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        INTERNATIONAL PROGRAMME ON CHEMICAL SAFETY

        WORLD HEALTH ORGANIZATION



        TOXICOLOGICAL EVALUATION OF CERTAIN
        VETERINARY DRUG RESIDUES IN FOOD



        WHO FOOD ADDITIVES SERIES 41





        Prepared by:
          The 50th meeting of the Joint FAO/WHO Expert
          Committee on Food Additives (JECFA)



        World Health Organization, Geneva 1998




    EPRINOMECTIN

    First draft prepared by
    M.E.J. Pronk and G.J. Schefferlie
    Centre for Substances and Risk Assessment
    National Institute of Public Health and the Environment
    Bilthoven, The Netherlands

    1.   Explanation
    2.   Biological data
         2.1  Biochemical aspects
              2.1.1  Absorption, distribution, and excretion
              2.1.2  Biotransformation
         2.2  Toxicological studies
              2.2.1  Acute toxicity
              2.2.2  Short-term toxicity
              2.2.3  Genotoxicity
              2.2.4  Reproductive toxicity
              2.2.5  Special studies on target animals
              2.2.6  Toxicity of emamectin
         3.   Comments
         4.   Evaluation
         5.   References

    1.  EXPLANATION

         Eprinomectin has not been evaluated previously by the Committee.

         The chemical name of eprinomectin is 4"-deoxy-4"-epiacetylamino-
    avermectin B1. It is a semi-synthetic member of the avermectin
    family of macrocyclic lactones and consists of a mixture of two
    homologous components, B1a (not less than 90%) and B1b (not more
    than 10%), which differ by a single methylene group at C26. The
    structure is shown in Figure 1. The purity of the compound used in the
    studies of toxicity was determined to be 95.1-99.6% by
    high-performnace liquid chromatography (HPLC).

    FIGURE 1

         Eprinomectin is active in animals against internal and external
    parasites. Its precise mode of action, in common with other
    avermectins, is unknown, despite many years of investigation of a
    variety of compounds in this class. The effect of avermectins,
    including eprinomectin, is mediated via a specific, high-affinity
    receptor present in the target organisms. The physiological response
    to avermectin binding is increased membrane permeability to chloride
    ions, which is independent of gamma-aminobutyric acid (GABA)-mediated
    chloride channels. Although avermectins interact with the GABA-gated
    channels, they do so only at very high concentrations, i.e. about
    three orders of magnitude greater than that necessary to activate the
    high-affinity receptor. Therefore, the action of the avermectins at
    the GABA-gated chloride ion channels is probably not involved in their
    nematocidal and insecticidal activity at therapeutic doses. Activation
    of the specific avermectin high-affinity receptor ultimately results
    in paralysis and death of the target organism (Turner & Schaeffer,
    1989). The fact that much higher concentrations of these compounds are
    needed in mammals than in nematodes to affect neurological function
    may be due to lack of a specific, high-affinity site associated with
    neuronal function or to the relatively poor penetration of these
    high-compounds into the central nervous system (Lankas & Gordon,
    1989).

    2.  BIOLOGICAL DATA

    2.1  Biochemical aspects

    2.1.1  Absorption, distribution, and excretion

          Rats

         [5-3H]Eprinomectin (specific activity, 7400 dpm/µg) was
    administered orally by gavage in 0.5% aqueous methylcellulose to
    Crl:CD (SD) BR VAF rats at a dose of 6 mg/kg bw per day for one week.
    Three rats of each sex were sacrificed 7 h and one, two, and five days
    after the final dose. Urine and faeces were collected immediately
    before treatment and daily until sacrifice. After sacrifice, samples
    of blood, liver, kidneys, abdominal and/or back fat tissue (females)
    and/or testicular fat pad (males), hind leg muscles, and
    gastrointestinal tract (including contents) were collected. The
    radiolabel in each sample was determined by scintillation
    spectrometry. The study was certified for compliance with GLP and
    quality assurance.

         During treatment and the five days thereafter, 90% of the
    administered dose was excreted in the faeces and less than 1% in the
    urine. The route and rate of excretion were independent of sex. At 7 h
    after treatment, the highest total residue concentrations were found
    in the gastrointestinal tract (55.6 mg/kg eprinomectin equivalents),
    followed by liver (10.7 mg/kg), fat (8.6 mg/kg), kidney (7.6 mg/kg),
    and muscle (2.2 mg/kg). Significantly lower concentrations were found
    in plasma (0.89 mg/kg) and erythrocytes (0.31 mg/kg). Similar patterns

    of distribution were seen at later times. By five days after
    treatment, the total residue concentration had declined to           
    < 0.1 mg/kg in all samples. The depletion pattern was comparable in
    male and female rats (Halley  et al., 1995).

          Cattle

         Angus and Hereford beef cattle received single topical
    applications of [5-3H]-eprinomectin (as the commercial formulation
    Eprinex Pour-On; specific activity, 0.061 mCi/mg or 135 dpm/ng) at a
    dose of 0.5 mg/kg bw. Three cattle of each sex were slaughtered 7, 14,
    21, and 28 days after treatment. Blood samples were collected from all
    animals before treatment and at several times after treatment. Urine
    and faeces were collected several times only from cattle slaughtered
    at 28 days. After sacrifice, samples of liver, kidney, hindquarter
    muscle, muscle beneath the application site, and perirenal fat were
    collected; samples of hide at the site of applications were collected
    only from those killed at 28 days. The radioactivity in each sample
    was determined by scintillation spectrometry; the tissue and plasma
    samples were also analysed for eprinomectin B1a by reverse-phase
    HPLC. The study was certified for compliance with GLP and quality
    assurance.

         Eprinomectin was slowly absorbed, as evidenced by a slow rise and
    a broad plateau in plasma concentrations over two weeks rather than a
    sharp peak. In plasma, the highest total residue concentrations were
    in the range 4.4-21.1 ng/ml eprinomectin equivalents and the highest
    concentrations of B1a in the range 7.3-20 ng/ml. Only a small
    portion of the applied dose was found in the urine (0.35%), and
    excretion was mostly in the faeces (14% of the dose after 28 days).
    Analysis of the hide samples revealed that 54% of the initially
    applied dose remained. By seven days after treatment, the highest
    concentrations of total residue were found in liver (980 µg/kg
    eprinomectin equivalents), followed by kidney (180 µg/kg), fat
    (34 µg/kg), and muscle beneath the application site (24 µg/kg); the
    lowest concentrations were found in hindquarter muscle (8 µg/kg). At
    later times, the total residue concentrations declined but the
    relative concentrations remained the same. By 28 days after treatment,
    the total residue concentrations had declined to 185 µg/kg in liver,
    30 µg/kg in kidney, 5 µg/kg in fat, 22 µg/kg in muscle beneath the
    application site, and 2 µg/kg in hindquarter muscle. The depletion
    half-lives for total residues in the different tissues were 7.8ś8.6
    days, but that in muscle beneath the application site was 36.1 days;
    however, the last value is probably unreliable owing to large
    interanimal variation and poor regression fit. In all tissues, the
    B1a concentrations accounted for more than 80% of the total
    radioactive residues. Depletion of B1a followed the same order as
    that of total residues at all times, the depletion half-lives varying
    from 7.5-9.6 days in liver, kidney, fat, and muscle to 29.4 days in
    muscle beneath the application site. These results indicate that B1a
    is depleted in parallel with the total residues in all tissues on days
    7-28. The depletion pattern was comparable in male and female cattle
    (Green-Erwin  et al., 1994).

         Holstein dairy cattle were given each of the following four
    treatments, with a period of 14 days between treatments: single
    intravenous doses of 25, 50, and 100 µg/kg bw eprinomectin in glycerol
    formal-propylene glycol and a single topical dose of 0.5 mg/kg bw
    eprinomectin in the commercial formulation along the back. Blood
    samples were collected from the jugular vein at several times after
    each treatment, and the plasma was assayed for eprinomectin by HPLC
    with fluorescence detection. The study was certified for compliance
    with quality assurance. After intravenous treatment, plasma clearance
    was independent of dose, indicating that the concentrations increased
    proportionally to dose. The volume of distribution decreased with
    increasing dose, corresponding to a decrease in mean residence time.
    After topical treatment, maximum plasma concentrations of 17-32 ng/ml
    (mean, 21 ng/ml) were reached after 2-5 days (mean, 3.5 days). The
    mean residence time was 165 h. The bioavailability was only 29%. Most
    of the absorption occurred within 7-10 days after treatment, following
    an initial lag of 24 h, but continued for 17-21 days after treatment
    (Faidley, 1995).

    2.1.2  Biotransformation

          Rats

         In the study of Halley  et al. (1995), described above,
    metabolites were identified in all tissue, plasma, and faecal samples
    by reverse-phase HPLC with mass spectroscopic analysis. The parent
    drug, comprised of B1a and B1b, was the major residue in all
    tissues and plasma at 7 h (89-94% in males, 75-93% in females), and in
    faeces after one day (87% in males, 82% in females). At these times,
     N-deacetylated B1awas the major metabolite in all samples (tissues
    and plasma: 0.6-5.2% in males, 2.3-20% in females; faeces: 1.2% in
    males, 5.8% in females) and was usually the main residue at later
    times (26 and 73% in liver and kidney at two days and 20 and 63% in
    faeces at five days in males and females, respectively). Other minor
    metabolites, each representing < 7% of the total radiolabel, were
    also present in the samples. Three were identified as the 24a-
    hydroxymethyl, 24a-hydroxy, and 26a-hydroxymethyl metabolites of
    B1a. These results indicate that the primary route of metabolism of
    eprinomectin in rats is via  N-deacetylation and that eprinomectin is
    metabolized more extensively in female than in male rats.

          Cattle

         The nature of the residues in tissues, plasma, and faeces of
    cattle after pour-on administration of [5-3H]eprinomectin at 0.5
    mg/kg bw was investigated by reverse-phase HPLC. The study was
    certified for compliance with GLP and quality assurance. Eprinomectin
    is not extensively metabolized in cattle, as the parent drug was the
    main residue at all slaughter times in all tissues (90-95%), plasma
    (95%), and faeces (86%). The parent drug contained 78-87% B1a and
    7.2-9.3% B1b.  N-Deacetylated B1a was a minor metabolite in these
    samples (< 1.3%, except for hindquarter muscle which contained 3.9%).
    Other minor metabolites present in the samples represented 0.1-2.4% of

    the total radiolabel in tissues and plasma and 0.5-7.4% of that in
    faeces. The metabolite profile was qualitatively and quantitatively
    independent of sex, slaughter time, and tissue type. Thus, shortly
    after drug administration, the metabolism of eprinomectin in cattle is
    very similar to that in rats, the parent compound representing most
    the residue. In rats, however, the amount of the  N-deacetylated
    metabolite increases relative to total residue at later times, while
    in cattle the concentration of this metabolite to total residue
    remains relatively constant over time. The profile of other minor
    metabolites is qualitatively similar in the two species (Venkataraman
    & Narasimhan, 1995).

    2.2  Toxicological studies

    2.2.1  Acute toxicity

         The acute oral and intraperitoneal toxicity of eprinomectin was
    studied in groups of three female Crl:CD-1 (ICR) BR mice and female
    Crl:CD (SD) BR rats given 9.8, 20, 39, or 78 mg/kg bw. The oral doses
    were given by gastric intubation and the intraperitoneal doses by
    injection through the ventral abdominal wall. In both cases, the
    vehicle was 0.5% aqueous methylcellulose. The study was certified for
    compliance with GLP and quality assurance. The approximate value for
    the oral LD50 was 70 mg/kg bw for mice and 55 mg/kg bw for rats; in
    both species, the approximate intraperitoneal LD50 value was 35
    mg/kg bw. The toxic symptoms observed were ataxia, tremors, loss of
    righting reflex, ptosis, and bradypnoea. The surviving animals
    recovered within four to five days (Bagdon & McAfee, 1990).

    2.2.2  Short-term toxicity

          Rats

         In a 23-day exploratory toxicity study, groups of five male and
    five female Crl:CD (SD) BR albino rats received eprinomectin in the
    diet at doses of 0, 0.5, 2.5, 5, or 10 mg/kg bw per day. The low dose
    was increased to 20 mg/kg bw per day from day 15 onwards. No
    treatment-related effects were seen on mortality or clinical signs.
    Decreases in weight gain and feed efficiency were observed in female
    rats at 20 mg/kg bw per day but not in females at lower doses. No
    adverse effects were observed in male rats (Kloss & Morrissey, 1990a).

         In a second exploratory study, groups of five male and five
    female Crl:CD (SD) BR albino rats received eprinomectin in the diet at
    doses of 0, 20, 40, or 60 mg/kg bw per day for 26 days. Owing to
    severe clinical signs (ataxia, tail and whole-body tremors, a hunched,
    unthrifty appearance, and piloerection), body-weight loss, and
    decreased food consumption, the groups at 40 and 60 mg/kg bw per day
    were terminated after one week of treatment, and a new group receiving
    30 mg/kg bw per day was started. In this group, clinical signs similar
    to but milder than those in animals at the two higher doses were
    observed, in addition to decreases in body-weight gain and food

    consumption. At 20 mg/kg bw per day, male rats were unaffected, but
    female rats had moderate reductions in body-weight gain and food
    consumption (Kloss & Morrissey, 1990b).

         Groups of 20 male and 20 female Crl:CD (SD) BR albino rats
    received eprinomectin in the diet for 90 days at nominal doses of 0,
    1, 5, or 30 mg/kg bw per day; however, owing to low food consumption
    by animals at the highest dose, the actual intake was 25 mg/kg bw per
    day. As this dose resulted in a high incidence of whole-body tremors
    and large decreases in body-weight gain, the dose was lowered to 20
    mg/kg bw per day in week 4 for females and in week 5 for males. The
    actual mean intakes throughout study were 0, 1, 5 and 22 mg/kg bw per
    day. The study was of conventional design, with GLP and quality
    assurance certification.

         Two rats at the high dose died under anaesthesia, and one rat
    died of trauma due to a maxillofacial fracture. Aside from tremors, no
    treatment-related clinical or ophthalmoscopic signs were noted in rats
    at 30/20 mg/kg bw per day. Treatment-related effects in males and
    females at the high dose included decreased food consumption and
    body-weight gain and increased blood urea nitrogen without a
    corresponding increase in creatinine. Females also showed decreased
    mean lymphocyte values. Additionally, slight increases in urine
    specific gravity (males and females), haematocrit and erythrocyte
    count (males), serum protein and albumin (females), and slight
    decreases in urine volume (males and females) suggest
    haemoconcentration at the high dose, probably as a secondary effect of
    the decreased food and water intake. Females at the high dose showed
    increased absolute and relative (to body and brain weight) weights of
    the liver, uterus, pituitary, and adrenal and decreased ovarian,
    spleen, and thymic weights. Males at this dose had increased adrenal
    weights and reduced weights of thymus, spleen, and prostate.
    Histopathological examination showed arrest of normal ovarian
    follicular maturation in 15 of 20 females at the high dose, and the
    uteri of four animals showed endometrial squamous metaplasia. These
    effects are indicative of oestrogen-progesterone imbalance, which was
    also manifested in decreased remodelling of the femora (primary
    spongiosa) in 12 of 20 females at the high dose. No remarkable changes
    were seen in the brain or spinal cord, but slight degeneration of the
    sciatic nerves was noted in three males and three females at the high
    dose. There were no other morphological changes related to treatment.
    The NOEL was 5 mg/kg bw per day (Kloss  et al., 1990a).

          Dogs

         In a six-week exploratory study, groups of two male and two
    female beagle dogs received eprinomectin at doses of 0, 0.5, 1, 2, or
    4 mg/kg bw per day. For the first 13 days of the study, eprinomectin
    was given in the diet; however, because of its unpalatability in
    milled dog food, resulting in reduced food consumption and body-weight
    loss in the groups at the two highest doses, it was given by gavage in
    0.5% aqueous methylcellulose from day 14 onwards. Treatment with the
    highest dose was discontinued after the first gavage dose because of

    severe clinical effects, consisting of mydriasis, salivation, ataxia,
    decreased activity, and the death of one animal. Mydriasis was
    occasionally seen in dogs at 2 mg/kg bw per day, and these animals
    also had decreased food intake and body weight. No drug-related
    changes were seen in dogs at the lower doses (Kloss & Bagdon, 1990).

         Groups of four male and four female beagle dogs received
    eprinomectin by gavage for 90 days at nominal doses of 0, 0.5, 1, or 3
    mg/kg bw per day in 0.5% aqueous methylcellulose. The high dose was
    lowered to 2 mg/kg bw per day from week 2 onwards because of toxicity.
    The actual doses administered, on the basis of analytical results,
    were about 80% of the nominal, resulting in 0, 0.4, 0.8, or
    2.4/1.6 mg/kg bw per day. The study had a conventional design, with
    GLP and quality assurance certification. During week 1 of treatment,
    the dose of 2.4 mg/kg bw per day induced the death of two males,
    mydriasis, emesis, ataxia, salivation, lateral recumbency, and
    body-weight loss. Once this dose was lowered to 1.6 mg/kg bw per day,
    no treatment-related clinical signs or mortality were observed, but
    decreased food consumption and body-weight gain were still seen. The
    body-weight gain and food consumption of animals at the intermediate
    and low doses were comparable to those of controls. No
    treatment-related effects were seen on ophthalmoscopic,
    electrocardio-graphic, haematological, blood biochemical, or urinary
    parameters or on organ weights or gross appearance. Apart from slight
    axonal degeneration in the sciatic nerves of two females at the high
    dose, no treatment-related microscopic changes were seen in any
    tissue, including brain and spinal cord. The NOEL was 0.8 mg/kg bw per
    day on the basis of axonal degeneration in the sciatic nerve and
    body-weight loss (Kloss  et al., 1990b).

         In a one-year study, groups of four male and four female beagle
    dogs received eprinomectin by gavage at doses of 0, 0.5, 1, or 2 mg/kg
    bw per day in 0.5% aqueous methylcellulose. The study had a
    conventional design, with GLP and quality assurance certification. The
    only clinical sign attributable to treatment was mydriasis in dogs at
    the high dose. One animal at this dose became less active, with
    salivation and ataxia progressing to lateral recumbency, and was
    therefore necropsied in week 13. This animal also had decreased food
    intake and weight loss, while no changes in food consumption or body
    weight were seen in any other treated dog. Ophthalmoscopic and
    electrocardiographic examinations, haematology, blood biochemistry,
    urinalysis, and measurement of organ weights indicated no drug-related
    changes. Gross findings were limited to pin-point dark-brown or black
    foci in the mucosa of the neck of the gall-bladder, which was found
    microscopically to be related to inspissated bile, with no changes in
    the histology of the gall-bladder or liver. This finding was observed
    in 1/8, 1/8, 1/8, and 3/8 animals at 0, 0.5, 1, and 2 mg/kg bw per
    day, respectively, and was considered not to be related to treatment.
    Histopathological examination showed very slight focal degeneration of
    one to three neurons per dog in the pons area and/or the cerebellar
    nuclei in three of eight dogs at the high dose. This degenerative
    change was characterized by neuronal enlargement resulting from
    increased eosinophilic, vacuolated cytoplasm with nuclear

    displacement, and was not seen in other treated dogs or controls. No
    other remarkable histopathological findings were seen in other
    tissues, including spinal cord and sciatic nerves. The NOEL was 1
    mg/kg bw per day on the basis of mydriasis and focal neuronal
    degeneration in the brain (Kloss  et al., 1994).

    2.2.3  Genotoxicity

         The results of studies of the genotoxicity of eprinomectin are
    summarized in Table 1. The studies were of conventional design, with
    GLP and quality assurance certification.

    2.2.4  Reproductive toxicity

          (i)  Multigeneration reproductive toxicity

          Rats

         In a range-finding study of reproductive toxicity, groups of 15
    female Crl:CD (SD) BR rats received eprinomectin at dietary
    concentrations of 0, 7, 36, or 180 mg/kg feed per day for 16 days
    before cohabitation, during cohabitation, and from day 0 of gestation
    through day 21 of lactation. When cohabitation lasted more than one
    night, eprinomectin was administered once daily by oral gavage in 0.5%
    aqueous methylcellulose; this occurred only in rats at the low and
    intermediate doses. On the basis of food intake, the overall mean
    intake of eprinomectin was 0, 0.7, 3.3, and 13 mg/kg bw per day,
    respectively. Females were mated with untreated males and were allowed
    to deliver naturally. Dams and pups were killed within two days of day
    21 of lactation. The study was certified for compliance with GLP and
    quality assurance.

         Dams showed no treatment-related deaths, abortions, or physical
    signs, and no effects were seen on length of gestation, the percent of
    females with live pups, or the percent of live pups at birth. Females
    at the intermediate dose had increased body-weight gain during days
    0ś20 of lactation because of failure to lose weight on days 8ś12 of
    lactation, as is normal. In comparison with controls, dams at the high
    dose had decreased body-weight gain throughout treatment and slightly
    decreased food consumption on gestation days 0-8 and lactation days
    0ś4. These animals were killed before lactation day 8 because of
    excessive pup mortality. They also showed significantly decreased
    fecundity indexes, number of implants per female, percent
    postimplantation survival, and number of live pups per litter.
    External examination of the pups revealed no treatment-related
    effects, but increased pup mortality was observed at the highest dose,
    particularly during lactation days 4-7. The remaining pups, which all
    had tremors, were therefore killed on lactation day 8. At the
    intermediate dose, toxicity in pups was evidenced by decreased body
    weight and fine tremors during the middle and end of lactation
    (Cukierski, 1990a).



        Table 1. Results of assays for genotoxity with eprinomectin

                                                                                                    

    End-point         Test object                  Concentration     Result         Reference
                                                                                                    

    In vitro
    Reverse           S. typhimurium TA97a,        100-10 000        Negativea      Sina (1990,
    mutation          TA98, TA100, TA 1535         µg/plate                         1994)
                      E. coli WP2, WP 2 uvrA,
                      WP2 urA pKM101

    Gene mutation     V-79 Chinese hamster         1-40 µmol/        Negativeb      DeLuca (1991)
                      lung cells (hprt locus)      plate (-S9)
                                                   10-40 µmol/
                                                   plate (+S9)

    Cytogenetic       Chinese hamster ovary        8-12 µmol/        Negativeb      Galloway
    alterations       cells                        plate (-S()                      (1990)
                                                   5-7 µmol/
                                                   plate (+S9)

    DNA damage        Primary rate hepatocytes     10-51 µmol/       Negative       Storer (1990)
                                                   plate

    In vivo
    Micronucleus      Mouse bone marrow            10-40 mg/kg       Negativec      Galloway
    formation                                      bw, once by                      (1994)
                                                   oral gavage
                                                                                                    

    a   With and without rate liver S9 fraction; precipitation on all plates at 10 000 µg/plate
    b   Dose-related cytotoxicity with and without rate liver S9 fraction
    c   At all doses and all times, the ratio of polychromatic to normochromatic erythrocytes did not 
        deviate from that in controls; however, clinical signs of toxicity (including decreased 
        activity, ataxia, and tremors) were observed at the highest dose.
    


         In a two-generation study of reproductive toxicity, groups of 32
    male and 32 female Crl:CD (SD) BR VAF/Plus rats received diets
    containing eprinomectin at 0, 6, 18, or 54 mg/kg feed. Treatment
    started 10 (males) or two (females) weeks before mating and was
    continued until all litters had been weaned. An F1 generation of 28
    animals of each sex per dose was selected and treated directly from
    four weeks of age. These animals were mated at 16 weeks of age to
    produce the F2a generation. After being allowed to rear their
    litters, the F1 animals were remated at 27 weeks of age to produce
    the F2b generation. In order to investigate body tremors in the
    offspring, the dietary concentrations of eprinomectin for the F1
    animals were reduced to 50% of their initial values during lactation
    of the F2b offspring. A contingent of 24 F2b animals of each sex
    per dose (except for those at 54 mg/kg, owing to inadequate numbers)
    was treated directly during weeks 4-7 of age, after which they were
    killed. The brain, spinal cord, and sciatic nerves of F0 and F1
    adults killed at about 27 and 38 weeks of age, respectively, and of
    F2b pups killed on day 21  post partum were examined
    histologically. The study was of conventional design, with GLP and
    quality assurance certification.

         F0 animals at all doses had slightly increased food consumption
    only during the first two weeks of treatment, resulting in slightly
    increased body weights. As this effect was transient and small, it is
    not considered toxicologically significant. Treatment at 6 mg/kg feed
    had no adverse effects on parents or their offspring. Treatment at 18
    mg/kg feed resulted only in body tremors in F2a pups in four of 26
    litters after day 8 of lactation. Treatment at 54 mg/kg feed had
    adverse effects on the dams, their reproduction, and their litters. No
    treatment-related deaths or physical signs occurred among the parental
    animals. The F1 animals had lowered body weights at week 4,
    reflecting their impaired growth during the pre-weaning period. During
    the first weeks of treatment, the food consumption and body weights of
    F1 animals were decreased, but these differences tended to be
    abolished or even reversed in later phases of the study. Although
    within each treated group, food consumption during lactation was
    increased over that during gestation, the food consumption of F0 and
    F1 (first mate) females was reduced during the first two weeks of
    lactation in comparison with controls; a similar effect, although less
    marked, was observed after the second mate of the F1 animals at the
    reduced dose of 27 mg/kg feed. Sexual maturation was delayed in F1
    animals, consistent with their delayed physical development. After the
    first mating of the F1 generation the pregnancy rate was slightly
    reduced, and at the second mating of these animals there was marked
    impairment of mating performance and a 50% reduction in pregnancy
    rate, resulting in a reduction in the number of females producing live
    litters. Litter sizes were not affected by treatment. Signs of
    toxicity in F1 and F2a pups were markedly increased mortality
    after day 8  post partum, decreased litter and mean pup weights from
    day 8  post partum through to weaning, and body tremors in all pups
    in all litters. In F2b pups, no body tremors were observed at any
    dose when the dietary concentrations were reduced to 0, 3, 9, or 27
    mg/kg feed, and the pup losses were not different from those of

    controls; however, at 27 mg/kg feed, the litter and mean pup weights
    were decreased, but to a lesser degree than for F1 and F2a pups.

         The NOEL for maternal toxicity was 18 mg/kg feed, equal to 2.5
    mg/kg bw per day, on the basis of decreased food intake during the
    first two weeks of lactation in F0 and F1 dams. The NOEL for
    reproductive toxicity was 18 mg/kg feed, equal to 1.6 mg/kg bw per
    day, on the basis of impaired reproductive performance in the F1
    animals. On the basis of tremors in F2a pups and decreased body
    weights in F2b pups, the NOEL for pup toxicity was 9 mg/kg feed,
    equal to 1.3 mg/kg bw per day (Brooker  et al., 1992).

         In a follow-up study to determine the concentrations of
    eprinomectin in maternal plasma and milk, groups of 12 mated female
    Crl:CD (SD) BR rats received eprinomectin at dietary concentrations of
    0, 6, 54/27, or 54 mg/kg feed from day 15 of gestation through day 21
    of lactation. The group at the intermediate dose received 54 mg/kg
    feed from day 15 of gestation through parturition but 27 mg/kg feed
    from day 0 of lactation through sacrifice to compensate for increased
    maternal food consumption during lactation. The actual mean intakes of
    eprinomectin during gestation were 0, 0.4, 4.0, and 4.1 mg/kg bw per
    day, and those during lactation were 0, 1.2, 4.5, and  6.6 mg/kg bw
    per day, respectively. All females were allowed to deliver naturally,
    and dams and pups were killed within four days of day 21 of lactation.
    The study was certified for compliance with GLP and quality assurance.

         No treatment-related deaths, abortions, or physical signs were
    seen among the dams, and there were no effects on the length of
    gestation or the number of live pups per pregnant female. In
    comparison with controls, the body-weight gain of dams at the high
    dose was increased during days 15-21 of gestation and days 0-21 of
    lactation, and the food consumption of dams at the intermediate and
    high doses was decreased during lactation days 8-21. Within the groups
    at the intermediate and high doses, however, food consumption was
    increased from gestation day 15 through lactation day 4. Eprinomectin
    was well absorbed by all rats, sustained concentrations being detected
    in milk and maternal plasma during lactation days 7-21 with a direct
    doseśconcentration relationship: the overall milk:plasma ratio was
    approximately 3:1. Treatment with eprinomectin resulted in
    dose-dependent toxicity in pups at the intermediate and high doses
    starting on or after day 5 of lactation. The signs of toxicity were
    decreased body-weight gain and intermittent body tremors in pups at
    the intermediate and high doses and increased mortality (mainly on
    lactation days 8-14) among pups at the high dose. As these effects
    were observed during a period when the only route of exposure was
    through milk, they are probably due to postnatal exposure, as
    evidenced by the sustained concentrations of eprinomectin in milk and
    as further supported by the results reported below (Mattson, 1992).

         In a multigeneration study of reproductive toxicity in rats with
    the related compound ivermectin at oral doses of 0.05-3.6 mg/kg bw per
    day, ivermectin had no effect on mating, fertility, or pregnancy up to
    the highest dose tested. Similar neonatal toxicity, characterized by

    decreased weight gain and pup mortality during lactation, was,
    however, observed in offspring at doses > 0.4 mg/kg bw per day, with
    a NOEL of 0.2 mg/kg bw per day. In a cross-fostering study, it was
    shown that the neonatal toxicity was not related to exposure  in 
     utero but to postnatal exposure through the milk. The concentrations
    of ivermectin (a highly lipophilic compound) in milk were three to
    four times those in plasma. These relatively high concentrations of
    ivermectin in milk resulted in significantly higher concentrations in
    the brain and plasma of nursing offspring, and the period of enhanced
    neonatal sensitivity correlated with the increased plasma:brain ratios
    of ivermectin, consistent with postnatal formation of the blood-brain
    barrier in this species. In other mammalian species, including humans,
    the blood-brain barrier is formed prenatally. Therefore, the toxicity
    of ivermectin in neonatal rats is probably the result of a combination
    of excessive exposure through maternal milk and the increased
    permeability of the blood-brain barrier during the early postnatal
    period in this species (Lankas & Gordon, 1989; Lankas  et al., 1989).

          (ii)  Developmental toxicity

          Rats

         In a range-finding study, eprinomectin was administered by gavage
    in 0.5% aqueous methylcellulose at doses of 0, 0.5, 1.5, 5, 10, or 15
    mg/kg bw per day to groups of 10 mated female Crl:CD (SD) BR rats on
    days 6-17 of gestation. Serum biochemical and haematological
    examinations were performed on day 14 of gestation. On day 20 of
    gestation, the dams were killed and necropsied, and the fetuses were
    weighed and examined for external abnormalities. One dam at the high
    dose was killed on day 14 of gestation because of severe weight loss;
    this animal also had slight tremors, ptosis, decreased activity, and
    abnormal posture and had increased erythrocyte count, haemoglobin, and
    haematocrit. One rat at the low dose died on day 14 of gestation due
    to anaesthesia overdose. There were no abortions. Some of the animals
    at the high dose had fine tremors, abnormal posture, and reluctance to
    be handled. Maternal body weight gain was significantly increased at 5
    and 10 mg/kg bw but significantly decreased at 15 mg/kg bw. The
    concentration of urea nitrogen and the activity of alanine
    aminotransferase were increased in rats at the two highest doses. No
    effects were observed on haematological parameters or on the number of
    implants, resorptions, or live or dead fetuses. Fetal body weights
    were significantly decreased at 1.5, 5, 10, and 15 mg/kg bw, but there
    was no dependence on dose. This effect was not seen in the main study,
    with larger groups (see below). External examination of the fetuses
    showed no evidence of teratogenicity (Cukierski, 1990b).

         In the main study, groups of 25 mated female Crl:CD (SD) BR rats
    were treated orally by gavage with eprinomectin in 0.5% aqueous
    methylcellulose at doses of 0, 0.5, 1, 3, or 12 mg/kg bw per day on
    days 6-17 of gestation. On day 20 of gestation, the dams were killed
    and necropsied, and the fetuses were weighed, sexed, and examined for
    external, visceral, and skeletal abnormalities. The study was of
    conventional design, with GLP and quality assurance certification.

    There were no treatment-related physical signs, deaths, abortions, or
    gross lesions. Increased weight gain and food consumption were
    observed during treatment with the two highest doses, followed by
    decreases during days 18-20, resulting in slightly increased total
    weight gain on days 6-18 of gestation. There was no evidence of
    developmental toxicity or teratogenicity at doses up to 12 mg/kg bw
    per day on the basis of postimplantation survival, fetal weight, and
    external, visceral, and skeletal examination. The NOEL for maternal
    toxicity was 1 mg/kg bw per day, on the basis of changes in body
    weight and food consumption. The NOEL for developmental toxicity was
    12 mg/kg bw per day, the highest dose tested (Cukierski, 1991).

          Rabbits

         In a range-finding study, groups of six female New Zealand white
    rabbits received eprinomectin in 0.5% aqueous methylcellulose by
    gavage at doses of 0, 1.5, 4, 10, or 25 mg/kg bw per day for 14 days.
    Owing to excessive weight loss and poor condition, the animals at 10
    and 25 mg/kg bw per day were killed on days 8 and 3 of treatment,
    respectively. There were no deaths and no effects on body weight at
    the lower doses. Dilated pupils and slowed pupillary reflexes were
    observed at doses > 4 mg/kg bw per day, and mild tremors and
    decreased food consumption were seen at doses > 10 mg/kg bw per
    day. At 25 mg/kg bw per day, some animals neither urinated nor
    defaecated (Clark, 1990).

         In a second range-finding study, eprinomectin was administered by
    gavage in 0.5% aqueous methylcellulose at doses of 0, 2, 4, or 8 mg/kg
    bw per day to groups of eight inseminated female New Zealand white
    rabbits on days 6-18 of gestation. Serum biochemical and
    haematological examinations were performed on day 19 of gestation. On
    day 28 of gestation, the dams were killed and necropsied, and the
    fetuses were weighed and examined for external abnormalities.
    Treatment with eprinomectin was associated with mydriasis and slowed
    pupillary reflex in all groups, and unresponsive mydriasis was found
    in the groups at the two highest doses. On day 12 of gestation, one
    rat at the high dose died from an intubation accident, and two others
    at this dose were killed on days 19 and 27 of gestation because of
    severe weight loss after not eating for one week. Slightly decreased
    food consumption and weight gain were also observed in the remaining
    rats at the high dose and in those at the intermediate dose. No
    effects were found on haematological or blood biochemical parameters
    or on the numbers of implants, resorptions, or live or dead fetuses,
    or on fetal body weights. External examination of the fetuses revealed
    no treatment-related findings (Minsker, 1990).

         In the main study, groups of 18 inseminated female New Zealand
    white rabbits were treated orally by gavage with eprinomectin in 0.5%
    aqueous methylcellulose at doses of 0, 0.5, 2, or 8 mg/kg bw per day
    on days 6-18 of gestation. On day 28 of gestation, the dams were
    killed and necropsied, and the fetuses were weighed, sexed, and
    examined for external, visceral, and skeletal abnormalities. The study
    was of conventional design, with GLP and quality assurance

    certification. There were no treatment-related deaths, abortions, or
    gross lesions. Maternal toxicity was evidenced by slowed pupillary
    reflex at the intermediate and high doses and mydriasis non-responsive
    to light and a slight decrease in body-weight gain in rabbits at the
    high dose. The numbers of implants and live fetuses per pregnant
    female were decreased at 2 and 8 mg/kg bw per day (significantly only
    at the highest dose), but these findings were considered not to be
    treatment-related, because the values were still within the range in
    historical controls and the lower values were a consequence of fewer
    corpora lutea per female at these doses. Likewise, the apparent
    increase in the percent preimplantation loss in animals at the
    intermediate and high doses was due to the smaller number of implants
    and was considered not to be treatment-related. There was no effect on
    live fetal weight, and there was no indication of teratogenicity at
    doses up to 8 mg/kg bw per day. The NOEL for maternal toxicity was 0.5
    mg/kg bw per day on the basis of slowed pupillary reflex. The NOEL for
    developmental toxicity was 8 mg/kg bw per day, the highest dose tested
    (Wise, 1991).

         In order to re-examine the possible effects of eprinomectin on
    embryo and fetal viability, a second study was conducted with larger
    groups. Eprinomectin was administered by gavage in 0.5% aqueous
    methylcellulose at doses of 0, 1.2, 2, or 8 mg/kg bw per day to groups
    of 24 mated female New Zealand white rabbits on days 6-18 of
    gestation. After sacrifice of the dams on day 28 of gestation, the
    numbers of corpora lutea, implants, resorptions, and live or dead
    fetuses were counted. The fetuses were not examined further. The study
    was certified for GLP and quality assurance. There were no
    treatment-related deaths or abortions. Maternal toxicity was seen only
    in rabbits at the high dose, which showed slowed pupillary reflex
    and/or mydriasis and decreased body-weight gain during treatment. No
    effects were found on embryonic or fetal survival. The NOEL for
    maternal toxicity was 2 mg/kg bw per day on the basis of physical
    signs and decreased body-weight gain. The NOEL for developmental
    toxicity was 8 mg/kg bw per day (Cukierski, 1994).

    2.2.5  Special studies on target animals

         The safety of the commercial formulation Eprinex Pour-On
    (containing eprinomectin in Myglyol 840 and 0.01% butylated
    hydroxytoluene) was tested by topical application to calves and
    breeding animals. Eight-week-old calves were treated at once, three
    times, or five times the recommended dose three times at seven-day
    intervals, while 12-month-old calves were treated once at 10 times the
    recommended dose. Breeding bulls were treated once at three times the
    recommended dose, and breeding cows were treated with at least three
    times the recommended dose throughout the reproductive cycle. The
    studies were certified for compliance with GLP and quality assurance.
    In all studies, eprinomectin was well tolerated and was without
    adverse effects (Gogolewski, 1994; Bierschwal, 1995; Bridi, 1995;
    Pitt, 1995).

    2.2.6  Toxicity of emamectin

         The toxicology of emamectin has also been reviewed (Department of
    Health and Family Services, 1997). Like eprinomectin, emamectin is an
    amino-substituted avermectin; the only difference between the two
    compounds is the presence of an epi-methylamino group at the C4
    position on the emamectin molecule, rather than an epi-acetylamino
    group at that position in the case of eprinomectin. The following
    studies of the short-term and long-term toxicity of emamectin were
    extracted directly from the review.

          Mice

         Groups of mice were given emamectin at doses of 0.5, 2.5, or
    12.5 mg/kg bw per day in the diet for 547-550 days. The dose of 12.5
    mg/kg bw per day was reduced to 7.5 mg/kg bw per day in females during
    week 48, to 7.5 mg/kg bw per day in males during week 9, and further
    reduced to 5.0 mg/kg bw per day in males during week 31. The mortality
    rate was increased in males and females at 12.5/7.5/5.0 mg/kg bw per
    day. Tremors and vocalization was seen in three to four male mice
    treated with 12.5 mg/kg bw per day between weeks 5 and 8-9, but these
    adverse clinical signs abated after the dose was reduced to 7.5 mg/kg
    bw per day. Vocalization occurred in female mice treated with 12.5
    mg/kg bw per day after week 16, but was not evident after week 34.
    Several animals treated with 12.5/7.5/5.0 mg/kg bw per day developed
    minor neurological abnormalities, e.g. fine forelimb fasciculations,
    after week 14, which persisted until the end of the study. Two males
    given 12.5 mg/kg bw per day had sciatic nerve degeneration,
    characterized by vacuolation and the presence of myelin balls in the
    nerve fibres. The body-weight gain of males and females was reduced
    after one to two weeks of treatment with 12.5/7.5/5.0 mg/kg bw per
    day. Emamectin showed no carcinogenic potential. The NOEL was 2.5
    mg/kg bw per day on the basis of neurological abnormalities and
    decreased weight gain in mice receiving higher doses.

          Rats

         Groups of rats were given emamectin at 0, 0.5, 2.5, or 12.5/8/5
    mg/kg bw per day in their diet for 14 weeks. The dose of 12.5 mg/kg bw
    per day was reduced to 8 mg/kg bw per day during week 3 and
    subsequently to 5 mg/kg bw per day during week 9. During weeks 3-11 of
    treatment, nine of 20 males receiving 12.5/8/5 mg/kg bw per day were
    killed because of ill health. Genera-lized body tremors were noted in
    most animals receiving 12.5/8/5 mg/kg bw per day, but the incidence
    decreased as the dose was lowered. During week 7, splaying of the
    hindlimbs was seen in a number of males and females receiving 8 mg/kg
    bw per day, which was associated with histological lesions in nervous
    tissue. Significant reductions in body weight and food consumption
    were seen in animals receiving 12.5/8/5 mg/kg bw per day. Decreased
    serum glucose concentration and a slight increase in blood urea
    nitrogen were seen at all sampling times in males and females
    receiving 12.5/8/5 mg/kg bw per day. Decreased urine output and an
    increase in urine specific gravity were seen in groups receiving

    12.5/8/5 mg/kg bw per day; at the same dose, neuronal cytoplasmic
    vacuolation and degeneration were noted. The NOEL was 2.5 mg/kg bw per
    day on the basis of neurotoxicity, weight loss, and decreased food
    consumption in rats receiving higher doses.

         Groups of rats were given emamectin at doses of 0, 0.1, 1.0, 2.5
    (males), or 5/2.5 (females) mg/kg bw per day in the diet for 53 weeks.
    The dose of 5 mg/kg bw per day in female rats was reduced to 2.5 mg/kg
    bw per day in study week 18. No treatment-related deaths were seen.
    Generalized body tremors were seen in females treated with 5/2.5 mg/kg
    bw per day, starting during week 9 and increasing in frequency up to
    week 18; tremors were not seen after week 21 and were not reported in
    males at doses < 2.5 mg/kg bw per day. A reduction in body weight
    was seen in females given 5 mg/kg bw per day, but after the dose was
    reduced to 2.5 mg/kg bw per day the body-weight gain gradually
    returned to that of controls, to which it was comparable by week 25.
    From week 37, females given 1.0 or 2.5 mg/kg bw per day had a slight
    increase in body weight. In general, the weight changes parallelled
    the minor decreases and increases in food consumption. The females
    given 5 mg/kg bw per day showed decreased forelimb grip strength by
    week 14, which decreased in frequency up to and including 24 weeks; no
    neurological abnormalities were seen beyond 24 weeks. Neuronal
    degeneration of the brain was seen in 19 of 20 females receiving 5/2.5
    mg/kg bw per day and 9 of 20 males given 2.5 mg/kg bw per day, and
    degeneration of the spinal cord was seen in 2 of 20 females and 4 of
    20 males given 5/2.5 and 2.5 mg/kg bw per day, respectively. The NOEL
    was 1 mg/kg bw per day on the basis of neurological toxicity in rats
    receiving higher doses.

         Groups of rats were given emamectin at doses of 0.25, 1, or 5/2.5
    mg/kg bw per day in the diet for 105 weeks. The dose of 5 mg/kg bw per
    day was reduced to 2.5 mg/kg bw per day in week 6 for males and week
    10 for females. Weight gain and food consumption were increased in
    females given doses > 1 mg/kg bw per day. Serum triglyceride
    concentrations were elevated in animals fed 1 and 5/2.5 mg/kg bw per
    day for most of the study, and elevated serum bilirubin concentrations
    were seen in the latter half of the study in females fed 1 or 5/2.5
    mg/kg bw per day. Males at the highest dose showed reduced weight gain
    and food intake in the latter half of the study. Neuronal vacuolation
    was seen in the brain and spinal cord of male and female rats given
    5/2.5 mg/kg bw per day, and an increased incidence of diffuse
    vacuolation of hepatocytes was seen in female rats fed 1 or 5/2.5
    mg/kg bw per day. Emamectin had no carcinogenic potential. The NOEL
    was 0.25 mg/kg bw per day on the basis of increased weight gain, food
    consumption and serum triglyceride and bilirubin concentrations in
    rats receiving higher doses.

          Dogs

         Groups of beagle dogs were given emamectin at doses of 0, 0.5, 1,
    or 1.5 mg/kg bw per day for 14 weeks, but the doses were reduced to
    0.25, 0.5, and 1 mg/kg bw per day respectively, at the start of week
    3. Three animals in the group receiving 1.5/1 mg/kg bw per day were
    killed during weeks 3-6 of treatment after showing tremors, mydriasis,
    anorexia, and lethargy. Six of eight animals receiving 1.5/1 mg/kg bw
    per day had tremors, mostly beginning during week 2 of treatment.
    Animals at 1.5 mg/kg bw per day had reduced weight gain and food
    consumption, but these parameters returned to normal when the dose was
    reduced to 1 mg/kg bw per day. Treatment-related histological changes
    were seen in the brain, spinal cord, sciatic and optic nerves, and
    skeletal muscle. Neuronal degeneration was seen in the brains of all
    animals receiving 1.5/1 mg/kg bw per day and in 50% of animals
    receiving 1/0.5 mg/kg bw per day. Scattered neuronal vacuolation was
    noted in the spinal cords of all animals treated with 1.5/1 mg/kg bw
    per day and of one of eight animals treated with 1/0.5 mg/kg bw per
    day. Sciatic and optic nerve lesions consisting of scattered
    vacuolation were seen in most animals receiving 1.5/1 mg/kg bw per
    day. Very slight to moderate skeletal muscle atrophy was seen in seven
    of eight animals receiving 1.5/1 mg/kg bw per day and two of eight
    animals receiving 1/0.5 mg/kg bw per day. The NOEL was 0.25 mg/kg bw
    per day on the basis of neuronal degeneration and skeletal muscle
    atrophy in dogs receiving higher doses.

         Groups of dogs were given emamectin at doses of 0, 0.25, 0.5,
    0.75, or 1 mg/kg bw per day by gavage for 53 weeks. Owing to evidence
    of overt toxicity, in the form of body tremors, mydriasis, decreased
    motor activity, and reduced food consumption and body weight, all
    animals receiving 1 mg/kg bw per day were killed on day 23 of
    treatment. Most of the males at 0.75 mg/kg bw per day developed
    tremors, stiffness of gait, mydriasis, and weight loss and were killed
    on day 50 of the study. Similar signs to those in males were seen in
    females treated with 0.75 mg/kg bw per day, but they were of decreased
    severity. One of eight dogs in the group treated with 0.5 mg/kg bw per
    day had fine tremors. Those given 1 mg/kg bw per day, particularly the
    female animals, showed weight loss associated with decreased food
    intake. Three of four males treated with 0.75 mg/kg bw per day had
    weight loss and decreased food consumption. Neuronal degeneration in
    the central nervous system was reported in males treated with 0.75
    mg/kg bw per day and males and females treated with 1 mg/kg bw per
    day. Axonal degeneration in the central and peripheral nervous systems
    was seen at doses > 0.5 mg/kg bw per day in animals of each sex.
    Degeneration of the retinal ganglionic cells and axonal degeneration
    of the optic nerve were reported at doses of 0.75 and 1 mg/kg bw per
    day. The NOEL was 0.25 mg/kg bw per day on the basis of neurotoxicity
    in dogs receiving higher doses.

    3.  COMMENTS

         The Committee considered the results of studies on the
    pharmacokinetics, metabolism, acute and short-term toxicity,
    genotoxicity, and reproductive toxicity of eprinomectin. All of the
    pivotal studies were carried out according to appropriate standards
    for study protocol and conduct.

         When radiolabelled eprinomectin was administered orally to rats,
    the radiolabel was found mainly in the gastrointestinal tract,
    followed by liver, fat, and kidney, while lower levels were found in
    muscle and blood. Elimination occurred almost exclusively in the
    faeces. For up to 24 h after drug administration, the major residue in
    tissues, plasma, and faeces was unchanged eprinomectin. After two to
    five days, the major residue was  N-deacetylated B1a. The primary
    route of metabolism of eprinomectin in rats is thus  N-deacetylation,
    and minor routes are hydroxylation and hydroxymethylation. Metabolism
    is more extensive in female than in male rats. In cattle,
    radiolabelled eprinomectin was absorbed slowly after topical
    administration. The absorbed radiolabel was taken up mainly by the
    liver and to a lesser extent by the kidney, fat, and muscle. The
    radiolabel disappeared from these tissues with half-lives of 7.8ś8.6
    days, except for muscle beneath the application site in which the
    half-life was 36 days. Elimination occurred mostly in the faeces. At
    all times of slaughter, the main residue in tissues, plasma, and
    faeces was unchanged eprinomectin (85%), B1a representing more than
    80%. B1a disappeared in parallel with the total residues in all
    tissues at all slaughter times, with half-lives of 7.5ś9.6 days in
    liver, kidney, fat, and muscle and 29 days in muscle beneath the
    application site. The profile of metabolites in cattle was
    qualitatively similar to that in rats.

         After oral administration of eprinomectin, the approximate LD50
    values were 70 mg/kg bw for mice and 55 mg/kg bw for rats.
    Eprinomectin is moderately hazardous after acute oral exposure.

         In a 90-day study of toxicity, rats received eprinomectin in the
    diet at nominal doses of 0, 1, 5, or 30/20 mg/kg bw per day. Male and
    female rats at the high dose had tremors, slight degeneration of the
    sciatic nerves, decreased body-weight gain, and changes in organ
    weights. Females at this dose also had arrest of normal ovarian
    follicular maturation, endometrial squamous metaplasia, and decreased
    remodelling of the femora (primary spongiosa), indicative of
    oestrogenśprogesterone imbalance. The NOEL was 5 mg/kg bw per day on
    the basis of effects on the central nervous system and other effects.

         In a 90-day study of toxicity, dogs received eprinomectin by
    gavage at doses of 0, 0.4, 0.8, or 2.4/1.6 mg/kg bw per day. The
    highest dose induced mydriasis, emesis, ataxia, salivation, lateral
    recumbency, body-weight loss, or death. After reduction of this dose
    to 1.6 mg/kg bw per day, decreased food consumption and decreased
    body-weight gain were observed in males and females. Females at this
    dose had slight axonal degeneration of the sciatic nerves. The NOEL

    was 0.8 mg/kg bw per day on the basis of sciatic nerve axonal
    degeneration and body-weight loss.

         In a one-year study of toxicity, dogs received eprinomectin by
    gavage at doses of 0, 0.5, 1, or 2 mg/kg bw per day. Treatment-related
    effects were observed only at the highest dose; these included
    mydriasis and slight focal neuronal degeneration in the pons and the
    cerebellar nuclei of the brain. On the basis of these effects, the
    NOEL was 1 mg/kg bw per day.

         Eprinomectin has been tested  in vitro for its ability to induce
    reverse mutations in  Salmonella typhimurium and  Escherichia coli, 
    gene mutations in Chinese hamster lung cells, chromosomal aberrations
    in Chinese hamster ovary cells, and DNA single-strand breaks in
    primary rat hepatocytes. It has been tested  in vivo for its ability
    to induce micronuclei in mouse bone marrow. The results of all tests
    were negative. On the basis of these data, the Committee concluded
    that eprinomectin is unlikely to be genotoxic.

         Rats were exposed to eprinomectin at dietary concentrations of 0,
    6, 18, or 54 mg/kg feed in a two-generation study of reproductive
    toxicity. On the basis of decreased food intake by the dams during the
    first two weeks of lactation, the NOEL for maternal toxicity was 18
    mg/kg feed, equal to 2.5 mg/kg bw per day. The NOEL for reproductive
    toxicity was 18 mg/kg feed, equal to 1.6 mg/kg bw per day, on the
    basis of delayed sexual maturation and a reduced pregnancy rate in
    first-generation animals. Toxicity to pups was the most sensitive
    indicator of the effects of eprinomectin, which consisted of decreased
    weights, body tremors, and increased mortality at 54 mg/kg feed in the
    first-generation pups and in the second-generation pups of the first
    mating. The second-generation pups also had body tremors when treated
    at 18 mg/kg feed. No body tremors or deaths occurred in the second-
    generation pups of the second mating at any dose when the dietary
    levels were reduced to 0, 3, 9, or 27 mg/kg feed, but decreased pup
    weights were still seen at 27 mg/kg feed. The NOEL for pup toxicity
    was 9 mg/kg feed, equal to 1.3 mg/kg bw per day. Furthermore, no
    histopathological changes were observed in the brain, spinal cord, or
    sciatic nerves of animals treated up to 38 weeks of age. A follow-up
    study suggested that the toxicity to pups is probably due to postnatal
    exposure through maternal milk, as there were high, sustained
    concentrations of eprinomectin in milk. This conclusion was further
    supported by the results of a cross-fostering study with ivermectin, a
    closely-related compound, which was reviewed by the Committee at its
    thirty-sixth meeting (Annex 1, reference 91).

         In studies of developmental toxicity in rats and rabbits,
    eprinomectin caused maternal toxicity, evident in rat dams as changes
    in body-weight gain and food consumption at oral doses of 3 and 12
    mg/kg bw per day, with a NOEL of 1 mg/kg bw per day. Rabbit dams had
    slowed pupillary reflexes, mydriasis, and decreased body-weight gain
    at oral doses of 2 and 8 mg/kg bw per day, giving a NOEL of 1.2 mg/kg
    bw per day. Eprinomectin did not cause embryotoxicity, fetotoxicity,

    or teratogenicity in either species at oral doses up to 12 mg/kg bw
    per day in rats and 8 mg/kg bw per day in rabbits.

         No long-term studies were available on eprinomectin; however, the
    long-term toxicity of emamectin, another amino-substituted avermectin
    structurally very similar to eprinomectin, has been reported. The
    Committee noted that dogs are the most sensitive species to both
    emamectin and eprinomectin and that the toxicological end-point for
    both compounds is neurodegeneration. It further noted that the
    neurotoxic effects of both compounds did not progress with prolonged
    treatment, resulting in the same NOELs in 90-day and one-year studies
    in dogs. On the basis of this information, the Committee concluded
    that it was unnecessary to request long-term studies of the toxicity
    of eprinomectin.

         Because the chemical structure of eprinomectin contains no
    structural alerts, and the structurally closely related avermectins,
    emamectin and abamectin, are not carcinogenic in mice or rats, the
    Committee concluded that eprinomectin is unlikely to be carcinogenic.
    This conclusion was supported by the negative findings in studies of
    genotoxicity with eprinomectin  in vitro and  in vivo.

    4.  EVALUATION

         The Committee considered that the most relevant effect for
    evaluating the safety of residues of eprinomectin is the effect on the
    mammalian nervous system. An ADI of 0-10 µg/kg bw was established on
    the basis of the NOEL of 1 mg/kg bw per day for mydriasis and focal
    neuronal degeneration in the brain in the one-year study in dogs and a
    safety factor of 100.5.

    5.  REFERENCES

    Bagdon, W.J. & McAfee, J.L. (1990) L-653,648: Acute toxicity studies
    in mice and rats. Unpublished report (studies no. TT #90-2512,
    TT #90-2513, TT #90-2526, and TT #90-2527) from Merck Sharp & Dohme
    Research Laboratories, West Point, Pennsylvania, USA. Submitted to WHO
    by MSD Sharp & Dohme GmbH, Haar, Germany.

    Bierschwal, C.J. (1995) MK-397, cattle, safety, toxicity,
    reproduction, breeding bulls. Unpublished report (trial no. ASR 14148)
    from Merck & Co., Inc., Fulton, Missouri, USA. Submitted to WHO by MSD
    Sharp & Dohme GmbH, Haar, Germany.

    Bridi, A.A. (1995) MK-397, cattle, safety, toxicity, reproduction,
    breeding cows. Unpublished report (trial no. 13639) from MSDRL
    Uruguaiana Veterinary Research Center, Uruguaiana, RS, Brazil.
    Submitted to WHO by MSD Sharp & Dohme GmbH, Haar, Germany.

    Brooker, A.J., Myers, D.P. & Parker, C.A. (1992) L-653,648:
    Two-generation dietary reproduction study in the rat. Unpublished
    report (study no. TT #90-9010) from Huntingdon Research Centre Ltd,
    Huntingdon, Cambridgeshire, United Kingdom (report No. MSD 203/911574)
    for Merck Research Laboratories, West Point, Pennsylvania, USA.
    Submitted to WHO by MSD Sharp & Dohme GmbH, Haar, Germany.

    Clark, R.L. (1990) L-653,648 Oral range-finding study in non-pregnant
    rabbits. Unpublished report (study no. TT #90-719-2) from Merck Sharp
    & Dohme Research Laboratories, West Point, Pennsylvania, USA.
    Submitted to WHO by MSD Sharp & Dohme GmbH, Haar, Germany.

    Cukierski, M.A. (1990a) L-653,648: Dietary range-finding study in
    female rats. Unpublished report (study no. TT #90-709-0) from Merck
    Sharp & Dohme Research Laboratories, West Point, Pennsylvania, USA.
    Submitted to WHO by MSD Sharp & Dohme GmbH, Haar, Germany.

    Cukierski, M.A. (1990b) L-653,648: Oral range-finding study in
    pregnant rats. Unpublished report (study no. TT #90-718-1) from Merck
    Sharp & Dohme Research Laboratories, West Point, Pennsylvania, USA.
    Submitted to WHO by MSD Sharp & Dohme GmbH, Haar, Germany.

    Cukierski, M.A. (1991) L-653,648: Oral developmental toxicity study in
    rats. Unpublished report (study no. TT #90-718-0) from Merck Sharp &
    Dohme Research Laboratories, West Point, Pennsylvania, USA. Submitted
    to WHO by MSD Sharp & Dohme GmbH, Haar, Germany.

    Cukierski, M.A. (1994) MK-0397: Oral embryo/fetal viability study in
    rabbits. Unpublished report (study no. TT #94-707-0) from Merck
    Research Laboratories, West Point, Pennsylvania, USA. Submitted to WHO
    by MSD Sharp & Dohme GmbH, Haar, Germany.

    DeLuca, J.G. (1991) L-653,648: V-79 mammalian cell mutagenesis assay.
    Unpublished report (studies no. TT #91-8502, TT #91-8510, and TT #91-
    8503) from Merck Sharp & Dohme Research Laboratories, West Point,
    Pennsylvania, USA. Submitted to WHO by MSD Sharp & Dohme GmbH, Haar,
    Germany.

    Department of Health and Family Services (1997) Emamectin.
    Toxicological evaluation report, prepared by the Chemical Products
    Assessment Section, Therapeutic Goods Administration, Department of
    Health and Family Services, Australia. Submitted to WHO by Department
    of Health and Family Services, Australia.

    Faidley, T. (1995) MK0397, topical, cattle, safety, metabolism,
    bioavailability, pharmacokinetics. Unpublished report (trial no. ASR
    14640) from Merck Research Laboratories, Somerville, New Jersey, USA.
    Submitted to WHO by MSD Sharp & Dohme GmbH, Haar, Germany.

    Galloway, S.M. (1990) L-653,648: Assay for chromosomal aberrations in
    vitro in Chinese hamster ovary cells. Unpublished report (studies no.
    TT #90-8611 and TT #90-8614) from Merck Sharp & Dohme Research
    Laboratories, West Point, Pennsylvania, USA. Submitted to WHO by MSD
    Sharp & Dohme GmbH, Haar, Germany.

    Galloway, S.M. (1994) MK-0397: Assay for micronucleus induction in
    mouse bone marrow. Unpublished report (study no. TT #93-8719) from
    Merck Research Laboratories, West Point, Pennsylvania, USA. Submitted
    to WHO by MSD Sharp & Dohme GmbH, Haar, Germany.

    Gogolewski, R.P. (1994) MK397, cattle, safety, tolerance, reactions.
    Unpublished report (trial no. 14291) from MSD Veterinary Research &
    Development Laboratory, Ingleburn, NSW, Australia. Submitted to WHO by
    MSD Sharp & Dohme GmbH, Haar, Germany.

    Green-Erwin, M., Venkataraman, K. & Narasimhan, N.I. (1994) Depletion
    of radioresidues in tissues of cattle dosed topically with a single
    dose of radiolabeled MK-0397 (trial CA-368). Unpublished report (study
    no. 93767) from Merck Research Laboratories, Rahway, New Jersey, USA.
    Submitted to WHO by MSD Sharp & Dohme GmbH, Haar, Germany.

    Halley, B.A., Andrew, N.W., Green-Erwin, M.L. & Zeng, Z. (1995) The
    distribution, excretion and metabolism of MK-0397 (L-653,648) in rats
    (ADMES-1). Unpublished report (study no. 93587) from Merck Research
    Laboratories, Rahway, New Jersey, USA. Submitted to WHO by MSD Sharp &
    Dohme GmbH, Haar, Germany.

    Kloss, M.W. & Bagdon, W.J. (1990) L-653,648: Exploratory six-week oral
    toxicity study in dogs. Unpublished report (study no. TT #89-118-0)
    from Merck Sharp & Dohme Research Laboratories, West Point,
    Pennsylvania, USA. Submitted to WHO by MSD Sharp & Dohme GmbH, Haar,
    Germany.

    Kloss, M.W., Bagdon, W.J. & Gordon, L.R. (1994) L-653,648:
    Fifty-three-week oral toxicity study in dogs. Unpublished report
    (study no. TT #92-116-0) from Merck Research Laboratories, West Point,
    Pennsylvania, USA. Submitted to WHO by MSD Sharp & Dohme GmbH, Haar,
    Germany.

    Kloss, M.W., Coleman, J.B. & Allen, H.L. (1990a) L-653,648:
    Fourteen-week oral toxicity study in rats. Unpublished report (study
    no. TT #90-037-0) from Merck Sharp & Dohme Research Laboratories, West
    Point, Pennsylvania, USA. Submitted to WHO by MSD Sharp & Dohme GmbH,
    Haar, Germany.

    Kloss, M.W., Coleman, J.B. & Ching, S.V. (1990b) L-653,648:
    Fourteen-week oral toxicity study in dogs. Unpublished report (study
    no. TT #89-141-0) from Merck Sharp & Dohme Research Laboratories, West
    Point, Pennsylvania, USA. Submitted to WHO by MSD Sharp & Dohme GmbH,
    Haar, Germany.

    Kloss, M.W. & Morrissey, R.E. (1990a) L-653,648: Exploratory four-week
    oral toxicity study in rats. Unpublished report (study no.
    TT #89-119-0) from Merck Sharp & Dohme Research Laboratories, West
    Point, Pennsylvania, USA. Submitted to WHO by MSD Sharp & Dohme GmbH,
    Haar, Germany.

    Kloss, M.W. & Morrissey, R.E. (1990b) L-653,648: Exploratory 4-week
    oral toxicity study in rats. Unpublished report (study no.
    TT #90-022-0,-1) from Merck Sharp & Dohme Research Laboratories, West
    Point, Pennsylvania, USA. Submitted to WHO by MSD Sharp & Dohme GmbH,
    Haar, Germany.

    Lankas, G.R. & Gordon, L.R. (1989) Toxicology. In: Campbell, W.C.,
    ed.,  Ivermectin and Abamectin, New York, Springer-Verlag, pp.
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    Lankas, G.R., Minsker, D.H. & Robertson, R.T. (1989) Effects of
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     Chem. Toxicol., 27, 523-529.

    Mattson, B.A. (1992) L-653,648: Secretion in rat milk study.
    Unpublished report (study no. TT #91-26-0) from Merck Research
    Laboratories, West Point, Pennsylvania, USA. Submitted to WHO by MSD
    Sharp & Dohme GmbH, Haar, Germany.

    Minsker, D.H. (1990) L-653,648: Oral range-finding study in pregnant
    rabbits. Unpublished report (study no. TT #90-719-1) from Merck Sharp
    & Dohme Research Laboratories, West Point, Pennsylvania, USA.
    Submitted to WHO by MSD Sharp & Dohme GmbH, Haar, Germany.

    Pitt, S.R. (1995) MK 397, cattle, safety, toxicity, reactions.
    Unpublished report (trial no. ASR 14578) from MSDRL Veterinary
    Laboratory, Hertford, Hertfordshire, United Kingdom. Submitted to WHO
    by MSD Sharp & Dohme GmbH, Haar, Germany.

    Sina, J.F. (1990) L-653,648: Microbial mutagenesis assay. Unpublished
    report (study no. TT #90-8004) from Merck Sharp & Dohme Research
    Laboratories, West Point, Pennsylvania, USA. Submitted to WHO by MSD
    Sharp & Dohme GmbH, Haar, Germany.

    Sina, J.F. (1994) L-653,648: Microbial mutagenesis; Cytotoxicity/
    range-finding assay. Unpublished report (study no. TT #89-8059) from
    Merck Research Laboratories, West Point, Pennsylvania, USA. Submitted
    to WHO by MSD Sharp & Dohme GmbH, Haar, Germany.

    Storer, R.D. (1990) L-653,648:  In vitro alkaline elution/rat
    hepatocyte assay. Unpublished report (studies no. TT #90-8305,
    TT #90-8309, and TT #90-8314) from Merck Sharp & Dohme Research
    Laboratories, West Point, Pennsylvania, USA. Submitted to WHO by MSD
    Sharp & Dohme GmbH, Haar, Germany.

    Turner, M. & Schaeffer, J. (1989) Mode of action of ivermectin. In:
    Campbell, W.C., ed.,  Ivermectin and Abamectin, New York,
    Springer-Verlag, pp. 73-88.

    Venkataraman, K. & Narasimhan, N.I. (1995) Metabolism of
    [3H]-MK-0397 in cattle following a topical application (ADMES-3).
    Unpublished report (study no. 93993) from Merck Research Laboratories,
    Rahway, New Jersey, USA. Submitted to WHO by MSD Sharp & Dohme GmbH,
    Haar, Germany.

    Wise, L.D. (1991) L-653,648: Oral developmental toxicity study in
    rabbits. Unpublished report (study no. TT #90-719-0) from Merck Sharp
    & Dohme Research Laboratories, West Point, Pennsylvania, USA.
    Submitted to WHO by MSD Sharp & Dohme GmbH, Haar, Germany.
    


    See Also:
       Toxicological Abbreviations
       EPRINOMECTIN (JECFA Evaluation)