Decree of the Ministry of Health No. 251 / 1998 Coll.
Decree of the Ministry of Health laying down methods for the detection of toxicity of chemicals and preparations
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251
DECLARATION
Ministry of Health
of 16 October 1998
laying down methods for the detection of toxicity to 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:
This Decree sets out methods for the detection of hazardous properties of chemicals and chemical preparations of highly toxic, toxic, health harmful, corrosive, irritating, sensitising, carcinogenic, mutagenic and toxic for reproduction ("toxicity of chemicals and preparations') .1).
(1) The toxicity of chemicals and preparations shall be determined by the tests carried out using the methods set out in the Annex to this Decree.
(2) Other methods may also be used to detect toxicity of chemicals and preparations if they are at least as sensitive, accurate and reproducible as those referred to in paragraph 1, but preferably without the use of experimental animals (2) and methods recommended by the Organisation for Economic Cooperation and Development (OECD) .3)
This Decree shall take effect on 1 January 1999.
Minister:
Dr. David, CSc.
Annex to Decree No 251 / 1998 Coll.
Methods for detection of toxicity of chemicals and preparations
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.
B.1. ACUTE TOXICITY ORAL (PER OS)
1. METHOD
1.1 Principle of test method
The test substance is administered in graduated doses to several groups of experimental animals using an oral probe, one dose level per group. The selected doses may be determined according to the results of the indicative test. The observed effects and deaths are registered. Animals that die during the experiment, even animals that survive the end of the experiment, are dissected. This method is mainly used in rodent experiments.
Animals with severe and persistent symptoms of stress and pain 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.
1.2. Description of method
1.2.1 Preparation
At least 5 days before the test, the animals are kept under the breeding and feeding conditions in which they will be kept during the experiment. Before the test, healthy young adult animals are randomly assigned to individual experimental groups.
If necessary, the solution or suspension of the test substance should be prepared in an appropriate vehicle. It is recommended that the use of an aqueous solution be considered first, the next choice being a solution in vegetable oil, then as another possibility of a solution in other suitable vehicles or suspension. In non-aqueous vehicles, the relevant toxicological properties shall be known or determined prior to or during the test. In rodents, the volume should normally not exceed 10 ml.kg.1 body weight; except for aqueous solutions which may be administered up to 20 ml.kg.1 The variability of the volumes of the substance administered in the tests should be minimised by adjusting concentrations to ensure a constant volume at all dose levels. At the same time, appropriate concentrations of the substance in the vehicle should be selected to minimise local irritation.
1.2.2 Experimental conditions
1.2.2.1. Test animals
It is preferable to rats if there are no known reasons against this. Young healthy animals should be used from commonly used test strains. At the beginning of the test for both sexes, the animal weight variation margin (for each sex separately) should not exceed ± 20% of the mean value.
1.2.2.2. Number and sex
Use at least 5 same-sex rodents for each dose level. When using females, they must be nulliparous and not pregnant. If information is available that one of the two sexes is significantly more sensitive, animals of this sex should be used.
Note: If animals of species higher than rodents are used in acute toxicity tests, fewer animals should be considered. Doses should be carefully selected and care should be taken to ensure that the median dose is not exceeded. In such tests, lethal doses of the test substance should be avoided.
1.2.2.3 Selection of benefits
There should be a sufficient number of dose levels, at least three, and should be suitably graduated in such a way that the range of toxic effects and mortality for test groups is visible. The data obtained shall be sufficient to show the relationship between dose and effect and, where possible, shall allow the determination of LD50 with acceptable reliability.
1.2.2.4. Limit test
The use of rodents may result in a single dose limit test of at least 2000 mg.kg-1 body weight in groups of 5 males and 5 females using the above procedures. If the substance administered causes death, a full study should be considered.
1.2.2.5. Observation time
The observation period should be at least 14 days. However, it cannot be determined rigorously. It should be determined by the picture of poisoning, the rate of development of poisoning and the duration of the recovery phase. The observation period may be extended as necessary. An important time is when symptoms of poisoning appear and disappear, and time to death, especially where there is a tendency to delay mortality.
Description of procedure
Animals should be hungry before administration of the test substance. The rats get their food the night before the test. For animals with a higher rate of metabolism, shorter starvation is sufficient; access to drinking water is not restricted. On the day of the test, the animals are weighed and the test substance is administered orally in a single dose. If a dose cannot be administered at the same time, the dose may be administered in smaller amounts within a maximum of 24 hours. After administration of the dose, access to food should be avoided for 3-4 hours. If the substance is administered fractionated during a certain period, it may be desirable to provide food and water to animals depending on the length of this period.
After administration of the substance, observations shall be made and recorded systematically and separate records shall be kept for each animal. During the first day, frequent observations should be made. It is necessary to monitor and detect the local effect of the applied substance and the immediate reaction of the animals to the application.
Careful clinical examinations should be carried out at least once during each working day. Other observations shall be made on a daily basis with appropriate measures to minimise the loss of animals for the study, such as autopsy or freezing of dead animals, and isolation or killing of weak or dying animals. Observations include changes in skin, hair, eyes, mucous membranes, breathing and circulation, changes in the function of the autonomic and central nervous system, somatomotor activity and behaviour. Particular attention should be paid to tremor, convulsions, salivation, diarrhoea, lethargy, sleep and coma. The moment of death should be recorded as accurately as possible.
Animals that die during the experiment, even those that survive until the end of the experiment, are being dissected. All pathological findings shall be recorded. If indicated, tissues should be taken for histological examination.
1.2.4. Evaluation of other sex toxicity
After completion of the experiment on one sex, a substance shall be administered to at least one group of five animals of the other sex to determine whether the animals of the other sex are significantly more sensitive to the test substance. In individual cases, the use of a smaller number of animals may be justified. If reliable information is available that animals of the sex tested are significantly more sensitive, testing on other sex animals may be omitted.
2.
The data shall be compiled into a table. It must show for each experimental group the number of animals at the beginning of the experiment, the time of death of individual animals, the number of animals with other signs of poisoning, the description of toxic effects and autopsy findings. The determination of the weight of each animal shall be done immediately prior to administration of the test substance, at weekly intervals and at the time of death. Changes in weight should be determined and recorded if the animals survive for more than one day. Animals that have been humanely killed in relation to the stress and pain caused by the substance shall be recorded as dying from the substance. LD50 is calculated using a recognised method. Data evaluation should also include the relationship, if any, between exposure of animals to the test substance and the occurrence and degree of all abnormalities including behavioural changes, clinical symptoms, macroscopic lesions, weight changes, mortality and other toxic effects.
3. FINAL REPORT
3.1 Test progress report
If possible, the test progress report shall contain the following information:
- species, strain, origin, breeding conditions, food, etc.,
- experimental conditions,
- dose levels (including vehicle, if applicable) and concentrations,
- the sex of the animals,
- tabular data on responses by sex and dose level (number of animals dead, number of animals showing poisoning, number of animals exposed),
- time of death following administration of the test substance, reasons and criteria for human culling of animals - all other observations,
- the LD50 value for the sex for which a complete experiment has been conducted for 14 days of observation (indicating the method of calculation),
- 95% confidence limits for LD50 if they can be calculated,
- the dose mortality curve and its guidance (if any),
- autopsy findings,
- all histopathological findings,
results of all tests on the second sex,
- analysis of the results (particular attention should be paid to the effect that the human culling of animals during the test may have on the calculated LD50),
- interpretation of results.
B.1.bis ACUTE TOXICITY ORAL (PER OS) - METHOD OF THE FIXED BENEFIT
1. METHOD
1.1 Principle of test method
The acute oral toxicity test provides information on adverse effects that may follow oral administration of a single dose of test substance within a short period of time.
The fixed dose method is performed in two stages.
In a preliminary indicative study, several doses are sequentially monitored for oral application by a probe in animals of the same sex, each animal per dose. The indicative study provides information on dose and toxicity relationship, including an estimate of the minimum lethal dose. Normally, no more than five animals are used in this first stage.
In the main study, the substance is administered orally to groups of five males and five females at one of the following fixed doses: 5, 50, 500 or 2000 mg.kg.1 body weight. The dose used is derived from a preliminary indicative study as a dose likely to cause "obvious toxicity '(see section B - General introduction point 1.2), but not death.
Effects are observed after administration. If the selected starting dose level causes obvious toxicity but no mortality, no further testing is required. When no apparent toxicity is observed at the selected dose level, the substance is re-tested at the nearest higher dose level. If animals die or if a serious toxic reaction requires human killing, the substance should be re-tested at the nearest lower dose level.
This procedure allows the detection of a "discriminatory dose '(see Section B - General introduction point 1.2), i.e. the highest of pre-established dose levels that can be administered without mortality (including cases of humane killing).
Animals with severe and persistent symptoms of stress and pain should be humanely killed. The test substances must not be administered at doses such as those known to cause significant pain and stress due to their corrosive or irritating properties.
1.2. Description of method
1.2.1 Preparation
1.2.1.1 Test animals
It is preferable to rats if there are no grounds against this.
Commonly used strains of experimental animals should be used. At the beginning of the test for both sexes, the animal weight variation margin (for each sex separately) should not exceed ± 20% of the mean value.
At least 5 days before the test, the animals are kept under housing and feeding conditions in which they will be during the experiment. Before the test, healthy young adult animals are randomly assigned to individual experimental groups to guide studies and main studies. Normally, one group of both sexes is sufficient for the main study.
1.2.1.2 Preparation and administration of the dose
If necessary, the solution or suspension of the test substance should be prepared in an appropriate vehicle. It is recommended that the use of an aqueous solution be considered first, the next choice being a solution in vegetable oil, then as another possibility of a solution in other suitable vehicles or suspension. In non-aqueous vehicles, the relevant toxicological properties shall be known or determined prior to or during the test. In rodents, the volume should normally not exceed 10 ml.kg.1 body weight; except for aqueous solutions which may be administered up to 20 ml.kg.1 The variability of the volumes of the substance administered in the tests should be minimised by adjusting concentrations to ensure a constant volume at all dose levels.
Animals should be hungry before administration of the test substance. The rat shall be fed in the evening before the test; access to drinking water is not restricted. On the day of the experiment, the animals are weighed and the test substance is administered orally in a single dose. If a dose cannot be administered at the same time, the dose may be administered in smaller amounts within a maximum of 24 hours. After administration of the dose, access to food should be avoided for 3-4 hours. If the substance is administered fractionated during a certain period, it may be desirable to provide food and water to animals depending on the length of this period.
Description of procedure
1.2.2.1 Indicative studies
The effects of different doses on individual animals are investigated. Normally, females are used unless there are indications to the extent that males are more sensitive to sex. Doses are given sequentially, waiting at least 24 hours before the next animal is given. All animals are carefully observed to detect signs of poisoning for at least seven days; if signs of mild poisoning persist on the seventh day, the animal should be observed for another seven days. The following starting dose levels are considered: 5, 50, 500 and 2000 mg.kg-1. If the selected starting dose does not cause severe poisoning and the next higher cause death, then one or more specified dose levels should be administered as necessary. This method should provide information on the level of dose (s) that causes symptoms of poisoning and the smallest dose that causes mortality.
The starting dose should be tried according to data on related chemicals. If not available, it is recommended to use 500 mg.kg-1. as the first dose If signs of poisoning are not observed after the initial dose, additional higher dose levels are investigated. If no death occurs at 2000 mg.kg-1, the indicative study is complete and the main study should be performed at this dose level. If severe effects requiring human killing are observed when using a starting dose (e.g. 500 mg.kg-1), the next animal is given the nearest lower dose (e.g. 50 mg.kg-1). If this animal survives, appropriate doses between fixed doses may be used for other animals. Under normal conditions, more than 5 animals are not expected to be required in this procedure.
1.2.2.2 Main studies
At least 10 animals (5 females and 5 males) should be used for each dose level tested. The females should be nulliparous and not pregnant.
The principle of the fixed dose method is to use only slightly toxic doses for the main study. The administration of lethal doses of the test substance should be avoided.
The dose to be taken in the test should be selected from one of the 4 fixed dose levels, namely 5, 50, 500 or 2000 mg.kg-1 body weight. The chosen starting dose level should be one that is likely to cause obvious toxicity but not mortality (including human spending); random deaths are not included but are to be recorded. If this dose causes obvious toxicity but does not cause mortality, further testing is not required.
When administration of the selected dose does not cause obvious toxicity, the substance should be re-tested at another higher dose level. However, animals should be further observed to complete the observation period. If a severe toxic response requires human culling of animals or if mortality is manifested by the substance, the substance should be re-tested at a further lower dose level. Again, animals that do not need to be humanely killed should be observed throughout the observation period.
After administration of the substance, observations are made and recorded systematically. Separate records shall be kept for each animal. It is necessary to monitor and detect the local effect of the applied substance and the immediate reaction of the animals to the application.
The observation period should be at least 14 days. However, it cannot be determined in rigorously. It should be determined by the picture of poisoning, the rate of development of poisoning and the duration of the recovery phase. The observation period may be extended as necessary. An important time is when signs of poisoning appear and disappear and time to death, especially where there is a tendency to develop toxic symptoms late.
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Regulation Information
| Citation | 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 | 10.11.1998 |
|---|---|
| Effective from | 01.01.1999 |
| Effective until | - |
| Status | Valid |
Legal Areas:
Administrative law
Health
The regulation text is for informational purposes only.
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