GB2408934A

GB2408934A - Method of screening candidate rodenticidal materials

Info

Publication number: GB2408934A
Application number: GB0425340A
Authority: GB
Inventors: William Dawson
Original assignee: Natrocell Technologies Ltd
Current assignee: Natrocell Technologies Ltd
Priority date: 2001-01-17
Filing date: 2002-01-16
Publication date: 2005-06-15
Anticipated expiration: 2022-01-16
Also published as: GB2408934B; GB0425340D0

Abstract

A laboratory method of screening candidate water-retentive materials such as cellulosic materials for rodenticidal activity in the field is disclosed, in which the rodents are tested to determine whether or to what extent the water-retentive material has disrupted water transport through the wall of the gut. The method involves examination of the gut (for compaction and bloating) and the caecum (for impaction). Rodenticides which act by interference with water transport through the gut wall, particularly in the caecum (where cellulose is digested in rats) have unique selective toxicity, since the caecum is vestigial in humans, who are not therefore affected by such materials.

Description

Method of screening for rodenticidal activity The present invention

relates to methods of screening for rodenticidal activity.

This application is divided from [JK patent application GB 0200943.9 which is directed to methods of screening candidate rodenticidcs on the basis of weight loss in rodents fed the candidate materials ad lithium under laboratory conditions.

Tn our patent GB 2,311,464B a novc] class of rodenticides is disclosed, based on l O cellulosic material derived from (eg the coTe of the cobs of) certain hybrids of maize, namely the Dekalb DK 446 hybrid and agonists thereof. T he ce]]ulosic material constitutes the active rodenticidal material and the disclosed rodenticides are non toxic to humans, domestic animals and livestock.

Although some symptoms of the test animals are given in the above patent specification, and excretion of fat and adipose fissure was presented as a particularly important symptom, the precise mode of action of the above rodenticides has remained unclear. Yet other hybrids besides the Dekalb DK 446 hybrid have been found by the present applicants to be effective as rodenticides in the field, and, like 2 0 the rodenticide based on the DK 446 hybrid, are non-toxic to humans and livestock.

Surprisingly, some hybrids which are effective as rodenticides in the field are not effective in killing rats under laboratory conditions.

An object ol the present invention is to provide a method of screening candidate materials (particularly carbohydrate materials and especia]]y maize hybrids) for rodenticidal activity.

It has now been found that the above rodenticides retain water in the gut and that the resulting disruption of water transport through the gut wall in turn interferes with the 3 0 normal digestion of the rodent. Among the various methods of water transport into and out of cells, passage thTougl1 ion chaTlilels is one of the important processes.

Phese channels var y amongst mammals-de there is a species variation such that mice we Il!)St 'iC!'',i\'e, i'i was le<i;t i<, .'!'.!-!2""'' i!1:tiac-!l'!l Or ail;.; A., iiiiii;.,ii with rats intermediate. It has now been found that cellulosic material is particularly effective at modifiying ion transport in rodents. Consequently the rodent is weakened and dies.

Furthermore in rats, digestion of cellulose takes place mainly in the caecum as a result of the action of bacteria in that region of'the guts whereas in humans the caecum is vestigial and has no digestive action on cellulose. J-t is now considered that water- retentive ce]lulosic materials, when ingested by rats, compromise their digestion in the caecum. Such materials have no effect on humans or livestock.

It has been unexpectedly found that the thymus is reduced in size in rats which have been fed the above cellulosic rodenticides, indicative of a compromised immune system, and it is envisaged that other water-retentive carbohydrate materials, particularly cellulose will have a similar effect. It is considered that their water retentive effect disrupts ion transport through ion channels in the wall of the gut.

Cellulose is the most water-retentive of the complex carbohydrates-it is more water- retentive than starch for example. Moreover cellulose in particular retains its three- dimensional structure in the gut and can thereby provide centres for microbial 2 0 proliferation. In practical terms, it has now been found that such materials reduce the number of immuno-competent cells when ingested by rodents. in particular the generation of T-lymphocytes in the thymus of rodents is inhibited by ingestion of water-retentive materials.

2 5 liny of these effects can be used as a basis for a method of screening candidate rodenticides in accordance with an aspect of the invention.

As noted above. i?1 rats, digestion of cellulose takes place mainly in the caecum as a result of' the action of bacteria in that region of the gut, whereas in humans the caecum 3 0 is vestigial and has no digestive action on cellulose. It is ?10W considered that water- retentive materials, when i?7gcsted by rats, compromise their digestion in the caecum and weaken their immu?1c system. However ur1dcr laboratory conditions a weakened.

i?77?777-C(?77j1177it't,? !! ii! r>< !7C7,7>ic il.ri! it amp < t;J,,?'' , '\ !. .i.iCG in the laboratory cage food and water will be readily available, there will be no predators and there will be a reduced possibility of infection.

It is envisaged that a wide variety of water-retentive materials may be suitable for use as rodenticides. The most useful materials are expected to be non-toxic (to humans) materials of natural origin such as celluloses whose water-retentive properties arise from their macromolecular structure. ()ther suitable materials can be found by experiment.

Accordingly the invention provides a method of screening water-retentive materials for rodenticidal activity, wherein a water-retentive material is fed to rodents (preferably laboratory rats) and the rodents are tested to determine whether or to what extent the water-retentive material has disrupted water transport through the wall of the gut.

Such disruption of water transport can be detected by its effects, eg inhibition of digestion, which can be determined by examination of the gut.

Preferably the rodents are tested to determine whether or to what extent the water- 2 0 retentive material has disrupted ion transport through the wall of the gut.

Preferably tile effect of ingesting the water-retentive material on the size or condition of the thymus gland is tested.

2 S Preferably said water-retentive material is of natural origin.

Prefcrably said water-retentive material is cellulosic material.

Preferably the water-retentive material is derived from coin-cobs.

preferably the r adepts are examined pool nlorlem.

F'!C. t,1 111!1!!S -it. !' ! 1' : S1; ,] til,.itTit Preferably the waterretentive material is substantially non-toxic to humans.

In view of the newly discovered mode of action, the invention is not limited to water- retentive materials derived from corn-cobs.

Other preferred features are defined in the dependent claims.

Methods of testing in accordance with the invention are described below with reference to the fo]]owing examples which detai] separate studies performed by independent testing laboratories.

EXAMPLE 1

1 5 PURPOSE The purpose of this study was to determine the effectiveness of ERADIMOUSE for control of Norway rats fRat/us norvegicus9. This was 1 5-day no-choice test, with a 5- day post-test observation period, to determine the laboratory efficacy.

TEST SUBSTANCE

The test substance was F,RAOIMOUSE, a pelleted rodenticide (5 mm (3/16") diameter pellets) which is a commercial product of the applicants that is non-kxic to 2 5 humans or livestock and which has proven effective in the field. It consists of 95 wt % white core corn cob material derived from "Corn ('obb 2()-4() grind" obtained commercially from Mount Pulaski Products inc., 908 N. Vine St., Mount Pulaski, L]l.

62548, USA and 5 wt % black strap molasses and is made in accordance with the process described in our GB 2,31 1,464B patent.

TEST SYSTEM

Species - Norway rat (Rattus norvegicu).

Strain - Wild types originally fiom the Chicago, 111. area, trapped during spring and summer of 2000, and placed in outdoor colony.

Source of Supply - 'I'he rats were live trapped using appropriate size live traps (Tomahawks) or similar) at a captixc colony in Colorado. The rats were placed in holding cages. 111 rats in holding were monitored daily to assure feed and water were available ad lihifum, and bedding changes were performed twice each week.

Justification for Selection - The Norway rat (wild type) was used because the source population was identified as having a high proportion of rats with genetic resistance to warfarin rodenticides.

Size - The mean rat weight on Day 0 was 297.1 g (+ 13.4 SE). Among the test groups, the females averaged 246.2 g (+ 20.0 SE) and males averaged 331.8 g (+ 16.9 g), whereas the control males were 313.4 g (+ 24.2 SE).

Age - Any ages of rats was acceptable, as long as the minimum weight criterion was met.

Sex and Number - The treatment group consisted of 10 males and 10 females. The control group was al] males (n = 10).

Physica] Condition - The test animals were healthy and active.

HOt]SIN(J, IDEN'r[FICA-l'lON ANI) ENVIRONMI'NTAI, CONDITIONS OF THE 3 0 TEST SYSTEM I-lousing and Fnvironment-A Lest room was reserved exclusively for the rats used in Finis solids. 17 Sue [,1e'i'lf.tit!t'(] , l] -t li'c'!] 1,,,!l,5!, I,',-! l,''t, I'.'S.'-._ I, ...ii I.'' Albert of approximately 972 cm2 during the test. All rats were kept in individual cages for the duration of this study. Cages were labeled so that the cage number corresponds will-,? the test animal number 0?1 the data collection l'orms.

photoperiod of] 2 hours light: l 2 hours dark was maintained for the duration of the test. 'I'he room was lighted with red incandescent bulbs, since research has shown that red light lessens stress of captive wild rats (Fal], l 974). 'I'he high and low temperature and humidity readings were recorded daily using a Fisher Scientific Thermo-Hygro measuring device.

FEED AND WATER OF THE TEST SYSTEM

Acclimation - Fresh Tek]ad F6 Rodent Diet (W) 8664 (Harlan Teklad, Madison, Wis.) was available ad lihitum to all rats during acclimation and the post-test period. Water from bottles equipped with stainless steed sipper tubes was available ad libitum throughout the acclimation, test phase, and post-test period.

Test - The XradiMouse test substance was provided to the treatment group rats during the l 5-day test period. Control group rats were offered the Teklad F6 Rodent Diet.

2 0 Water was available ad lihitum.

Post-test - The Fresh Tek]ad F6 Rodent Diet was provided at all times during the 5- day post-test observation period or any extended period. Water from bottles equipped with stainless steed watering tubes was provided ad libidos.

EXPERIMF,N'rAI, DESIGN Number of Animals Tiler Group - 'I'he test group consisted of 20 Norway rats (] 0 males and I () i'en?aies). An additional gr cup of I () made rats served as the control 3 0 group.

Group Assignment - The trials rats inhere assigned to treatmc,?lt and control groups tISi,?' .i Ciji??!-!ltli/ l ti'-idttT-?i,\.ti(? !^)'>^??? (,1?--()N Feed Consumption - Feed consumption was monitored during the 5day acclimation period, 1 5-day exposure period, and 5-day post-treatnent recovery period.

Body Weight - Body weights were recorded during acclimation, when dead animals were found, and when rats were euthanized.

Necropsy, Microbiological Analysis, and Histology - Animals from the treatment group that died during study were subjected to gross necropsy, on the day of death, for signs of toxicosis or otler cause of death. I, ikewise, whenever a male rat died a control rat was randomly.selected and euthani;<ed. The controls were also necropsied.

Organ tissues including the gastrointestinal tract (from esophagus to colon, with the ends tied offto prevent spillage), liver, and kidneys were extracted and placed in labeled plastic bags, and flee organ samples were taken to the Colorado State University Veterinary Hospital Diagnostic Laboratory. Contents from the Gl tract were analyzed:for C] ostridium species, as well as for other anaerobic and aerobic microbes. Histological tissues from the Gl tract, liver, and kidney were analyzed to evaluate for any lesions or abnormalities.

Observations - All rats were observed daily during acclimation, exposure, and the 2 0 post-test period for signs of toxicosis, morbidity, or mortality.

STATISTiCAl, METT-IODS Data on the environment, observations, mortality and necropsy were tabulated and 2 5 presented descriptively. Daily food consumption data for acclimation and test period were analyzed separately, using a 2-wa,N repeated measures ANOVA, with food consumption as the dependent variable, diet group (made treatment group, female treatment group, and ma]c control) as the test factor? and time as the repeated factor.

RESULTS AND DISCUSSION

Temperatures within the test room were maintained between 2() and 29 C, and 4t,}]i(]i7-\ or,,-,., r7''\) ) I\ 7./'! iiLlli'= i'.' ":.!2. I'_1i l.

Food C'onsumption - During acclimation, the male (15.4 g/day + 0.8 SE) and female 17.3 g/day 1.2 Sly)) rats from the treatment groups consumed slightly less feed than the male rats from the control group (20.7 g/day --I 1.4 SE) (F2, 27 = 5.21, P = 0.01).

Post hoc comparison (Tukey's test) indicated that the male treatment group consumed less food per day than the control (P < 0.05), but the there was no difference between females and either the control group those that were scheduled for treatment. Note that the one of the males fi om the treatment group (rat #15) consumed < 10 g of food during the first four days of acclimation, and this lack of consumption skewed the data. This rat consumed 26 g on days 5 and 6 of acclimation, thins we chose to include 1 0 this anima] with the treatment group.

Evaluation of food consumption during the definitive phase indicated a significant effect from the treatment (172,405 = 3.11, P = 0.046), time (F14,405 = 4.68, P < 0.001), and the interaction oftreatment and time (F28,405 = 3.19, P < 0.001). The troth males and females from the Eradimouse test groups appeared to consume less than controls during the first flour days after beginning the test. Food consumption among groups was relatively equivalent during days 5- 10, but the Eradimouse rats appeared to eat more than controls during days] 1 - ] 5 of the test (FIG. 2). Post hoc analysis with Tukey's test indicate that the females consumed less than the controls on 2 0 Days], 2, and 4 (P < 0.05), whereas males consumed less than controls on Day 1 (PO.05), and the patterns of food consumption for each sex were similar (FIG. 2).

These data appear to suggest that the rats initially rejected the palatability of the Eradimouse bait, but because of hunger they compensated for the loss by the end of the 1 S-day trial.

Bodv Weight - Of the animals that died during the test period (2 females and 5 males), the females lost an average of 35.5% of' pre-te.st body weight, and the males lost 33.9% (combined = 34.4% -t 4.3 SE). The 6 Eradimouse rats that were euthanized on Day 15 also lost substantial weight (22.1% + 2.4 SE), but the weight 3 0 loss of the euthanized was not as much as those that died (t = 2.39, ] I df, P = 0.0356).

the 5 control males that were sacrificed during' the test lost 1.9% of pretest body weight, and the additional 3 that were eutllanized on Day gained 0.7%. Summary data 4 i lathic '.

Observations - The rats remained active through most of the triad. According to observation records one of the treated rats (#28) appeared moribund on day l 4, and this animal was found dead 5.75 hours after initial observations. Other than appearing to lose weight, the rats did not display any other clinical signs of illness.

Mortality - A total of 7 rats (35%) that were fed the Eradimouse bait died during the test and recovery period (5 males and 2 females). All 5 of tile male rats died during the actual test phase. One of the females (#7) died on the first day of recovery the other died on Day of the lest.

Necropsy - Most of the rats did not exhibit any external abnormalities, although rat #l 5 had some lesions on its leg and chest and rat #20 had a small wound on its foot Table 2). Of the 7 rats from the Eradimouse test group that died during the study, 6 exhibited empty but bloated gastrointestinal tracts. One of the rats (#26) had liver parasites. Three of the ten control rats that were sacrificed also had bloated or gasfilled gastrointestinal tracts, but these were fill of food. Two control rats showed liver abnormalities, including one with yellow spots and the other with parasites.

At the termination of the feeding test (Day 15), n=9 rats were euthanized (6 from the 2 0 Eradimouse treatment group and 3 controls), necropsied, and tissue samples were sent to the Veterinary Diagnostic Laboratory. 'lithe three controls showed no internal abnormalities. Three of the six Prom the Eradimouse group had discoloration of the livers and 3 of 6 had bloated gastrointestinal tracts. (:)ne rat exhibited no internal abnormalities.

RF,F;ERENCISS Fall, M. W. l 974. The use of red light for handling wild rats. Laboratory Animal Science. 24:4.

TABLE l

Body Weigllts (BOO) of Norway rats during,, l 5 day no choice test with Eradimouse bait.

ID Sex Diet Died/Sac BW before (g) BW after (g) 1 * F E Sac 209.4 5G.0 2 F E Sac 323.3 319.9 3 P E Sac 128.8 135.7 4 F E Sac 220.6 229.6 F E Sac 177.6 187.S 6 F E Sac 246.3 250.8 7 F E Died 260.0 159.1 8* F E Sac 287.9 22G.4 9* F E Sac 282.1 25.4 26 F E Died 325.5 221.0 10* M E Sac 365.3 267.0 11* M E Sac 264.9 228.9 13 M E Sac 369.4 346.5 1 5 14 M E Sac 312.2 294.8 M E Died 336.1 302.3 17 M E Died 279.5 150.7 19 M E Died 299.6 189.3 22* M E Sac 362.1 251.8 23 M E Died 440.6 270.2 2 0 28 M E Died 288.5 168.4 12* M C Sac 320.5 320.5 16 M C Sac 253.1 270.5 18 M C Sac 285.7 314.1 20* M C Sac 355.8 352.3 21 M C Sac 394.6 292.4 24* M C Sac 299.6 309.0 M C Sac 435.7 416.1 27 M C Sac 178.1 198.1 29 M C Sac 249.8 232.5 M C Sac 361.4 332.4 *Rats were sacrificed on Day 15.

TABLE 2

Results of gross necropsy performed on Norway rats Luring test of Eradimouse l.ui.

Necrcpsy 1D TO Do. Eternal 1 1 M Gf;smybba.ted;nokod I7 Dactyl d&l=i;mfo(xi 23 Gr lV1 ma Died " " G[bloat, Qty 26 I' fib. Died " " 28 PLUIlS;tE;g Died " " GI bid -11 blade- Mod in Gl 12 M Cad Sac " ' Ndabrn11 16 Cow. Sac " " 18 Cow Sac " " ScmeyUbwotscbsvedcnEva Cow Sac Snullrxicnibot:.lii Sac flies " " 24 Cow Sac cc cc M Cal Sac " " " " 27 M Cone Sac " " 29 Hi Cow; Sac " jvas(<tb M Control Sac " " r Sac Dkl8) C: CI 3. LIvRS(b,fi fit Sac C cd GIuasbgy,l , fit Sad cc 2 0 vim Joanne Sac cc LIwas dished, '['ut All L Bat Sa:: " C: LIvas(Egaspsr'tl 9 I1vDi (jibe) It 1 See " " Nbcxli; tvI fight " ' LI-Iaewlumeofdiet 3 o 11 1vI Exam 1C " " 13 He Sac " " Il-.nebloabrgobNi(<trtal) I4 h r lasdied Hi vI ED Sac " " 22 fig= Sac " " WSIebcdisbaldga.sy lea C-rstrxbe4lL lh=o for 1^ (it = Urinal t act =i= Exclusion I'he study shows that ERADIMOUSE interferes with the digestion of wild type rats ailed leads to weight loss but not necessarily death under laboratory conditions.

Lest ion of the product causes intestinal bloating as a result of inhibition of normal digestion, in turn considered to be caused by disruption of water transport throtg.'l, talc wall of the gut.

EXILE 2

OBJECTIVE

To determine the effectiveness of the test substance to produce death in the treated rats when administered as supplied, ad libitum, for a period of 14 days.

METHODS AND MAT:ER]ALS 1. Test Substance: Identification::ERADIRAT, lot#020901.

This sample was pelleted feed in a white bag.

2 5 EIlADAT a pelleted rodenticide (9 mm (3/8" ) diameter pellets) which is a commercial product of the applicants that is non-toxic to humans or livestock and which has proven effective in the field. It consists of 94 wt% white core corn cob material obtained commercially from "Corn Cobb 2040 grind" obtained commercially from Mount Pulaski Products Inc., 908 N. Vine Sit., Mount Pulasl'.i, Illinois 62548, USA 5wt% black strap molasses and lwt% vLeatnour.

2. Test Animal: Species: Rat (raffus norvegicus) Strain: Sprague Dawley Supplier:Harlan Sprague Dawley Indianapolis, IN Number/Sex: 10 males 10 females (nulliparous and non-pregnant) controls (5 males and 5 female) :During the acclimation and testing period, each animal was housed and maintained according to the "Guide for the Care and Use of Laboratory Animals" (NRC, 1996).

All animals were identified by the ear hole-notch method and had cage cards wick provided the individual animal and project numbers. The animals were singly housed in suspended wire cages and fed certified Purina Rodent Chow and county water, ad libitum. Note: The first day the rats were fed non-certified rodent chow.

All animals were acclimated for 7 days prior to testing. Animals were observed for general health and suitability for testing during this period. Beginnimg the day aver arrival, both feed and water consumption analyses were performed.

3. Experimental Procedure: Aider the acclimation period, 20 Sprague Dawley rats (10 males, 10 females) were selected as the test system and 10 Sprague Dawley rats (5 males and 5 females) were 2 0 selected as controls. Rats for each sex's test system were selected by calculating the meals body weight and determining the acceptable weight limit range (+/- 20% of the mean weight). The initial body weights of the test animals ranged from 130.7 g to 148.2 g in the test females, 153.0 g to 169.6 g iri the test males, 134.0 g to 145.2 g in the control females and 155.5 g to 162.5 g in the control males.

2 5 The test substance was administered orally ad libiturn. The control animals received certified Purina Rodent Chow ad libitum.

Feed and water consumption were calculated daily. Eacl, day the amount of food r emaining in the hopper was weighed and recorded. If necessary more food was added to ensure ad libitum feeding and this total was recorded. Additionally, tlc 3 Rod which dropped onto the absorbent pad under each cage was removed weiglicd and recorded for each animal. The amount consulned for each animal was calculatcrJ li ones these values.

Previous day's total food amount returned to hopper (g) - weight of food remaining in hopper (g) - weight of food recovered from abs_bent pad fg) = Amount of Food Consumed (g) ITach day the water bottles were weighed and recorded. If necessary, more water Ads added to ensure ad libitutn watering. The total amount was weighed and recorlcd.

Amount consumed was calculated from these values.

Previous day's total water amount in bottle (g) - weig1Lofwater remain Envy in bottle for current day = Amount of Water Consumed (g) Note: l.0 g is equivalent to 1.0 ml of water.

Averages for the acclimation and test period for.boLh food and water consumption can be seen in Tables 5, and 7.

Note: Care was taken to the best of our ability not to lose test substance, feed or water. Some loss was inevitable during procedures due to spillage.

The animals were examined for signs of toxicity twice daily throughout the 14-Day observation period. On Day 14 the animals were observed only once. The observations included the following: circulatory, respiratory, autonomic and central nervous systems; somatomotor activity; behaviour patterns; onset of tremors, convulsions, salivation, lethargy, diarrhea; skin and fur; and eyes and mucous 2 5 membranes.

Fecal samples for both the test and control animals were collected on Days 1,3, 7' i vl and 14. They were stored frozen for a possible future analysis.

Body weights of the rats (recorded to the nearest tenth of a gram) were recorded 3 0 daily. Initial and final weights as well as the change in total body weight are recorded in Tables 3 and 4.

At the evil of the study, on Day 14, all rats were euthanized (by carbon dioxide) and gross secropsies were performed. Tissue samples for possible future histological examination were taken from each of the test animals. T issues takes and preserved in 1 Oslo formalin were both kidneys, the full gastrointestinal tract and sections of bloc liver. Nccropsy observations are documented in Table 1. Additionally, the stomach contents were collected and stored frozen for possible future exarnisation.

R:ESIJLTS 111 20 test animals survived the 14 day observation period; however, all experienced weight loss. See Table 3 for specific weight losses. Observations were as follows: Rat b1: Appeared unremarkable on Days 1 through 3. On Days 4 through 6, the animal had slight dehydration. On Days 7 through 14, the animal appeared unremarkable.

Rat #2: Appeared unremarkable on Days 1 through 3. On Day 4, the animal appeared dehydrated. On Day 5, the animal appeared unremarkable. On Day 6, the Dirndl had slight dehydration. On Days 7 through 14, the animal appeared 2 0 uinresnarkable ' Rat 43: Appeared unremarkable on Days 1 through 3. On Day 4, the animal lead dull eyes and appeared dehydrated. On Day 5, the dehvdratios remained. Oat Day 6, I:he animal was only slightly dehydrated. On Days 7 through 14, the animal appeared us2resnas-kable.

Rat #4: Appeared unremarkable on Days I through 3. On i)ays 4- and 5, the aiiial was dehydrated. On Days 6 through 14, the animal appeared unremarkable.

Nat #5: Appeared unremarkable on Days] through 4. On Day 5, Alec animal exhibited slight dehydration. On Days 6 through 14, the animal appeared unremarkable.

Rat #6: Appeared unremarkable on.Oays 1 through,' 3. Oil Day 4' lice 3nimc!i was ii.hl-i r1;hydrated f)!.2!" 5, the znira' clad I'll" eyed,.d vitas uch;ira,el. . Day 6, the animal had slight dehydration. On Days 7 through 14, the animal appeared unremarkable.

Rat #8: Appeared unremarkable on Days 1 through 3. On Days 4 arid S. the animal was dehydrated. On Days 6 through 14, the animal appeared unremarkai;)le.

Rat #'3: Appeared unremarkable on Days] through 3. On Days 4 and S. the animal was dehydrated. On Days 6 through 14, the animal appeared unremarkallc.

Rat #16: Appeared unremarkable throughout the 14 Day observation periocl.

Rat #17: Appeared unremarkable on Days 1 through 3. On Days 4 and 5, the animal appeared slightly dehydrated. On Days 6 through 14, the animal appeared unremarkable.

Rat #21: Appeared unremarkable on Days 1 through 3. On Day 4, the animal had dull eyes and appeared dehydrated. On Day 5, the animals had tremors, dull eyes and was dehydrated. On Days 6 ttu'ough 14, the animal appeared unremarkable.

Rat #24: Appeared unremarkable on Days 1 through 3. On Days 4 and 5, tle animal appeared dehydrated. On Days 6 through 14, the animal appeared unremarkable., Rat #26: Appeared unremarkable on Days l through 3. On Day 4, the animal had dull eyes and was dehydrated. On Day 5, the animal had rapid breathing, dull eyes and was dehydrated. On Day 6 the animal was slightly dehydrated. On:E)ays 7 through 14, the animal appeared unremarkable.

Rat #27: Appeared unremarkable on Days 1 through 3. On Day 4, the animal appeared dehydrated. On Days 5 through 14, the animal appeared unremarkable.

stat #29: Appeared unremarkable on Days I througl1 3. On Days 4 and 5, the animal was dehydrated. On Days 6 through 14, the Dirndl appeared unremarlcalle.

Rat #30: Appeared unremarkable on Days 1 through 3. On Days 4 and 5, tile animal had slight dehydration; On Days 6 through 14, the animal appeared unremarkable. ] 7

Rat #31: Appeared unremarkable on Days I through 3. On Days 4 and 5, the animal had slight dehydration. OnDays 6 through 14, the animal appeared unremarkable.

Rat #32: Appeared um'emarkable on Days I through 3. On Day 4, the animal had tremors, dull eyes, and was dehydrated. On Day 5, the animal had dull eyes and dehydration. On Days 6 through 14, the animal appeared unremarkable.

Itat #33: Appeared unremarkable on Days 1 through 4. On Day 5, the animal had slight dehydration. On Days 6through 14, the animals appeared unremarkable.

Rat #35: Appeared unremarkable on Days 1 through 3. On Days 4 and 5, the animal had slight dehydration. On Days 6 through 14, the animal appeared unremarkable.

All 10 of the control animals survived, gained weight, and appeared unremarkable during the 14 day observation period. See Table 4 for specific weight gains.

On Day I 4, all 20 test rats were euthanized and necropsied. Three of the test animals lead fluid- filled small intestines, one of which had a I x I cm) pale yellow area on the stomach lining. All other test animals appeared unremarkable. Ten control 2 0 animals were euthanized and necropsied. All control animals appeared normal. Statistical evaluation of the study data was not deemed necessary.

J

TABI,E 3 3 0 (inRatBoyWeightsforTestAf=,a s Initial Foul AnimallDNo. See DOOM) Dy14() d3einbocwt I M 153.6 136.5 -17.1 2 M 160.6 125.4 -352 3 M 156.5 131.4 -25.1 4 M]69.6 151.8 -17.8 M 161.0 139.0 -220 6 M 154.5 1321 -224 8 M 153.0 123.8 -292 9 M 163.6 135.3 -28.3 16 M 165.0 1372 -27.8 17 M 163.6 151.4 -122 21 F 141.7 1223 -19.4 24 F 138.2 125.4 -12.8 26 F]33.4 129.1 -4.3 27 F 140.0 135.0 -5.0 29 F 141.8 131.5 -10.3 F 1482 131.8 -16.4 31 F 134.0 123.7 -10.3 32 F 145.1 131.5 -13.6 33 F: 13Q7 1241.6 F 136.9 124.1 -12.8

TABLE 4

Change in Rat Body Weights for Control Animals Initial Fmal ArnrnallDNd. , DyO() :D14 c; 3 0 13 M 1582 245.4 +879: 14 M 155.5 256.1 +100.6 M 162.5 263.1 +100.6 18 M 155.5 270.1 Al 14.6 19 M 160.0 263.7 +103.7 23 F 1452 187.6 -t42.4 28 F. 139.5 179.5 140.0 34 F 134.0 170.0 +36.0 36 F B92 1862 7.0 37 F 134.3 183.6 -149.3

TABLE 5

Average Food and Water Consumption for Test Animals Asagewtof Avgewtof Avgevold: Awageolof Arilrl See Fined Food Gamed WatrCcd WatCo IDS P[ilDTe31 While()nTest()2 PloTest(ml) WlileTed:(m9 3 0 1 M 16.0 293 21.8 39.8 2 M 17.1 26.1 Z7.1 37.1 3 M 16.9 26.0 22.8 362 4 [4 18.1 25.3 23.8 35.9 M 1/.4 I.4 249 38.0 6 M 16.5 27.4 Z.2 37.5 M 14.3 27.8 19.1 37.0 9 M 16.6 25.9 21.5 33.7 16 M 18.2 27.8 2I 6 35.3 17 M 162 27.8 22.9 37.1 21:li 14.1 273 22.3 39.6 24 F 147 25.8 20.8 35.53 26 F 15.0 27.2 19.3 40.3 27 F 15.7 26.7 21.9 39.3 29 F 14.4 29.0 18.7 39.4 1 0 30 F 16.8 25.6 25.9 35.7.

31 F 163 293 19.3 37.6 32 F 16.1 30.1 27.64 39.9 33 F 14.9 30.2 21.8 41.6 F 14.3 24.7 22.0 34.0 1 5 Rats received Certified Purina Rodent Chow 2Rats received: the test substance, Eradirat, Lot #020901 3Calculation based on 13 days due to leaky water bottle Calculation based on 6 days due to leaky water bottle

TABLE 6

Average Food and Water Consumption for Control Animals Amagewtof Amagewt Amage;of Avagewtaf Animal See Food Canned Food CQ W=Oc'ned WaterCawrr IDIt Piarto'.fest()* WeTest()*RiortD'r'llileTed (rn) 3 0 13 M 14.4 21.6 23.0 29.3 14 M 15.6 24.7 21.9 28.3 M 18.3 25.4 252 30.4 18 162 25.3 21.0 29.1 19 M 15.0 26.8 21.4 26.6 23 F 15.4 17.7 218 2 28 F 141 17.1 18.0 9.4 34 F 16.8 16.1 24.5. 26.0 36 F 13.5 16.1 16.4 21.6 37 F 16.5 17.7 21.4 23.3 Rats received certified Purina Rodent Chow

TABLE 7

Food and Water Consumption Averages for All Animals AvofAII AvofAllFeed AmaofAU:FeedWrr - AvageofAIL (Pricr Cnp PrtoTe Utafr(n ] 5 loTeston( Du[irTest(0 (no lurirgTest(rti9 l estates 16.7 27.1 2Z7 3G. 8 riestFenales 152 27.6 220 38.3 Corm 15.9 248 22.5 28.7 Co'clFemales is. 3 16.9 20.6 225 S'Y Eradirat, lot #020901, was administered to a group ot 20 white rats (10 males and 10 females) to evaluate its toxic characteristics in accordance with federal requirements as listed in 40 CF 158, Subdivision F. Series 81-1. The animals were observed for 14 days. Any and all behavioral/clinical abnormalities, including mortalities, were recorded. On Day 14, all 20 rats were necropsied for gross pathology. At the time of ;ecropsy, seventeen of the test rats appeared unremarkable, while three had fluid filled small intestines, one of which had a yellowish patch on the stomach lining.

C:onclusion The above study shows that ERADIRAT interferes with tile digestion of laboratory r ats and leads to dehydration and weight loss but not death under laboratory conditions.

In other laboratory studies, (on wild-caught Rattus norvegius rats) impaction of the caecum and intestine was noted on post mortem analysis following ingestion of a similar pelletised rodenticide in accordance with the invention. Caecal and intestinal compaction was noted in other laboratory studies following ingestion of a similar pelletised rodenticide in accordance with the invention.

Claims

CLAIMS: 1. A method of screening water-reteutive materials for

rodenticidal activity, wherein a water-retentive material is ted to rodents and the rodents are tested to determine whether or to what extent the water- retentive material has disrupted water transport through the wall of the gut.
2. A method according to claim I wherein the rodents are tested to determine whether or to what extent the water-retentive material has disrupted ion transport through the wall of the gut.
3. A method according to claim 1 or claim 2 wherein the effect of ingesting the water-retentive material on the size or condition of the thymus gland is tested.
4. A method according to any preceding claim wherein the rodents are examined for one or more of: bloating of the gut, impaction or compaction of the caecum and impaction or compaction of the intestine.
5. A method according to any preceding claim wherein said water-retentive 2 0 material is of natural origin.
6. A method according to any preceding claim wherein said water-retentive material is cellulosic material.

2 5
7. A method according to claim 12 wherein the water-retentive material is derived from corn-cobs.
8. A method according to any preceding claim wherein the rodents are examined post mortem.
9. ^\ method according to any rcc.eciing claim Nrvhcrein the IN atcrreteiZtive matci-ial is subs an,iail: noi-tt><ic l.CJ hUr'-idl:,>.

-

I (). /\ method according to any preceding claim wherein the rodents are examined for intestinal bloating.

9. method according to any preceding claim wherein the water-retentive material is substantially non-toxic to humans.

I 0. A method according to any preceding claim wherein the rodents are examined for intestinal bloating.

AmendmCn! 10 the claims hone beets fi]cd as fo]lo s CLAIMS: 1. A method of screening water-retentiN-e materials for rodenticidal activity, wherein a water-retentive material is fed to rodents and the rodents are tested to s determine whether or to what extent the watcr- retentiNe material has disrupted water transport through the wall of the got.

2. A method according to claim 1 wherein the rodents are tested to determine NN;hether or to what extent the water-retentive material has disrupted ion transport lo through the wall of tile gut:.

3.' method according to claim 1 or claim 2 wherein tile effect of ingesting tile vvat:er-;ctei?tivc material oil the size or condition of iDe thymus gilled is tested.

4. A method according to any preceding claim whc rein the rodents are cxami?Zed for one or Lore of: bloating of the gut, impaction or compaction of'he caecum and impaction or compaction of the intestine.

5. A method according to any preceding claim wherein said water-retentive material is of natural origin.

6. A method according to any preceding claim wherein said wat:erret:entiNre material its cellulosic material.

7. A method according to claim 6 wherein the water-?etentive material is d en i -v ad fro i? ? c orl? - co b s.

8. ,) nf.thod accrTdit} to,)r:!^r c: -lip Cl'-i? .,. j, life 'r> :!ts:I'{""'3]i7]c'( preset I1?olten7.