Hydroxyl-2-pyridone

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

1994-10-20 to 1994-11-30

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

The conclusions of this study are to be considered with the external review from CTL, as described in the section "overall remarks, attachements"

Data source

Reference

Reference 1

Reference Type:

study report

Title:

Unnamed

Year:

1995

Report date:

1995

Reference 2

Reference Type:

other: review of available toxicological data by external laboratory

Title:

Unnamed

Year:

1995

Report date:

1995

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

Original Guideline published in 1983 - 100 cells were scored per culture (200 per concentration), and percentage of mitotic inhibition for dose selection for metaphase evaluation appears to be too high comapred to current guideline

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

EEC Annex V Test B10

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: UK EMS Test Guidelines

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

other: in vitro mammalian chromosome aberration test

Test material

Test material information

Constituent 1

Reference substance name:

Hydroxyl-2-pyridone

EC Number:

212-506-0

EC Name:

Hydroxyl-2-pyridone

Cas Number:

822-89-9

Molecular formula:

C5H5NO2

IUPAC Name:

1-hydroxy-1,2-dihydropyridin-2-one

Test material form:

solid: particulate/powder

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 93110060
- Purity: 99.6%
- Date received: 1994-10-12

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature in the dark

TREATMENT OF TEST MATERIAL PRIOR TO TESTING: Test article solutions were prepared by dissolving the test item in culture medium to give the top doses described below.
The stock solutions were membrane filter-sterilized and dilutions made using sterile culture medium. The test article solutions were used within 2.5h of initial formulation.

OTHER SPECIFICS:
- Appearance: beige crystalline powder

Method

Species / strain

Species / strain / cell type:

lymphocytes: human

Details on mammalian cell type (if applicable):

not applicable

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254 induced rat liver post-mitochondrial fraction (S9)

Test concentrations with justification for top dose:

Dose range finding assay: 0, 9.722, 14.96, 23.01, 35.40, 54.46, 83.79, 128.9, 198.3, 305.1, 469.4, 722.2, 1111 µg/mL (top dose equivalent to 10 mM)

Experiment 1:
- 20h treatment +0h, without S9: 0, 9.722, 14.96, 23.01, 35.40, 54.46, 83.79, 128.9, 198.3, 305.1, 469.4, 722.2, 1111 µg/mL (0, 23.01, 35.4, 54.46 µg/mL were selected for metaphase analysis)
- 3h treatment +17h, with S9: (0, 305.1, 469.4, 722.2 µg/mL were selected for metaphase analysis)

Experiment 2:
- 20h treatment +0h, without S9: 0, 25.10, 29.53, 34.47, 40.87, 48.09, 56.57, 66.56, 78.30, 92.12, 108.4, 127.5 and 150.0 µg/mL (0, 34.74, 40.87, 48.09 µg/mL were selected for metaphase analysis)
-3h treatment +17h, with S9: 0, 356.2, 419.0, 493.0, 579.9, 682.3, 802.7, 744.4 and 1111 µg/mL (0, 682.3, 802.7, 944.4 µg/mLwere selected for metaphase analysis)
- 3h treatment +41h, with S9: 0, 356.2, 419.0, 493.0, 579.9, 682.3, 802.7, 744.4 and 111 1µg/mL (0, 944.4 µg/mL was selected for metaphase analysis)

Justification for top dose
- in dose range finding assay: Information provided by the sponsor indicated that 10 mM, considered a suitable top dose for in vitro aberration assay, according to Scott et al, would be equivalent to a final concentration in culture medium of 1111 µg/mL.
- for chromosome analysis from 20+0 hour without S9 and 3+17 hour with S9, in Experiments 1 and 2, was to be one at which a 50-80 % reduction in MI occured or was to be the highest dose treated.
- for delayed harvest: If a negative or equivocal result was obtained in Experiment 1, a single dose from the delayed harvest (44+0, without S9 and 3+41 with S9) was to be scored in Experiment 2. The dose level selected corresponding to the top dose scored following the 20+0 hour treatment wihtout S9 or 3+17 hour treatment with S9, unless mitotic inhibition was too severe in which case the highest scorable dose was to be selected.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: no solvent (dissolved in exposure medium directly
- Justification for choice of solvent/vehicle: Preliminary (small volumes) solubility determinations indicated that 1-Hydroxy-2(1H)-Pyridione was soluble directly in culture medium at aproximately 6 mg/mL, which reduced medium to pH 5.83.

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

without S9; 1.25, 2.5 and 5 µg/mL; 5 and 2.5 µg/mL were selected for chromosome aberration analysis for respectively experiments 1 and 2 because they gave satisfactory responses in terms of quality and quantity of mitoses and extent of chromosomal damage.

Controls 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

with S9; 12.5 and 25 µg/mL; 25 µg/mL was selected for chromosome aberration analysis because cells treated with this dose level gave satisfactory responses in terms of quality and quantity of mitoses and extent of chromosomal damage.

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

Prior to treatment, culture tubes were centrifuged (200 x 'g', 10 minutes) and 1.9 mL of medium removed to allowfor addition of solvent, test article concentration or positive control. S9-mix or KCl (0.5 mL) were added appropriately. One set of quadruplicate cultures for each of the treatment regimes was then treated with the solvent and one set of duplicate cultures with the test article (2 mL per culture). Additional duplicate cultures for 20+0 hour treatments in the absence of S9 and 3+17 hour treatment in the presence of S9 were treated wth 0.1 mL f the positive control chemicals (+1.9 mL fresh culture medium).
The delayed sample (44h fixation time) was adopted for test and negative control cultures only, not for positive controls.

DURATION
- Exposure duration: 3h (Experiment 1 with and without S9 + Experiment 2 with S9), or 20h (Experiment 2 with S9)
- Expression time (cells in growth medium): 15.5h or 39.5h (after 3h treatment, Experiment 1+2 and 2 only, respectively); 0h (after 20h continuous treatment in Experiments 1 and 2)
- Fixation time (start of exposure up to fixation or harvest of cells): 20h (Experiment 1+2) or 44h (Experiment 2)

SPINDLE INHIBITOR (cytogenetic assays): colchicine (added 1.5h before harvesting).

STAIN (for cytogenetic assays): After the slides had dried, the cells were stained for 5 minutes in 4% (v/v) Giemsa stain in buffer. The slides were rinsed, dried and mounted with coverslips

NUMBER OF REPLICATIONS: triplicate

NUMBER OF CELLS EVALUATED: at least 1000 cells counted per slide

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells): 100 metaphases from each code were analyzed for chromosome aberrations (only cells with 44-46 chromosomes were considered acceptable for analysis). At least 25 cells from each of the selected positive control cultures were analysed to ensure that the system was operating satisfactorily.

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index or percentage of cells in mitosis, based on 1000 cells counted

OTHER EXAMINATIONS:
- Determination of polyploidy: yes
- Determination of endoreplication: yes
- other: hyperdiploidy
- Because of the drop in pH observed during the solubilty determination, and treatment at a top dose equivalent to 10 mM, measurement on post treatment media for pH and osmolality were performed in the main study (experiment 1 only).

Evaluation criteria:

The test article was to be considered as positive in this assay if:
1) a statistically significant increase in the proportion of cells with structural aberrations (excluding gaps) occurred at one or more concentrations,
and
2) the proportion of cells with structural aberrations at such doses exceeded the normal range,
and
3) the results were confirmed in the second experiment.

Statistics:

The proportion of cells with structural aberrations excluding gaps for each test treatment condition was compared with the proportion in concurrent negative controls using Fisher's exact test. Probability values of p=<0.05 were accepted as significant. The proportion of cells with structural aberrations including and excluding gaps, and of polyploid, endoreduplicated or hyperdiploid cells, was examined in relation to historical negative control (normal) ranges.

Results and discussion

Test results

Test results 1

Species / strain:

lymphocytes: human

Remarks:

Experiments 1 + 2

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

see mitotic inhibition in section below

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

lymphocytes: human

Remarks:

Experiments 1 + 2

Metabolic activation:

without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

see mitotic inhibition in section below

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no marked effects at top dose tested (1111 µg/mL)
- Effects of osmolality: no marked effects at top dose tested (1111 µg/mL)
- Precipitation: not observed
- Definition of acceptable cells for analysis: Only cells with 44-46 chromosomes were considered acceptable for analysis of structural aberrations.

RANGE-FINDING/SCREENING STUDIES: The results of a preliminary toxicity test were used to set the concentration range for the chromosome aberration test. Dose selection for the chromosome aberration test was based on toxicity.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: the positive control chemicals induced statistically significant increases in the number of cells with structural aberrations
- Negative (solvent/vehicle) historical control data: the proportion of cells with structural aberrations (excluding gaps- in negative control cultures fell within the normal range

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: mitotic index

- Experiment 1: 73% and 57 % mitotic inhibition at the highest dose level for the 20h continuous treatment and the 3h-treatment, repectively
Mitotic index was severely depressed at several doses of the test item, following continuous treatment in the absence of S9. MI in cultures treated in teh presence of S9 was only markedly depressed at the top dose of test item. The differing response between cultures treated with the test item in the absence of S9 and those treated in the presence of S9 was probably, to some extent, a result of the differing exposure period to the test item; continuous for 20h without S9, 3h only with S9.
- Experiment 2: 66%, 54% and 0% mitotic inhibition at the highest dose level for the 20h continuous treatment and the 3h-treatment (20 and 44h fixation, repectively)
Because of the MI profile seen in Experiment 1, different dose ranges were treated in the absence and in the presence of S9. Severe inhibition of MI was again observed at several dose levels of test item, treated in the absence of S9. Treatment in the presence of S9 for 3+17 hours resulted in similar levels of mitotic inhibition to those seen in Experiment 1, although a slightly higher scorable dose was achieved in Experiment 2 (944.4 µg/mL, which induced approximately 54% mitotic inhibition). At the delayed harvest, inhibition of MI was much less marked, with 944.4 µg/mL having no apparent effect on mitosis.

OTHER:
- the binomal dispersion test demonstrated acceptable heterogeneity between the majroity of replicate cultures and at least 160 cells out of the intended 200 were anlysed at each dose levels

Any other information on results incl. tables

Structural aberrations

Treatment of cultures with the test item in the absence of S9 resulted in several cultures exhibiting frequencies of aberrant cells which exceeded historical negative control (normal) ranges. Normal ranges were exceeded in both replicate cultures treated at 54.46 µg/mL (Experiment 1), 40.87 and 48.09 µg/mL (Experiment 2). Statistical analysis of these data indicated that the increased frequency of aberrant cells were significantly different from those seen in concurrent solvent controls. These results were sufficient to fulfil the criteria for definition of a positive response. It is noteworthy, however that the positive effects were only seen at doses which induced greater that 50% mitotic inhibition and the majority of aberrations observed were deletion-types.

The majority of cultures treated with the test item, in the presence of S-9, exhibited frequencies of aberrant cells which were similar to those seen in concurrent and historical negative controls. Only one replicate (Experiment 1) treated with 469.4 µg/mL (3h+17h) exhibited frequencies of aberrant cells exceeding normal ranges. The combined frequency of aberrant cells, for both cultures at this dose, was statistically different from the frequency seen in concurrent controls, however, no biological importance was attributed to this observation as it did not fulfil all the criteria for definition of a positive response.

Numerical aberrations:

The majority of cultures treated with the test item, both in the absence and presence of S-9, exhibited acceptable frequencies of cells with numerical aberrations. Only one culture (722.2 µg/mL, 3h treatment+17h, with S9, Experiment 1) exhibited frequencies of aberrant cells outside the normal range. No biological importance was attributed to this result as the increase was small, not reproduced and not associated with any statistically significant increase in aberrant cells.

Applicant's summary and conclusion

Conclusions:

Interpretation of results:
inconclusive

The initial report concluded that 1-Hydroxy-2(1H)-Pyridione was able to induce structural chromosomal aberrations in cultured human peripheral blood lymphocytes. The effect was, however, only seen in the absence of metabolic activation at doses inducing freater than 50% mitotic inhibition, which may be of dubious biological importance. Since top dose levels for scoring of the metaphases for chromosome aberrations were chosen based on dose levels inducing marked toxicity (70-80%), and considering the fact that excessive levels of cytotoxicity could lead to artifactual positive results, these results are considered inconclusive.