition. The proportion of micronucleated binucleate (MNBN) cells in these cultures fell within the 95thpercentile of the current observed historical vehicle control (normal) ranges. Mitomycin C (MMC) and Vinblastine (VIN) were employed as clastogenic and aneugenic positive control chemicals respectively in the absence of rat liver S-9. Cyclophosphamide (CPA) was employed as a clastogenic positive control chemical in the presence of rat liver S-9. Cells receiving these were sampled in the Micronucleus Experiment at 24 hours (CPA, MMC) or 48 hours (VIN) after the start of treatment. All positive control compounds induced statistically significant increases in the proportion of cells with micronuclei.
All acceptance criteria were considered met and the study was therefore accepted as valid.
3+21 hour treatment in the absence of S-9
Treatment of cells withBernel EsterDCMfor 3+21 hours in the absence of S-9 resulted in frequencies of MNBN cells that were similar to and not significantly (p≤0.05) higher than those observed in concurrent vehicle controls for three of the four concentrationsanalysed. A weak but statistically significant increase was observed at the highest concentration analysed (32.5 µg/mL, inducing 50% cytotoxicity). This increase was only marginally above the 95thpercentile of the observed (normal) range (mean frequency of 1.03% MNBN cells against a range of 0.2 to 1.0% MNBN cells) and fell within the current observed range (0.1 to 1.2% MNBN cells). Furthermore the increase was noted in a single replicate only at this concentration.
With the exception of a single replicate at an intermediate concentration (25 µg/mL, giving 18% cytotoxicity) all other treated cultures demonstrated MNBN cell frequencies within the normal range. A statistically significant linear trend (p≤0.05) was observed. The weak but statistically significant increase noted at 32.5 µg/mL was poorly reproduced between replicates and is therefore considered of questionable biological relevance.
3+21 hour treatment in the presence of S-9
Treatment of cells withBernel EsterDCMfor 3+21 hours in the presence of S-9 resulted in frequencies of MNBN cells which were similar to and not significantly (p≤0.05) higher than those observed in concurrent vehicle controls for all concentrationsanalysed. With the exception of a single culture at the lowest concentrationanalysed(10 µg/mL, giving 12% cytotoxicity) all test article treated cultures exhibited MNBN cell frequency that fell within the normal range. These data indicate a negative result.
24+24 hour treatment in the absence of S-9
Following 24+24 hour treatment in the absence of S-9, an increase in the frequency of MNBN cells, which was significantly (p≤0.05) higher than that observed in the concurrent vehicle controls, was observed at the highest concentration analysed (27.5 µg/mL). The observed increase exceeded the normal range in both replicate cultures and a statistically significant linear trend (p≤0.05) was observed, indicating a positive result under this treatment condition.
Conclusion
It is concluded thatBernel EsterDCMshowed evidence of micronucleus induction in cultured human peripheral blood lymphocytes following 24+24 hour treatment in the absence of an Aroclor-induced rat liver metabolic activation system (S‑9). In the same test system,Bernel EsterDCMdid not induce biologically relevant increases in micronuclei following 3+21 hour treatment in the absence and presence of S-9. Sporadic increases were noted following 3+21 hour treatment in the absence of S-9 but there was poor reproducibility between replicates and therefore this observation was considered of questionable biological relevance. Maximum concentrations analysed were limited by toxicity under all treatment conditions (in line with current regulatory test guidelines for thein vitromicronucleus assay).
- Endpoint:
- in vitro gene mutation study in mammalian cells
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- To be defined
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- in vitro mammalian cell micronucleus test
- Species / strain / cell type:
- lymphocytes:
- Metabolic activation:
- with and without
- Metabolic activation system:
- Aroclor 1254-induced rat liver post-mitochondrial fraction (S-9)
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- yes
- Positive controls:
- yes
- Positive control substance:
- 4-nitroquinoline-N-oxide
- benzo(a)pyrene
- Species / strain:
- lymphocytes:
- Metabolic activation:
- with and without
- Genotoxicity:
- not determined
- Cytotoxicity / choice of top concentrations:
- not determined
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- other: awaiting final result
Referenceopen allclose all
RESULTS
Selection of Concentrations for Micronucleus Analysis
The results of the RI determinations from the cytotoxicity Range-Finder Experiment were as follows:
Text Table 1: Data for 3+21 Hour Treatments -S-9, Range-Finder - Female Donors
Treatment (µg/mL) |
Replicate |
Mono |
Bi |
Multi |
Total |
RI |
Cytotoxicity Based on RI (%) |
Vehicle |
A |
35 |
158 |
7 |
200 |
0.86 |
|
|
B |
45 |
148 |
7 |
200 |
0.81 |
|
|
Total |
80 |
306 |
14 |
400 |
0.84 |
- |
UTC |
A |
55 |
140 |
5 |
200 |
0.75 |
- |
0.7256 |
A |
NSc |
- |
- |
- |
- |
- |
1.209 |
A |
NSc |
- |
- |
- |
- |
- |
2.016 |
A |
NSc |
- |
- |
- |
- |
- |
3.359 |
A |
NSc |
- |
- |
- |
- |
- |
5.599 |
A |
43 |
148 |
9 |
200 |
0.83 |
1 |
9.331 |
A |
47 |
147 |
6 |
200 |
0.80 |
5 |
15.55 |
A |
46 |
147 |
7 |
200 |
0.81 |
4 |
25.92 |
A |
78 |
122 |
0 |
200 |
0.61 |
27 |
43.20 |
A |
172 |
28 |
0 |
200 |
0.14 |
83 |
72.00 |
A |
194 |
6 |
0 |
200 |
0.03 |
96 P |
120.0 |
A |
184 |
16 |
0 |
200 |
0.08 |
90 P |
200.0 |
A |
186 |
14 |
0 |
200 |
0.07 |
92 P |
Text Table 2: Data for 3+21 Hour Treatments +S-9, Range-Finder - Female Donors
Treatment (µg/mL) |
Replicate |
Mono |
Bi |
Multi |
Total |
RI |
Cytotoxicity Based on RI (%) |
Vehicle |
A |
35 |
152 |
13 |
200 |
0.89 |
|
|
B |
30 |
155 |
15 |
200 |
0.93 |
|
|
Total |
65 |
307 |
28 |
400 |
0.91 |
- |
UTC |
A |
30 |
160 |
10 |
200 |
0.90 |
- |
0.7256 |
A |
NSc |
- |
- |
- |
- |
- |
1.209 |
A |
NSc |
- |
- |
- |
- |
- |
2.016 |
A |
NSc |
- |
- |
- |
- |
- |
3.359 |
A |
NSc |
- |
- |
- |
- |
- |
5.599 |
A |
28 |
153 |
19 |
200 |
0.96 |
0 |
9.331 |
A |
39 |
142 |
19 |
200 |
0.90 |
1 |
15.55 |
A |
40 |
152 |
8 |
200 |
0.84 |
7 |
25.92 |
A |
44 |
148 |
8 |
200 |
0.82 |
10 |
43.20 |
A |
56 |
141 |
3 |
200 |
0.74 |
19 |
72.00 |
A |
159 |
40 |
1 |
200 |
0.21 |
77 P |
120.0 |
A |
193 |
7 |
0 |
200 |
0.04 |
96 P |
200.0 |
A |
194 |
6 |
0 |
200 |
0.03 |
97 P |
UTC = Untreated control NSc = Not scored P = Precipitation observed at treatment
Mono = Mononucleate Bi = Binucleate Multi = Multinucleate
RI = Replication index
Text Table 3: Data for 24+24 Hour Treatments -S-9, Range-Finder - Female Donors
Treatment (µg/mL) |
Replicate |
Mono |
Bi |
Multi |
Total |
RI |
Cytotoxicity Based on RI (%) |
Vehicle |
A |
18 |
155 |
27 |
200 |
1.05 |
|
|
B |
19 |
155 |
26 |
200 |
1.04 |
|
|
Total |
37 |
310 |
53 |
400 |
1.04 |
- |
UTC |
A |
18 |
142 |
40 |
200 |
1.11 |
- |
0.7256 |
A |
14 |
155 |
31 |
200 |
1.09 |
0 |
1.209 |
A |
19 |
148 |
33 |
200 |
1.07 |
0 |
2.016 |
A |
23 |
155 |
22 |
200 |
1.00 |
4 |
3.359 |
A |
25 |
150 |
25 |
200 |
1.00 |
4 |
5.599 |
A |
38 |
154 |
22 |
214 |
0.93 |
11 |
9.331 |
A |
27 |
142 |
31 |
200 |
1.02 |
2 |
15.55 |
A |
61 |
129 |
10 |
200 |
0.75 |
28 |
25.92 |
A |
79 |
116 |
5 |
200 |
0.63 |
39 |
43.20 |
A |
178 |
22 |
0 |
200 |
0.11 |
89 |
72.00 |
A |
189 |
11 |
0 |
200 |
0.06 |
95P |
120.0 |
A |
NE |
- |
- |
- |
- |
-P |
200.0 |
A |
NE |
- |
- |
- |
- |
-P |
UTC = Untreated control
NE = Not evaluated - no scoreable cells
P = Precipitation observed at treatment
Mono = Mononucleate
Bi = Binucleate
Multi = Multinucleate
RI = Replication index
No marked changes in osmolality or pH were observed at the highest concentration tested (200 µg/mL) as compared to the concurrent vehicle controls (individual data not reported).
The results of the cytotoxicity Range-Finder Experiment were used to select suitable concentrations for the Micronucleus Experiment.
The results of the RI determinations from the Micronucleus Experiment were as follows:
Text Table 4: Data for 3+21 Hour Treatments -S-9, Micronucleus Experiment - Female Donors
Treatment (µg/mL) |
Replicate |
Mono |
Bi |
Multi |
Total |
RI |
Cytotoxicity Based on RI (%) |
Vehicle |
A |
177 |
316 |
7 |
500 |
0.66 |
|
|
B |
182 |
310 |
8 |
500 |
0.65 |
|
|
C |
163 |
328 |
9 |
500 |
0.69 |
|
|
D |
155 |
339 |
6 |
500 |
0.70 |
|
|
Total |
677 |
1293 |
30 |
2000 |
0.68 |
- |
UTC |
A |
163 |
317 |
20 |
500 |
0.71 |
|
|
B |
128 |
353 |
19 |
500 |
0.78 |
|
|
Total |
291 |
670 |
39 |
1000 |
0.75 |
- # |
5.000 |
A |
189 |
304 |
7 |
500 |
0.64 |
|
|
B |
136 |
357 |
7 |
500 |
0.74 |
|
|
Total |
325 |
661 |
14 |
1000 |
0.69 |
0 # |
10.00 |
A |
186 |
310 |
4 |
500 |
0.64 |
|
|
B |
171 |
322 |
7 |
500 |
0.67 |
|
|
Total |
357 |
632 |
11 |
1000 |
0.65 |
3 |
15.00 |
A |
179 |
316 |
5 |
500 |
0.65 |
|
|
B |
155 |
340 |
5 |
500 |
0.70 |
|
|
Total |
334 |
656 |
10 |
1000 |
0.68 |
0 |
20.00 |
A |
181 |
316 |
3 |
500 |
0.64 |
|
|
B |
169 |
331 |
0 |
500 |
0.66 |
|
|
Total |
350 |
647 |
3 |
1000 |
0.65 |
3 |
25.00 |
A |
243 |
255 |
2 |
500 |
0.52 |
|
|
B |
204 |
294 |
2 |
500 |
0.60 |
|
|
Total |
447 |
549 |
4 |
1000 |
0.56 |
18 # |
27.50 |
A |
260 |
239 |
1 |
500 |
0.48 |
|
|
B |
235 |
265 |
0 |
500 |
0.53 |
|
|
Total |
495 |
504 |
1 |
1000 |
0.51 |
25 |
30.00 |
A |
305 |
194 |
1 |
500 |
0.39 |
|
|
B |
324 |
175 |
1 |
500 |
0.35 |
|
|
Total |
629 |
369 |
2 |
1000 |
0.37 |
45 # |
32.50 |
A |
317 |
183 |
0 |
500 |
0.37 |
|
|
B |
347 |
153 |
0 |
500 |
0.31 |
|
|
Total |
664 |
336 |
0 |
1000 |
0.34 |
50 # |
35.00 |
A |
408 |
92 |
0 |
500 |
0.18 |
|
|
B |
359 |
141 |
0 |
500 |
0.28 |
|
|
Total |
767 |
233 |
0 |
1000 |
0.23 |
66 |
37.50 |
A |
445 |
55 |
0 |
500 |
0.11 |
|
|
B |
435 |
65 |
0 |
500 |
0.13 |
|
|
Total |
880 |
120 |
0 |
1000 |
0.12 |
82 |
40.00 |
A |
471 |
29 |
0 |
500 |
0.06 |
|
|
B |
441 |
59 |
0 |
500 |
0.12 |
|
|
Total |
912 |
88 |
0 |
1000 |
0.09 |
87 |
45.00 |
A |
486 |
14 |
0 |
500 |
0.03 |
|
|
B |
484 |
16 |
0 |
500 |
0.03 |
|
|
Total |
970 |
30 |
0 |
1000 |
0.03 |
96 |
60.00 |
A |
498 |
2 |
0 |
500 |
0.00 |
|
|
B |
497 |
3 |
0 |
500 |
0.01 |
|
|
Total |
995 |
5 |
0 |
1000 |
0.01 |
99 |
Table continued overleaf
Text Table 4 Continued: Data for 3+21 Hour Treatments -S-9, Micronucleus Experiment - Female Donors
Treatment (µg/mL) |
Replicate |
Mono |
Bi |
Multi |
Total |
RI |
Cytotoxicity Based on RI (%) |
MMC, 0.20 |
A |
220 |
280 |
0 |
500 |
0.56 |
|
|
B |
240 |
259 |
1 |
500 |
0.52 |
|
|
Total |
460 |
539 |
1 |
1000 |
0.54 |
20 |
MMC, 0.30 |
A |
285 |
215 |
0 |
500 |
0.43 |
|
|
B |
259 |
240 |
1 |
500 |
0.48 |
|
|
Total |
544 |
455 |
1 |
1000 |
0.46 |
32 # |
UTC = Untreated control
Mono = Mononucleate
Bi = Binucleate
Multi = Multinucleate
RI = Replication index
# Highlighted concentrations selected for analysis
Text Table 5: Data for 3+21 Hour Treatments +S-9, Micronucleus Experiment - Female Donors
Treatment (µg/mL) |
Replicate |
Mono |
Bi |
Multi |
Total |
RI |
Cytotoxicity Based on RI (%) |
Vehicle |
A |
79 |
372 |
49 |
500 |
0.94 |
|
|
B |
73 |
392 |
35 |
500 |
0.92 |
|
|
C |
77 |
388 |
35 |
500 |
0.92 |
|
|
D |
76 |
393 |
31 |
500 |
0.91 |
|
|
Total |
305 |
1545 |
150 |
2000 |
0.92 |
- |
UTC |
A |
130 |
339 |
31 |
500 |
0.80 |
|
|
B |
96 |
364 |
40 |
500 |
0.89 |
|
|
Total |
226 |
703 |
71 |
1000 |
0.85 |
- # |
10.00 |
A |
123 |
355 |
22 |
500 |
0.80 |
|
|
B |
115 |
361 |
24 |
500 |
0.82 |
|
|
Total |
238 |
716 |
46 |
1000 |
0.81 |
12 # |
20.00 |
A |
122 |
351 |
27 |
500 |
0.81 |
|
|
B |
102 |
378 |
20 |
500 |
0.84 |
|
|
Total |
224 |
729 |
47 |
1000 |
0.82 |
11 |
30.00 |
A |
138 |
341 |
21 |
500 |
0.77 |
|
|
B |
138 |
353 |
9 |
500 |
0.74 |
|
|
Total |
276 |
694 |
30 |
1000 |
0.75 |
18 |
35.00 |
A |
115 |
369 |
16 |
500 |
0.80 |
|
|
B |
141 |
342 |
17 |
500 |
0.75 |
|
|
Total |
256 |
711 |
33 |
1000 |
0.78 |
16 |
40.00 |
A |
141 |
355 |
4 |
500 |
0.73 |
|
|
B |
135 |
354 |
11 |
500 |
0.75 |
|
|
Total |
276 |
709 |
15 |
1000 |
0.74 |
20 |
45.00 |
A |
177 |
318 |
5 |
500 |
0.66 |
|
|
B |
177 |
321 |
2 |
500 |
0.65 |
|
|
Total |
354 |
639 |
7 |
1000 |
0.65 |
29 # |
50.00 |
A |
192 |
300 |
8 |
500 |
0.63 |
|
|
B |
147 |
349 |
4 |
500 |
0.71 |
|
|
Total |
339 |
649 |
12 |
1000 |
0.67 |
27 |
55.00 |
A |
225 |
274 |
1 |
500 |
0.55 |
|
|
B |
219 |
280 |
1 |
500 |
0.56 |
|
|
Total |
444 |
554 |
2 |
1000 |
0.56 |
40 |
60.00 |
A |
263 |
236 |
1 |
500 |
0.48 |
|
|
B |
255 |
241 |
4 |
500 |
0.50 |
|
|
Total |
518 |
477 |
5 |
1000 |
0.49 |
47 |
65.00 |
A |
316 |
184 |
0 |
500 |
0.37 |
|
|
B |
289 |
209 |
2 |
500 |
0.43 |
|
|
Total |
605 |
393 |
2 |
1000 |
0.40 |
57 # |
70.00 |
A |
388 |
112 |
0 |
500 |
0.22 |
|
|
B |
423 |
77 |
0 |
500 |
0.15 |
|
|
Total |
811 |
189 |
0 |
1000 |
0.19 |
80 |
80.00 |
A |
423 |
77 |
0 |
500 |
0.15 |
|
|
B |
433 |
67 |
0 |
500 |
0.13 |
|
|
Total |
856 |
144 |
0 |
1000 |
0.14 |
84 |
100.0 |
A |
487 |
13 |
0 |
500 |
0.03 |
|
|
B |
484 |
14 |
0 |
498 |
0.03 |
|
|
Total |
971 |
27 |
0 |
998 |
0.03 |
97 |
Table continued overleaf
Text Table 5 Continued: Data for 3+21 Hour Treatments +S-9, Micronucleus Experiment - Female Donors
Treatment (µg/mL) |
Replicate |
Mono |
Bi |
Multi |
Total |
RI |
Cytotoxicity Based on RI (%) |
CPA, 2.00 |
A |
150 |
348 |
2 |
500 |
0.70 |
|
|
B |
139 |
353 |
8 |
500 |
0.74 |
|
|
Total |
289 |
701 |
10 |
1000 |
0.72 |
22 |
CPA, 3.00 |
A |
143 |
350 |
7 |
500 |
0.73 |
|
|
B |
128 |
367 |
5 |
500 |
0.75 |
|
|
Total |
271 |
717 |
12 |
1000 |
0.74 |
20 # |
UTC = Untreated control
Mono = Mononucleate
Bi = Binucleate
Multi = Multinucleate
RI = Replication index
# Highlighted concentrations selected for analysis
Text Table 6: Data for 24+24 Hour Treatments -S-9, Micronucleus Experiment - Female Donors
Treatment (µg/mL) |
Replicate |
Mono |
Bi |
Multi |
Total |
RI |
Cytotoxicity Based on RI (%) |
Vehicle |
A |
134 |
331 |
35 |
500 |
0.80 |
|
|
B |
107 |
349 |
44 |
500 |
0.87 |
|
|
C |
105 |
353 |
42 |
500 |
0.87 |
|
|
D |
106 |
358 |
36 |
500 |
0.86 |
|
|
Total |
452 |
1391 |
157 |
2000 |
0.85 |
- |
UTC |
A |
83 |
377 |
40 |
500 |
0.91 |
|
|
B |
68 |
367 |
65 |
500 |
0.99 |
|
|
Total |
151 |
744 |
105 |
1000 |
0.95 |
- |
5.000 |
A |
104 |
371 |
25 |
500 |
0.84 |
|
|
B |
102 |
373 |
25 |
500 |
0.85 |
|
|
Total |
206 |
744 |
50 |
1000 |
0.84 |
1 |
10.00 |
A |
97 |
379 |
24 |
500 |
0.85 |
|
|
B |
71 |
397 |
32 |
500 |
0.92 |
|
|
Total |
168 |
776 |
56 |
1000 |
0.89 |
0 |
15.00 |
A |
115 |
361 |
24 |
500 |
0.82 |
|
|
B |
103 |
368 |
29 |
500 |
0.85 |
|
|
Total |
218 |
729 |
53 |
1000 |
0.84 |
2 # |
17.50 |
A |
125 |
362 |
13 |
500 |
0.78 |
|
|
B |
134 |
357 |
9 |
500 |
0.75 |
|
|
Total |
259 |
719 |
22 |
1000 |
0.76 |
10 |
20.00 |
A |
169 |
322 |
9 |
500 |
0.68 |
|
|
B |
131 |
358 |
11 |
500 |
0.76 |
|
|
Total |
300 |
680 |
20 |
1000 |
0.72 |
16 |
22.50 |
A |
162 |
332 |
6 |
500 |
0.69 |
|
|
B |
179 |
319 |
2 |
500 |
0.65 |
|
|
Total |
341 |
651 |
8 |
1000 |
0.67 |
22 # |
25.00 |
A |
259 |
238 |
3 |
500 |
0.49 |
|
|
B |
265 |
234 |
1 |
500 |
0.47 |
|
|
Total |
524 |
472 |
4 |
1000 |
0.48 |
44 |
27.50 |
A |
294 |
203 |
3 |
500 |
0.42 |
|
|
B |
271 |
227 |
2 |
500 |
0.46 |
|
|
Total |
565 |
430 |
5 |
1000 |
0.44 |
48 # |
30.00 |
A |
340 |
160 |
0 |
500 |
0.32 |
|
|
B |
384 |
115 |
1 |
500 |
0.23 |
|
|
Total |
724 |
275 |
1 |
1000 |
0.28 |
68 |
32.50 |
A |
400 |
100 |
0 |
500 |
0.20 |
|
|
B |
413 |
86 |
1 |
500 |
0.18 |
|
|
Total |
813 |
186 |
1 |
1000 |
0.19 |
78 |
35.00 |
A |
451 |
49 |
0 |
500 |
0.10 |
|
|
B |
404 |
96 |
0 |
500 |
0.19 |
|
|
Total |
855 |
145 |
0 |
1000 |
0.15 |
83 |
40.00 |
A |
472 |
28 |
0 |
500 |
0.06 |
|
|
B |
479 |
21 |
0 |
500 |
0.04 |
|
|
Total |
951 |
49 |
0 |
1000 |
0.05 |
94 |
Table continued overleaf
Text Table 6 Continued: Data for 24+24 Hour Treatments -S-9, Micronucleus Experiment - Female Donors
Treatment (µg/mL) |
Replicate |
Mono |
Bi |
Multi |
Total |
RI |
Cytotoxicity Based on RI (%) |
45.00 |
A |
475 |
25 |
0 |
500 |
0.05 |
|
|
B |
485 |
15 |
0 |
500 |
0.03 |
|
|
Total |
960 |
40 |
0 |
1000 |
0.04 |
95 |
60.00 |
A |
492 |
8 |
0 |
500 |
0.02 |
|
|
B |
493 |
7 |
0 |
500 |
0.01 |
|
|
Total |
985 |
15 |
0 |
1000 |
0.02 |
98 |
VIN, 0.04 |
A |
220 |
231 |
49 |
500 |
0.66 |
|
|
B |
207 |
240 |
53 |
500 |
0.69 |
|
|
Total |
427 |
471 |
102 |
1000 |
0.68 |
21 # |
VIN, 0.06 |
A |
287 |
168 |
45 |
500 |
0.52 |
|
|
B |
323 |
139 |
38 |
500 |
0.43 |
|
|
Total |
610 |
307 |
83 |
1000 |
0.47 |
45 |
UTC = Untreated control
Mono = Mononucleate
Bi = Binucleate
Multi = Multinucleate
RI = Replication index
# Highlighted concentrations selected for analysis
Micronucleus Analysis
Raw Data
The raw data for the observations on the test article plus positive and vehicle controls are retained by Covance Laboratories Ltd. A summary of the number of cells containing micronuclei is given inTable 8.1toTable 8.3.
Validity of Study
The data inTable 8.1toTable 8.6, Section7.3andText Table 4toText Table 6confirm that:
1. The binomial dispersion test demonstrated acceptable heterogeneity (in terms of MNBN cell frequency) between replicate cultures (Table 8.4 to Table 8.6)
2. The frequency of MNBN cells in vehicle controls fell within the normal ranges (Section 7.3)
3. The positive control chemicals induced statistically significant increases in the proportion of MNBN cells. Both replicate cultures at the positive control concentration analysed under each treatment condition demonstrated MNBN cell frequencies that clearly exceeded the normal range (Table 8.1 to Table 8.3)
4. A minimum of 50% of cells had gone through at least one cell division (as measured by binucleate + multinucleate cell counts) in vehicle control cultures at the time of harvest (Text Table 4 to Text Table 6)
5. The maximum concentration analysed under each treatment condition met the criteria specified in Section 4.7.
Analysis of Data
3+21 hour treatment in the absence of S-9
Treatment of cells with Bernel Ester DCM for 3+21 hours in the absence of S-9 resulted in frequencies of MNBN cells that were similar to and not significantly (p≤0.05) higher than those observed in concurrent vehicle controls for three of the four concentrations analysed (Table 8.1andTable 8.4) . A weak but statistically significant increase was observed at the highest concentration analysed (32.5 µg/mL, inducing 50% cytotoxicity). This increase was only marginally above the 95thpercentile of the observed (normal) range (mean frequency of 1.03% MNBN cells against a range of 0.2 to 1.0% MNBN cells) and fell within the current observed range (0.1 to 1.2% MNBN cells) (Section7.3). Furthermore the increase was noted in a single replicate only at this concentration.
With the exception of a single replicate at an intermediate concentration (25 µg/mL, giving 18% cytotoxicity) all other treated cultures demonstrated MNBN cell frequencies within the normal range. A statistically significant linear trend (p≤0.05) was observed. The weak but statistically significant increase noted at 32.5 µg/mL was poorly reproduced between replicates and is therefore considered of questionable biological relevance.
No test article related increases in cells with NPBs were observed (data not reported).
3+21 hour treatment in the presence of S-9
Treatment of cells with Bernel Ester DCM for 3+21 hours in the presence of S-9 resulted in frequencies of MNBN cells which were similar to and not significantly (p≤0.05) higher than those observed in concurrent vehicle controls for all concentrations analysed (Table 8.2andTable 8.5). With the exception of a single culture at the lowest concentration analysed (10 µg/mL, giving 12% cytotoxicity) all test article treated cultures exhibited MNBN cell frequency that fell within the normal range (Section7.3). These data indicate a negative result.
No test article related increases in cells with NPBs were observed (data not reported).
24+24 hour treatment in the absence of S-9
Following 24+24 hour treatment in the absence of S-9, an increase in the frequency of MNBN cells, which was significantly (p≤0.05) higher than that observed in the concurrent vehicle controls, was observed at the highest concentration analysed (27.5 µg/mL) (Table 8.3andTable 8.6). The observed increase exceeded the normal range in both replicate cultures and a statistically significant linear trend (p≤0.05) was observed, indicating a positive result under this treatment condition (Section7.3).
No test article related increases in cells with NPBs were observed (data not reported).
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Genetic toxicity in vivo
Endpoint conclusion
- Endpoint conclusion:
- no study available
Additional information
Justification for classification or non-classification
The registered substance did not induce mutations in the AMES test and the micronucleus study was considered to be inconclusive. It was therefore considered that the registered substance failed did not meet the criteria for classification as a mutagen under the Classification, Labelling, and Packaging (CLP) regulation (1272/2008).
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