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    DIHYDROSTREPTOMYCIN

    First draft prepared by
    Dr Leena Gajjar
    Veterinary Medicines Directorate
    Ministry of Agriculture, Fisheries and Food
    Addlestone, Surrey, United Kingdom

    1.  EXPLANATION

         Both dihydrostreptomycin and streptomycin were evaluated at the
    twelfth meeting of the Committee (Annex 1, Reference 17).  An ADI was
    not established for either compound at that time.

         The two compounds are aminoglycoside antibiotics, and are closely
    related in structure as shown in Figures 1 and 2 below.

    FIGURE 1

         Both dihydrostreptomycin and streptomycin are used for treatment
    of bacterial infections in food-producing animals.

         Dihydrostreptomycin is formed by reduction of streptomycin.  As
    a result, pharmacokinetic properties, toxicological profiles, and
    spectrum of antimicrobial and biological activity of the two compounds
    are similar.  Therefore, data on the two compounds were considered
    together for the purpose of establishing a single ADI.  The Committee
    noted that the two compounds are generally considered together in the
    published literature and in the report of the twelfth meeting of the
    Committee. Complete toxicological monographs were prepared for each
    compound and are provided in their entirety below.

    2.  BIOLOGICAL DATA

    2.1  Biochemical aspects

    2.1.1  Absorption, distribution and excretion

    2.1.1.1  Rats

         The sulfate and calcium chloride complex salts of streptomycin,
    and the sulfate and hydrochloride salts of dihydrostreptomycin, were
    administered orally in solution to groups of 5 rats at a single dose
    of 7.5 g of antibiotic base per kg bw. Heart blood was withdrawn at
    0.5, 1, 2, 3, and 4 hours, and assayed microbiologically using
     Staphylococcus aureus. Peak blood levels of all four compounds were
    measured at 1-2 hours. Streptomycin calcium chloride complex and
    dihydrostreptomycin hydrochloride were better absorbed (peak blood
    levels of 3200 to 4300 g/ml, respectively) compared to the two
    sulfate salts of each compound (peak blood levels of 200-300 g/ml).
    The elimination half-lives and AUCs were not calculated for this study
    (Edison  et al., 1951).

    2.1.1.2  Cats

         Nine female cats aged 4 to 6 months and weighing approximately 2
    to 3 kg were given single intramuscular injections of 10 mg/kg bw each
    of penicillin G and dihydrostreptomycin. Serum levels of each drug
    were assayed by microbiological agar diffusion methods up to 32 hours
    after injection.  Dihydrostreptomycin was assayed using  Bacillus
     pumilis, and penicillin was assayed using either  Bacillus subtilis
    or  Bacillus stearothermophilus.  One cat had detectable penicillin 
    levels in the pre-treatment sample  (0.04 g/ml).  Another cat died
    within two minutes of the six-hour blood sample, which was taken by
    heart puncture.  Death was considered to be caused by the blood
    sampling method, and therefore was not treatment-related.

         Peak plasma levels of approximately 25 g/ml were reached at
    approximately 1 hour. The plasma elimination half-life was
    approximately 1 hour, and the AUC was 61.4  15.1 g/ml/hour.
    Dihydrostreptomycin was below the limit of detection (0.6 g/ml) in
    all animals by 24 hours after injection (Rudd & Silley, 1987d).

    2.1.1.3  Dogs

         Ten dogs (six females, four males, aged 1.5 to 4 years and
    weighing approximately 11 to 22 kg) were given single intramuscular
    injections of 10 mg/kg bw each of penicillin G and
    dihydrostreptomycin. Serum levels of each drug were assayed by
    microbiological agar diffusion methods up to 72 hours after injection.
    Dihydrostreptomycin was assayed using  Bacillus pumilis, and
    penicillin was assayed using either  Bacillus subtilis  or  Bacillus
     stearothermophilus.

         Peak plasma levels of approximately 41 g/ml were reached at
    approximately 1 hour. The plasma elimination half-life was
    approximately 1 hour, and the AUC was 74.1  8.50 g/ml/hour.
    Dihydrostreptomycin was below the limit of detection (0.6 g/ml) in
    all animals by 8 hours after injection (Rudd & Silley, 1987c).

    2.1.1.4  Sheep

         Ten female sheep aged 15 to 18 months and weighing approximately
    60 to 90 kg were given single intramuscular injections of 10 mg/kg bw
    each of penicillin G and dihydrostreptomycin. Serum levels of each
    drug were assayed by microbiological agar diffusion methods up to 72
    hours after injection. Dihydrostreptomycin was assayed using  Bacillus
     pumilis, and penicillin was assayed using either  Bacillus subtilis
    or  Bacillus stearothermophilus.

         Peak plasma levels of approximately 45 g/ml were reached at
    approximately 1 hour. The plasma elimination half-life was
    approximately 2 hours, and the AUC was 151.5  35.9 g/ml/hour.
    Dihydrostreptomycin was below the limit of detection (0.3 g/ml) in
    all animals by 24 hours after injection (Rudd & Silley, 1987b).

    2.1.1.5  Cattle

         Ten cattle (one male, nine females, weighing approximately 200
    to 450 kg) were given single intramuscular injections of 10 mg/kg bw
    each of penicillin G and dihydrostreptomycin. Serum levels of each
    drug were assayed by microbiological agar diffusion methods up to 72
    hours after injection. Dihydrostreptomycin was assayed using  Bacillus
     pumilis, and penicillin was assayed using either  Bacillus subtilis
    or  Bacillus stearothermophilus.

         Peak plasma levels of approximately 44 g/ml were reached at
    approximately 1 hour. The plasma elimination half-life was
    approximately 5 hours, and the AUC was 207.2  33.8 g/ml/hour.
    Dihydrostreptomycin was below the limit of detection (0.3 g/ml) in
    all animals by 48 hours after injection (Rudd & Silley, 1987a).

         Two young Holstein-Friesian steers weighing approximately 300 kg
    each were injected intramuscularly with uniformly tritiated
    dihydrostreptomycin (specific activity 262 mCi/mmol) plus carrier drug
    at a single dose of 25 mg/kg bw, given as three separate injections
    into the scapular region.  Plasma, urine, saliva and lachrymal
    secretions were sampled up to 5 days after the injection.  The maximal
    concentrations in plasma and urine were 78 and 1875 g/ml,
    respectively, and were achieved at 3-4 hours after injection. Low drug
    concentrations (1.7 g/ml) were still detectable in urine at 96 hours
    after injection.  No radioactivity was detected in saliva or lachrymal
    secretions.  The apparent volume of distribution was 23%, 

    corresponding to the extra-cellular fluid. The absorption and
    excretion rates were similar when assayed microbiologically with
     Staphylococcus aureus (Stalheim, 1970).

         Dihydrostreptomycin was administered intramuscularly to 21
    heifers and 6 dairy cows at 5 and 7.5 mg/lb bw (11 and 16.5 mg/kg bw),
    respectively. Peak blood levels were reached at 1-2 hours after
    injection and averaged approximately 45 g/ml (with a range of 8.5-
    100 g/ml) for the 11 mg/kg bw dose, and 65.0 g/ml (with a range of
    40-122 g/ml) for the 16.5 mg/kg bw dose. Dihydrostreptomycin was
    assayed in the milk of 7 cows which had been injected with 11 mg/kg
    bw. The drug was detected in 4 cows only, at concentrations ranging
    from trace levels to 6.25 g/ml, at 4 to 18 hours after injection. The
    limit of detection of the assay was not stated for either blood or
    milk (Hammond, 1953).

         After intramuscular administration of 0.5 g dihydrostreptomycin
    per 100 lb (11.1 mg/kg bw) to 26 dairy cows, drug concentrations were
    detectable in the milk from 11 animals. At 36 hours milk levels were
    at the limit of detection of 0.04 g/ml for 8 cows.  At 48 hours the
    drug was undetectable in all animals.  When administered at  1 to  2
    g per 100 lb bw (22 to 44 mg/kg bw) peak levels of 0.07 to 0.12 g/ml
    were reached in milk at 24 hours, but were below the limit of
    detection at 48 hours (Blobel & Burch, 1960).

    2.1.1.6  Pigs

         Three cross-bred barrows weighing approximately 100 kg each were
    injected intramuscularly with uniformly tritiated dihydrostreptomycin
    (specific activity 262 mCi/mmol) plus carrier drug at a single dose of
    25 mg/kg bw/day, given as three separate injections. Plasma and urine
    were sampled up to 5 days after the injection. The maximal
    concentrations in plasma and urine were approximately 90 and 7100
    g/ml, respectively, and were achieved 1-2 hours after injection. Low
    drug concentrations (approximately 3 g/ml) were still detectable in
    urine at 86 hours after injection. The apparent volume of distribution
    was 21%, corresponding to the extracellular fluid. The absorption and
    excretion rates were similar when assayed microbiologically with
     Staphylococcus aureus (Stalheim, 1970). 

         The results of the foregoing studies are summarized in Table 1.

    2.1.1.7  Humans

         As a class, aminoglycoside antibiotics cross membranes very
    poorly and only about 1% of an oral dose is absorbed, even when there
    is intestinal inflammation or ulceration (Pratt & Fekaty, 1986).

         Dollery (1991) reported that after oral administration of
    streptomycin, 60%-100% of the drug can be recovered unchanged from the
    faeces. 


        Table 1.  Pharmacokinetics parameters for dihydrostreptomycin after intramuscular administration to various species

    (Values represent  standard deviation or range)
                                                                                                                                     
    Species   Dose (i.m.)    Cmax (plasma)      Tmax hours    AUC mcg/        Cmax urine     t hours      Vd     Reference
              (mg/kg bw)     (g/ml)                          (ml/hour)       (g/ml)
                                                                                                                                     
    Cats          10         25.1  8.6         1.2  0.4      61.4  15.1        -          1.4  0.1     -      Rudd & Silley, 1987d
    Dogs          10         41.2  8.07        0.8  0.26     74.1  8.50        -          1.0  0.17    -      Rudd & Silley, 1987c
    Sheep         10         44.8  14.3        0.8  0.3     151.5  35.9        -          2.3  0.3     -      Rudd & Silley, 1987b
    Cattle        10         44.3  15.2        1.1  0.4     207.2  33.8        -          4.9  0.5     -      Rudd & Silley, 1987a
    Cattle        11         44.7 (8.5-100)     1 - 2         -                   -          -             -      Hammond, 1953
    Cattle        16.5       65.0 (40.0-122)    1 - 2         -                   -          -             -      Hammond, 1953
    Cattle        25         78                 3 - 4         -                   1875       -             23%    Stalheim, 1970
    Pigs          25         87                 1 - 2         -                   7120       -             21%    Stalheim, 1970
                                                                                                                                     
    

         The aminoglycosides are reported to have a volume of distribution
    of 25% of lean body mass in adults, corresponding to the extracellular
    fluid volume. The volume of distribution in neonates may average 60%
    of body weight. The volume of distribution in obese individuals is
    less than in those with normal body weight. The volume of distribution
    also varies with edema, which tends to reduce serum concentrations,
    and dehydration which increases serum concentrations. The presence of
    fever reduces serum concentrations by up to 40%, due to increased
    renal blood flow and glomerular filtration. 

         Aminoglycosides accumulate in the kidney relative to plasma
    levels, and detectable concentrations of aminoglycosides persist in
    urine for several weeks, suggesting binding of the drug to kidney.
    Accumulation also occurs in the perilymph of the inner ear, where the
    elimination half-life is much longer than in serum (10-12 hours in
    perilymph compared to 2 hours in serum).

         Aminoglycoside levels in interstitial fluid approximate serum
    levels. In bile, 25-50% of serum levels are obtained in the absence of
    biliary obstruction. In sputum and bronchial secretions 25-67% of
    serum levels may be achieved; in pericardial, pleural and ascitic
    fluids approximately 50% of serum levels are obtained; and synovial
    fluid may contain 50-100% of serum levels. Penetration into the eye,
    cerebrospinal fluid and ventricular fluid is very poor. Placental
    transfer occurs and fetal serum concentrations range from 20-40% of
    maternal serum levels (Pratt & Fekaty, 1986).

         Approximately 20% of a parenteral dose of streptomycin cannot be
    accounted for in urine, but no metabolites have yet been identified.
    Approximately 1% is excreted in bile (Dollery, 1991).

         The excretion rate after parenteral administration of
    amino-glycosides is dependent on renal function and is linearly
    related to the creatinine clearance rate. The elimination half-life in
    adults is 2 hours, but 5-6 hours in neonates due to their lower
    glomerular filtration rate (Pratt & Fekaty, 1986).

    2.1.2  Biotransformation

         The aminoglycosides are reported not to be metabolized in humans
    and are excreted in their active forms by glomerular filtration (Pratt
    & Fekaty, 1986).

    2.1.3  Effects on enzymes and other biochemical parameters

         No information available

    2.2  Toxicological studies

    2.2.1  Acute toxicity studies

    2.2.1.1  Mice

         The oral LD50 of different dihydrostreptomycin salts was compared
    in male Carworth CF1 mice weighing 20 g on average.
    Dihydrostreptomycin hydrochloride gave an LD50 of 12 500 mg/kg bw,
    whereas the sulfate salt gave an LD50 of greater than 30 000 mg/kg bw
    (Edison  et al., 1951).

    2.2.1.2  Chickens

         The LD50 for dihydrostreptomycin sulfate administered
    intramuscularly to groups of fifty 1, 2, 3, or 4 week-old chicks was
    743, 1090, 1738, and 1868 mg/kg bw, respectively. Clinical signs of
    toxicity in all birds were depression, drooping wings, closed eyes,
    muscular weakness and slowed respiration (Huebner  et al., 1956). 

         Dihydrostreptomycin sulfate was administered intramuscularly to
    groups of 3 white Leghorn chickens weighing 600 g, at doses of 800 to
    2500 mg/kg bw. Clinical signs of toxicity included ataxia,
    prostration, gasping, and coma. The LD50 was 2167 mg/kg bw (Gray &
    Purmalis, 1958).

    2.2.1.3  Pigeons

         Dihydrostreptomycin sulfate was administered intramuscularly at
    doses of 400 to 1000 mg/kg bw to groups of 5 racing pigeons weighing
    an average of 360 g. Clinical signs of toxicity included emesis,
    somnolence, ataxia, depression, and coma. The LD50 was determined as
    715 mg/kg bw (Gray & Purmalis, 1958).

    2.2.1.4  Gerbils

         A group of 10 gerbils (5/sex), 7-10 weeks of age and weighing
    55-65 g were given single intramuscular injections of 50 mg
    dihydrostreptomycin sulfate (equivalent to 769-909 mg/kg bw).
    Ascending flaccid paralysis was observed in 10/10 animals at 2 to 5
    minutes after injection, and 8/10 animals died within 4 to 25 minutes
    after injection. The 2 surviving animals gradually regained muscle
    control and awareness with apparently complete recovery at 65 and 120
    minutes post-injection, respectively (Wightman  et al., 1980).

         The results of acute toxicity studies on dihydrostreptomycin are
    summarized in Table 2.

        Table 2.  Acute toxicity of dihydrostreptomycin
                                                                                       
    Species        Number         Route     LD50 mg/kg bw       Reference
                   (age/weight)             (salt)
                                                                                       
    Mice           -              oral      >30 000             Edison  et al., 1951
                   (20g)                    (sulfate)
                                            12 500
                                            (hydrochloride)

    Chickens       50/group       i.m.      743 - 1868          Huebner  et al., 1956
                   (1-4 weeks)              (sulfate)

    Chickens       3/group        i.m.      2167                Gray & Purmalis,
                   (600g)                   (sulfate)           1958

    Pigeons        5/group        i.m.      715                 Gray & Purmalis,
                   (360g)                   (sulfate)           1958

    Gerbil         10             i.m.      < 769               Wightman  et al., 1980
                   (7-10 weeks,
                    55-65g)
                                                                                       
    
    2.2.2  Short-term toxicity studies

    2.2.2.1  Guinea-pigs

         Three groups of 30 young adult guinea-pigs (15/sex/group) were
    treated for 90 days with either 40 mg dihydrostreptomycin/kg bw/day in
    distilled water by gavage, an equal volume of distilled water alone by
    gavage (negative control), or 200 mg kanamycin/kg bw/day, s.c.
    (positive control). 

         The kanamycin-treated animals exhibited spastic twitching
    immediately after dosing, injection site reactions and some animals
    became ataxic. Body-weight gain was depressed in the kanamycin group,
    but was comparable to controls in the dihydrostreptomycin group. 

         Auditory function was assessed using the pinna twitch reflex
    (Preyer reflex) in response to a whistle. Hearing loss was observed in
    all the animals in the kanamycin group after week 6, and in two
    negative control animals after weeks 7 to 9, which were later found to
    be suffering from an inner ear infection. No hearing loss was observed
    in the dihydrostreptomycin treated animals.

         A large number of animals died during the study (7 negative
    controls, 6 positive controls, and 6 treated with
    dihydrostreptomycin). These were necropsied, as were all surviving
    animals at the end of the study. All tissues with observed gross
    lesions, plus the organ of Corti from all animals, were examined
    histologically.

         Of the animals treated by gavage, several animals died of a
    ruptured oesophagus (3 negative controls and 3 in the
    dihydrostreptomycin group). Almost all of the remaining animals dying
    on test had lesions of the lungs due to pneumonia.

         The authors reported that because the organs of Corti were
    inadequately preserved they showed signs of degeneration,
    histologically.  However, the architecture of the cell layers and the
    hair cells were still discernible. The positive control animals
    (kanamycin group) demonstrated either no hair cells at any level of
    the cochlea, or very few and damaged hair cells. The majority of
    dihydrostreptomycin-treated animals had hair cells with largely normal
    architecture, although hair cell damage was noted as being more severe
    in males than females treated with dihydrostreptomycin (Tisdel &
    Harris, 1976).

    2.2.2.2  Cats 

         Dihydrostreptomycin was administered orally in a food vehicle
    (liver cat food) to 3 groups of 10 cats (5/sex/group) weighing between
    1.5 and 4.6 kg, for 90 days at doses of 1, 5 or 10 mg/kg bw/day. Ten
    cats of each sex were used as controls.

         One male cat in the mid-dose group died during the ninth week of
    treatment. A watery red urethral discharge and a distended bladder
    were observed in this animal the day prior to death, and pathological
    examination revealed acute cystitis with concomitant peritonitis. All
    other cats survived the 90 days of treatment and no compound-related
    effects were observed on body-weight gain, food consumption,
    haematology, blood chemistry, urinalysis, gross pathology or
    histopathology (only liver and kidneys of controls and 10 mg/kg/day-
    treated cats were examined) (Wazeter, 1970a).

         Vestibular function was assessed in 10 cats (5/sex) treated daily
    with oral doses of  40 mg  dihydrostreptomycin/kg bw/day for  90 days. 
    Streptomycin sulfate was used as the  positive control (200 mg/kg
    bw/day, s.c.).  Animals in the negative control group were
    administered water by gavage.

         Three cats died, one in each of the negative control, positive
    control, and dihydrostreptomycin groups, due to urinary obstruction
    which was considered unrelated to treatment.

         For the dihydrostreptomycin group, no treatment-related effects
    were observed on body weight, haematology, blood chemistry,
    ophthalmoscopic examination, bone marrow smears, or gross pathology.
    Vestibular function remained normal throughout the treatment period
    based on results of righting reflex tests in which animals were
    dropped from an inverted position two feet above a cushioned mat.  The
    NOEL was 40 mg/kg bw/day dihydrostreptomycin in this study.  The
    results with streptomycin are described in section 2.2.2.5 of the
    monograph on streptomycin (Tisdel  et al., 1976).

    2.2.2.3  Sheep

         A target animal safety study was conducted with
    dihydro-streptomycin in 3 groups of 4 sheep (2/sex/group) aged 13 to
    16 months and weighing approximately 45 kg to 70 kg. Animals were
    treated with daily intramuscular injections of 10 mg procaine
    penicillin G plus 10 mg dihydrostreptomycin/kg bw/day for 5
    consecutive days  (the recommended therapeutic treatment regime), the
    second group received 30 mg of each drug/kg bw/day for 5 days, and the
    third group received 10 mg of each drug/kg bw/day for 15 days. A
    treatment-free control group was not included, although pre-treatment
    parameters were measured in each animal.

         Behaviour was normal and there were no clinical signs of
    neuromuscular blockade. No localized injection site reactions were
    observed. Pathological changes observed in skeletal muscle and liver
    at post-mortem examination were largely due to parasitic infestations.
    Slight congestion/consolidation was observed in the lungs at necropsy,
    which was not examined further. There was no evidence of ototoxicity
    based on results of a crude hearing test and normal histology of the
    VIIIth cranial nerve. There was no pathological evidence of
    nephrotoxicity at post-mortem examination.  Urinalyses were not
    performed. 

         Some haematological and biochemical parameters were altered
    during and after treatment.  Differences reached statistical
    significance in some groups compared with pre-treatment values,
    whereas other parameters were altered then subsequently recovered to
    pre-treatment values. The haematological changes included a reduction
    in haemoglobin, red cells, haematocrit and mean cell volume.
    Biochemical changes included a decrease in sodium, phosphorus,
    creatinine, and bilirubin, and an increase in chloride. The only
    consistent dose-related trends were decreased sodium and increased
    chloride levels during prolonged treatment (15-day group), and a lower
    haematocrit in all treatment groups. These changes, although
    statistically significant were small and were considered by the
    authors not to be clinically relevant (Brooks & Rolph, 1989b).

    2.2.2.4  Pigs  

         A target animal safety study was conducted with
    dihydrostreptomycin in 3 groups of 4 pigs (2/sex/group in groups 2 and
    3, 1 female and 3 males in group 1) approximately 8 weeks of age and
    weighing between 23 and 29 kg. Animals were treated with daily i.m.
    injections of 10 mg procaine penicillin G plus 10 mg
    dihydrostreptomycin/kg initial bw/day for 5 consecutive days (the
    recommended therapeutic treatment regime, group 1), the second group
    received 30 mg of each drug/kg initial bw/day for 5 days (group 2),
    and the third group received 10 mg of each drug/kg initial bw/day for
    15 days (group 3). An untreated control group was not included,
    although pre-treatment parameters were measured in all animals.

         Body weights increased rapidly in all groups (in group 3 body
    weights had increased by 50% after 15 days). Therefore the dose
    administered on a body-weight basis was reduced with time,
    particularly in group 3. A crude hearing test revealed no auditory
    impairment, and behaviour was normal, except for 2 animals in group 3
    that showed symptoms that were attributed to stress (vomiting,
    inactivity, haematemesis and slight cyanosis of ears and legs) on days
    1 and 15, respectively. 

         Changes were observed compared with pretreatment values in
    haematology and serum chemistry values (reduced haemoglobin, red cells
    and haematocrit, reduced neutrophils, increased mean corpuscular
    haemoglobin, decreased creatinine, and increased calcium), some of
    which reached statistical significance. However, there were no obvious
    drug-related or time-related trends, and although statistically
    significant, the changes were small and were considered by the authors
    not to be clinically relevant.

         The VIIIth cranial nerve was histologically normal in all but one
    animal in group 1 in which focal chronic inflammation was observed.
    Focal interstitial nephritis was observed in one pig in group 1 and
    two pigs in group 2. These effects were not considered by the authors
    to be treatment-related. No local injection site reactions were
    observed (Brooks  et al., 1989).

    2.2.2.5  Cattle  

         A target animal safety study was conducted with
    dihydrostreptomycin in 3 groups of 4 cattle (2/sex/group),
    approximately 6 months of age and weighing between 100 and 170 kg.
    Animals were treated with daily i.m. injections of 10 mg procaine
    penicillin G plus 10 mg dihydrostreptomycin/kg bw/day for 5
    consecutive days (the recommended therapeutic treatment regime), the
    second group received 30 mg of each drug/kg bw/day for 5 days, and the
    third group received 10 mg of each drug/kg bw/day for 15 days. An
    untreated control group was not included, although pre-treatment
    parameters were measured in each animal.

         No consistent treatment-related effects on haematological or
    biochemical parameters were observed. Behaviour was normal and there
    were no clinical signs of neuromuscular blockade. The few injection
    site reactions that were observed were minor in nature. The only
    pathological changes present in tissues and organs at post-mortem
    examination were in the lungs, which were attributed to respiratory
    tract infection. There was no evidence of ototoxicity based on results
    of a crude hearing test and normal histology of the VIIIth cranial
    nerve. There was no histological evidence of nephrotoxicity at
    post-mortem examination.  Urinalyses were not performed (Brooks &
    Rolph, 1989a).

    2.2.3  Long-term toxicity/carcinogenicity studies

    2.2.3.1  Rats

         Dihydrostreptomycin was administered in the diet to Charles River
    CD rats, (35/sex/group), to achieve dosages of approximately 1, 5, or
    10 mg/kg bw/day for up to 2 years (dietary concentrations were
    adjusted weekly to compensate for changes in food consumption and body
    weight). Two control groups were included in the study. Five
    rat/sex/group were scheduled for interim kills at 6 and 12 months. The
    remaining 25 animals/sex/group were administered dihydro-streptomycin
    for 2 years. Interim study reports were submitted at 6 and 18 months,
    and a final report at 2 years.

         In the 6-month interim report, it was reported that 2 rats died
    in the low-dose group during the first week of treatment (cause of
    death unknown), and were replaced. One male control rat died after its
    6-month blood sample was taken. There were no treatment-related
    effects observed on food consumption, haematology or urinalysis
    (analyses from 5 rats/ sex/group at 3 and 6 months), hearing or
    vestibular function (measured in all rats at 3 and 6 months). Serum
    chemistry was not evaluated in this study.  Body weights were slightly
    decreased in males in all treatment groups compared with controls,
    which reached statistical significance for all groups; however, no
    clear dose-response relationship was established. One male rat in the
    5 mg/kg bw/day group had a grossly abnormal posture from the seventh
    treatment onwards, and was found to have severe bilateral suppurative
    otitis media when necropsied at 6 months. One to two rats in all
    treated groups, but no controls, were also reported to have this
    condition at the 6 month necropsy when 5 animals per sex/group were
    killed. No other treatment-related effects were noted on gross
    pathology or histopathology.  Histological examination of the auditory
    system or associated nerves was not performed (Wazeter, 1970b).

         In the 18-month interim report, the body weights of male rats in
    the high-dose group were significantly reduced when compared to
    control group 1 but not control group 2. Body weights in all other
    male treatment groups were comparable to controls. No

    treatment-related effects were observed on food consumption,
    haematology, or urinalysis.  No effects were reported on hearing or
    vestibular function as defined by the tests used. One female rat in
    the high-dose group had an abnormal posture and occasional slowed
    righting reflex from week 73 onwards, and two males in the mid-dose
    group had "impaired use of the hindquarters" from week 64 onwards, one
    of which subsequently died in week 69.  Mortality rates were similar
    for all treatment and control groups at 78 weeks, ranging from 0 to 4
    deaths per group of 25 animals. None of the deaths were considered by
    the author to be treatment-related. Causes of death were reported as
    due primarily to spontaneous neoplasias (usually of the pituitary or
    mammary glands) and infectious processes; however, no gross pathology
    or histopathology data were provided with the report (Wazeter, 1971).

         In the two-year report, 12 to 17 out of 25 rats/sex in treated
    groups had survived up to two years. No treatment-related effects were
    observed on survival, food consumption, urinalysis, haematology, or
    hearing.  Body-weight gain was reduced in males in the high-dose group
    compared to control group 2, but not control group 1. Body weights
    were comparable to controls in all other groups.  Vestibular function
    was impaired in both control and treated groups (inability to remain
    on a bar or abnormal gait was observed in 5 control rats, 2 rats each
    in the low- and mid-dose groups, and 3 rats in the high-dose group, no
    sex differences were observed).  On necropsy, 2 rats in the high-dose
    group had gross lesions of the middle ear (purulent otitis media). All
    other gross pathological changes and histopathological changes
    (examined in controls and high-dose group only) occurred with similar
    frequency in all rats examined at two years.  Neoplasms were observed
    with comparable frequency in both treatment and control groups and
    were similar to the historical control incidence for this facility.

         Although this study did not meet current standards regarding the
    number of animals started on the two-year phase of the study
    (25/sex/group), the Committee concluded that survival of more than 50%
    of all treated animals in the 2-year phase of the study (24/50, 33/50,
    and 29/50 in the low-, mid-, and high-dose groups, respectively),
    represented an adequate test of the carcinogenic potential of the
    compound. The NOEL was 5 mg/kg bw/day based on decreased body-weight
    gain in males at the high dose (Wazeter & Goldenthal, 1972).

    2.2.4  Reproductive toxicity studies

         No information available

    2.2.5  Special studies on embryotoxicity/teratogenicity

    2.2.5.1  Guinea-pigs

         Streptomycin and dihydrostreptomycin were administered by daily
    i.m. injection to groups of 2 to 4 pregnant guinea-pigs at doses of
    10, 25, 50, 100 or 200 mg/kg bw/day. The duration of the treatment
    period was not stated. A dose of 100-200 mg/kg bw/day of both drugs
    caused abortion or death within 8-10 days. All fetusus were macerated,
    indicating that death occurred prior to abortion or death of dams. At
    doses of 25-50 mg/kg bw/day, some animals aborted, some survived, and
    some stillbirths were observed. A dose of 10 mg/kg bw/day for 24 days
    caused no abortions. The surviving F1 animals were clinically normal
    with respect to vestibular and auditory function, and the CNS was
    normal on histological examination. The placentae from streptomycin-
    or dihydrostreptomycin-treated animals showed marked hyperaemia,
    haemorrhage, and endometrial necrosis 24 hours after a single
    intramuscular dose of 100 mg/kg bw. These changes progressed with
    further treatment.  Serum concentrations of dihydrostreptomycin and
    streptomycin measured in the fetus were 2-4% of those in the dams
    (Riskaer  et al., 1952).

    2.2.5.2  Rabbits

         Groups of 31, 23, and 26 pregnant female Dutch rabbits, weighing
    approximately 2 to 4 kg, were treated by oral intubation with 0, 5, or
    10 mg dihydrostreptomycin/kg bw/day, respectively, from days 6 to 18
    of gestation. The vehicle control group received 0.5% aqueous
    methylcellulose.

         One rabbit in the 5 mg/kg bw/day group was found dead on
    gestation day 10. Four control rabbits and 1 in the 10 mg/kg bw/day
    treatment group aborted, all after gestation day 14.  No
    treatment-related effects were observed on maternal behaviour,
    appearance, or body-weight gain. On day 30, all animals were killed
    for pathological examination. There were no treatment-related effects
    on the pregnancy rate, number of corpora lutea, number of viable
    fetuses, percentage of abnormal fetuses, litter size, mean fetal body
    weight, or 24-hour survival rate. The number of abortions and
    resorptions/empty implantation sites were higher in controls compared
    to treated groups. Presence of a 13th rib was a frequently noted fetal
    abnormality; however the incidence was similar in all groups. One
    control fetus was acephalic with a ventral hernia, and one fetus in
    the 5 mg/kg bw/day group had agenesis of skin and soft tissue over the
    left lateral nasal and buccal area, and absent hind claws.  All of
    these findings were considered by the authors as unrelated to
    treatment.

         Because no maternal toxicity was observed, the Committee
    concluded that the dose levels may not have been high enough to fully
    exclude teratogenic potential (Wazeter, 1970c). 

    2.2.5.3  Humans

         Auditory toxicity has been reported in children whose mothers
    were treated with streptomycin and dihydrostreptomycin during
    pregnancy.  However, details of the dose administered and the timing
    in relation to the pregnancy are often not stated (Davies, 1991;
    Robinson & Cambon, 1964; Snider  et al., 1980; Varpela  et al.,
    1969; Warkany, 1979). 

         Organogenesis of the inner ear occurs at week 7 of pregnancy, and
    cochlear development continues up to mid-term (Varpela  et al.,
    1969). It is the only organ in the body to reach full adult size and
    complete differentiation by midterm (Robinson & Cambon, 1964).
    However, unlike teratogens, the toxic effects of streptomycin and
    dihydrostreptomycin on the fetus are independent of the critical
    periods of embryogenesis. Ototoxicity can occur throughout the
    gestation period (Snider  et al., 1980), as well as in adults (see
    effects in humans, section 2.3.1 below). In children of streptomycin-
    and dihydrostreptomycin-treated mothers, as well as in adults,
    high-tone hearing is primarily affected first, which is outside of the
    range of normal speech frequencies (Donald & Sellars, 1981).

         The results of a literature review of pregnancy outcomes in women
    receiving streptomycin or dihydrostreptomycin for tuberculosis is
    presented by Snider  et al., 1980. Of a total of 203 women, 206
    pregnancies were followed to term. Seventy-two women received
    streptomycin or dihydrostreptomycin during the first 4 months of
    pregnancy, 73 women received one or the other after the fourth month,
    and the timing was unknown for 61 women. The dose of streptomycin or
    dihydrostreptomycin administered in these cases, where stated, was
    between 1 g to 2 g daily or twice weekly, with total doses ranging
    from 2 g to 202 g. Several other drugs were administered concomitantly
    for tuberculosis, i.e. 88 women also received isoniazid, 1 received
    rifampicin, 1 received ethambutol, 83 received p-amino-salicylic acid,
    and for 59 women the other drug was not known. There were 2
    spontaneous abortions, no premature births and no stillbirths, and 170
    normal term infants were delivered. Abnormalities were observed in 35
    infants (a rate of 169 per 1000, roughly 1 in 6, is reported by the
    authors). The majority of these consisted of vestibular dysfunction
    and varying degrees of hearing loss. Hearing loss occurred in the
    high-tone range first, i.e. before the range of normal speech
    frequencies was affected. No other abnormalities were reported (Snider
     et al., 1980). 

    2.2.6  Special studies on genotoxicity

         Streptomycin and dihydrostreptomycin bind to and alter the
    configuration of the 30S subunit of ribosomes, thus inhibiting protein
    synthesis and causing misreading of the genetic code. RNA and DNA
    synthesis are unaffected (Goldberg, 1965; Davies  et al., 1964).

         The results of genotoxicity assays with dihydrostreptomycin are
    summarized in Table 3.

        Table 3.  Results of genotoxicity assays on dihydrostreptomycin
                                                                                
    Test system         Test object       Concentration    Results     Reference
                                                                                
     In vitro

    Cytogenetics     Human lymphocytes    4.7-13.7 mg/ml   Negative    Obe, 1970
                                                                                
    
    2.2.7  Special studies on immune responses

         The incidence of allergic reactions in humans to systemically
    administered aminoglycosides is reported to be very low (Pratt &
    Fekaty, 1986). 

    2.2.8  Special studies on ototoxicity

    2.2.8.1  Cats

         The ototoxicity of streptomycin and dihydrostreptomycin was
    investigated in cats after they had recovered from surgical
    destruction of one ear. Seven cats were treated with streptomycin at
    doses and treatment periods ranging from 200 mg/kg bw/day for 9.5 days
    to 25 mg/kg bw/day for 84 days. Six cats were treated with
    dihydrostreptomycin at doses and treatment periods ranging from 300
    mg/kg bw/day for 21 days to 100 mg/kg bw/day for 60 days. After the
    last dose, animals were kept treatment-free for 1 year then necropsied
    for histopathological examination of the inner ear and the brain stem
    nuclei. 

         Clinical signs of ototoxicity were similar in both groups, which
    included permanent high frequency hearing loss, loss of
    post-rotational nystagmus, and ataxia.  Weight loss was also noted as
    a treatment-related effect in both groups. With dihydrostreptomycin
    treatment, the hearing loss was delayed and occurred several weeks to
    several months after treatment.

         In the inner ear, loss of hair cells in the cochlea, and damage
    to the sensorimotor epithelium and hair loss in the cristae of the
    vestibular canals were observed. The cristae appeared to be more
    susceptable to toxic effects than the maculae of the saccule and
    utricle.

         In the brain stem, no lesions were found in the vestibular
    nuclei.  The vestibular portion of the VIIIth nerve was normal,
    despite the fact that the sensorimotor epithelium and supporting cells
    of the vestibular apparatus had been destroyed (surgically on one
    side, and due to streptomycin and dihydrostreptomycin treatment on the
    other side).

         Effects observed in the brain stem included degenerative changes
    in the ventral and dorsal cochlear nuclei, occurring primarily in
    those parts corresponding to the ear which had been surgically
    destroyed. In 2 animals treated with streptomycin, these lesions were
    observed on the side corresponding to the ear which had not been
    destroyed surgically. In these 2 animals, degeneration of the spiral
    ganglion was also observed. 

         The authors described these degenerative changes as being typical
    of trans-synaptic atrophy. The nuclei were smaller, containing smaller
    nerve cells arranged more compactly, and changes were observed in the
    myeloarchitecture. The cochlear branch of the VIIIth nerve appeared
    shrunken with only a few myelinated fibres. There were no lesions in
    the cerebellum or any other part of the brain stem. 

         The authors concluded that the lesions produced by both
    streptomycin and dihydrostreptomycin were limited to the vestibular
    end-organ of the inner ear in cats (McGee & Olszewski, 1962). 

         Dihydrostreptomycin was administered subcutaneously to groups of
    2 cats at a dose of 800 mg/kg bw/day for 15 days or 200 mg/kg bw/day
    for 120 days. Moderate to severe ataxia was observed from days 8 and
    10 in the 800 mg/kg bw/day group, and from days 28 and 78 in the 200
    mg/kg bw/day group. Animals were necropsied at the end of the
    treatment period and the inner ear examined. Scattered degeneration of
    the hair cells was observed in the cristae of the horizontal
    semicircular canals, and was more severe in the high-dose group. The
    cochlea of dihydrostreptomycin-treated cats was comparable to controls
    as judged by electrophysiological studies of voltage changes at the
    round window in response to tones of varying frequencies. The authors
    reported that streptomycin produced more severe vestibular and
    cochlear changes in a previous study than those produced by
    dihydrostreptomycin; however, the doses of streptomycin used were not
    stated. The authors also reported that cochlea of the cat appears to
    be more sensitive to streptomycin than dihydrostreptomycin, whereas
    the opposite is true in humans (Hawkins & Lurie, 1953).

    2.2.9  Special studies on microbiological effects

    2.2.9.1  Hamsters

         Groups of 6 hamsters were fed a diet containing 0 or 10 mg/kg
    dihydrostreptomycin. After 47 days, oxytetracycline (10 mg/kg) was
    added to the diet and a suspension of  E. coli (106 organisms/ml)
    susceptible to both drugs was added to the drinking-water for two
    weeks. Faecal samples were collected throughout the study and analyzed
    for lactose-fermenting enteric organisms resistant to either
    dihydrostreptomycin or oxytetracycline. No dihydrostreptomycin- or
    oxytetracycline-resistant organisms were detected in the faeces at any
    time (Rollins  et al., 1975). 

    2.2.9.2  Dogs

         Groups of 5 beagle dogs with an average weight of 15 kg were fed
    a diet containing 0, 2, or 10 mg dihydrostreptomycin per kg of feed
    (equivalent to 50 or 250 g/kg bw/day) for 35 days. In both treatment
    groups there was a shift in the faecal flora from predominantly
    dihydrostreptomycin-susceptible lactose fermenting coliforms to a
    resistant population after 15 days, which was significant (p < 0.01)
    in both groups. This persisted throughout the 28-day post-treatment
    phase of the study. A definitive response was not seen in the control
    group. The authors concluded that the NOEL for development of drug
    resistance in enteric bacteria was less than 2 mg/ kg of diet,
    equivalent to 50 g/kg bw/day (Gaines  et al., 1978).

    2.2.9.3  Humans

         Healthy human volunteers were treated with 0.75 mg of
    dihydrostreptomycin administered orally in a hamburger patty, 4 days
    per week, for 7 weeks. Faecal samples were examined weekly for the
    presence of coliforms bearing R-factor mediated resistance to
    dihydrostreptomycin. The presence of resistant coliforms fluctuated
    from week to week in about 30% of untreated controls. Fluctuations
    were also observed in treated subjects. Four of 25 treated subjects
    and 6/29 controls never excreted resistant coliforms; however, when
    resistant coliforms were acquired, they tended to persist longer and
    to multiply to greater numbers in the presence of dihydrostreptomycin
    than in controls (Snyder  et al., 1972). 

    2.2.9.4  Dairy starter cultures

         Five strains each of various bacterial species were tested for
    their sensitivity to dihydrostreptomycin by measuring zones of
    inhibition of bacterial growth around discs impregnated with 2 or 10
    g/disc. The bacteria used were strains of  Streptococcus (S.
     lactis, S. cremoris, S. diacetilactis, S. thermophilus, S. durans, S.
     faecalis) Lactobacillus bulgaricus, Brevibacterium linens,

     Leuconostoc dextranicum, Staphylococcus (S. aureus, S. epidermidis),
    and  Micrococcus varians. All were susceptible except all
    strains of  Streptococcus faecalis and 3/5 strains of  Streptococcus
     durans (Reinbold & Reddy, 1974).

    2.2.9.5  Minimum inhibitory concentrations in vitro

         The minimum inhibitory concentration (MIC) of dihydrostreptomycin
    sulfate was determined  in vitro for bacteria isolated from the
    intestines or faeces of healthy human adults. Over 100 isolates were
    tested which represented 17 species and covered the 10 most common
    genera of human intestinal microbes. The methods and controls used
    were those proposed by the National Council for Clinical Laboratory
    Standards (NCCLS) describing MIC assays for anaerobic bacteria.
     E. coli was additionally assayed under aerobic conditions. The agar
    dilution method was used, with Wilkins Chalgren agar or BHI agar
    supplemented with 5% rabbit's blood, and incubations were for 48 hours
    at 37  2 C. Dihydrostreptomycin was tested at serial double
    dilutions from 128 g/ml to 2 g/ml. At each concentration, bacterial
    cells were tested at two densities, 1 x 105 cells per spot, which is
    recommended by NCCLS for clinical isolates, and 1 x 107 cells per
    spot, which is considered to be more representative of the bacterial
    concentration within the intestinal ecosystem. The results of this
    study are summarized in Table 4.

          Bifidobacterium spp. were found to be the most sensitive. Data
    available for 5 strains of  Bifidobacterium adolescentis gave an
    MIC50 value of 32 g/ml and a MIC range of 16-64 g/ml, and data on
    5 strains of  Bifidobacterium longum gave an MIC50 value of 16 g/ml
    and a MIC range of 8-32 g/ml.

         The MIC50 value for dihydrostreptomycin in most of the other
    bacterial species tested (17 bacterial species, with 5-11 strains
    tested per species) was higher than 32 g/ml, with the highest MIC50
    values being more than 128 g/ml. 

         An MIC50 value of 32 g/ml was therefore established as the
    concentration without effect on the human gut flora (Carman, 1994).

        Table 4.  MIC values for bacterial isolates from human intestinal material
              (All values represent mcg/ml)

                                                                                       

    Species                     1 x 107 cells / spot          1 x 105 cells / spot
    n = no. of strains      MIC50      MIC90      Range     MIC50     MIC90     Range

                                                                                       

     E. coli                  16         64        8-64      16        32        8-64
    (aerobic) n=11

     E. coli                  64         128       32->128   32        64        16-128
    (anaerobic) n=11

     Bacteroides vulgatus     >128       >128      128-128   >128      >128      64->128
    n=5

     Bacteroides uniformis    >128       >128      >128      >128      >128      >128
    n=5

     Bifidobacterium          32         64        16-64     32        32        8-32
     adolescentis
    n=5

     Bifidobacterium          16         32        8-32      16        16        8-32
     longum n=5

     Lactobacillus            >128       >128      64->128   128       >128      32->128
     acidophilus n=10

     Fusobacterium            >128       >128      128->128  >128      >128      128->128
     russi n=5

     Fusobacterium            >128       >128      128->128  >128      >128      64->128
     prauznitii n=5

     Peptostreptococcus       >128       >128      64->128   >128      >128      64->128
     DZ n=5

     Peptostreptococcus       >128       >128      128->128  >128      >128      128->128
     productus 1 n=5

     Ruminococcus             64         64        64        64        64        32-64
     gnavus n=5

     Ruminococcus             64         64        64-128    64        64        64
     bromii n=5
                                                                                       

    Table 4 (contd)
                                                                                       

    Species                     1 x 107 cells / spot          1 x 105 cells / spot
    n = no. of strains      MIC50      MIC90      Range     MIC50     MIC90     Range

                                                                                       

     Eubacterium              >128       >128      128->128  >128      >128      64->128
     aerofaciens n=5

     Eubacterium              >128       >128      >128      >128      >128      128->128
     rectale n=5

     Coprococcus              64         64        64-128    32        64        32-64
     eutactus n=5

     Coprococcus              64         64        32-64     32        64        32-64
     comes n=5

     Gemmiger                 64         128       64->128   64        128       32->128
     formicilis n=10
                                                                                       
    
    2.3  Observations In humans

    2.3.1  Ototoxicity

         The aminoglycosides are all potentially toxic to the inner ear at
    therapeutic doses. Streptomycin-induced ototoxicity primarily affects
    the vestibular system before auditory function is affected, whereas
    dihydrostreptomycin primarily affects the cochlea before vestibular
    function is affected. With both streptomycin and dihydrostreptomycin
    hearing loss occurs in the high tone range first, i.e. before the
    range of frequencies associated with normal speech is affected
    (Martindale, 1993; Davies, 1991; Shambaugh  et al., 1959; Hawkins &
    Lurie, 1953; Pratt & Fekaty, 1986).

         The primary toxic effect of both streptomycin and
    dihydrostreptomycin is on the peripheral sensory portions of the inner
    ear rather than on the eighth cranial nerve itself, which results in
    progressive destruction of the hair cells of the Organ of Corti from
    the basal region (responsive to high-frequency sounds) to the apex of
    the cochlea (responsive to low-frequency sounds) as dosage and
    duration of treatment are increased (Davies, 1991).

         Dihydrostreptomycin causes delayed ototoxicity in humans
    manifested as a sensorineural hearing loss which may involve any or
    all frequencies. Hearing loss may occur from 1 to 6 months after drug
    treatment, it is permanent, and it is not related to the size of the
    dose (McGee & Olszewski, 1962; Shambaugh  et al., 1959).

         Aminoglycosides, including streptomycin and dihydrostreptomycin,
    accumulate in the perilymph of the inner ear (Davies, 1991). A large
    proportion of both drugs are excreted unchanged by the kidneys (Pratt
    & Fekaty, 1986), and the risk of ototoxicity is therefore increased
    when renal function is compromised (Erlansen & Lundgren, 1964).

         Patients presenting with ototoxic side effects after treatment
    with streptomycin, dihydrostreptomycin or kanamycin, for various
    clinical conditions, were studied in a retrospective study. The route
    of administration was not stated, although it was probably
    intramuscular injection.

         Two patients receiving a 50:50 mixture of streptomycin and
    dihydrostreptomycin at doses of 0.5 or 1.0 mg/kg bw/day of each drug
    (total dose 5 or 14.5 g of each drug) reported vertigo at the end of
    the course of treatment.  Four patients receiving dihydro-streptomycin
    at a dose of 1 g per day (equivalent to 14 to 20 mg/kg bw/day, total
    dose 6 to 36 g) reported tinnitus or impaired hearing (as assessed by
    tone audiometry) between 2 weeks and 3 months after the course of
    treatment. None of the patients in this group reported vertigo. Three
    of these patients had impairment of renal function which may have
    increased blood levels of dihydrostreptomycin and hence increased the
    risk of ototoxicity. It was not stated whether the renal impairment
    was pre-existing or caused by dihydrostreptomycin treatment (Erlanson
    & Lundgren, 1964).

         Dihydrostreptomycin has been reported to be more likely to cause
    partial or complete hearing loss than streptomycin and was therefore
    not recommended for use in humans (Martindale, 1993).

    2.3.2  Renal toxicity

         Nephrotoxicity due to aminoglycosides usually presents in humans
    as an acute tubular necrosis manifested as proteinuria, cylinduria and
    inability to concentrate the urine. This is followed by a reduction in
    glomerular filtration rate with a rise in serum creatinine and blood
    urea nitrogen. Aminoglycosides bind to the luminal surface of the
    proximal tubules, are internalized by pinocytosis and fuse with
    lysosomes. It is postulated that they become sequestered in lysosomes
    where they disrupt phospholipid metabolism (prominant myeloid bodies
    have been observed in renal tubular cells exposed to gentamicin), and
    renal cell death occurs consequent to changes in lysosomal
    biochemistry (Pratt & Fekaty, 1986).

    2.3.3  Neuromuscular blockade

         Aminoglycosides can produce a non-depolarizing type of
    neuromuscular blockade due to reduced acetylcholine release from motor
    nerve endings and reduced sensitivity of the post-junctional endplate;
    this effect is completely reversed by administration of calcium (Pratt
    & Fekaty, 1986). 

    2.3.4  Allergic reactions

         The incidence of allergic reactions to systemically administered
    aminoglycosides is reported to be very low. The incidence of allergic
    reactions to dihydrostreptomycin specifically was not reported (Pratt
    & Fekaty 1986).

    2.3.5  Other effects

         Aminoglycosides have been reported to cause neutropenia,
    agranulocytosis, and aplastic anaemia on rare occasions. Transient
    elevation of hepatic enzymes occurs occasionally with
    amino-glycosides. The incidence of specific effects associated with
    dihydrostreptomycin were not reported (Pratt & Fekaty, 1986).

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    See Also:
       Toxicological Abbreviations
       DIHYDROSTREPTOMYCIN (JECFA Evaluation)