A critical review of neonicotinoid insecticides for developmental neurotoxicity

Abstract A comprehensive review of published and previously unpublished studies was performed to evaluate the neonicotinoid insecticides for evidence of developmental neurotoxicity (DNT). These insecticides have favorable safety profiles, due to their preferential affinity for nicotinic receptor (nAChR) subtypes in insects, poor penetration of the mammalian blood–brain barrier, and low application rates. Nevertheless, examination of this issue is warranted, due to their insecticidal mode of action and potential exposure with agricultural and residential uses. This review identified in vitro, in vivo, and epidemiology studies in the literature and studies performed in rats in accordance with GLP standards and EPA guidelines with imidacloprid, acetamiprid, thiacloprid, clothianidin, thiamethoxam, and dinotefuran, which are all the neonicotinoids currently registered in major markets. For the guideline-based studies, treatment was administered via the diet or gavage to primiparous female rats at three dose levels, plus a vehicle control (≥20/dose level), from gestation day 0 or 6 to lactation day 21. F1 males and females were evaluated using measures of motor activity, acoustic startle response, cognition, brain morphometry, and neuropathology. The principal effects in F1 animals were associated with decreased body weight (delayed sexual maturation, decreased brain weight, and morphometric measurements) and acute toxicity (decreased activity during exposure) at high doses, without neuropathology or impaired cognition. No common effects were identified among the neonicotinoids that were consistent with DNT or the neurodevelopmental effects associated with nicotine. Findings at high doses were associated with evidence of systemic toxicity, which indicates that these insecticides do not selectively affect the developing nervous system.

The CHECKED (X) tissues will be evaluated for adult offspring.

INTRODUCTION
PNR 1427T and PNR 1427G are antiparasitic collars for flea and tick control for cats and dogs, respectively. Both collars contain 10% imidacloprid as an active ingredient. The collars will be used for an eight month period and therefore would be used once per year in many regions of the United States and no more than twice per year in the warmer regions of the country. The purpose of this exposure and risk assessment is to support the registration application for PNR 1427T and PNR 1427G.

PNR 1427 Release Kinetics
The flea collar contains a patented Bayer polymer matrix system that ensures the active ingredients are slowly and continuously released in low concentrations from the collar towards the animal. This minimizes peak concentrations and ensures that acaricidal / insecticidal concentrations are present in the dog or cat's haircoat during the entire eight month efficacy period. The active substances spread from the site of direct contact over the entire skin surface. Bayer conducted several studies to quantify the release rate of imidacloprid from the collar and also to measure residues on the animal's fur over time.

Toxicology (imidacloprid)
HED selected a NOEL of 10 mg/kg/day from the rat developmental toxicity study as the most appropriate endpoint for short-term oral, dermal, and inhalation exposure risk assessments. A NOEL of 9.3 mg/kg/day from the rat subchronic neurotoxicity toxicity study was selected as the most appropriate endpoint for intermediate-term oral, dermal, and inhalation exposure risk assessments. For the purpose of this assessment the intermediate-term NOEL of 9.3 mg/kg/day is used to address both short-term and intermediate-term exposures. Based on the results of the dermal absorption study a dermal absorption adjustment of 4.2% is used for the dermal exposure risk assessment.

POST-APPLICATION EXPOSURE AND RISK
Post-application exposure to imidacloprid contained in the flea collars is estimated based on the guidance provided in the "Current Guidance for Residential Exposure Risk Assessment for Pet Insecticide Treatments" of 14 January 2009. Post application exposure occurs from pet owner contact with the dog or cat wearing the flea collar. The guidance for pet flea collar assessments assumes that the daily dose on the day of application is based on 20% of the maximum application rate being available on the pet's body and transferred to adults and children as a dislodgeable residue.
As previously stated, the cat/small dog flea collar contains 1.25 grams of imidacloprid and the large dog flea collar contains 4.50 grams of imidacloprid. Based on the kinetic data, the daily release rates are 8.35 mg/day and 22.7 mg/day of imidacloprid from the small and large collars, respectively.
The small flea collar will be used to assess exposure to a 9 lb cat and the large flea collar will be used to assess exposure to a 30 lb dog. The HED defaults for the surface area of the cat and dog are 2737 cm 2 for the cat and 5986 cm 2 for the dog. The Agency uses the single hug method to assess post application dermal exposure with a 5625 cm 2 surface area for 70 kg adults and 1875 cm 2 for a 15 kg 3 year old child.
Post Application Dermal Exposure to Cats Adult: 8.35 mg a.i. x 0.2 ÷ 2737 cm 2 x 5625 cm 2 x 0.042 ÷ 70 kg = 2.1 x 10 -3 mg/kg/day Child: 8.35 mg a.i. x 0.2 ÷ 2737 cm 2 x 1875 cm 2 x 0.042 ÷ 15 kg = 3.2 x 10 -3 mg/kg/day Post Application Dermal Risk to Cats Adult: 9.3 mg/kg/day ÷ 2.1 x 10 -3 mg/kg/day = 4,429 Child: 9.3 mg/kg/day ÷ 3.2 x 10 -3 mg/kg/day = 2,906 Post Application Dermal Exposure to Dogs Adult: 22.7 mg a.i. x 0.2 ÷ 5986 cm 2 x 5625 cm 2 x 0.042 ÷ 70 kg = 2.6 x 10 -3 mg/kg/day Child: 22.7 mg a.i. x 0.2 ÷ 5986 cm 2 x 1875 cm 2 x 0.042 ÷ 15 kg = 4.0 x 10 -3 mg/kg/day Post Application Dermal Risk to Dogs Adult: 9.3 mg/kg/day ÷ 2.6 x 10 -3 mg/kg/day = 3,577 Child: 9.3 mg/kg/day ÷ 4.0 x 10 -3 mg/kg/day = 2,325 Post application incidental oral exposure is only calculated for the three year old child. An equilibrium approach is taken by HED and assumes that 20% of the available residues are available for transfer to the hands of the child. The surface area of the hands that provide oral contact is 20 cm 2 and that the saliva removes 50% of the residue that is transferred to the hands. The oral NOAEL of 9.3 mg/kg/day is used to calculate the MOEs for imidacloprid. The oral incidental exposure to the child is calculated as follows: Post Application Incidental Oral Exposure to Cats Child: 8.35 mg a.i. x 0.2 ÷ 2737 cm 2 x 20 cm 2 x 0.5 ÷ 15 kg = 4.07 x 10 -4 mg/kg/day Post Application Incidental Oral Risk to Cats Child: 9.3 mg/kg/day ÷ 4.07 x 10 -4 mg/kg/day = 22,850 Post Application Incidental Oral Exposure to Dogs Child: 22.7 mg a.i. x 0.2 ÷ 5986 cm 2 x 20 cm 2 x 0.5 ÷ 15 kg = 5.06 x 10 -4 mg/kg/day Post Application Incidental Oral Risk to Dogs Child: 9.3 mg/kg/day ÷ 5.06 x 10 -4 mg/kg/day = 18,380

Post Application Exposure and Risk Summary
Adult post application exposure resulting from contact with the treated animal is by the dermal route of exposure resulting from the transfer of imidacloprid residues from the animal's fur to the adult's skin. The imidacloprid exposures were calculated to be 2.1 x 10 -3 mg/kg/day for animals wearing the small collar and 2.6 x 10 -3 mg/kg/day for animals wearing the large collar. The resultant MOEs were 4,429 and 3,577 for the small and large collars, respectively.
The total post application exposure and risk to small children contacting pets wearing the flea collars results from both the dermal contact and the incidental ingestion of imidacloprid residues from the hands. For children contacting pets wearing the small collars, the aggregate post application exposure to imidacloprid is 3.61 x 10 -3 mg/kg/day and 4.51 x 10 -3 mg/kg/day with the large collar. The dermal and oral NOAEL for imidacloprid is 9.3 mg/kg/day and the aggregate MOEs for the small child are 2,576 with the small collar and 2,062 with the large collars.

CONCLUSIONS
An assessment of the potential exposure resulting from the use of PNR 1427 flea collars containing imidacloprid as an active ingredient was calculated for post application contact with treated cats, small dogs, and large dogs. The small flea collars weigh 12.5 grams and contain 1.25 grams imidacloprid and are intended for use on cats or small dogs up to 8 kg in weight. The large flea collar is intended for dogs weighing over 8 kg and weighs 45.0 grams and contains 4.5 grams of imidacloprid.
The daily application rate of imidacloprid from the collars to the pets was determined based on release rate kinetic studies. Approximately 40% of the imidacloprid is released over an eight month period at a rate that slightly decreases over the period. During the first month of use when the release rate is slightly higher the daily rate of release of imidacloprid from the small collar is 8.35 mg/day and from the large collar it is 22.7 mg/day. Fur kinetic data supported the release rate kinetics.
The revised HED guidelines for pet exposure and risk assessments were used with the release rate data to estimate the exposure and risks to adults applying the collars and to both adults and small children contacting pets wearing the collars. MOEs for adult post application contact with the treated pets ranged from 2,200 to 4,400. The aggregate oral and dermal MOEs for small children ranged from 1,300 to 2,600.

Suppl. Table 1. Coefficients of Variation (CV) for Total Session Average Peak Startle Amplitude from DNT-like studies published by EPA scientists
Note: DNT -developmental neurotoxicity IDPN -3,3'-iminodiproprionitrile (a within-litter study design) MAM -methylazoxymethanol PND -post-natal day a The startle reflex with blank prepulse in the reflex modification design is comparable to the startle reflex in the habituation design. The CVs were estimated from the figures which reported total session peak amplitude and the SEM. Sample size (n) was used to calculate SD from SEM [SD = SEM * SQRT(n)]. CV=SD x 100/mean Suppl.