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    PESTICIDE RESIDUES IN FOOD - 1997


    Sponsored jointly by FAO and WHO
    with the support of the International Programme
    on Chemical Safety (IPCS)




    TOXICOLOGICAL AND ENVIRONMENTAL
    EVALUATIONS 1994




    Joint meeting of the
    FAO Panel of Experts on Pesticide Residues
    in Food and the Environment
    and the
    WHO Core Assessment Group 

    Lyon 22 September - 1 October 1997



    The summaries and evaluations contained in this book are, in most
    cases, based on unpublished proprietary data submitted for the purpose
    of the JMPR assessment. A registration authority should not grant a
    registration on the basis of an evaluation unless it has first
    received authorization for such use from the owner who submitted the
    data for JMPR review or has received the data on which the summaries
    are based, either from the owner of the data or from a second party
    that has obtained permission from the owner of the data for this
    purpose.



    LINDANE (addendum)

    First draft prepared by
    P.H. van Hoeven-Arentzen and M.E. van Apeldoorn
    Centre for Substances and Risk Assessment
    National Institute of Public Health and the Environment
    Bilthoven, The Netherlands

         Explanation
         Evaluation for acceptable daily intake
              Toxicological studies
                   Short-term toxicity
                   Long-term toxicity and carcinogenicity
                   Genotoxicity
                   Reproductive toxicity
                   Special studies
                        Hyaline droplet formation in rat kidneys
                        Immune responses
         Comments
         Toxicological evaluation
         References

    Explanation

    Lindane was evaluated toxicologically by the JMPR in 1963, 1965, 1966,
    1970, 1973, 1977, and 1989 (Annex 1, references 2, 4, 6, 14, 20, 28,
    and 56). An ADI of 0-0.01 mg/kg bw was established in 1977. On the
    basis of additional data, the 1989 JMPR allocated an ADI of 0-0.008
    mg/kg bw. An Environmental Health Criteria monograph on lindane has
    been published (WHO, 1991). Additional short-term studies on toxicity
    after dermal exposure, long-term toxicity and carcinogenicity,
    genotoxicity, and reproductive toxicity have become available, which
    were evaluated at the present Meeting.

    Evaluation for acceptable daily intake

    1.  Toxicological studies

     (a)  Short-term toxicity

     Rats

    Groups of 49 male and 49 female Charles River rats (strain Crl:(WI)BR;
    5-7 weeks of age) received lindane (purity, 99.5%) formulated in 5%
    aqueous carboxymethylcellulose as dermal applications on the clipped
    dorsal skin under occlusive conditions for 6 h per day on five
    consecutive days per week for 13 weeks at doses of 0, 10, 60, or 400
    mg/kg bw per day in a volume of 4 ml/kg bw. In each group, 13 rats of
    each sex were selected for an interim kill after six weeks of
    treatment, and 13 rats of each sex were allowed to recover from the
    13-week treatment for another six weeks and were then killed. At each
    kill, three animals of each sex per group were selected for

    determination of the concentrations of lindane in brain, liver,
    kidney, and fat. The limits of quantification were 10 ng/ml in plasma,
    0.1 g/g in brain, 0.5 g/g in liver, 1 g/g in kidney, and 2 g/g in
    fat.

    Females at the high dose were more aggressive than controls, and the
    incidences of languor, piloerection, rapid respiration, ataxia, or
    tremors and convulsions were slightly higher. There were also more
    deaths in this group. Body-weight loss was observed in animals at the
    high dose (by 2% in males and 4% in females) during the first week of
    treatment, when food consumption was lower than control values (11 and
    17%, respectively). There were no changes in ophthalmoscopic or
    haematological parameters or in bone-marrow samples. The activities of
    alanine and aspartate aminotransferases in females at the intermediate
    and high doses exceeded (not significantly) the control values in
    weeks 7 and 14. In week 14, the total plasma cholesterol concentration
    in females at the high dose exceeded (significantly) the control
    value, and the concentrations in males and females at the intermediate
    dose and males at the high dose were slightly (not significantly)
    higher than in controls. At the end of the recovery period, the
    cholesterol concentration and the activities of alanine and aspartate
    aminotransferases were within the control range. In urinalyses, all
    treated males showed higher incidences of protein, blood, and
    turbidity in the urine in week 7, and males at the high dose showed a
    higher incidence of protein at week 14. At the time of the interim
    kill, significant increases in liver weight were seen in males at the
    intermediate dose (relative weight) and males and females at the high
    dose (absolute and relative). At terminal kill, significant,
    dose-dependent increases were observed in the absolute and relative
    liver weights in males and females and in the relative kidney weights
    in males at the intermediate and high doses. After the recovery
    period, only the absolute weights of the livers and kidneys of males
    at the high dose were increased.

    Dose-dependent centrilobular hypertrophy was present in the livers of
    most animals at the intermediate and high doses at the interim and
    terminal kills, and periportal vacuolation was seen in females at the
    intermediate and high doses at the terminal kill (Table 1). At the end
    of the recovery period, no hepatocellular alterations were seen. In
    all treated males, a dose-related increase in hyaline droplet
    deposition was seen in the proximal convoluted tubules of the kidney
    at weeks 6 and 13. A dose-related increase in regenerative and/or
    atrophic basophilic tubules was seen in males at the intermediate and
    high doses at weeks 6 and 13. Tubular vacuolar or granular necrosis
    and granular casts were observed in a few males at the low dose but
    were more frequent and more marked in animals at the intermediate and
    high doses at week 13. After six weeks of recovery, there was only a
    slight increase in the incidence of basophilic tubules and focal
    nephropathy in males at the intermediate and high doses, indicating
    that the changes in the male kidney were not fully reversible. The
    organs of females contained more lindane than those of males, with the
    exception of the kidney: kidneys from males at the low dose contained
    more lindane than the kidneys of females at the high dose. The

    formation of hyaline droplets in the kidneys of males and the
    associated renal effects were considered not to be toxicologically
    relevant for humans. Although this study was evaluated by the 1989
    JMPR, it was re-evaluated because more is now known about the
    toxicological relevance of the renal effects in males. Therefore, the
    NOAEL was 10 mg/kg bw per day, on the basis of increased alanine and
    aspartate aminotransferase activities, increased liver weight and
    histopathological changes in the liver (Brown, 1988).

     Rabbits

    Groups of 40 male and 40 female New Zealand white rabbits weighing
    2.0-2.5 kg received dermal applications of 2 ml/kg bw lindane (purity
    unspecified), formulated in 5% aqueous carboxymethylcellulose, on the
    shaved dorsal skin under occlusive conditions for 6 h per day on five
    days per week for 13 weeks at doses of 0, 10, 60, or 400 mg/kg bw per
    day; owing to marked toxicity, the high dose was reduced to 350 mg/kg
    bw per day from week 9 and further to 320 mg/kg bw per day from week
    11. Ten rabbits of each sex were selected from each group for an
    interim kill after six weeks of treatment, and 10 of each sex were
    allowed to recover from the 13-week treatment for another six weeks
    and were then killed. The interim kill and recovery phases were run
    concurrently, whereas the main study was started afterwards. Blood
    samples and samples of brain, fat, kidney, and liver were taken from
    all animals at necropsy. Blood samples were also taken from five
    animals of each sex per group during week 12 in the main study and
    from five animals of each sex in the control group and that at the
    high dose during weeks 13 and 14 in the recovery phase to study the
    absorption of lindane. The limits of quantification were 5 ng/ml in
    plasma, 0.1 g/g in brain, liver, and kidney, and 2 g/g in fat.

    Tremors and convulsions were observed in animals at the high dose, and
    the incidence of the signs tended to increase each week with the
    number of doses administered. At week 13, 10 males and five females in
    the main study and six males and three females in the recovery study
    died or were removed from the study because of the frequency and
    severity of the convulsions. The clinical signs disappeared rapidly
    during recovery. Animals at the high dose gained less weight and had
    lower food consumption than controls during treatment; the difference
    in body weight at the end of the main study was -7% for males and -9%
    for females. In the recovery phase, the body-weight differences were -
    6% for males and -4% for females both at week 13 and at the end of the
    recovery period. No changes in ophthalmoscopic or urinary parameters
    were seen, and them was no evidence of local irritation at the site of
    application.

    At week 13, erythrocyte and haemoglobin counts and packed cell volume
    were significantly reduced in males at the high dose, even at the time
    of the interim kill. The mean corpuscular volume was significantly
    reduced in males at the intermediate and high doses at week 6 but was
    slightly higher than the control value in males at the high dose at
    week 13. The only changes in haematological parameters seen after the
    recovery period were decreased total leukocyte and absolute lymphocyte

        Table 1. Histopathological changes in the livers of rats treated with lindane

                                                                                                 

    Lesion                        Dose (mg/kg bw per day)
                                                                                                 
                                  Males                           Females
                                                                                                 
                                  0       10      60      400     0       10      60      400
                                                                                                 

    Centrilobular hypertrophy
      Interim kill                0/9     0/9     8/10    10/10   0/7     0/9     4/10    8/8
      Terminal kill               0/20    0/19    10/18   20/20   0/16    0/14    8/17    13/13
      All animals                 0/40    0/40    18/40   31/40   0/40    0/40    12/40   27/40

    Periportal vacuolation
      Interim kill                3/9     1/9     4/10    2/10    4/7     4/9     7/10    6/8
      Terminal kill               2/20    1/19    3/18    5/20    4/16    6/14    8/17    12/13
      All animals                 7/40    2/40    9/40    11/40   16/40   15/40   20/40   23/40
                                                                                                 
    

    counts in males at 400 mg/kg bw. The activities in plasma of alkaline
    phosphatase at weeks 6 and 13 and of gamma-glutamyl transferase at
    week 13 were increased in animals of each sex at the high dose. By
    week 13, alkaline phosphatase activity was 144% of the control value
    in males and 153% in females (both statistically significant) and that
    of gamma-glutamyl transferase was increased by 122% in males (not
    significant) and 138% in females (significant). Owing to sampling
    errors, clinical chemical analyses were not carried out at the end of
    the recovery period.

    The absolute and relative weights of the kidney, liver, and adrenal
    were significantly increased and those of the thymus slightly
    decreased in animals at the high dose at the terminal kill. Similar
    but less marked changes were seen at the interim kill. At the
    intermediate dose, the weights of the livers of males and females and
    those of the adrenals of males were significantly increased at he
    terminal kill. After the recovery period, only the weights of the
    livers of females at the high dose were still slightly increased.
    Animals at the intermediate and high doses showed treatment-related
    centrilobular hypertrophy in the liver at both the interim and
    terminal kills, and this effect was not completely reversed during the
    recovery period. The concentrations of lindane in kidney, liver,
    brain, and fat increased with dose and between weeks 6 and 13. Lindane
    showed a marked tendency to accumulate in fat, the concentrations
    exceeding those in the other tissues examined by a factor of 20-50.
    The plasma concentrations of lindane were approximately proportional
    to the applied dose and increased with time. A rapid decline in the
    mean plasma concentration of about 50% was seen during the first week

    of recovery, and the pattern of clinical signs appeared to he related
    to the plasma concentration of lindane. By the end of the recovery
    period, the concentrations in plasma and all of the tissues examined
    were below the limit of quantification. Them was no apparent
    difference by sex. The NOAEL was 10 mg/kg bw per day on the basis of
    changes in liver and adrenal weights and histopathological changes in
    the liver (Brown, 1990).

     (b)  Long-term toxicity and carcinogenicity

     Rats

    Lindane (purity, 99.7%) was administered in the diet to groups of 60
    Wistar rats of each sex, aged 21-28 days, to provide concentrations of
    0, 1, 10, 100, or 400 ppm, equal to 0, 0.05, 0.47, 4.8, or 20 mg/kg bw
    per day for males and 0, 0.06, 0.59, 6.0, or 24 mg/kg bw per day for
    females. The toxicity of these doses was investigated in groups of 60
    rats of each sex, 15 males and 15 females from each group being killed
    after 30 days and 26 and 52 weeks of treatment. The remaining 15 rats
    of each sex were exposed to lindane for 52 weeks and were then
    maintained on basal diet for a further 26 weeks (recovery phase). The
    carcinogenicity of lindane at these concentrations was investigated in
    groups of 55 rats of each sex exposed for two years. The
    concentrations of lindane in blood, liver, kidney, and brain were
    measured in five rats of each sex from each group in the toxicity and
    carcinogenicity phases. Urinalysis was carried out in five rats of
    each sex after water loading, five of each sex after water
    deprivation, and 15 of each sex under the usual conditions; a terminal
    collection was made by suprapubic pressure before scheduled or
    unscheduled sacrifice. Haematological and clinical chemical
    measurements were made in 10 rats of each sex at various intervals
    during the toxicity phase and at the end of the recovery period and in
    all surviving rats (with a maximum of 20 rats of each sex at the end
    of the carcinogenicity phase. Although only 15 rats of each sex were
    used in the toxicity phase, and the blood samples for haematology and
    clinical chemistry were taken from rats at each sacrifice and
    therefore not from the same rats at each collection interval, the
    Meeting considered that the reliability of the study was not
    jeopardized.

    The incidence of convulsive episodes was significantly increased in
    females at 400 ppm. Most of the episodes occurred during the second
    year of treatment. A treatment-related effect on survival was seen in
    the carcinogenicity phase in females at 400 ppm (significant) and
    males at 100 and 400 ppm (not significant), the rates after 24 months
    being 36% in male controls, 36% in males at 1 ppm, 31% at 100 ppm, and
    17% at 400 ppm, and 49% in female controls, 35% in females at 1 ppm,
    44% at 100 ppm, and 18% at 400 ppm. At 22 months, the rates were 51,
    53, 49, 38, and 42% for males and 62, 56, 58, 55, and 31% for females,
    respectively. No difference in survival rates was seen in animals in
    the toxicity phase after one year. The body-weight gain of males and
    females at 400 ppm was reduced during weeks 0-88 of treatment, and the
    food consumption of animals at this dose was lower during the first

    three months; water consumption was slightly increased in males at 400
    ppm after 3, 24, 52, and 102 weeks of treatment.

    Ophthalmoscopy revealed no abnormalities. Haematological examinations
    showed decreased haemoglobin and erythrocyte counts and (sometimes) in
    packed cell volume throughout treatment in animals at 400 ppm.
    Platelet counts were higher during the first 24 weeks of treatment in
    males receiving 100 or 400 ppm. At the end of the recovery phase,
    however, all of the values for haematological parameters were
    comparable to those of controls. Changes in blood chemistry were
    observed during the first year only among animals receiving 400 ppm
    and were not seen at the end of recovery. The changes included
    generally higher plasma inorganic phosphorus and calcium
    concentrations in males and females and higher total plasma
    cholesterol and urea concentrations and lower albumin:globulin ratios
    in females. At routine collections and after water deprivation,
    greater urinary output and lower urinary pH were found in males at 400
    ppm during the first year and in males at 100 ppm at weeks 3 and 12.
    In addition, the specific gravity was lower in males at these doses
    during the first three months; females usually had higher specific
    gravity during the first six months at routine urinalysis and after
    water loading. Higher urinary urea and creatinine concentrations were
    observed in males (weeks 12 and 24) and females (week 24) at 400 ppm.
    There was a tendency to higher protein concentrations and larger
    numbers of epithelial cells in the urine during the first six months
    of treatment in males at 400 ppm and, occasionally, at 100 ppm. There
    was no effect on creatinine or urea clearance.

    The absolute and relative weights of the kidney in males and the liver
    in males and females and the relative weight of the spleen in females
    were high throughout treatment at 400 ppm, and the absolute brain
    weight was increased in females at this dose at weeks 26, 52, and 104.
    At 100 ppm, the absolute and relative kidney weights were slightly
    increased in males throughout treatment, and the relative liver
    weights were increased after 104 weeks only. Macroscopic examination
    revealed an increased incidence of pale kidneys in males at 100 and
    400 ppm after four weeks. Other macroscopic changes at this dose after
    104 weeks were increased incidences of large kidneys and large livers
    in males and swollen spleens in females. A decrease in the number of
    masses in the pituitary was observed in animals of each sex at 400
    ppm. Microscopic changes were seen in the kidneys of males and the
    livers of males and females. The main changes in the kidneys of males
    at 400 ppm and to a lesser extent in males at 100 ppm were higher
    incidences of necrosis, regeneration, and hyaline droplets in proximal
    tubules after 4 and 26 weeks and higher incidences of hyaline droplets
    in proximal tubules, papillary mineralization, and interstitial
    chronic nephritis after 52 and 104 weeks. Hyaline droplets were also
    observed in males at 10 ppm at weeks 4, 26, and 52 and in males at 1
    ppm only at week 26. Microscopy of the liver showed a dose-related
    increase in periacinar hypertrophy in males and females at 100 and 400
    ppm at all sacrifices. Hepatocytic hypertrophy was also observed in a
    few males at 1 or 10 ppm only after 52 weeks and in a few animals of
    each sex at 10 ppm after 104 weeks, but the increases were not

    statistically significant (Table 2). The nonsignificantly increased
    incidences of liver hypertrophy seen at 1 and 10 ppm are considered
    not to be adverse. The microscopic changes in the kidney were fully
    reversible after the recovery period, while those in the liver were
    partially reversed. There was no treatment-related increase in tumour
    incidence. Although mortality was > 50% at 24 months, reducing the
    reliability of a conclusion of lack of carcinogenicity, mortality at
    22 months was at or near 50% in animals at doses up to 100 ppm. As
    this is a toxic dose, lindane was considered not to be carcingenic in
    this study.

    Lindane was detected in the plasma, brain, kidney, and liver of all
    treated animals, the concentrations being dose-related. At high doses,
    the concentrations in brain were higher in females than males; and at
    all doses, the renal concentrations were higher in males than females.
    In males receiving 400 ppm, the concentrations in the kidney were
    about 100 times the concentrations in serum, brain, and liver and 5-20
    times those in female kidneys. Lindane was no longer present in plasma
    or tissues 26 weeks after withdrawal of treatment.

    The formation of hyaline droplets in the kidneys of males and the
    associated renal effects are considered not to be toxicologically
    relevant for humans. Therefore, the NOAEL was 10 ppm, equal to 0.47
    mg/kg bw per day, on the basis of increased liver weight and
    histopathological changes in the liver (Amyes, 1990).

     (c)  Genotoxicity

    Lindane has been tested for its ability to induce chromosomal
    aberration and unscheduled DNA synthesis. The results are summarized
    in Table 3.

     (d) Reproductive toxicity

    Technical-grade lindane (purity unspecified) was administered in the
    diet at concentrations of 0, 1, 20, or 150 ppm, equivalent to 0, 0.05,
    1, or 7.5 mg/kg bw per day, to groups of Charles River CD rats, about
    four weeks old, over two successive generations. The F0 and the F1
    generation each comprised 30 males and 30 females per group. Both
    generations received treatment 10 weeks before pairing to produce the
    F1 and F2 litters and until termination after breeding. The
    offspring were culled on day 4  post partum to four males and four
    females per litter. The F1 offspring that were not selected for the
    F1 parent generation and the F2 offspring were killed at about four
    weeks of age.

    No effects on general condition, mortality, oestrus cycle, mating
    performance, fertility, or gestation were seen in the F0 or F1
    parent generation. The body-weight gain of F0 females at 150 ppm was
    slightly lowered during the period before pairing and was
    significantly lowered at the end of gestation. The initial and
    terminal body weights of F1 males at 150 ppm were lowered and their


        Table 2. Incidences of liver hypertrophy in rats fed diets containing lindane

                                                                                                                                

    Length of treatment      Dose (ppm)
                                                                                                                                
                             Males                                             Females
                                                                                                                                
                             0         1         10        100       400       0         1         10        100       400
                                                                                                                                

    30 days                  0/10      0/10      0/10      7/10      10/10**   0/8       0/10      0/10      0/10      9/9**
    26 weeks                 0/9       0/10      0/10      4/10      10/10**   0/10      0/10      0/10      3/9       9/9**
    52 weeks                 0/10      3/10      3/10      9/10**    9/9**     0/10      0/10      0/9       5/9*      8/8**
    52 weeks plus 26         0/8       0/8       2/9       0/7       1/8       0/8       0/9       1/8       5/9*      2/9
    weeks' recovery
    104 weeks                1/50      0/50      6/50      25/50**   40/50**   1/50      1/50      4/50      19/50'*   43/50**
                                                                                                                                

    * p <0.05
    ** p < 0.001

    Table 3. Results of assays for the genotoxicity of lindane

                                                                                                                                

    End-point                  Test object             Concentration                     Purity   Results        Reference
                                                                                         (%)
                                                                                                                                

    Chromosomal aberration     Chinese hamster         25.4-305 g/ml in DMSO;           99.7     Negativea      Murli (1990)
                               ovary cells             harvest at 20 h; toxic from
                                                       152 g/m

    Chromosomal aberration     Chinese hamster         25-100 g/ml in DMSO;             99.7     Negativeb      Murli (1990)
                               ovary cells             harvest at 10 h; not toxic

    Chomosomal aberration      Chinese hamster         25-305 g/ml in DMSO;             99.7     Negativeb      Murli (1990)
                               ovary cells             harvest at 20 h; toxic from
                                                       102 g/ml

    Cbomosomal aberration      Chinese hamster         99.8 and 150 g/ml in             99.7     Negativeb;     Murli (1990)
                               ovary cells             DMSO; harvest at 30 h                      toxin at
                                                                                                  150 g/ml

    Unscheduled DNA            Fischer 344 rat         0.25-15 g/ml in DMSO             99.7     Negative       Cifone (1990)
    synthesis                  primary hepatocytes
                                                                                                                                

    DMSO, dimethyl sulfoxide
    a Without metabolic activation
    b With metabolic activation
    

    weight gain was slightly lowered at 20 and 150 ppm but in a dose-
    related fashion. The food intake of F0 females at 150 ppm was reduced
    only during the first week of treatment, and that of F1 males at 20
    or 150 ppm was slightly reduced from treatment week 3 onwards,
    although significantly so only at weeks 3 (20 and 150 ppm) and 4 (150
    ppm).

    No effects were seen on the general condition of F1 or F2 pups or on
    the sex ratios of their litters; however, the 'four-day viability
    index' and litter size on day 4  postpartum were slightly reduced at
    150 ppm. The postimplantation survival index and the four-day
    viability index were also slightly reduced in F2 pups at this dose,
    and the body weights of F1 and F2 pups on day 1 and their weight
    gain during lactation were significantly lower than those of controls.
    Development was impaired in F2 pups at 150 ppm, as demonstrated by a
    delay in the onset and completion of tooth eruption and the completion
    of hair growth. No effects were observed at necropsy of F1 pups. F2
    pups at 150 ppm that died before weaning showed increased incidences
    of hydronephrosis and hydroureter.

    A dose-related increase in absolute and relative kidney weights was
    observed in male and female F0 parents at 20 and 150 ppm; reductions
    in the absolute weights in females at 20 and 150 ppm and the relative
    weights in females at 20 ppm were not statistically significant. F1
    parents at 150 ppm also showed an increase in relative kidney weights.
    Relative liver weights were increased in both F0 and F1 males and
    females at 150 ppm and the absolute weights only in F0 females.
    Necropsy of male F0 and F1 parents at 150 ppm revealed an increased
    incidence of pale kidneys with areas of change; a slight increase was
    seen in F1 males at 20 ppm. F1 male parents at 150 ppm also had an
    increased incidence of hydronephrosis. These effects on the kidney and
    liver were confirmed histopathologically, with increased incidences of
    chronic interstitial nephritis, cortical tubular-cell regeneration,
    hyaline droplets in proximal tubules, tubular necrosis with
    exfoliation and cellular casts, and cortical tubular casts in the
    kidneys of F0 and F1 males at 20 and 150 ppm. In the livers, an
    increased incidence of periacinar hepatocytic hypertrophy was observed
    in F0 and F1 males and females at 150 ppm and in F1 males at 20 ppm
    (Table 4).

    The NOAEL for reproductive and developmental toxicity was 20 ppm,
    equivalent to 1 mg/kg bw per day. The formation of hyaline droplets in
    the kidneys of males and the associated renal effects were considered
    not to be toxicologically relevant for humans. The single finding of
    an increased incidence of liver hypertrophy in F1 males at 20 ppm was
    considered to be an adaptive effect and of no toxicological relevance
    at this dose. Therefore, the NOAEL for parental toxicity was 20 ppm,
    equivalent to 1 mg/kg bw per day, on the basis of effects on body-
    weight gain, increased kidney weights, and hepatic effects (King,
    1991).

        Table 4. Incidences of liver hypertrophy in rats given lindane in the diet over two generations

                                                                                                 

    Generation     Dose (ppm)
                                                                                                 
                   Males                                   Females
                                                                                                 
                   0         1         20        150       0         1         20        150
                                                                                                 

    FO             0/30      1/30      1/30      9/29**    0/29      1/30      1/30      14/3**
    F1             0/28      2/30      6/30*     6/30*     0/30      0/29      1/29      11/28**
                                                                                                 

    * p< 0.05
    ** p < 0.001
    

     (e) Special studies

     (i) Hyaline droplet formation in rat kidneys

    Weanling Wistar and Fischer 344 rats were given lindane in the diet at
    concentrations of 0 or 250 ppm for 13 weeks. Decalin was used as a
    positive control. Urinalysis after 2, 5, 8, and 13 weeks showed slight
    transient protein excretion in Fischer 344 rats and a decrease in
    creatinine clearance in both strains. Histopathological examination
    after 8 and 13 weeks revealed the presence of hyaline droplets in the
    proximal tubules of treated male rats, which was more pronounced in
    the Fischer 344 strain. With an antiserum against alpha2-globulin,
    immunoreactivity was selectively localized to tubules containing
    hyaline droplets. Comparison of the proteins induced by decalin and
    lindane revealed that the molecular mass of the latter differed from
    that of alpha2-globulin. The authors concluded that lindane induces
    a protein with an antigenic structure corresponding to
    alpha2-globulin by a mechanism closely resmbling that of the
    well-documented light hydrocarbon-induced nephropathy (Franken et al.,
    1987).

    Rats from the two-year study of toxicity and carcinogenicity described
    above were used to investigate whether the observed nephrotoxicity was
    due to binding of alpha2-globulin. Slides of the kidneys of all
    males and of female controls and those at the high dose that had been
    exposed for 30 days were stained by an immunohistochemical technique
    specific for alpha2-globulin, and staining was scored on a scale
    from 0-5, from none to markedly severe. Alpha2-globulin accumulated
    in the proximal tubules of males in a dose-dependent manner, with mean
    scores of 1.5 in controls, 1.7 for rats at 1 ppm, 3.2 at 10 ppm, 4.4
    at 100 ppm, and 4.9 at 400 ppm. No staining for alpha2-globulin was
    found in females at the high dose (Swenberg & Dietrich, 1989).

        Table 4. Incidences of liver hypertrophy in rats given lindane in the diet over two generations

                                                                                                 

    Generation     Dose (ppm)
                                                                                                 
                   Males                                   Females
                                                                                                 
                   0         1         20        150       0         1         20        150
                                                                                                 

    FO             0/30      1/30      1/30      9/29**    0/29      1/30      1/30      14/3**
    F1             0/28      2/30      6/30*     6/30*     0/30      0/29      1/29      11/28**
                                                                                                 

    * p< 0.05
    ** p < 0.001
    

     (ii)  Immune responses

    In adult Balb/c mice fed diets containing lindane (purity unspecified)
    at 0 or 150 mg/kg diet (equivalent to 0 or 22 mg/kg bw per day), from
    one month before initiation of immune function tests until termination
    of the study, no effect on the primary immunoglobulin (Ig) M response
    to sheep red blood cells was seen after a, single intraperitoneal
    immunization. After five consecutive daily intragastric doses of sheep
    red blood cells, specific IgA, IgG1, IgG2a, IgG3, and IgM levels were
    not affected, but specific IgG2b levels were significantly increased.

    The resistance of control Balb/c mice and of mice exposed to the same
    dose of lindane for 10 weeks before the immune function test to oral
    infection with  Giardia muris was assessed by counting the number of
    trophozoites in the small intestine on day 28 after inoculation and by
    determining anti- Giardia IgM, IgA, and IgG antibodies. An increased
    duration of giardasis (3-59  104 trophozoites per animal in
    comparison with < 1  104 in controls) was demonstrated in mice
    exposed to lindane. In addition, the lindane-treated mice more
    frequently developed systemic anti- Giardia antibodies (Andre et al.,
    1983).

    Male albino Hissar mice weighing 20-22 g were exposed to lindane
    (purity, 97%) at dietary concentrations of 0, 10, 30, or 50 mg/kg diet
    (equivalent to 1.5, 4.5, or 7.5 mg/kg bw per day) for 6-12 weeks. The
    animals showed depressed humoral immunity, as demonstrated by the
    primary and secondary direct splenic plaque-forming cell response
    after immunization with sheep red blood cells. After exposure for
    three weeks, a reduction was observed only in the secondary
    plaque-forming cell response in mice at 50 ppm. The primary antibody
    response to sheep md blood cells, as determined by haemagglutination
    tests, was affected only in mice at 50 ppm for 12 weeks. The secondary

    haemagglutinating antibody titres were decreased from three weeks of
    exposure onwards in mice at 50 ppm and after 12 weeks of exposure at
    30 ppm. No effects on plaque-forming cell responses of anti-sheep red
    blood cell haemagglutinating antibody titres were seen at 10 ppm
    (Banerjee et al., 1996).

    The immune stares of young female Swiss albino mice (weighing 15-16 g)
    was investigated by dietary exposure to lindane (purity, 97%) at
    concentrations of 0, 0.012, 0.12, or 1.2 mg/kg bw per day for up to 24
    weeks. Delayed-type hypersensitivity reactions, lymphocyte
    transformation (reaction to concanavalin A), mixed lymphocyte
    reactions, and haemolytic plaque-forming cells were assessed in
    separate groups of animals at monthly intervals. In addition,
    immunohistology was performed at weeks 4, 12, and 24. Both cellular
    and humoral immune functions to T-dependent and T-independent antigens
    were stimulated in a dose-dependent fashion by all doses up to weeks
    4-8 of exposure, followed by suppression until termination of the
    study. No changes were seen in the one-way mixed lymphocyte reaction.
    The bactericidal activity of lipopolysaccharide-activated peritoneal
    macrophages against  Staphylococcus aureus in vitro was not affected.
    Histological alterations in lymphoid organs were also noted,
    consisting initially of increased lymphoid follicular activity,
    followed by a depletion of cell populations in the thymus, lymph
    nodes, and spleen. These correlated with the observed biphasic
    functional modulation of the immune system (Meera et al., 1992).

    Humoral immune responses to  Salmonella typhimurium and 
     S. paratyphimurium A and B antigens were suppressed in weanling male
    and female Charles Foster albino rats (weighing 40-50 g) given lindane
    (purity unspecified) by gavage at 6.25 or 25 mg/kg bw per day for 35
    days and intramuscular injections of typhoid-paratyphoid vaccine on
    days 7 and 14. Control animals received the vehicle, olive oil, only.
    Antibody titres determined in serum samples collected at weekly
    intervals on days 14-35 indicated slightly lower specific antibody
    titres after primary dosing (day 14) and significantly suppressed
    responses after the booster injection (days 21-35) (Dewan et al.,
    1980).

    Young male Wistar albino rats (weighing 85-90 g) were fed diets
    containing lindane (purity, 97%) at 0, 5, 20, or 30 ppm (equivalent to
    0, 0.25, 1, or 1.5 mg/kg bw per day) for 8, 12, 18, or 22 weeks. In
    animals injected subcutaneously with tetanus toxoid in Freund's
    complete adjuvant 20 days before sacrifice, an increase in serum
    albumin/globulin was seen at weeks 18-22 in rats at 30 ppm and at week
    22 also in those at 20 ppm, which was due to decreased globulin
    concentrations. These differences correlated with an impaired increase
    in total IgM and IgG levels in response to the immunization. In
    addition, a significant decrease in tetanus toxoid-specific antibody
    titres was observed at weeks 12-22 in animals at 20 and 30 ppm.
    Cellular immune function was also altered after exposure to 20 ppm on
    weeks 12-22 and from week 8 onwards in rats at 30 ppm, as demonstrated
    by decreased inhibition of leukocyte and peritoneal macrophage
    migration. No immunomodulating effects were seen at 5 ppm. There were

    no signs of general toxicity or changes in body weight, food intake,
    or thymus or spleen weights in any treated group (Saha & Banerjee,
    1993).

    In male rabbits (weighing 2000-2500 g) given lindane (purity
    unspecified) at doses of 0, 1.5, 3, 6, or 12 mg/kg bw by capsule on
    five days per week for five to six weeks and weekly intravenous
    injections of a  S. typhimurium 'Ty-3' vaccine, a dose-dependent
    decrease in  S. typhimurium 'O'-specific agglutinating antibody
    titres was observed at all doses. The titres in the test groups were
    already lower at week 1. Although the decreases were reported to be
    statistically significant, the results of statistical analyses were
    not presented (Dsi et al., 1978).

    Comments

    In all of the studies in rats summarized below, the formation of
    hyaline droplets in the kidneys of males and the associated renal
    effects were specific to that sex and were characterized as so-called
    'alpha2-globulin nephropathy'. This type of nephropathy is
    considered not to be relevant for humans. Therefore, in dtermining the
    NOAELs in studies in rats, these male-specific renal effects were not
    taken into account.

    In a 13-week study of dermal toxicity, rats were exposed to doses of
    0, 10, 60, or 400 mg/kg bw per day. At the highest dose, clinical
    signs of neurological effects (convulsions) were observed. Other
    targets were the kidneys of male animals and the liver, as
    demonstrated by changes in organ weight and histopathological changes.
    Since the male-specific renal effects were not taken into account, the
    NOAEL for dermal exposure was 10 mg/kg bw per day, on the basis of
    increased liver weight and histopathological changes in the liver.
    In another 13-week study of dermal toxicity, rabbits were exposed to
    doses of 0, 10, 60, or 400 mg/kg bw per day. At the highest dose,
    clinical neurological effects (convulsions) were observed. The NOAEL
    for dermal exposure was 10 mg/kg bw per day on the basis of increased
    liver and adrenal weights and centrilobular hypertrophy of the liver.

    In a two-year study of toxicity and carcinogenicity, rats were exposed
    to dietary concentrations of lindane at 0, 1, 10, 100, or 400 ppm. At
    the highest dose, neurological effects (convulsions), reduced
    body-weight gain, decreased survival rates (also in males at 100 ppm),
    and changes in erythrocyte parameters were observed. Other changes
    seen at 400 ppm, and to a lesser extent at 100 ppm, were changes in
    weight and in the histological appearances of the liver and kidneys.
    The effects on the kidneys were confined to male rats, with a slight
    increase in hyaline droplet formation that was also observed in male
    rats at 1 and 10 ppm. Since the male-specific renal effects were not
    taken into account, the NOAEL was 10 ppm, equal to 0.47 mg/kg bw per
    day, on the basis of a slight increase in mortality and effects on the
    liver. There was no evidence of carcinogenicity.

    A two-generation study in rats given lindane at dietary concentrations
    of 0, 1,20, or 150 ppm did not indicate reproductive toxicity. The
    main effects found in progeny at 150 ppm were on weight gain;
    decreased viability of pups was seen up to day 4  post partum. In the
    pups of the second generation, there was a slight delay in tooth
    eruption and hair growth Pups of the F2 generation at 150 ppm that
    died before weaning showed increased incidences of hydronephrosis and
    hydroureter. The NOAEL for reproductive and developmental toxicity was
    20 ppm, equivalent to 1 mg/kg bw per day. Histopathological effects in
    the kidneys were observed only in male parents at 20 and 150 ppm.
    Since the male-specific renal effects were not taken into account, the
    NOAEL for parental toxicity was 20 ppm, equivalent to 1 mg/kg bw per
    day, on the basis of effects on body-weight gain and the liver and
    increased kidney weights in animals of each sex.

    Lindane did not induce chromosomal aberrations or unscheduled DNA
    synthesis  in vitro.

    Functional effects and histological changes in the immune system were
    induced by lindane (purity, 97% or unknown) in mice, rats, and
    rabbits. In rats and rabbits, effects were seen at doses equivalent to
    1 mg/kg bw per day and higher, but they were not seen in rats at 0.25
    mg/kg bw per day. In mice exposed to doses of 0.012 mg/kg bw per day
    and higher, an initial immunostimulation followed by immunosuppression
    was observed. It was noted, however, that the purity of the test
    material used in these studies was lower than that specified by
    current FAO specifications, namely > 99% gamma-hexachloro-
    cyclohexane.

    The toxicological effects that are relevant for estimating hazard for
    humans are those on the liver and the central nervous system. In
    published studies, however, lindane of a purity of 97% or of unknown
    purity has been found to affect the immune system. As immunotoxic
    effects were observed at doses close to or even lower than the NOAEL
    found in the two-year study in rats, the Meeting decided that
    additional data should be generated on immunotoxicity. Further, the
    Meeting recommended that, when the new results become available, a
    full re-evaluation be performed to consider the validity of the
    studies that have been reviewed previously and to consider any new
    information that becomes available.

    The Meeting established a temporary ADI at 0-0.001 mg/kg bw on the
    basis of the NOAEL of 0.5 mg/kg bw per day in the two-year study of
    toxicity and carcinogenicity in rats, using a safety factor of 500.
    Pending clarification of the immunotoxicity of lindane that meets FAO
    specifications, this ADI provides a 10-fold margin of safety over the
    LOAEL of 0.012 mg/kg bw per day in a study of immunotoxicity in mice.

    Toxicological evaluation

     Levels that cause no toxic effect

         Mouse:    300 ppm, equivalent to 15 mg/kg bw per day (26-week
                   study of effects on the liver)
                   50 ppm, equal to 7.8 mg/kg bw per day (80-week study of
                   carcinogenicity)
                   30 mg/kg bw per day (maternal and developmental
                   toxicity in a study of developmental toxicity)
                   < 0.012 mg/kg bw per day (24-week study of
                   immunotoxicity with 97% pure lindane)

         Rat:      10 ppm, equal to 0.75 mg/kg bw per day (13-week study
                   of toxicity)
                   4 ppm, equal to 0.29 mg/kg bw per day (three-month
                   study of toxicity, LOAEL= 20 ppm)
                   10 ppm, equal to 0.5 mg/kg bw per day (two-year study
                   of toxicity and carcinogenicity)
                   20 ppm, equivalent to 1 mg/kg bw per day
                   (two-generation study of reproductive toxicity)
                   5 mg/kg bw per day (maternal toxicity in a study of
                   developmental toxicity)
         Dog:      50 ppm, equal to 1.6 mg/kg bw per day (two-year study
                   of toxicity)
         Rabbit:   <5 mg/kg bw per day (maternal toxicity in a study of
                   developmental toxicity)

     Estimate of temporary acceptable daily intake for humans

         0-0.001 mg/kg bw

     Studies without which the determination of an ADI is impracticable,
     to be provided by 2000

    Confirmatory study of immunotoxicity in mice with lindane that meets
    the current FAO specification (> 99% gamma-hexachlorocyclohexane)

     Studies that would provide information useful for continued 
     evaluation of the compound

    Further observations in humans

    References

    Amyes, S.J. (1990). Lindane: Combined oncogenicity and toxicity study
    by dietary administration to Wistar rats for 104 weeks. Unpublished
    report LSR No. 90/CIL002/0839 from Life Science Research Ltd, United
    Kingdom. Submitted to WHO by Centre International d'Etudes du Lindane,
    Brussels, Belgium.


        Toxicological criteria for setting guidance values for dietary and non-dietary exposure to lindane

                                                                                                                                 

    Human exposure    Relevant route, study type, species                     Results, remarks
                                                                                                                                 

    Short-term        Oral, toxicity, dog                                     LD50 = 40 mg/kg bw
    (1-7 days)        Dermal, toxicity, rabbit                                LD50 = 900 mg/kg bw
                      Inhalation, 4 h, toxicity, rat                          LC50 = 1600 mg/m3
                      Skin, irritation, rabbit                                Not irritating
                      Eye, irritation, rabbit                                 Slightly irritating
                      Skin, sensitization, galnea-pig                         Not sensitizing

    Medium-term       Repeated inhalation, 90 days, toxicity, rat             NOAEL = 0.6 mg/m3 per day: clinical signs and
    (1-26 weeks)                                                              increased cytochrome P450 enzymes
                      Repeated dermal, 90 days, toxicity, rat/rabbit          NOAEL = 10 mg/kg bw per day: hepatic effects
                      Repeated oral, 90 days, toxicity, rat                   NOAEL = 0.75 mg/kg bw per day: changes in liver
                                                                              and kidney weight
                      Repeated oral, developmental toxicity, rabbit           No NOAEL for maternal toxicity; NOAEL = 
                                                                              10 mg/kg bw per day: fetuses with 13 ribs
                      Repeated oral, reproductive toxicity, rat               NOAEL = 1 mg/kg bw per day: developmental
                                                                              toxicity and hepatic effects

    Long-term         Repeated oral, 2 years, toxicity/carcinogenicity, rat   NOAEL = 0.5 mg/kg bw per day: hepatic changes
    (> 1 year)                                                                and decreased survival; no carcinogenicity
                                                                                                                                 
    

    Andre, F., Gillon, J.,Andr, C., Lafont, S. & Jourdan, G. (1983)
    Pesticide containing diets augment anti-sheep md blood cell
    nonreagenic antibody responses in mice but may prolong murine
    infection with  Gardia muris. Environ. Res., 32, 145-150.

    Banerjee, B.D., Koner B.C., Ray, A. & Pasha, S.T (1996) Influence of
    subchronic exposure to lindane on humoral immunity in mice.  Indian 
     J. Exp. Biol., 34, 1109-1113.

    Brown, D. (1988) Lindane; 13-week dermal toxicity study (with interim
    kill and recovery period) in the rat. Unpublished report HUK Project
    No. 580/2 from Hazleton UK. Submitted to WHO by Centre International
    d'Etudes du Lindane, Brussels, Belgium.

    Brown, D. (1990). Lindane; 13-week dermal toxicity study (with interim
    kill and recovery period) in the rabbit. Unpublished report HUK
    Project No. 580/6 from Hazleton UK. Submitted to WHO by Centre
    International d'Etudes du Lindane, Brussels, Belgium.

    Cifone, M.A. (1990) Lindane (technical) in the in vitro rat primary
    hepatocyte unscheduled DNA synthesis assay. Unpublished HLA study no.
    12024-0-447 from Hazleton Laboratories America. Submitted to WHO by
    Centre International d'Etudes du Lindane, Brussels, Belgium.

    Desi, L., Varga, L. & Farkas, I. (1978) Studies on the immune
    suppressive effect of organochlorine and organophosphoric pesticides
    in subacute experiments.  J. Hyg. Epidemiol. Microbiol. Immunol., 22,
    115122.

    Dewan, A., Gupta, S.K., Jani, J.P. & Kashyap, S.K. (1980) Effect of
    lindane on antibody response to typhoid vaccine in weanling rats. 
     J. Environ. Sci. Health, B15, 395-402.

    Franken, M.A.M., Kapteijn, R. & Krajnc, E.J. (1987) Nephrotoxicity of
    lindane (gamma-HCH) and decalin in male Wistar and Fisher-344 rats
    (abstract).  Pharm. Weekbl. Sci. Ed., 9, 41.

    King V.C. (1991) Lindane: Reproductive performance study in rats
    treated continuously through two successive generations. Unpublished
    LSR Report No. 91/CIL004/0948 from Life Science Research Ltd, United
    Kingdom. Submitted to WHO by Centre International d'Etudes du Lindane,
    Brussels, Belgium.

    Meera, P., Rao, P. R., Shanker, R. & Tripathi, O. (1992)
    Immunomodulatory effects of gamma-HCH (lindane) in mice.
     Immunopharmacol, Immunotoxicol., 14, 261-282.

    Murli, H. (1990). Lindane (technical) in an in vitro cytogenetic assay
    measuring chromosomal aberration frequencies in Chinese hamster ovary
    (CHO) cells with multiple harvests under conditions of metabolic
    activation. Unpublished HLA study no. 12024-0-437C from Hazleton
    Laboratories America. Submitted to WHO by Centre International
    d'Etudes du Lindane, Brussels, Belgium.

    Saha, S. & Banerjee, B.D. (1993) Effect of subchronic lindane exposure
    on humoral and cell-mediated immune responses in albino rats. 
     Bull. Environ. Contam. Toxicol., 51,795-802.

    Swenberg, J.A. & Dietrich, D.R. (1989) Immunohistochemical
    localization of alpha2-globulin in kidneys of rats treated with
    lindane. Unpublished report from Centre International d'Etudes du
    Lindane (document No. 464-001). Submitted to WHO by Centre
    International d'Etudes du Lindane, Brussels, Belgium.

    WHO (1991)  Lindane (Environmental Health Criteria 124), Geneva,
    International Programme on Chemical Safety.
    


    See Also:
       Toxicological Abbreviations
       Lindane (EHC 124, 1991)
       Lindane (HSG 54, 1991)
       Lindane (ICSC)
       Lindane (PIM 859)
       Lindane (FAO Meeting Report PL/1965/10/1)
       Lindane (FAO/PL:1967/M/11/1)
       Lindane (JMPR Evaluations 2002 Part II Toxicological)
       Lindane (FAO/PL:1968/M/9/1)
       Lindane (FAO/PL:1969/M/17/1)
       Lindane (WHO Pesticide Residues Series 3)
       Lindane (WHO Pesticide Residues Series 4)
       Lindane (WHO Pesticide Residues Series 5)
       Lindane (Pesticide residues in food: 1977 evaluations)
       Lindane (Pesticide residues in food: 1978 evaluations)
       Lindane (Pesticide residues in food: 1979 evaluations)
       Lindane (Pesticide residues in food: 1989 evaluations Part II Toxicology)