# Introduction: Investigation of Age - Dependent Pharmacodynamic Sensitivity to Pyrethroids Larry Sheets, Bayer CropScience on behalf of The Council for the Advancement of Pyrethroid Human Risk Assessment (CAPHRA)

# Hypothesis 1: Age - related Sensitivity to Pyrethroids in Rats is Limited to High Doses
• In vivo PD: Assess acute neurotoxic sensitivity in young & adult rats at defined internal dose levels • Acoustic startle response  Neurologic (reflex - based)  ↑ Type I and ↓ Type II  Suited to test young & adult rats  Sensitive: Low doses PYR in adults  Less known in young rats

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# Deltamethrin effect on ASR in PND 21 and Adult LE Rats (1 mL/kg corn oil)

• Similar dose - related decrease in ASR at 2 & 4 mg/kg
• PND 21: Acute neurotoxic signs at 4 mg/kg; 6 mg/kg was too toxic • Differential sensitivity led us to examine toxicity at higher doses 

### Sheets et al., 1994

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# Deltamethrin Lethality Assessment in PND 11, 21 and Adult LE Rats (1 mL/kg)*

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## Deltamethrin: Comparable Brain Concentrations in PND 21 and Adult LE Rats at Given Effect Level*

Exclusive Property of the Council for the Advancement of Pyrethroid Human Risk Assessment, LLC (CAPHRA) *Sheets et al., 1994 Same effect at similar brain conc. at 7x lower dose

~2x higher brain conc. at same dose & effect

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## Model Type I and II Pyrethroids: Acoustic Startle Response* Pyrethroid Doses (mg/kg) Adult PD21 Comment Cismethrin 0, 3, 6, 12 +300% - 24% (ns) Effect at top dose (unless otherwise stated) Permethrin 0, 30, 60, 120 +300% +65% (ns) (mid - dose) Deltamethrin 0, 1, 2, 4 - 50%* - 59%* Cypermethrin 0, 9, 19, 38 - 50%* - 55%* LE hooded rats; 2 - hr after gavage dose (corn oil; 1 mL/kg) Type I : Uncertain effect in PND 21 vs. adult ( ns = not statistically significant ); • Did not determine this was the optimal time to test pups; • Different strain, dose volume and lab / test equipment in CAPHRA studies Type II : Sensitivity ( ↓ASR ) comparable in adult & PD21; • Clinical signs in pups at much lower dose than adults * Sheets (2000): NeuroToxicology 21 (1 - 2): 57 - 64 (Research at EPA ORD). Exclusive Property of the Council for the Advancement of Pyrethroid Human Risk Assessment, LLC (CAPHRA) 5

## Model Type I and II Pyrethroids: Acute Oral LD 50 Sheets et al., 1994 

### Adult PND 21 PND 11 I: Cismethrin 55 32 119 I: Permethrin 406 201 (2x) 254 (<2x) II: Deltamethrin 81 11 (8x) 5 (16x) II: Cypermethrin 439 73 (6x) 18 (24x)

Cantalamessa , 1993 

### Adult PND 21 PND 8 I: Permethrin 1500 471 (3x) 340 (4x) II: Cypermethrin 250 49 (5x) 15 (17x) **LD50 (mg/kg), based on 6 or 7 male rats per dose group at each age; Magnitude of**  **the difference from adult shown in parentheses.**  Exclusive Property of the Council for the Advancement of Pyrethroid Human Risk Assessment, LLC (CAPHRA) 6

# Background: Sensitivity of Juvenile Rats
• Sensitivity of young rats to Type II pyrethroids raised concern for sensitivity of children

– Only evident with Type II PYR, at high dose levels & dose rates (e.g., not at 1 - 2 mg/kg DLM)

– Decreased rapidly with age: PND 11  21  Adult rats

– Deltamethrin: Evidence sensitivity associated with limited metabolic capacity of young rats (kinetics ) vs. tissue sensitivity (dynamics ) 

### • However: Limited data are available to evaluate

– ASR: Deltamethrin, Cypermethrin, Permethrin & Cismethrin

– Target - tissue levels (lethality & ASR) limited to Deltamethrin

– Some PYR with residential uses (potential exposures) have not been evaluated Exclusive Property of the Council for the Advancement of Pyrethroid Human Risk Assessment, LLC (CAPHRA) 7

# Pharmacodynamics – Targeted Testing Based on the Adverse Outcome Pathway

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# Why PND 15 to Model Sensitivity of 1 - 3 Year Old Children?
• No ideal age : Depends on system, region and “process” of interest • Neurobehavioral measures : Limited selection suited for rats <PND 12 - 15 (sensory and motor function)

– ASR : R espond to acoustic stimulus by PND 12 but motor function (coordination) is limited until ~PND 15;

– Motor activity : PND 13 low level of activity / highly variable; 

### • Brain morphology : General agreement “brain” development too immature at PND 10 and maybe too advanced at PND 21

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# Presentations In vivo measures to Assess Age - Related Sensitivity: The Acoustic Startle Response & Detailed Clinical Observations

– Charles Vorhees ; Cincinnati Children’s Research Foundation, University of Cincinnati 

### Evaluation of Age - Related Pharmacodynamics using Isolated Human (cloned) VGSC

– Steve Symington ; Salve Regina University 

### Targeted Evaluation of Age - Related Pharmacodynamics using Mammalian CNS Neurolemma Preparations

– John Clark ; University of Massachusetts Exclusive Property of the Council for the Advancement of Pyrethroid Human Risk Assessment, LLC (CAPHRA) 10

# In Vivo Rat: Acoustic Startle Response and Detailed Clinical Observations AOP : In vivo relevance but least specific

– Organism: Integrated response

– Brain, spinal cord + PNS; PK + PD; various ion channels 

### Working hypothesis

– If there is no age - related PD difference in sensitivity in rats, we would expect to see comparable neurologic effects at the same internal dose (PYR concentration in the brain). 

### Results : Support hypothesis & previous results

– Deltamethrin : NOAEL ~1 mg/kg in PND 15 and adult; Higher brain tissue levels on PND 15 vs. adult at a given effect level;

– Permethrin : PND 15 only modest effect at high doses (produces tremor in PND 15 and adult rats) – NO difference in dose associated with tremor Exclusive Property of the Council for the Advancement of Pyrethroid Human Risk Assessment, LLC (CAPHRA) 1 3 5 7 9 11 15 19 23 27 31 35 39 43 47 51 55 59 63 0 200 400 600 800 1000 1200

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# Isolated Human VGSCs • Model : Mammalian cells transfected with TTX - sensitive human VGSC’s co expressed with β 1 and β 2 subunits ( Na v 

### 1.1, 1.2, 1.3 and 1.6) • Working Hypothesis: If there is no age - dependent PD sensitivity, VGSCs expressed in children will not be more sensitive than VGSCs in adults. • Results (7 core actives)

– Confirm reports that hVGSCs are less sensitive than rat VGSCs & the response of Na v 1.1 vs. 1.3 indicates children would be less sensitive than adults

Exclusive Property of the Council for the Advancement of Pyrethroid Human Risk Assessment, LLC (CAPHRA) Cheah et al., 2013 (human brain) 12

# Rat Neurolemma Model • Effect of PYR on TTX - sensitive VGSCs in brain tissue from PND 15 & adult rats incorporated into the plasma membrane of Xenopus oocytes 

### Working Hypothesis • If there is no age - dependent PD sensitivity, VGSCs from PND 15 rat neurolemma will not express greater sensitivity than from adults 

### Results (deltamethrin & permethrin) • The VGSCs expressed in PND 15 rats are not more sensitive to deltamethrin or permethrin than those expressed in adult rats 

### Assessment • This system lends itself well to evaluate age - dependent sensitivity of VGSCs at defined stages of development Exclusive Property of the Council for the Advancement of Pyrethroid Human Risk Assessment, LLC (CAPHRA) 13

# Pharmacodynamics - Conclusions
• In Vivo Rat (DLM and PRM)

– At the common NOAEL for neurotoxicity (1 mg/kg ) the predicted brain Cmax levels of deltamethrin is ~4x higher in PND 15 than in adults.

– The results indicate PD sensitivity is less in PND 15 rats than in adults . 

### • Isolated human ion channel (7 core actives)

– The channel most prevalent in juveniles aged 1 - 2 years (and most likely, adults) is less sensitive than the isoform more prevalent at birth.

– The evidence argues against any PD difference likely to favor increased sensitivity of the young (older than ~6 months) .

– These results also confirm reports that human VGSS’s are less sensitive than rat channels to pyrethroids. 

### • Rat neurolemma studies (DLM and PRM)

– No age - related difference in sensitivity evident at a wide range of concentrations. Exclusive Property of the Council for the Advancement of Pyrethroid Human Risk Assessment, LLC (CAPHRA) 14