Clinical and other observations in animal toxicity studies

Koen Van Deun

Beltox Advanced Toxicology Seminar

Outline

• Clinical observations• Body weight , weight gain & food

consumption• Ophthalmology• Other observations• Discussion & conclusion

Clinical observations (1)

OECD 407, 408, 409, 451, 452, 453• General clinical observations (health condition):

at least once daily– preferably at the same time(s) each day – considering peak period of anticipated effects (peak time?)

• Moribundity & mortality: at least twice daily• Detailed clinical observations (outside the home cage):

once before exposure & at least once a week – Changes in skin, fur, eyes, mucous membranes, secretions ...– Autonomic activity (e.g. piloerection, pupil size, respiration) – Changes in gait, posture, response to handling– Bizarre behaviour (e.g. self-mutilation, walking backwards)

•

Clinical observations (2)

• Additional observations in rodents: – Sensory reactivity to different stimuli: towards the end of the study

auditory, visual & proprioceptive stimuli assessment of grip strength motor activity assessment

– Functional observations (90-day study; optional in 4-week study)

BalanceComputer

Volumetric pump

Observation at dosing

Dosing

Observation at peak period

Clinical observations (3)

FDA Redbook 2000• Individual records should be maintained for each animal

– the time of onset and the characteristics and progression of any effects should be recorded, preferably using a scoring system.

• Tumor development (long-term studies)– the time of onset, location, dimensions, appearance and progression

of each grossly visible or palpable tumor should be recorded.

• Toxic and pharmacologic signs during the study – may suggest the need for additional clinical tests or expanded post-

mortem examinations.

Examples• from the cage

Clinical observations (4)

https://www.aalaslearninglibrary.org

From the cage exteriorRats are sleeping. Hair coat normal.

Cage Wirelid offRats are alert. Normal reaction.

Examples• Handling

MalocclusionNot test article related!

ChromodacryorrheaDirty skinSkin lesionsTest article related!Adverse!

Clinical observations (5)

Normal rat

SedationTest article related!Pharmacodynamic?Adverse?

Clinical observations (6)

Examples • Total occurence/ No. of animals with observation ( T=20)

GROUP (MALES)

BEHAVIOR/CNS CONTROL LOW MEDIUM HIGH

TIME OF DOSE

PARTIAL CLOSURE EYES (PTOSIS) 0/0 0/0 0/0 7/1 EXCESSIVE STRUGGLING DURING DOSING 1/1 0/0 0/0 0/0

1-2 HOUR POST-DOSING

PARTIAL CLOSURE EYES (PTOSIS) 0/0 0/0 0/0 5/1

EYES/EARS/NOSE

PUPIL DILATED RIGHT EYE 0/0 2/2 0/0 0/0 DRIED RED MATERIAL AROUND NOSE 0/0 2/2 2/1 2/2 COMPLETE CLOSURE RIGHT EYE 0/0 0/0 0/0 6/1

EXCRETA SOFT FECES 1/1 1/1 0/0 1/1 INCREASED URINATION 0/0 2/1 21/13 25/15

ORAL/DENTAL UPPER INCISOR(S) BROKEN 0/0 1/1 0/0 1/1 UPPER INCISOR(S) LONG, TRIMMED 1/1 0/0 0/0 0/0 UPPER INCISOR(S) MALALIGNED ... 7/1 0/0 0/0 5/1

Target organ relation?

• Relation to target organs? *

* The toxicologist’s pocket handbook M.J. Derelanko, 2008 (ISBN 978-1-4200-5138-4)

Clinical observations (7)

Group Example Organs/systems likely involved

1. Respiratory Cyanosis (Cardio)pulmonary insufficiency

2. Motoric Somnolence CNS sleep center

3. Convulsions Clonic CNS, respiratory failure, neuromuscular, autonomic

4. Reflexes No normal righting CNS, sensory, neuromuscular

5. Ocular Myosis Autonomic (parasympathomimetic)

6. Cardiovascular Tachycardia Autonomic (sympathomimetic, (Cardio)pulmonary insufficiency

• Relation to target organs? *

* The toxicologist’s pocket handbook M.J. Derelanko, 2008 (ISBN 978-1-4200-5138-4)

Clinical observations (8)

Group Example Organs/systems likely involved

7. Salivation Excessive Autonomic (parasympathomimetic)

8. Piloerection Rough hair Autonomic (sympathomimetic)

9. Analgesia Decreased reaction to pain (e.g. hot plate)

Sensory, CNS

10. Muscle tone Hypertonia Autonomic (sympathomimetic)

11. Excretion Red urine (hematuria) Damage in kidney

12. Skin Redness (erythema) Immunity (inflammation, sensitization...)

• Signs indicative of pain, suffering & distress in animals *– Appearance & physical signs:

chromodacryorrheapiloerection, dehydratation, dirty skin, skin lesionsrapid & laboured respirationexcessive salivationhunched posture

– Movement & behaviour gait irregularity, ataxiaabnormal aggressive behaviour, restlessness, agitationabnormal reaction to handling, vocalisation on handlingtremors, writhinglethargy, decreased activity, reluctance to movelicking, scratching, self mutilation

* The toxicologist’s pocket handbook M.J. Derelanko, 2008 (ISBN 978-1-4200-5138-4) Picture: AALAS Learning Library https://www.aalaslearninglibrary.org

Clinical observations (9)

• Signs indicative of moribundity as criteria for sacrifice *– Prolonged impairment of locomotion, preventing access to food/water – Continued difficult, laboured breathing– Continued severe diarrhea– Severe dehydration– Eyes fixed or sunken– Severe self mutilation– Significant blood loss– Excessive weight loss (> 25% over 7 days or more)– Evidence of irreversible major organ failure (see further)– Sustained 10% decrease in body temperature

* The toxicologist’s pocket handbook M.J. Derelanko, 2008 (ISBN 978-1-4200-5138-4)

Clinical observations (10)

Body weight and food consumption (1)

OECD 407, 408, 409, 451,452, 453• Body weight: measured at least once a week (up to week 13; every 4 weeks thereafter)• Food consumption: measured at least every weekly (up to week 13; every 4 weeks

thereafter)• water consumption: measured at least weekly if the test substance is administered via

the drinking water

FDA redbook 2000•Attempt to quantify spillage of feed by test animals, and to determine if spillage is greater with test diets than with control diets. Appropriate discussions of feed spillage should be included in the study report.

• Mean body weight in male rats (g)

Mean adult male body weight: 350-400 g/day

Body weight (2)

g C L M H week 0 118 118 118 118week 1 156 155 154 153week 2 203 200 197 193 *week 3 241 237 234 226 ***week 4 270 263 260 250 ***week 5 293 286 277 ** 267 ***week 6 315 304 293 ** 281 ***week 7 331 319 304 *** 294 ***week8 345 333 316 *** 302 ***week 9 351 341 324 *** 311 ***week 10 361 351 335 ** 319 ***week 11 369 359 341 ** 324 ***week 12 375 365 345 *** 328 ***week 13 379 369 347 *** 329 ***

*; **; *** statistical significance (p<0,05; 0,01; 0,001)

• Mean body weight in male rats (% vs C)

Body weight (3)

% vs C C L M H week 0 100 100,0 100 100week 1 100 99,4 99 98week 2 100 98,0 99 95 *week 3 100 97,9 99 93 ***week 4 100 97,4 99 93 ***week 5 100 97,3 97 ** 91 ***week 6 100 96,5 96 ** 89 ***week 7 100 96,4 95 *** 89 ***week8 100 96,8 95 *** 88 ***week 9 100 97,2 95 *** 89 ***week 10 100 97,2 95 ** 88 ***week 11 100 97,3 95 ** 88 ***week 12 100 97,1 95 *** 87 ***week 13 100 97,4 94 *** 87 ***

*; **; *** statistical significance (p<0,05; 0,01; 0,001)

• Mean body weight in female rats

Mean adult female body weight: 180-200 g/day

Body weight (4)

g C L M Hweek 0 100 100 100 100week 1 123 122 122 122week 2 137 136 135 143 *week 3 155 155 150 159week 4 165 164 164 174 *week 5 178 173 173 184 *week 6 185 182 180 190week 7 190 186 186 196week 8 198 193 192 202week 9 203 196 197 206week 10 207 202 204 212week 11 210 207 208 214week 12 212 207 208 213week 13 214 211 212 216

* statistical significance (p<0,05)

• Mean body weight gain in male rats

Weight gain (5)

g/week C L M Hweek 1 39 38 37 * 35 ***week 2 46 44 43 * 41 ***week 3 40 38 37 ** 33 ***week 4 28 26 26 ** 23 ***week 5 23 21 * 17 *** 17 ***week 6 22 19 * 16 *** 15 ***week 7 15 16 * 10 *** 13 ***week 8 14 12 13 *** 8 ***week 9 7 9 7 *** 8 ***week 10 10 11 12 *** 8 ***week 11 9 8 5 *** 6 ***week 12 5 4 5 *** 3 ***week 13 3 5 1 *** 2 ***

*; **; *** statistical significance (p<0,05; 0,01; 0,001)

• Mean body weight gain in female rats

Weight gain (6)

g/week C L M Hweek 1 23 22 22 22week 2 14 14 13 21 ***week 3 17 18 15 16 *week 4 11 10 13 15 ***week 5 13 8 10 11 **week 6 7 10 7 5week 7 6 4 7 6 *week 8 7 7 5 5week 9 5 3 5 5week 10 4 6 7 6week 11 3 6 4 2week 12 2 -1 -1 1week 13 2 4 5 1

*; **; *** statistical significance (p<0,05; 0,01; 0,001)

• Mean weekly & total food consumption in male rats

Mean adult food consumption: 10-20 g/day

Food consumption (7)

g/week C L M Hweek 1 147 143 143 148week 2 165 164 163 168week 3 179 179 175 180week 4 179 177 174 177week 5 173 173 168 170week 6 177 178 170 * 173week 7 173 170 161 ** 165 *week 8 170 170 164 164week 9 156 156 148 * 155week 10 162 165 159 163week 11 157 158 155 159week 12 163 164 159 163week13 146 148 141 150Total 2147 2145 2080 2135

*; ** statistical significance (p<0,05; 0,01)

• Mean weekly & total food consumption in female rats

Mean adult food consumption: 10-20 g/day

Food consumption (8)

g/week C L M Hweek 1 117 116 116 123 *week 2 115 115 115 132 ***week 3 120 118 118 133 ***week 4 119 118 117 136 ***week 5 120 117 117 128 *week 6 124 123 123 136 **week 7 118 116 118 130 **week 8 124 123 124 133 *week 9 115 112 113 124 **week 10 121 118 122 132 *week 11 118 115 118 128 **week 12 125 121 123 131weel 13 110 113 109 118 **Total (g) 1546 1525 1533 1684 **

*; **; *** statistical significance (p<0,05; 0,01; 0,001)

Opthalmology (1)

OECD 408, 409:• Opthalmological examination: prior to the administration and at termination of the

study– Preferably in all animals of control and high dose group – If changes are detected, all animals should be examined

• Practically:– Visual inspection:

conjunctiva, sclera, cornea, iris, lens and fundus

– After induction of mydriasis (e.g. atropine sulphate 1%), eyes are examined by use of a slit lamp biomicroscope.

•

Opthalmology (2)

Examples:• Visual inspection:

– Corneal opacity– Pronounced scleral vessels– Phtisis bulbi

• Slit lamp examination:– Fundus hyperreflectivity– Small tapetal fundus– Retinal folds non-tapetal fundus

Other observations (1)

• Electrocardiography (E.C.G.) non-rodents (standard)

• Blood pressure non-rodents (optional)

• Water consumption: test articles administered in drink water, or if dehydrationis suspected

• Temperature: in case of bad condition/hypothermia/fever

• Otoscopy: for otitis (e.g. dogs)• Behaviour / Reflexes / Motor activity ... E.g.

• tachycardia, bradycardia, arythmia

• QT prolongation, heart block ...

Other observations (2)

Example E.C.G. dogs:

* Statistically significant p<0.05 a Van de Water correction of QT due to high heart rate in dogs

! Preferably: add a 4-week interim measurement

Parameter Week Control Low Medium High

PQ (msec) 012

73.271.4

74.176.1

75.275.9

73.977.8

QRS (msec) 012

36.135.4

36.535.4

37.934.2

37.535.4

QT (msec) 012

157.5160.5

152.1155.4

151.4164.8

153.2172.3 *

QTc (msec) a

012

232.8240.1

237.0230.1

224.9242.8

240.8245.1

R (mvolt) 012

1.7561.742

1.6841.854

1.8901.603

1.5591.529

Rate (bpm) 012

132.0135.4

148.6135.8

135.7134.7

151.4131.2

Thank You!

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Discussion & Conclusion