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

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.

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 µg/g in brain, 0.5 µg/g in liver, 1 µg/g in kidney, and 2 µg/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 µg/g in brain, liver, and kidney, and 2 µg/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 µg/ml in DMSO; 99.7 Negative^a Murli (1990) ovary cells harvest at 20 h; toxic from 152 µg/m Chromosomal aberration Chinese hamster 25-100 µg/ml in DMSO; 99.7 Negative^b Murli (1990) ovary cells harvest at 10 h; not toxic Chomosomal aberration Chinese hamster 25-305 µg/ml in DMSO; 99.7 Negative^b Murli (1990) ovary cells harvest at 20 h; toxic from 102 µg/ml Cbomosomal aberration Chinese hamster 99.8 and 150 µg/ml in 99.7 Negative^b; Murli (1990) ovary cells DMSO; harvest at 30 h toxin at 150 µg/ml Unscheduled DNA Fischer 344 rat 0.25-15 µg/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 alpha_2µ-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 alpha_2µ-globulin. The authors concluded that lindane induces a protein with an antigenic structure corresponding to alpha_2µ-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 alpha_2µ-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 alpha_2µ-globulin, and staining was scored on a scale from 0-5, from none to markedly severe. Alpha_2µ-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 alpha_2µ-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 (Dési 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 'alpha_2µ-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

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    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

Andre, F., Gillon, J.,André, 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 alpha_2µ-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.