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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 39





    Prepared by:
    The forty-eighth meeting of the Joint FAO/WHO Expert
    Committee on Food Additives (JECFA)



    World Health Organization, Geneva 1997


    DIHYDROSTREPTOMYCIN AND STREPTOMYCIN (addendum)

    First draft prepared by
    Dr P.L. Chamberlain
    Center for Veterinary Medicine
    Food and Drug Administration
    Rockville, Maryland, USA

    1. Explanation
    2. Biological data
       2.1 Toxicological studies
           2.1.1  Reproductive toxicity
       2.2 Observations in humans
    3. Comments
    4. Evaluation
    5. References


    1.  EXPLANATION

         Dihydrostreptomycin and streptomycin are aminoglycoside
    antibiotics used for the treatment of bacterial infections in
    food-producing animals. The two compounds were evaluated together at
    the twelfth (Annex 1, reference 17) and forty-third (Annex 1,
    reference 113) meetings of the Committee. The forty-third Committee
    established a temporary ADI of 0-30 g/kg bw for the combined residues
    of dihydrostreptomycin and streptomycin on the basis of a NOEL for
    toxicological effects of 5 mg/kg bw per day in a two-year study of
    toxicity in rats treated orally. The toxicological effect was a
    decrease in the body weight of males at the highest dose. A safety
    factor of 200 was used. The forty-third Committee determined that
    additional information to assess the potential for effects on
    fertility and peri- and postnatal toxicity and information on residues
    would be required for re-evaluation of dihydrostreptomycin and
    streptomycin in 1997: (i) an evaluation of the results of experimental
    studies on the metabolism of dihydrostreptomycin and streptomycin;
    (ii) data on residues of dihydrostreptomycin and streptomycin in eggs;
    and (iii) results of studies to determine the relationship between the
    antimicrobial activity of the residues and their concentration, as
    measured by specific chemical methods.

         No new studies were conducted in laboratory animals to assess the
    reproductive or peri- or postnatal toxicity of dihydrostreptomycin and
    streptomycin. Instead, an expert report prepared by a recognized
    expert in reproductive toxicology was provided. This report was
    supplemented by published articles that were not available for review
    by the forty-third Committee and field reports from veterinarians in
    the United Kingdom and Ireland. None of the studies or reports met
    current standards for quality assurance, but they did provide some
    useful information for the evaluation of these compounds. The new data
    are summarized in this monograph addendum.

    2.  BIOLOGICAL DATA

    2.1  Toxicological studies

    2.1.1  Reproductive toxicity

         Pigs

         An EEC Directive of 26 June 1990 (90/429/EEC) listed the health
    requirements related to the import and trade of porcine semen. The
    Directive required that in the absence of a negative result in an
    agglutination test for  Leptospira spp., boars must be treated with
    two doses of streptomycin at 25 mg/kg bw given 14 days apart during a
    30-day isolation period prior to their admission to an approved semen
    collection centre. Adoption and implementation of this Directive would
    imply that there are no concerns about the reproductive safety of
    streptomycin; however, the EEC Directive did not specify the extent to
    which reproductive safety was considered when the decision was made to
    include this requirement.

         In one field report, 18 mature boars received two injections, 14
    days apart, of a combination drug containing 150 mg/ml streptomycin
    and 150 mg/ml dihydrostreptomycin, as directed by the EEC Directive.
    The dose administered at each injection was 25 mg/kg bw streptomycin
    and dihydrostreptomycin. Treatment was administered on weeks 4 and 6,
    and data were collected for nine weeks. Semen was collected once or
    twice per week before and after treatment and evaluated for overall
    motility, percentage of sperm showing progressive forward motility,
    and average number of 90-ml insemination doses produced (at a
    concentration of 28  106 sperm per ml).

         The quality of sperm motility was decreased in comparison with
    pretreatment values in two boars in week 5 and in one of these animals
    in week 7. By week 8, the motility quality score of one of the boars
    had returned to the average for the group, while the other remained
    slightly below. Values were missing for three boars in week 5 and two
    boars in week 7, and the quality of sperm motility was not evaluated
    for any boar in week 9. Percentage forward motility was decreased in
    week 5 for four boars, and one also showed a decrease in week 7.
    Values were missing for three boars in week 5 and two in week 7. One
    boar showed a decrease inweek 7 but not in week 5, and another showed
    a progressive decrease in weeks 8 and 9. In week 9, the values for all
    but one boar were at pretreatment levels. Decreases in the number of
    semen doses per ejaculate were seen in week 5 for seven boars. Semen
    from two boars was reported as 'not used' at this time; semen was 'not
    used' for one during weeks 1 and 7 and for the other in weeks 5-8.
    Values for week 5 were missing for three boars. The number of semen
    doses was decreased in eight boars in week 7, but values at this time
    were missing for two boars. In week 9, the values for all but one boar
    were within the range of values observed up to and including week 4.
    In week 9, semen from one boar was 'not used', and the values for two
    boars were missing.

         The veterinarian who submitted this field report considered that
    the quality of the semen from all of these boars, with the possible
    exception of one, was not adversely affected by treatment. The quality
    of motility in the semen from one boar appeared to have been reduced
    by treatment, and the semen from this boar was 'not used' for four
    weeks. This effect may have been an incidental occurrence or
    treatment-related, but the latter cannot be reliably determined from
    this study owing to the lack of an untreated control group, the
    absence of statistical analysis of the data, and numerous missing data
    entries (Glossop, 1996).

         In another field report, five young (age not stated) boars at an
    artificial insemination facility were treated as required by the EEC
    Directive. Records of semen collections four to six weeks later were
    evaluated for volume of ejaculate, raw motility score, and
    concentration. No pretreatment values and no untreated control group
    were included in this study. All of the parameters evaluated were
    within the normal range (Glossop, 1996).

         In a field report, six boars, six to seven months of age
    (approximately 100 kg bw) at an artificial insemination facility were
    treated as required by the EEC Directive. Sperm morphology was
    assessed in two ejaculates collected after the first injection and in
    two to six ejaculates collected after the second injection.
    Pretreatment morphology was also assessed. No control group was used
    in this study. One boar showed a notable increase in the proportion of
    abnormal sperm after treatment with streptomycin, the predominant
    abnormalities being bent tails and distal protoplasmic droplets.
    Another boar showed high levels of abnormal sperm before treatment
    with streptomycin, and the levels increased after treatment. Such
    abnormalities can result from external stress. Owing to the small
    sample size and lack of an untreated control group, it could not be
    determined with certainty whether the observed abnormalities were
    induced by treatment or by stress (Glossop, 1996).

         In another field report, 11 boars, six months of age
    (approximately 100 kg bw), were given one injection of
    dihydrostreptomycin at 25 mg/kg bw as routine procedure prior to entry
    into a boar facility for the prevention and control of  Leptospira 
    spp. infection. Semen from four post-treatment collections over about
    one month was evaluated. Semen quality was assessed on a scale of 1 to
    5 (1 being the best). Pre-injection evaluations were not available,
    nor was an untreated control group included in this study. The semen
    quality scores improved with time after treatment for all boars
    (Wager, 1996).

         In a field report, 25 sows and two gilts were each given one
    injection of 15 mg/kg bw of a streptomycin/dihydrostreptomycin
    combination drug before breeding. The treatment was intended to remedy
    a repeated breeding problem thought to be caused by  Leptospira spp.
    in this herd. Service records (return rates) and litter-size records
    were compared with those of a group of 25 untreated sows. The return

    rate for the treated group was 33%, while that of the untreated group
    was 40%. Of the nine sows in the treated group that returned to
    oestrus, five were successfully bred after the second service. All 10
    in the untreated group that returned to oestrus were successfully bred
    after the second service; however, of these 10, six were treated with
    a 15 mg/kg bw dose of streptomycin and dihydrostreptomycin before the
    second service. Only one of the nine treated animals was so treated,
    and this animal was successfully bred after the second service.

         The records of litter size showed that the percentage born dead
    of the total number of pigs born to sows and gilts that conceived at
    first service was 6.8% for the treated pigs and 2.3% for the untreated
    pigs. For those that conceived after the second service, the
    corresponding values were 7 and 3.2%. In this herd, a value of < 7%
    stillborn piglets is desirable, and action is taken when the value
    exceeds 10%. The reporting veterinarian commented that although the
    treated group had a higher percentage of stillborn piglets, it was
    still within the normal range of variability for this parameter. The
    clinical or toxicological significance of this finding is uncertain as
    six of 10 animals that returned to oestrus in the untreated group were
    treated before the second breeding (Wager, 1996).

         Sheep

         In a field report, it was stated that an injection of
    streptomycin or a combination of streptomycin and dihydrostreptomycin
    had been used in clinical veterinary practice for the treatment of
     Leptospira hardjo infections in ewes in late pregnancy, as two doses
    of 25 mg/kg bw streptomycin given 14 days apart. No apparent adverse
    effects on reproduction were observed or reported in the treated
    animals. The basis for this conclusion was not provided (McKeown,
    1996).

         Cattle

         Nine bulls, approximately 18 months of age, were injected
    subcutaneously with two doses of 22 mg/kg bw dihydrostreptomycin given
    12 h apart. Nine additional bulls served as untreated controls. Two
    samples of semen were collected 5 min apart by electroejaculation on
    post-treatment days 3 and 7. The samples were evaluated for pH,
    volume, percent motile spermatozoa, rate of sperm motility,
    concentration of spermatozoa, and capability to withstand freezing.
    The post-treatment results were compared with those of samples taken
    on days 5 and 1 before treatment. The investigators noted that in view
    of the time required for sperm to pass through the bovine epididymis
    -- about eight days -- any treatment-related effects on the parameters
    evaluated would reflect an effect of the antibiotic on epididymal
    physiology, accessory sex gland function, and/or the ejaculatory
    response to electrical stimulation. On each of days 8, 17, and 26
    after treatment, one treated and one control bull underwent surgical
    removal of the testes. Testicular size and weight were recorded, and
    the samples were examined histologically at all stages of

    spermatogenesis. The results showed no treatment-related effects, and
    it was concluded that treatment with streptomycin had no adverse
    effect on spermatogenesis in the bulls in this study (Abbitt  et 
     al., 1984).

         In a field report, the number of doses of semen obtained from an
    ejaculate were recorded for four bulls used for artificial
    insemination over the course of one year. After six months, each bull
    received two injections of 25 mg/kg bw of a streptomycin/
    dihydrostreptomycin combination drug given approximately three weeks
    apart. The mean number of doses in seven ejaculates collected after
    treatment increased slightly for three of the bulls, in comparison
    with the mean for the seven ejaculates collected before treatment. For
    the fourth bull, the mean number of doses decreased but was still
    within the normal range. Treatment thus had no apparent detrimental
    effect on the number of doses produced by the bulls (Kennedy, 1996).

         About 1500 bulls intended for artificial insemination which were
    housed at Danish centres were treated with two intramuscular doses of
    25 mg/kg bw streptomycin given 14 days apart, as directed by the
    Danish Bull Service Directive (88/407/EEC), which became effective in
    January 1990. No clinical anomalies or deficits in semen production or
    quality were reported to the Danish Veterinary Service during the year
    after implementation of the Directive. As the fertility of animals in
    artificial insemination centres is carefully monitored, any adverse
    effects of streptomycin on semen quality would probably have been
    reported (personal communication from Jorgen Bent Anderson to Kennedy,
    1996).

         Routine use of streptomycin or streptomycin and
    dihydrostreptomycin by one veterinarian in clinical veterinary
    practice for the control of infections with  Leptospira spp. in about
    50 dairy herds comprising 120 head per herd did not result in adverse
    effects on the fertility of the treated animals. The treatment regime
    described for use in pregnant cows and stock bulls included a
    combination of penicillin and 250 mg/ml dihydrostreptomycin (or
    streptomycin), which was injected intramuscularly at 20 ml (5 g) for
    three or four consecutive days. Another regime reported was two
    injections of 25 mg/kg bw 14 days apart, given as a 'blanket
    treatment' over a short period of time on a given farm to all adult
    breeding stock, including pregnant cows, non-pregnant cows, and stock
    bulls. No adverse effects on fertility were associated with these
    treatment regimes (McKeown, 1996).

         Council Directive 88/407/EEC of 14 June 1988 on health
    requirements for intra-community trade and imports of frozen bovine
    semen stated that finally diluted semen must contain no less than 500
    IU/ml streptomycin. This implies a lack of any direct adverse effects
    of streptomycin on bovine semen; however, the data on which this
    conclusion was based were not provided. Streptomycin in combination
    with penicillin was shown to facilitate the preservation of motility
    and acrosome morphology of stored boar semen by controlling bacterial
    growth (Bamba & Sone, 1981).

         Horses

         Field reports from two veterinarians describe the use of
    penicillin and streptomycin (or dihydrostreptomycin) in pregnant mares
    and breeding stallions, with no obvious adverse effects on fertility
    or pregnancy outcomes. The treatment regime described was 20 ml (5 g)
    of the combination (containing 250 mg of streptomycin or
    dihydrostreptomycin per ml) given intramuscularly once daily for five
    consecutive days. Details of the examinations conducted on the
    fertility of individual animals were not provided (Dillon, 1996;
    Mills, undated).

    2.2  Observations in humans

         In a brief published article on the treatment of tuberculosis
    during pregnancy, use of streptomycin was discussed. According to the
    authors, streptomycin is the only licensed anti-tubercular drug with
    known harmful effects on the fetus. The specific effect described was
    congenital ototoxicity, attributed to eighth cranial nerve damage. No
    other congenital defects due to streptomycin have been reported (Addis
    & Blowey, 1996).

         In a published review of the use of antibiotics for ear, nose,
    and throat disorders in mothers during pregnancy or lactation, Niebyl
    (1992) discussed the use of aminoglycosides. He noted that clinical
    deafness has occurred in the offspring of mothers who took
    streptomycin or kanamycin during pregnancy. Doses as low as 1 g
    streptomycin twice weekly for eight weeks during the first trimester
    caused ototoxicity in offspring. He also reported that no other
    teratogenic effects are associated with the use of aminoglycosides in
    the first trimester. In a study of 135 infants exposed to
    streptomycin, no teratogenic effects were observed. Additional
    evidence cited to support this conclusion was a study of 1619 newborns
    whose mothers were treated for tuberculosis with multiple drugs,
    including streptomycin, during pregnancy. The incidence of congenital
    defects was the same as that in the control group. Niebyl noted that
    limited information existed about the excretion of gentamicin into
    breast milk, but low levels of amikacin, kanamycin, streptomycin, and
    tobramycin have been detected. He concluded that ototoxicity would not
    be expected to occur in infants exposed in this way in view of the low
    level of excretion into breast milk and the poor oral absorption of
    these drugs.

         In a published review of antituberculosis therapy during
    pregnancy, Scheinhorn and Angelillo (1977) addressed the use of
    streptomycin by reviewing several studies, with a total of 690 cases,
    in which streptomycin was administered to women during pregnancy.
    Evaluation of the children born to these mothers showed 16 with loss
    of high-frequency hearing, eight with vestibular defects, and one with
    clinical disability. Since organogenesis of the inner ear occurs
    during the seventh intrauterine week and differentiation of the
    cochlear cells continues up to mid-gestation, the authors recommend
    avoidance of the use of streptomycin during this period.

    3.  COMMENTS

         The present Committee considered data from published references
    and unpublished field reports on the reproductive toxicity of
    dihydrostreptomycin and streptomycin. Although none of the studies or
    reports met current standards for quality assurance, they did provide
    some useful information for evaluation of the safety of the compounds.

         The field reports consisted of empirical observations by
    veterinarians and evaluations of fertility records for cattle, pigs,
    sheep, and horses treated parenterally with streptomycin or a
    formulation of dihydrostreptomycin and streptomycin for the treatment
    or control of infections by  Leptospira spp. In most cases, the
    treatment consisted of two injections of 25 mg/kg bw streptomycin
    given 14 days apart. Treatment regimes for cattle and horses
    consisting of daily injections of 5 g of streptomycin or of the
    combination for three to five consecutive days were also described.
    None of these reports indicated that treatment with streptomycin or
    the combination had an adverse effect on the fertility or reproductive
    performance of the treated animals.

         The Committee was also asked to consider European directives that
    required bulls and boars to be injected with streptomycin for the
    control of  Leptospira spp. upon entry to artificial insemination
    facilities. The treatment regimes consisted of two injections 14 days
    apart of 25 mg/kg bw of streptomycin or of a combination of
    dihydrohydrostreptomycin and streptomycin. In one country alone, 1500
    bulls were treated over the course of one year, with no reported
    adverse effects on the quality of the semen produced. The Committee
    recognized that the fertility of animals in artificial insemination
    centres is carefully monitored and therefore any adverse effects of
    streptomycin on semen quality would probably have been reported.

         In a controlled study, nine bulls were injected subcutaneously
    with two doses of 22 mg/kg bw of dihydrostreptomycin 12 h apart. Semen
    was collected and evaluated three and seven days after treatment, and
    the results were compared with those for samples collected five and
    one days before treatment. Semen samples were evaluated for pH,
    volume, percentage of motile spermatozoa, rate of spermatozoal
    motility, concentration of spermatozoa, and capacity to withstand
    freezing. Because it takes about eight days for bovine sperm to pass
    through the epididymis, these post-treatment samples represented sperm
    that were in the epididymis at the time of treatment. On each of days
    8, 17, and 26 after treatment, one treated and one control bull
    underwent surgical removal of the testes. Testicular size and weight
    were recorded, and the samples were examined histologically for all
    stages of spermatogenesis. The results showed no treatment-related
    effects on any of the parameters evaluated. The Committee concluded
    that treatment with streptomycin had no adverse effect on
    spermatogenesis in the bulls in this study.

         The Committee reviewed new information on the pregnancy outcomes
    of women treated parenterally with streptomycin for tuberculosis. The
    new information supported the conclusion that streptomycin damages the
    eighth cranial nerve in offspring born to treated mothers. Rigourous
    literature searches revealed no other reported adverse reproductive
    effect associated with the use of streptomycin in humans.

         At the forty-third meeting, the Committee evaluated a study of
    reproductive toxicity in ICR mice treated with streptomycin at 250
    mg/kg bw per day intraperitoneally on days 12-18 of gestation.
    Behavioural tests were conducted on the offspring. No treatment-
    related adverse effects on activity or functional development were
    observed. The present Committee concluded that, although the protocol
    of this study allowed an evaluation of prenatal effects, the results
    could not be used to evaluate the peri- or postnatal effects of
    streptomycin because the dams were not treated throughout lactation.

    4.  EVALUATION

         The Committee noted the long history of use of
    dihydrostreptomycin and streptomycin in veterinary and human medicine
    and the notable absence of reports in the literature of adverse
    effects on fertility or peri- or postnatal toxicity other than
    ototoxicity. Furthermore, the Committee recognized that very small
    amounts of aminoglycosides are absorbed by adults and infants after
    ingestion. It concluded that consumption of residues of
    dihydrostreptomycin or streptomycin in foods derived from animals
    treated in accordance with good practice in the use of veterinary
    drugs presents essentially no risk to peri- or postnatal human health.

         The Committee considered the potential risk for infants consuming
    milk containing residues of dihydrostreptomycin or streptomycin during
    the first weeks of life, before closure of the gut. The temporary MRL
    for the combined residues of dihydrostreptomycin and streptomycin in
    cows' milk recommended by the forty-third Committee was 100 g/litre.
    By comparison, streptomycin is administered parenterally to infants
    for therapeutic purposes at doses of 10-20 mg/kg bw per day. The
    present Committee concluded that the low levels of these compounds
    permitted in cows' milk present essentially no risk to newborns.

         The forty-third Committee determined that the ADI based on the
    antimicro-bial activity of the combined residues of
    dihydrostreptomycin and streptomycin is 0-80 g/kg bw. The equation
    used by the forty-third Committee was modified at the forty-seventh
    meeting (Annex 1, reference 125) by replacing the value for daily
    faecal bolus (150 g) with a value for colonic content (220 g). This
    increases the ADI based on the antimicrobial activity of the combined
    residues of dihydrostreptomycin and streptomycin to 0-120 g/kg bw.

         The forty-third Committee determined that the most sensitive
    toxic effect observed in studies on dihydrostreptomycin and
    streptomycin was that obtained with dihydrostreptomycin in a two-year
    study of toxicity in rats treated orally. The NOEL was 5 mg/kg bw per
    day on the basis of a decrease in the body-weight gain of males at the
    high dose. The present Committee confirmed this as the appropriate
    NOEL, applied a safety factor of 100, and established a group ADI of
    0-50 g/kg bw for the combined residues of dihydrostreptomycin and
    streptomycin.

    5.  REFERENCES

    Abbitt, B., Berndtson, W.E. & Seidel, G.E., Jr (1984) Effect of
    dihydrostreptomycin or oxytetracycline on reproductive capacity of
    bulls. Am. J. Vet. Res., 45, 2243-2246.

    Addis, A. & Blowey, D. (1996) Tuberculosis during pregnancy. Motherisk
    Newsl., 5, 4.

    Bamba, K. & Sone, M. (1981) Factors affecting the quality of boar
    semen stored by means of dialysis. J. Reprod. Fertil., 66, 193-197.

    Dillon, H. (1996)  Unpublished report from Hugh Dillon at the practice
    of Edward C. Gowing, The Curragh, Kildare. Submitted to WHO by
    Norbrook Laboratories Ltd, Newry, Northern Ireland, United Kingdom.

    Glossop, C.E. (1996) Data on the use of streptomycin in AI boars.
    Unpublished reports from Christianne E. Glossop, Merton Farmhouse, The
    Street, Lea, Malmesbury, Wiltshire. Submitted to WHO by Norbrook
    Laboratories Ltd, Newry, Northern Ireland, United Kingdom.

    Kennedy, B.W. (1996) Review of semen collection records for four long
    stay bulls for the period March 1989 to April 1990. Unpublished report
    submitted by Brian W. Kennedy, Company Veterinary Surgeon, Ai Services
    (Northern Ireland) Ltd, Antrim Road, Newtownabbey, Co. Antrim.
    Submitted to WHO by Norbrook Laboratories Ltd, Newry, Northern
    Ireland, United Kingdom.

    McKeown, D.M. (1966) Unpublished report from David M. McKeown,
    Knockanboy Veterinary Clinic, Dervock, Ballymoney, Antrim, Northern
    Ireland. Submitted to WHO by Norbrook Laboratories Ltd, Newry,
    Northern Ireland, United Kingdom.

    Mills, N.J. (undated) Unpublished report from Nicholas J. Mills,
    Cinque Ports Veterinary Associated, Hawkhurst, Kent. Submitted to WHO
    by Norbrook Laboratories Ltd, Newry, Northern Ireland, United Kingdom.

    Niebyl, J.R. (1992) Use of antibiotics for ear, nose, and throat
    disorders in pregnancy and lactation. Am. J. Otolaryngol., 13, 
    187-192.

    Scheinhorn, D.J. & Angelillo, V.A. (1977) Antituberculous therapy in
    pregnancy. Risks to the fetus. West. J. Med., 127, 195-198.

    Wager, A. (1996) Data on the use of dihydrostreptomycin and
    streptomycin in pigs. Unpublished report from Annika Wager, Garth
    Veterinary Group, Straight Lane, Beeford, Nr. Driffield, East
    Yorkshire. Submitted to WHO by Norbrook Laboratories Ltd, Newry,
    Northern Ireland, United Kingdom.

    


    See Also:
       Toxicological Abbreviations