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EC number: 812-111-9 | CAS number: 188959-24-2
- 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
- Biodegradation
- Bioaccumulation
- Transport and distribution
- 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 mutagenic activity of acetylxylan esterase has not been determined but another enzyme (Lipase, IUBMB 3.1.1.3) of comparable genotoxic profile has been analyzed and used as a source substance for read-across.
Lipase did not show mutagenic activity in the Ames assay and did not induce chromosomal aberrations in the in vitro mammalian chromosome aberration test performed with human lymphocytes. These results are also supported by an in vitro gene mutation studies in L5178Y mouse lymphoma cells.
Therefore, acetylxylan esterase is considered to be non mutagenic.
Link to relevant study records
- Endpoint:
- in vitro gene mutation study in bacteria
- Type of information:
- read-across from supporting substance (structural analogue or surrogate)
- Adequacy of study:
- other information
- Justification for type of information:
- According to ECHA Guidance Chapter R 7a: Endpoint specific guidance (version 6.0, July 2017), the following studies on genetic toxicity are required: In vitro gene mutation study in bacteria and one of the following, in vitro cytogenicity study in mammalian cells or an in vitro micronucleus study. In case these studies are both negative, an in vitro gene mutation study in mammalian cells is requested in addition.
The present target substance, Acetylxylan esterase IUBMB 3.1.1.72, has not been tested but another enzyme (Lipase, IUBMB 3.1.1.3) has been investigated in the Ames test and in the in vitro chromosome aberration test. Both tests have been performed according to current OECD guidelines, and in compliance with GLP. No evidence for genetic toxicity was observed. These results are also supported by read-across from an in vitro gene mutation studies in L5178Y mouse lymphoma cells performed on lipase.
The conclusion is that the target substance Acetylxylan esterase IUBMB 3.1.1.72 is considered to be non-mutagenic.
Justification for read-across: see attached justification. - Reason / purpose for cross-reference:
- read-across source
- Reason / purpose for cross-reference:
- read-across source
- Reason / purpose for cross-reference:
- read-across: supporting information
- Endpoint:
- in vitro gene mutation study in bacteria
- Type of information:
- experimental study
- Adequacy of study:
- key study
- 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:
- EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
- Deviations:
- no
- GLP compliance:
- yes
- Type of assay:
- bacterial reverse mutation assay
- Species / strain / cell type:
- S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
- Species / strain / cell type:
- E. coli WP2 uvr A
- Metabolic activation:
- with and without
- Metabolic activation system:
- Aroclor 1254 induced rat liver S9
- Test concentrations with justification for top dose:
- In the pre-experiment the concentration range of the test item was 3 - 5000 µg/plate. The pre-experiment is reported as Experiment I. Since no relevant toxic effects were observed, 5000 µg/plate was chosen as maximal concentration. Due to observed minor contamination at higher concentrations eight concentrations were tested in Experiment II.
The concentration range included two logarithmic decades. The following concentrations (based on the total protein for this test item) were tested in Experiment II: 3, 10, 33, 100, 333, 1000, 2500, and 5000 µg/plate. - Vehicle / solvent:
- Deionized water
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- Remarks:
- Deionized water
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- sodium azide
- methylmethanesulfonate
- other: 4-Nitro-o-phenylenediamine: TA 1537 and TA 98 without S9 Aminoanthracene (2-AA): TA 98, TA 100, TA 1535, TA 1537, and E. coli
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in agar (plate incorporation)
- Cell density at seeding (if applicable): 10E8-10E9 cells/mL
DURATION
- Preincubation period: 60 minutes
- Exposure duration: 48 hours
NUMBER OF REPLICATIONS: 3 plates per strain and dose, both with and without metabolic activation, and the same number of plates for the controls
DETERMINATION OF CYTOTOXICITY: Performed between 3 to 5000 µg/plate - Evaluation criteria:
- The assay is considered to be valid if all of the following criteria are met:
• The negative and positive control data are consistent with the historical control data for the testing laboratory
• The positive control data show marked increases over the concurrent negative control values
• The evaluation of the test data is not restricted by the loss of plates (e.g., through contamination).
A test item is considered as a mutagen if all of the following criteria are met:
• A biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA 98, TA 100, and WP2 uvrA) or thrice (strains TA 1535 and TA 1537) the colony count of the corresponding solvent control is observed.
• A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration.
• The increases are reproducible between replicate plates
• An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment. - Statistics:
- According to the OECD guideline 471, a statistical analysis of the data is not mandatory.
- Key result
- 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:
- not examined
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 1537
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 98
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- In Experiment I, a minor reduction in the number of revertants (below the indication factor of 0.5), was observed in strain TA 98 at 5000 µg/plate with S9 mix.
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 100
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- Positive controls validity:
- valid
- Additional information on results:
- No toxic effects, evident as a reduction in the number of revertanst (below the indication factor of 0.5), occurred in the test groups with and without metabolic activation. only in experiment I a minor reduction in the number of revertants (below the indication factor of 0.5), was observed in strain TA98 at 5000 µg/plate with S9 mix.
No substantial increase in revertant colony numbers of any of the five testerr strains was observed following treatment with the test substance at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.
Appropriate reference mutagens were used as positive controls. They showed a distinct increase of induced revertant colonies.
In experiment I with S09 mix, the data in the negative control of strian WP2 uvrA were slightly above the test facility historical control range. Since the deviation is rather small, the effect is considered to be based upon biologically irrelevant fluctuations in the number of colonies. - Conclusions:
- The test substance is considered to be non-mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.
- Executive summary:
This study was performed to investigate the potential of the test substance to induce gene mutations in the plate incorporation test (Experiment I) and the pre-incubation test (Experiment II) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, and TA 100, and the Escherichia coli strain WP2 uvrA in accordance with OECD Test Guideline 471. The assay was performed in two independent experiments both with and without liver microsomal activation. Each concentration, including the controls, was tested in triplicate. The test item was tested at the following concentrations (based on the total protein for this test item): Pre-Experiment/Experiment I and II: 3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate.
The plates incubated with the test item showed normal background growth up to 5000 µg/plate with and without metabolic activation in both independent experiments. No toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred in the test groups with and without metabolic activation. Only in experiment I a minor reduction in the number of revertants (below the indication factor of 0.5), was observed in strain TA 98 at 5000 µg/plate with S9 mix. No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with the test substance at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance. Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies. In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test substance did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used. Therefore, the test substance is considered to be non-mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.
- Endpoint:
- in vitro cytogenicity / chromosome aberration study in mammalian cells
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- 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
- GLP compliance:
- yes
- Type of assay:
- other: Chromosome aberration test in human lymphocytes
- Species / strain / cell type:
- lymphocytes: Human
- Details on mammalian cell type (if applicable):
- Blood samples were obtained from healthy donors not receiving medication. For this study, blood was collected from a male donor (46 years old). Blood samples were drawn by venous puncture and collected in heparinized tubes. The tubes were sent to Harlan CCR to initiate cell cultures within 24 hours after blood collection. If necessary, the blood was stored before use at 4°C.
- Metabolic activation:
- with and without
- Metabolic activation system:
- Aroclor-1254 induced rat liver S9
- Test concentrations with justification for top dose:
- Experiment I (with and without S9): 32.5, 56.8, 99.5, 174.1, 304.6, 533.1, 932.9, 1632.7, 2857.1, 5000 µg/mL
Experiment II (without S9): 32.5, 56.8, 99.5, 174.1, 304.6, 533.1, 932.9, 1632.7, 2857.1, 5000 µg/mL
Experiment II (with S9): 533.1, 932.9, 1632.7, 2857.1, 5000 µg/mL
With respect to the current OECD Guideline 473 and protein content of the test item 5000 µg/mL was applied as the top concentration for the pre-test. No cytotoxic effects were observed after 4 hours treatment in the absence and presence of S9 mix. Therefore, the highest required concentration, 5000 µg/mL, was chosen as top treatment concentration for Experiment II. - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: Deionized water
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- Remarks:
- Deionized water
- True negative controls:
- not specified
- Positive controls:
- yes
- Positive control substance:
- cyclophosphamide
- ethylmethanesulphonate
- Details on test system and experimental conditions:
- Details on test system and conditions
METHOD OF APPLICATION: In medium
DURATION
Exposure time 4 hours: About 72 hours after seeding for each test group 2 blood cultures (10 mL each) were set up in parallel in 25 cm2 culture flasks. The culture medium was replaced with serum-free medium containing the test substance. For the treatment with metabolic activation 50 µL S9 mix per mL medium were used. Concurrent solvent and positive controls were performed. After 4 hours the cells were spun down by gentle centrifugation for 5 minutes. The supernatant with the dissolved test substance was discarded and the cells were re-suspended in “saline G.” The washing procedure was repeated once. After washing the cells were re-suspended in complete culture medium and cultured until preparation.
Exposure time 22 hours (without S9 mix): About 73 hours after seeding for each test group 2 blood cultures (10 mL each) were set up in parallel in 25 cm2 cell culture flasks. The culture medium was replaced with complete medium (with 10% FCS) containing the test substance without S9 mix. The culture medium at continuous treatment was not changed until preparation of the cells. Concurrent solvent and positive controls were performed.
All cultures were incubated at 37°C in a humidified atmosphere with 5.5% CO2 (94.5% air).
- Culture harvesting: Three hours before harvesting, colcemid was added to the cultured (final concentration of 0.2 μg/mL). The cultures were harvested by centrifugation 22 hours after beginning of treatment. The supernatant was discarded and the cells were re-suspended in approximately 5 mL of 0.0375 M KCl. The cell suspension was then allowed to stand at 37°C for 20 to 25 minutes. After removal of the hypotonic solution by centrifugation the cells were fixed with a mixture of methanol and glacial acetic acid (3 parts plus 1 part). At least 2 slides per experimental group were prepared by dropping the cell suspension onto a clean microscope slide. The cells for evaluation of cytogenetic damage were stained with Giemsa.
NUMBER OF REPLICATIONS: 2
SLIDE ANALYSIS: The slides were evaluated using NIKON microscopes with 100x oil emersion objectives. Breaks, fragments, deletions, exchanges, and chromosomal disintegrations were recorded as structural chromosome aberrations. Gaps were recorded as well, but they were not included in the calculation of aberration rates. 100 well-spread metaphase plates per culture were scored for cytogenetic damage on coded slides, except for the positive controls in Experiment I with metabolic activation, where only 50 metaphase plates were scored. Only metaphases with 46±1 centromer regions were included in the analysis. To describe a cytotoxic effect the mitotic index (% cells in mitosis) was determined. - Evaluation criteria:
- The chromosomal aberration assay is considered acceptable if it met the following criteria:
The number of aberrations found in the solvent controls falls within the range of historical laboratory control data range. The positive control substances should produce significant increases in the number of cells with structural chromosome aberrations, which are within the range of the laboratory’s historical control data.
A test item is classified as non-mutagenic if:
- the number of induced structural chromosome aberrations in all evaluated dose groups is in the range of our historical control data.
- no significant increase of the number of structural chromosome aberrations is observed.
A test item is classified as mutagenic if:
- the number of induced structural chromosome aberrations is not in the range of our historical control data.
and
- either a concentration-related or a significant increase of the number of structural chromosome aberrations is observed.
Although the inclusion of the structural chromosome aberrations is the purpose of this study, it is important to include the polyploids and endoreduplications. The following criteria is valid:
A test item can be classified as aneugenic if:
- the number of induced numerical aberrations is not in the range of our historical control data. - Statistics:
- Statistical significance was confirmed by means of the Fisher´s exact test (p < 0.05). However, both biological and statistical significance should be considered together. If the above mentioned criteria for the test item are not clearly met, the classification with regard to the historical data and the biological relevance is discussed and/or a confirmatory experiment is performed.
- Key result
- Species / strain:
- lymphocytes: Human
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- Positive controls validity:
- valid
- Additional information on results:
- No precipitation of the test substance in the culture medium was observed. No relevant increase in the osmolarity or pH value was observed (e.g. Exp. I: solvent control: 233 mOsm, pH 7.4 versus 278 mOsm and pH 7.4 at 5000 µg/mL. At both preparation intervals, in the absence as well as in the presence of S9 mix, no biologically relevant cytotoxity indicated by clearly reduced mitotic indices could be observed. In both experiments, in the absence and presence of S9 mix, no biologically relevant increase in the number of cells carry structural chromosomal aberrations was observed. The aberration rates of the cells after treatment with the test substance (0.0-2.0% aberrant cells, excluding gaps) were close to the range of the solvent control values (0.5-2.0% aberrant cells, excluding gaps) and within the range of the laboratory’s historical solvent control data (0.0-4.0% aberrant cells, excluding gaps). No evidence of an increase in polyploid metaphases was noticed after treatment with the test substance as compared to the control cultures. In both experiments, either EMA or CPA were used as positive controls and showed distinct increases in cells with structural chromosome aberrations.
- Conclusions:
- The test substance did not induce structural chromosomal aberrations in human lymphocytes in vitro when tested up to the highest required concentration.
- Executive summary:
The test substance, dissolved in deionized water, was assessed for its potential to induce chromosomal aberrations in human lymphocytes in vitro in the absence and presence of metabolic activation by S9 mix in accordance with OECD Test Guideline 473. Two independent experiments were performed. In Experiment I, the exposure period was 4 hours with and without S9 mix. In Experiment II, the exposure period was 4 hours with S9 mix and 22 hours without S9 mix. The chromosomes were prepared 22 hours (Exp. I & II) after start of treatment with the test substance. In each experimental group two parallel cultures were analyzed. 100 metaphase plates per culture were scored for structural chromosomal aberrations, except for the positive controls in Experiment I, in the presence of S9 mix, where 50 metaphases were scored. 1000 cells were counted per culture for determination of the mitotic index. With respect to the current OECD Guideline 473 and protein content of the test item 5000 µg/mL were applied as top concentration in this study.
In this study, no precipitation of the test item in the culture medium was observed. No relevant increase in the osmolarity or pH value was observed (e.g. Exp. I: solvent control: 233 mOsm, pH 7.4 versus 278 mOsm and pH 7.4 at 5000 µg/mL). At both preparation intervals, in the absence as well as in the presence of S9 mix, no biologically relevant cytotoxicity indicated by clearly reduced mitotic indices was observed. In both experiments, in the absence and presence of S9 mix, no biologically relevant increase in the number of cells carrying structural chromosomal aberrations was observed. The aberration rates of the cells after treatment with the test substance (0.0 - 2.0 % aberrant cells, excluding gaps) were close to the range of the solvent control values (0.5 – 2.0 % aberrant cells, excluding gaps) and within the range of the laboratory´s historical solvent control data (0.0 - 4.0 % aberrant cells, excluding gaps). No evidence of an increase in polyploid metaphases was noticed after treatment with the test substance as compared to the control cultures. In both experiments, either EMS (660 and 825 µg/mL) or CPA (22.5 µg/mL) were used as positive controls and showed distinct increases in cells with structural chromosome aberrations. In conclusion, it can be stated that under the experimental conditions reported, the test substance did not induce structural chromosomal aberrations in human lymphocytes in vitro when tested up to the highest required concentration.
- Endpoint:
- in vitro gene mutation study in mammalian cells
- Remarks:
- Type of genotoxicity: gene mutation
- Type of information:
- experimental study
- Adequacy of study:
- supporting study
- Study period:
- 14 June 1989 - 11 October 1989
- 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
- GLP compliance:
- yes
- Type of assay:
- mammalian cell gene mutation assay
- Target gene:
- HGPRT (6-thioguanine resistance)
- Species / strain / cell type:
- mouse lymphoma L5178Y cells
- Details on mammalian cell type (if applicable):
- - Type and identity of media: Fishers medium (10% and 20% horse serum)
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes - Metabolic activation:
- with and without
- Metabolic activation system:
- S9 mix
- Test concentrations with justification for top dose:
- The highest concentration tested was 5000 µg/mL, tested as supplied.
- Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: Distilled water
- Justification for choice of solvent/vehicle: Substance is water-soluble and any human exposure is in aqueous solutions. - Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- Positive controls:
- yes
- Positive control substance:
- other: 4-nitroquinoline-1-oxide (without S-9) and benzo(a)pyrene (with S-9)
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in medium, growth suspension. Selection phase was performed in microtitre plates.
DURATION
- Exposure duration: 2 hours
- Expression time (cells in growth medium): With exception of experiment 1 treatments in the absence of S-9 (where cultures were maintained for eight days) cultures were maintained for 7 days.
- Fixation time (start of exposure up to fixation or harvest of cells): At least 7 days after treatment.
SELECTION AGENT (mutation assays): 6-TG
NUMBER OF REPLICATIONS: duplicate
DETERMINATION OF CYTOTOXICITY
- Method: Cell density by counting viable cells, expressed as percentage relative survival (RS%) - Evaluation criteria:
- A test article was considered positive if:
- The assay was valid, and
- Significant induced mutation (i.e. the lower 95 percentile of a treated culture exceeded the upper 95 percentile of a control culture) occurred at consecutive doses in at least one experiment, and
- Dose-related increases in mutation could be confirmed by regression analysis in both experiments.
- Statistics:
- The mutation frequency was evaluated statistically by using logarithmic transformation of the variances of the number of clones observed on viability and mutation plates as described by E.E. Furth et al., Anal Biochem 110: 1-8, 1981
- Key result
- Species / strain:
- mouse lymphoma L5178Y cells
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- No Lipase treatment in the presence of S-9 in either experiment, or in the absence of S-9 in experiment 1, resulted in a statistically significant increase in mutation frequency. Experiment 2 treatments in the absence of S-9, did result in a small but statistically significant increase in mutation frequency. However, this significant increase was observed only at the lower dose plated for determination of 6-thioguanine resistance, and furthermore, showed no dose-correlation by linear-regression analysis. The data therefore cannot be considered evidence of mutation induction at the HGPRT locus of LS178Y mouse cells.
- Conclusions:
- It was concluded that Lipase had no mutagenic activity in the present test system.
- Executive summary:
Lipase was assayed for its ability to induce mutation at the HGPRT locus (6-thioguanine resistance) in mouse lymphoma cells using a fluctuation protocol. The study consisted of two independent experiments, each conducted in the absence and presence of metabolic activation (S-9 mix).
Following a wide range of treatments, separated by half-log intervals and reaching 5000 µg/mL, cells survived this dose of 5000 µg/mL (93% survival) in the absence and 500 µg/mL (11% survival) in the presence of S-9. This, together with the next three lower doses without S-9 and the next five lower doses with S-9, were plated for viability and 6-thioguanine resistance seven days after treatment (with the exception of experiment 1 treatments in the absence of S-9, plated after eight days). In the second experiment a narrower dose range was used to maximise the chance of detecting any lose related effects. The top doses plated in this experiment were 5000 µg/mL in the absence and 500 µg/mL in the presence of S-9, which yielded 104% and 5% survival, respectively.
Negative (solvent) and positive control treatments were included in each experiment in the absence and presence of S-9. Mutation frequencies in negative control cultures fell within normal ranges, and statistically significant increases in mutation were induced by the positive control chemicals 4-nitroquinoline-1-oxide (without S-9) and benzo(a)pyrene (with S-9). Therefore the study was accepted as valid.
No Lipase treatment in the presence of S-9 in either experiment, or in the absence of S-9 in experiment 1, resulted in a statistically significant increase in mutation frequency. Experiment 2 treatments in the absence of S-9, did result in a small but statistically significant increase in mutation frequency. However, this significant increase was observed only at the lower dose plated for determination of 6-thioguanine resistance, and furthermore, showed no dose-correlation by linear-regression analysis. The data therefore cannot be considered evidence of mutation induction at the HGPRT locus of LS178Y mouse cells.
It was concluded that Lipase had no mutagenic activity in this test system.
Referenceopen allclose all
Table 1a - Summary of Results Pre-Experiment and Experiment I (Without activation)
Revertant Colony Counts (Mean ±SD)
Substance |
µg/plate |
TA 98 |
TA 100 |
TA 1535 |
TA 1537 |
WP2 uvrA |
Vehicle |
|
29 ± 2 |
155 ± 3 |
22 ± 3 |
12 ± 3 |
69 ± 7 |
Untreated |
|
35 ± 6 |
138 ± 10 |
19 ± 8 |
11 ± 5 |
68 ± 4 |
TS |
3 |
32 ± 3 |
155 ± 8 |
18 ± 10 |
13 ± 4 |
77 ± 5 |
TS |
10 |
35 ± 7 |
145 ± 2 |
22 ± 3 |
14 ± 1 |
80 ± 5 |
TS |
33 |
36 ± 1 |
139 ± 7 |
23 ± 4 |
12 ± 2 |
73 ± 4 |
TS |
100 |
37 ± 7 |
155 ± 19 |
23 ± 6 |
13 ± 3 |
72 ± 9 |
TS |
333 |
29 ± 1 |
150 ± 2 |
20 ± 3 |
15 ± 3 |
71 ± 6 |
TS |
1000 |
38 ± 7 |
154 ± 8 |
21 ± 4 |
13 ± 6 |
65 ± 8 |
TS |
2500 |
21 ± 4* |
111 ± 8* |
15 ± 4* |
7 ± 2* |
53 ± 5* |
TS |
5000 |
14 ± 1* |
106 ± 5* |
16 ± 2* |
7 ± 3* |
55 ± 6* |
NaN3 |
10 |
|
2161 ± 7 |
2141 ± 41 |
|
|
4-NOPD |
10 |
306 ± 13 |
|
|
|
|
4-NOPD |
50 |
|
|
|
94 ± 3 |
|
MMS |
3.0 µL |
|
|
|
|
1541 ± 141 |
TS = Test substance
* = Contaminated and Manual count
Table 1b - Summary of Results Pre-Experiment and Experiment I (With activation)
Revertant Colony Counts (Mean ±SD)
Substance |
µg/plate |
TA 98 |
TA 100 |
TA 1535 |
TA 1537 |
WP2 uvrA |
Vehicle |
0 |
39 ± 6 |
170 ± 11 |
26 ± 7 |
12 ± 3 |
79 ± 6 |
Untreated |
|
31 ± 5 |
172 ± 2 |
22 ± 6 |
16 ± 6 |
87 ± 6 |
TS |
3 |
39 ± 6 |
164 ± 8 |
23 ± 9 |
15 ± 2 |
84 ± 11 |
TS |
10 |
38 ± 3 |
146 ± 17 |
17 ± 4 |
13 ± 1 |
76 ± 5 |
TS |
33 |
40 ± 3 |
167 ± 4 |
20 ± 1 |
13 ± 3 |
87 ± 7 |
TS |
100 |
40 ± 2 |
161 ± 2 |
22 ± 3 |
14 ± 2 |
72 ± 8 |
TS |
333 |
34 ± 2 |
161 ± 17 |
25 ± 8 |
11 ± 4 |
77 ± 8 |
TS |
1000 |
38 ± 5 |
149 ± 9 |
21 ± 7 |
16 ± 3 |
76 ± 4 |
TS |
2500 |
23 ± 4* |
140 ± 7* |
16 ± 5* |
11 ± 1* |
57 ± 7* |
TS |
5000 |
14 ± 4* |
117 ± 6* |
18 ± 4* |
13 ± 2* |
71 ± 7* |
2-AA |
2.5 |
1363 ± 198 |
1368 ± 100 |
189 ± 22 |
155 ± 12 |
|
2-AA |
10 |
|
|
|
|
219 ± 3 |
TS = Test substance
* = Contaminated and Manual count
Table 2a - Summary of Experiment II (Without activation)
Revertant Colony Counts (Mean ±SD)
Substance |
µg/plate |
TA 98 |
TA 100 |
TA 1535 |
TA 1537 |
WP2 uvrA |
Vehicle |
0 |
34 ± 9 |
127 ± 13 |
21 ± 2 |
13 ± 4 |
46 ± 9 |
Untreated |
|
33 ± 8 |
142 ± 10 |
28 ± 9 |
16 ± 2 |
52 ± 5 |
TS |
3 |
30 ± 7 |
145 ± 15 |
29 ± 6 |
13 ± 3 |
52 ± 7 |
TS |
10 |
34 ± 5 |
143 ± 15 |
20 ± 5 |
13 ± 8 |
48 ± 8 |
TS |
33 |
36 ± 10 |
155 ± 32 |
23 ± 5 |
19 ± 7 |
55 ± 9 |
TS |
100 |
44 ± 11 |
140 ± 8 |
24 ± 4 |
16 ± 6 |
53 ± 3 |
TS |
333 |
29 ± 11 |
154 ± 33 |
29 ± 12 |
14 ± 1 |
48 ± 4 |
TS |
1000 |
34 ± 5 |
145 ± 14 |
24 ± 3 |
11 ± 2 |
51 ± 2 |
TS |
2500 |
31 ± 6 |
151 ± 17 |
31 ± 8 |
13 ± 4 |
67 ± 17 |
TS |
5000 |
39 ± 9 |
158 ± 18 |
29 ± 1 |
17 ± 1 |
59 ± 7 |
NaN3 |
10 |
|
1766 ± 85 |
1615 ± 160 |
|
|
4-NOPD |
10 |
354 ± 17 |
|
|
|
|
4-NOPD |
50 |
|
|
|
114 ± 23 |
|
MMS |
3.0µL |
|
|
|
|
1453 ± 79 |
TS = Test substance
Table 2b - Summary of Experiment II (With activation)
Revertant Colony Counts (Mean ±SD)
Substance |
µg/plate |
TA 98 |
TA 100 |
TA 1535 |
TA 1537 |
WP2 uvrA |
Vehicle |
|
40 ± 4 |
166 ± 8 |
38 ± 6 |
17 ± 5 |
63 ± 14 |
Untreated |
|
37 ± 4 |
156 ± 4 |
30 ± 11 |
18 ± 7 |
68 ± 11 |
TS |
3 |
42 ± 9 |
185 ± 27 |
38 ± 6 |
22 ± 4 |
67 ± 9 |
TS |
10 |
34 ± 14 |
161 ± 17 |
31 ± 4 |
22 ± 2 |
62 ± 6 |
TS |
33 |
43 ± 11 |
171 ± 1 |
28 ± 8 |
20 ± 3 |
68 ± 1 |
TS |
100 |
37 ± 8 |
160 ± 2 |
39 ± 15 |
23 ± 4 |
71 ± 6 |
TS |
333 |
45 ± 9 |
170 ± 15 |
42 ± 5 |
22 ± 10 |
61 ± 7 |
TS |
1000 |
43 ± 6 |
167 ± 21 |
30 ± 6 |
24 ± 3 |
72 ± 14 |
TS |
2500 |
46 ± 4 |
194 ± 26 |
43 ± 8 |
24 ± 3 |
83 ± 31 |
TS |
5000 |
50 ± 8 |
169 ± 17 |
43 ± 1 |
24 ± 1 |
70 ± 3 |
2-AA |
2.5 |
482 ± 20 |
828 ± 35 |
748 ± 95 |
127 ± 6 |
|
2-AA |
10 |
|
|
|
|
314 ± 21 |
TS = Test substance
Table 1a: Cytotoxicity of the test substance to the cultures of human lymphocytes (Experiment I; Without S9 mix)
Concentration (µg/mL) |
Exposure time |
Preparation interval |
Mitotic cells per 1000 cells* |
% of solvent controls |
Solvent control |
4 hours |
22 hours |
12.4 |
100 |
32.5 |
4 hours |
22 hours |
Not determined |
Not determined |
56.8 |
4 hours |
22 hours |
Not determined |
Not determined |
99.5 |
4 hours |
22 hours |
Not determined |
Not determined |
174.1 |
4 hours |
22 hours |
Not determined |
Not determined |
304.6 |
4 hours |
22 hours |
Not determined |
Not determined |
533.1 |
4 hours |
22 hours |
14.7 |
118.1 |
932.9 |
4 hours |
22 hours |
13.8 |
111.3 |
1632.7 |
4 hours |
22 hours |
13.1 |
105.6 |
2857.1 |
4 hours |
22 hours |
13.4 |
108.1 |
5000 |
4 hours |
22 hours |
13.9 |
112.1 |
Experimental groups evaluated for cytogenetic damage are shown in bold characters
* Mean value of two cultures in %
Table 1b: Cytotoxicity of the test substance to the cultures of human lymphocytes (Experiment I; With S9 mix)
Concentration (µg/mL) |
Exposure time |
Preparation interval |
Mitotic cells per 1000 cells* |
% of solvent controls |
Solvent control |
4 hours |
22 hours |
13.7 |
100 |
32.5 |
4 hours |
22 hours |
Not determined |
Not determined |
56.8 |
4 hours |
22 hours |
Not determined |
Not determined |
99.5 |
4 hours |
22 hours |
Not determined |
Not determined |
174.1 |
4 hours |
22 hours |
Not determined |
Not determined |
304.6 |
4 hours |
22 hours |
Not determined |
Not determined |
533.1 |
4 hours |
22 hours |
13.3 |
97.1 |
932.9 |
4 hours |
22 hours |
12.3 |
90.1 |
1632.7 |
4 hours |
22 hours |
14.2 |
104.0 |
2857.1 |
4 hours |
22 hours |
14.0 |
102.6 |
5000 |
4 hours |
22 hours |
15.4 |
112.5 |
Experimental groups evaluated for cytogenetic damage are shown in bold characters
* Mean value of two cultures in %
Table 2a: Cytotoxicity of the test substance to the cultures of human lymphocytes (Experiment II; Without S9 mix)
Concentration (µg/mL) |
Exposure time |
Preparation interval |
Mitotic cells per 1000 cells* |
% of solvent controls |
Solvent control |
4 hours |
22 hours |
16.0 |
100 |
32.5 |
4 hours |
22 hours |
Not determined |
Not determined |
56.8 |
4 hours |
22 hours |
Not determined |
Not determined |
99.5 |
4 hours |
22 hours |
Not determined |
Not determined |
174.1 |
4 hours |
22 hours |
Not determined |
Not determined |
304.6 |
4 hours |
22 hours |
Not determined |
Not determined |
533.1 |
4 hours |
22 hours |
16.4 |
102.5 |
932.9 |
4 hours |
22 hours |
15.1 |
94.4 |
1632.7 |
4 hours |
22 hours |
15.1 |
94.4 |
2857.1 |
4 hours |
22 hours |
11.4 |
70.0 |
5000 |
4 hours |
22 hours |
11.4 |
71.3 |
Experimental groups evaluated for cytogenetic damage are shown in bold characters
* Mean value of two cultures in %
Table 2b: Cytotoxicity of the test substance to the cultures of human lymphocytes (Experiment II; With S9 mix)
Concentration (µg/mL) |
Exposure time |
Preparation interval |
Mitotic cells per 1000 cells* |
% of solvent controls |
Solvent control |
4 hours |
22 hours |
14.4 |
100 |
533.1 |
4 hours |
22 hours |
13.9 |
96.5 |
932.9 |
4 hours |
22 hours |
15.0 |
104.2 |
1632.7 |
4 hours |
22 hours |
12.5 |
86.8 |
2857.1 |
4 hours |
22 hours |
14.4 |
100.3 |
5000 |
4 hours |
22 hours |
14.6 |
101.4 |
Experimental groups evaluated for cytogenetic damage are shown in bold characters
* Mean value of two cultures in %
Table 3: Structural chromosome aberrations Experiment I; preparation interval 22 hours with S9 mix: exposure period 4 hours
Aberrations
% Aberrant cells Gaps Chromatid type Chromosome type Other
Slide # |
Cells scored |
Incl. gaps* |
Excl. gaps* |
With exchanges |
g ig |
b f d ex |
ib if id cx |
ma cd |
Solvent control 1 2 1+2 |
100 100 200 |
2.0 |
1.5 |
0.0 |
1 0 0 0 1 0 |
1 0 0 0 1 0 0 0 2 0 0 0 |
0 0 0 0 0 1 0 0 0 1 0 0 |
0 0 0 0 0 0 |
Positive control: CPA 22.5 µg/mL 1 2 1+2 |
50** 50** 100 |
39.0 |
35.0 |
3.0 |
7 2 1 0 8 2 |
26 3 0 3 15 0 0 0 41 3 0 3 |
2 0 0 0 0 0 0 0 2 0 0 0 |
1 0 0 0 0 0 |
TS: 1632.7 µg/mL 1 2 1+2 |
100 100 200 |
1.5 |
1.5 |
0.0 |
0 0 0 0 0 0 |
2 0 0 0 1 0 0 0 3 0 0 0 |
0 0 0 0 0 0 0 0 0 0 0 0 |
0 0 0 0 0 0 |
TS: 2857.1 µg/mL 1 2 1+2 |
100 100 200 |
1.0 |
1.0 |
0.0 |
0 0 0 0 0 0 |
0 1 0 0 1 0 0 0 1 1 0 0 |
0 0 0 0 0 0 0 0 0 0 0 0 |
0 0 0 0 0 0 |
TS: 5000 µg/mL 1 2 1+2 |
100 100 200 |
1.0 |
0.5 |
0.0 |
0 0 1 0 1 0 |
1 0 0 0 0 0 0 0 1 0 0 0 |
0 0 0 0 0 0 0 0 0 0 0 0 |
0 0 0 0 0 0 |
* Inclusive cells carrying exchanges
** 50 metaphase plates per culture were evaluated due to strong clastogenic effects
TS = Test substance
Abbreviations
g = gap, ig = iso-gap (gaps are achromatic lesions of chromatid or chromosome type where no or only a minimal misalignment of chromosomal material is visible), b = break, ib = iso-break, f = fragment, if = iso- fragment, d = deletion, id = iso-deletion, ma = multiple aberration (= more than 4 events in one cell [excluding gaps]), ex = chromatid type exchange, cx = chromosome type exchange, cd = chromosomal disintegration (= pulverization)
Table 4: Structural chromosome aberrations Experiment I; preparation interval 22 hours without S9 mix: exposure period 4 hours
Aberrations
% Aberrant cells Gaps Chromatid type Chromosome type Other
Slide # |
Cells scored |
Incl. gaps* |
Excl. gaps* |
With exchanges |
g ig |
b f d ex |
ib if id cx |
ma cd |
Solvent control 1 2 1+2 |
100 100 200 |
2.5 |
2.0 |
0.0 |
0 0 1 0 1 0 |
1 1 0 0 2 0 0 0 3 1 0 0 |
0 0 0 0 0 0 0 0 0 0 0 0 |
0 0 0 0 0 0 |
Positive control (EMS 825 µg/mL) 1 2 1+2 |
100 100 200 |
12.0 |
11.5 |
1.0 |
1 0 1 0 2 0 |
9 2 0 2 8 0 0 0 17 2 0 2 |
0 3 0 0 1 0 0 0 1 3 0 0 |
0 0 0 0 0 0 |
TS: 1632.7 µg/mL 1 2 1+2 |
100 100 200 |
3.0 |
1.5 |
0.0 |
2 0 1 0 3 0 |
0 0 0 0 3 0 0 0 3 0 0 0 |
0 0 0 0 0 0 0 0 0 0 0 0 |
0 0 0 0 0 0 |
TS: 2857.1 µg/mL 1 2 1+2 |
100 100 200 |
0.0 |
0.0 |
0.0 |
0 0 0 0 0 0 |
0 0 0 0 0 0 0 0 0 0 0 0 |
0 0 0 0 0 0 0 0 0 0 0 0 |
0 0 0 0 0 0 |
TS: 5000 µg/mL 1 2 1+2 |
100 100 200 |
0.5 |
0.5 |
0.0 |
0 0 0 0 0 0 |
1 0 0 0 0 0 0 0 1 0 0 0 |
0 0 0 0 0 0 0 0 0 0 0 0 |
0 0 0 0 0 0 |
* Inclusive cells carrying exchanges
Table 5: Structural chromosome aberrations Experiment II; preparation interval 22 hours without S9 mix: exposure period 22 hours
Aberrations
% Aberrant cells Gaps Chromatid type Chromosome type Other
Slide # |
Cells scored |
Incl. gaps* |
Excl. gaps* |
With exchanges |
g ig |
b f d ex |
ib if id cx |
ma cd |
Solvent control 1 2 1+2 |
100 100 200 |
0.5 |
0.5 |
0.0 |
0 0 0 0 0 0 |
0 0 0 0 1 0 0 0 1 0 0 0 |
0 0 0 0 0 0 0 0 0 0 0 0 |
0 0 0 0 0 0 |
Positive control (EMS 660.0 µg/mL) 1 2 1+2 |
100 100 200 |
13.0 |
10.0 |
2.0 |
3 0 8 0 11 0 |
10 0 0 1 9 0 0 3 19 0 0 4 |
0 1 0 0 0 1 0 0 0 2 0 0 |
0 0 0 0 0 0 |
TS: 1632.7 µg/mL 1 2 1+2 |
100 100 200 |
1.5 |
1.5 |
0.0 |
0 0 0 0 0 0 |
1 0 0 0 1 0 0 0 2 0 0 0 |
0 0 0 0 0 1 0 0 0 1 0 0 |
0 0 0 0 0 0 |
TS: 2857.1 µg/mL 1 2 1+2 |
100 100 200 |
2.5 |
2.0 |
0.0 |
0 0 1 0 1 0 |
0 0 0 0 4 0 0 0 4 0 0 0 |
0 0 0 0 0 0 0 0 0 0 0 0 |
0 0 0 0 0 0 |
TS: 5000 µg/mL 1 2 1+2 |
100 100 200 |
1.5 |
0.5 |
0.0 |
1 0 1 0 2 0 |
0 0 0 0 1 0 0 0 1 0 0 0 |
0 0 0 0 0 0 0 0 0 0 0 0 |
0 0 0 0 0 0 |
* Inclusive cells carrying exchanges
Table 6: Structural chromosome aberrations Experiment II; preparation interval 22 hours with S9 mix: exposure period 4 hours
Aberrations
% Aberrant cells Gaps Chromatid type Chromosome type Other
Slide # |
Cells scored |
Incl. gaps* |
Excl. gaps* |
With exchanges |
g ig |
b f d ex |
ib if id cx |
ma cd |
Solvent control 1 2 1+2 |
100 100 200 |
1.0 |
1.0 |
0.0 |
0 0 0 0 0 0 |
1 0 0 0 1 0 0 0 2 0 0 0 |
0 0 0 0 0 1 0 0 0 1 0 0 |
0 0 0 0 0 0 |
Positive control (CPA 22.5 µg/mL) 1 2 1+2 |
100 100 200 |
14.0 |
13.5 |
2.5 |
3 0 1 0 4 0 |
10 2 0 6 18 2 0 0 28 4 0 6 |
0 0 0 0 0 0 0 0 0 0 0 0 |
0 0 0 0 0 0 |
TS: 1632.7 µg/mL 1 2 1+2 |
100 100 200 |
0.5 |
0.5 |
0.0 |
0 0 0 0 0 0 |
0 0 0 0 1 0 0 0 1 0 0 0 |
0 0 0 0 0 0 0 0 0 0 0 0 |
0 0 0 0 0 0 |
TS: 2857.1 µg/mL 1 2 1+2 |
100 100 200 |
0.0 |
0.0 |
0.0 |
0 0 0 0 0 0 |
0 0 0 0 0 0 0 0 0 0 0 0 |
0 0 0 0 0 0 0 0 0 0 0 0 |
0 0 0 0 0 0 |
TS: 5000 µg/mL 1 2 1+2 |
100 100 200 |
1.0 |
0.0 |
0.0 |
1 0 1 0 2 0 |
0 0 0 0 0 0 0 0 0 0 0 0 |
0 0 0 0 0 0 0 0 0 0 0 0 |
0 0 0 0 0 0 |
* Inclusive cells carrying exchanges
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Additional information
Justification for classification or non-classification
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