Decree of the Ministry of Health No. 208 / 2001 Coll.
Decree of the Ministry of Health amending Decree of the Ministry of Health No. 251 / 1998 Coll., laying down methods for the detection of toxicity of chemicals and preparations
Valid
Order
Effective from 22.06.2001
Text versions:
22.06.2001
Zobrazeno prvních 200 z celkem 1178 ustanovení tohoto předpisu.
Zobrazit celý předpis →
Pro stažení celého znění použijte tlačítko Stáhnout výše.
208
DECLARATION
Ministry of Health
of 8 June 2001
amending Decree No. 251 / 1998 of the Ministry of Health Coll., laying down methods for the detection of toxicity of chemicals and preparations
The Ministry of Health provides pursuant to § 4 (1) (c) of Act No. 157 / 1998 Coll., on Chemicals and Chemical Products and amending certain other laws:
The Annex to Decree No 251 / 1998 Coll., laying down methods for the detection of toxicity of chemicals and preparations, is amended as follows:
1. the following part "B. General Introduction" is inserted before Method B.1.:
"B. GENERAL INTRODUCTION
1. BASIC INJURY
1.1 ACUTE TOXICITY
involves adverse effects that occur within a certain period (mostly 14 days) after a single dose of a substance.
1.2 EXAMINATION TOXICITY
is a general term describing clear signs of toxicity after application of the test substance. These symptoms are sufficient to assess the risk and should be such that severe toxic symptoms and possibly death may be expected following an increase in the dose administered.
1.3 DOSES
is the amount of test substance administered. The dose is expressed as weight (grams or milligrams) or weight of the test substance per unit weight of the test animal (e.g. milligrams per kilogram body weight), or as constant dietary concentration (parts per million parts or milligrams per kilogram of food).
1.4. DISCRIMINATING DOSES
is the highest of the four fixed dose levels that can be administered without causing death (including humane culling) associated with the administered substance.
1.5 DOSAGE
is the general term comprising the dose, frequency and total duration of administration.
1.6 LD50 (MEDIUM DEATH DOSES)
is a statistically calculated single dose of a substance which is likely to cause death at a defined time of 50% of the animals to which it was administered. The LD50 is given as the mass of the test substance per unit of weight of the test animal (mg.kg-1 body weight).
1.7 LC50 (MEDIUM DEATH CONCENTRATION)
is the statistically calculated concentration of a substance likely to cause death within a certain time after exposure in 50% of the test animals exposed for a defined period. The LC50 is given as the mass of the test substance in the standard volume of air (mg.l-1).
1.8 NOAEL
is the abbreviation for "no observed adverse level 'and is the highest in the test dose or exposure concentration at which no detectable toxic signs occur.
1.9 TOXICITY AFTER REPEATED DOSE / SUBCHRONIC TOXICITY
includes adverse effects that occur in experimental animals due to repeated daily administration or exposure to the chemical for a period representing a short period of expected life expectancy of the species concerned.
1.10 MAXIMUM TOLERATED DOSE
is the highest dose that causes clear signs of toxicity in animals but without significant impact on survival with respect to the effect that is tested.
_
is the induction of inflammatory changes on the skin after application of the test substance.
_
is the induction of eye changes after application of the test substance to the surface of the eye.
1).
is an immunologically induced skin reaction to the test substance.
_
is the induction of irreversible tissue damage by action of the test substance for a period of 3 minutes to 4 hours.
TOXICOKINETIC
is the study of absorption, distribution, metabolism and excretion of test substances.
ABSORPTIONS
denotes the process (s) by which the applied substance enters the body.
_
denotes the process (s) by which the applied substance or its metabolites are removed from the body.
1.18 DISTRIBUTION
denotes the process (s) by which the absorbed substance or its metabolites are distributed in the body.
METABOLISMUS
denotes the process (s) by which the chemical structure of the applied substance is changed in the body by enzymatic or non-enzymatic reactions.
2. ACUTE - REPEATED APPLIANCES / SUBCHRONIC AND CHRONIC TOXICITY
Acute toxic effects and organ or systemic toxicity may be evaluated using a large number of different toxicity tests (methods B.1 - B.5) from which a preliminary estimate of toxicity may be obtained after single administration.
Depending on the toxicity of the substance, different procedures can be chosen from the limit test to the complete determination of LD50. For inhalation studies, the limit test is not designed because it was not possible to define the limit value of a single inhalation exposure.
Consideration should be given to methods that use the smallest number of animals and minimise the suffering of the animal, such as the fixed dose method (Method B.1 bis) and the method for determining the acute toxicity class (Method B.1 tris). When testing on one species, the study on the other may complement the conclusions drawn from the first study. In this case, a standard test method may be used or fewer animals may be used.
The repeated application toxicity test (methods B.7, B.8 and B.9) is assessed for toxic effects resulting from repeated exposure. Clinical observations of animals are important to obtain as much information as possible. These tests should help identify target organs of toxicity and toxic and non-toxic doses. Long-term studies require further research into these aspects (methods B.26 - B.30 and B.33).
3. MUTAGENITA - GENOTOXICITY
Mutagenicity refers to the induction of permanent transmissible changes in the amount or structure of genetic material of cells or organisms. These changes ("mutations") may affect a single gene or segments of genes, a block of genes or whole chromosomes. Effects on whole chromosomes may manifest themselves by changing their structure or number.
The mutagenic activity of the substance is determined in vitro for bacterial gene (point) mutations (method B.13 / 14) or structural chromosome aberrations in mammalian cells (method B.10).
In vivo procedures such as micronucleus test (method B.12) or bone marrow chromosome metaphase analysis (method B.11) are also acceptable. However, in vitro methods should be clearly preferred if they are not contraindicated.
Further studies may be required for substances manufactured in large quantities or for the determination and control of risk to detect mutagenicity or for preliminary carcinogenicity testing. These studies may be used for several purposes: confirmation of the results obtained from the baseline test set; examination of the effects of the non-detected basic set of methods; initiation or extension of in vivo studies.
For this purpose, methods B.15 to B.25 include both in vivo and in vitro eucaryonte systems and extend the range of biological effects. These tests provide information on point mutations and other effects in organisms more complex than bacteria used in the baseline test set.
In general, further mutagenicity studies considered should be planned to provide relevant additional information on the mutagenic or, where appropriate, carcinogenic potential of the test substance.
A specific study, suitable for the case, will depend on numerous factors, including the chemical and physical characteristics of the substance, the results of basic bacterial and cytogenetic tests, the metabolic profile of the substance, the results of further toxicity tests and known uses of the substance.
For the assessment of the risk of hereditary effects in mammals, methods are available to detect hereditary effects throughout the mammalian organism, whether caused by gene (point) mutations, e.g. a specific locus test in mice detecting germ cell mutations in the first generation (not included in this Annex), or chromosome aberrations such as a mouse translocation test (method B.25). These methods can be used to estimate the potential genetic risk of a substance to humans. Due to the complexity of these tests and the very large number of animals needed, especially for a specific locus test, it is only for serious reasons to decide on such a study.
The determination of genotoxicity shall be carried out in accordance with the standard methodological protocol developed for the methodology after consultation with the National Reference Laboratory of Genetic Toxicology.
4. CARCINOGENITY
Chemicals may be characterised as genotoxic or non-genotoxic carcinogens, depending on the intended mechanism of action.
Preliminary information on the genotoxic carcinogenic potential of the substance is obtained from mutagenicity / genotoxicity studies. Further information is provided by repeated application toxicity tests and subchronic or chronic toxicity tests. The repeated application toxicity test, method B.7, and longer-term repeated dose studies include an assessment of histopathological changes such as hyperplasia in certain tissues, which could also be significant. These studies and toxickinetic information may help identify chemicals with carcinogenic potential that require further, more detailed examination of this aspect by a carcinogenicity test (method B.32) or often in a combined chronic toxicity / carcinogenicity study (method B.33).
There are also mammalian cell transformation tests that determine the ability of the substance to induce morphologic changes and behavioural changes in cell cultures that are expected to be related to malignant transformation - in vivo (method B.21). Several different cell types and transformation criteria are used.
5. REPRODUCTION TOXICITY
Reproductive toxicity is assessed in various ways, e.g. due to deterioration of reproductive function or ability of males and females (effect on fertility) or non-hereditary harmful effects on offspring (developmental toxicity) including teratogenicity and effects during lactation.
The test method (method B.31) for teratogenicity studies as part of developmental toxicity mainly involves oral administration. Alternatively, other applications may be used depending on the physical properties of the test substance or the likely human exposure. In such cases, the test method should be adapted accordingly.
If a three-generation reproductive test is required, the described method for the two-generation reproductive test (method B.35) may be extended to cover the third generation.
6. NEUROTOXICITY
Neurotoxicity is determined in different ways, e.g. by functional changes or structural and biochemical changes in the central or peripheral nervous system. Preliminary warning for neurotoxicity may result from acute toxicity tests. The repeated application toxicity test (Method B.7) also includes an assessment of the neurotoxic effect. The method should help detect chemicals with neurotoxic potential that require further in-depth examination of this aspect. In addition, it is important to take into account specific neurotoxic effects that cannot be detected in other toxicity studies. For example, certain organic compounds of phosphorus have been found to cause late toxicity, which is assessed by methods B.37 and B.38 following single or repeated administration of the substance.
7. IMMUNOTOXICITY
Immunotoxicity is assessed in different ways, for example by immunosuppression or by increasing the response of the immune system, resulting in either hypersensitivity or induced autoimmune. The repeated application toxicity test (Method B.7) includes the determination of immunotoxic effects. The method should help detect chemicals with immunotoxic potential requiring further in-depth examination of this aspect.
_
Toxickinetic studies help to interpret and evaluate toxicity data, clarify specific aspects of toxicity of the test chemical and the results may help in the design of further toxicity studies. All parameters are not expected to need to be determined in any case. A full sequence of toxickinetic studies (absorption, distribution, metabolism and excretion study) will be necessary only in isolated cases. For some compounds, changes in this sequence may be appropriate or may prove to be a sufficient single-dose study (Method B.36).
Information on the chemical structure and physicochemical properties may also provide data to permit estimation of absorption characteristics in the planned method of application, metabolism and distribution to tissues. Information on toxicological parameters from previous toxicological and toxicological studies may also contribute.
9. CHARACTORISTS OF THE TESTED SUBSTANCE
The composition of the test substance, including major impurities, and its relevant physicochemical properties, including stability, should be known before any toxicological study is initiated.
The physical-chemical properties of the test substance provide information relevant to the choice of route of administration, to the design of a specific study and to the handling and storage of the substance.
The development of an analytical method for the qualitative and quantitative determination of the test substance (and, if possible, larger impurities) in the dosing medium and biological material should prevent the study from starting.
All information concerning identification, physicochemical properties, purity and behaviour of the test substance should be included in the final test report.
_
For toxicological tests, strict control of the conditions of the environment in which the animals are kept and proper care of the animals is essential.
10.1 CONDITIONS OF THE TERRITORY
The living conditions of experimental animals should be adapted to each species. For rats, mice and guinea pigs, the room temperature is 22 ° C ± 3 ° C at 30 - 70% relative humidity; for rabbits, the temperature shall be 20 ° C ± 3 ° C at 30 - 70 per cent relative humidity.
Some experimental research techniques are particularly sensitive to temperature. For these cases, details of the relevant conditions are given in the description of the test method. In all toxic tests, the temperature and humidity should be monitored, recorded and reported in the final test report.
The lighting should be artificial with light and dark changes after 12 hours. Details of the lighting shall be recorded and reported in the test course.
If the method does not require any other method, animals may be kept individually or in small groups of individuals of one sex, not more than 5 animals per cage.
Data on caging methods and number of animals kept in a single cage are an essential part of the animal test report, both during exposure to the substance and during the subsequent observation period.
10.2 CONDITIONS OF FEED
Feed shall meet all nutritional requirements for the species used. If the animals are fed the nutritional value may be reduced by the interaction of the study substance and certain food ingredients. The possibility of such a reaction should be taken into account when interpreting the results. Conventional laboratory diets with unlimited access to drinking water are used. The choice of diet should be adapted when the test substance is administered in food.
Food additives which have a proven effect on toxicity must not be present in the concentrations at which this effect would occur.
11. ANIMAL PROTECTION
The necessary attention must be paid to the protection of animals when developing test methods.
Reductions in the number and reduction of animal pain and stress may be achieved e.g. by using a fixed dose method (B.1.bis) or an acute toxicity class (B.1.tris). The fixed dose method does not use death as specific criteria and requires a smaller number of animals. The method of determining the acute toxicity class on average requires 70% less animals than Method B.1.
Animals with severe and persistent symptoms of stress should be humanely killed; test substances must not be administered at doses and in ways known to cause significant pain and stress due to their corrosive or irritating properties.
The use of limit tests, not only in acute toxicity tests (methods B.1, B.2 and B.3), but also in in vivo mutagenicity tests (methods B.11 and B.12), makes it possible to avoid testing at unnecessarily large doses.
When testing irritation, the test may not be performed or limited to a study in a single animal if this is scientifically justified. Such scientific justification may be based on the physicochemical properties of the substance, the results of other tests already carried out or the results of well-validated in vitro tests. For example, if an acute skin toxicity study with the dose used in the limit test (method B.3) has been conducted with the substance and no skin irritation has been observed, further skin irritation testing (method B.4) may be unnecessary; substances that have been shown to cause corrosion or severe skin irritation in a skin irritation study (method B.4) must not be further tested for eye irritation.
Alternative procedures must be constantly developed and verified which can provide the same level of information as the current animal testing, using fewer animals, causing less suffering or completely avoiding the use of animals.
Where such methods are available, their use for risk characterisation, subsequent hazard classification and labelling shall be taken into account wherever possible.
12. ASSESSMENT AND INTERPRETATION
In assessing and interpreting animal experiments and in vitro tests, it should be considered that direct extrapolation to humans is possible to a limited extent; evidence of adverse effects in humans, where available, may serve to verify the results of the testing.
Test results may be used for classification and labelling of new and existing substances based on human health effects based on the characteristics identified and quantified by these methods.
These results may also be used for studies aimed at assessing the risk of both new and existing substances. '
2. methods B.10., B.11. and B.12 are as follows:
"B.10. MUTAGENITY - CHROMOSIUM ABERATIONS TEST IN VITRO
1. METHOD
The method is a replication of OECD TG 473, In Vitro Mammalian Chromosome Aberration Test (1997).
1.1 INTRODUCTION
The purpose of the in vitro chromosome aberration analysis is to find substances that cause structural chromosome aberrations in mammalian cell cultures (1) (2) (3). Structural aberrations are of two kinds: chromosomes and chromatids. The aberrations induced by chemical mutagens are mostly chromatids, but also chromosome-type aberrations occur. The formation of polyploidy may indicate that the chemical is capable of causing numerical aberrations. However, this method is not designed to analyse numerical aberrations and is not commonly used for this purpose. Chromosomal mutations and related phenomena are the cause of a number of genetic diseases in humans and there are compelling evidence that chromosomal mutations and related phenomena that cause changes in oncogenes and somatic cell tumorsuppressor genes play a role in the formation of cancer in humans and experimental animals.
This test is used to detect possible mammalian mutagens and carcinogens. Although a number of chemicals that are positive in this test are carcinogenic to the female, there is not enough correlation between this test and carcinogenicity. This correlation depends on the nature of the substance and there is increasing evidence that there are chemicals whose carcinogenic properties are not detected by this test because they appear to be acting by a mechanism other than direct DNA damage
In vitro chromosome aberrations may be analysed using stable cell lines, cell strains or primary cell cultures. The cells used are selected based on the ability to grow in culture, the stability of the karyotype, the number of chromosomes, their diversity and the frequency of spontaneous chromosome aberrations.
In vitro tests generally require an exogenous source of metabolic activation. This metabolic activation system cannot perfectly mimic mammalian conditions in vivo. Conditions that may lead to positive results that do not reflect internal mutagenicity but are due to changes in pH, osmolality or high cytotoxicity should be avoided (4) (5).
1.2 DEFINITIONS
Chromatid type aberration: structural damage to the chromosome, which manifests itself as a fraction of one or both chromatids, or a break and reconnection between chromatids.
Chromosome type aberration: structural damage of the chromosome expressed as a break, respectively, and reconnection of both chromatids in the same place (included are adrenal chromosome type aberrations, nipple chromosome ring)
Chromatid break: A break in the continuity of one or both chromatids can be evaluated as a break provided that the resulting gap in the chromatid structure is greater than the width of the damaged chromatid. Furthermore, in cases where the terminal (distal) fragment at the breaking point is dislocated (or outside the chromatid axis), or one chromatid of the evaluated chromosome is shorter due to deletion.
Endoreplication: a process where, after the S-phase of DNA replication, the nucleus does not pass into mitosis but begins another S-phase. The result is chromosomes with 4, 8, 16... chromatids.
Gap: Achromatic lesion less than the width of one chromatid and with minimal deflection of the chromatids.
Mitotic index: the ratio of cells in metaphase divided by the total number of cells in the cell population; indicates the degree of proliferation of this population.
Numerical aberration: change in chromosome count compared to normal cell characteristics.
Polyploidy: multiple haploid number of chromosomes (n) other than diploid number (i.e. 3n, 4n, etc.).
Structural aberration: change in the chromosome structure detectable by microscopic analysis of cell division at metaphase stage, observed as deletion and fragments and exchanges.
1.3. PRINCIPLE OF METHODS
Cell cultures are exposed to the test substance with or without metabolic activation. At specified intervals after exposure to cell culture to the test substance, for example, metaphase is stopped by Colcemide ® or colchicine, cells are harvested, dyed, and chromosomal aberrations are microscopically detected in metaphasic cells.
1.4 DESCRIPTION OF THE METHOD
1.4.1 Preparation
1.4.1.1 Cells
A number of cell lines, strains or primary cell cultures, including human cells (e.g. Chinese hamster fibroblasts or peripheral blood lymphocytes or other mammals) may be used.
1.4.1.2 Media and culture conditions
Appropriate culture media and incubation conditions (culture vessels, CO2 concentration, temperature and humidity) should be used to maintain cultures. For cell lines and strains, the stability of the modal number of chromosomes and the absence of mycoplasmas in cells should normally be checked. In case of contamination, cells are not used. Normal cell cycle time and culture conditions should be known.
1.4.1.3. Culture preparation
stabilised cell lines and strains: cells are multiplied from stock cultures by sifting into the culture medium in such a density that the cultures do not reach confluent layers before harvest time and incubate at 37 ° C.
Lymphocytes: the culture medium containing mitogen (e. g. phytohaemegglutinin) is either added full blood with an appropriate anticoagulant (e. g. heparin) or separated lymphocytes obtained from healthy individuals and cultured at 37 ° C.
1.4.1.4. Metabolic activation
The cells have a test substance in the presence and absence of an appropriate metabolic activation system. The most common cofactors are the supplemented mitochondrial fraction (S9) prepared from rodent liver, affected by enzymes such as Aroclor 1254 (6) (7) (8) (9), or a mixture of phenobarbital and β -naphthoflavon (10) (11) (12).
The final concentration of mitochondrial fraction in the medium is usually 1-10% v / v. The activity of the metabolic activation system will depend on the type of test substance. In some cases it is appropriate to use mitochondrial fraction at several concentrations.
For endogenous activation, genetically modified cell lines expressing specific activation enzymes may also be used. The selection of cell lines should be scientifically verified (e.g. by the relationship of cytochrome P450 isoenzyme to the test substance metabolism).
1.4.1.5 Preparation of test substance
If the test substance is solid, the substance is dissolved or prepared in an appropriate solvent before action on the cells and diluted if necessary. Liquid test substances may be added directly or diluted. Fresh preparations should be used unless stability data show that longer storage does not affect their properties.
1.5. TEST CONDITIONS
1.6 SOLVENT
The chosen solvent shall not be suspected to react chemically with the test substance and shall not interfere with cell survival and S9 activity. If a less well-known solvent is used, it shall be supported by references demonstrating its compatibility. It is recommended that, wherever possible, use as water solvents be considered first. If the test substance is unstable in water, the organic solvent used shall be completely anhydrous. Water can be removed by a molecular sieve.
1.6.1. Strength
The criteria to be considered in determining the highest concentration include cytotoxicity, solubility in the system and changes in pH or osmolality.
Cytotoxicity is determined with or without metabolic activation in the main experiment using appropriate indication of cellular integrity and growth, such as degree of confluence, number of live cells, or mitotic index. It is appropriate to establish cytotoxicity and solubility in preliminary experiments.
A minimum of three analysable concentrations should be used. If cytotoxicity occurs, these concentrations shall cover the range from maximum to low or zero toxicity. This usually means that the individual concentrations will not differ more than the multiple that lies between the values of 2 and --10. A significant decrease in confluent growth, cell count, or mitotic index (always by more than 50%) must be observed at the time of cell harvesting at the highest concentration. The mitotic index is only an indirect measure of cytotoxic / cytostatic effects and is dependent on the time elapsed after exposure to the test substance. However, it is acceptable for suspension cultures where other toxicity detection would be difficult and impractical. Data on cell division kinetics such as average generation time may also be used as additional information; This, however, represents an overall average that does not always reveal existence in the division of delayed subpopulations of cells. Thus, even a small extension of the average generation time may mean a significant departure from the appropriate harvest time in terms of optimal yield of aberrations.
For relatively non-cytotoxic substances, the maximum concentration of the test substance is the lowest of 5 μl / ml, 5 mg / ml or 0,01 M.
For relatively insoluble substances which are non-toxic at concentrations below the concentration at which the substance is no longer dissolved, the maximum concentration used should be higher than the limit of solubility in the final culture medium at the end of the culture with the test substance. In some cases - for example, when taking toxic effects at concentrations above the lowest insoluble concentration - it is recommended to test at several concentrations with visible precipitation. It may be useful to evaluate the solubility at the beginning and end of exposure, as solubility may change during exposure in the test system due to the presence of cells, serum, S9 etc. Insolubility can be detected with a simple eye. The clot must not be an obstacle to cell evaluation.
1.6.2 Negative and positive controls
Concomitant positive and negative controls (solvent or vehicle) should be included in each experiment, with or without metabolic activation. If metabolic activation is applied, the positive control shall be a substance requiring metabolic activation to induce mutagenic action.
A known clastogen is used for positive control at exposure levels where it is expected to provide a reproducible and detectable increase in background aberrations, demonstrating sensitivity of the test system.
The concentration for positive control should be chosen so that the effect is obvious but does not reveal directly to the evaluator the identity of the encoded preparations. Positive control substances include, for example:
| Metabolická aktivace | Látka | CAS | EINECS |
|---|---|---|---|
| Ne | methyl methansulfonát | 66-27-3 | 200-625-0 |
| ethyl methansulfonát | 62-50-0 | 200-536-7 | |
| ethyl nitrosomočovina | 759-73-9 | 212-072-2 | |
| mitomycin C | 50-07-7 | 200-008-6 | |
| 4-nitrochinolin-N-oxid | 56-57-5 | 200-281-1 | |
| Ano | benz[a]pyren | 50-32-8 | 200-028-5 |
| cyklofosfamid monohydrát cyklofosfamidu | 50-18-0 6055-19-2 | 200-015-4 |
Other appropriate substances may also be used for positive control. Where possible, it is appropriate to use a substance from the same chemical group for positive control.
A negative control in which a solvent or vehicle is used in the culture medium, treated in the same way as the experimental culture, should be used for each culture harvest time interval. In addition, control should also be included without any influence.
Procedure
1.6.3.1. Exposure to test substance
The proliferation cells are treated with the test substance in the presence and absence of the metabolic activation system. Lymphocytes should be treated about 48 hours after mitogen stimulation.
Usually two cultures are used for each concentration; and the same is recommended for negative solvent or vehicle controls. If historical data show that the differences between duplicate cultures are minimal (13) (14), it is acceptable that only one culture should be used for each concentration.
In the case of gaseous or volatile substances, appropriate procedures such as the use of airtight closed culture vessels (15) should be applied in the test.
1.6.3.2. Culture harvesting period
In the first experiment, cells are exposed to the test substance with or without metabolic activation for 3-6 hours and samples are taken at a time equivalent to approximately 1.5 times the normal cell cycle length since the beginning of exposure (12). If the results are negative when using the activation system and without it, an additional experiment shall be conducted without activation, with continuous exposure up to the time of completion of the culture being approximately 1.5 times the normal cell cycle. Some substances are more easily evaluated at exposure times longer than 1.5 times the length of the cell cycle. Negative results with metabolic activation shall be assessed on a case-by-case basis. Where confirmation of negative results is not considered necessary, this Decision should be justified.
1.6.3.3 Preparation of chromosomes
Colcemide ® or colchicine is usually applied to cell cultures for 1-2 hours prior to completion of culture. Each cell culture is processed separately for chromosome preparation. The preparation of chromosomes consists of affecting cells with hypotonic solution, fixation and dyeing.
1.6.3.4 Analysis
All preparations, including positive and negative controls, shall be encoded independently before microscopic analysis. As fixation procedures often lead to a certain proportion of metaphasic cells and a loss of chromosomes, for all cell types, the number of centromer in the investigated cells must be equal to the modal number ± 2. For each concentration and control, at least 200 well spread metaphases should be evaluated evenly from both cultures if used. If the number of aberrations is high, the number of metaphases analysed may be reduced.
Although the purpose of the test is to detect structural chromosome aberrations, it is also important to note possible polyploidy and endoreplication.
2.
2.1. PROCESSING OF RESULTS
Since the experimental unit is a cell, the percentage of cells with structural chromosome aberrations should be evaluated. Individual types of structural aberrations, including their number and frequency of experimental and control cultures, should be registered. Gaps are recorded separately, but are usually not included in the overall aberration frequency.
At the same time, cytotoxicity detected in both experimental and control cultures should also be recorded in the test.
Data are given for individual cultures. Finally, all data shall be shown in the summary table.
Verification of clearly positive results is not required. The inconclusive results are explained by further testing or modification of experimental conditions. The need to confirm negative results was discussed in paragraph 1.4.3.3. In subsequent experiments, the modification of the parameters used should be considered in order to extend the scope of the conditions considered. The parameters that can be modified include concentration intervals and metabolic activation conditions.
2.2 EVALUATION AND INTERPRETATION OF RESULTS
Several criteria are used to determine a positive result, such as the concentration of the test substance dependent cell frequency with chromosome aberrations. The biological significance of the results should be considered first. Statistical methods (3) (13) may be used to evaluate test results. However, statistical significance should not be the only determining factor for a positive outcome.
An increase in the number of polyploid cells may indicate that the test substance has the ability to inhibit mitotic processes and induce numerical chromosome aberrations. An increase in the number of cells with endosuppressed chromosomes may indicate that the test substance has the ability to inhibit the phases of the cell cycle (17) (18).
A test substance in which the results do not meet the above criteria shall be considered non-mutagenic in this system.
Although most experiments show clearly positive or negative results, in exceptional cases the data obtained do not allow a clear assessment of the effect of the test substance. The results may be uncertain or questionable regardless of the number of repeated experiments. Positive in vitro chromosome aberration test results indicate that the test substance induces structural chromosome aberrations in mammalian somatic cell culture. Negative results indicate that under given experimental conditions the test substance does not induce chromosome aberrations in mammalian somatic cell culture.
3. FINAL REPORT
_
The following information shall be provided in the report on the conduct of the experiment:
Solvent / vehicle:
- justification for choice of solvent / vehicle
- the solubility and stability of the test substance in the solvent / vehicle, if known.
Cells:
- cell type and source,
- caryotype and suitability of the cell type,
- absence of mycoplasmas, if necessary
- information on cell cycle length,
- the sex of the donor blood, whether full blood or separated lymphocytes, used mitogen,
- number of cell passages, if necessary - methods of maintaining cell culture, if necessary
Sign in for notes, favorites and notifications
Regulation Information
| Citation | Decree of the Ministry of Health No. 208 / 2001 Coll., amending Decree of the Ministry of Health No. 251 / 1998 Coll., laying down methods for the detection of toxicity of chemicals and preparations |
|---|---|
| Regulation Type | Order |
| Author | - |
| Collection | Code of Laws |
| Date of Promulgation | 22.06.2001 |
|---|---|
| Effective from | 22.06.2001 |
| Effective until | - |
| Status | Valid |
Legal Areas:
Administrative law
Health
The regulation text is for informational purposes only.
Comments 0