# Summary: 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)1 Exclusive Property of the Council for the Advancement of Pyrethroid Human Risk Assessment, LLC (CAPHRA)

# In Vivo Rat: Acoustic Startle Response and Detailed Clinical Observations • Working hypothesis

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

# • Results : Support hypothesis & previous results

– ASR: Comparable effect in PND 15 and adult rats at “ low” doses

– DCO: PND 15 more sensitive than adults to higher doses (>2 - 4 mg/kg)

– Sensitivity of PND 15 rats associated with higher brain tissue levels

– Permethrin : ASR is a less sensitive assay in PND 15 than adult rats; Sensitivity to tremor is the same at both ages 

# • Assessment : In vivo relevance but relatively nonspecific

– Requires considerably more animals and time to investigate, with less certainty regarding the neurobiologic basis for comparison (AOP) Exclusive Property of the Council for the Advancement of Pyrethroid Human Risk Assessment, LLC (CAPHRA) 2

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

– Functional assessment of CNS & PNS

– PK and PD elements

– Complex of channels involved: Na + 

# , Ca 2+ 

# , Cl - 

# • PD sensitivity based on neurobehavior

– Results consistent with previous findings • No difference in age - dependent sensitivity at “low” dose • Acute neurotoxic signs in young at higher doses (2 - 4 mg/kg)



# – NOAEL for PND 15 (~1 mg/kg) comparable to adult (1 or <2 mg/kg)

– Brain tissue levels of deltamethrin are higher in PND 15 rat than adult at given effect level Exclusive Property of the Council for the Advancement of Pyrethroid Human Risk Assessment, LLC (CAPHRA) 3 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

TIME (msec)A/D Units Peak

OnsetAdult Male SD Rats PND 15 SD Rats (Vorhees) Dose (mg/kg)* ASR FOB / DCO / MA Brain Conc. (ng/ml) Dose (mg/kg) ASR FOB / DCO Brain Conc. (ng/ml) 1 ( Wolansky 2006) Threshold effect for MA (0.99 mg/kg) 1 Equivocal No effect 27 (4 h) 2 - 41% (2 h)

- 6% (4 h) No effect 9 (2 h) 14 (4 h) 2 - 28% Impaired mobility (87%), tremor (17%) 48 (4 h)

-- -- -- 3 - 37% Impaired mobility (93%), tremor (27%), salivation (33%), 15% mortality -- 4 - 44% (2 h)

- 31% (4 h) No effect 12 (2 h) 21 (4 h ) 28 (6 h) 4 - 59% Impaired mobility (100%), tremor (100%), salivation (73%), 6.7% mortality 110 (4 h) 5 ( WIL/Vorhees) -- No effect (FOB or MA; ASR) 28 (2 h) 20 (4 h) 5 - 78% Impaired mobility (100%), tremor (100%), salivation (93%) 15% mortality -- 6 -- Impaired mobility (100 %),tremor ( - ), salivation (100%)

73% mortality -- 8 - 48% (2 h)

- 31% (4 h) No effect 18 (2 h) 33 (6 h) 8 -- 100% mortality -- 12.5 (PWG) -- Salivation (10%) & splayed hind limbs (20%) -- 15 (WIL) -- Salivation, impaired mobility, decreased MA -- 25 (PWG) -- Salivation (50%), flattened posture (10%), splayed hind limbs (30%) -- 25 (Vorhees) -- Impaired mobility (75%), tremor (25%), salivation (75 %); decreased ASR 44 (2 h) 153 (4 h) 35 (PWG) -- Convulsions (30%), salivation (10%), flattened posture (60%), splayed hind limbs (70%) -- 35 (Vorhees) -- Impaired mobility (100%), tremor (12%), salivation (88%), -- 50 (WIL) -- Convulsions, writhing, salivation, impaired mobility & 1 death -- 55 - 75 (Vorhees) -- 1 of 8 (12.5%) mortality

95 (Aventis) -- LD50 -- Exclusive Property of the Council for the Advancement of Pyrethroid Human Risk Assessment, LLC (CAPHRA) 4 

# Overview: **In Vivo** Rat (Deltamethrin)

Wolansky **et al** ., 2006 (MA relative potency) WIL = Acute Neurotoxicity Study (OPPTS 870.6200) PWG = FOB comparison 12 PYR (Weiner et al., 2009) Vorhees = CAPHRA - sponsored research ASR & DCO Aventis = Acute oral LD50 (adult male rats) 

### NOAEL ~1 mg/kg in PND15 & adult PND 15 rat has higher brain levels of DLM than adult at a given effect level

# Isolated Human VGSCs Working Hypothesis • If there is no age - dependent PD sensitivity, VGSCs expressed in children will not express greater sensitivity than in adults. 

# Results (7 core actives) • The VGSC most prevalent as early as 1 - 2 y/o ( Na v 

# 1.1) is less sensitive to PYR than the VGSC most prevalent at birth ( Na v 

# 1.3); • These results confirm reports hVGSCs are less sensitive than rat VGSCs & the response of Na v 

# 1.1 vs. Na v 

# 1.3 favors children being less sensitive than adults. 

# Assessment • Uncertain how the response of single cloned channels compares to in vivo sensitivity (lacking elements that could impact their response).
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# Rat Neurolemma

• Working Hypothesis

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

# • Results (deltamethrin and 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

– We propose to use this system to evaluate the remaining five core actives for age - dependent pharmacodynamic sensitivity. Exclusive Property of the Council for the Advancement of Pyrethroid Human Risk Assessment, LLC (CAPHRA) 6

# Overview: Pharmacodynamic Sensitivity • In Vivo Rat (DLM & PRM) : Acoustic Startle Response & DCOs

– Adult : Confirmed differential Type I and Type II effect on the ASR and sensitivity as a measure of acute neurotoxicity;

– PND 15 : Overlap of clinical signs with effects on ASR • DLM: Combination ASR & DCO suited to compare PND 15 and adult; • PRM: Only modest increase ASR at 120 mg/kg (t remor PND 15 & 90)

– Adult & PND 15 (DLM) : NOAEL ~1 mg/kg at both ages, with higher brain conc. at a given effect level in the PND 15 rat 

# • Isolated Human VGSC (7 core actives)

– Human channels are less sensitive than rat VGSC;

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

# • Rat Neurolemma (DLM & PRM)

– Relative sensitivity of VGSCs from PND 15 and 90 rats is best suited for comparison of relative sensitivity at defined stages of development. Exclusive Property of the Council for the Advancement of Pyrethroid Human Risk Assessment, LLC (CAPHRA) 7

# Conclusions: Pharmacodynamic Sensitivity

• Results to date : Consistent with Sheets et al . ( 1998; 2000) & Meacham et al . ( 2008), which indicate:

– The age - dependent increased sensitivity to Type II PYR observed in young rats is likely due to PK factors; and

– Children >6 mo would not be more sensitive than adults to PYR due to PD factors. 

# • Future Plans (proposed):

– In vivo rat : Finish the studies with DLM and PRM (PND 15 & adult rats)

– Isolated human channels (7 core actives): No additional work

– Neurolemma (DLM and PRM): Evaluate the remaining five core actives

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