Decree No. 443 / 2004 Coll.
Decree laying down the basic methods for testing the toxicity of chemicals and chemical products
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443
DECLARATION
of 16 July 2004
laying down the basic methods for testing the toxicity of chemicals and chemical products
The Ministry of Health provides pursuant to § 8 (5) (b) of Act No. 356 / 2003 Coll., on Chemicals and Chemical Products and amending certain laws:
Subject matter
(1) This Decree incorporates the relevant provisions of the European Communities (1), (2), (3), (4), (5), (6), (7), (7a) and establishes basic methods for testing the hazardous properties of a chemical or a chemical preparation for which a substance or a preparation is classified as highly toxic, toxic, harmful, corrosive, irritating, sensitising, carcinogenic, mutagenic or toxic to reproduction8 (hereinafter referred to as "toxicity of chemicals and preparations").
(2) This decree is without prejudice to the provisions of specific legislation governing the protection of animals (9), (10).
Test methods
(1) The list of basic methods for testing the toxicity of chemicals or chemical preparations is set out in Annex 1.
(2) The basic methods for testing the toxicity of chemicals or chemical products are set out in Annex 2.
Repeal
The following shall be deleted:
1. Decree No 251 / 1998 Coll., laying down methods of detection of toxicity of chemicals and preparations.
2. Decree No. 208 / 2001 Coll., amending Decree No. 251 / 1998 Coll., laying down methods for the detection of toxicity of chemicals and preparations.
Efficacy
This Decree shall take effect on 1 August 2004.
Minister:
Dr. Kubinyi, Ph.D. v. r.
Příloha č. 1
Annex No 1 to Decree No 443 / 2004 Coll.
List of basic methods for testing the toxicity of chemicals or preparations
| B.1 bis | Akutní toxicita orální - metoda fixní dávky |
| B.1 tris | Akutní toxicita orální - metoda stanovení třídy akutní toxicity |
| B.2 | Akutní toxicita inhalační |
| B.3 | Akutní toxicita dermální |
| B.4 | Akutní toxicita: dráždivé a leptavé účinky na kůži |
| B.5 | Akutní toxicita: dráždivé a leptavé účinky na oči |
| B.6 | Senzibilizace kůže |
| B.7 | Subakutní toxicita orální (per os) (28 denní opakovaná aplikace) |
| B.8 | Subakutní toxicita inhalační (28 denní opakovaná aplikace) |
| B.9 | Subakutní toxicita dermální (28 denní opakovaná aplikace) |
| B.10 | Mutagenita - test chromozómových aberací u savčích buněk in vitro |
| B.11 | Mutagenita - test chromozómových aberací v kostní dřeni savců in vivo |
| B.12 | Mutagenita - in vivo mikronukleus test v savčích erytrocytech |
| B.13/14 | Mutagenita - test reverzních mutací u baktérií |
| B.15 | Genové mutace - Saccharomyces cerevisiae |
| B.16 | Mitotické rekombinace - Saccharomyces cerevisiae |
| B.17 | Mutagenita - test genových mutací v savčích buňkách in vitro |
| B.18 | Poškození DNA reparace - neplánovaná syntéza DNA - savčí buňka in vitro |
| B.19 | SCE - výměna sesterských chromatid in vitro |
| B.20 | Recesivní letální mutace vázané na pohlaví u Drosophila melanogaster |
| B.21 | Test transformace savčích buněk in vitro |
| B.22 | Dominantní letální test u hlodavců |
| B.23 | Analýza chromozómových aberací u savčích spermatogonií |
| B.24 | Spot test u myší |
| B.25 | Přenosné translokace u myší |
| B.26 | Test subchronické orální toxicity (90 denní studie orální toxicity s opakovanou aplikací hlodavcům) |
| B.27 | Test subchronické orální toxicity (90 denní studie orální toxicity s opakovanou aplikací nehlodavcům) |
| B.28 | Subchronická toxicita dermální (90 denní opakovaná aplikace, studie na hlodavcích) |
| B.29 | Subchronická toxicita inhalační (90 denní opakovaná aplikace, studie na hlodavcích) |
| B.30 | Testování chronické toxicity |
| B.31 | Studie prenatální vývojové toxicity |
| B.32 | Test karcinogenity |
| B.33 | Kombinovaný test chronické toxicity/karcinogenity |
| B.34 | Reprodukční toxicita - jednogenerační test |
| B.35 | Dvougenerační studie reprodukční toxicity |
| B.36 | Toxikokinetika |
| B.37 | Pozdní neurotoxicita organických sloučenin fosforu |
| B.38 | Pozdní neurotoxicita organických sloučenin fosforu - 28 denní opakovaná expozice |
| B.39 | Test neplánované syntézy DNA v savčích jaterních buňkách in vivo |
| B.40 | Leptavé účinky na kůži |
| B.41 | Fototoxicita - test fototoxicity 3T3 NRU - in vitro |
| B.42 | Senzibilizace kůže: zkouška s vyšetřením lokálních lymfatických uzlin |
| B.43 | Zkouška neurotoxicity na hlodavcích“. |
Příloha č. 2
Annex No 2 to Decree No 443 / 2004 Coll.
Basic concepts and methods for testing toxicity of chemicals or chemical products
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.
Obvious 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
is the amount of the test substance administered. The dose is expressed as the weight of the substance (grams or milligrams) or as the weight of the test substance per unit weight of the test animal (e.g. milligrams per kilogram body weight), or as a constant dietary concentration (parts per million parts - ppm, or milligrams per kilogram of food).
1.4. Discrimination dose
is the highest of the four fixed dose levels that can be administered without causing death (including humane culling) related to the administered substance.
1.5 Posology
is the general term comprising the dose, frequency and total duration of administration.
1.6 LD50 (mean lethal dose)
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 (mean mortality 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 an abbreviation for "No Observed Adverse Effect Level 'and is the highest dose or exposure concentration of a substance at which no statistically significant adverse effect on the body is observed compared to the control group.
1.9 LOAEL
is an abbreviation for "Lowest Observed Adverse Effect Level" and is the lowest dose or exposure concentration of a substance at which a statistically significant adverse effect on the body is still observed compared to the control group.
Repeated dose toxicity / 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 (tens of days) of expected life expectancy of the species concerned.
1.11 Maximum tolerated dose (MTD - 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.
1.12 Skin irritation
is the induction of inflammatory changes on the skin after application of the test substance.
1.13 Eye irritation
is the induction of eye changes after application of the test substance to the surface of the eye.
1.14 Skin sensitisation (allergic contact dermatitis)
is an immunologically induced skin reaction to the test substance.
1.15 Skin corrosion
is the induction of irreversible tissue damage by action of the test substance for a period of 3 minutes to 4 hours.
Toxickinetics
is the study of absorption, distribution, metabolism and excretion of test substances.
Absorption
denotes the process (s) by which the applied substance enters the body.
1.18 Excretion
denotes the process (s) by which the applied substance or its metabolites are removed from the body.
Distribution
denotes the process (s) by which the absorbed substance or its metabolites are distributed in the body.
Metabolism
denotes the process (s) by which the chemical structure of the applied substance is changed in the body by enzymatic or non-enzymatic reactions.
GHS
A globally harmonised classification system for chemicals and mixtures. Joint project of the Organisation for Economic Cooperation and Development (human health and the environment), the United Nations Expert Committee on the Transport of Dangerous Goods (Physico-chemical Properties) and the International Labour Organisation (Hazard Information), coordinated by the Inter-Organisation Programme for Good Chemical Management (IOMC).
1.22 Approaching death
when a condition of agony or death is expected before the next scheduled observation period. Symptoms indicative of this condition in rodents may include convulsions, side position, lying position and tremor. 7a)
1.23.
denotes the maximum permissible dose (2000 or 5,000 mg / kg).
1.24 Agony
indicates the condition before death or inability to survive even when the test animal is being treated. 7a)
1.25 Predictable mortality
the presence of clinical signs indicative of imminent death, the time at which the mortality can be predicted and less than the time of the expected termination of the experiment. 7a)
1.26 Late mortality
means that the test animal does not die or show signs of dying within 48 hours, but dies later within a 14-day observation period.
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.52), 3), 4), 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 shall be given to methods which use the smallest number of animals and minimise the suffering of the animal, such as the fixed dose method (method B.1 bis) 2) and the method for determining the acute toxicity class (method B.1 tris) 4). 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 shall be used or fewer animals may be used.
The repeated application toxicity test (methods B.7,4) B.82 and B.92) shall be assessed for toxic effects resulting from repeated exposure. Clinical observations of animals are important to obtain as much information as possible. These tests will help identify target organs of toxicity and toxic and non-toxic doses. Long-term studies require examination of these aspects to greater depth (methods B.26,7) B.27,7) B.28,1) B.29,1) B.301) and B.331).
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 and / or number.
The mutagenic activity of the substance is determined in vitro for bacterial gene (point) mutations (method B.13 / 14) 5) or structural chromosome aberrations in mammalian cells (method B.10) 5.
In vivo procedures such as micronucleus test (method B.12) 5) or bone marrow chromosome metaphase analysis (method B.11) 5 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.15,1) B.16,1) B.17,5) B.18,1) B.19,1) B.20,1) B.21,1) B.22,1) B.23,5) B.241) and B.251) both eukaryote systems in vivo and in vitro 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.
Certain general principles for testing strategy are laid down in Directive 93 / 67 / EEC.
Methods for further research are listed below according to their main genetic effect:
Genetic (point) mutation detection studies
(a) Forward or reverse mutation study using eukaryotic micro-organisms (Saccharomyces cerevisiae) (method B.15) .1)
b) In vitro test for forward mutations in mammalian cells (method B.17). 5)
c) Sex-bound recessive lethal mutation test in Drosophila melanogaster (method B.20) .1)
d) In vivo somatic cell mutation test, mouse spot test (method B.24) .1)
Chromosome aberration detection studies
a) Cytogenetic test in in vivo mammals. The in vivo analysis of bone marrow cell metaphase chromosomes should be considered if not included in the baseline test (method B.11) .5) Another option is cytogenetic analysis of germ cells in vivo (method B.23). 5)
b) Cytogenetic test in mammalian cells in vitro, unless it has been included in the baseline test (B.10). 5)
c) Dominant lethal test in rodents (method B.22) .1)
(d) Hereditary translocation test in mice (method B.25) .1)
Genotoxic effects - DNA effects
Genotoxicity is a general concept for potentially harmful effects on genetic material which are not necessarily associated with mutagenicity. Genotoxicity indicator may be caused by DNA damage without direct evidence of mutation.
The following methods using eukaryotic microorganisms or mammalian cells are suitable for such studies:
a) Mitotic recombination in Saccharomyces cerevisiae (method B.16) .1)
b) Damage to DNA repair - unplanned DNA synthesis in mammalian cells - in vitro (method B.18) .1)
c) Exchange of sister chromatids in mammalian cells - in vitro (method B.19) .1)
Alternative methods for investigating carcinogenic potential
There are 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) .1).
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 chromosomal aberrations such as a mouse translocation test (method B.25) .1) of these methods can be used to estimate the potential genetic risk of the substance to humans. Due to the complexity of these tests and the large number of animals needed, especially for a specific locus test, it is only for serious reasons that a decision should be made for 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.7,4) and a longer-term repeated dose study 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) 1) or often in a combined chronic toxicity / carcinogenicity study (method B.33) .1)
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) .1).
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) 1 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 appropriately taking into account the relevant 28-day test method points.
If a three-generation reproductive test is required, the described method for the two-generation reproductive test (method B.35) 1 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) (4) also includes an assessment of the neurotoxic effect: this method stresses the need for careful clinical observation of animals in order to obtain as much information as possible. The method helps to 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 phosphorus compounds were found to cause late neurotoxicity, which is assessed by methods B.374) and B.384) after single or repeated administration of the substance.
7 IMMUNOTOXICITY
Immunotoxicity is assessed in different ways, such as immunosuppression or enhanced response to the immune system, resulting in either hypersensitivity or induced autoimmune. The repeated application toxicity test (method B.7), 4) includes the determination of immunotoxic effects. The method helps to detect chemicals with immunotoxic potential requiring further in-depth examination of this aspect.
8 TOXICOKINETIC
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) .1)
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.
10 SUBSTANCES TESTED
The composition of the test substance, including significant 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 where possible significant impurities) in the dosing medium and biological material is preceded by the initiation of the study.
All information concerning the identification, physicochemical properties, purity and behaviour of the test substance shall be included in the final test report.
11 FOR ANIMAL TREATMENT
For toxicological tests, strict monitoring of the conditions of the environment in which experimental animals are kept and proper care of animals is a fundamental condition. The conditions of protection, breeding, use including feeding of animals are laid down in specific legislation governing the protection of animals. 9)
11.1 Breeding conditions
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 toxicity studies, the temperature and humidity should be 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 the same 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.
11.2 Conditions of feeding
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 ingredients which have a proven effect on toxicity must not be present in the concentrations in which this effect would occur.
12 ANIMAL PROTECTION
When developing testing methods, the necessary attention shall be paid to the protection of animals in accordance with the legislation governing the protection of animals. 9) The following are briefly given some examples, this list is not exhaustive. Alternative methods should be taken into account for the determination of acute oral toxicity: a fixed dose procedure and an acute toxicity class method. A fixed dose procedure does not use death as specific criteria and requires a smaller number of animals. The method of determining the acute toxicity class requires on average 70% of animals less than Method B.12) for acute oral toxicity. These and alternative methods result in less pain and stress than classical methodology.
13 ALTERNATIVE TESTS
Alternative methods 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.
In accordance with the relevant legislation governing the protection of animals (9), it is necessary to verify in registries of internationally validated and recognised alternative methods whether there is an alternative method for the intended experiment where an animal may not be used.
Where such methods are available, their use for risk characterisation, subsequent hazard classification and labelling shall be taken into account wherever possible.
14 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 according to human health effects based on the properties 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 bis ACUTE TOXICITY ORAL - METHOD OF FIXED BENEFIT
1 The methodology is in accordance with the law of the European Communities. 7a)
1.1 INTRODUCTION
The use of only moderate toxic doses in the main study is essential and doses that are expected to be lethal should not be administered. It is also not necessary to administer doses known to cause significant pain and suffering due to corrosive or severely irritating effects. Dying experimental animals or experimental animals which appear to show signs of pain or significant and persistent suffering shall be humanely killed and evaluated as dead animals in the interpretation of the results in testing 7a).
The method provides information on hazardous properties and allows classification and classification of the substance according to the Globally Harmonised System (GHS) classification of chemicals that cause acute toxic7a.
The test laboratory should take into account all available information on the test substance before carrying out the study. This information includes the identity and chemical structure of the substance, its physicochemical properties, the results of other in vitro or in vivo toxicity tests, toxicological data on structurally related substances and the expected use of the substance. This information is necessary to ensure that all concerned are convinced that the test is relevant for the protection of human health and helps in the selection of the appropriate starting dose.
1.2 DEFINITIONS
Acute oral toxicity: refers to adverse effects occurring after oral administration of one or more doses of the substance within 24 h.
1.3. TERMS OF THE TEST METHOD
A fixed dose of 5, 50, 300 and 2,000 mg / kg should be administered to a group of same-sex experimental animals (exceptionally, a further fixed dose of 5,000 mg / kg may be considered, see section 1.6.2). The initial dose level should be chosen on the basis of an indicative study as a dose that is expected to induce some signs of toxicity without causing serious toxic effects or mortality. 7a) Other groups of experimental animals may receive higher or lower doses depending on the presence or absence of signs of toxicity or death. According to this procedure, it is continued until the dose causes obvious toxicity or more than one death or if no effects are observed at the highest dose or if mortality occurs at the lowest dose.
1.4 DESCRIPTION OF THE TEST METHOD
1.4.1. Selection of experimental animals
The preferred rodent species is the rat, but other rodent species may also be used. They are usually used by females (7a). This is because the review of literature on conventional LD50 tests shows that there is usually a small difference in sensitivity between sexes, but in cases where differences are observed, females are generally slightly more sensitive 7a). However, if knowledge of the toxicological or toxickinetic properties of structurally related chemicals indicates that males are likely to be more sensitive, this sex shall be used. If the test is carried out in males, appropriate justification shall be provided.
Young healthy adult experimental animals of commonly used laboratory strains are used. The female must be nulliparous and not pregnant. Each test animal must be 8 to 12 weeks old at the beginning of dosing and its weight should be within ± 20% of the mean weight of the test animals given the previous dose.
1.4.2. Breeding and feeding conditions
The temperature in the experimental animal room should be (22 ± 3) ° C. Although the relative humidity should be at least 30% and preferably not exceed 70% except for room cleaning time, the target should be 50-60%. The lighting should be artificial, 12 h light and 12 h dark should be alternated, unless daylight is in the breeding room. The normal feed intended for experimental animals from a registered manufacturer with an unlimited supply of drinking water may be used for feeding. Test animals may be kept in cages in groups by dose in accordance with the relevant legislation10), unless otherwise specified by the experiment project, but the number of test animals in the cage must not prevent the uninterrupted observation of each test animal.
1.4.3 Preparation of experimental animals
Experimental animals shall be selected with regard to species, sex and age in accordance with the provisions of Section 15 of the Specific Legislation (10) and identified in such a way as to identify individual experimental animals. Paragraph 10 of the Specific Legislation (10) provides for the period of curtailment.
1.4.4 Preparation of doses
In general, the test substance should be administered in constant volume at all levels of the test dose by adjusting the concentration of the dosed product. However, in cases where a liquid end product or mixture is to be tested, the use of an undiluted test substance, i.e. with a constant concentration, may have greater severity for the assessment of the subsequent risk of that substance. In either case, the maximum dose volume must not be exceeded. The maximum volume of liquid that can be administered at one time depends on the size of the test animal. In rodents, the volume should normally not exceed 1 ml / 100 g body weight: however, for aqueous solutions, a dose of 2 ml / 100 g body weight is considered. Taking into account the composition of the dose preparation, the use of aqueous / suspension / emulsion is recommended in all cases where possible, then, in order of preference, the use of the solution / suspension / emulsion in oil (e.g. maize oil) and, finally, the solution in other vehicles. For vehicles other than water, their toxicological characteristics shall be known. Doses must be prepared shortly before administration if the stability of the product is not known during the period of use and does not appear acceptable.
1.5. PROCEDURE
1.5.1.
The test substance is administered by a single dose probe using a stomach tube or a suitable intubation tube. If, exceptionally, a single dose cannot be administered, the dose may be administered in smaller quantities over a maximum period of 24 h.
Prior to administration of the test substance, test animals should not receive feed (e.g. rats should not be administered overnight, mice 3 - 4 h), but water is kept. After the starvation period, the test animals are weighed and given the test substance. After administration of the substance, access to feed is avoided for another 3-4 h in rats or 1-2 h in mice. If the substance is administered in parts over a period of time, it may be necessary to provide feed and water to experimental animals according to the length of the period.
1.5.2 Indicative studies
The aim of the indicative study is to allow the choice of an appropriate starting dose for the main study. The test substance is administered gradually to individual animals according to the development diagram in Table 1. The indicative study is completed as soon as the starting dose for the main study can be determined (or if mortality is observed at the lowest fixed dose).
The initial dose of the indicative study shall be chosen from fixed dose levels of 5, 50, 300 and 2000 mg / kg as a dose expected to cause obvious toxicity, based, if possible, on evidence from in vivo and in vitro data from the same chemical and structurally related substances. In the absence of such information, the starting dose is 300 mg / kg.
A minimum of 24 h should be allowed before administration of the dose to any other experimental animal. All test animals are observed for at least 14 days.
In exceptional cases and only if justified by specific regulatory needs, the use of an additional highest dose level of 5000 mg / kg may be considered (see Table 3). In view of the good treatment of experimental animals, experiments in experimental animals within GHS category 5 (2,000 - 5,000 mg / kg) are not recommended and should only be considered if there is a strong likelihood that the results of such an experiment are of direct importance for the protection of human health, animal health or the environment.
In cases where the test animal in which the substance is tested at the lowest fixed dose level (5 mg / kg) dies in an indicative study, the study usually ends and assigns the substance to GHS Category 1 (as shown in Table 1). However, if further confirmation of classification is required, the following optional supplementary procedure may be carried out: a dose of 5 mg / kg is administered to the second test animal. If this second test animal dies, GHS Category 1 will be confirmed and the study terminated immediately. If the second test animal survives, a dose of 5 mg / kg should be administered to up to three other test animals. Since the risk of mortality will be high, the substance should be administered gradually to experimental animals to ensure good treatment of experimental animals. The time interval between doses administered to individual experimental animals should be sufficient to determine whether the test animal to which the substance was previously administered is likely to survive. If the second test animal dies, the sequence of administration is immediately terminated and the substance will not be administered to any other test animal. Since the occurrence of a second mortality (regardless of the number of test animals on which the substance was tested at the time of termination) falls within the result A (2 or more mortality), the classification rule in Table 2 at a fixed dose of 5 mg / kg (Category 1 if two or more mortality or Category 2 if there is no more than 1 mortality) is followed. In addition, Table 4 provides guidance on the classification under the EU scheme until a new globally harmonised system (GHS) is introduced.
1.5.3 Main studies
1.5.3.1. Number of test animals and dose levels
The steps to be taken after the initial dose level tests are shown in the development chart in Table 2. It will be necessary to select one of the three procedures: either terminate the test and determine the appropriate hazard classification class, perform higher fixed dose tests or lower fixed dose tests. However, in view of the protection of experimental animals, the main study does not reapply the dose level that led to death in the indicative study (see Table 2). Experience has shown that the most likely result at the starting dose level will be that the substance can be classified without further testing.
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Regulation Information
| Citation | Decree No. 443 / 2004 Coll., laying down the basic methods for testing the toxicity of chemicals and chemical products |
|---|---|
| Regulation Type | Order |
| Author | - |
| Collection | Code of Laws |
| Date of Promulgation | 30.07.2004 |
|---|---|
| Effective from | 01.08.2004 |
| Effective until | - |
| Status | Valid |
The regulation text is for informational purposes only.
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