Registration Dossier
Registration Dossier
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EC number: 269-041-1 | CAS number: 68186-45-8
Endpoint summary
Administrative data
Key value for chemical safety assessment
Genetic toxicity in vitro
Description of key information
Based on the QSAR prediction for Ames test as well as results of the read across studies, the test substance is considered to be non-genotoxic, with and without metabolic activation.
Link to relevant study records
- Endpoint:
- in vitro gene mutation study in bacteria
- Type of information:
- experimental study
- Adequacy of study:
- supporting study
- Study period:
- From June 14, 2017 to July 17, 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)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- bacterial reverse mutation assay
- Species / strain / cell type:
- S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
- Additional strain / cell type characteristics:
- not specified
- Metabolic activation:
- with and without
- Metabolic activation system:
- Rat liver homogenate metabolizing system (10% liver S9 in standard co-factors)
- Test concentrations with justification for top dose:
- Experiment 1: 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate (Presence and absence of S9)
Confirmatory Experiment 1: 15, 50, 150, 300, 500, 750, 1500 µg/plate (Presence and absence of S9)
Experiment 2: 5, 15, 50, 150, 500, 1500, 5000 µg/plate (Presence and absence of S9)
Confirmatory Experiment 2: 15, 50, 150, 300, 500, 750, 1500 µg/plate (Presence and absence of S9)
The maximum concentration was 5000 µg/plate (the maximum recommended dose level). - Vehicle / solvent:
- The test substance was insoluble in sterile distilled water at 50 mg/mL but was fully soluble in dimethyl sulphoxide at the same concentration and acetone at 100 mg/mL in solubility checks performed in house, although the test substance came out of solution after standing at room temperature in acetone. Dimethyl sulphoxide was therefore selected as the vehicle.
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- Remarks:
- Identity: Dimethyl sulphoxide, Supplier: Fisher Scientific, Batch number (purity): 1690734 (>99%), Expiry: 03/2022 (Experiment 1) 1710280 (>99%), Expiry: 05/2022 (Experiment 2 and Confirmatory tests)
- Positive controls:
- yes
- Positive control substance:
- 4-nitroquinoline-N-oxide
- 9-aminoacridine
- N-ethyl-N-nitro-N-nitrosoguanidine
- benzo(a)pyrene
- other: Identity: 2-Aminoanthracene (2AA), CAS No.: 613-13-8, Batch number: STBB1901M9, Purity: 97.5%, Expiry date: 08 October 2017, Solvent: DMSO, Concentration: 1 µg/plate for TA100, 2 µg/plate for TA1535 and TA1537 , 10 µg/plate for WP2uvrA
- Details on test system and experimental conditions:
- Bacteria
The five strains of bacteria used, and their mutations, are as follows:
1) Salmonella typhimurium
Strains - Genotype - Type of mutations indicated
TA1537 - his C 3076; rfa-; uvrB-: - frame shift
TA98 - his D 3052; rfa-; uvrB-; - R-factor
TA1535 - his G 46; rfa-; uvrB-: - base-pair substitution
TA100 - his G 46; rfa-; uvrB-;R-factor
2) Escherichia coli
Strain - Genotype - Type of mutations indicated
WP2uvrA - trp-; uvrA-: - base-pair substitution
All of the Salmonella strains are histidine dependent by virtue of a mutation through the histidine operon and are derived from S. typhimurium strain LT2 through mutations in the histidine locus. Additionally due to the "deep rough" (rfa-) mutation they possess a faulty lipopolysaccharide coat to the bacterial cell surface thus increasing the cell permeability to larger molecules. A further mutation, through the deletion of the uvrB- bio gene, causes an inactivation of the excision repair system and a dependence on exogenous biotin. In the strains TA98 and TA100, the R factor plasmid pKM101 enhances chemical and UV-induced mutagenesis via an increase in the error prone repair pathway. The plasmid also confers ampicillin resistance which acts as a convenient marker (Mortelmans and Zeiger, 2000). In addition to a mutation in the tryptophan operon, the E. coli tester strain contains a uvrA- DNA repair deficiency which enhances its sensitivity to some mutagenic compounds. This deficiency allows the strain to show enhanced mutability as the uvrA repair system would normally act to remove and repair the damaged section of the DNA molecule (Green and Muriel, 1976 and Mortelmans and Riccio, 2000).
The bacteria used in the test were obtained from:
1) University of California, Berkeley, on culture discs, on 04 August 1995.
2) British Industrial Biological Research Association, on a nutrient agar plate, on 17 August 1987.
All of the strains were stored at approximately -196 °C in a Statebourne liquid nitrogen freezer, model SXR 34. In this assay, overnight sub-cultures of the appropriate coded stock cultures were prepared in nutrient broth (Oxoid Limited; lot number 1865318 05/21) and incubated at 37 °C for approximately 10 h. Each culture was monitored spectrophotometrically for turbidity with titres determined by viable count analysis on nutrient agar plates. - Evaluation criteria:
- There are several criteria for determining a positive result. Any, one, or all of the following can be used to determine the overall result of the study:
1) A dose-related increase in mutant frequency over the dose range tested (De Serres and Shelby, 1979).
2) A reproducible increase at one or more concentrations.
3) Biological relevance against in-house historical control ranges.
4) Statistical analysis of data as determined by UKEMS (Mahon et al., 1989).
5) Fold increase greater than two times the concurrent solvent control for any tester strain (especially if accompanied by an out of historical range response (Cariello and Piegorsch, 1996)).
A test substance will be considered non-mutagenic (negative) in the test system if the above criteria are not met.
Although most experiments will give clear positive or negative results, in some instances the data generated will prohibit making a definite judgment about test substance activity. Results of this type will be reported as equivocal. - Statistics:
- Statistical significance was confirmed by using Dunnetts Regression Analysis (* = p < 0.05) for those values that indicate statistically significant increases in the frequency of revertant colonies compared to the concurrent solvent control.
- Key result
- Species / strain:
- other: S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2 uvr A
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Conclusions:
- Under study conditions, the test substance was considered to be non-mutagenic with and without metabolic activation.
- Executive summary:
An in vitro study was conducted to determine the genotoxic potential of the test substance, mono- and di- C8-10 PSE, DEA, according to the OECD Guideline 471 (Bacterial Reverse Mutation Test - Ames Test), EU Method B13/14 and the USA, EPA OCSPP harmonized Guideline, in compliance with GLP. In this study, Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA were treated with the test substance using both the Ames plate incorporation and pre-incubation methods at up to eight dose levels (i.e., 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate), in triplicate, both with and without the addition of a rat liver homogenate metabolizing system (10% liver S9 in standard co-factors). The dose range for Experiment 1 was pre-determined to range between 1.5 to 5000 µg/plate. The experiment was repeated on a separate day using fresh cultures of the bacterial strains and fresh test substance formulations. The dose range was amended (from 5 to 5000 µg/plate) following the results of Experiment 1. Seven test substance concentrations were selected in Experiment 2 in order to achieve both four non toxic dose levels and the potential toxic limit of the test substance following the change in test methodology. Two further experiments (Confirmatory Test 1 and Confirmatory Test 2) were performed using the pre-incubation method, in triplicate, in order to assess the reproducibility of non dose related increases in TA1535 revertant colony frequency, seen in both the absence and presence of S9, in Experiment 2. The vehicle (dimethyl sulphoxide) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated. The maximum dose level of the test substance in the first experiment was selected as the maximum recommended dose level of 5000 µg/plate. There was no visible reduction in the growth of the bacterial background lawn at any dose level, either in the presence or absence of metabolic activation (S9-mix), in the first mutation test (plate incorporation method). However, substantial reductions in revertant colony frequency were noted to all of the Salmonella strains at 5000 µg/plate in both the presence and absence of S9-mix. These results were not indicative of toxicity sufficiently severe enough to prevent the test substance being tested up to the maximum recommended dose level of 5000 µg/plate in Experiment. The test substance induced a much stronger toxic response in Experiment 2 after employing the pre-incubation modification with weakened bacterial background lawns initially noted in the absence of S9 mix from 500 µg/plate (TA1535 and TA1537), 1500 µg/plate (TA100) and at 5000 µg/plate (TA98). In the presence S9-mix, weakened bacterial background lawns were initially noted from 1500 µg/plate (TA1535 and TA1537) and at 5000 µg/plate (TA100). No toxicity was noted to Escherichia coli strain WP2uvrA at any test substance dose level in either the absence or presence of S9-mix or Salmonella strain TA98 dosed in the presence of S9. Further toxicity (weakened background lawns) were noted to TA1535 from 750 µg/plate in the additional confirmatory tests. The sensitivity of the bacterial tester strains to the toxicity of the test substance varied slightly between strain type, exposures with or without S9-mix and experimental methodology. No test substance precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix. No biologically relevant, reproducible or dose-related increases in revertant colony numbers over control counts were observed with any of the tester strains (in any of mutation or confirmation tests) following exposure to the test substance at any concentration, in either the presence or absence of metabolic activation (S9-mix). Under study conditions, the test substance was considered to be non-mutagenic in the Ames test, with and without metabolic activation (Envigo, 2017).
- Endpoint:
- in vitro cytogenicity / chromosome aberration study in mammalian cells
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- From July 29, 2011 to January 04, 2012
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- other: in vitro mammalian chromosome aberration test (migrated information)
- Target gene:
- Not applicable.
- Species / strain / cell type:
- lymphocytes: human
- Details on mammalian cell type (if applicable):
- For each experiment, sufficient whole blood was drawn from the peripheral circulation of a volunteer who had been previously screened for suitability. The volunteer had not been exposed to high levels of radiation or hazardous chemicals and had not knowingly recently suffered from a viral infection. The cell-cycle time for the lymphocytes from the donors used in this study was determined using BrdU (bromodeoxyuridine) incorporation to assess the number of first, second and third division metaphase cells and so calculate the average generation time (AGT). The average AGT for the regular donors used in this laboratory has been determined to be approximately 16 h under typical experimental exposure conditions. Cell Culture: Cells were grown in Eagle's minimal essential medium with HEPES buffer (MEM), supplemented "in-house" with L-glutamine, penicillin/streptomycin, amphotericin B and 10% foetal bovine serum (FBS), at 37°C with 5% CO2 in humidified air. The lymphocytes of fresh heparinised whole blood were stimulated to divide by the addition of phytohaemagglutinin (PHA).
- Additional strain / cell type characteristics:
- not applicable
- Metabolic activation:
- with and without
- Metabolic activation system:
- phenobarbitone and beta-naphthoflavone induced rat liver, S9
- Test concentrations with justification for top dose:
- Preliminary Toxicity Test (Cell Growth Inhibition Test): The dose range of test substance used was 19.53 to 5000 µg/mL
Experiment 1: 4 (20) h without S9: 0*,6.25, 12.5, 25, 50*, 75*, 100*, 150, 200 µg/mL, 4 (20) h with S9: 0*, 6.25, 12.5, 25*, 50*, 75*, 100*, 150, 200, µg/mL
Experiment 2: 24 h without S9: 0*,12.5, 25, 50*, 75*, 100*, 200 µg/mL, 4 (20) h with S9: 0*, 12.5, 25, 50*, 75*, 100*, 200, µg/mL
*Dose levels selected for metaphase analysis - Vehicle / solvent:
- Dimethyl sulphoxide
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- cyclophosphamide
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- mitomycin C
- Details on test system and experimental conditions:
- Methods of application: In medium
Duration- Pre-incubation period: 48 h Exposure duration:
Experiment 1 – 4 h with and without S9.
Experiment 2 – 24 h without S9, 4 h with S9.
Expression time (cells in growth medium): 20 h for 4 h exposure
Selection time (in incubation with a selective agent): Not applicable
Fixation time (start of exposure up to fixation or harvest of cells): 24 h
Spindle inhibitor (Cytogenetic assays): Demecolcine
Stain (for cytogenetic assays): When slides were dry they were stained in 5% giemsa for 5 minutes, rinsed, dried and cover slipped using mounting medium.
Number of replications: Duplicate cultures
Number of cells evaluated: 100/culture
Determination of cytotoxicity - Method
Mitotic index – A total of 2000 lymphocyte cell nuclei were counted and the number of cells in metaphase recorded and expressed as the mitotic index as a percentage of the vehicle control value.
Scoring of Chromosome Damage
Where possible the first 100 consecutive well-spread metaphases from each culture were counted, where there were approximately 30 to 50% of cells with aberrations, slide evaluation was terminated at 50 cells. If the cell had 44-48 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. Cells with chromosome aberrations were reviewed as necessary by a senior cytogeneticist prior to decoding the slides.
Other examinations
Determination of polyploidy: In addition, cells with 69 chromosomes or more were scored as polyploid cells and the incidence of polyploid cells (%) reported. - Evaluation criteria:
- A positive response was recorded for a particular treatment if the % cells with aberrations, excluding gaps, markedly exceeded that seen in the concurrent control, either with or without a clear dose-relationship. For modest increases in aberration frequency a dose response relationship is generally required and appropriate statistical tests may be applied in order to record a positive response.
- Statistics:
- The frequency of cells with aberrations excluding gaps and the frequency of polyploid cells was compared, where necessary, with the concurrent vehicle control value using Fisher's exact test.
- Key result
- Species / strain:
- lymphocytes: human
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- Refer to information on results and attached tables.
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Additional information on results:
- Results
Preliminary toxicity test (cell growth inhibition test): The mitotic index data are presented in Appendix 1 (5) and (6) (see attached background material - Appendix 1). The test substance exhibited clear evidence of dose-related toxicity in all three exposure groups. A precipitate was observed in the parallel blood-free cultures at the end of the exposure period initially at 156.25 µg/mL and 312.5 µg/mL without and with S9-mix, respectively. Microscopic assessment of the slides prepared from the treatment cultures showed that metaphase cells were present up to 78.13 µg/mL in the continuous exposure and 4(20) h with S9, whereas metaphase cells were present at 156.25 µg/mL in the 4(20) h without S9 exposure group. Therefore, the dose selection for Experiments 1 and 2 was based on toxicity in accordance with the OECD 473 test guideline.
Chromosome aberration test - Experiment 1
The dose levels of the controls and the test substance are given in the table below:
Group Final concentration of Esterification products of Phosphorus Pentoxide and Alcohols C6-C10 (Even numbered) (µg/mL)
4 (20) h without S9: 0*, 6.25, 12.5, 25, 50*, 75*, 100*, 150, 200, MMC 0.4*
4 (20) h with S9: 0*, 6.25, 12.5, 25*, 50*, 75*, 100*, 150, 200, CP 5**
Dose levels selected for metaphase analysis
MMC: Mitomycin CCP: Cyclophosphamide
The qualitative assessment of the slides determined that the toxicity was similar to that observed in the Preliminary Toxicity Test and that there were metaphases suitable for scoring present up to the test substance dose level of 100 µg/mL in the absence and presence of metabolic activation (S9). The results of the mitotic indices (MI) from the cultures after their respective treatments are presented in Form 1, Appendix 2 (see attached background material - Appendix 2). These data show an approximate 50% growth inhibition was achieved at 100 µg/mL in the presence of S9. However, in the absence of S9, an 18% growth inhibition was observed at 100 µg/mL but there were no metaphases present above this dose level. The selection of the maximum dose level for metaphase analysis was based on toxicity, and was 100 µg/mL both in the absence and presence of S9, because there were no metaphases present above this dose level. The chromosome aberration data are given in Form 1, Appendix 2 (see attached background material - Appendix 2). All of the vehicle control cultures had frequencies of cells with chromosome aberrations within the expected range. The positive control substances induced statistically significant increases in the frequency of cells with aberrations. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated. The test substance did not induce any statistically significant increases in the frequency of cells with aberrations in either the absence or presence of metabolic activation (S9). The polyploid cell frequency data are given in Form 1, Appendix 2. The test substance did not induce a statistically significant increase in the numbers of polyploid cells at any dose level in either of the exposure groups.
Chromosome aberration test - Experiment 2:
The dose levels of the controls and the test substance are given in the table below:
Group Final concentration of Esterification products of Phosphorus Pentoxide and Alcohols C6-C10 (Even numbered) (µg/mL)
24 h without S9: 0*, 12.5, 25, 50*, 75*, 100*, 200, MMC 0.2*
4 (20) h with S9: 0*, 12.5, 25, 50*, 75*, 100*, 200, CP 5**
Dose levels selected for metaphase analysis
MMC: Mitomycin CCP: Cyclophosphamide
The qualitative assessment of the slides determined that there were metaphases suitable for scoring present at the maximum test substance dose level of 100 µg/mL in the presence and absence of S9. The results of the mitotic indices (MI) from the cultures after their respective treatments are presented in Form 2, Appendix 2 (see attached background material - Appendix 2). These data show 29% and 21% growth inhibition was achieved at 100 µg/mL in the absence and presence of S9, respectively. The selection of the maximum dose level for metaphase analysis was similar to Experiment 1, and was based on toxicity. The chromosome aberration data are given in Form 2, Appendix 2 (see attached background material - Appendix 2). All of the vehicle control cultures had frequencies of cells with chromosome aberrations within the expected range. The positive control substances induced statistically significant increases in the frequency of cells with aberrations. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated. The test substance did not induce any statistically significant increases in the frequency of cells with chromosome aberrations in either the absence or presence of metabolic activation. The polyploid cell frequency data are given in Form 2, Appendix 2 (see attached background material - Appendix 2). The test substance did not induce a significant increase in the numbers of polyploid cells at any dose level in either of the exposure groups. - Conclusions:
- Under study conditions, the test substance was considered to be non-clastogenic to human lymphocytes in the chromosomal aberration assay, with and without metabolic activation
- Executive summary:
An in vitro study was conducted to determine the clastogenic potential of the test substance, mono-, di- and tri- C6-10 PSE, in cultured human lymphocytes, according to the OECD Guideline 473, EU Method B10, EPA OPPTS 870.5375, in compliance with GLP. Duplicate cultures of human lymphocytes, treated with the test substance, were evaluated for chromosome aberrations at up to four dose levels, together with vehicle and positive controls. Four treatment conditions were used for the study, i.e. In Experiment 1, a 4 h exposure in the presence of an induced rat liver homogenate metabolising system (S9), at a 2% final concentration with cell harvest after a 20 h expression period (tested at 0, 6.25, 12.5, 25, 50, 75, 100, 150, 200 µg/mL test concentrations) and a 4 h exposure in the absence of metabolic activation (S9) with a 20 h expression period (tested at: 0, 6.25, 12.5, 25, 50, 75, 100, 150, 200 µg/mL test concentrations). In Experiment 2, the 4 h exposure with addition of S9 was repeated (using a 1% final S9 concentration) (tested at: 0, 12.5, 25, 50, 75, 100, 200 µg/mL test concentrations, mitomycin C 0.2 µg/mL); whilst in the absence of metabolic activation the exposure time was increased to 24 h (tested at: 0, 12.5, 25, 50, 75, 100, 200 µg/mL test concentrations, cyclophosphamide 5 µg/mL). The dose levels used in the main experiments were selected using data from the preliminary toxicity test. All vehicle (solvent) control groups had frequencies of cells with aberrations within the range expected for normal human lymphocytes. All the positive control substances induced statistically significant increases in the frequency of cells with aberrations indicating that the sensitivity of the assay and the efficacy of the S9-mix were validated. The test substance did not induce any statistically significant increases in the frequency of cells with aberrations, in either of two separate experiments, using a dose range that included a dose level that induced or exceeded approximately 50% mitotic inhibition. Under study conditions, the test substance was considered to be non-clastogenic to human lymphocytes in the chromosomal aberration assay, with and without metabolic activation (XXXX, 2012).
- Endpoint:
- in vitro gene mutation study in mammalian cells
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- From August 11, 2011 to December 23, 2011
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
- 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:
- EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- other: in vitro mammalian cell gene mutation test using the Hprt and xprt genes (migrated information)
- Target gene:
- Hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus of Chinese hamster ovary (CHO) cells
- Species / strain / cell type:
- Chinese hamster Ovary (CHO)
- Details on mammalian cell type (if applicable):
- - Properly maintained: yes
- Periodically checked for Mycoplasma contamination:yes
- Periodically checked for karyotype stability: no
- Periodically "cleansed" against high spontaneous background: yes
Cell Line: The Chinese hamster ovary (CHO-K1) cell line was obtained from ECACC, Salisbury, Wiltshire.
Cell Culture: The stocks of cells were stored in liquid nitrogen at approximately -196°C. Cells were routinely cultured in Ham's 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: Cell stocks spontaneously mutate at a low but significant rate. Before the stocks of cells were frozen down they were cleansed of HPRT- mutants by culturing in HAT medium for 4 d. This is Ham's F12 growth 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). After 4 d in medium containing HAT, the cells were passaged into HAT-free medium and grown for 4 to 7 d. 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 applicable
- Metabolic activation:
- with and without
- Metabolic activation system:
- phenobarbitone/beta-naphthoflavone induced rat liver, S9
- Test concentrations with justification for top dose:
- Preliminary cytotoxicity test:
The preliminary cytotoxicity test was initially performed with a test substance dose range of 19.53 to 5000 µg/mL. However due to excessive toxicity seen in the initial experiment the test was repeated with modified ranges, 0.5 to 24 µg/mL in the absence of metabolic activation (S9) and 2 to 64 µg/mL in the presence of S9.
Mutagenicity test - Experiment 1
The dose levels of the controls and the test substance are given in the table below:
Group Final concentration of test substance (µg/mL) 4 h without S9: 0*, 0.5*, 1*, 2*, 4*, 6*, 8*, 12, EMS 750*
4 h with S9 (2%): 0*, 8*, 16*, 32*, 40*, 44*, 48, DMBA 0.5* and 1*
Mutagenicity test - Experiment 2
The dose levels of the controls and the test substance are given in the table below:
Group Final concentration of test substance (µg/mL) 24 h without S9: 0*, 1*, 2*, 4*, 6*, 8*, 10*, 12*, EMS 200* and 300*
4 h with S9 (1%): 0*, 2*, 4*, 8*, 16*, 32*, 40, 48, DMBA 0.5* and 1**
Dose levels plated for mutant frequency
EMS = Ethyl methanesulphonate
DMBA = Dimethyl benzanthracene - Vehicle / solvent:
- The test substance was considered to be a mixture and therefore the maximum recommended dose was 5000 µg/mL. The test substance was dissolved in DMSO as it formed a solution with the test substance suitable for dosing and appropriate dilutions made.
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- ethylmethanesulphonate
- other: Dimethyl benzanthracene
- Details on test system and experimental conditions:
- Method of application:
Plate assay using tissue culture flasks and 6-thioguanine (6-TG) as the selective agent.
Duration
- Exposure duration: 4 h (experiment 1 with and without S9 and experiment 2 with S9), 24 h (experiment 2 without S9).
- Expression time (cells in growth medium): 7 d.
Selection agent (mutation assays): 6-thioguanine (6-TG)
Number of replications: Duplicate culture
Determination of cytotoxicity
Cytotoxicity flasks were incubated for 7 d then fixed with methanol and stained with Giemsa. Colonies were manually counted and recorded to estimate cytotoxicity.
Other examinations:
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.
Assay acceptance criteria
An assay will normally be considered acceptable for the evaluation of the test results only if all the following criteria are satisfied. The with and without metabolic activation portions of mutation assays are usually performed concurrently, but each portion is, in fact, an independent assay with its own positive and negative controls.
Activation or non-activation assays will be repeated independently, as needed, to satisfy the acceptance criteria.
i) The average absolute cloning efficiency of negative controls should be between 70 and 115% with allowances being made for errors in cell counts and dilutions during cloning and assay variables. Assays in the 50 to 70% range may be accepted but this will be dependent on the scientific judgement of the Study Director. All assays below 50% cloning efficiency will be unacceptable.
ii) The background (spontaneous) mutant frequency of the vehicle controls are generally in the range of 0 to 25 x 10-6. The background values for with and without-activation segments of a test may vary even though the same stock populations of cells may be used for concurrent assays. Assays with backgrounds greater than 35 x 10-6 will not be used for the evaluation of a test substance.
iii) Assays will only be acceptable without positive control data (loss due to contamination or technical error) if the test substance clearly shows mutagenic activity. Negative or equivocal mutagenic responses by the test substance must have a positive control mutant frequency that is markedly elevated over the concurrent negative control.
iv) The test substance with little or no mutagenic activity, should include an acceptable assay where concentrations of the test substance have reduced the clonal survival to approximately 10 to 15% of the average of the negative controls, reached the maximum recommended dose (10 mM or 5 mg/mL) or twice the solubility limit of the test substance in culture medium. Where a test substance is excessively toxic, with a steep response curve, a concentration that is at least 75% of the toxic dose level should be used. There is no maximum toxicity requirement for test substances that are clearly mutagenic.
v) Mutant frequencies are normally derived from sets of five dishes for mutant colony count and three dishes for viable colony counts. To allow for contamination losses it is acceptable to score a minimum of four mutant selection dishes and two viability dishes.
vi) Five dose levels of test substance, in duplicate, in each assay will normally be assessed for mutant frequency. A minimum of four analysed duplicate dose levels is considered necessary in order to accept a single assay for evaluation of the test substance. - Evaluation criteria:
- Please see 'Assay Acceptance criteria', in details on test system and conditions section.
- Key result
- Species / strain:
- Chinese hamster Ovary (CHO)
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Remarks:
- The test substance demonstrated no significant increases in mutant frequency at any dose level, either with or without metabolic activation, in either the first or second experiment.
- 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 test:
A dose range of 19.53 to 5000 µg/mL was used initially in the preliminary cytotoxicity test but due to the excessive toxicity seen in this experiment the dose range was modified and the preliminary toxicity test repeated. The modified dose range was 0.5 to 24 µg/mL for the 24 h and 4 h exposure groups in the absence of S9 and 2 to 64 µg/mL for the 4 h exposure group in the presence of S9. The results of the individual flask counts and their analysis are presented in the attached Table 1 (see attached background material). It can be seen that there was a steep toxicity curve in all three exposure groups. There were no surviving colonies at and above 12 µg/mL in both the 4 h and the 24 h exposure groups in the absence of S9. In the 4 h exposure group in the presence of S9 the toxicity was less severe, probably due to the protective effects of the S9 with a reduction in cloning efficiency of 31% at 40 µg/mL when compared to the vehicle control. Cloudy precipitate was noted at the end of the exposure period in the 4 h exposure group in the presence of S9 at and above 40 µg/mL.
Mutagenicity test - experiment 1
The dose levels of the controls and the test substance are given below:
Group final concentration of test substance (µg/mL)
4 h without S9: 0*, 0.5*, 1*, 2*, 4*, 6*, 8*, 12, EMS 750*
4 h with S9 (2%): 0*, 8*, 16*, 32*, 40*, 44*, 48, DMBA 0.5* and 1*
A precipitate of the test substance was seen at the end of exposure at and above 40 µg/mL in the 4 h exposure group in the presence of S9 only. The Day 0 and Day 7 cloning efficiencies are presented in Table 2 and Table 3 (see attached background material). The Day 0 cloning efficiencies were comparable with those seen in the Preliminary Cytotoxicity Test. The dose level of 12 µg/mL in the 4 h exposure group in the absence of S9 was too toxic for Day 0 plating and the dose level of 48 µg/mL in the 4 hour exposure group in the presence of S9 was excluded from the Day 7 plating due to toxicity. The dose levels of 8 µg/mL in the absence of S9 and 44 µg/mL in the presence of S9 both had cloning efficiencies of less than 10% but were selected for plating as they provided an intermediate dose level in an otherwise steep toxicity curve. The Day 0 vehicle cloning efficiencies of the 4 h exposure group in the absence and presence of S9 did not achieve 70% but were considered to be acceptable as they did achieve at least 50% and there was no evidence of a positive response. The Day 7 viability, mutation frequency counts and mean mutation frequency per survivor values are presented in the attached Table 2 and Table 3 (see attached background material). There was no marked reduction in viability in the test substance dose levels plated out in either exposure group. There were no increases in mutation frequency per survivor which exceeded the vehicle control value by 20 x 10-6 with or without the presence of S9.It can be seen that the vehicle control values were all within the maximum upper limit of 25 x 10E-6 mutants per viable cell, and that the positive controls all gave marked increases in mutant frequency, indicating the test and the metabolic activation system were operating as expected.
Mutagenicity test - experiment 2
The dose levels of the controls and the test substance are given table below:
Group final concentration of test substance (µg/mL)
24 h without S9: 0*, 1*, 2*, 4*, 6*, 8*, 10*, 12*, EMS 200* and 300*
4 h with S9 (1%): 0*, 2*, 4*, 8*, 16*, 32*, 40, 48, DMBA 0.5* and 1*
A precipitate of the test substance was seen at the end of exposure in the 4 h exposure group in the presence of S9 at and above 40 µg/mL. The Day 0 and Day 7 cloning efficiencies are presented in the attached Tables 4 and 5 (see attached background material). It can be seen that the 24 h exposure group demonstrated similar toxicity at Day 0 to that seen in the Preliminary Cytotoxicity Test. The 4 h exposure group in the presence of S9 had a reduction in the Day 0 cloning efficiency of 83% at 32 µg/mL when compared to the vehicle control and the dose levels of 40 and 48 µg/mL were too toxic for plating at Day 0. The increased toxicity seen in the 4 h exposure group in the presence of S9 in Experiment 2 is considered to be due to the reduced S9 concentration. The 24 h exposure group demonstrated a reduction in cloning efficiency at Day 0 of 28% at 8 and 10 µg/mL when compared to the vehicle control group. The dose level of 12 µg/mL exceeded the 90% toxicity limit but was plated since it provided an intermediate dose level in the toxicity curve. The Day 0 vehicle control counts for the both the 4 h exposure group in the presence of S9 and the 24 h exposure group did not achieve 70% but were considered to be acceptable as they did achieve the 50% minimum required and there was no evidence of a positive response. The Day 7 viability counts, mutation frequency counts and mean mutation frequency per survivor values are presented in the attached Tables 4 and 5 (see attached background material). There was no marked reduction in viability in the test substance dose levels plated out in either exposure group There were no increases in mutation frequency per survivor which exceeded the vehicle control value by 20 x 10E-6 with or without the presence of S9.It can be seen that the vehicle control values were all within the maximum upper limit of 25 x 10E-6 mutants per viable cell in both Experiment 1 and Experiment 2, and that the positive controls all gave marked increases in mutant frequency, indicating the test and the metabolic activation system were operating as expected. - Conclusions:
- Under study conditions, the test substance was therefore considered to be non-mutagenic to CHO cells at the HPRT locus, with and without metabolic activation
- Executive summary:
An in vitro study was conducted to determine the mutagenic potential of the test substance, mono-, di- and tri- C6-10 PSE, on the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus of Chinese hamster ovary (CHO) cells, according to the OECD Guideline 476, EU Method B17 and US EPA OPPTS 870.5300, in compliance with GLP. The technique used was a plate assay using tissue culture flasks and 6-thioguanine (6TG) as the selective agent. Chinese hamster ovary (CHO) cells were treated with the test substance at up to seven dose levels, in duplicate, together with vehicle (solvent) and positive controls. Four treatment conditions were used for the test, i.e. In Experiment 1, a 4 h exposure in the presence of an induced rat liver homogenate metabolising system (S9), at a 2% final concentration (tested at: 0, 8, 16, 32, 40, 44, 48 µg/mL test concentrations) and a 4 h exposure in the absence of metabolic activation (S9) (tested at: 0, 0.5, 1, 2, 4, 6, 8, 12 µg/mL test concentrations). In Experiment 2, the 4 h exposure with addition of S9 was repeated (using a 1% final S9 concentration) (tested at: 0, 2, 4, 8, 16, 32, 40, 48 µg/mL test concentrations), whilst in the absence of metabolic activation the exposure time was increased to 24 h (tested at: 0, 1, 2, 4, 6, 8, 10, 12 µg/mL test concentrations). The dose ranges selected for Experiment 1 and Experiment 2 were based on the results of the preliminary cytotoxicity test. The vehicle (solvent) controls gave mutant frequencies within the range expected of CHO cells at the HPRT locus. The positive control treatments, both in the presence and absence of metabolic activation, gave significant increases in the mutant frequency indicating the satisfactory performance of the test and of the metabolising system. The test substance demonstrated no significant increases in mutant frequency at any dose level, either with or without metabolic activation, in either the first or second experiment. Under study conditions, the test substance was therefore considered to be non-mutagenic to CHO cells at the HPRT locus, with and without metabolic activation (XXXX, 2012).
Referenceopen allclose all
Results
Prior to use, the master strains were checked for characteristics, viability and spontaneous reversion rate (all were found to be satisfactory). The amino acid supplemented top agar and the S9-mix used in both experiments was shown to be sterile. The test substance formulation was also shown to be sterile. Results for the negative controls (spontaneous mutation rates) were considered to be acceptable. These data are for concurrent untreated control plates performed on the same day as the Mutation Test. The vehicle (sterile distilled water) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated. There was no visible reduction in the growth of the bacterial background lawn at any dose level, either in the presence or absence of metabolic activation (S9-mix), in the first mutation test (plate incorporation method). Similarly, there was no visible reduction in the growth of the bacterial background lawn at any dose level, either in the presence or absence of metabolic activation (S9-mix), in the second mutation test (pre-incubation method). No test substance precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix.
Experiment 1 (plate incorporation)
The maximum dose level of the test substance in the first experiment was selected as the maximum recommended dose level of 5000 µg/plate. In both the absence and presence of S9, there were no biologically relevant increases in the frequency of revertant colonies at any test substance dose level. In light of these results, Experiment 2 was conducted using the pre-incubation method.
Table 1: Test Results: Experiment 1 – Without Metabolic Activation (Plate Incorporation)
Test Period |
From:1 June 2017 |
To:4 June 2017 |
||||||||||
S9-Mix (-) |
Dose Level Per Plate |
Number of revertants (mean) +/- SD |
||||||||||
Base-pair substitution strains |
Frameshift strains |
|||||||||||
TA100 |
TA1535 |
WP2uvrA |
TA98 |
TA1537 |
||||||||
Solvent Control (DMSO) |
111 92 118 |
(107) 13.5# |
17 17 15 |
(16) 1.2 |
40 39 38 |
(39) 1.0 |
22 36 23 |
(27) 7.8 |
15 11 13 |
(13) 2.0 |
||
1.5 µg |
85 122 109 |
(105) 18.8 |
17 9 14 |
(13) 4.0 |
32 33 36 |
(34) 2.1 |
31 23 36 |
(30) 6.6 |
15 12 12 |
(13) 1.7 |
||
5 µg |
90 98 105 |
(98) 7.5 |
10 14 13 |
(12) 2.1 |
35 36 40 |
(37) 2.6 |
24 30 27 |
(27) 3.0 |
14 16 15 |
(15) 1.0 |
||
15 µg |
104 95 111 |
(103) 8.0 |
13 13 13 |
(13) 0.0 |
47 33 47 |
(42) 8.1 |
23 14 29 |
(22) 7.5 |
12 15 16 |
(14) 2.1 |
||
50 µg |
107 106 118 |
(110) 6.7 |
14 14 14 |
(14) 0.0 |
37 36 39 |
(37) 1.5 |
28 27 24 |
(26) 2.1 |
12 12 12 |
(12) 0.0 |
||
150 µg |
90 94 99 |
(94) 4.5 |
15 15 10 |
(13) 2.9 |
40 42 36 |
(39) 3.1 |
28 21 28 |
(26) 4.0 |
9 12 14 |
(12) 2.5 |
||
500 µg |
94 83 105 |
(94) 11.0 |
16 13 12 |
(14) 2.1 |
32 39 42 |
(38) 5.1 |
28 21 26 |
(25) 3.6 |
12 12 12 |
(12) 0.0 |
||
1500 µg |
100 93 78 |
(90) 11.2 |
12 12 12 |
(12) 0.0 |
34 33 38 |
(35) 2.6 |
17 18 18 |
(18) 0.6 |
7 12 7 |
(9) 2.9 |
||
5000 µg |
51 50 46 |
(49) 2.6 |
5 8 9 |
(7) 2.1 |
38 37 34 |
(36) 2.1 |
6 9 6 |
(7) 1.7 |
2 3 3 |
(3) 0.6 |
||
Positive controls S9-Mix (-) |
Name Dose Level No. of Revertants |
ENNG |
ENNG |
ENNG |
4NQO |
9AA |
||||||
3 µg |
5 µg |
2 µg |
0.2 µg |
80 µg |
||||||||
753 769 885 |
(802) 72.0 |
140 165 167 |
(157) 15.0 |
490 480 479 |
(483) 6.1 |
288 284 289 |
(287) 2.6 |
476 484 516 |
(492) 21.2 |
|||
Table 2: Test Results: Experiment 1 – With Metabolic Activation (Plate Incorporation)
Test Period |
From:1 June 2017 |
To:4 June 2017 |
||||||||||
S9-Mix (+) |
Dose Level Per Plate |
Number of revertants (mean) +/- SD |
||||||||||
Base-pair substitution strains |
Frameshift strains |
|||||||||||
TA100 |
TA1535 |
WP2uvrA |
TA98 |
TA1537 |
||||||||
Solvent Control (DMSO) |
139 131 141 |
(137) 5.3# |
16 8 13 |
(12) 4.0 |
51 38 48 |
(46) 6.8 |
31 29 22 |
(27) 4.7 |
16 12 12 |
(13) 2.3 |
||
1.5 µg |
120 146 141 |
(136) 13.8 |
13 9 8 |
(10) 2.6 |
52 43 36 |
(44) 8.0 |
28 22 31 |
(27) 4.6 |
14 12 16 |
(14) 2.0 |
||
5 µg |
134 139 130 |
(134) 4.5 |
11 10 8 |
(10) 1.5 |
44 49 44 |
(46) 2.9 |
20 25 17 |
(21) 4.0 |
12 13 10 |
(12) 1.5 |
||
15 µg |
131 143 142 |
(139) 6.7 |
15 12 10 |
(12) 2.5 |
55 51 49 |
(52) 3.1 |
28 25 20 |
(24) 4.0 |
12 13 12 |
(12) 0.6 |
||
50 µg |
124 140 138 |
(134) 8.7 |
10 16 9 |
(12) 3.8 |
47 42 51 |
(47) 4.5 |
27 26 17 |
(23) 5.5 |
12 14 12 |
(13) 1.2 |
||
150 µg |
131 129 144 |
(135) 8.1 |
8 12 13 |
(11) 2.6 |
50 44 45 |
(46) 3.2 |
16 23 23 |
(21) 4.0 |
12 15 12 |
(13) 1.7 |
||
500 µg |
143 138 141 |
(141) 2.5 |
15 15 13 |
(14) 1.2 |
47 46 34 |
(42) 7.2 |
16 19 23 |
(19) 3.5 |
11 12 8 |
(10) 2.1 |
||
1500 µg |
135 133 117 |
(128) 9.9 |
15 16 7 |
(13) 4.9 |
33 46 43 |
(41) 6.8 |
19 20 18 |
(19) 1.0 |
12 12 12 |
(12) 0.0 |
||
5000 µg |
51 36 19 |
(35) 16.0 |
6 4 5 |
(5) 1.0 |
43 49 47 |
(46) 3.1 |
12 12 10 |
(11) 1.2 |
2 3 0 |
(2) 1.5 |
||
Positive controls S9-Mix (+) |
Name Dose Level No. of Revertants |
2AA |
2AA |
2AA |
BP |
2AA |
||||||
1 µg |
2 µg |
10 µg |
5 µg |
2 µg |
||||||||
1089 1175 1451 |
(1238) 189.1 |
302 283 259 |
(281) 21.5 |
524 540 526 |
(530) 8.7 |
193 202 207 |
(201) 7.1 |
459 542 461 |
(487) 47.4 |
|||
Experiment 2 (pre-incubation)
The maximum dose level of the test substance in the second experiment was the same as for Experiment 1 (5000 µg/plate).In the absence of S9, there were statistically significant increases in the mean number of revertant colonies at 500 µg/plate with strain TA1535. This increase was considered to have no biological relevance because weakened bacterial background lawns were also noted. Therefore the response would be due to additional histidine being available to His-bacteria allowing these cells to undergo several additional cell divisions and presenting as non-revertant colonies.In the presence of S9, there were statistically significant increases in the mean number of revertant colonies at 15, 50, 150, 500 and 1500 µg/plate with strain TA1535 (counts at 150 and 500 µg/plate were in excess of twofold the concurrent vehicle control). The mean values at 150 and 500 µg/plate were also in excess of the maximum historical control value for the strain, therefore the results for TA1535 were investigated in further experiments. The counts at 1500 µg/plate were considered to have no biological relevance as weakened bacterial background lawns were also noted. These increases observed with TA1535 were considered to be of no biological relevance, as there was no clear dose-response relationship and, more importantly, the results could not be reproduced in two confirmatory tests discussed below, both of which contained extra intermediate test substance concentration levels, in an attempt to qualify the response.
There are no obvious reasons for these increases noted with the TA1535 culture. Many different sized colonies were noted at the statistically significant dose levels in this tester strain, which may confirm the possibility that there may have been underlying contamination present which was indistinguishable from the revertant colonies; however, the culture was considered valid on the day of scoring as the untreated and vehicle control counts were within the in-house untreated/vehicle historical control data.
Table 3: Test Results: Experiment – Without Metabolic Activation (Pre-Incubation)
Test Period |
From: 03 July 2017 |
To: 06 July 2017 |
||||||||||
S9-Mix (-) |
Dose Level Per Plate |
Number of revertants (mean) +/- SD |
||||||||||
Base-pair substitution strains |
Frameshift strains |
|||||||||||
TA100 |
TA1535 |
WP2uvrA |
TA98 |
TA1537 |
||||||||
Solvent Control (DMSO) |
80 84 61 |
(75) 12.3# |
16 29 20 |
(22) 6.7 |
22 15 20 |
(19) 3.6 |
17 11 9 |
(12) 4.2 |
24 14 16 |
(18) 5.3 |
||
5 µg |
95 61 78 |
(78) 17.0 |
19 15 24 |
(19) 4.5 |
26 18 24 |
(23) 4.2 |
12 12 18 |
(14) 3.5 |
21 13 21 |
(18) 4.6 |
||
15 µg |
66 69 85 |
(73) 10.2 |
29 27 25 |
(27) 2.0 |
22 27 24 |
(24) 2.5 |
17 17 17 |
(17) 0.0 |
8 8 18 |
(11) 5.8 |
||
50 µg |
75 71 96 |
(81) 13.4 |
20 18 19 |
(19) 1.0 |
21 17 21 |
(20) 2.3 |
12 19 12 |
(14) 4.0 |
7 8 15 |
(10) 4.4 |
||
150 µg |
62 65 79 |
(69) 9.1 |
28 26 40 |
(31) 7.6 |
23 21 22 |
(22) 1.0 |
15 17 11 |
(14) 3.1 |
11 10 13 |
(11) 1.5 |
||
500 µg |
70 65 61 |
(65) 4.5 |
24 S 125 S 121 S |
** (90) 57.2 |
13 25 23 |
(20) 6.4 |
9 12 16 |
(12) 3.5 |
6 S 8 S 7 S |
(7) 1.0 |
||
1500 µg |
54 S 43 S 39 S |
(45) 7.8 |
0 V 0 V 0 V |
(0) 0.0 |
16 16 22 |
(18) 3.5 |
11 6 11 |
(9) 2.9 |
0 V 0 V 0 V |
(0) 0.0 |
||
5000 µg |
25 S 29 S 26 S |
(27) 2.1 |
0 V 0 V 0 V |
(0) 0.0 |
19 14 29 |
(21) 7.6 |
6 S 11 S 5 S |
(7) 3.2 |
0 V 0 V 0 V |
(0) 0.0 |
||
Positive controls S9-Mix (-) |
Name Dose Level No. of Revertants |
ENNG |
ENNG |
ENNG |
4NQO |
9AA |
||||||
3 µg |
5 µg |
2 µg |
0.2 µg |
80 µg |
||||||||
432 432 485 |
(450) 30.6 |
508 539 646 |
(564) 72.4 |
395 373 535 |
(434) 87.9 |
217 249 197 |
(221) 26.2 |
441 466 440 |
(449) 14.7 |
|||
Table 4: Test Results: Experiment – With Metabolic Activation (Pre-Incubation)
|
|||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Experiment 3 (Confirmatory Test 1, (pre-incubation)
The dose range for this experiment was determined by the results of Experiment 2 in tester strain TA1535 (with S9-mix).More intermediate dose levels (300 and 750 µg/plate) were included in an effort to confirm and/or enhance the results obtained previously in Experiment 2. There were no biologically relevant increases in revertant colony frequency observed at any of the dose levels tested. Statistical values were noted at 750 and 1500 µg/plate, however these counts were considered to have no biological relevance as weakened bacterial background lawns were also noted.
Table 5: Test Results: Experiment 3 – (Confirmatory Test 1 – Pre-Incubation) With Metabolic Activation
Test Period |
From: 07 July 2017 |
To: 10 July 2017 |
||
Dose Level Per Plate |
Number of revertants (mean) +/- SD |
|||
Base-pair substitution strain |
||||
With S9-mix |
||||
TA1535 |
||||
Solvent Control (DMSO) |
12 15 13 |
(13) 1.5# |
||
15 µg |
17 15 13 |
(15) 2.0 |
||
50 µg |
10 10 12 |
(11) 1.2 |
||
150 µg |
15 13 19 |
(16) 3.1 |
||
300 µg |
12 15 17 |
(15) 2.5 |
||
500 µg |
10 15 12 |
(12) 2.5 |
||
750 µg |
36 S 54 S 47 S |
*** (46) 9.1 |
||
1500 µg |
333 S 284 S 334 S |
*** (317) 28.6 |
||
Positive controls S9-Mix (+) |
Name Dose Level No. of Revertants |
2AA |
||
2 µg |
||||
288 226 233 |
(249) 34.0 |
|||
Experiment 4 (Confirmatory Test, pre-incubation)
A second confirmatory test was performed in TA1535 (with S9-mix) in an effort to attain reproducibility between two conflicting results employing the same dose range as Experiment 3. Again, no increases in revertant colony frequency were observed at any dose level tested.
Table 6: Test Results: Experiment 4 – (Confirmatory Test – Pre-Incubation) With Metabolic Activation
Test Period |
From: 13 July 2017 |
To: 16 July 2017 |
||
Dose Level Per Plate |
Number of revertants (mean) +/- SD |
|||
Base-pair substitution strain |
||||
With S9-mix |
||||
TA1535 |
||||
Solvent Control (DMSO) |
10 22 12 |
(15) 6.4# |
||
15 µg |
14 13 14 |
(14) 0.6 |
||
50 µg |
17 15 15 |
(16) 1.2 |
||
150 µg |
13 17 4 |
(11) 6.7 |
||
300 µg |
7 13 10 |
(10) 3.0 |
||
500 µg |
11 14 8 |
(11) 3.0 |
||
750 µg |
11 S 8 S 11 S |
(10) 1.7 |
||
1500 µg |
16 S 11 S 15 S |
(14) 2.6 |
||
Positive controls S9-Mix (+) |
Name Dose Level No. of Revertants |
2AA |
||
2 µg |
||||
205 258 243 |
(235) 27.3 |
|||
ENNG : N-ethyl-N'-nitro-N-nitrosoguanidine
4NQO : 4-Nitroquinoline-1-oxide
9AA : 9-Aminoacridine
BP : Benzo(a)pyrene
2AA : 2-Aminoanthracene
S : Sparse bacterial background lawn
N/T : Not tested at this dose level
* : p≤0.05
** : p≤0.01
*** : p≤0.001
# : Standard deviation
Conclusion
No biologically relevant, reproducible or dose-related increases in revertant colony numbers were observed in any strain tested, after exposure to the test substance at up to 5000 µg/plate. It was, therefore, concluded that the test substance was non-mutagenic at concentrations up to 5000 μg/plate, in the absence or presence of metabolic activation under the conditions of this test.
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Genetic toxicity in vivo
Endpoint conclusion
- Endpoint conclusion:
- no study available
Additional information
Study 1: The Ames test results for the test substance, mono- and di- C8-10 PSE, were predicted using the Danish EPA MultiCASE commercial model A2H for Ames test prediction (MC4PC v. 2005). Since the test substance is a UVCB, the Ames test predictions were determined for the individual components, followed by overall assessment for the test substance. SMILES codes were used as the input parameter. The individual constituents were all predicted to be negative in the Ames test (Danish EPA, 2018). The predictions for all the constituents are considered to be reliable without restrictions, as they fall within of the applicability domain. Similar negative predictions were also obtained with the US EPA T.E.S.T v.4.2.1 QSAR model. Therefore, based on the Ames test predictions for the individual constituents, the test substance is considered to be not mutagenic.
Study 2: An in vitro study was conducted to determine the genotoxic potential of the read across substance, mono- and di- C8-10 PSE, DEA, according to the OECD Guideline 471 (Bacterial Reverse Mutation Test - Ames Test), EU Method B13/14 and the USA, EPA OCSPP harmonized Guideline, in compliance with GLP. In this study, Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA were treated with the test substance using both the Ames plate incorporation and pre-incubation methods at up to eight dose levels (i.e., 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate), in triplicate, both with and without the addition of a rat liver homogenate metabolizing system (10% liver S9 in standard co-factors). The dose range for Experiment 1 was pre-determined to range between 1.5 to 5000 µg/plate. The experiment was repeated on a separate day using fresh cultures of the bacterial strains and fresh test substance formulations. The dose range was amended (from 5 to 5000 µg/plate) following the results of Experiment 1. Seven test substance concentrations were selected in Experiment 2 in order to achieve both four non toxic dose levels and the potential toxic limit of the test substance following the change in test methodology. Two further experiments (Confirmatory Test 1 and Confirmatory Test 2) were performed using the pre-incubation method, in triplicate, in order to assess the reproducibility of non dose related increases in TA1535 revertant colony frequency, seen in both the absence and presence of S9, in Experiment 2. The vehicle (dimethyl sulphoxide) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated. The maximum dose level of the test substance in the first experiment was selected as the maximum recommended dose level of 5000 µg/plate. There was no visible reduction in the growth of the bacterial background lawn at any dose level, either in the presence or absence of metabolic activation (S9-mix), in the first mutation test (plate incorporation method). However, substantial reductions in revertant colony frequency were noted to all of the Salmonella strains at 5000 µg/plate in both the presence and absence of S9-mix. These results were not indicative of toxicity sufficiently severe enough to prevent the test substance being tested up to the maximum recommended dose level of 5000 µg/plate in Experiment. The test substance induced a much stronger toxic response in Experiment 2 after employing the pre-incubation modification with weakened bacterial background lawns initially noted in the absence of S9 mix from 500 µg/plate (TA1535 and TA1537), 1500 µg/plate (TA100) and at 5000 µg/plate (TA98). In the presence S9-mix, weakened bacterial background lawns were initially noted from 1500 µg/plate (TA1535 and TA1537) and at 5000 µg/plate (TA100). No toxicity was noted to Escherichia coli strain WP2uvrA at any test substance dose level in either the absence or presence of S9-mix or Salmonella strain TA98 dosed in the presence of S9. Further toxicity (weakened background lawns) were noted to TA1535 from 750 µg/plate in the additional confirmatory tests. The sensitivity of the bacterial tester strains to the toxicity of the test substance varied slightly between strain type, exposures with or without S9-mix and experimental methodology. No test substance precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix. No biologically relevant, reproducible or dose-related increases in revertant colony numbers over control counts were observed with any of the tester strains (in any of mutation or confirmation tests) following exposure to the test substance at any concentration, in either the presence or absence of metabolic activation (S9-mix). Under study conditions, the read across substance was considered to be non-mutagenic in the Ames test, with and without metabolic activation (Envigo, 2017). Based on the results of the read across study, the test substance, mono- and di- C8-10 PSE can also be considered to be non-mutagenic in the Ames test, with and without metabolic activation.
Study 3: An in vitro study was conducted to determine the clastogenic potential of the test substance, mono-, di- and tri- C6-10 PSE, in cultured human lymphocytes, according to the OECD Guideline 473, EU Method B10, EPA OPPTS 870.5375, in compliance with GLP. Duplicate cultures of human lymphocytes, treated with the test substance, were evaluated for chromosome aberrations at up to four dose levels, together with vehicle and positive controls. Four treatment conditions were used for the study, i.e. In Experiment 1, a 4 h exposure in the presence of an induced rat liver homogenate metabolising system (S9), at a 2% final concentration with cell harvest after a 20 h expression period (tested at 0, 6.25, 12.5, 25, 50, 75, 100, 150, 200 µg/mL test concentrations) and a 4 h exposure in the absence of metabolic activation (S9) with a 20 h expression period (tested at: 0, 6.25, 12.5, 25, 50, 75, 100, 150, 200 µg/mL test concentrations). In Experiment 2, the 4 h exposure with addition of S9 was repeated (using a 1% final S9 concentration) (tested at: 0, 12.5, 25, 50, 75, 100, 200 µg/mL test concentrations, mitomycin C 0.2 µg/mL); whilst in the absence of metabolic activation the exposure time was increased to 24 h (tested at: 0, 12.5, 25, 50, 75, 100, 200 µg/mL test concentrations, cyclophosphamide 5 µg/mL). The dose levels used in the main experiments were selected using data from the preliminary toxicity test. All vehicle (solvent) control groups had frequencies of cells with aberrations within the range expected for normal human lymphocytes. All the positive control substances induced statistically significant increases in the frequency of cells with aberrations indicating that the sensitivity of the assay and the efficacy of the S9-mix were validated. The test substance did not induce any statistically significant increases in the frequency of cells with aberrations, in either of two separate experiments, using a dose range that included a dose level that induced or exceeded approximately 50% mitotic inhibition. Under study conditions, the test substance was considered to be non-clastogenic to human lymphocytes in the chromosomal aberration assay, with and without metabolic activation (XXXX, 2012).Based on the results of the read across study, the test substance, similar non-clastogenic behaviour in the chromosomal aberration assay can be expected for the test substance,mono- and di- C8-10 PSE, with or without metabolic activation.
Study 4: An in vitro study was conducted to determine the mutagenic potential of the read across substance, mono-, di- and tri- C6-10 PSE, on thehypoxanthine-guanine phosphoribosyl transferase (HPRT) locus of Chinese hamster ovary (CHO) cells,according to the OECD Guideline 476, EU Method B17 and US EPA OPPTS 870.5300, in compliance with GLP.The technique used was a plate assay using tissue culture flasks and 6-thioguanine (6TG) as the selective agent.Chinese hamster ovary (CHO) cells were treated with the test substance at up to seven dose levels, in duplicate, together with vehicle (solvent) and positive controls. Four treatment conditions were used for the test, i.e. In Experiment 1, a 4 h exposure in the presence of an induced rat liver homogenate metabolising system (S9), at a 2% final concentration (tested at: 0, 8, 16, 32, 40, 44, 48 µg/mL test concentrations) and a 4 h exposure in the absence of metabolic activation (S9) (tested at: 0, 0.5, 1, 2, 4, 6, 8, 12 µg/mL test concentrations). In Experiment 2, the 4 h exposure with addition of S9 was repeated (using a 1% final S9 concentration) (tested at: 0, 2, 4, 8, 16, 32, 40, 48 µg/mL test concentrations), whilst in the absence of metabolic activation the exposure time was increased to 24 h (tested at: 0, 1, 2, 4, 6, 8, 10, 12 µg/mL test concentrations). The dose ranges selected for Experiment 1 and Experiment 2 were based on the results of the preliminary cytotoxicity test. The vehicle (solvent) controls gave mutant frequencies within the range expected of CHO cells at the HPRT locus. The positive control treatments, both in the presence and absence of metabolic activation, gave significant increases in the mutant frequency indicating the satisfactory performance of the test and of the metabolising system. The test substance demonstrated no significant increases in mutant frequency at any dose level, either with or without metabolic activation, in either the first or second experiment. Under study conditions, the read across substance was therefore considered to be non-mutagenic to CHO cells at the HPRT locus, with and without metabolic activation (XXXX, 2012). Based on the results of the read across study, a similar non-mutagenic behaviour in the HPRT assay can be expected for the test substance, mono- and di- C8-10 PSE, with or without metabolic activation.
Justification for classification or non-classification
Based on the negative predictions/results ofin vitroAmes, chromosomal aberration and/or HGPRT tests available with the read across substance, the test substance, mono- and di- C8-10 PSE, does not warrant classification for genotoxicity, according to the EU CLP criteria (Regulation 1272/2008/EC).
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