SUPPLEMENTAL MATERIAL, LP Sheets, AA Li, DJ Minnema, RH Collier, MR Creek

Tags: imidacloprid, Neonicotinoid Insecticides, DNT, PND, cerebellum, Aroclor 1254, flea collars, flea collar, Piriform Cortex, Crofton, optic nerves, Goldey, gastrocnemius muscle, Frontal Cortex, Crofton KM, Goldey ES, gasserian ganglion, Fundam Appl Toxicol, Imidacloprid Neuropathology, optic chiasm, Post-application, application rate, granular layer, coronal section, forebrain, thickness, medulla oblongata, Body Weight, Critical Review
Content: Review of Neonicotinoid Insecticides for DNT (Sheets et al., 2015) SUPPLEMENTAL MATERIAL A Critical Review of Neonicotinoid Insecticides for Developmental Neurotoxicity by LP Sheets, AA Li, DJ Minnema, RH Collier, MR Creek and RC Peffer Critical reviews in Toxicology, 2015 Page 1 of 16
Review of Neonicotinoid Insecticides for DNT (Sheets et al., 2015) Appendix I: Neuropathology and Morphometry Procedures for the guideline DNT study with Imidacloprid Neuropathology and Morphometry ­ PND 11 Brains were divided into eight coronal blocks (including forebrain, cerebrum, midbrain, cerebellum, pons and medulla oblongata) for microscopic examination (Fig. 1). Each block was processed according to standard procedures for paraffin embedding and sectioned at 5 µm, with slides prepared with hematoxylin and eosin (H&E), luxol fast blue/cresyl violet and Sevier-Munger stains. Seven linear measurements were taken from brain sections (levels 4, 5 and 7), as follows: (1) Frontal cortex thickness (forebrain); the dorsal portion of the cerebral cortex within the coronal section passing through the region of the optic chiasm; (2) Parietal cortex thickness (forebrain); the dorsolateral portion of the cerebral cortex within the coronal section taken through the optic chiasm; (3) Caudate putamen horizontal width (forebrain; maximum cross-sectional width) - performed on the coronal section, taken at the level of the optic chiasm; (4) corpus callosum thickness (forebrain) - at the mid-point, from the section taken at the level of the optic chiasm; (5) Hippocampal gyrus thickness (midbrain) - the full width of the hippocampal gyrus from the ventral tail of the dentate gyrus to the overlying subcortical white matter; (6) Cerebellum height (cerebellum/pons) - from the roof of the fourth ventricle to the dorsal surface; and (7) External germinal layer (cerebellum) thickness (cerebellum/pons; PND 11 only) - due to considerable regional differences in thickness, multiple areas were measured over the dorsum of the cerebellum. Neuropathology and Morphometry ­ PND 75 The brain and Spinal Cord, both eyes (with optic nerves) and selected (bilateral) peripheral nerves (sciatic, tibial, and sural), the gasserian ganglion, gastrocnemius muscle, and both forelimbs were collected from each animal and post-fixed in 10% buffered formalin. The fixed brain was weighed upon removal from the skull, before placement into formalin. Vernier calipers were used to obtain two linear measurements of the brain, as described for the PND 11 pups. The following tissues from control and high-dose perfused animals, and any gross lesions collected at necropsy, were routinely processed for microscopic examination: · 8 coronal sections of the brain, including forebrain, center of cerebrum, midbrain, cerebellum, pons, and medulla oblongata; · 4 sections from 3 levels of the spinal cord (cervical, thoracic, and lumbar) and the cauda equina were embedded in paraffin and stained with H&E, luxol fast blue/cresyl violet, and Sevier-Munger stains; · Eyes, optic nerves, and gastrocnemius muscle were embedded in paraffin and stained with H&E; · Dorsal root ganglia (including dorsal and ventral root fibers) from the cervical and lumbar swellings, gasserian ganglia, and peripheral nerve tissues (sciatic, tibial, and sural) were embedded in glycol methacrylate resin (GMA), sectioned at 2-3 µm, and stained with a modified Lee's stain. Microscopic evaluation and measurements of brain tissue were performed as described for the PND 11 animals, with modifications appropriate for the adult. Page 2
Review of Neonicotinoid Insecticides for DNT (Sheets et al., 2015)
The CHECKED (X) tissues will be evaluated for adult offspring.
CENTRAL nervous system BRAIN (8 levels, including) x Forebrain x Center of cerebrum x Midbrain x Cerebellum x Pons x Medulla oblongata SPINAL CORD x Cervical swelling x Thoracic x Lumbar swelling OTHER x Gasserian Ganglion x Optic nerves x Eyes x Cauda equina
PERIPHERAL NERVOUS SYSTEM PERIPHERAL NERVES x Sciatic x Tibial x Sural OTHER x Lumbar dorsal root ganglion x Lumbar dorsal root fibers x Lumbar ventral root fibers x Cervical dorsal root ganglion x Cervical dorsal root fibers x Cervical ventral root fibers x Gastrocnemius muscle
Dorsal and ventral root fibers were evaluated as they were generally included with the ganglion.
Page 3
Review of Neonicotinoid Insecticides for DNT (Sheets et al., 2015) Levels 4 and 5 (Level 7 is not shown) Page 4
Review of Neonicotinoid Insecticides for DNT (Sheets et al., 2015) Page 5
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Review of Neonicotinoid Insecticides for DNT (Sheets et al., 2015) Appendix II: Occupational and Residential Exposure and Risk assessment for Imidacloprid and Flumethrin Formulated Flea Collars (from Lunchick, 2010). SUMMARY 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, with 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 greater, 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 both adults and small children contacting pets wearing the collars. Margins of Exposure (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. 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. Page 7
Review of Neonicotinoid Insecticides for DNT (Sheets et al., 2015) 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 cm2 for the cat and 5986 cm2 for the dog. The Agency uses the single hug method to assess post application dermal exposure with a 5625 cm2 surface area for 70 kg adults and 1875 cm2 for a 15 kg 3 year old child. Post Application Dermal Exposure to Cats Adult: 8.35 mg a.i. x 0.2 ч 2737 cm2 x 5625 cm2 x 0.042 ч 70 kg = 2.1 x 10-3 mg/kg/day Child: 8.35 mg a.i. x 0.2 ч 2737 cm2 x 1875 cm2 x 0.042 ч 15 kg = 3.2 x 10-3 mg/kg/day Post Application Dermal Risk to Cats Adult: 9.3 mg/kg/day ч 2.1 x 10-3 mg/kg/day = 4,429 Child: 9.3 mg/kg/day ч 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 ч 5986 cm2 x 5625 cm2 x 0.042 ч 70 kg = 2.6 x 10-3 mg/kg/day Child: 22.7 mg a.i. x 0.2 ч 5986 cm2 x 1875 cm2 x 0.042 ч 15 kg = 4.0 x 10-3 mg/kg/day Post Application Dermal Risk to Dogs Adult: 9.3 mg/kg/day ч 2.6 x 10-3 mg/kg/day = 3,577 Child: 9.3 mg/kg/day ч 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 cm2 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 ч 2737 cm2 x 20 cm2 x 0.5 ч 15 kg = 4.07 x 10-4 mg/kg/day Post Application Incidental Oral Risk to Cats Page 8
Review of Neonicotinoid Insecticides for DNT (Sheets et al., 2015) Child: 9.3 mg/kg/day ч 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 ч 5986 cm2 x 20 cm2 x 0.5 ч 15 kg = 5.06 x 10-4 mg/kg/day Post Application Incidental Oral Risk to Dogs Child: 9.3 mg/kg/day ч 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. Lunchick C (2010). Occupational and residential exposure and risk assessment for PNR 1427 dog and cat collars formulated with imidacloprid and flumethrin. Bayer HealthCare Report No. 33861. Page 9
Review of Neonicotinoid Insecticides for DNT (Sheets et al., 2015)
Suppl. Table 1. Coefficients of Variation (CV) for Total Session Average Peak Startle Amplitude from DNT-like studies published by EPA scientists
DNT Study
CV Weaning
CV Adult
(Habituationa) (Blank Prepulse)
(percent)
(percent)
Reference
Maternal Separation (n=8)
42
Stanton et al. (1992)
IDPN control (n=12)
39
MAM control (n=8)
Methylmercury control (n=8)
56
Crofton et al. (1993)
42
Goldey et al. (1994)
58
Goldey et al. (1994)
Methanol control (n=6)
Propylthiouracil control (n=6)
27
58
Stanton et al. (1995)
58
Goldey et al. (1995a)
Aroclor 1254 (n=8)
27
Aroclor 1254 & thyroxine replacement (n=8)
27
Iodine levels (assume n=12 for males based on figure legend for cognitive test)
55
Goldey et al. (1995b)
27
Goldey and Crofton (1998)
Gilbert et al. (2013)
60
Average
30
51
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
REFERENCES Crofton KM, Peele DB, Stanton ME. Developmental neurotoxicity following neonatal exposure to 3,3'-iminodipropionitrile in the rat. Neurotoxicol Teratol 1993; 15:117-129. Gilbert ME, Hedge JM, Valentнn-Blasini L, Blount BC, Kannan K, Tietge J, Zoeller RT, Crofton KM, Jarrett JM, Fisher JW. An animal model of marginal iodinedeficiency during development: the thyroid axis and neurodevelopmental outcome. Toxicol Sci. 2013; 132(1):177-195. Goldey ES, O'Callaghan JP, Stanton ME, Barone S, Crofton KM. Developmental neurotoxicity: Evaluation of testing procedures with methylazoxymethanol and methylmercury. Fundam Appl Toxicol 1994; 23:447-464. Page 10
Review of Neonicotinoid Insecticides for DNT (Sheets et al., 2015) Goldey ES, Kehn LS, Rehnberg GL, Crofton KM. Effects of developmental hypothyroidism on auditory and motor function in the rat. Toxicol Appl Pharmacol 1995a; 135: 67-76. Goldey ES, Kehn LS, Lau C, Rehnberg GL, Crofton KM. Developmental exposure to polychlorinated biphenyls (Aroclor 1254) reduced circulating thyroid hormone concentrations and causes hearing deficits in rats. Toxicol Appl Pharmacol 1995b; 135: 77-88. Goldey ES and Crofton KM. Thyroxine replacement attenuates hypothyroxinemia, hearing loss, and motor deficits following developmental exposure to Aroclor 1254 in rats. Toxicol Sci 1998; 45(1): 94-105. Stanton ME, Crofton KM, Gray LE, Gordon CJ, Boyes WK, Mole ML, Peele DB, Bushnell PJ. 1995. Assessment of offspring development and behavior following gestational exposure to inhaled methanol in the rat. Fund. Appl. Toxicol. 1995; 28:100-110. Stanton ME, Crofton KM, Lau C. Behavioral development following daily episodes of mother-infant separation in the rat. Fundam Appl Toxicol. 1992;19(3):474-477. Page 11
Review of Neonicotinoid Insecticides for DNT (Sheets et al., 2015)
Suppl. Table 2. Brain Morphometry Measurements Adjusted for Terminal body weight ­
Thiamethoxam DNT Study
Level 2 ­ Frontal Cortex ­ Height Level 2 ­ Frontal Cortex ­ Width Level 3 ­ Dorsal Cortex 1Thickness Level 3 ­ Dorsal Cortex 2Thickness Level 3 ­ Piriform Cortex ­ Thickness Level 3 ­ Hippocampus ­ Length from Midline Level 4 ­ Dorsal Cortex ­ Thickness Level 4 ­ Piriform Cortex ­ Thickness Level 4 ­ Corpus Callosum ­ Thickness Level 4 ­ Thalamus ­ Height Level 4 ­ Thalamus ­ Width Level 4 ­ Thalamus/Cortex ­ Overall Width Level 4 Hippocampus ­ Length from Midline Level 4 ­ Hippocampus ­ Width Dentate Gyrus Level 4 ­ Hippocampus ­ Length Dentate Gyrus
Day 11 ­ Adjusted for Body Weight
0 ppm Males
4000 ppm Males
0 ppm Females
4000 ppm Females
5.50
5.83
5.67
6.04
4.29
4.53
4.44
4.74
1.29
1.38
1.32
1.39
1.43
1.48
1.46
1.47
1.02
1.12
1.03
1.07
2.64
2.62
2.58
2.46
1.19
1.18
1.21
0.96
1.02
0.95
0.61
0.52
0.57
4.91
4.94
7.43
7.46
12.31 12.21
5.05 7.58 12.43
1.19 1.01 0.50 4.97 7.30 12.08
3.89
3.91
4.00
3.81
0.48
0.46
0.47
0.48
1.53
1.44
1.41
1.43
Day 62 ­ Adjusted for Body Weight
0 ppm Males
4000 ppm Males
0 ppm Females
4000 ppm Females
6.68
6.51
6.41
6.47
4.84
5.00
4.72
4.79
1.58 1.40**
1.54
1.41*
1.89
1.73
1.79
1.70
1.51
1.38
1.40
1.42
2.50
2.45
2.50
2.64
1.54
1.35
1.40
1.30
1.29
1.33
1.28
1.38
0.42
0.40
0.39
0.42
5.41
5.26
8.83
8.53
14.79 14.12
5.18
5.26
8.44
8.04**
14.44 13.55**
4.13
4.04
4.04
3.91
0.62
0.60
0.60
0.60
1.63
1.71
1.70
1.64
Page 12
Review of Neonicotinoid Insecticides for DNT (Sheets et al., 2015)
Level 5 ­ Dorsal Cortex ­ Thickness Level 5 ­ Piriform Cortex ­ Thickness Level 5 ­ Thalamus Width Level 5 ­ Hippocampus Width Dentate Gyrus Level 5 ­ Hippocampus Width Overall Cerebellum ­ Height Cerebellum ­ Length Cerebellum ­ Preculminate Fissure ­ Thickness of Outer Granular Layer Cerebellum ­ Preculminate Fissure ­ Thickness of Molecular Layer Cerebellum Preculminate Fissure ­ Thickness of Inner Granular Layer Cerebellum ­ Prepyramidal Fissure ­ Thickness of Outer Granular Layer Cerebellum ­ Prepyramidal Fissure ­ Thickness of Molecular Layer Cerebellum ­ Prepyramidal Fissure ­ Thickness of Inner Granular Layer
Day 11 ­ Adjusted for Body Weight
0 ppm Males
4000 ppm Males
0 ppm Females
4000 ppm Females
1.08
1.09
1.10
1.11
1.05
1.10
1.08
1.10
6.77
6.50
6.59
7.04*
0.61
0.64
0.61
0.66
1.19
1.23
1.26
1.29
3.68
3.93
3.82
4.01
4.26
4.24
4.11
4.36
40.1
41.5
42.1
39.3
68.6
73.7
73.1
73.0
126
141
138
141
47.7
48.4
45.9
45.0
59.8
59.1
59.2
60.5
128
133
122
137
Day 62 ­ Adjusted for Body Weight
0 ppm Males
4000 ppm Males
0 ppm Females
4000 ppm Females
1.41
1.31
1.40
1.33
1.24
1.23
1.23
8.10 7.51** 7.86
1.20 7.31**
0.76
0.69
0.74
0.75
1.56 1.45*
1.53
1.49
5.59
5.36
5.48
5.37
7.18
6.94
6.75
6.75
ND
ND
ND
ND
213.8 202.1 214.1
196.4
179
161
185
189
ND
ND
ND
ND
218.7 198.9
211.5
196.2
170
156
151
158
Page 13
Review of Neonicotinoid Insecticides for DNT (Sheets et al., 2015) ND = not determined. Values in mm, except for thickness of cerebellum inner granular layer, outer granular layer and molecular layer (m) *p<0.05; **p<0.01 (ANCOVA on terminal body weight + Student's t-test) Page 14
Review of Neonicotinoid Insecticides for DNT (Sheets et al., 2015)
Suppl. Table 3. Historical Control Data ­ Brain Morphometry Measurements at Day 62 ­
Thiamethoxam DNT Study
Males ­ Range
Females ­ Range
Level 3 ­ Dorsal Cortex 1 ­ Thickness 0 ppm ­ Mean
1.25 ­ 1.58 1.58
1.31 ­ 1.51 1.51
4000 ppm ­ Mean
1.40
1.46
Level 4 ­ Thalamus ­ Width
NA
0 ppm ­ Mean
NA
4000 ppm ­ Mean
NA
7.6 ­ 8.46 8.46 8.01
Level 4 - Thalamus/Cortex ­ Overall Width
NA
0 ppm ­ Mean
NA
4000 ppm ­ Mean
NA
13 ­ 14.5 14.5 13.5
Level 5 ­ Thalamus Width 0 ppm ­ Mean 4000 ppm ­ Mean
7.33 ­ 8.11 8.11 7.49
7 ­ 7.88 7.88 7.28
Level 5 ­ Hippocampus ­ Width Overall
1.34 ­ 1.55
NA
0 ppm ­ Mean
1.55
NA
4000 ppm ­ Mean
1.45
NA
Values (mm) are not adjusted for terminal body weight. Range of historic controls = means from 5 studies that were
initiated in 2000 ­ 2002, including the control group from the thiamethoxam study. Mean values from control (0
ppm) and high-dose (4000 ppm) groups in the thiamethoxam study (unadjusted) are also shown, for comparison.
NA = not applicable.
Page 15
Review of Neonicotinoid Insecticides for DNT (Sheets et al., 2015)
Suppl. Table 4. Selected literature studies for the effects of postnatal or gestational nicotine exposure in rats on motor activity
Huang et al. 2007 Thomas 2000
Exposure PND 1-7 PND 4-9
Dose 6 mg/kg/day (2 mg/kg 3x/day) 6 mg/kg/day (1.5 mg//kg 4x/day)
Route
weight loss?
Age of testing
Activity Test
Gavage
Yes
PND 35 and 10 min
70
open field
Gastronomy tube
No1
PND 18-19
1 hr open field
Motor activity results
Horizontal activity
Habituation Rearing
(35) or (70)
N.M.



N.M.
Peters et al. 1979
2 mo prior to mating until birth (cross fostered) or weaning (not cross fostered)
6 mg/kg/day
drinking water
Yes (males only Birth weight reported)
PND 60-80
24-hr home cage
Not cross- fostered: day; night; cross-fostered day
N.M.2
N.M.
Newman et al. 1999
Gestation (19 days) until PND 16
0.75, 1.5, 3.0 mg/kg/day
Osmotic minipumps in dams
Yes at birth ( PND 14 due to Litter size)
PND 14 and 21
40 min open field
0.75 and 3.0 No habituation
at PND 14 but in controls or
not PND 21
treated
N.M.
LeSage 2006
GD 4 -delivery
2 mg/kg/day (0.03 mg/kg every 14 min 16 hr/day)
i.v. cannula to jugular vein
Yes
PND 19-21
30 min open field
(n.s.)
activity first 5-min
(n.s.)
Gestation: 3
Yes, and pair- PND 25-40
Less
Schneider 2012
weeks before mating until
4.2 mg/kg
Drinking Water
fed control group
(testing on 9 1 hr open (4th to 9th
consecutive
Field Day of testing
habituation after first 15
N.M.
delivery
included
days)
min period
Tizabi 20003
GD 4 to delivery
9 mg/kg/day (previous study no effects 3 & 6 mg/kg/day)
s.c. via Osmotic mini pump
Yes
PND 20-24 (daily for 4 consecutive days)
1 hr open field
4
N.M.

n.s. not statistically significant; N.M. not measured
signifies endpoint was measured but no effects were reported 1 Some adjustments to diet were made 2 day and night pattern within 24-hr session was measured so "habituation", as defined by EPA guidelines within a shorter session, was measured
3 Litter was not used as the experimental unit.
4 The authors state there was a numerical increase in horizontal activity that was not statistically significant, but based on inspection of figure and final
conclusions there are no overall effects.
Page 16

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