WO1993007749A1

WO1993007749A1 - Pest control compositions

Info

Publication number: WO1993007749A1
Application number: PCT/EP1992/002422
Authority: WO
Inventors: Roger Valentine Short; Yuan Gao
Original assignee: Akzo Nobel N.V.
Priority date: 1991-10-23
Filing date: 1992-10-16
Publication date: 1993-04-29
Also published as: AU2769092A

Abstract

Disclosed are stable chemosterilant compositions useful in the control of pest populations comprising an orally active progestogenic compound of formula (I), wherein R1 is selected from the group consisting of OR2, OH and F, and R2 is carbacyl of 1 to 18 carbon atoms, and a paraffin wax. The compositions are palatable to roden species and also cause temporary or permanent sterility in the rodents. Preferred compositions contain 16α-ethyl-21-hydroxy-δ4,9-19-norpregnadiene-3,20-dione in amounts ranging from 0.4 to 20 milligrams per kilogram of composition. A method of controlling the number of animal pest species in an area involves distributing the described compositions in the area. Animals eating the pests are not effected by the described progestogens, thereby causing fewer problems with the food chain.

Description

PEST CONTROL COMPOSITIONS

Technical Field. This invention relates to pest control generally and to chemical methods and composi- tions useful for reversibly or irreversibly sterilizing rodent populations specifically.

Background Art. Animal pest species (e.g. rats, mice, rabbits, pigeons, foxes, opossums and others) cause untold damage each year. Rodents are some of the worst pests. Not only are they vectors of many human diseases, such as plague, leptospirosis typhus and salmonellosis, but they also destroy an estimated 20% of the world's harvested crops during storage, and can do major damage to buildings and equipment. Globally, rodents do billions of dollars of damage a year.

In the past, plague populations of rats or mice have been controlled by use one of a number of commer¬ cially available poisons. Under optimal conditions such poisoning campaigns can be extremely effective. Unfor- tunately however, a few animals usually survive the poi¬ soning. They may be ones that failed to ingest a suffi¬ cient amount of poison due to "bait shyness" or some other reason, or animals in the population that are genetically resistant to the particular poison. What- ever the reason, these survivors are capable of repro¬ ducing to produce new plagues, and a strong selection pressure exists in favor of developing a genetic resis¬ tance to the poison. This has certainly occurred in the case of warfarin resistance, and resistance is now developing to difenacoum, a "second generation" anticoagulant.

An alternative to poisoning is to use a chemosteri- lant which prevents the target species from reproducing. A practical disadvantage here is that one must "free feed" the animals until the end of their natural lifes- pans before the population begins to decline, and in the meantime they can continue to do much damage. This is probably why so little research has been done on chemosterilaήts in the past (Bomford, 1990). The idea of using chemosterilants for rodent con¬ trol was discussed as a theoretical concept by Knipling and McGuire in 1972; many potential chemosterilants were screened in the late 1960's and early 1970's. But until now, only one substance, α-chlorohydrin, has been found to-be of any use, and then only for rats. It has been shown to be highly toxic to male and female rats, killing 85% and leaving the adult male survivors ster¬ ile. α-Chlorohydrin is not palatable to rats, but microencapsulation increases its acceptability, although the consumption of a diet containing micro-encapsulated α-chlorohydrin was only half that of the basal diet, a- Chlorohydrin has no antifertility effect in female rats, or in mice of either sex. Other compounds which have been considered for use include estrogens (e.g. EP-A-192,598, DT-2216495, DE- 1,910,122), estrogens ?lso having progestational charac¬ teristics (e.g. US 4,308,265), androstane derivatives (DT 2940132), "claudogenic" compounds (e.g. GB 961,502), and cytostatic agents optionally with progesterone (FR 2,641,943).

One problem with the use of chemosterilants is that, at a baiting station, one must ensure that the bait is highly palatable to the target species through- out the treatment period, especially when an abundance of alternative foods may exist. Rodents may develop a distaste for the prior art chemosterilants after prolonged administration.

For example, although it is described in Yuan Gao, "POSSIBLE USE OF ANDROGEN TO CONTROL RAT POPULATIONS," Proceedings of the 22nd Annual Meeting of the Australian Society for Reproductive Biology, p. 73 (Sept. 24 - 26 1990) that androgens may be used to control rat popula¬ tions, it is further described that the amount of andro- gen the rats will readily consume decreases with an increasing concentration of androgen. Furthermore, in "AVERSION OF FEMALE RATS TO PARAFFIN BLOCKS CONTAINING METHYL TESTOSTERONE DURING PREGNANCY AND LACTATION," Proceedings of the 22nd Annual Meeting of the Australian Society for Reproductive Biology_f p. 47 (Sept. 24 - 26 1990), it was shown that pregnant female rats actually had an aversion to paraffin blocks containing the andro- gen methyl testosterone. Clearly pregnant female rats should be a high priority target group for rat pest con¬ trol, and the described method therefore leaves something to be desired.

A need exists for a highly effective chemosterilant which is effective in controlling pests, especially rodent populations, and which also remains palatable to the pest species throughout a prolonged treatment period.

Disclosure of the Invention.

The invention includes a method of controlling the number of animal pest species in a geographical area. Such a method includes distributing a composition con¬ taining an orally active progestogen associated with an ingestible carrier in the area. The carrier is selected for its desirability or palatability to the pest species, its suitability for the local environment, and the ease with which it can be admixed or otherwise asso¬ ciated with the progestogen while still producing a bioavailable product. The progestogen remains palatable to the members of the pest species when associated with the carrier.

The invention also includes new compositions which have been found to be eminently suited for use as chemosterilants in controlling rat and mouse plagues. The compositions include orally active progestogenic compounds such as Org 5933, its derivatives, and mix¬ tures thereof incorporated into paraffin blocks. The compositions may further contain cereal grains or other components which act as attractants for the particular rodent.

The compositions are spread about infested areas and left for the infesting animals to ingest. Option¬ ally, the compositions can be used in conjunction with an acute poisoning campaign, using conventional rodenti- cides or other poisons, with chronic provision of a chemosterilant to prevent the surviving animals from reproducing, thus decreasing the likelihood of spreading genetic resistance to the poison or chemosterilant.

The invention also includes methods of making and using such compositions.

Brief Description of the Figures.

Fig 1. Average daily consumption of steroid treated paraffin blocks vs. untreated control paraffin blocks by female rats in a free-choice feeding trial in the presence of their standard laboratory diet (unpaired t-test) .

A: The ethinyl estradiol blocks containing differ¬ ent concentrations of steroid were given to the animals for 3 periods of 6 days at intervals of 2 days.

B: The methyl testosterone blocks containing dif¬ ferent concentrations of steroid were given to the animals for 3 periods of 6 days at intervals of 2 days.

C: The Org 5933 blocks were given to animals for 8 ssuucccceessssiive days. (**:p< 0.01; ***:p< 0.001; ****:p< 0.0001).

Detailed Description of the Preferred Embodiments

Compounds useful for practicing the invention are orally active progestogenic compounds which are suffi¬ ciently potent to induce temporary or permanent steril¬ ity in rodents in the amounts used. The compounds are highly palatable to rodents when incorporated into the paraffin.

Progestogens useful in the practice of the inven¬ tion include compounds disclosed in U.S. Patent No. 4,223,030 to de Winter, the contents of which are incor- porated by this reference. These compounds have the formula:

wherein R is selected from the group consisting of F, OH and OR , and R is carbacyl of one to eighteen carbon atoms.

A preferred such progestogen is "Org 5933". Org 5933 is 16α-ethyl-21-hydroxy-rS '⁹-19-norpregnadiene- 3,20-dione. Methods of preparing these compounds are described in U.S. Patent No. 4,223,030. Org 5933 is available from Organon International bv of Oss, NL.

Org 5933 at a concentration of 4 mg/kg paraffin block is highly palatable to female rats and mice, and at doses of about 420 ng/g body weight/day (daily inges- tion of about 20 grams of paraffin block containing 4 mg/kg of the compound) inhibits ovulation in rats within 3 to 4 days after the start of treatment. This infer¬ tility persists throughout the treatment period, but the animals conceived within 5 days after cessation of treatment.

A dose of about 930 ng/g body weight/day or Org 5933 was not completely effective in inhibiting ovula¬ tion in mice, but the females which were pregnant during treatment all gave birth to dead young. When this pro¬ gestogen was given to female rats and mice in the last days of pregnancy, the duration of gestation was signif¬ icantly prolonged, and most young were born dead; some of the females died in labour.

The great advantage of the progestogen Org 5933 is its high palatability and high potency. The optimal concentration was found to be 4 milligrams per kilogram of paraffin block, although lower and higher amounts may be used. The high palatability is shown by the fact that rats will consume up to 20 grams of block per day, and mice about 5 grams of block per day.

Another advantage of this particular progestogen is that there appears to be no aversion to it by pregnant or lactating animals. Thus all the individuals in a population could theoretically start to consume the com¬ pound at the commencement of baiting. This results in a suppression of estrus and ovulation within a few days in any animals that were cycling, and it would effectively prevent parturition or neonatal survival in any animals that were pregnant. The compound is also highly likely to inhibit spermatogenesis.

Female fertility rapidly resumes following cessa¬ tion of treatment with ORG 5933 meaning that little chance of secondary poisoning exists if a treated animal is eaten by an avian or mammalian predator.

Paraffin blocks are ideally suited to chronic administration since the chemosterilant is not leached out by rain, or decomposed by exposure to sunlight, moisture, oxidation or bacterial contamination, and the block is unlikely to be consumed by humans, carnivores, herbivores or birds. Paraffin blocks containing a mix¬ ture of cereal grains are highly palatable to rats and mice, and an ideal way of delivering a steroidal chemosterilant to rodent populations. No chance exists for the progestogen to leak out, and it will be pro¬ tected from decomposition in the block, thereby acting to stabilize the compound. The blocks can, if desired, be secured inside baiting stations, e.g. drainpipes, to further reduce exposure to non-target species.

The compositions are conveniently prepared by sim¬ ply melting the desired amount of a selected paraffin, ■ and then admixing therewith the desired progestogen. Cereal grains and specific attractants (e.g. other types of food or the urine of the particular rodent) can like¬ wise be incorporated into the compositions. Paraffins can be selected for their melting point to take into account local weather conditions. Once made, the inventive compositions may be broken up and distributed throughout the geographical area where the pest species is to be controlled. For rodents, this may be near dumps, landfills, sewers, grain silos, drainage pipes, refuge areas like hedge rows, and other places in the geographicalarea where rodents frequent. The compositions can be monitored, and supplies replenished throughout the erradication period. The treatment period will preferably span at least one natural life span of the pest species. More prefer¬ ably the eradication period will extend over three or four generations of the pest species to insure complete erradication in the geographical area. In the case of rats and mice, this period will typically last one to two years.

The inventive compositions are also eminently suited for use prophylactically to prevent a build up of significant numbers of the target species. It is pre- ferred to initiate long term preventative strategies to stop the population ever reaching plague proportions, and hence chronic baiting with a palatable composition has much to offer as a control strategy. The composi¬ tions are then preferably used when the population is at a nadir, e.g. during winter,.

Furthermore the inventive compositions can be used in an alternate manner with a conventional acute poison¬ ing campaign. The target population is first poisoned to reduce the numbers of infesting animals as rapidly and effectively as possible. The survivors are then chronically exposed to the chemosterilant in a different bait, thereby not only preventing a population rebound, but also inhibiting the development of strains geneti¬ cally resistant to the initial poison. With the rapidly reversible effect of the Org 5933, the inventive compo¬ sitions also overcome the potential problem of secondary poisoning of non-target predators who might feed on the target species. Another potential advantage of chemosterilants for rodent control relates to the humanitarian issue. Animal welfare groups are increasingly concerned about the way in which many conventional poisons kill rodents. A chemosterilant, which does not kill the animal, but rather stops it from breeding, therefore has some ethical advantages.

The invention is further explained by the following illustrative Examples. EXAMPLES

Laboratory studies were carried out to evaluate three synthetic steroids as chemosterilants for rodent control. Ethinyl estradiol, methyl testosterone or Org 5933 were incorporated into paraffin blocks containing cereal grains and offered to laboratory rats and mice in addition to their standard laboratory diet.

EXAMPLE I. Ethinyl estradiol at a concentration of as low as 50 mg/kg paraffin blocks was so highly unpalatable to female rats that the amount of steroid ingested was not sufficient to interfere with their estrous cycles or inhibit ovulation.

EXAMPLE II. Methyl testosterone at a concentration of 5000 mg/kg paraffin block, although not as palatable as untreated blocks, was effective in inducing almost immediate infertility in female rats and mice at an ingested dose for rats of about 35 μg/g body weight/day. This infertility persisted throughout the duration of treatment, and lasted for several weeks after the cessa¬ tion of treatment. Male rats became infertile after 3 months of treatment due to suppression of spermatogene- sis. Female rats developed a specific aversion to methyl testosterone when they were pregnant or lactat- ing, so it was not possible to masculinize the brains of their female offspring. In mice, the androgen treatment induced high levels of aggression in the females so that they fought with and occasionally even defeated males. Some females died of their wounds.

Methyl testosterone was found to suffer from the disadvantage that the minimal effective concentration in the paraffin block needs to be about 5 grams per kilo¬ gram, giving a daily ingested dose of about 35 μg/g body weight for a rat. At this concentration, the bait begins to lose its palatability, although a rat will consume about 1.2 grams a day of the treated paraffin block. Any animals that are pregnant or lactating at the start of baiting show an aversion to the bait, and hence give birth to normal litters of young.

EXAMPLE III. Org 5933 at a concentration of 4 mg/kg paraffin block was highly palatable to female rats and mice, and at doses of around 420 ng/g body weight/day was effective in inhibiting ovulation in rats within 3 to 4 days after the start of treatment. This infertility persisted throughout the duration of treat- ment, the animals conceived within 5 days after cessa¬ tion of treatment. A dose of about 930 ng/g body weight/day was not completely effective in inhibiting ovulation in mice, but those females which were pregnant during treatment all gave birth to dead young. When the progestogen was given to female rats and mice in the last days of pregnancy, the duration of gestation was significantly prolonged, and most young were born dead; some of the females also died in labour. The progesto¬ gen did not appear to inhibit lactogenesis, since the few animals that gave birth to live young reared them normally for the first 5 days of life.

These results show that Org 5933 in paraffin blocks is useful as a chemosterilant for the control of rat and mouse populations. The examples are hereinafter described in more detail.

Materials and Methods.

Animals. Sexually mature male and female Long Evans rats and Balb-C mice were kept in a light:dark cycle of 12:12 hours and.given ad lib, water and a stan¬ dard laboratory diet consisting of wheat meal, soybean meal, linseed meal, rice pollard, meat meal, molasses, vitamins, minerals, salts, calcium and phosphorus. Steroids. Ethinyl estradiol (EE) and methyl testosterone (MeT) were purchased from Sigma Chemical Company, St. Louis, MO, USA. A potent orally active progestogen, Org 5933, was supplied by the Pharma Division of Organon International, Oss, The Netherlands. Baits and diets. Paraffin wax (Paraplast, melting point 56°C, Monoject Scientific, St. Louis, MO, USA) was first melted in a oven at 65°C. The required amount of steroid was added to the melted paraffin which was stirred on a hot plate for 20 minutes. Then a mixture of blended cereals was added to give a ratio of 26% paraffin: 74% mixed cereals (wheat meal 29%, corn meal 28%, oat meal 28%, canary seed 7%, sugar 7% and defatted milk powder 1%) together with blue dye (3 μl dye: 1 gram cereal-paraffin mixture; Queen Fine Foods Pty. Ltd., Queensland, Australia) for the purpose of identifica¬ tion. The paraffin mixture was then poured into plastic dishes 7 cm² and 1 cm deep and allowed to set, to give individual paraffin blocks weighing approximately 35 grams when containing EE or MeT, or 25 grams when containing Org 5933.

Control paraffin blocks were prepared in the same way but without the addition of steroids.

Treatment Groups.

Ethinyl estradiol. Eighteen female rats were weighed and assigned at random to 6 groups of 3 animals per cage. In addition to the standard laboratory diet, they were given untreated paraffin blocks, or treated blocks containing either 50, 100, 200, 500 or 1000 mg of EE/kg for 3 periods of 6 days, separated by no-treatment periods of 2 days. This 2 day discontinuation is the conventional way of assessing re-acceptability of a bait. The block was suspended inside the cage by a wire, and sawdust bedding was excluded from the area beneath the block by a wooden partition so that any chewed but non-ingested block could be weighed. Food intake of the laboratory diet and paraffin blocks was recorded every other day throughout the treatment period by weighing the food remaining in the hoppers and weigh¬ ing the paraffin blocks. Vaginal smears were taken daily to see if ingestion of EE could inhibit ovulation. On day 23 after the start of treatment, untreated male rats were caged with the females for 19 days. The females were then separated and caged singly, and observed daily for signs of pregnancy or birth. Dates of birth and number of offspring were recorded as the index of female fertility. Methyl testosterone. In a dose-seeking experiment,

18 female rats were weighed and assigned at random to 6 groups of 3 animals per cage. In addition to the stan¬ dard laboratory diet, they were given untreated paraffin blocks or paraffin blocks containing either 200, 500, 1250, 2500 or 5000 mg MeT/kg for 3 periods of 6 days, separated by no-treatment periods of 2 days. Food intake and female fertility were monitored as in the EE experiment. In addition, fertile males were caged with any females that had not produced young in the first 40 days after cessation of treatment, to determine when their fertility eventually returned.

In a long-term study of contraceptive efficacy, 18 female rats were weighed and randomly assigned to 3 con¬ trol or treatment groups of 6 animals which were given untreated paraffin blocks or paraffin blocks containing 500 or 5000 mg MeT/kg in addition to the standard labo¬ ratory diet for 98 days. For the first 8 days of the treatment they were individually caged. On day 9, females were housed 2 to a cage and a fertile male was put into each cage for 90 days. The males also had access to the blocks throughout this period.

During the treatment period, any females that had become pregnant at lower dosages of MeT and were close to parturition were individually caged, and the dates of birth and number of young were recorded. For the first 20 days of lactation, daily intake of the paraffin blocks was recorded. If any of the treated females pro¬ duced young, on day 21 of lactation one female from each litter was randomly selected and kept for fertility tests in adulthood. At about 90 days of age, vaginal smears from the surviving young were examined daily for 6 days to see if estrouε cycles were occurring. The females were then paired with fertile males to test their fertility.

After the end of the MeT treatment period, all the adult females were killed. Their ovaries, uteri and pituitaries were weighed and fixed in Bouin's solution for histological study. The males that had been caged with the experimental females were caged with untreated females to check male fertility after exposure to the androgen for 90 days. Then the males were killed. Their testes, epididymides, seminal vesicles, ventral prostates and pituitary glands were weighed and fixed in Bouin's solution for histolog¬ ical study; the left testis and epididymis were not fixed, but were used for assessment of sperm content and motility.

The left testis was decapsuled and homogenised in Triton buffer (0.9% sodium chloride, 0.05% Triton-X-100, 0.01% sodium azide) for 20 seconds using a tissue homogeniser (Ultra-Turrex, Janke and Kunkel GmbH & Co., KG, Ika-Werk Staufen, F.R.G). Nuclei of elongated sper- matids and spermatozoa in an aliquot of the suspension were counted using a haemocytometer. Spermatozoa from the caput, corpus and cauda of the left epididymis were also counted. Motility of the cauda sperm was assessed according to the World Health Organization Laboratory Manual (1987). In a long-term study of female mice, 12 females were weighed and assigned at random to control or treat¬ ment groups of 6 animals. . In addition to the standard laboratory diet, the animals were given untreated paraf- • fin blocks or paraffin blocks containing 5000 mg MeT/kg for 98 days. For the first 8 days the animals in each group were caged together, and from day 9, they were housed 2 to a cage and an untreated, fertile male was introduced into each cage and any young subsequently produced were recorded. The males were replaced ταonthly by fresh, fertile males. The animals were weighed on days 39, 69 and 99 of treatment. At the end of the treatment period, the females were killed and their ovaries and uteri weighed and fixed in Bouin's solution for histological study. Six non-pregnant female mice were used to provide additional histological controls.

The progestogen Org 5933. In a study of non-preg¬ nant rats, 12 females were caged singly and vaginal smears were examined daily for 5 days to assess the nor- mality of their estrous cycles. Then they were weighed and randomly assigned to control or treatment groups of 6 animals which were given untreated paraffin blocks or paraffin blocks containing 4 mg Org 5933/kg for 38 days of the treatment, and during this period vaginal smears were also examined daily to see if ovulation had been inhibited. From day 9 of treatment, an untreated male was introduced into each cage overnight without being offered the paraffin blocks. Dates of birth and number of live young were recorded. On day 39, the treatment was stopped. The adult females were then housed contin¬ uously with the same males until they gave birth; dates of birth and number of young were recorded in order to monitor how long it took fertility to return following cessation of treatment. In a study of pregnant rats, 14 non-pregnant females were weighed and assigned at random to control (n=6) or treatment (n=8) groups, and caged singly. A male rat was then put into each cage and the animals were checked daily for vaginal plugs as evidence of pre- ganancy. The day of mating was defined as day 0 of pregnancy. From day 18 of pregnancy, the females were offered untreated paraffin blocks containing 4 mg Org 5933/kg, in addition to the standard laboratory diet, until day 5 of lactation (the day of birth was defined as day 0 of lactationO. Consumption of the paraffin blocks was recorded daily between day 18 and 20 of preg¬ nancy. Dates of birth and number of live young were recorded. In a long-term study of non-pregnant mice, 18 ani¬ mals were caged singly and vaginal smears were examined daily for 5 days to assess the normality of their estrous cycles before treatment started. Then they were weighed and assigned randomly to 3 groups of 6 animals which were given, in addition to the standard laboratory diet, untreated paraffin blocks or paraffin blocks con¬ taining 0.4 or 4 mg Org 5933/kg for 65 days. Vaginal smears were examined for a further 5 days to see if the treatment had inhibited ovulation, an food intake was recorded daily for 5 days and subsequently at 15 day in¬ tervals. From day 6 of treatment, the females were caged with untreated males which also had access to the paraffin blocks. The males were replaced monthly by fresh, untreated males. Dates of birth and number of young were recorded.

In a study of pregnant mice, 12 non-pregnant female were weighed and assigned at random to control or treat¬ ment groups of 6 animals, and caged singly. The experi- mental design was similar to that for pregnant rats, but treatment started from day 16 instead of day 18 of preg¬ nancy, and the consumption of paraffin blocks was recorded daily during days 16 and 18 of pregnancy.

RESULTS

Ethinyl estradiol. The average intake of treated blocks was very low in all treatment groups during the first two days of treatment; less than 1 g/rat/day vs. 17.4 g/rat/day of the untreated blocks, giving an aver¬ age consumption of EE of 0.08, 0.49, 0.92, 1.68 or 3.49 μg/g body weight/day respectively for the animals given paraffin blocks containing either 50, 100, 200, 500 or 1000 mg EE/kg. During day 3 and 4, consumption declined even further in the treatment groups, and after day 4, none of the treated animals consumed any of the blocks throughout the remainder of the treatment period. The average daily consumption of the blocks for all treat¬ ment periods is shown in Fig. 1. Ingestion of EE during the first 2 days of treatment did not obviously affect the length of subsequent estrous cycles, as judged by vaginal smears.

All treated females gave birth 27.1±5.5 (S.E.M.) days after introduction of untreated males in the post- treatment phase. This did not differ significantly in the timing of birth from the controls (29.0±5.6 days, p>0.05, unpaired t-test) . The average litter size did not differ between the treated (8.5±3.2 young/litter) and control (9.7±4.0 young/litter) animals (p>0.05, unpaired t-test) .

It was concluded that EE even at a concentration as low as 50 mg/kg immediately rendered the paraffin blocks extremely unpalatable to female rats, so that no animal ingested sufficient estrogen to interfere with the estrous cycle or depress fertility. No studies were therefore undertaken in mice.

Ethinyl estradiol is highly unpalatable to female rats in paraffin blocks, even at a concentration as low as 50 mg/kg. The animals only consumed small amounts of the bait during the first 2 days of treatment, and the amount of steroid ingested was insufficient to interfere with subsequent estrous cycles or inhibit ovulation. Therefore EE is of no use as a chemosterilant for rat control. Methyl testosterone. The mean daily intake of the control and treated blocks containing different concen¬ trations of MeT by female rats is shown in Fig. 1. The animals given the higher concentration blocks showed decreased bait consumption. The mean consumption of MeT ranged from 9.8±2.l (S.E.M.), 16.6±2.0, 17.5±2.5 or 15±2.7 μg/g body weight/day respectively for the animals given paraffin blocks containing either 200, 500, 1250 or 2500 mg MeT/kg. It was not possible to measure con¬ sumption of the paraffin blocks containing 5000 mg MeT/kg since after the first 2 days the animals gnawed the whole block into small pieces, scattering them around the cage. This phenomenon was also observed in other MeT experiments when a high concentration of steroid was used, but it was not observed at the lower MeT concentrations. The average intake of the blocks during the first 2 days of treatment was 1.2 g/rat, giv¬ ing a consumption of 34.5 μg/kg body weight/day of MeT. The mean intake of the standard laboratory diet for the controls or the treated animals given paraffin blocks containing either 200, 500, 1250, 2500, or 5000 mg MeT/kg was 1.6±0.3, 5.5±0.9, 7.2±0.5, 10.4±0.6, 10.8±0.7 or 12.7±1.1 g/rat/day respectively, indicating a high palatability of the untreated bait and showing that as the concentration of MeT in the blocks was increased, the animals increasingly preferred the standard laboratory diet to the blocks.

When daily consumption of the blocks was analyzed within a group across the 3 treatment periods there was no significant difference within groups between the treatment periods in the control and the two low dose treatment groups (p>0.05, unpaired t-test), but the intake in the third treatment period was significantly depressed in animals given paraffin blocks containing 1250 or 2500 mg MeT/kg (p<0.05 and p<0.01 respectively), indicating a poor reacceptability of the MeT blocks at high MeT concentrations.

After the cessation of treatment with the 5000 mg MeT/kg paraffin blocks conception was prevented for at least 19 days, and fertility of the females returned within 3 weeks. The controls and almost all the females in the lower dose treatment groups conceived within one or two weeks of being caged with the males following cessation of treatment. There was no statistical difference in litter size between the groups.

In a long-term study of female rats, the animals given paraffin blocks containing 500 mg MeT/kg gave birth 31.2±2.9 (S.E.M.) days after introduction of males; this was significantly later than 24.5±1.0 days in the untreated controls (p<0.05, unpaired t-test). No significant difference in litter size existed between the 2 groups. Fertility of all 6 females given 5000 mg MeT/kg blocks was totally suppressed throughout the 3 months that they were caged with males. The weights of the ovaries, uteri and pituitary glands of the control and treated females given higher concentration blocks after 98 days of treatment are pre¬ sented in Table 1. The ovaries and pituitary glands of the treated rats were half the weight of those of the control females, but there was no significant difference in uterine weight between the two groups. Table 1. Weights of organs of female rats fed paraffin blocks containing 5000 mg MeT/kg for 98 days (mg, Mean±S.E.M.)

Histological examination showed that the ovaries of the treated animals were quiescent, with no large Graafian follicles or corpora lutea. Only one of 6 treated animals had ovaries with corpora albicantia. There were no new corpora lutea in any of the ovaries of the treated animals, indicating that the treated females probably had not been ovulating since the treatment started.

The males that had been exposed to the treated blocks were caged with untreated females after the end of the 3 month treatment period. However, they failed to sire any young as a result of matings in the first 5 days post treatment. Spermatogenesis was severely depressed, as assessed by testicular weight (Table 2), histological appearance and testicular content of sper¬ matozoa. In contrast, all the males that had been caged with the control females successfully sired young and their spermatogenesis appeared normal. _Ta_ble 2. Weights of organs of male rats fed paraffin blocks containing 5000 mg MeT/kg for 90 days (mg, Mean±S.E.M.)

Groups Testis Epididymis Seminal Ventral Pituitary vesicle prostate gland

An aversion of female rats to paraffin blocks con- taining 500 mg MeT/kg was noted during lactation. The females in the control group consumed untreated blocks at about the same rate as non-pregnant animals. How¬ ever, the treated rats consumed only 0.34 g/rat/day of the treated blocks during the first 16 days of lacta- tion, followed by a sharp increase in consumption of the blocks, which might have been due to the young starting to eat the blocks. In comparison, non-pregnant females consumed about 5 g/rat/day of the treated blocks. This aversion had problably started during pregnancy, but since the female was caged with the male until late pregnancy it was not possible to monitor the food intake of the females. This aversion of female rats to treated blocks during pregnancy and lactation was confirmed in later experiments. No evidence existed to suggest that the fertility of the female offspring of rats treated during pregnancy and lactation was inhibited, as they all had normal estrous cycles as judged by their vaginal smears at about 90 days of age, and they all subsequently gave birth to young after being caged with males.

The progestogen Pro 5933. In a study of non-preg¬ nant rats, the mean daily consumption of paraffin blocks containing 4 mg/kg Org 5933 by females during the first 8 days of treatment was significantly higher (p<0.01) than that of untreated blocks (Fig. l) giving an average consumption of 419 ng/g body weight/day of Org 5933.

All female rats showed normal oestrous cycles, as judged by vaginal smears, during the 5 days before the start of treatment. But 4 days after the start of treatment, the smears of the treated animals contained strings of mucus containing large numbers of metoe- strous-type cells. The first 4 days of treatment failed to prevent females progressing from proestrous to estrous smears.

When caged with males, all the 6 control females gave birth to litters of live young 24.5±0.6 (S.E.M.) days after the introduction of the males, but none of the 6 treated females gave birth, and none appeared to be pregnant one month after the introduction of the males.

Treatment was stopped after 38 days and fresh males were introduced the following day. All the controls gave birth to litters of live young 31.2+0.5 (S.E.M.) days later, whereas all the treated animals gave birth to live young 26.6±1.5 days later, significantly earlier than the controls (p<0.05, unpaired t-test).

These results show that paraffin blocks containing 4 mg/kg Org 5933 were highly palatable to female rats, and consumption of the steroid at a dosage of about 420 ng/g body weight/day completely inhibited ovulation. But this antifertility effect was rapidly reversible following cessation of treatment.

In a study of pregnant rats, the consumption of both untreated paraffin blocks and paraffin blocks con¬ taining 4mg/kg Org 5933 declined in the last few days of pregnancy. The control animals consumed 21.2±1.6, 11.2±1.9 and 5.5±1.6 g/rat/day on days 18, 19 and 20 of pregnancy, while the treated animals consumed 20.9±1.0, 11.3±0.9 and 4.4±1.3 g/rat/day during those 3 days. This difference was not statistically significant between the 2 groups (p>0.05, unpaired t-test), and probably reflects a decrease in feeding activity of rats in the last stages of pregnancy. The average consump- tion of Org 5933 was 246 ng/g body weight/day during days of 18, 19, and 20 of pregnancy.

All the 6 control females gave birth to litters of live young. In the treatment group, 2 of the 8 animals died in labour on day 25 of pregnancy, 3 gave birth to litters of dead young between day 24 and 26 of preg¬ nancy, and the other 3 gave birth to live litter on day 22 of pregancy and all the young were still alive on day 5 of lactation, when the experiment was terminated. The length of gestation, as judged by the time elapsed from presence of a copulatary plug to the date of birth, was 21.7±0.2 days in the control animals vs. 23.5±0.7 days in the 6 suvivors in the treatment gorup (p<0.01, unpaired t-test). These results show that ingestion of Org 5933 dur¬ ing the last few days of pregnancy can postpone parturi¬ tion, leading to neonatal and maternal deaths in labour. Surprisingly however, ingestion of the compound did not seem to inhibit lactation, as the young which were born alive survived throughout the first 5 days of lactation. In a long-term study of non-pregnant mice, there was no statistically significant difference in mean daily intake of the control or 0.4 mg/kg or 4 mg/kg Org 5933 blocks during the first 5 days of treatment ( .78±0.2 vs. .7l±0.3 vs. 5.06±0.3 g/mouse/day) , and there was no significant change in consumption of the blocks throughout the remaining treatment period, indi¬ cating that the treated blocks were as palatable as untreated blocks. The mean intake of standard labora- tory diet in all the groups was about 1 g/mouse/day, indicating that the treated paraffin blocks were very palatable to female mice. The average consumption of the steroid in the 2 treatment groups was 87 ng/g body weight/day or 929 ng/g body weight/day. All the female mice showed normal estrous cycles, as judged by vaginal smears, before the treatment started, but 3 days after the start of treatment the smears from the treated females contained strings of mucus containing large numbers of metestrous-type cells. The first 3 days of treatment failed to prevent females progressing from poestrous to estrous smears.

When caged with male mice for a month, all the 6 control females gave birth to litters of live young, whereas in the 0.4 mg/kg treatment group only 4 of the 6 females gave birth, and half the young were dead. One female died in labour and one was not pregnant. In the 4 mg/kg treatment group, again only 4 of the 6 gave birth, but all the young were dead. One female died in labour and one animal was not pregnant. The control females gave birth 26.0±2.7 (S.E.M.) days after intro¬ duction of the males, and the 0.4 mg/kg group gave birth after 25.313.3 days, whereas the 4 mg/kg group gave birth after 35.811.5 days, a significant delay (p<0.05, unpaired t-test) .

After the first births, all the animals remained on treatment. Five of the 6 control females had second births of live young 23.211.8 days after the introduc¬ tion of males; 2 of the surviving females from the 0.4 mg/kg Org 5933 group gave birth to young 21 or 28 days after the introduction of males, but only 25% of the young survived. However, in the 4 mg/kg group only one of the 5 surviving females gave birth 28 days after the introduction of the male, and none of the young survived.

Treatment was stopped after -65 days, and the fol¬ lowing day fresh males were introduced. The 6 controls all gave birth to live young 29.712.0 days later; 4 out of 5 of the 0.4 mg/kg group gave birth to live young 26.511.9 days later, and all 5 of the 4 mg/kg group gave birth to live young 22.410.7 days later, a significant advance on the controls (p<0.05, unpaired t-test).

Thus the progestogen was less effective in mice than in rats for inhibiting ovulation, but there was a pronounced adverse effect on parturition and neonatal survival. As in rats, the effects of the steroid were rapidly reversible following cessation of treatment.

In a study of pregnant mice, the control animals consumed 6.610.7 (S.E.M.), 4.710.5 and 4.310.9 g/mouse/day of the untreated blocks on days 16, 17, and 18 of pregnancy, while the treated animals consumed 4.611.1 (p>0.05, unpaired t-test) . 6.610.5 (p<0.05) and 5.711.0 (p>0.05) g/mouse/day of the blocks containing 4 mg/kg of Org 5933 on those 3 days, and unlike rats, there was no decline in the block intake in the last stages of pregnancy. The average intake of the progestogen was 1053 ng/g body weight/day.

All the 6 control mice gave birth to litters of live young, whereas 5 of the 6 treated females gave birth to dead litters. None of the females died in labour. One treated mouse gave birth to live young on day 20 of pregnancy. She then successfully suckled them until day 5 of lactation, when the experiment was terminated.

The controls gave birth 19.310.2 days after detec¬ tion of vaginal plugs, while the treated females gave birth 21.8+0.4 days after detection of vaginal plugs, a highly significant delay (p<0.θl, unpaired t-test). This clearly shows that ingestion of Org 5933 interfered with parturition, leading to death of neonates. But again as in rats, it did not seem to inhibit lactation.

Claims

CLAIMSWhat is claimed is:

1. A composition useful as a chemosterilant comprising an

and 0R₂, and R is carbacyl of one to eighteen carbon atoms, and a paraffin wax, said compound being palatable to rodent species in the amounts used and also causing temporary or permanent sterility in said rodents.

2. The composition of claim 1 wherein said compound is 16α-ethyl-21-hydroxy-fS⁴'⁹-19-nor-pregnadiene-3,20-dione present in amounts of about 0.4 to 20 milligrams per kilogram composition.

3. The composition of claim 2 wherein said compound is present in amounts ranging from 4 to 9 milligrams per kilogram of composition.

4. The composition of claim 1, 2 , or 3 further comprising an attractant.

5. The composition of claim 4, wherein said attractant comprises cereal grain.

6. A method of rodent control comprising feeding a composition of any of claims 1 to 5 to rodents so that said rodents consume progestogen in an amount equivalent to at least 420 nanograms of 16α-ethyl-21- hydroxy-5⁴'⁹-19-nor-pregnadiene-3,20-dione per gram body weight.

7. The method of claim 6 further comprising feeding poisoned bait to said rodents.

8. The method of claim 7 wherein the feeding of the composition of any of claims 1 to 5 is alternated with the feeding of the poisoned bait to said rodents.

9. A method of controlling the number of animal pest species in an area comprising distributing a composition containing an orally active progestogen associated with an ingestible carrier in the area, said carrier selected for its desirability to the pest species, and said progestogen being palatable to the members of the pest species when associated with said carrier, and being present in sufficient quantities to reduce the fertility of said animal pest species.

10. A composition useful as a chemosterilant comprising an amount of a paraffin wax and an orally active proge a:

wherein R^ is selected from the group consisting of F, OH and 0R₂, and R₂ is carbacyl of one to eighteen carbon atoms, and present in a sufficient amount to cause temporary or permanent sterility in rodents ingesting the composition, said compound further being palatable to rodent species in the amounts used.