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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

An in vitro gene mutation assay and an in vitro chromosome aberration test are available. Both key studies have a negative result.

Also the in vitro mammalian cell gene mutation test was negative.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study with GLP.
Qualifier:
according to guideline
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
Version / remarks:
. EC directive 92/69, method B.10., considering the draft of the update, DOC.ECB/TM/8. This is equivalent to 2000/32/EC, B.10.
Deviations:
no
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test
Species / strain / cell type:
mammalian cell line, other: Human lymphocytes.
Details on mammalian cell type (if applicable):
Whole blood was obtained from healthy males (aged 43 years for experiments "A" and 47 years for experiments "B") into Na-heparinized Vacutainers (Becton-Dickinson). The volunteers were non-smokers and were not suspected of any virus infection nor had been exposed to high levels of radiation or toxic chemicals. Primary cultures were established within one hour after venipuncture by adding 0.7 mL of whole blood to 9.3 mL of complete culture medium
Metabolic activation:
with and without
Metabolic activation system:
S-9 mix from Aroclor 1254 induced rat livers.
Test concentrations with justification for top dose:
Concentration range in the main test (with metabolic activation): 4 to 100 µg/mL.
Concentration range in the main test (without metabolic activation): 4 to 100 µg/mL.
The test substance is of low solubility in aqueous media. 0.1 mg test substance per mL of culture medium were the highest technically feasible concentration in medium.
Vehicle / solvent:
Culture medium: RPMI 1640 medium with L-glutamine.
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
cyclophosphamide
methylmethanesulfonate
Details on test system and experimental conditions:
Four experiments were performed: two of them without and two with the use of a metabolic activation system (liver microsomes from Aroclor 1254 induced rats, with a co-factor solution).
Primary lymphocyte cultures were established from whole blood freshly obtained from male donors. After 48 hours of incubation, the test substance was added.
In the experiments with the use of a metabolic activation system the test substance was washed out three hours later, and the cultures were cultivated for another 15 hours. Colcemid was added for 2 hours, and then cells were fixed and slides prepared.
For cultures without addition of a metabolic activation system, the treatment time was 3 hours in the first experiment and 20 hours in the second experiment.
For each concentration of the test substance two cultures were established.
Evaluation criteria:
Mitotic indices:
The mitotic indices were determined by counting a total of 2000 lymphocytes per cell culture and by recording the number of lymphocytes in any stage of mitosis.
Selection of cultures for analysis:
In all experiments the cultures with the highest test substance concentrations and with the next lower two concentrations were analysed. By analysing 3 consecutive concentrations, an about tenfold range of concentrations was covered in each experiment.
Coding of slides
Slides from the selected treatments were coded by assigning random numbers before analysing for aberrations. Self adhesive labels covered the identification marks on the slides. The code list was not available to the analysing persons until the last slide was analysed.
Chromosome analysis
ZEISS microscopes (Universal R and Photomicroscope III) were used for analysis (magnification factor of about 600 to 1000). For each culture evaluated 100 metaphases were analysed for structural and numerical chromosomal aberrations (except for cultures with obviously high numbers of metaphases with aberrations), i.e. 200 per concentration. Only well spread cells with 44 to 47 chromosomes, polyploid and endoreduplicated cells were acceptable for analysis.
Statistics:
The Chi2-Test (two-tailed, p=0.05) was used for the comparison between the negative control and the 7-AMCA cultures. If the results were positive, comparisons were made separately between the negative control and each concentration. If conditions for the Chi2-Test were not met, Fisher's Exact Test was used. Chi2-Test or Fisher's Exact Test were also used for the comparison between the negative and the positive controls.
The following parameters were evaluated:
• the number of metaphases with numerical aberrations ( < 46 chromosomes, > 46 chromosomes, tetraploidy, endoreduplication)
• the number of metaphases with structural aberrations, excluding gaps
• the number of chromatid-type aberrations, excluding gaps
• the number of chromosome-type aberrations, excluding gaps
• the number of gaps (chromatid- and isochromatid-type).
If there were more than one structural aberration of the same type within one metaphase, these aberrations were referred to as ,multiple". For statistical comparisons of the numbers of a specific aberration, such aberrations were counted as two aberrations.
A reproducible increase in the number of metaphases with aberrations or a concentration related increase in this number is considered as a positive result. However, a result can also be regarded as positive when other than merely statistical considerations, for example the kind of aberrations observed, are taken into account.
Species / strain:
lymphocytes: human
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
lymphocytes: human
Metabolic activation:
with
Genotoxicity:
not determined
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
lymphocytes: human
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
Concurrent positive controls: The mitotic indices were between 66.4 and 88.2 %of the negative controls. The positive control substances caused clearly higher numbers of metaphases with structural aberrations (statistically significant) than found in the negative controls, without as well as with the use of a metabolic activation system, thus demonstrating the validity of both experiments.
Additional statistically significant differences to the negative controls like the number of gaps, the number of chromatid-type aberrations, etc., are indicated in the attached Tables.

Mitotic index: 7-AMCA did not cause marked cytotoxicity in any experiment performed at any concentration tested. The mitotic indices of the test substance treated cultures were between 85.5 and 103.5 % of those of the corresponding negative controls.

Structural aberrations: No statistically significant increases in the number of metaphases with structural aberrations were noted at any concentration analysed compared to the concurrent negative controls, neither after treatment for 3 hours nor for 20 hours and neither without nor with the use of a metabolic activation system. All figures were within the range of historical negative controls.

Numerical aberrations: A statistically significantly higher number of metaphases with less than 46 chromosomes was noted in one experiment with a metabolic activation system at each tested concentration. This result was not confirmed by a second experiment with a metabolic activation system and was therefore regarded as a random event. There were no indications for a raise in numerical aberrations in the test substance treated cultures in any other experiment.

Gaps: No statistically significant increases in the number of gaps were noted at any concentration analysed compared to the concurrent negative controls, neither after treatment for 3 hours nor for 20 hours and neither without nor with the use of a metabolic activation system. All figures were within the range of historical negative controls.
Remarks on result:
other: other: treatment length 3 h.
Remarks:
Migrated from field 'Test system'.
Conclusions:
Interpretation of results (migrated information):
negative with metabolic activation
negative without metabolic activation

The test substance did not induce chromosomal aberrations in cultured human lymphocytes.
Executive summary:

Possible mutagenic properties of 7-AMCA were investigated by means of an in vitro mammalian chromosome aberration test in human lymphocytes, according to the EC-method B.10.

Methods

Four experiments were performed: two of them without and two with the use of a metabolic activation system (liver microsomes from Aroclor 1254 induced rats, with a co-factor solution).

Primary lymphocyte cultures were established from whole blood freshly obtained from male donors. After 48 hours of incubation, the test substance was added. In the experiments with the use of a metabolic activation system the test substance was washed out three hours later, and the cultures were cultivated for another 15 hours. Colcemid was added for 2 hours, and then cells were fixed and slides prepared. For cultures without addition of a metabolic activation system, the treatment time was 3 hours in the first experiment and 20 hours in the second experiment.

For each concentration of the test substance two cultures were established. One negative control (medium) and one positive control (methanesulfonic acid methyl ester (MMS) for cultures without metabolic activation system and cyclophosphamide (CP) for cultures with a metabolic activation system) were set up concurrently in each experiment.

The concentrations of 7-AMCA ranged from 0.004 to 0.1 mg/mL.

100 metaphases were analysed for structural and numerical chromosomal aberrations, i.e. 200 per concentration.

Results

Cytotoxicity:

7-AMCA did not cause marked cytotoxicity in any experiment.

Numerical aberrations, gaps:

There were no indications for a raise in numerical aberrations in the test substance treated cultures in any other experiment. Neither with nor without the use of a metabolic activation system a statistically significant increase in the number of gaps compared to the concurrent negative controls was noted at any of the concentrations analysed in any experiment performed.

Structural aberrations:

No statistically significant increases in the number of metaphases with structural aberrations were noted at any concentration analysed, neither after treatment for 3 hours nor for 20 hours and neither without nor with the use of a metabolic activation system. The figures were also within the range of historical negative controls.

Conclusion

7-AMCA did not cause any marked cytotoxic effect at any treatment. Under the conditions of this study there was no relevant evidence that 7-AMCA did induce chromosomal aberrations in cultured human lymphocytes. The conclusion is based on a lack of a statistically significant increase of metaphases with structural aberrations in any experiment and on a lack of a concentration-dependent increase of metaphases with structural aberrations.

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study with GLP.
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
Principles of method if other than guideline:
Direct plate incorporation method.
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Target gene:
Histidine
Species / strain / cell type:
S. typhimurium, other: TA97a, TA98, TA100, TA102, TA1535
Metabolic activation:
with and without
Metabolic activation system:
Microsomal fraction of rat liver, induced with Aroclor 1254.
Test concentrations with justification for top dose:
21 to 5000 µg/plate.
In a preliminary experiment the strain TA100 was mixed with phosphate buffer, the test substance solutions and top-agar and plated on standard agar. The growth of the bacterial lawn was recorded. At 5000 µg/plate a reduced number of revertant colonies was detected. 5000 µg per plate were therefore selected as the highest concentration for the main test.
Vehicle / solvent:
Na-bicarbonate solution.
The solubility of the test substance in water or suitable organic solvents is not sufficient for preparing a stock solution of 50 mg/mL (data from the sponsor). Therefore the test substance - which contains an acid group - was dissolved in a sodium-bicarbonate solution. 250 mg of the test substance were moistened with 0.9 mL deionised water. Then 4.1 mL of a 8 % solution of NaHCO3 were added. This resulted in a clear solution with a pH between 7.1 and 7.4. The amount of NaHCO3, which is necessary to dissolve this amount of 7-AMCA, was determined in preliminary experiments. According to the sponsor, the test substance is stable in solutions as long as the pH is not greater than 7.8. This solution was used for the highest concentration group. The solutions for the lower concentrations were prepared by subsequent dilution of one volume of the higher concentrated solution with two volumes of water.
Negative solvent / vehicle controls:
yes
Remarks:
water was used and not the bicarbonate solution, to avoid high pH.
Positive controls:
yes
Positive control substance:
7,12-dimethylbenzanthracene
sodium azide
other: 2-aminoanthracene; 1,8-dihydroxy-anthraquinone; 4-nitro-o-phenylenediamine; t-butyl-hydroperoxide; 2-nitrofluorene
Details on test system and experimental conditions:
The exposure was performed according to the 'Plate Incorporation Assay', in which bacteria, test substance (and microsomes) are in contact on the plate without preceding incubation in the liquid state. The number of viable cells in the overnight-cultures is in the range of 2 x 10^8 cells per mL.
The plates were incubated at 37 °C for 2 days and the growth of the bacterial background and the density of revertant colonies was determined.
The test substance was tested without as well as with an external metabolising system (S9-mix). The results were verified by a second, independent experiment. Three plate were used per concentration and per strain for the test substance; 6 for the negative control.
Evaluation criteria:
The plates with less than about 50 revertant colonies, i.e. the plates of TA98 and TAI535 with the exception of the positive controls, were counted visually by marking the colonies with a felt tipped pen. The other plates were photographed with a video camera and the picture files were scanned for colonies by a computer program.
The criteria for a positive result are:
A reproducible increase of the number of revertants to more than the following threshold values for at least one of the concentrations:
• For the strains with a low spontaneous revertant rate i.e. TA98 and TA1535: The 2.5 fold
of the amount of the spontaneous revertants.
• For the strains with a high spontaneous revertant rate i.e. TA97a, TA100 and TA102: The 1.67 fold of the amount of the spontaneous revertants.
These threshold values were derived from the variations in the control samples of the historic data of the Ames test.

Statistics:
Means and standard deviation were calculated for the number of mutants in every concentration group.

For comparison of the control group and the test substance groups the analysis of variance was used followed by the test of Scheffé. For comparison of the control group and the positive control group, the t-test was used. p = 0.05 was used as the significance level.
Species / strain:
S. typhimurium, other: TA97a, TA98, TA100, TA102, TA1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
The used strains of Salmonella typhimurium showed the expected genetic properties and were sensitive against several mutagenic chemicals.

All positive control substances increased the mutation frequency to more than the threshold values. As 2-aminoanthracene, 1,8-dihydroxy-anthraquinone and 7,12-dimethyl-benz[a]anthracene require metabolic activation for mutagenicity, the results of these substances demonstrate also the efficiency of the metabolising system.

Solubility
No precipitation of the test substance was seen in any of the concentration groups.

Toxicity
In the preliminary test with strain TA100 a reduced number of revertants was obtained at 5000 µg/plate. In the main test, reduced numbers of revertant colonies were observed in all strains in the higher concentrations. The bacterial background lawn was only affected in strain TA97a in the first experiment. Metabolic activation did not significantly change the toxicity.

Mutagenicity:
No increase in the number of mutants was observed in any of the tested bacterial strains at any of the tested concentrations. The addition of an external metabolising system did not change these results.

Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Conclusions:
The substance is not mutagenic in the Ames test with the strains of Salmonella typhimurium TA97a, TA98, TA100, TA102 and TA1535 up to the concentration of 5000 µg per plate.
Executive summary:

7-AMCA was tested for mutagenic activity with the Salmonella typhimurium reverse mutation test (Ames Test). The study was conducted in accordance with the OECD guideline 471 and directive 92/69/EEC, part B.14. The test substance, dissolved in a sodium hydrogencarbonate solution and diluted with water, was tested at concentrations ranging from 21 µg to 5000 µg per plate according to the "direct plate incorporation method" without external metabolisation as well as with an external metabolising system (S9-mix). As test system the bacterial strains Salmonella typhimurium TA97a, TA98, TA100, TA102 and TAI535 were used. Negative and positive controls were included. An independent repetition of the experiment was performed.

Results

Positive controls: All positive control groups showed significantly increased mutation frequencies which demonstrate the sensitivity of the test system.

Test substance:

Toxicity: In the preliminary test with strain TA100 a reduced number of revertants was obtained at 5000 µ/plate. In the main test, reduced numbers of revertant colonies were observed in all strains in the higher concentrations. The bacterial background lawn was only affected in strain TA97a in the first experiment.

Mutagenicity:

In none of the concentrations tested and with none of the strains used an increase of the mutation frequency to more than the threshold values (250 % of the controls for strains TA98 and TA1535, 167 % of the controls for strains TA97a, TA100 and TA102) was obtained. Metabolic activation did not change these results.

 

 

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2016
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)
Version / remarks:
2015
Deviations:
no
Principles of method if other than guideline:
Mutant cells deficient in Hprt enzyme activity in the HPRT test are resistant to the cytostatic effects of the purine analogue 6-thioguanine (TG). The Hprt proficient cells are sensitive to TG, which causes the inhibition of cellular metabolism and halts further cell division. Thus, mutant cells are able to proliferate in the presence of TG, whereas normal cells, which contain the Hprt enzyme, are not.
GLP compliance:
yes (incl. QA statement)
Type of assay:
other: HPRT test
Target gene:
Hypoxanthine-guanine phosphoribosyl transferase enzyme locus.
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):
CHO K1: Sub-line (K1) of Chinese hamster ovary cell line CHO
Lot. No.: 12G006
Supplier: ECACC (European Collection of Cell Cultures)

Each batch of frozen cells was purged of HPRT mutants and was free for mycoplasma infections. For each experiment the cells were thawed rapidly, the cells diluted in Ham's F12 medium containing 10 % foetal bovine serum and incubated at 37 °C in a humidified atmosphere of 5 % CO2 in air. Growing cells were subcultured in an appropriate number of flasks. The CHO K1 cells for this study were grown in Ham's F12 medium (F12-10) supplemented with 1 % Antibiotic-antimycotic solution (containing 10000 U/mL penicillin, 10 mg/mL streptomycin and 25 μg/mL amphotericin-B) and heat-inactivated bovine serum (final concentration 10 %). During the 5 treatments with the test item, solvent (negative control) and positive controls, the serum content was reduced to 5 % (F12-5). The selection medium for TG resistant mutants contained 3.4 μg/mL 6-thioguanine (6-TG) (EX-CELL® CD CHO Serum-Free Medium for CHO Cells-SEL).
Metabolic activation:
with and without
Metabolic activation system:
S9 mix of phenobarbital and β-naphthoflavone induced rat liver.
Test concentrations with justification for top dose:
5-hour treatment period without S9-mix: 125, 250, 500, 1000 and 2000 μg/mL
5-hour treatment period with S9-mix: 125, 250, 500, 1000 and 1250 μg/mL
The concentrations were chosen based on the cytotoxicity and the maximum recommended concentration.
Vehicle / solvent:
The test item was prepared in DMSO.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
7,12-dimethylbenzanthracene
ethylmethanesulphonate
Details on test system and experimental conditions:
A pre-test on toxicity was performed to select treatment concentrations for the mutation assay.

A 5-hour treatment in the presence and absence of S9-mix was performed. For the 5-hour treatment, 5 x10E6 cells were each placed in sterile dishes and incubated for approximately 24 hours before treatment at 37 °C in a humidified atmosphere of 5 % CO2. Duplicate cultures were used at each test item concentration, for negative (solvent) controls and the positive controls for treatment without and with S9-mix. On the day of treatment the culture medium of exponentially growing cell cultures were replaced with medium (F12-5) containing the test item. The exposure period was 5 hours. Following the exposure period the cells were washed with F12-5 medium and incubated in fresh F12-10 medium for 19 hours. After the 19-hour incubation period, cells were washed twice with F12-10 medium and suspended by treatment with trypsin-EDTA solution and counted using a Bürker chamber. Solubility of the test item in the cultures was assessed by the naked eye, at the beginning and end of treatment. In samples where sufficient cells survived, cell number was adjusted to 10E5 cells/mL. Throughout the expression period, cells were transferred to dishes for growth or diluted to be plated for survival.

The pH and osmolality of the negative (solvent) control and test item solutions were determined in Experiment 1 and Experiment 2.
Plating for survival: Following adjustment of the cultures to 10E5 cells/mL, samples from these cultures were diluted to 40 cells/mL.
A total of 5 mL (200 cells/dish) of the final concentration of each culture was plated into 3 parallel dishes (diameter is approx. 60 mm). The dishes were incubated at 37 °C in a humidified atmosphere of 5 % CO2 in air for 6 days for growing colonies. Then, colonies were fixed with methanol, stained with Giemsa and counted. Survivals were assessed by comparing the cloning efficiency of the test item treated groups to the negative (solvent) control.
Expression of the mutant phenotype: During the phenotypic expression period the cultures were subcultured. Aliquots of approximately 2x10E6 cells were taken on days 1, 3, 6, and evaluated on day 8.
Selection of the mutant phenotype: At the end of the expression period, cultures from each dose level were adjusted to 2 x 10E5 cells / dish ( 4 x five dishes) in selection medium (hypoxanthine Ham's F12-SEL medium) containing 3.4 μg/mL of thioguanine (6-TG).
Plating for viability: At the end of the expression period cell number in the samples were adjusted to 2 × 10E5 cells/mL. Cells were plated in 3 parallel dishes (diameter is approx. 60 mm) for a viability test as described in “Plating for Survival“ section for the survival test.
Fixation and staining of colonies: After the selection period, the colonies were fixed with methanol for five minutes, stained with Giemsa and counted for either mutant selection or cloning efficiency determination.
Rationale for test conditions:
See above.
Evaluation criteria:
Calculation of mutation frequency: The mutation frequency was calculated by dividing the total number of mutant colonies by the number of cells selected (10E6 cells: 5 plates at 2 x 10E5 cells/plate), corrected for the cloning efficiency of cells prior to mutant selection (viability), and was expressed as 6-TG resistant mutants per 10E6 clonable cells.
Key result
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Clear treatment solutions were obtained and no precipitation in the medium was noted at the test item concentrations used. No relevant changes in pH or osmolality were found after treatment with the test item.

On Day 1, there was very clear evidence of toxicity with the test item in presence of metabolic activation (S9 mix) when compared to the negative (solvent) controls, confirming the response seen in the dose selection cytotoxicity assays. The Day 8 cloning efficiency data indicate that in general the cells had recovered during the expression period.
In the absence of the metabolic activation, no cytotoxicity was observed after test item treatment up to the highest applied concentration, confirming the response seen in the dose selection cytotoxicity assays.
There were no significant differences between treatment and control groups and no dose-response relationships were noted. All values were within the range of the laboratory historical solvent control data and no dose-related increase was observed in any of the cultures.

The sensitivity of the tests and the efficacy of the S9 mix were demonstrated by large and statistically significant (p < 0.01) increases in mutation frequency in the positive control cultures with Ethyl methanesulfonate (1.0 μL/mL) and 7,12-Dimethyl benz[a]anthracene (20 μg/mL). The mutation frequencies of the positive and negative control cultures were consistent with the historical control data from the previous studies performed at this laboratory). Thus, the study is considered valid.
The osmolality and pH values of test item solutions did not show any significant alterations compared to the concurrent control groups in the Pre-test on Toxicity and Main Mutation Assay.
Conclusions:
The test item was not mutagenic in this in vitro mammalian cell gene mutation test.
Executive summary:

The test item, applied up to the maximum recommended concentration (2000 μg/mL) concentration without metabolic activation system and up to the cytotoxic concentration with metabolic activation system over a 5 hour treatment period did not induce statistically and biologically significant increases in mutant frequency over the background (negative solvent control).

It is concluded that the test item was not mutagenic in this in vitro mammalian cell gene mutation test performed with in Chinese hamster ovary cells.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vitro:

Justification for selection of genetic toxicity endpoint
The tests, required in REACH, were performed.

Justification for classification or non-classification

No classification is derived from the results.