Experimental toxicology

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EXPERIMENTAL TOXICOLOGY

Növényvédelmi higiéniai és toxikológiai ismeretek

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1. Introduction to experimental toxicology 2. Acute toxicity test on laboratory animals 3. Subacute and subchronic toxicity tests on

laboratory animals

4. Chronic toxicity tests on laboratory animals 5. Mutagenicity tests

6. Ecotoxicological tests on terrestrial organisms

7. Ecotoxicological tests on aquatic organism Topics

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Animal experimentation

Any use of an animal for experimental or other scientific purposes which may cause it pain, suffering, distress or lasting harm.

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Purposes of animal experimentations:

 the development, manufacture, quality, effectiveness and safety testing of drugs, foodstuffs and other substances or products

 for the avoidance, prevention, diagnosis or treatment of disease, ill-health or other abnormality or their effects on man, animals or plants

 the protection of the natural environment in the interests of the health or welfare of man or animal

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Laws

 European Convention for the Protection of Vertebrate Animals used for Experimental and Other Scientific Purposes (ETS No. 123)

 Council directive of 24 November 1986 on the approximation of laws, regulations and administrative provisions of the Member States regarding the protection of animals used for experimental and other scientific purposes (86/609/EEC)

 Directive 2010/63/EU of the European Parliament and of the Council of 22 September 2010 on the protection of animals used for scientific purposes

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Protection of any live non-human vertebrate (but excluding foetal or embryonic forms).

Straying animals of a domesticated species shall not be used in procedures.

Restrictions of the primates using.

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Persons who carry out procedures, or take part in procedures, or take care of animals used in procedures, including supervision, shall have had appropriate education and training.

It must be requested permission to do any experiment  valid for 5 years (Animal Testing Work Committee, Government Office Food Chain Safety and Animal Health Directorate, National Animal Ethics Advisory Committee).

Checking is the responsibility (task) of the state.

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Internationally accepted experimental methods

 OECD guidelines

 EPA guidelines

 GLP requirements

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General rules of animal experimentations

 user establishment must be registered

 experiments with animals are replaced, wherever possible, by an alternative method which is scientifically satisfactory

 the number of animals used in projects is reduced to a minimum without compromising the quality of results

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General rules of animal experimentations

 a procedure shall be performed under general or local anaesthesia or analgesia designed to eliminate as far as practicable pain, suffering, distress or lasting harm applied throughout the procedure unless he use of anaesthesia or analgesia is incompatible with the aim of the procedure

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Experimental animal

Any live vertebrate animal used or intended for use in research, testing, or teaching.

Laboratory animal

These refer to any animal bred or maintained for use in animal experiments (including those during transport to facilities).

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Ideal animal model

 visible circulatory system and vital process

 similarity

 extrapolating ability

 reproducibility

 availability

 tractability

 size and fecundity

 lifetime

 keeping

 inexpensive

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Ideal experimental animal (IEA)

  30 g

 hairless

 transparent skin

 rudimentary claw, tooth

 tame, friendly

 big ear and easy blood sampling

 temperature control

 homesickness

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Selecting Experimental Animals

An animal model is a living organism in which normal biological processes can be studied, or in which a spontaneous or induced pathological process can be investigated. To be effective, the process being modelled should resemble closely the analogous process in humans (or some other species) in one or more ways.

Some important criteria for animal models are:

 relevance to the problem being studied

 the accuracy with which the model reflects all or some important aspects of the problem

 the model's predictivity

 the model's availability

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Selecting Experimental Animals

Species selection has important implications for the quality of animal-based science, animal welfare and the 3Rs.

The two most important factors in the selection of species to be used are:

- the objectives of the research - the species chosen should be the most likely to produce satisfactory results - the need to minimise potential harm to the animals involved, in accordance with relevant legislation

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Frequent laboratory animals

 mouse or white mouse (NUDE, SCID)

 brown rat or white rat

 syrian (golden) hamster

 guinea pig

 european rabbit or domestic rabbit

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Ecotoxicological test organisms

Terrestrial environment:

birds (mallard duck, quail…)

non target arthropods (bees…)

soil organisms (earthworms…)

Aquatic environment:

fishes (trout…)

invertebrates (water fleas…)

algae

aquatic plants

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Alternatives to animal experimentation

 in vitro techniques

 use of lower species

 immunological techniques

 quantitative structure-activity relationship analysis

 mathematical modelling of physiological processes

 human models

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1. In vitro techniques

 organotypic cultures

 cell cultures

Eg HET-CAM test

2. Use of lower species

Bacteria, moulds, insects, molluscs Eg AMES test, LAL test

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3. Immunological techniques Eg ELISA

4. Quantitative structure-activity relationship analysis It is well known that a relationship exist between molecular structure, the physical-chemical properties and the biological activities of compounds. Using this knowledge, it is possible to predict the biological activities of many types of new compounds.

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5. Mathematical modelling of physiological processes Many of the processes which take place in living organism can be expressed as mathematical equations, so mathematical models of many physiological, biochemical, pathological and toxicological events can be developed.

6. Human models

There are some occasions when humans can justifiably be used as a test subjects. One such example is in drug development (clinical trial phase).

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Housing of experimental animals

Creation of animal welfare

(suitable air, temperature controlled, regulated light, free of germs or aseptic environment ),

Shutting out of pathogens (viruses, bacteriums, parasites).

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The role of hygienic barrier

 pathogen free feed, drinking water and bedding material

 sterilized cages and equipments

 pathogen free air

 pathogen free human

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Hygiene levels

1. Axenic

Refers to animals derived by caesarean section (or embryo transfer) reared and maintained in an isolator by gnotobiotic techniques. It implies that the animals demonstrably free of associated forms of life, including viruses, bacteria, fungi, protozoa, and other saphrophytic or parasitic organisms.

2. Specific Pathogen Free (SPF)

Refers to animals with no evidence of certain

microorganisms. The term should be related to a specific list of organisms and a specific set of tests/methods used to

detect the organisms.

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Hygiene levels

3. Caesarean Originated Barrier Sustained (COBS)

Refers to animals obtained by caesarean rederivation and maintained behind a barrier against specific rodent

pathogens. It is a form of SPF.

4. Virus Antibody Free

Implies that the animals free of antibodies to viruses,

specifically rodent viruses. The term is a variation of SPF, as it is related to a specific list of viruses, and the method of

detection implied is serology.

5. Conventional (MD)

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Elements of laboratory environment

1. Abiotic elements

1.1. Physical conditions Environmental elements

 light: intensity, colour, periodicity

 temperature sensation: temperature, humidity, ventilation

 odours: animals, chemicals

Space allowance

 cage (material and construction)

 motion and play

 bedding

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1.2. Chemical conditions Feed

2. Biotic elements 1. Intraspecies

 density of population

 socio-psychological (Lee-Boot-, Whitten-, Bruce-effect) 2. Interspecies

 animal - human

 animal - microorganism

 animal - animal

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mouse rat Guinea pig

Temperature, ^oC 20-24 20-24 20-24

Relative humidity, % 50-60 60 50

Light/dark, hour 14/10 12-14/10-12 14/10

Min. cage size, cm² 180 350 600

Body weight adult male/female, g

20-40/25-40 300-500/250-300 900-1000/700-900

Lifetime, year 1-2 2-3 5-6

Body temperature, ^oC 36,5-38 37,5-38,5 38-40

Gestation period, day 19 (18-21) 21-23 68 (59-72)

Litter size 6-12 6-12 1-6

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rabbit dog pig

Temperature, ^oC 15-21 15-21 17-24

Relative humidity, % 50-60 40-60 40-60

Light/dark, hour 12/12 -- --

Min. cage size, cm² 1400-3600 0,75-1,75 0,35-0,8 Body weight adult

male/female, kg

2-5/2-6 10-80/10-60 200-300/150-220

Lifetime, year 5-6 10-15 14-18

Body temperature, ^oC 38,5-39,5 38-39 38-40

Gestation period, day 30 (28-35) 63-67 110-118

Litter size 4-10 3-6 11-16

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Hygiene of animal laboratory

Hygiene

Cleanliness to preserve the health.

Disinfection

A process that eliminates a specific species of infectious or other undesired microorganism, but not necessarily bacterial spores, in the inanimate environment only.

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Levels of disinfection

Bacteriostat effect

An effect that prevents the growth of bacteria but that does not necessarily kill them or their spores.

Sporocidal effect

An effect that kills the spores of bacteria.

Bactericidal effect

An effect that kills bacteria.

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Levels of disinfection

Virucidal effect

An effect that cause the loss of virulence.

Fungicidal effect

An effect that destroys fungi and/or fungal spores pathogenic to humans or other animals in the inanimate environment.

Parasitocidal effect

An effect that destroys pathogenic parasites.

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Methods of disinfection

 physical

 chemical

 combined

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The sources of infection

 other laboratory animals

 biological materials (production)

 pet animals

 stuff (vector)

 visitors (veterinarian, inspectors…)

 materials and equipments (feed, bedding material)

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Feeding of experimental animals

Any animal used or intended for use in a procedure shall be provided with accommodation, an environment, at least a minimum degree of freedom of movement, food, water and care, appropriate to its health and well-being.

(ETS No. 123)

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Effects of food intake on the results of experiments

1. Direct

 difference in the intake of test material

2. Indirect

 the effect of food intake on parameters (eg tumor development, lifetime)

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Organization of work in the animal laboratory

 animal needs

 space allowance (cage)

 feed, drinking water and bedding material

 recording

 order of works

 labour organization

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Routes of administration

1. Enteral administration

2. Parenteral administration 3. Topical administration

4. Inhalation administration

5. Extraordinary administration

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Enteral administration

 oral (p.o.)

 gavage, tube

 rectal (p.r.)

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Parenteral administration

 percutaneous (p.c.)

 intradermal (i.d.)

 subcutaneous (s.c.)

 intramuscular (i.m.)

 intravenous (i.v.)

 intraperitoneal (i.p.)

 intracerebral (i.c.)

 intraocular (i.oc.)

 sub or epidural (e.du.)

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Parenteral administration

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Parenteral administration

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Topical administration

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Inhalation administration and extraordinary administration

 intranasal (i. nas.)

 intratracheal (i.trach.)

 transplantation

 implantation

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Blood sampling

 generally:

jugular vein (v. jugularis) and saphenous vein (v.

saphena) (v. femoralis)

 rabbit: ear vein (v. auricularis marginalis)

 rat, mouse: tail vein (v. coccigea lat.) (v. caudalis)

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Durations of animal experiments

 acute: max. 14 days

 subchronic: max. 90 days

 chronic: 90 days - 2 years

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Euthanasia

Euthanasia is the act of humanely killing animals by methods that induce rapid unconsciousness and death without pain or distress.

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Animals are euthanased in laboratories or breeding establishments for the following reasons:

 at the termination of studies, to provide tissues for scientific purposes

 when pain, distress or suffering are likely to exceed designated levels

 where the health or welfare of animals are grounds for concern

 when animals are no longer used for breeding

 when stock are not required for certain reasons, such as sex preference utilisation

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To meet the objectives of euthanasia procedures must:

avoid distress and produce rapid loss of consciousness until death occurs

 be reliable, reproducible and irreversible

 be appropriate for age, species, and health of the animal

 require minimum restraint

 be compatible with the objectives of the study

 be simple to administer

 be safe for the operator

 be aesthetically acceptable to the operator, where at all possible

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Methods of Euthanasia

1. Physical methods

 penetrative and non-penetrative captive bolt device

 firearm with free projectile

 percussive blow to the head

 electrical stunning

 cervical dislocation

 decapitation

 maceration

 microwave radiation

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Methods of Euthanasia

2. Chemical methods

 carbon dioxide

 non-explosive inhalant anesthetics (halothane, enflurane, isoflurane)

 benzocainum,

 trikain methane sulfonate

 ethomidate and methomidate

 kvinaldin

 barbiturates

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Purpose

• In the assessment and evaluation of the toxic characteristics of a substance, determination af acute toxicity is useful where exposure by the oral, dermal or inhalation route is likely.

• It provides information on health hazard likely to arise from a short-term exposure by the oral, dermal or inhalation route.

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Purpose

• Data from an acute toxicity study may serve as a basis for classification and labelling.

• It is an initial step in establishing a dosage regimen in subchronic and other studies and may provide information on the absorption and the mode of toxic action of a substance by oral, dermal or inhalation route.

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Definitions

• Acute oral toxicity is the adverse effects occurring within a short time of oral application of a single dose of a test substance.

• Acute dermal toxicity is the adverse effects occuring within a short time of dermal application of a single dose of a test substance.

• Acute inhalation toxicity is the adverse effects occuring within a short time of inhalation application of a single dose of a test substance.

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Definitions

• The LD₅₀ (median lethal dose), oral or dermal, is a statistically derived single dose of a substance that can be expected to cause death in 50 per cent of treated animals when applied by oral or dermal route. The LD₅₀ value is expressed in terms of weight of the test substance per unit weight of test animal (mg/body weight kg).

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Definitions

• The LC₅₀ (median lethal concentration) is a statistically derived single concentration of a substance that can be expected to cause death in 50 per cent of treated animals when applied by inhalation route. The LC₅₀ value is expressed in terms of weight of the test substance per unit cubical contents of the air (mg/m³).

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Acute oral toxicity test (for determining the LD₅₀value) Experimental animals:

 rats

 mice

 rabbits

Dose levels:

 at least 3

 control group (vehicle)

Number of animals:

 5/sex/group

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Route of administration:

 oral administration

by gavage using a stomach tube

The observation period should be at least 14 days after the treatment.

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Dose-response relationship

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Parameters

 body weight

 food consumption

 clinical signs

 gross pathology:

• died animals

• surviving animals

• measuring organ weight (optional)

• histopatholpgy (optional)

A study of acute toxicity by the oral route and determination of an oral LD₅₀provides an estimate of the relative toxicity of a substance by the oral route of exposure.

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Alternative acute oral toxicity tests

1. Up and Down Method

• starting dose: preliminary estimate of the LD₅₀

• the first animal receives a dose a step below the level of the best estimate of the LD₅₀

• if the first animal survives, the dose for the next animal is increased by a factor of 1.3 times the original dose

• if the first animal dies, the dose for the next animal is decreased by a similar dose progression

• each animal should be observed for up to 48 hours

• the testing stops:

• 3 consecutive animals survive at the upper bound

• 5 reversals occur in any 6 consecutive animals tested

• At least 4 animals have followed the first reversal and specified likelihood-ratios exceed the critical value

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Alternative acute oral toxicity tests

2. The Fixed Dose Procedure

• sighting study with several dose levels 1-1 animal/sex/dose,

• suggested doses: 5, 50, 300, 2000 mg/b. w. kg

• main study 5-5 animal/sex in the selected dose

• this study avoids using death as an endpoints, and relies instead on the observation of clear signs of toxicity at one of a series of fixe dose levels

• the action to be taken following testing at the starting dose level indicated by the flow charts

• the method provides information on the hazardous properties and allows the substance to be ranked and classified

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Acute dermal toxicity test Experimental animals:

 rats

 rabbits

 guinea pigs

Dose levels:

 at least 3 (limit test 2000 mg/kg b. w.)

 control group (vehicle)

Number of animals:

 5/sex/group

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 dermal administration

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Parameters

 body weight

 clinical signs

• died animals

• histopathology (optional)

A study of acute toxicity by the dermal route and determination of a dermal LD₅₀ provides an estimate of the relative toxicity of a substance by the dermal route of exposure.

Acute toxicity test on laboratory animals

86

(87)

TÁMOP-4.1.2.A/2-10/1-2010-0012

Acute toxicity test on laboratory animals

87

(88)

TÁMOP-4.1.2.A/2-10/1-2010-0012

Acute toxicity test on laboratory animals

88

(89)

TÁMOP-4.1.2.A/2-10/1-2010-0012

Acute inhalation toxicity test Experimental animals:

 rats

 rabbits

 mice

Dose levels:

 at least 3

 (control group vehicle)

Number of animals:

 5/sex/group

Acute toxicity test on laboratory animals

89

(90)

TÁMOP-4.1.2.A/2-10/1-2010-0012

 nose-only administration

4 (up to 6) hours exposure period

Acute toxicity test on laboratory animals

90

(91)

TÁMOP-4.1.2.A/2-10/1-2010-0012

Inhalation chamber

Acute toxicity test on laboratory animals

91

(92)

TÁMOP-4.1.2.A/2-10/1-2010-0012

Exposure conditions

 particle size distribution

To allow for exposure of all relevant regions of the respiratory tract, aerosols with mass median aerodynamic diameters (MMAD) ranging from 1 to 4m with a geometric standard deviation in the range of 1.5 to 3.0 are recommended.

Acute toxicity test on laboratory animals

92

(93)

TÁMOP-4.1.2.A/2-10/1-2010-0012

Monitoring of exposure conditions

 chamber airflow

 chamber temperature and relative humidity

 nominal concentration

 actual concentration

Acute toxicity test on laboratory animals

93

(94)

TÁMOP-4.1.2.A/2-10/1-2010-0012

Rotation Brush Generator

Acute toxicity test on laboratory animals

94

(95)

TÁMOP-4.1.2.A/2-10/1-2010-0012

Cascade impactor

Acute toxicity test on laboratory animals

95

(96)

TÁMOP-4.1.2.A/2-10/1-2010-0012

Acute toxicity test on laboratory animals

96

(97)

TÁMOP-4.1.2.A/2-10/1-2010-0012

Parameters

 body weight

 clinical signs

• died animals

• histopathology (optional)

A study of acute toxicity by the inhalation route and determination of the LC₅₀ provides an estimate of the relative toxicity of a substance by the inhalation route of exposure.

Acute toxicity test on laboratory animals

97

(98)

TÁMOP-4.1.2.A/2-10/1-2010-0012

Acute toxicity test on laboratory animals

98

(99)

TÁMOP-4.1.2.A/2-10/1-2010-0012

Alternative acute inhalation toxicity test

1. Acute Toxic Class Method

• a stepwise procedure

• main study 3-3 animal/sex in the selected dose

• the testing schemes for gases 100, 500, 2500, 20000 ppm/4h

• the testing schemes for vapours 0.5, 2, 10, 20 mg/L/4h

• the testing schemes for aerosols 0.05, 0.5, 1, 5 mg/L/4h)

• depending on the number of dead animals, the test procedure follows the flow charts until a categorisation can be made

• the method provides information on the hazardous properties and allows the substance to be ranked and classified

Acute toxicity test on laboratory animals

99

(100)

TÁMOP-4.1.2.A/2-10/1-2010-0012

Acute eye irritation/corrosion test

Purpose

• In the assessment and evaluation of the toxic characteristics of a substance, determination of the irritant and/or corrosive effect on eyes of mammals is an important initial step.

• Information derived from this test serves to indicate the possible existence of hazards likely to arise from exposure of the eyes and associated mucous membranes to the test substance.

Acute toxicity test on laboratory animals

10 0

(101)

TÁMOP-4.1.2.A/2-10/1-2010-0012

Acute eye irritation/corrosion test

Experimental animals:

• albino rabbits Dose levels:

• 0.1 ml / g

• untreated eye  control Number of animals: 3

Physical chemical properties pH ≤ 2

pH ≥ 11,5

Acute toxicity test on laboratory animals

10 1

(102)

TÁMOP-4.1.2.A/2-10/1-2010-0012

Both eyes of each experimental animal provisionally selected for testing should be examined within 24 hours before testing starts.

Application: in the conjunctival sac.

Irrigation: at 24 hours a washout may be used if considered appropriate.

Measuring body weight.

Acute toxicity test on laboratory animals

10 2

(103)

TÁMOP-4.1.2.A/2-10/1-2010-0012

Clinical observations: 1, 24, 48, 72 hours, 4 and 7 days.

Gross pathology.

Histopathology 3 eyes/group (optional).

Acute toxicity test on laboratory animals

10 3

(104)

TÁMOP-4.1.2.A/2-10/1-2010-0012

Grades for ocular lesions

Score

I. Conjunctivae

A. Redness

• Blood vessels normal 0

• Some blood vessels definitely hyperaemic 1

• Diffuse, crimson colour, individual vessels not easily

discernible 2

• Diffuse beefy red 3

Acute toxicity test on laboratory animals

10 4

(105)

TÁMOP-4.1.2.A/2-10/1-2010-0012

Grades for ocular lesions

Score B. Chemosis

• No swelling 0

• Any swelling above normal 1

• Obvious swelling with partial eversion of lid 2

• Swelling with lids about half closed 3

• Swelling with lids more than half closed 4

Acute toxicity test on laboratory animals

10 5

(106)

TÁMOP-4.1.2.A/2-10/1-2010-0012

Grades for ocular lesions

Score

C. Discharge

• No discharge 0

• Any amount different from normal 1

• Discharge with moistening of the lids and hairs just

adjacent to lids 2

• Discharge with moistening of the lids and hairs, and

considerable area around the eye 3

Evaluation: (A + B + C) x 2 Max.: 20 point

Acute toxicity test on laboratory animals

10 6

(107)

TÁMOP-4.1.2.A/2-10/1-2010-0012

Acute toxicity test on laboratory animals

10 7

(108)

TÁMOP-4.1.2.A/2-10/1-2010-0012

Grades for ocular lesions

Score II. Cornea

A. Degree of density

• No ulceration or opacity 0

• Scattered or diffuse areas of opacity, details of iris

clearly visible 1

• Easily discernible translucent area, details of iris

slightly obscured 2

• Nacreous area, no details of iris visible 3

• Opaque cornea, iris not discernible through the

opacity 4

Acute toxicity test on laboratory animals

10 8

(109)

TÁMOP-4.1.2.A/2-10/1-2010-0012

Acute toxicity test on laboratory animals

10 9

(110)

TÁMOP-4.1.2.A/2-10/1-2010-0012

Grades for ocular lesions

Score B. The area of corneal opacity

• One quarter or less 1

• Greater than one quarter, but less than half 2

• Greater than half, but less than three quarter 3

• Greater than three quarter, up to whole area 4

Evaluation: A x B x 5 Max.: 80 point

Acute toxicity test on laboratory animals

11 0

(111)

TÁMOP-4.1.2.A/2-10/1-2010-0012

Grades for ocular lesions

Score III. Iris

• Normal 0

• Markedly deepened rugae, congestion, swelling, moderate circumcorneal hyperaemiai, or injection, any of these combination of any thereof, iris still

reacting to light 1

• No reaction to light, haemorrhage, gross

destruction 2

Evaluation: A x 5 Max.: 10 point

Acute toxicity test on laboratory animals

11 1

(112)

TÁMOP-4.1.2.A/2-10/1-2010-0012

Evaluation of the acute eye irritation

Total score (irritation index):

I + II + III (20+80+10) = 110 At each observation time.

Ratings:

80 – 110 very severely (super) irritating 50 - 79 severely irritating

20 - 49 moderately irritating

0 - 19 non-irritating or slightly irritating

Acute toxicity test on laboratory animals

11 2

(113)

TÁMOP-4.1.2.A/2-10/1-2010-0012

Acute dermal irritation/corrosion test

Purpose

• In the assessment and evaluation of the toxic characteristics of a substance, determination of the irritant and/or corrosive effect on the skin of mammals is an important initial step.

• Information derived from this test serves to indicate the possible existence of hazards likely to arise from exposure of the skin and associated mucous membranes to the test substance.

Acute toxicity test on laboratory animals

11 3

(114)

TÁMOP-4.1.2.A/2-10/1-2010-0012

Acute dermal irritation/corrosion test

• albino rabbits Dose levels:

• 0.5 ml / g

• untreated area of the skin

 control

Number of animals: 3

Physical chemical properties pH ≤ 2

pH ≥ 11,5

Acute toxicity test on laboratory animals

11 4

(115)

TÁMOP-4.1.2.A/2-10/1-2010-0012

Approximately 24 hours before the test, fur should be removed by close-clipping the dorsal area of the trunk of the animals.

Application: 4 hours exposure period.

Acute toxicity test on laboratory animals

11 5

(116)

TÁMOP-4.1.2.A/2-10/1-2010-0012

Clinical observations: 24, 48, 72, 96 hours.

Gross pathology.

Histopathological examination may be carried out to clarify doubtful reactions.

Acute toxicity test on laboratory animals

11 6

(117)

TÁMOP-4.1.2.A/2-10/1-2010-0012

Grading of skin reaction

A. Erythema and Eschar Formation Score

• No erythema 0

• Very slight erythema 1

• Well defined erythema 2

• Moderate to severe erythema 3

• Severe erythema (beet redness) to eschar formation preventing grading of erythema 4 Acute toxicity test on laboratory animals

11 7

(118)

TÁMOP-4.1.2.A/2-10/1-2010-0012

Grading of skin reaction

B. Oedema Formation Score

• No oedema 0

• Very slight oedema 1

• Slight oedema (edges of area well defined by definite

raising) 2

• Moderate oedema (raised approximately 1mm) 3

• Severe oedema (raised more than 1 mm and extending

beyond area of exposure) 4

Acute toxicity test on laboratory animals

11 8

(119)

TÁMOP-4.1.2.A/2-10/1-2010-0012

Evaluation of the acute dermal irritation

Total score (irritation index):

A + B = 8

At each observation time.

Ratings:

0.0 – 0.5 non-irritating

0.5 – 2.0 slightly irritating

2.0 – 5.0 moderately irritating 5.0 – 8.0 severely irritating

Acute toxicity test on laboratory animals

11 9

(120)

TÁMOP-4.1.2.A/2-10/1-2010-0012

Skin sensitisation test

• albino guinea pigs

Dose levels:

• the appropriate concentration can be determined from a preliminary study

Number of animals: 20 (+ 10 control) Application: topical

24 hours before the test, fur should be removed by close- clipping.

Subacute and subchronic toxicity tests on laboratory animals

12 0

(121)

TÁMOP-4.1.2.A/2-10/1-2010-0012

Induction: topical application

• daily, 3 times (day 0, 6-8, 13-15), 6 hours exposition (sensibilisation)

Challenge: topical application

• on day 27-29 the untreated flank of treated and control animals is cleared of hair  topical application for 6 hours

• Observation on day 29-30 and 24 hours after 

All skin reactions and any unusual findings, including systemic reactions, resulting from induction and challenge procedures should be observed and recorded.

12 1

(122)

TÁMOP-4.1.2.A/2-10/1-2010-0012

Grading of topical skin reaction

Score Reaction:

• no reaction 0

• very slight erythema 1

• slight diffuse erythema 2

• moderate to severe erythema

(with oedema or Ø) 3

Gross pathology

Histopathological examination (optional).

12 2

(123)

TÁMOP-4.1.2.A/2-10/1-2010-0012

Evaluation of the skin sensitisation

Dermal sensitisation:

0 not sensitizing

0,1-0,4 very slight sensitizer 0,5-1,9 slight sensitizer

2,0-4,9 moderate sensitizer 5,0-8,0 severe sensitizer

12 3

(124)

TÁMOP-4.1.2.A/2-10/1-2010-0012

Purpose

• In the assessment and evaluation of the toxic characteristics of a substance, determination of toxicity using repeated doses is useful where repeated exposure by the oral, dermal or inhalation route is likely.

• These tests provide information on the possible health hazard likely to arise from repeated exposure over a prolonged period of time converting post-weaning maturation and growth well into adulthood.

• These studies provide information on the major toxic effects, indicate target organs and the possibility of accumulation, and can provide an estimate of a NOAEL of exposure which can be used in selecting dose levels for chronic studies and for establishing safety criteria for human exposure.

12 4

(125)

TÁMOP-4.1.2.A/2-10/1-2010-0012

Subacute inhalation toxicity test

Experimental animals:

• rats

• rabbits

• mice

Dose levels:

• at least 3

• (control groupvehicle).

Number of animals:

• 5/sex/group

12 5

(126)

TÁMOP-4.1.2.A/2-10/1-2010-0012

Route of administration:

 nose-only administration

 for 6 hours per day on a 5 day

per week basis a period of 4 weeks

The animals should be clinically observed before, during and after the exposure period. Total study duration of 28 days.

12 6

(127)

TÁMOP-4.1.2.A/2-10/1-2010-0012

Inhalation chamber

12 7

(128)

TÁMOP-4.1.2.A/2-10/1-2010-0012

Exposure conditions

To allow for exposure of all relevant regions of the respiratory tract, aerosols with mass median aerodynamic diameters (MMAD) ranging from 1 to 4m with a geometric standard deviation in the range of 1.5 to 3.0 are recommended.

12 8

(129)

TÁMOP-4.1.2.A/2-10/1-2010-0012

Monitoring of exposure conditions

 chamber airflow

 chamber temperature and relative humidity

 nominal concentration

 actual concentration

12 9

(130)

TÁMOP-4.1.2.A/2-10/1-2010-0012

Rotation Brush Generator

13 0

(131)

TÁMOP-4.1.2.A/2-10/1-2010-0012

13 1

(132)

TÁMOP-4.1.2.A/2-10/1-2010-0012

13 2

(133)

TÁMOP-4.1.2.A/2-10/1-2010-0012

Parameters

 body weight

 food (water) consumption

 clinical signs

 clinical patology:

 haematology

 clinical chemistry

 urinalysis (optional)

• died animals

• measuring organ weight

• histopathology

13 3

(134)

TÁMOP-4.1.2.A/2-10/1-2010-0012

Cumulative toxicity test by Kagan Experimental animals:

• rats

• rabbits

• mice

Dose levels:

• at least 3

• (control groupvehicle)

Number of animals:

• 10/sex/group

13 4

(135)

TÁMOP-4.1.2.A/2-10/1-2010-0012

Administration:

• oral by gavage

Exposition period:

• 4 weeks (once a day)

Parameters:

• body weight

• food consumption

• clinical signs

13 5

(136)

TÁMOP-4.1.2.A/2-10/1-2010-0012

Parameters:

• gross pathology:

 died animals

 survived animals

 measuring organ weight

• histopathology

13 6

(137)

TÁMOP-4.1.2.A/2-10/1-2010-0012

Cumulative toxicity test by Lim Experimental animals:

• rats

• rabbits

• mice

Dose levels:

• 9 % of p.o. LD₅₀

• once a day for 4 days

• dose increasing (1.5 times)

• (control groupvehicle)

Number of animals:

• 10/sex/group

13 7

(138)

TÁMOP-4.1.2.A/2-10/1-2010-0012

Administration:

• oral by gavage

Exposition period:

• 4 weeks (once a day)

Parameters:

• body weight

• food consumption

• clinical signs

13 8