Guidelines for ovine lousicides efficacy submissions
Guideline 23
Contents:
1. Introduction
These guidelines apply to insecticidal substances to be submitted to the National Registration Authority (NRA) for clearance where claims are made for the control of Bovicola ovis infestation of sheep. Claims for strains of varying resistance may be made if adequate efficacy data is presented. Where claims are made against a particular resistant strain, the resistance status of the strain must be quantified by in vitro LC 50 testing. If claims are to be made for other species of lice, comparable data should be presented.
The applicant must show that, using the examination methods and intervals described herein, the product is capable of eliminating lice from all sheep in a treated mob. Claims for long wool products must demonstrate greater than 95% reduction in lice population at 90 days after treatment.
No part of these guidelines should be taken to be an alternative to recognised statistical design and analytical methods, but should be used as a basis for the design of trials.
It is the responsibility of the applicant to devise the appropriate experimental procedures which will yield significant, repeatable results. Any departures from the guidelines should be discussed well in advance with the NRA secretariat.
2. Laboratory data
Basic lousicidal data should be presented and where feasible should include the MLD 100 (minimum lethal dose required to kill all susceptible lice under normal conditions) for the active ingredient for use on sheep. The method used for this determination is selected by the applicant and described in the submission.
3. Trials
The following information should be detailed for each trial:
- location;
- date;
- officer-in-charge and other staff or co-operators involved;
- product identity;
- formulation and batch number;
- breed of sheep used;
- time since last shearing and length of wool;
- the method of application (dipping, jetting, backline or others) and any departures from label recommendations proposed for the product;.
- where artificial rainfall is used to test the efficacy of the product after heavy rain, the method of wetting used and the equivalent in terms of natural rainfall should be stated (eg artificial rain applied via inverted sprinklers, equivalent to a rainfall of 20mm in a storm lasting 30 minutes);
- any other chemical treatment or medication of the treated sheep during the course of the trial;
- use of animals in trials must be in accordance with the principles set down in the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes (1990) and the requirements of any State or Territory legislation controlling the use of animals in research.
3.1 Pen trials
For each new product at least two pen trials are required.
Purpose of the trials
- To demonstrate that the minimum dose rate of concentration of the product that is recommended for commercial use, is capable of reliably eliminating lice from all sheep that are treated under the proposed conditions for use. This could involve trials to show product efficacy under a variety of conditions of use (breed, length of wool, type of equipment, quality of water used as diluent etc.).
- To demonstrate what, if any, length of protection against reinfestation is provided.
- To identify peculiarities, contraindications, side effects etc when the commercial formulation of the product is used on sheep.
- To demonstrate that heavy rainfall immediately before or after treatment does not effect the efficacy of the product.
Each trial should consist of an untreated control group and treated groups including at least one treated at the recommended rate with the proposed commercial formulation.
Each group should contain at least 5 sheep with a naturally acquired or stable, artificially induced infestation of lice. Sheep included in these groups should be acclimatised to their environment for at least 14 days before the trials begin.
The groups should be maintained in isolation for the duration of each trial so that there is no contact with sheep from any other group.
3.2 Field trials
(a) Off-shears or short wool (0-42 days) products
In addition to pen trials, a minimum of 5 field trials are to be conducted, 3 of which are to be conducted using fine wool Merino sheep. Each mob to be treated should have at least 1,000 sheep and trials will run for at least 20 weeks.
(b) Long wool (43-300 days) products
In addition to pen trials a minimum of 6 field trials will be required, two each at fleece lengths equivalent to about 3, 6 and 9 months growth of wool. Three (3) of the trials (1 at each wool length) are to be conducted in fine wool Merino sheep. Each mob to be treated should have at least 500 sheep. Where the product is claimed to reduce lice to non-detectable levels, trials will run for at least 20 weeks or until the next shearing, whichever occurs first. Where the product is claimed to reduce louse numbers by ³ 95% for at least 30, 60 or 90 days, trials will run for a minimum of 40, 70 or 100 days, respectively, or until the next shearing, whichever is the sooner.
(c) Numbers of sheep to be examined
In each field trial at least 25 tagged sheep from the mob should be examined before treatment and lice counted using the prescribed technique. All tagged sheep should have at least a moderate infestation of lice at the pre-treatment examination. After treatment the tagged sheep are examined on at least 2 occasions after treatment. Where a claim for a protective period is being sought, one of these examinations should coincide with the protective period determined in earlier pen trials. The timing of the second inspection is determined by the claim made for the product.
At the final examination, if no live lice are found, another 25 are selected and examined. Any sheep with signs of fleece derangement should be included among the extras. If there is no fleece derangement evident, the additional sheep should be randomly selected from the treated mob.
(d) Untreated control groups
Untreated control mobs need not be included in trials conducted on farms belonging to co-operators who are commercial wool growers.
(e) Climatic conditions
Records should be kept of rainfall and temperature on the farm during the 24 hours preceding and the 24 hours after treatment and monthly rainfall on the farm should be recorded for the duration of the trial.
3.3 Trials to establish protective period
These trials are optional and need only be carried out where the applicant wishes to register a claim for a specific period of protection
Purpose of the trials
To establish the period of protection afforded by lousicides against reinfestation of treated sheep by B. ovis.
(a) Selection of sheep
Sheep used in the trial will be of known lice free status and will be thoroughly inspected. Merinos should be used and all sheep in the trial should be from the same mob.
(b) Number of sheep
The initial pen challenges will consist of at least 5 treated sheep and 5 untreated controls per challenge. Fresh groups of treated and control sheep will be used for each challenge. When a protection period is established, a confirmatory trial will be done with at least 10 treated sheep and 5 untreated controls challenged.
(c) Treatment
The commercial formulation of the product will be applied to the sheep to be challenged according to the directions for use on the draft label and in a manner which parallels the method used to apply the product in the field.
(d) Method of challenge
Treated and untreated control sheep should remain in natural conditions with groups isolated from each other in separate paddocks, until the start of the trial, when the sheep may need to be housed. Separation of treated and control groups should be maintained in the shed.
Either of the following methods of challenge may be used:
(i) A sheep infested with lice is partially shorn and the wool containing live lice is placed tip uppermost on a mesh screen over a collecting tray and held over night at about 30-37°C and 70%-80% humidity. A strong light is placed above the wool and live lice are collected next morning. Lice could also be collected by a simple vacuum technique.
Approximately 50 live lice are placed at skin level on the right shoulder and a similar number on the right flank of treated sheep and untreated control sheep. The site of application is identified by marking cross reference points on the wool adjacent to the site.
OR,
(ii) One donor sheep with a known infestation of lice determined using the standard inspection technique is introduced for every two treated or control sheep and confined in a pen for 10 days. If sufficient donors are available, those in contact with the treated recipients may be replaced with fresh sheep every 2 days. Louse counts on donor sheep must be made using the prescribed method before and after challenge.
Applicants may vary these methods of challenge provided the technique used is fully described in the submission and its reliability is demonstrated.
(e) Timing of challenges
Challenges should be made at regular intervals to fresh groups of recipient sheep. Groups may be challenged at any time after treatment.
(f) Assessments
Prior to challenging all sheep, an inspection will be conducted to confirm lice free status. Challenged sheep will be subjected to regular inspections at intervals
nominated by the applicant but a final inspection for lice 20 weeks after challenge is essential. The inspections will consist of 20 x 10cm fleece partings per side, including 4 each on the neck, shoulder, withers, rump and flank with particular attention to the regions where lice were applied, if challenge technique (i) was used. Numbers of live adult lice and nymphs will be recorded. (Numbers of dead lice found may also be included separately in the results, but this is not essential). Valid results will depend on live lice being found on the untreated control sheep.
The break in protection is taken as the first day on which sheep were challenged, prior to an inspection revealing live lice on treated sheep. The period of protection is therefore the number of weeks between treatment and the last day of the challenge immediately preceding the one for which the break was recorded.
(g) Label statement
The protection period should be expressed as "This product can be expected to protect sheep against reinfestation with lice for x weeks".
3.4 Overseas trials
While results of pen and field trials conducted outside Australia may be submitted and can be used in support of the submission, they will not be considered as alternatives to the Australian trials. Sufficient Australian trials which demonstrate efficacy in different classes of wool and different climate zones are necessary.
4. Techniques
4.1 Inspection of sheep
In all trials, to determine the level of infestation, 20 sites on each side of the body should be examined by opening the fleece about 10cm and counting all live lice seen. The total count from the 40 sites will constitute the body count for each animal. An alternative method is the inspection of 80 partings each about 5cm in length, per sheep. The sites examined should be spaced so that they are representative of the full area of the body covered by the fleece on each side of the sheep, as illustrated in Figure 1.
Lice should be counted as late as possible before treatment. If the period between examination and treatment is more than 24 hours then the interval should be stated.
After treatment with products which are claimed to eliminate lice, final inspections should be made in at least 2 pen trials 12 months after treatment. In field trials final inspections must be made at least 20 weeks after treatment.
FIGURE 1
Sites on the body where partings of the fleece should be made during examination of sheep for infestations of lice in pen and field trials.
4.2 Treatment of sheep
(a) Plunge or shower dip
In pen and field trials animals should be treated in the manner recommended on the draft label. After charging the dip at the recommended rate the concentration of active ingredient in the dip fluid should be measured by appropriate analytical techniques before, during and at the end of dipping. The concentration of active constituent in the dip fluid that is used to determine any protective period should be the minimum concentration measured under these conditions.
The method of application should be clearly detailed:
For plunge dips: the length, depth, number of times sheep are dunked after entering the dip, range of times in the dip and average time in the dip should be recorded. Sheep should be dunked at least twice.
For shower dip: the brand and model of dip used should be stated together with the operating pressure and output of the pump. The length of time that sheep are showered and the combination of upper and lower nozzles used should be stated.
For plunge and shower dips: the method of mixing the chemical in the sump, the method of addition of fresh chemical and amounts added should be recorded.
(b) Backlines
The MLD 100 should form the basis of the proposed treatment regime, taking into consideration the disposition of the chemical as a consequence of the delivery system (solvent and hardware). Claims made in respect of this point should be supported by appropriate data.
(c) Insect growth regulators (IGRs)
Applicants wishing to register IGR based products or any product based on compounds that work over a protracted period must nominate the period of time required to kill all lice on treated sheep. The time nominated must be based on valid scientific data. Recommendations must be made on the time of application of the product in relation to lambing and time which must elapse before contact can be permitted with untreated, lice-free sheep.
(d) Other systems
Submissions for the registration of other classes of insecticides applied in other ways will be based on these guidelines except where the applicant is of the opinion that the new chemical or treatment method cannot be properly assessed in this way. In such cases discussions should be held with the NRA before the application for registration is completed.
5. Formulation changes
All formulation changes require NRA clearance. All such submissions require stability data.
5.1 Minor formulation changes
For example changes in inert ingredients (apart from any that control the disposition of the active) or changes in the concentration of active ingredients require:
- Stability data and
- Pen trials.
5.2 Major formulation changes
For example, changes to a solvent system controlling the disposition of the active ingredient require:
- Stability data
- Pen trials
- For products applied to short wool - Two field trials
- For products applied to long wool - Six field trials, 2 each in sheep with about 3, 6 or 9 months growth of wool.
- Residue data to demonstrate compliance with existing MRLs.
Please Note: Any change of isomers of active ingredients or changes of active ingredients, constitutes a new product and requires a complete submission for registration of a new end use product. This would incorporate data covering all sections of these guidelines.
5.3 Dye changes
Require:
- Stability data;
- Scourability data based on fleeces from at least 5 sheep.
6. Adverse reactions
In addition to the class of adverse reactions related to the user or the animal treated (data required for all products), the following should be examined for all products:
(a) Wool staining or damage
The scourability using normal commercial processing for any stains and the extent of any wool damage affecting woolen or worsted processing.
(b) Hide/skin damage
Examination of sheep in pen and field trials should include thorough inspection of the wool and skin for any apparent reaction to the product. If any reaction is seen, the effect of factors such as breed, age, climate, geography and repeated treatment should be investigated and histopathological examinations of affected skin should be made (Britt et al, 1984).
Sheep with visible or palpable reactions should be slaughtered and the affected skins tanned and processed. The effects of treatment should be assessed following scouring, woolskin tanning and processing and fellmongering. This assessment should reflect the variety of processes used commercially. At least 30 tanned and processed woolskins from at least 4 different farms should be examined. The applicant should seek to define the aetiology and pathogenesis of any adverse effects of the product and the duration of damage (eg time for skin to return to normal).
References
Australian Code of Practice for the Care and Use of Animals for Scientific Purposes (1990)
Australian Government Publishing Service, Canberra
Britt A G, Cotton C L, Trask J A and Pitman I H (1984)
Effects of a pour-on insecticidal formulation on sheep skin.
Australian Veterinary Journal 61: 329-330
Terminology
As in Appendix V Labeling Requirements of Ectoparasiticides for Sheep and Goats
Off-Shears: Within 24 hours after shearing
Short wool: >24 hours to 6 weeks after shearing
Long wool: >6 to 43 weeks after shearing
Low infestation of lice: <1 louse per 10cm parting
Moderate infestation: 1-5 lice per 10cm parting
Heavy infestation: >5 lice per 10cm parting
Eradication: Elimination of all live lice and viable eggs from treated animals (sheep or goats).
For sheep, definition is applicable only to products applied off-shears or in short wool and will be determined by inspection of sheep using the standard technique (or any other method approved by the NRA) 52 weeks after treatment as well as sheep in field trials inspected at 20 weeks.
Control:
(i) For products applied to sheep off-shears or in short wool:
Reduction of the lice population to non-detectable levels 20 weeks after treatment in pen and field trials.
ii) For products applied to sheep 6 to 43 weeks after shearing:
Reduction of the lice population by >95% after 90 days (or less as supported by data) in sheep examined in pen and field trials.
If unequivocal evidence of a reduction in fleece damage caused by the lice at any time period is presented, then the 95% figure may be modified.