Current Status of Behavioural Toxicology Animal and Human Data

1
Current Status of Behavioural ToxicologyAnimal
and Human Data

• Jochen Buschmann Asher Ornoy
• Fraunhofer Institute of Toxicology Hebrew
  University
• and Aerosol Research Jerusalem, Israel
• Hannover, Germany

2
Current Status of Behavioural ToxicologyAnimal
and Human Data

• Past or history and background
• Present or current procedures
• Test Batteries
• Example open field
• Future or Some Alternatives
• Ontogeny and Reflexes
• Learning
• Social Behaviour
• Guideline News
• Conclusions
• Critical Questions

3
History (Vorhees, 1986)
1. Hamilton (1944) Sodium bromide Locomotor
activity Problem solving Seizures Irreversibil
ity Increase in Variability Dose-response
(not in all tests) 2. Werboff (1963) etc.
etc. "Behavioural teratology" Tranquillizers
influence offspring behavoiur
4
History
3. JOFFE (1969) "Prenatal Determinants of
Behaviour" "Methodological deficiencies in many
of the studies of the behavioural effects of
prenatally administered drugs make it
inappropriate to attempt any statement of
substantive findings. The one point which emerges
clearly ... is that a wide variety of
pharmacological agents are capable of producing
effects on the behavior of offspring in the
absence of overt morphological effects." (p.
94) "In most of the studies ... methodological
and procedural defects have prevented the
extraction of general statements on the effects
of particular treatments on offspring behaviour
..." (p. 152)
5
History
4. BUELKE-SAM KIMMEL (1979) Development and
Standardization of ScreeningMethods for
Behavioral Teratology Test batteries Apical
testing 5. CBTS (1978-1984)..... .... EIBT
(ongoing) Interlaboratory studies
6
Biological Bases (Clausing Buschmann, 1984)
1. Epidemiological Aspect 2-3 of newborn with
(morphological) anomalies at birth Increase
during the first years of life up to 7.5 due
to functional anomalies recognized
later (Schumacher Fanghänel, 1977) 20 of
pupils suffer from failure to concentrate during
the first school year (Klaus Heinecke, 1981)
7
Biological Bases
2. Biocybernetical Aspect (Tembrock, 1978) 3
vectors of behaviour input vector condition
vector output vector Functional systems and
interactions sensory system (central)
nervous system endocrine system effector
system BACK TO THE ROOTS ! False positive
/ negative results specific vs. unspecific
effects
8
Biological Bases
3. Ontogenetic Aspect "Critical periods of
teratogenic sensitivity (Wilson, 1973) "Brain
growth spurt" (Dobbing Smart, 1974) 4.
Subjective Aspect there is some intuitive
feeling that behaviour must be a subtle and
sensitive way to monitor early damage from
chemicals (Norton, 1980, Zbinden, 1981)
9
Current Status of Behavioural ToxicologyAnimal
and Human Data
Past or history and background Present or
current procedures Test Batteries Example
open field Future or Some Alternatives Ontog
eny and Reflexes Learning Social
Behaviour Guideline News Conclusions Critical
Questions
10
Test Batteries
apical tests they grossly analyse the
integrative response of the organism rather than
specific behavioural functions (Buelke-Sam
Kimmel, 1979) Equivalent of feeling ill in
humans (Silvermann, 1988) Instrument to make
sure that as many endpoints as possible are not
influenced by substance treatment
11
Test Batteries (Jensh, 1983)
1. Simple 2. Comprehensive, variety of
behavioral functions (global) 3. Sensitive to
slight alterations, to alterations by different
agents 4. The behavioral schedule should be
simple 5. Minimum of time 6. Reliable,
reproducible, and valid (intra-/interlab) 7.
Proven past history of positive results 8.
Economical (financial expense, animals) 9.
Within the framework of existing guidelines 10.
Graded steps from elementary to more
sophisticated tests 11. Data should be
quantifiable 12. Predictive of effects in
humans 13. Longitudinality
12
Postnatal Ontogeny
Developmental milestones Reflexes Body
weight Surface righting Pinna detachment Grasp
reflex Incisor eruption Pivoting Fur
develoment Negative geotaxis (slope) Eye
opening Cliff drop avoidance Homing (nest
preference) Swimming ontogeny
13
Example of a Test Battery
Day p.p. Test Day p.p. Test ------------------
--------------------------------------------------
---------------------------------- 0 Day and
hour of birth 21 Pup weight Litter
size Weaning Pup weight 50 Body
weight 1 - 4 Righting reflex around 50 Open
field Grasp reflex Locomotor
activity Pinna detachment Learning 7 Pup
weight 100 Body weight 8 - 10 Negative
geotaxis around150 Social interactive
behaviour Swimming ontogeny 14 Pup
weight 14-16 Eye opening 15-17 Swimming
ontogeny
14
Open Field (Hall, 1934)
15
Locomotor Activity
Time in rest Time in movement Path length
Rearing Rearing time
16
Current Status of Behavioural ToxicologyAnimal
and Human Studies
Past or history and background Present or
current procedures Test Batteries Example
open field Future or Some Alternatives Ontog
eny and Reflexes Learning Social
Behaviour Guideline News Conclusions Critical
Questions
17
Swimming Ontogeny in Rodents (Klaus Hacker,
1978)
18
Learning
19
Learning
20
Learning - Morris Water Maze
Test- Design Learning 5 consecutive days, 4
trials per day, platform position
unchanged Memory after approx. 4 weeks 2 days
platform position unchanged 2 days opposite
platform position 1 day plat form in centre
21
Elements of Social Interactive Behaviour (1)

• ELEMENT DESCRIPTION
• --------------------------------------------------
  -
• Sideway Approaching or circling the opponent in
  an arched back attitude.
• Upright Standing on the hindlegs in front of and
  in close contact with the opponent.
• On Top Standing or lying over the opponent, which
  has adopted the On Back posture.
• On Back Lying on the back, belly exposed to the
  opponent.
• Fight Rapid sequence of various behavioural
  elements, involving very short upright postures,
  bites and kicks. Terminated by the retreat of one
  animal. Frequently accompanied by audible cues.

22
Elements of Social Interactive Behaviour (2)

• ELEMENT DESCRIPTION
• --------------------------------------------------
  -
• Stretched Standing with slightly arched back,
  one or both forelimbs lifted from the ground,
  head oriented to the opponent.
• Autogroom Licking or nibbling fur or tail, face
  washing with the forepaws.
• Unspecified All bodily contacts with the
  opponent except the contacts during the above
  defined elements. All kinds of snout contacts,
  sniffing or licking the opponent.

23
Elements of Social Interactive Behaviour (3)

• ELEMENT DESCRIPTION
• --------------------------------------------------
  -
• Lying Lying in a corner of the cage without
  interactive contact to the opponent.
• Ambulation Undirected locomotion without
  interactive contact with the opponent. Includes
  sniffing substrate or cage components and rearing
  at cage walls.
• --------------------------------------------------
  -
• (Haemisch Buschmann, 1996)

24
Current Status of Behavioural ToxicologyAnimal
and Human Data
Past or history and background Present or
current procedures Test Batteries Example
open field Future or Some Alternatives Ontog
eny and Reflexes Learning Social
Behaviour Guideline News Conclusions Critical
Questions
25
What is new critical in OECD 426 ?
Litter standardisation day 4 p.p., randomly, 8
pups Selection of pups different pups for
different learning tasks humanely dispose of
surplus pups at weaning Number of pups 20 per
sex group learning, brain weight 10
neuropathology 6 Route of exposure oral,
consider others exposure of pups via milk
(gavage of pups ??!!) Physical development
reflexes
26
What is new critical in OECD 426 ?
Neuropathology facilitate correlations to
function fix by perfusion (gt PND
22) in situ fixation (termination) plast
ic embedding preferred qualitative
examination
27
Placement of behavioural tests

• Special Study
• One-Generation Study
• (Poor old) Two-Generation Study
• Practicality ?

28
days p.p. 416 426 _____________________________
__________________________________ 0 body
weight body weight clinical signs clinical
signs (landmarks) gt 2 landmarks 11 in
terim sacrifice (neuro) (sensory
functions) (motor activity) motor
activity 21 interim sacrifice 1
1 weaning weaning
29
days p.p. 416 426 _____________________________
__________________________________ 21 weaning
weaning body weight body weight food
consumption sexual maturation (sexual
maturation) (motor activity) motor
activity (sensory function) motor / sensory
function learning memory 35
30
days p.p. 416 426 _____________________________
__________________________________ 60 body
weight body weight food consumption mo
tor activity motor / sensory
function learning memory 75 termin
al sacrifice (neuro) 90 oestrus
cycle mating terminal sacrifice (repro)
sperm analysis
31
Too much for the 2-gen-study ?

• Other goals
• male ( female) fertility
• sperm parameters
• hormone activities

32
Current Status of Behavioural ToxicologyAnimal
and Human Data

• Past or history and background
• Present or current procedures
• Test Batteries
• Example open field
• Future or Some Alternatives
• Ontogeny and Reflexes
• Learning
• Social Behaviour
• Guideline News
• Conclusions
• Critical Questions

33
Conclusions (1)
1. Behaviour can be used in two ways specific
indicator of central functions equivalent of
(unspecific) feeling ill. 2. Physiological
function includes much more than (measurable)
behaviour, e.g. immune, digestive, circulatory
systems etc. 3. Behavioural endpoints
represent apical tests. 4. Use test
batteries.
34
Conclusions (2)

• 5. Extend behavioural testing until adolescence.
• 6. Investigate more complex forms of behaviour
• (standardized stimuli / standardized output).
• 7. Keep in mind biological background of
• the applied tests.
• meaning, predictive value, limitations
• influence of side factors
• reference data

35
Conclusions (3)
8. Dont test behaviour in isolation, include
other functions hormone status metabolic
functions organ functions neuropathology,
histochemistry of pituitary etc. etc. 9. At
present, it seems difficult to createbehavioural
guidelines, due to the contradiction complexne
ss and necessary flexibility of behavioural
testing vs. rigidity of guidelines (an FOB
vs. the FOB)
36
Current Status of Behavioural ToxicologyAnimal
and Human Data

• Past or history and background
• Present or current procedures
• Test Batteries
• Example open field
• Future or Some Alternatives
• Ontogeny and Reflexes
• Learning
• Social Behaviour
• Guideline News
• Conclusions
• Critical Questions

37
Some critical questions

• 1. Interpretation of behavioural data in animals
• 1.1 What is an effect ?
• 1.1.1. Dealing with asterisks
• 1.1.1.1. Significance of preweaning data,
  acceleration vs. retardation
• 1.1.1.2. Dose response relationships
• 1.1.1.3. Sexual differences
• 1.1.1.4. Biological relevance vs. statistical
  significance (which test, test criteria)
• 1.1.2. Are there criteria for an effect ?
• 1.1.3. Correlation between pathology and function

38
More critical questions

• 1. Interpretation of behavioural data in animals
• 1.2. What should be tested and when are there
  sensitive periods ?
• 1.2.1. Usefulnesss of testing during adulthood ?
• 1.2.2. Usefulness and practicality of more
  complex forms of behaviour ?
• 1.2.3. Testing behaviour in obviously otherwise
  affected animals vs. stepwise downwards testing
  ?
• 1.3. Why are there so many controversial results
  ?
• 1.3.1. Side effects
• 1.3.2. Standardization vs. flexibility

39
Even more critical questions

• 2. Extrapolation of animal data to humans
• 2.1. What animal behaviour is a model for what
  human equivalent ?
• 2.2. How predictive are our tests, are we always
  certain ourselves ?
• 2.3. Animal vs. human learning, familiarization
  vs. learning, etc. ?
• 2.4. What does safety mean in terms of
  behavioural teratology ?
• 2.5. How to get on ? (Guidelines now ?)

40
Environment and brain plasticity Experimental
studies

• Charles Darwin Domesticated animals have smaller
  brains than their wild counterparts that live in
  more enriched environment (1859, 1874).
• Donald O. Hebb use-dependent plasticity of the
  nervous system (1949).
• Mark R. Rosenzweig et al. Rats in enriched
  environments have increased brain weight,
  increased thickness of cerebral cortex and
  acetylcholine esterase activity (1962,1996,1999).
• David Hubel Torsten Wiesel Cytoarchitecture of
  the visual cortex in kittens is responsive to
  visual manipulations (1962,1965).

41
Environment and brain plasticity in man
Speculations

• Ramon J Cajal Learning and enriched mental
  experience increase the number of neuronal
  branches.
• Rosenzweig M. Men of distinction are usually
  found to have larger brains than those of
  inferior intellect. However, nutrition and
  diseases might affect brain weight and intellect.

42
Training affects human brain

• Violinists have increased representation of
  somatosensory cortex of left hand digits (Ellbert
  et al, 1995)
• Positive correlation between education and
  dendritic branching in Wernickes area Jacobs et
  al, 1993)

43
Environment and development in children with
slight brain damage

• It is presumed that the environment affects
  experience-dependent processes, and perhaps
  experience-expectant.
• The effects might be more pronounced than in
  children with a normal brain.
• Insufficient data in the neuropediatric
  literature and in experimental animals.

44
Developmental tests that are used on children
from birth to puberty

• Bayley scales for infant development 0-3 years
• WIPSI test for children at ages 4-6 years
• WISC-R Performance, Verbal and other subscales
  for ages 6-14
• Bender and Goodenough draw a person
• Conners questionnaire for hyperactivity and
  inattention (ADHD)
• Achenbachs questionnaire (ADHD and behavior)
• Pollack Taper audio-visual test (ADHD, LD)
• Touwen Prechtl neurological test (ADHD)

45
Intrauterine exposure to heroin effects at
school age

• Slightly or moderately impairs intellectual
  ability, arithmetic and reading skills (causing
  learning impairment)
• Induces a high rate of inattention and
  hyperactivity (ADHD)
• The effects are, to a large extent,
  environmentally-dependent, and can be corrected
  by good environment

46
Conclusions 2effect of heroin

• Intrauterine exposure to heroin induces
• slight brain damage manifested by increased
  inattentionhyperactivity (ADHD) and behavioral
  problems.
• Basic intellectual functions of the children
  are not damaged, as good environment (i.e.
  adoption) may result in almost normal function.
• Motor functions are not impaired by heroin.

47
General Conclusions 2

• The environment of the child born with slight
  brain damage may
• Affect intellectual and learning functions
• Affect the childs behavior, emotions, attention
  and activity span, but has little effect on motor
  development
• It is the duty of our society to improve the
  environment of such children as good environment
  may overcome slight brain damage

48
General Conclusions Ipostnatal environment

• The environment of the child at early age affects
  his intellectual abilities. This is more evident
  in children with slight brain damage, as
  deprivation and neglect increases the damage
• We could prove this hypothesis in children born
  to drug dependent parents, in very low birth
  weight infants and in twins discordant (over 25)
  for birth weight.
• In a different study this was also shown by us in
  children born to diabetic mothers

49
Postnatal Brain Development is Dependent on
Genetic Factors

• AND
• Nutrition
• Health
• Experience and learning
• Emotional support
• Damaged brain is dependent on similar factors

50
Environmentally-dependent brain maturation
(GreenoughBlack, 1992).

• Experience-expectant Development is
  predetermined but will occur if experience
  exists experience is predetermined and is occurs
  in any specific species. Its absence would
  contribute to synapse elimination
• Experience-dependent Experience is not
  predetermined It will generate in response to
  increased experience more synapses, and is
  variable among individuals

51
Behavioral treatment in animals (rats)Bennet,
Diamond, Krech and Rosenzweig, 1996

• Comparisons were made among males of similar age,
  parentage etc.
• Informal and formal training.
• Controls 3 in a cage, exposed to ongoing
  activity.
• Enriched 10-12 in large cages with toys.
• Reduced experience single in a cage, quite
  environment.

52
Animal studies showing the positive effects of
training

• In genetically identical animals it was shown
  that environmental enrichment
• alters brain morphology after lesions cortical
  thinning associated with early
• frontal cortex lesions is attenuated in rats by
  rearing them in enriched
• environment (Elliot et al, 1987,1991)

53
Experiments in behavioral treatment in normal
animals Rosenzweig et al, 1996)

• In enriched environments the rat brain has
• 1. Increased acetylcholinesterase activity.
• 2. Heavier cerebral cortex in enriched vs
  reduced environments .
• 3.Younger animals perhaps respond better than
  older animals, but they too are very responsive.

54
Early Postnatal Brain Development (Danya Glaser,
2000)

• Is dependent on genetic factors responsible for
  (over)production of neurons, axons, dendrites and
  synapses.
• Is modified by environmental influences
  experience regulates the persisting synapses and
  those that are not utilized gradually disappear.
• Emotional and physical neglect are hazardous to
  brain development

55
Developmental areas in infants and children

• Gross motor
• Fine motor
• Cognitive
• Language
• Social
• Adaptive
• Various tests are used to monitor these
  developmental areas

56
Bayley Scales for Infant Development

• Mental scale (MDI)
• Psychomotor (motor) scale (PDI)
• Socioemotional maturity (Behavioral Record)
• From birth - 30 months of age.
• No good correlation with later measures of
  intelligence in normal children.

57
Stanford-Binet Intelligence Scales

• For preschool children, 2-6 years
• Relies more on verbal skills than on motor
  skills.
• Only mental scores

58
Mc Carthy Scales of Childrens Abilities

• Measures mental and motor functions for 3-8 years
  old children.
• Good for children with learning difficulties.
• General cognitive (GCI) 5 major areas verbal,
  perceptual performance, quantitative, memory,
  motor.

59
Wechsler Primary and Preschool Test of
Intelligence (WPPSI)

• Preschool measure - 4-6 years of age
• 11 subtests verbal and performance scales
• Good for learning disabled, mentally retarded and
  inattentive children.

60
Wechsler Intelligence Scale for Children
Revised (WISC-R)

• For children aged 6-16 years.
• Good reliability and validity.
• Verbal scale - language related skills.
• Performance scale - perceptual motor skills
• Good for learning disabled, mentally retarded and
  inattentive children.

61
Bender Gestalt Test for young children.

• Measures visual motor skills for children above 5
  years. Can also serve as an IQ test.
• Test may demonstrate abnormal grasp, motor
  control, speed, efficiency and organization.
• Goodenough draw-a-man test is similar

62
Conners Rating Scales and DSMIV questionnaire

• Parents and Teachers Questionnaire
• Measures three symptoms of ADHD activity,
  attention, anxiety.
• Good for school age children.
• Various forms from 93 to 10 questions.
• DSM IV measures similar symptoms to those
  measured by Conners.

63
Child behavior check list (CBCLAchenbach
questionn).

• Measures various areas of behavioral dysfunction
  attention, activity level, depressive, schizoid
  and delinquent behavior.
• Measures school and social performance.
• Good validity and reliability.

64
Frequent developmental delay at preschool age

• Speech delay/deficits
• Motor delay/deficits
• Behavioral problems
• Attention deficit and/or hyperactivity
• Mental retardation
• Visual and hearing impairment

65
General Conclusions I postnatal environment

• The environment of the child at early age affects
  his intellectual abilities. This is more evident
  in children with slight brain damage, as
  deprivation and neglect increases the damage
• We could prove this hypothesis in children born
  to drug dependent parents, in very low birth
  weight infants, in twins discordant (over 25)
  for birth weight and in offspring of diabetic
  mothers.
• Similar findings were also reported by others

66
General Conclusions 2

• The environment of the child born with slight
  brain damage may
• Affect intellectual and learning functions
• Affect the childs behavior, emotions, attention
  and activity span, but has little effect on motor
  development
• It is the duty of our society to improve the
  environment of such children as good environment
  may overcome slight brain damage