Grete Østergaard, DVM, Ph.D. Department of Experimental Medicine Panum Institute

1. Repeated dose toxicity Grete Østergaard, DVM, Ph.D. Department of Experimental Medicine Panum Institute

2. OECD guidance documents • No. 35: Guidance Notes for Analysis and Evaluation of Chronic Toxicity and Carcinogenicity Studies • No. 32: Guidance Notes for Analysis and Evaluation of Repeat-Dose Toxicity Studies • No. 20: Guidance Document for Neurotoxicity Testing • Freely avaiable for download at OECD website. www.oecd.org.

3. Definitions: Short-term, subchronic, chronic • Subchronic Toxicity Studies 13 weeks (90 days) duration (rodent). • Short-term repeat-dose studies periods less than this • defined (WHO, 1990) as “having a duration lasting up to 10% of the animal’s lifespan, 90 days in rats and mice, or 1 year in dogs” • The US EPA considers a 1-year dog study to be a chronic study. • Chronic Toxicity Studies, or long-term studies, are defined as studies lasting for the greater part of the lifespan of the test animals, usually 18 months in mice, 2 years in rats (WHO, 1987; 1990).

4. OECD guidelines: short-term studies • 407 Repeated Dose 28-day Oral Toxicity Study in Rodents (Updated Guideline, adopted 27th July 1995) • 410 Repeated Dose Dermal Toxicity: 21/28-day Study (Original Guideline, adopted 12th May 1981) • 412 Repeated Dose Inhalation Toxicity: 28-day or 14-day Study (Original Guideline, adopted 12th May 1981) • 422 Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test (Original Guideline, adopted 22nd March 1996)

5. OECD guidelines: Subchronic studies • 408 Repeated Dose 90-Day Oral Toxicity Study in Rodents (Updated Guideline, adopted 21st September 1998) • 409 Repeated Dose 90-Day Oral Toxicity Study in Non-Rodents (Updated Guideline, adopted 21st September 1998) • 411 Subchronic Dermal Toxicity: 90-day Study (Original Guideline, adopted 12th May 1981) • 413 Subchronic Inhalation Toxicity: 90-day Study (Original Guideline, adopted 12th May 1981)

6. OECD guidelines: Chronic studies • 452 Chronic Toxicity Studies (Original Guideline, adopted 12th May 1981) • 453 Combined Chronic Toxicity/Carcinogenicity Studies (Original Guideline, adopted 12th May 1981): oral, dermal, inhalation

7. EU guidelines • B.7 repeated dose (28 days) toxicity (oral) • B.8 repeated dose (28 days) toxicity (inhalation) • B.9 repeated dose (28 days) toxicity (dermal) • B.26 sub-chronic oral toxicity test. Repeated dose 90 - day toxicity study in rodents • B.27 sub-chronic oral toxicity test: repeated dose 90 - day toxicity study in non-rodents • B.28 sub-chronic dermal toxicity test: 90-day repeated dermal dose study using rodent species • B.29 sub-chronic inhalation toxicity test: 90-day repeated inhalation dose study using rodent species • B.30 chronic toxicity test • B.33 combined chronic toxicity/carcinogenicity test • Freely available at http://ecb.jrc.it/testing-methods/

8. What information is gained? • Hazard classification and labelling requirements • Risk assessment for human health and the environment • - relevant for exposure of humans to single high dose • Longer duration: More time for effects to become manifest – larger group sizes (5,10,20 per sex) and more endpoints means more sensitive

9. EU classification and labelling • The results of repeated dose toxicity studies form the basis for classification as • Very toxic T+ - Toxic T - Harmful Xn • If swallowed, in contact with skin, by inhalation • R48: danger of serious damage to health by prolonged exposure

10. General considerations • At least 3 dose levels + controls • Limit test at 1000 mg/kg (28-day and 90-day oral rodent or non-rodent) • Largest dose: Some toxicity, but should not compromise biological interpretability of the observed responses – i.e. not be severely toxic • Lowest dose should not produce any evidence of toxicity (i.e. allows the establishment of a NOAEL).

11. NOAEL and LOAEL Ex. In a 28-day rat study increased incidence of small liver necroses was identified as the harmful effect. • Points to note • Some incidence in the control group • Statistically significant difference from control in a biologically meaningful parameter (adverse) is relevant

12. NOEL, NOAEL, LOEL, LOAEL • Adverse – Non-adverse • Physiological: Only considered adverse if correlation with other signs of toxicity • Pharmacological: Interaction of xenobiotic with receptor site, limited duration, reversible • Toxic: Reversible or irreversible, injury, adverse, harmful

13. NOAEL, LOAEL • NOAEL the highest dose which does not cause an adverse effect in a given study • LOAEL correspondingly the lowest dose which does cause an adverse effect • Exercise: • Three studies with NOAEL and LOAEL for chemical x. The studies are equally valid, i.e. none of them can be disregarded. • Study 1: NOAEL 100 LOAEL 500 • Study 2: NOAEL 200 LOAEL 300 • Study 3: NOAEL 50 LOAEL 150 • Please suggest an overall NOAEL and LOAEL for chemical x based on the combined information from the three studies

14. Solution: Select the highest NOAEL which is not in conflict with any LOAEL. Select the lowest LOAEL. Highest NOAEL not in conflict with any LOAEL Lowest LOAEL The NOAEL 100 is the highest NOAEL which does not conflict with any LOAEL. The LOAEL 150 is the lowest LOAEL.

15. Common early/low-dose effects • According to OECD, • in repeated dose toxicity studies, the most common quantitative effects that drive the setting of the NOEL are • body weight - REDUCTION • kidney weight, liver weight - INCREASE • and testis weight - REDUCTION

16. Number of animals • 28-day study: 5 males, 5 females per dose level • 90-day study: • Rodent: 10 males, 10 females per dose level • Non-rodent: 4 males, 4 females per dose level • Chronic study: • Rodent: 20 males, 20 females • Non-rodent: 4 males, 4 females

17. Toxicity at the top dose level • 28-day study, 90 day study: ”Toxic effects but not death or severe suffering” • Chronic toxicity: ”Frank toxicity” • (Carcinogenicity: ”minimal toxicity…less than 10% depression of body weight gain”) • - If a compound has been investigated at a top dose below the limit (1000 mg/kg/day) • - and there is no toxicity • The study is inadequate • However, you can still use the top dose for the LOAEL considerations

18. Administration of doses • Gavage: 1, max 2 ml/100 g body weight. Same time every day. Preferably same technicians. Some loss of animals common, especially during first weeks. • Prefer constant volume: To give higher dose, use more concentrated solution • Substance in food/drinking water: Not interfere with normal nutrition or water balance • Dogs: oral studies – vomition not uncommon – conditioned reflex

19. Elements of repeated dose studies • General clinical observations • Ophthalmological examination (not 28-day) • Body weight and food/water consumption • Haematology • Clinical biochemistry • Pathology

20. Local effect – Systemic effect • Local : Observed at the site of first contact, caused irrespective of whether a substance can become systemically available. • Systemic: Normally observed distant from the site of first contact. Can also appear on surfaces. • Exercise: In a 28-day study, the test chemical caused severe degeneration of the kidney epithelium in rats. Gastrointestinal problems (diarrhoea) was also observed, and necropsy findings included gastritis and enteritis. Please discuss the findings in terms of local vs. systemic effects.

21. Immunotoxicity • 1. Hypersensitivity: Skin sensitisation tested in sensitisation tests, no recognised test for respiratory or oral sensitisation • 2. Immunosuppression: Several useful parameters, but no functional test in OECD guideline tests • 3. Autoimmune diseases: At present there are no specific assays to assess substances for their potential to induce autoimmune reactions.

22. Guideline study parameters useful for assessment of immunotoxicity • Organ weight of thymus and spleen, • Histopathological examination of thymus, spleen, draining and distant lymph nodes, Peyer’s patches, bone marrow section • Leukocyte status - WBC

23. Indications of immunotoxicity • • morphological changes of lymphoid organs and tissues including bone marrow • • weight changes of lymphoid organs • • changes in haematology parameters (e.g. white blood cell number, differential cell counts) • • changes in clinical chemistry parameters (e.g. serum protein levels, immunoglobulin concentrations). • NOT necessarily primary immunotoxicity, but can be secondary effects

24. Functional assays of immune function • T-cell function test for substances which cause histopathological changes in the thymus • Host resistance models • Other tests: mitogen stimulation tests, leucocyte • phenotyping • - May give valuable information to identify immunotoxic effects • May be more sensitive than non-functional assays (Lower NOAEL/LOAEL) • BUT functional tests are NOT standardised or validated

25. Neurotoxicity • OECD guideline test parameters • Clinical signs - Histopathology of nervous tissue • Not tested: Learning, memory • Superficially tested: Sensory function • Need for particular test species: - Pyridine derivatives in primates, Organophosphorus compounds in adult hens

26. Indications of neurotoxicity • morphological (structural) changes in the central or peripheral nervous system or in special sense organs; • neurophysiological changes (e.g. electroencephalographic changes); • behavioural (functional) changes; • neurochemical changes (e.g. neurotransmitter levels)

27. The nervous system is very complex • Look for pattern of related effects • Irreversible effects usually involve structural changes • - But in humans severe lasting functional effects exist for which morphological abnormalities have not (yet) been identified. • Compensation may be suspected if a neurotoxic effect slowly resolves during the lifespan. • Effects observed in experimental animals that appear harmless might be of high concern in humans, e.g. sleepiness

28. Acetylcholinesterase inhibitors WHO/FAO • Primary end-points of concern - Inhibition of brain acetylcholinesterase activity and clinical signs • Erythrocyte acetylcholinesterase inhibition (surrogate for NS enzyme) is justified in acute studies, but not for repeated exposures (slow resynthesis) • A statistically significant inhibition by 20% or more represents a clear toxicological effect • Plasma acetylcholinesterase inhibition is considered not relevant.

29. Lung overload (inhalation studies) • Irreversible impairment of lung clearance • Lung fibrosis • Lung tumours • Caused by inhalation of insoluble substances which are not cytotoxic – in HIGH concentrations. • If lung fibrosis is detected in an animal study it is important to test the substance for cytotoxicity. • Substances which cause lung overload should not be tested for systemic toxicity at levels causing lung overload.

30. Mortality/Survival • When you check reports: Always check the number of animals entering and the number finishing the study!!! • Important to separate substance-related deaths from deaths caused by other factors. You cannot set a LOAEL based on a group with unexplained deaths. • High mortality could make a study inconclusive. Also in confict with guideline requirements, besides unethical.

31. Clinical Observations • Signs do not always correlate with pathology or biochemistry • Does not mean they are valueless • e.g. Diarrhoea, tremor, ataxia • probably will not show up in pathology or biochemistry but are still important and must be included in the risk assessment (LOAEL/NOAEL determination) • Often difficult to do statistics on this kind of data

32. Body Weight Changes, Food and Water Consumption • Usually related to food intake • - Analysis of body weight without an analysis of food/water intake is of limited value • - Weight loss may not always be related to toxicity

33. Diet dilution by test substance • Animals eat according to their energy need • => Animals receiving diluted diet must eat more gram food per kg body weight • Animals get more test substance High dose group get more test substance than intended – you see more toxicity AND possibly effects of dietary imbalance • Necessary to keep close and accurate track of feed consumption

34. Vehicle of caloric/nutritional value (corn oil) • If the vehicle is of nutritional value => animals will eat less gram food per kg body weight • => Nutritional imbalance • Solution: Adjust nutritional content of feed. • Food must contain the same amount of nutrients per energy unit in every group.

35. If you suspect a dietary imbalance • An extra control group can be added: • Control diet supplemented with an inert filler at a percentage equal to the highest percentage of the test substance in the diet • Differences between this group and the conventional control group are due to nutritional imbalance. • And can be used to separate toxic effects from nutritional imbalance effects in the dosed group

36. Palatability • Common problem. Animals refuse to eat or drink compound-containing food/water. Animals may quickly become very ill (dehydration, body weight loss) for this reason – not a toxicity problem, but certainly a major welfare and scientific problem. • Anticipate, do pilot test. • Often possible to solve by including e.g. a one-week pre-study period where concentration is gradually increased to desired level. • On-going study: Lower concentration - try again • Sometimes PAIR-FEEDING is used when palatability is a recognized problem which cannot be overcome

37. Pair-feeding I • Pairs of rats of the same sex and approximate size are selected and kept single-caged. • In each pair, one is fed the control diet, the other the experimental diet. • Food consumption is determined daily. • The control animal is fed an amount of food equal to that which the paired experimental animal ate on the preceding day.

38. Pair-feeding II • If the test substance is non-nutritive and composes a significant proportion of the diet • the pair-fed control animals should not be fed exactly the same amount as the dosed animals • But be fed a nutritionally equivalent amount of diet as the paired experimental animal • Question : Does this mean a smaller or larger volume of food to the control animals?

39. Haematological, Clinical Chemistry, and Urinary Measurements • Subject of other lectures • But generally have to be correlated to other measures of toxicity to gain toxicological importance

40. Absolute and Relative Organ Weights • Method of killing – Exsanguination - Timing of necropsy • Calculation of the organ weight/body weight gives a figure which can be compared among animals. • When body weight is lost, some organs lose weight, while other organs do not. • Question: When an animal loses body weight - what happens to the relative organ weight for organs which lose weight? For organs which do not?

41. Influence of reduced growth on organ growth • Organs which grow at a reduced rate: Liver, kidney, intestine, heart, pituitary, seminal vesicles, prostate, uterus • Organs which are spared: Brain, thyroid, adrenals, testes • Data from rats which have been fed 25-80% of control for 2-13 weeks

42. Pathology • Subject of other lecture • But generally the most important toxicological endpoint • Hard evidence • Standardisation in nomenclature and severity rating necessary • Several Societies of toxicological pathologists are active in the standardisation process

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