Pesticide residues in food - 2002 - Joint FAO/WHO Meeting on Pesticide Residues

First draft prepared by
E. Mendez
Office of Pesticide Programs, United States Environmental Protection Agency,
Washington DC, USA

Lindane (1alpha,2alpha,3beta,4alpha,5alpha,6beta-1,2,3,4,5,6-hexachlorocyclohexane) is a broad-spectrum organochlorine compound used against a wide range of soil-dwelling and plant-eating insects. It is commonly used on numerous crops, as a seed treatment, in warehouses and to control insect-borne diseases. Lindane is also used in the treatment of scabies and lice in humans. Lindane was last evaluated by the 1997 JMPR (Annex 1, reference 80), when a temporary ADI of 0–0.001 mg/kg bw was established on the basis of deaths and hepatic toxicity in a 2-year study of toxicity and carcinogenicity in rats. The ADI was made temporary because of concern about immunotoxic effects reported in mice given lindane (purity, 97%) at doses of 0.12 mg/kg bw per day and above. Lindane was re-evaluated at the present Meeting within the periodic review programme of the Codex Committee on Pesticide Residues.

Lindane is the gamma isomer of hexachlorocyclohexane. Five other isomers of hexachlorocyclo-hexane are commonly found in technical-grade lindane, but the gamma isomer is the predominant one, comprising at least 99% of the mixture.

1.1 Absorption, distribution, and excretion

Mice

Single doses of [¹⁴C]lindane were administered by gavage to female ICR mice that had been fasted for 12 h at a dose of 1 mg/kg bw (1 µCi of labelled compound). About 54% of the administered dose was absorbed through the gastrointestinal tract within 15 min, and 71% had been absorbed within 1 h. Urinary excretion of the radiolabelled compound reached a maximum of 4.1% of the dose 60 min after administration (Ahdaya et al., 1981).

In a published study, five female C57 Bl/6 and six DBA/2 mice received lindane (purity, 99.9%) at a single oral dose of 20 mg/kg bw by gavage. Blood samples were collected 0, 5, 10, 15, 20, 30, 40, 50 and 60 min after administration to determine the concentration of lindane or its metabolites. A time-dependent increase in the blood concentration of lindane was seen in both strains within 40 min of treatment. After 40 min, the concentration in blood reached a plateau of 500 ng/ml (Liu & Morgan, 1986).

In a study designed to assess the accumulation of residues, groups of five female Swiss mice were given diets containing lindane (purity, 99.8%) at a concentration providing a dose of 1.5 mg/kg bw per day for 4 weeks, 1 mg/kg bw per day for 6 weeks or 3.1 mg/kg bw per day for 2, 4 or 6 weeks. Lindane was measured in whole brain, ovary, adrenal gland, liver, kidney, abdominal fat and femoral muscle. The accumulation of lindane residues in tissues displayed a time- and dose-related increase in all treated groups. For example, the accumulation was greater in animals receiving 3.1 mg/kg bw per day for 6 weeks than in animals given the same dose for shorter periods (2 or 4 weeks). Although the total intake of the compound remained constant at 43 mg, accumulation of lindane residues increased in a time-dependent manner (Table 1), suggesting that a steady state had not been reached. For all treatment groups, the highest lindane content was in fat, followed by brain, kidney, muscle, liver, adrenal and ovary (Lahiri et al., 1990).

Lindane (purity, 99.9%) was administered by gavage to six B6 and six D2 female mice at a dose of 20 mg/kg bw per day for 10 consecutive days. Blood samples were collected daily 1 h after administration or at the time of sacrifice after blood collection on day 10. Brain, liver, spleen and kidneys were collected for determination of lindane, and metabolites were measured in all tissues except brain. In order to determine the time course of lindane concentrations in blood, three additional B6 mice were given lindane at the same dose for 20 days.

Table 1. Lindane content (ppm) of various tissues of mice given diets containing lindane

From Lahiri et al. (1990)

The concentration of lindane in blood increased to a similar extent in the two strains during the first 4 days of the study. Subsequently, however, the concentration was higher in D2 mice than in B6 mice. All of the B6 mice but none of the D2 mice survived to the end of the study on day 10. One death was reported on day 6 of the study, and deaths occurring daily thereafter within 1 h after the last dose; the clinical signs of toxicity at that time included tremors, rapid respiration, spasms and convulsions. At the end of the study, the concentration of lindane in the blood of B6 mice was 41% lower than that in D2 mice. Furthermore, the concentration of lindane in the brains of D2 mice was statistically significantly higher (by 78%; p < 0.001) than in B6 mice, while the concentrations in liver, kidney and spleen were comparable in the two strains. In B6 mice dosed for 20 days, the concentration of lindane in blood continued to increase until the end of the study (Liu & Morgan, 1986).

Groups of 72 F₁ female mice of the obese yellow, lean pseudoagouti and lean black phenotypes, 4 weeks of age, were given diets containing lindane (purity, > 99%) at a concentration of 0 or 160 ppm. Eighteen mice of each phenotype at each dose received a single oral dose of 18 mg/kg bw of [¹⁴C]lindane (55 µCi; specific activity, 82 mCi/mmol; radiochemical purity, 97%) for 13, 26, 52 or 82 weeks, were then transferred to metabolism cages for collection of urine, faeces and expired air for 24 h and were killed 24 h later. Liver, kidney, blood and adipose tissue were collected at necropsy for determination of their radioactivity content.

Mice that had received lindane in their diet before administration of radiolabelled compound (for < 52 weeks; i.e. 56 weeks of age) excreted less radioactivity than concurrent controls, consistent with the reported increase in radioactivity in adipose tissue induced by lindane. Mice aged 86 weeks (treated for 82 weeks), regardless of phenotype, showed an excretion pattern similar to that of concurrent controls, representing 30–47% of the administered dose. In general, obese yellow mice (control and treated) excreted substantially less radioactivity than their black and pseudoagouti counterparts. In tissues, the highest lindane content was that of adipose tissue at all times (Table 2). The main route of excretion was urine, which contained 35–40% of the administered dose, while faeces contained 3–7% (Chadwick & Copeland, 1987).

Table 2. Content of radiolabel in tissues (per cent administered dose per organ [liver, kidney] or per dose [blood, adipose tissue])

From Chadwick & Copeland (1987)

Rats

Six female Holtzman rats were given [¹⁴ C]lindane (purity, 99%) at a single oral dose of 1.7 mg and were killed 24 h later. Urine, faeces, liver, kidneys and adipose tissues were collected. By 24 h after administration, 13% of the administered dose was recovered in the excreta, urinary excretion accounting for 12% and faeces efor only 0.3%. Urine samples were further analysed to ascertain the metabolite profile. Most of the radioactivity recovered in urine was in the form of chlorophenols (both free and conjugated) and polar metabolites; the metabolites included 2,3,5-trichlorophenol, 2,4,6-trichlorophenol and 2,3,4,6-tetracholorophenol. Evaluation of the distribution of radioactivity indicated that adipose tissue accumulated lindane and/or its metabolites to a greater extent than kidney or liver (Chadwick et al., 1977).

The absorption, distribution, metabolism and excretion of [¹⁴C]lindane (radiochemical purity, 98%) were studied in groups of six female Sprague-Dawley rats given a single oral dose of 1.9 mg of lindane with or without pretreatment (in the diet) with 500 ppm Aroclor 1254, 360 ppm phenobarbital or beta-naphthoflavone 1 week before lindane administration. The excretion and distribution pattern of radioactivity were determined 24 h after treatment. Animals that had not been pretreated excreted a total of 22% of the administered dose in urine, faeces and expired air within 24 h. Urinary excretion accounted for 20% of the administered dose, while that in faeces and expired air represented 2% and 0.02%, respectively. Liver, kidneys and adipose tissue were collected from each animal. The highest concentration of radiolabel was found in adipose tissue, accounting for 10% of the administered dose, while the radioactivity detected in liver and kidneys amounted to 0.03% and 0.02%, respectively.

In animals pretreated with either Aroclor 1254 or phenobarbital, 83% and 78% of the administered dose was excreted, respectively, within 24 h, representing a fourfold increase over that in animals given lindane alone. In contrast, pretreatment with beta-naphthoflavone did not significantly alter the pattern of excretion of radioactivity when compared with the control group receiving no pretreatment (Chadwick et al., 1981).

Male Wistar rats received diets containing lindane (purity, 99.99%) at a concentration of 0 or 25 ppm for 6 months, and an additional group of rats received lindane in the diet for 6 months, followed by a 1-month recovery period. After the appropriate treatment period, four rats receiving 25 ppm lindane for 6 months and two rats each receiving 25 ppm lindane for 6 months with a 1-month recovery or basal diet for 6 months were given a single dose of 5 mg/kg bw per day of [³H]lindane (specific activity, 57 mCi/mmol per l) by gavage. The animals were killed 8 h after administration of radiolabelled compound, and samples were collected from the liver, kidney, renal cortex, fat, brain and testes. The highest concentration of radioactivity in the tissue samples was found in the renal cortex (31% of recovered radioactivity), followed by fat, liver, brain, testes and kidneys (Zhu et al., 1986).

In a study to compare the concentration of radioactivity in blood and urine after oral, intraperitoneal and intravenous administration of [¹⁴C]lindane in rats, intragastric administration resulted in rapid absorption, as evidenced by observation of peak concentrations in blood 5–10 h after treatment. Rapid absorption was coupled with rapid elimination from the blood compartment, where no radioactivity was detected 35 h after treatment. Approximately 80% of the administered radioactivity was excreted in the urine within the first 8 days (Sulik et al., 1988).

Six male Wistar rats received lindane (purity unspecified) at a dose of 8 mg/kg bw per day for 10 days, followed by [¹⁴C]lindane at a single oral dose of 90 µg/kg bw (2.5 µCi). The rats were killed 24 or 72 h after administration of the radioactive dose. Urine, blood and faeces and 14 organs (e.g., brain, heart, lung, liver, spleen, kidney, muscle, fat) were collected at necropsy for determination of excretion and radiolabel deposition.

The highest concentration of radioactivity was detected in adipose tissue at 24 and 72 h (37% and 17% of the administered dose, respectively), followed by the kidneys (3.7%) and muscle (3.5%) at the 24-h evaluation period only. The remaining organs examined contained < 1% of the administered radioactivity. Urine contained 31% of the administered radiolabel at 24 h and 46% at 72 h after treatment. Most of the radioactivity recovered in excreta (92% in urine and 57% in faeces) was in the form of conjugated metabolites, while unmodified lindane comprised 3–6% of the administered dose (Seidler et al., 1971).

Lindane (purity, 99.9%) was administered to Chbb:THOM (SPF) rats by gavage at a dose of 15 mg/kg bw per day for 14, 28 or 56 days to investigate the effect of dosing duration on the distribution of lindane. Additionally, the distribution of lindane in blood, brain, liver, kidney and fat tissue was evaluated 5, 8 and 15 days after cessation of administration. The concentration in tissue samples increased during the first 2 weeks of treatment but declined thereafter. Although all the tissues had this pattern of accumulation and elimination, the liver and kidneys showed a more dramatic change in lindane deposition, as evidenced by a 10-fold decrease in radioactivity at the end of the study (day 71) when compared with the value at day 14. This suggests that lindane induces self-metabolism by induction of metabolic enzymes (Eichler et al., 1983).

In a study of the distribution of lindane, groups of five Wistar rats were given diets containing lindane (purity, > 99%) at a concentration of 100 or 800 ppm for 5, 10 or 15 days. Brain, liver, blood, kidney, heart, lung, spleen, muscle and epidydimal fat samples were collected at necropsy to determine the lindane content. In a concurrent set of experiments, the role of nutritional state on the distribution of lindane was evaluated. Animals received lindane for 2 weeks and were then assigned to three groups: one for immediate sacrifice, one given basal diet ad libitum for 1 week, and one given a restricted diet.

The experiments designed to yield information on the tissue distribution of lindane showed that, with the exception of fat tissue, the concentration of lindane did not increase with dose or duration. In the case of adipose tissue, a twofold increase in lindane content was reported in animals at 800 ppm as compared with those given 100 ppm. An increase was also noted with duration of treatment in the groups at 100 and 800 ppm, although the increase at the lower dose was slight.

Food restriction after administration of lindane did not result in a significant redistribution of lindane residues. As would be expected during a period of partial starvation, fat depots were mobilized (Srinivasan & Radhakrishnamurty, 1983).

In a study of the effects of a protein-deficient diet on the distribution of hexachlorocyclo-hexanes in rat tissues, groups of 120 male Druckrey rats received diets containing technical-grade material containing 6.5% lindane at a concentration providing a dose of 50 mg/kg bw per day, for 30 days and were then killed. The animals received a diet containing 5, 10 or 21% casein starting 28 days before initiation of dosing (a diet containing 21% casein is considered to be normal).

The pattern of distribution of lindane to various tissues was affected by the protein content of their diet. The concentrations in adipose tissue and adrenal glands were twofold higher in animals that received a protein-deficient diet (5% casein) than in those receiving a basal diet (21% casein). Animals receiving the basal diet had less overall distribution of lindane (relative to animals receiving the protein-deficient diet) to all tissues examined (kidney, thymus, adipose, adrenals, muscle, lungs, spleen, testes and blood), whereas the liver, heart and brain showed slight increases (Table 3) (Khanna et al., 1995).

Table 3. Effect of dietary protein content on distribution of lindane in tissues of rats

From Khanna et al. (1995)

Six female Sprague-Dawley rats were each given 2 mg of lindane for 6 days and, on day 7, 2 mg of lindane containing [¹⁴C]lindane (1.6 µCi). The rats were then placed in metabolism cages and their urine and feces collected for 24 h and analysed for radioactivity. Within the first 24 h, 58% of the administered dose was found in the excreta (Chadwick & Freal, 1972).

Groups of 36 female Sprague-Dawley rats received diets containing lindane (purity, 99.8%) at a concentration of 0, 130, 220 or 350 ppm. Six rats per dose were killed after 1, 2, 4, 8, 16 and 24 weeks of treatment, 24 h after receiving a single oral dose of lindane (2.1 mg after 1 and 2 weeks and 19 mg thereafter). A dose-dependent increase in the lindane content of fat and liver over that in concurrent controls was reported. However, the hepatic content declined with prolonged treatment. A plausible explanation for this effect is that lindane induces its own metabolism (Copeland & Chadwick 1979).

Lindane (20% emulsifiable concentrate) spiked with [¹⁴C]lindane was applied topically to groups of four male Crl:CD®(SD)BR rats at a dose of 0.1, 1 or 10 mg. Four rats at each dose were bled and killed 0.5, 1, 2, 4, 10 and 24 h after treatment. As the area was not rinsed, the animals were exposed continuously until sacrifice. Urine was collected at the time of sacrifice, and radioactivity was measured in urine, faeces, blood, application site (skin), skin wash, carcass, dose applicator and the cover of the application site.

No clinical signs of toxicity were observed during the study period. The absorption of [¹⁴C]lindane in the group given 0.1 mg ranged from 0.6% after 30 min to 28% 24 h after administration. The percentage of the dose absorbed appeared to decrease with increasing dose. The absorption in animals given 1 mg of lindane ranged from 1% at 30 min to 21% at the 24-h sacrifice, while animals given the highest dose of lindane (10 mg) had an absorption rate of 0.7% at 30 min and 5.1% at 24 h (Bosch, 1987).

Rabbits

Five male New Zealand white rabbits received [¹⁴C]lindane (purity, > 99%) in gelatin capsules twice a week for 26 weeks, resulting in doses of 3 mg/kg bw per day during the first 4 weeks of treatment, 6 mg/kg bw per day in weeks 5–15 and 12 mg/kg bw per day thereafter. Urine and faeces were collected daily for 32 weeks. After a 6-week recovery period (week 32 of the study), the animals were killed, and radioactivity was measured in various tissues. At the end of the dosing regimen (week 26), 54% of the administered radiolabel was recovered in the urine, while the faecal output amounted to 13% of the administered dose. During the recovery period, 3% and 1% of the administered radiolabel was recovered in urine and faeces, respectively. The highest concentration of lindane or its metabolites was found in fat tissue, followed by liver, kidney, muscle and brain (Karapally et al., 1973).

Lindane (20% emulsifiable concentrate containing ¹⁴C-lindane) was administered topically once to groups of four male Hra:(NZW)SPF rabbits at a dose of 0.5, 5 or 50 mg. Four rabbits at each dose were bled and killed 0.5, 1, 2, 4, 10 and 24 h after application. As the area had not been rinsed, they were exposed continuously until sacrifice. Urine was collected at the time of sacrifice, and radioactivity was measured in urine, faeces, blood, application site (skin), skin wash, carcass, dose applicator and the cover of the application site. The only signs of toxicity seen during the study were soft, mucoid faeces in 2/24 animals given 0.5 mg, 6/24 given 5 mg and 1/24 given 50 mg.

The absorption of [¹⁴C]lindane in animals given a topical application of 0.5 mg ranged from 6% 30 min after application to 57% after 24 h. The percentage of the dose absorbed appeared to decrease with increasing dose, as evidenced by absorption of 7–40% (30 min–24 h after exposure) of the dose of 5 mg and absorption of 2–17% (30 min–24 h after exposure) of the dose of 50 mg (Bosch, 1987).

Sheep

In a study designed to examine the potential transfer of lindane across the placenta, lindane (purity unspecified) was administered to groups of four ewes at a dose of 1 or 5 mg/kg bw per day for 10 days at mid-gestation. The ewes lambed 7–11 weeks after initiation of dosing. The concentrations of lindane in blood and fat were assayed at various times. Omental fat from ewes at the two doses contained 10 and 54 ppm lindane, respectively, during the first and second week after administration, while these values declined substantially after lambing, to 0.3 and 0.6 ppm. The concentrations of lindane in lambs (0.2–0.4 ppm) were slightly lower than those in ewes 4 weeks after parturition (Harrison & Mol, 1968).

Goats

Alpine goats were given lindane (purity, 99.8%) containing [¹⁴C]lindane in gelatine capsules at a dose of 1 (two goats) or 10 mg/kg bw per day (one goat) for 4 days. The animals were killed 24 h after the last administration, and radioactivity was measured in tissues, milk, urine, faeces, gut content and expired air. Within 4 days of treatment, 40% of the administered radiolabel had been excreted in the urine, while the faecal output accounted for 6% of the administered dose. The concentration of lindane in milk was 1–2% and reached a plateau after 3 days. It is noteworthy that 85% of the ¹⁴C-labelled residues found in milk were partitioned into fat. As seen in other species, the highest concentration of radioactivity in tissues was detected in adipose tissue (about 2% of the administered dose), followed by liver (Wilkes et al., 1987).

Pigs

Four Durok and Yorkshire boars were given diets containing lindane (purity unspecified) at concentrations providing a dose of 0, 1 or 2 mg/kg bw per day for 21 days. During the last week of the study, the animals were housed in metabolism cages for collection of urine and faeces. At necropsy, liver, kidney, back fat and longissimus dorsi samples were collected for analysis of their lindane content. The highest concentration was found in back fat, 0.19, 20 and 44 ppm being detected after treatment at 0, 1 and 2 mg/kg bw per day, respectively (Davey & Gerrits, 1969)

Groups of three pigs of each sex were given diets containing lindane (purity unspecified) at concentrations providing a dose of 0, 2 or 40 mg/kg bw per day. Blood and back fat were collected at 6-week intervals for determination of the lindane content. During most of the study, the concentrations in blood remained at < 0.01 ppm for animals at both doses. A time- and dose-dependent increase in lindane content was reported in the back fat samples. The highest concentration was detected in adipose tissue, followed by brain, kidney and muscle (Davey & Johnson, 1974).

Cattle

Four milking cows received diets containing lindane (purity unspecified) in gelatine capsules at doses ranging from 0.07 to 6.2 mg/kg bw per day for 70–180 days. The lindane concentration in the milk was measured daily. A dose-dependent increase was reported, the concentration ranging from 0.07 to 10 ppm (Ely et al., 1952).

1.2 Biotransformation

Mice

Five C57Bl/6 and six DBA/2 female mice received lindane (purity, 99.9%) at a dose of 20 mg/kg bw per day once or for 10 consecutive days, and the time course of the blood concentration of the metabolite 2,4,6-trichlorophenol was assayed over 1 h. On day 10 of the study, the animals were killed, and brain, liver, spleen, and kidney samples were collected for determination of metabolites. In animals given the single oral administration of lindane, the blood concentration of 2,4,6-trichlorophenol increased in both strains up to 100 ng/ml within 1 h. Two principal metabolites were identified in the blood after repeated oral administration of lindane: 2,4,6-trichlorophenol and 2,3,4,6-tetrachlorophenol, the former predominating (Liu & Morgan, 1986).

Rats

The synthesis of chlorinated phenols resulting from metabolism of lindane was examined in eight male Wistar rats that received a single oral dose of lindane (purity unspecified) at 0 or 68 mg/kg bw. Urine samples were collected daily for 4 days after treatment and analysed for lindane metabolites. In addition, the conjugation rates of metabolites were investigated. During the collection period, four major metabolites were identified consistently: 2,3-dichlorophenol, 2,4,6-trichlorophenol, 2,4,5-trichlorophenol and 2,3,5,6-tetrachlorophenol. The degree of conjugation was extensive, particularly during the first 24 h after administration of the test article. The conjugation rate ranged from 72% for 2,4,6-trichlorophenol to 42% for 2,4,5-trichlorophenol and declined in a time-dependent fashion. Under the conditions of this study, 2,3,5,6-tetrachloro-phenol was the predominant metabolite (1–2 µmol/l), followed by 2,4,6-trichlorophenol (0.5–1.5 µmol/l), 2,3-dichlorophenol (0.25–1 µmol/l) and 2,4,5-trichlorophenol (0.1–0.5 µmol/l) after 2–4 days. The phenolic metabolites were excreted primarily in the urine within 24–48 h of administration (Baliková et al., 1989).

The profile of lindane metabolites in the brain was investigated in male Wistar rats given lindane (purity unspecified) at a single dose of 0, 30 or 60 mg/kg bw. Animals receiving the lower dose were killed 5 h after treatment, while those given the higher dose were killed 24 h after treatment. Brain samples were collected, and homogenates were analysed for the presence and identity of metabolites. The predominant metabolite was 2,4,6-trichlorophenol. After administration of 60 mg/kg bw, six major metabolites (1,2,3,5-tetrachlorobenzene, 1,2,4,5-tetrachlorobenzene, 1,2,3,4-tetrachlorobenzene, 2,3,4,5,6-pentachlorocyclohexene, pentachlorobenzene, 3,6/4,5-hexachlorocyclohexene) and lindane were detected. The metabolites in the cerebella of animals at the lower dose were investigated. In this structure, metabolism occurred primarily through dehydrochlorination, resulting in 3,6/4,5- and 3,5/4,6-pentachlorocyclohexene. Hexachlorobenzene was detected in all samples at concentrations of 0.5–1 ppb (Artigas et al., 1988).

Six female Holtzman rats received 1.7 mg of lindane (purity unspecified) containing 1.5 µCi [¹⁴C]lindane. Urine samples were collected 24 h later and analysed for metabolite content, and the animals were killed. Liver samples were homogenized and analysed for microsomal enzyme activity in vitro. Of the radioactivity excreted in the urine 24 h after treatment, only 4.4% was identified as neutral metabolites, while 26%, 27% and 45% of the radioactivity was associated with free, conjugated and polar metabolites, respectively. The predominant phenols excreted in the urine were 2,4,6-trichlorophenol and 2,3,4,6-tetrachlorophenol, in a ratio of 2.7 (Chadwick et al., 1977).

Groups of male Wistar rats received lindane (purity unspecified) or its metabolites gamma-2,3,4,5,6 pentachlorocyclohexene, pentachlorobenzene and pentachlorophenol at a dose of 8 mg/kg bw per day orally for 19 days At necropsy, blood, liver, kidneys, adrenals, heart, spleen, brain, muscle and intestinal adipose tissue were collected and analysed. Excreta were pooled weekly and analysed with and without glucuronidase treatment.

The metabolites found in urine were pentachlorophenol, 2,3,4,6-tetrachlorophenol, 2,3,5,6-tetrachlorophenol and 2,4,6-trichlorophenol, as well as lindane. Only lindane was identified in faeces. The pattern of metabolites in blood paralleled that in urine, while the metabolites in liver consisted of gamma-pentachlorocyclohexene, pentachlorophenol, 2,3,4,6-tetrachlorophenol and 2,3,5,6-tetrachlorophenol. The predominant metabolite varied among the organs examined. While gamma-pentachlorocyclohexene was the predominant metabolite in kidneys, that in spleen was pentachlorophenol, those in the heart were 2,4,6-trichlorophenol, 2,3,4,6-tetrachlorophenol and gamma-pentachlorocyclohexene, and gamma-pentachlorocyclohexene was the only metabolite identified in brain. Glucuronidase treatment showed that trichlorophenols, 2,3,4,6-, 2,3,5,6- and 2,3,4,5-tetrachlorophenol and pentachlorophenol were conjugated, although not extensively (Engst et al., 1976).

Lindane (purity unspecified) was administered to six female Fischer 344 rats at a dose of 2 mg for 13 days, and on day 14 [¹⁴C]lindane (2 µCi) was added to the dose. Urine and faeces were collected daily and assayed for metabolite content. Urine contained increasing concentrations of glucuronic acid conjugates from day 4 of treatment; sulfate conjugation appeared to be slightly inhibited during the first 5 days of treatment but increased to the level in concurrent controls thereafter (Chadwick et al., 1971).

Six female Sprague-Dawley rats received 0 or 2 mg of lindane (purity unspecified) orally for 6 days and 2 mg of lindane containing 1.6 µCi [¹⁴C]lindane on day 7. Urine samples were collected at 24-h intervals throughout the study and analysed for metabolite content. In general, the pretreated animals excreted more radioactivity than concurrent controls and excreted substantially more trichlorophenols (65%, with 30% in controls). On day 8, the tetrachlorophenols excreted by controls consisted of approximately 25% 2,3,4,6-tetrachlorophenol, while pretreated animals excreted 45% (Chadwick & Freal, 1972).

Three novel lindane metabolites were identified in female Sprague-Dawley rats given diets containing lindane (purity unspecified) at a concentration of 400 ppm for 1 month. Urine samples were collected daily and analysed for lindane metabolites after acidification and extraction. Analysis of the urinary metabolite profile resulted in the identification of 2,3,4,5,6-pentacychlorocyclohexene-(2)-ol-1, 2,3,4,6-tetrachlorocyclohexenol and 2,4,5,6-tetrachlorocyclo-hexenol. These metabolites were excreted primarily as sulfate and glucuronide conjugates (Chadwick et al., 1978).

Rats received lindane (purity unspecified) at an oral dose of 40 mg/kg bw per day on days 1, 3 and 5, and urine and faeces were collected daily until study termination on day 7 and analysed for metabolites. Hexachlorobenzene was detected exclusively in the faeces. Formation of hexachlorobenzene is the result of sequential dehydrogenation of lindane (Gopalaswamy & Aiyar, 1986).

Humans

Human metabolism of lindane was investigated during biomonitoring of lindane production and forestry workers, by analysing the urine of workers potentially exposed to technical-grade material. The analysis revealed the presence of alpha-, beta-, gamma- and delta-hexachlorocyclohexane, traces of hexachlorobenzene and pentachlorobenzene, gamma- and delta-pentachlorocyclohexene, pentachlorophenol, 2,3,4,5-, 2,3,4,6- and 2,3,5,6-tetrachlorophenol, several trichlorophenols as well as glucuronides of the aforementioned metabolites. In addition, pentachlorocyclohexanes, tetrachlorophenol, hexachlorobenzene and pentachlorobenzene were identified in blood samples (Engst et al., 1978).

During biomonitoring, the urine of workers involved in the production of gamma-hexachlorocyclo-hexane (purity, 99.8%) from technical-grade material was examined. The major metabolites identified were 2,4,6-, 2,3,5- and 2,4,5-trichlorophenol, which were excreted in urine in equal proportions (Angerer et al., 1983).

Figure 1 shows the proposed metabolism of lindane.

Figure 1. Major steps in the biotransformation of lindane

DCB, dichlorobenzene; DCP, dichlorophenol; HCB, hexachlorobenzene; HCCH, hexachlorocyclohexene;
HCCOL, hexachlorocyclohexenol; alpha-HCH, alpha-1,2,3,4,5,6-hexachlorocyclohexane; gamma-HCH,
gamma-1,2,3,4,5,6-hexachlorocyclohexane; PCB, pentachlorobenzene; gamma-PCCH, gamma-2,3,4,5,6-pentachlorocyclohexene;
PCCOL, 2,3,4,5,6-pentachlorocyclohexene-(2)-ol-(1); PCP, pentachlorophenol; TCB, trichlorobenzene; TCP, tricholorophenol;
TeCCH, 3,4,5,6-tetrachlorocyclohexene; TeCCOL, tetrachlorocyclohexenol; TeCB, tetrachlorobenzene; TeCP, tetrachlorophenol;
PG, glucuronide conjugate; PMA, sulfate conjugate

1.3 Effects on enzymes and other biochemical parameters

The effects of lindane on drug metabolizing enzymes were investigated in rodents. In an attempt to ascertain if various strains and species had different enzyme activities, CF1 and B6C3F₁ mice and Mendel rats received diets containing lindane (purity unspecified) at concentrations of 50–300 ppm for 3 days or 3 months. The animals were killed after dosing and the livers collected for assay of the specific activities of enzymes implicated in lindane metabolism.

In all three rodent strains, the basal glutathione-S-transferase activity was higher in males than in females. This difference in enzyme activity did not appear to correlate with susceptibility to lindane. In comparison with the other strains investigated, CF1 mice had the highest glutathione-S-transferase activity. Furthermore, female CF1 mice showed five- to sixfold induction of this enzyme after treatment with lindane at the highest dose (300 ppm). Treatment of animals with high doses of lindane induced more UDP-glucuronosyl transferase activity in rat liver microsomes than in mouse liver microsomes. Enhanced activity of this enzyme could result in an increase in the conjugation of phenol metabolites derived from lindane. In contrast, CF1 mice had more monooxygenase activity than rats, regardless of treatment. CF1 mice given lindane, however, had less epoxide hydrolase activity than rats. Coupled with the high monooxygenase activity, the reduction in epoxide hydroxylase activity could result in accumulation of the reactive epoxides produced during lindane metabolism (Oesch et al., 1982).

The potential cytotoxic and cell-transforming effects of lindane on BALB/c 3T3 cells were studied in vitro in the presence and absence of an exogenous metabolic activation system obtained from rats treated with phenobarbital. To determine the cytotoxic concentration, the cells were exposed to 10, 50, 100 and 200 µg/ml of lindane (purity, 99%), while doses of 10, 50 and 100 µg/ml were used to test for transformation.

In the absence of metabolic activation, lindane was not cytotoxic at any dose. However, in the presence of the metabolizing system, a dose-dependent increase in cytotoxicity was observed. Moreover, lindane showed statistically significant, dose-dependent cell transformation capacity at all doses tested, regardless of metabolic activation. This result suggests that lindane may act as a promoter in carcinogenesis (Perocco et al., 1995).

In a study designed to compare the formation of chlorophenol metabolites from hexachlorocyclohexane, male Swiss mice and female Wistar rats received a single intraperitoneal injection of 500 mg/kg bw Aroclor 1254 and were killed 5 days later. Microsome fractions were prepared from the liver to determine the extent of formation of chlorophenol metabolites after addition of lindane to the microsome fractions. In both rodent species, 2,6-dichlorophenol, 2,3,5-, 2,3,6- and 2,4,6-trichlorophenol, 2,3,4,5- and 2,3,5,6-tetrachlorophenol and pentachlorophenol were produced. While the rat microsome preparation produced more 2,4,6-trichlorophenol than that of the mice, the two species produced comparable amounts of 2,6-dichlorophenol (the predominant metabolite) (Munir et al., 1984).

The potential role of a hepatic microsomal mixed-function oxidase system in lindane dehydrogenation was evaluated in vitro. Female Sherman and Sprague-Dawley rats were pretreated with 2 mg of DDT (to stimulate lindane metabolism) for 2 weeks. They were then killed, and the livers were used to prepare microsomal fractions. Microsomal fractions from untreated rats were used as concurrent controls. Lindane was incubated with the microsomal fractions and NADPH-generating systems in the presence and absence of inhibitors. Molecular oxygen and reduced pyridine nucleotide coenzyme were required to attain maximum lindane dehydrogenation. Inhibition by SKF 525-A and carbon monoxide implicated the cytochrome P450 system in the dehydrogenation process. Conversely, the failure of cyanide to inhibit dehydrogenation suggested that the cytochrome b desaturase system is not involved. It was shown that DDT could enhance dehydrogenase activity (Chadwick et al., 1975).

The saturable, dose-dependent nature of lindane metabolism was studied by incubation of lindane with rat liver microsomes in vitro. The dehydrogenation of lindane to hexachlorocyclohexene and further hydroxylation to 2,3,4,6-tetrachlorocyclohexenol showed non-linear increases with dose and a significant non-linear decrease with time, indicating that the metabolism of lindane is both saturable and dose-dependent (Copeland, 1985).

In a study designed to examine the metabolism of lindane by liver microsomes, male Wistar rats were given phenobarbital, 3-methylcholanthrene, cobalt chloride, SKF 525-A or piperonyl butoxide. Rats given saline solution were used as controls. The rats were killed after treatment, and their livers were used to prepare a microsomal fraction which was subsequently incubated with lindane in the presence of NADPH.

The metabolism proceeded by dehydrogenation, dehydrochlorination and dechlorination. Inhibition by SKF 525-A, piperonyl butoxide, N₂ or the absence of NADPH indicated that cytochrome P450 was involved in this reaction. This assertion was further supported by the fact that cyanide had no effect on dehydrogenation. Furthermore, the ability of phenobarbital to induce dehydrogenation suggests that the cytochrome b system is not involved in this reaction. Conversely, dehydrochlorination was inhibited by N₂, carbon monoxide, piperonyl butoxide, potassium cyanide and the absence of NADPH, but not by SKF 525-A. In fact, SKF 525-A and cobalt chloride induced this reaction, while phenobarbital had no effect, suggesting that a specific species of cytochrome P450 and the cytochrome b5 system or another enzyme system are responsible for the dehydrochlorination of lindane (Yamamoto et al., 1983).

The metabolism of lindane by human liver microsomes was studied by incubating them with lindane and an NADPH generating system. The microsomes metabolized lindane to four major metabolites: gamma-hexachlorocyclohexene, gamma-pentachlorocyclohexene, beta-pentachlorocyclohexene and 2,4,6-trichlorophenol. The two major secondary metabolites identified were 2,3,4,6-tetrachlorophenol and pentachlorobenzene (Fitzloff et al., 1982).

In a study conducted to examine the anaerobic metabolism of lindane, rat liver microsomes were prepared and incubated with lindane in the presence of NADPH and nitrogen. Under the conditions of this study, lindane was dechlorinated to 3,5,6/5-tetrachlorocyclohexene, which may be an intermediate between lindane and 4-chloromercapturic acid, thereby indicating that dechlorination has a pivotal role in lindane metabolism in vivo (Kurihara et al., 1979a).

The formation of mercapturic acid in rats exposed to lindane was evaluated in vitro by administering lindane (purity unspecified) intraperitoneally at a dose of 17 or 34 µmol. A crude soluble enzyme fraction was obtained from the livers of the animals and incubated with glutathione. Under the conditions of this study, glutathione conjugation occurred directly on polychlorocyclohexenes. While most polychlorocyclohexenes are metabolized similarly in vivo and in vitro, hexachlorocyclohexene might be dechlorinated and dehydrochlorinated before glutathione conjugation in the cytosol (Kurihara et al., 1979b).

2.1 Acute toxicity

Studies of the acute toxicity of lindane are summarized in Table 4. Mice given lindane orally showed clinical signs indicative of effects on the central nervous system, including but not limited to hypoactivity, dyspnoea, ataxia and convulsions. Rats given lindane showed similar signs of toxicity within 30 min of treatment.

Table 4. Acute toxicity of technical-grade lindane

Lindane of > 99 % purity was used in all studies.

Rats to which lindane was applied dermally at the LD₅₀ showed signs of toxicity including dyspnoea, hunched posture and hypoactivity. Hypoactivity, hunched posture and emaciation were observed in rats exposed to lindane by inhalation. In mice and rats treated intraperitoneally with lindane, hypoactivity, staggering, tremors and dyspnoea were observed. The signs of toxicity shown by mice after intramuscular administration of lindane included hypoactivity and staggering.

2.2 Short-term studies of toxicity

Mice

In a 14-week study of toxicity, groups 45 CD1 mice of each sex were exposed (whole-body) to lindane (purity, 99.6%) by inhalation at a concentration of 0, 0.3, 1 or 10/5 mg/m³ for 6 h on 5 days/week. Owing to a high mortality rate, the high concentration was reduced from 10 to 5 mg/m³ at the beginning of the second week. The particle size achieved for the dust was 3.2 µm, with a geometric standard deviation of 1.7. Groups of 15 mice of each sex at each dose were killed during week 7 of the study, at the end of exposure in week 14 and at the end of the recovery period in week 20.

A substantial number of deaths occurred among animals at 10 mg/m³ during the first week of the study (12/45 females and 2/45 males), and three males and three females died after the concentration had been lowered to 5 mg/m³. No clinical signs of toxicity, changes in ophthalmic parameters or consistent changes in body weight were reported during the study. Females showed dose-dependent increases in glucose concentration, which were first detected during week 7 and attained statistical significance (p < 0.001) in those at 5 mg/m³ by week 20 of the study (177% of control). At this time, females at concentrations > 1 mg/m³ also showed a statistically significant increase in blood urea nitrogen (133% and 144% of control at 1 and 5 mg/m³, respectively). An increase in urinary potassium concentration was reported in animals at the highest concentration.

Bone-marrow myelograms from males exposed to 5 mg/m³ showed significant increases in sternum megarubricytes, total erythrocyte series, total eosinophils and myeloid:erythrocytic ratios as well as decreases in progranulocytes during week 7. Females at this concentration for 7 weeks showed decreased eosinophilic myelocytes in femoral smears and decreased eosinophilic metamyelocytes in sternum smears. During week 14, several parameters were affected in both males and females, including decreases in lymphocyte counts, increased total lymphocyte cell counts in femoral smears and increased rubiblast and polychromatophilic cell counts in both sternum and femoral smears. Gross necropsy of animals that were killed on schedule showed no remarkable findings. Males that died intercurrently had an increased incidence of kidney lesions (cysts, colour changes, hydronephrosis and increased size), distension of the urinary bladder and distension of the urethra in those at the highest concentration.

Males at the highest concentration showed histological evidence of fibronecrotic thymus regions and mediastinitis in week 7, ceroid degeneration of the adrenal gland cortex (3/10 and 1/10 control) and nodular cortical-cell hyperplasia in week 14. At the lowest concentration, 3/9 males had fibronecrotic thymus regions and mediastinitis. No treatment-related effects were seen in females killed in week 7, but females exposed to the highest concentration and killed in week 14 had a statistically significant increase in the incidence of spindle-cell hyperplasia in the adrenal glands (71%, with 10% in controls).

The absolute weight of the testes and those relative to body weight and brain weight were increased in animals at the highest concentration that were killed at interim sacrifice. Females at concentrations > 0.3 mg/m³ that were killed in weeks 7 and 20 showed dose-dependent increases in thymus weights. The NOAEL was 0.3 mg/m³, equivalent to 0.003 mg/l, on the basis of increased thymus weight, mediastinitis and fibronecrotic thymus regions (Klonne & Kintigh, 1988).

Rats

In a 28-day range-finding study, groups of 15 Wistar rats of each sex received diets containing lindane (purity, 99.5%) at a concentration of 0, 1, 10, 100 or 400 ppm and were observed twice daily for clinical signs of toxicity or death. Individual body weights, food consumption and food use efficiency were assessed at the beginning of the study and weekly thereafter. Haematological and clinical chemical parameters were evaluated only at the end of the study.

No deaths were reported during the study. An increased incidence of convulsions was the only clinical sign of toxicity observed and was found only in females at the highest concentration. A statistically significant decrease in body-weight gain was seen in females (by 56%) and males (by 25%) at this concentration, and a slight decrease in body-weight gain (8.5%) were seen in females at 100 ppm. Males at concentrations > 100 ppm had a higher urinary output with lower specific gravity and pH than controls, but the effects were inconsistent. Statistically significant (p < 0.05 or p < 0.001) decreases in haemoglobin concentration, erythrocyte count and packed cell volume were reported for males and females at 400 ppm, and males at concentrations > 100 ppm had increased (p < 0.05) platelet counts from the third week of the study. The clinical chemical parameters affected by lindane at 400 ppm included marginal increases in phosphorus, calcium, cholesterol and urea concentrations and a decreased albumin:globulin ratio in both males and females.

The concentration of lindane in serum, liver, kidney and brain, measured at the time of sacrifice, showed a dose-dependent increase in all tissues. Females at dietary concentrations > 100 ppm had higher concentrations in the brain than males; however, males had higher renal concentrations of lindane than females at all dietary concentrations. Pale kidneys and increased absolute and relative weights of the kidney were reported in males at concentrations > 100 ppm. At the highest concentration, males and females had increased absolute and relative weights of the liver and females had an increase in the relative spleen weight. Males given lindane showed a dose-dependent increase in the severity of hyaline droplet accumulation in the renal proximal tubule, which led to interstitial chronic nephritis and necrosis coupled with tubule regeneration at concentrations > 100 ppm. Changes in the histological appearance of the livers of animals that died both during the study and at scheduled sacrifice consisted of an increased incidence of periacinar hepatocytic hypertrophy in males at 100 and 400 ppm (2/10 and 10/16, respectively, with 0/10 controls) and in 12/12 females at 400 ppm (0/10 in controls). The NOAEL was 10 ppm, equal to 0.98 mg/kg bw per day, on the basis of a statistically significantly increase in the incidence of periacinar hepatocytic hypertrophy in males and increased platelet counts at 100 ppm, equal to 9.6 mg/kg bw per day (Amyes, 1990).

In a 6-week study, groups of five Crl:CD(SD)Br rats of each sex received diets containing lindane (purity, 99.6%) at a concentration of 0, 80, 200, 400 or 800 ppm. Animals were observed daily for signs of toxicity and twice a day for deaths or moribundity. Body weights, food consumption and food use efficiency were assessed at the beginning of the study and weekly thereafter. At the end of the study, the animals were killed and necropsied grossly. The adrenals, kidneys, liver and testes were weighed, and the kidneys, liver and gross pathological lesions were examined histologically.

Two females at 800 ppm died during the study, but the cause of death could not be determined as the only sign of toxicity was rough fur. Dose-dependent decreases in body-weight gain were reported for males, reaching a maximum (decrease of 15% in comparison with concurrent controls) in those at 800 ppm. The decreases in body-weight gain were paralleled by decreases in food consumption (9–15% lower than control) throughout the study. The absolute and relative weights of the kidneys were increased in a dose-dependent manner in males; females also showed an increased relative kidney weight, although the increase in males was more dramatic (36% increase in males, 5% in females at 800 ppm). The absolute and relative weights of the liver increased in a dose-dependent manner in males and females. The increases in relative liver weight reached 40% and 57% and attained statistical significance in males and females at 800 ppm, respectively. Gross necropsy revealed no concentration-related abnormalities. Hyaline droplet nephropathy was observed at all concentrations only in males. Liver hypertrophy and leukocyte foci were reported almost all treated animals. Males at concentrations > 200 ppm showed an increased incidence of mottled kidneys, and animals of each sex showed an increased incidence of mottled livers. The NOAEL was 80 ppm, equivalent to 8 mg/kg bw per day, on the basis of an increased incidence of hepatotoxicity and mottled kidneys at 200 ppm, equivalent to 20 mg/kg bw per day (Jones, 1988).

Groups of 20 Wistar rats of each sex received diets containing lindane (purity, 99.85%) at a concentration of 0, 0.2, 0.8, 4, 20 and 100 ppm for 90 days. Five rats of each sex per dose were maintained on basal diet for 6 weeks after the end of the dosing period for a recovery phase. Animals were observed twice a day for signs of toxicity and deaths, with comprehensive examinations (including palpation) and measurements of body weight and food consumption weekly. Ophthalmic and auditory evaluations were conducted before treatment, in week 12 and at the end of the recovery period. Haematological, clinical chemical and urinary analyses were carried out before initiation of the study, in weeks 6 and 13 and at the end of the recovery period. Brain cholinesterase activity was assessed in five rats of each sex per dose at the end of treatment. Cytochrome P450, N-demethylase, and carboxylesterase activities were measured in 10 rats of each sex per dose at the end of treatment and in five rats of each sex per dose after the recovery phase. All animals that died intercurrently or were killed on schedule were examined grossly and histologically. All gross lesions and 33 organs or tissues were examined microscopically, and the adrenal glands, brain, ovaries, testes, heart, kidneys, liver and thyroid glands were weighed.

One death was reported in the group at 4 ppm, which was considered to be incidental to treatment. No clinical signs of toxicity or changes in body weight, food consumption, food use efficiency or ophthalmic or auditory parameters were reported. No consistent changes in haematological or urine end-points were recorded. A statistically significant, dose-dependent, 12–26% increase in urea concentration was reported in males at concentrations > 4 ppm in weeks 6 and 13, but this effect was not observed after the 6-week recovery period. Brain cholinesterase activity was not affected by treatment. Animals at the highest concentration showed increased activity of cytochrome P450, while N-demethylase activity was unaffected. The cytochrome P450 activity had returned to control values by the end of the recovery period.

A pattern of diffuse renal discolouration was observed in all males at concentrations > 20 ppm, severity increasing in a dose-dependent manner. This finding was correlated with hyaline droplet accumulation, tubule degeneration and distension, and an increased incidence of interstitial nephritis. The tubule-cell degeneration had resolved by the end of the recovery period. The absolute weight of the liver was slightly but statistically significantly increased in males at 100 ppm (by 13%; p < 0.05), while females showed a 10% increase (p < 0.05) at concentrations > 20 ppm. A statistically significant (p < 0.05 or 0.01) increase was found in the relative (to body weight) weights of the liver in males at 20 and 100 ppm (10 and 14%, respectively) and females at 100 ppm (10%; p < 0.01). These increases were consistent with the observations made during histopathological examination, in which a dose-related (but reversible) increase in the incidence and severity of hepatocellular hypertrophy was found in both sexes at 4, 20 and 100 ppm (2/30, 14/30 and 21/30 animals, respectively). The NOAEL was 100 ppm, equal to 7.6 mg/kg bw per day, the highest dose tested (Suter, 1983).

Lindane (purity, 99.9%) was administered by inhalation to groups of 12 male and 12 female Wistar rats at a nominal concentration of 0, 0.02, 0.1, 0.5 or 5 mg/m³ for 6 h/day for 90 days. Additional groups of 12 rats of each sex at 0 and 5 mg/m³ were treated for 90 days and allowed to recover for 6 weeks before they were killed. The analytical atmospheric concentrations were 0, 0.02, 0.12, 0.6 and 4.5 mg/m³, respectively. The arithmetic mean particle size of the aerosol was 1.1 ± 0.39 µm, and the geometric mean was 1.0 ± 1.4 µm. The animals were observed for deaths, signs of toxicity and food and water consumption once a day on 5 days/week. Haematology, clinical chemistry, urine analysis and measurements of cholinesterase inhibition and enzyme induction were conducted before, during and after exposure. The concentration of lindane was measured in the liver, brain, serum and peritoneal fat of five rats of each sex per dose at the end of the treatment and recovery periods. Gross and histological (31 organs or tissues) examinations were conducted on all animals at the end of treatment and recovery.

Lindane was detected in brain, liver, fat and serum of all treated rats. The chemical accumulated in fat, reaching concentrations of 130 mg/g and 58 mg/g in females and males at the highest dose, respectively. After the recovery period, traces of lindane were still detectable in these tissues. All rats survived to scheduled sacrifice. ‘Slight’ diarrhoea and piloerection were observed in all males and females exposed to the highest concentration, beginning during week 3 of exposure and persisting until day 41 of the study. No exposure-related effects were seen on body-weight gain, food or water consumption or urine parameters. Although haematological parameters did not appear to be affected by treatment, data for individual animals were not provided, and the statistics could not be verified. The clinical chemical results, especially for Na⁺, K⁺ and Ca⁺⁺, were highly variable. The cytochrome P450 activity was 340% and 170% of the control values in males and females at 5 mg/m³, respectively, after 90 days but similar to control levels after the recovery period. In animals at lower doses, the cytochrome P450 activity was comparable to that of concurrent controls. Bone-marrow myelograms from animals exposed to 5 mg/m³ showed significantly (p < 0.05) increased reticulocyte (+110%), stem cell (+31%) and myeloblast (+33%) counts in males, increased reticulocyte count (+55%) in females and decreased lymphocyte count (–45%) in females. No dose–response relationship could be established for these changes, however, as bone marrow from the other groups was not assayed.

Males exposed to 5 mg/m³ had significantly (p < 0.05 or 0.01) increased absolute (+7.8% to +12%) and relative (+19%) kidney weights as compared with controls, and the absolute and relative kidney weights of males exposed to 0.5 mg/m³ were increased by 8–9.8% and 6.9–8.2%, respectively, which were considered to be biologically significant even though they are not statistically significant. After the recovery phase, the kidney weights of exposed males were similar to those of controls. In females exposed to 5 mg/m³, the absolute and relative kidney weights were increased (p < 0.05) by 9.2–9.9% and 7.9–8.2%, respectively, as compared with controls. A statistically significant increase in relative testis weight was also reported at all doses, but the toxicological relevance of this observation is uncertain, as the absolute testis weight was statistically significantly increased (p < 0.01) only at the low concentration and no compound-related changes were seen in the histopathological evaluation. The absolute liver weights of males at the highest dose were not affected, but the relative liver weights were slightly (6.9%) higher than those of controls. In females at the highest dose, the absolute and relative liver weights were 12% and 11% higher, respectively, than those of controls. No differences in absolute and relative liver weights were seen between the exposed and control groups after the recovery period.

Kidney lesions were observed in 17% of control males, none of those at 0.02 mg/m³, 25% of those at 0.1 mg/m³, 83% of those at 0.50 mg/m³ and 82% of those at 5 mg/m³. The lesions included cloudy swelling of the tubule epithelium, dilated renal tubules containing protein and proliferated tubules. After the recovery phase, only cloudy swelling of the tubule epithelium was observed in two control animals and one at the highest concentration The NOAEL was 0.5 mg/m³, equal to 0.12 mg/kg bw per day, on the basis of diarrhoea, piloerection and changes in the bone-marrow myelogram at 5 mg/m³ (Hertel et al., 1983).

Rabbits

Groups of 40 male and 40 female New Zealand white rabbits received dermal applications of lindane (purity, 99.5%) in 5% aqueous carboxymethyl cellulose at a dose of 0, 10, 60 or 400 mg/kg bw per day under occlusion for 6 h/day, 5 days/week for 13 weeks. Owing to excessive toxicity, the highest dose was reduced to 350 mg/kg bw per day in week 9 and to 320 mg/kg bw per day from week 11 until the end of the study. Within each group, 10 animals of each sex per group were killed at week 6, 20 were killed at 13 weeks and 10 were dosed for 13 weeks and allowed a 6-week recovery.

Tremors and convulsions were observed in animals at the highest dose, from day 16 in males and day 19 in females. One female at 60 mg/kg bw per day showed these clinical signs on day 50 only. Clinical signs of toxicity were not observed in animals at the lowest dose. Reactions at the site of application were not reported. Of animals at the highest dose, 17 males and eight females died before scheduled sacrifice. Deaths were first observed after week 5. All animals at lower doses survived to scheduled sacrifice.

The body weights and body-weight gains of animals at the two lower doses were similar to those of controls throughout the study. Males and females at the highest dose began to lose weight after the first week of the study, so that their absolute body weights were 3–7% and 3–10%, respectively, lower than those of controls during the 13 weeks of treatment. During recovery, the body weights of the males remained 3–8% lower, while those of females were only 1–3% lower than those of controls. The data on body weights were not analysed statistically. The body-weight loss by rabbits at the highest dose correlated with generally reduced food consumption during treatment.

No treatment-related effects were observed on ophthalmic, urine or leukocyte parameters. Alkaline phosphatase activity was significantly increased in females at the highest dose at interim sacrifice (+34%; p < 0.05), and in males (+44%; p < 0.01) and females (+53%; p < 0.01) at terminal sacrifice. Females at this dose also had significantly increased gamma-glutamyl transferase activity (+38%; p < 0.01) at terminal sacrifice. Males at the highest dose showed significant (p < 0.05 or 0.01) reductions in haemoglobin concentration (–7%), erythrocyte count (–8.6%) and packed cell volume (–5.7%) at terminal sacrifice, but these erythrocyte parameters were comparable to those of controls after recovery and were not affected in females.

At terminal sacrifice, males and females at the highest dose had slightly increased absolute kidney weights and significantly (p < 0.01) increased relative kidney weights as compared with controls. The absolute and relative kidney (left and right) weights were increased by 104–106% and 112–114%, respectively, in males and 105–106% and 115–116%, respectively, in females. Females at the highest dose also had significantly (p < 0.01) increased absolute (+27%) and relative (+30–45%) liver weights at both sacrifices, which remained slightly (+13–17%) elevated after recovery. The relative liver weights were significantly (+37%; p < 0.01) increased in males at the highest dose at terminal sacrifice. The absolute adrenal weights (left and right) were significantly (p < 0.05 or 0.01) increased at terminal sacrifice in males at the intermediate dose (+20–23%) and highest dose (+40–46%) and in females at the highest dose (+33–34%). The relative adrenal weights were increased (p < 0.05 or 0.01) by 19–22% in males at the intermediate dose and by 46–57% in males and females at the highest dose. After the recovery period, the organ weights of the treated groups were similar to those of controls.

No treatment-related gross or histopathological lesions were observed in the kidneys, adrenals or skin. The incidence and severity of centrilobular hypertrophy of the liver was increased in males and females at the two higher doses at all sacrifices. At both the interim and terminal sacrifices, centrilobular hypertrophy was observed in 20% of males and 25–30% of females at the intermediate dose and 80–100% of males and 73–90% of females at the highest dose. After recovery, this lesion was seen in 30% of males and 40% of females at the intermediate dose and in 50% of males and 29% of females at the highest dose. The NOAEL was 10 mg/kg bw per day on the basis of lesions in the liver and increased adrenal weights in males at 60 mg/kg bw per day (Brown, 1988).

Dogs

In a 90-day study, groups of four beagle dogs of each sex received diets containing lindane (purity, > 99%) at a concentration of 0, 25, 50 or 100 ppm. The animals were observed daily for signs of toxicity and deaths. The haematological parameters evaluated at the end of the study included erythrocyte count, haemoglobin concentration, mean corpuscular volume, mean corpuscular haemoglobin, mean corpuscular haemoglobin concentration, total leukocyte count, reticulocyte count, platelet count, thromboplastin time and partial thromboplastin time. The clinical chemistry included measurements of glucose, urea, creatinine, total cholesterol, total bilirubin, alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, lactate dehydrogenase, total protein and electrolytes. Urine was also analysed.

No changes were found in the parameters evaluated. The NOAEL was 100 ppm, equivalent to 1 mg/kg bw per day, the highest dose tested (Noel et al., 1969).

Groups of four beagle dogs of each sex received diets containing lindane (purity, > 99%) at a concentration of 0, 25, 50 or 100 ppm for 104 weeks. The animals were observed daily for signs of toxicity and deaths; food consumption was measured twice a day and body weights were recorded weekly. Ophthalmic evaluations were performed before dosing and at 1, 3, 6, 12 and 24 months. Haematology, clinical chemistry and urine analysis were conducted twice before initiation of dosing and at 1, 3, 6, 12 and 24 months. At the end of the study, all animals were examined grossly and histologically (34 tissues or organs), and 15 organs were weighed. The concentrations of lindane were determined in fat, liver and brain samples from all animals.

One animal at the highest concentration died 393 days after the second of two brief convulsive episodes occurring 200 days apart. Each episode was followed by rapid recovery. Convulsions in 1/4 female controls and 2/4 males at 25 ppm were the only clinical sign of toxicity seen during the study. These observations are considered incidental, as similar effects were not seen at higher doses. Body weights, food consumption, urine parameters and electroencephalograms were unchanged. The only haematological change was a dose-dependent increase (> 17%) in platelet count in animals at concentrations > 50 ppm during week 4. In terms of clinical chemistry, a statistically significant increase in alkaline phosphatase activity (by 36–53%) was found in animals at 100 ppm from week 25, which persisted until the end of the study. Alkaline phosphatase activity increased in a time-dependent fashion.

At gross necropsy, an increased incidence (3/8 animals) of enlarged liver was reported in dogs at 100 ppm, and the animals in this group had darker, more friable livers (6/8) than controls (0/8). Increased absolute and relative spleen weights (150–160% and 140–160% of control, respectively) were found in all treated groups. Histopathological changes were observed in the pituitary and adrenal glands but not in the spleen. In the pituitary, the changes consisted of an increased incidence of cysts in the pars distalis (2/8, 4/8, 5/8 and 3/8 animals at 0, 25, 50 and 100 ppm, respectively). The changes in the adrenals consisted of increased incidences of cytoplasmic vacuolation (4/8 and 3/8 animals at 50 and 100 ppm, respectively, with 1/8 in the control group). The NOAEL was 25 ppm, equal to 0.83 mg/kg bw per day, on the basis of an increased incidence of cytoplasmic vacuolation in the adrenal glands at 50 ppm, equal to 1.6 mg/kg bw per day (Rivett et al., 1971).

2.3 Long-term studies of toxicity and carcinogenicity

Mice

Groups of 20–40 male dd mice received diets containing various isomers (purity, > 99%) alone or in combination at a concentration of 0, 100, 250 or 500 ppm for 24 weeks. The animals were weighed weekly and necropsied grossly at the end of the study, when the livers were weighed and examined by histopathology and electron microscopy.

Body weights were unaffected by lindane at any concentration, but the absolute and relative liver weights were increased by 16% and 33%, respectively, in animals at 500 ppm. Microscopic evaluation of these livers revealed hypertrophy but no tumours. The NOAEL for lindane was 500 ppm, equivalent to 25 mg/kg bw per day, the highest dose tested (Ito et al., 1973a).

In a study of the role of polychlorinated biphenyls in promoting liver tumorigenesis induced by benzene hexachloride and the effect of exposure to various benzene hexachloride isomers on liver tumorigenesis, groups of 20–30 dd male mice were given diets containing alpha-, beta or gamma-benzene hexachloride (purity, > 99%) at a concentration of 0, 50, 100 or 250 ppm in the presence or absence of 250 ppm Kanechlor-500 (pentachlorobenzene-5) for 24 weeks. The animals were weighed weekly. At the end of the study, they were killed and necropsied grossly; the livers were weighed and examined microscopically. Animals that died intercurrently were discarded without further evaluation.

Exposure to pentachlorobenzene-5 in conjunction with all three isomers of benzene hexachloride at any concentration resulted in increases in absolute and relative liver weights of 87–370% and 83-370%, respectively. In the absence of pentachlorobenzene-5, however, only alpha-benzene hexachloride at a concentration of 250 ppm elicited a 120% and 110% increase in absolute and relative liver weights, respectively. Histopathological evaluation revealed 27–81% increases in the incidences of nodular hyperplasia and hepatocellular carcinoma after administration of alpha-benzene hexachloride at 250 ppm in the presence or absence of pentachlorobenzene-5. beta-Benzene hexachloride increased the incidence of liver tumours only at concentrations > 100 ppm and only in the presence of pentachlorobenzene-5. gamma-Benzene hexachloride (lindane) did not cause liver tumours at any concentration. The NOAEL for lindane was 250 ppm, equivalent to 12 mg/kg bw per day, the highest dose tested (Ito et al., 1973b).

Groups of 50 Chbb:NMRI (SPF) mice of each sex received diets containing lindane (purity, 99.5%) at a concentration of 12, 25 or 50 ppm for 80 weeks. The control group consisted of 100 mice of each sex. Food consumption and the body weights of 15 mice of each sex per dose were measured weekly for the first 26 weeks and then every 2 weeks. All animals were subjected to a full gross examination, and the brain, adrenals, gonads and urinary bladder and any gross lesions were examined histologically.

Mortality rates, food consumption and body weights were not affected by treatment. Animals at 50 ppm that died intercurrently had a higher incidence of enlarged spleens (5/19, with 1/28 in the control group), and those killed at the end of the study had a 10% increase in the incidence of pulmonary lesions, including mottled and pale lungs (5/41) over that in controls (2/86). There were two incidences of polymorphonuclear sarcoma and one of spindle-cell sarcoma among animals at the highest concentration, with none in the control group. The NOAEL was 25 ppm, equal to 3.9 mg/kg bw per day, on the basis of enlarged spleens and lung lesions at 50 ppm, equal to 7.8 mg/kg bw per day (Köllmer, 1975).

Groups of 50 B6CF3 mice of each sex were given diets containing lindane at a concentration of 0, 80 or 160 ppm, equal to 0, 11 and 23 mg/kg bw per day, for 80 weeks and observed for an additional 10–11 weeks. Food consumption was determined weekly for the first 16 weeks and every 4 weeks thereafter. Animals were observed for signs of toxicity and deaths twice daily. Detailed examinations, including palpation, were conducted once a week. A full gross examination and histopathological examination of gross lesions, adrenal glands, bone (with marrow), brain, bronchi, colon, duodenum, heart kidneys, liver, lungs, lymph nodes, mammary glands, ovaries, pancreas, parathyroid, pituitary, prostate, salivary glands, spleen, stomach, testes, thymus, thyroid, trachea and uterus were conducted for mice that were killed before or at the end of the study. All organs examined histopathologically were weighed.

Survival and body weights were unaffected by treatment. Alopecia, rough fur and distended abdomens were observed in treated animals. During the second year of the study, the females appeared to be more excitable and males showed more aggressive behaviour (fighting). High incidences of hepatocellular carcinomas were reported in all groups of males, including controls (20%, 39% and 20% of controls and at the two concentrations, respectively). In females, the incidences of this lesion were 0%, 4% and 7% for controls and treated animals, respectively (Table 5). Under the conditions of this study, lindane was not carcinogenic (Steinberg et al., 1977).

Table 5. Incidences of neoplastic lesions in the livers of B6C3F₁ mice fed diets containing lindane

From Steinberg et al. (1977)

The carcinogenic potential of lindane in mice was further investigated in groups of 50 Chbi:NMRI (SPF) mice of each sex given diets containing lindane (purity unspecified) at a concentration of 12, 25 or 50 ppm for 80 weeks. The concurrent control group consisted of 100 mice of each sex. The animals were observed daily for signs of toxicity or tumours. Animals killed at the end of the study and those dying intercurrently were necropsied, and the brain, heart, lungs, liver, spleen, kidneys, suprarenal glands, gonads, bladder and any gross lesions identified at necropsy were examined histologically. In addition, 10–15 liver samples from four mice of each sex per dose were examined by electron microscopy.

Clinical signs of toxicity, body weight, food consumption and mortality rates were unaffected by treatment. The tumour incidence in the treated groups (13–23%) was comparable to that of concurrent controls (24%). The commonest neoplastic lesions were lymphocytic leukaemia or lymphosarcoma and primary lung tumours. These two lesions occurred at comparable frequencies in treated and control groups: 4–7% versus 8.5% for lymphocytic leukaemia or lymphosarcoma and 8–11% versus 10% for primary lung tumour. The incidences of liver-cell adenomas were also comparable in treated (2%) and control groups (2.5%). The only malignancy found in the liver was a malignant hæmangioendothelioma in an animal at 12 ppm (equal to 2 mg/kg bw per day). The histological findings in the liver were confirmed by electron microscopy, which showed no difference between the treated groups and the control. The NOAEL was 50 ppm, equal to 7.8 mg/kg bw per day, the highest dose tested (Weisse & Herbst, 1977).

Groups of 36–96 female mice of the agouti, pseudoagouti and black strains were given diets containing lindane at a concentration of 0 or 160 ppm, equivalent to 0 and 23 mg/kg bw per day, for up to 24 months. These concentrations were selected on the basis of a preliminary study in which no deaths occurred after 1 month. Additional groups of 48–96 agouti and black mice were fed treated or control diets for 6 months and then fed control diet for 6 or 18 months for evaluation of recovery.

Clinical signs of toxicity and information on survival were not reported. No apparent effects on body weight or food consumption were observed, but limited data were presented. At 6 and 12 months, the benzo[a]pyrene monooxygenase activity in the liver was increased by 1.6–1.8 times in the agouti, 2.7–2.8 times in the pseudoagouti and 2.1 times in the black strains in comparison with controls. The weights of the liver were increased by 15–31% in the agouti, 14–22% in the pseudoagouti and 12–16% in the black strains at interval sacrifices up to 24 months. After the recovery period, the weights of the liver in treated mice were similar to those of controls.

No evidence was found for an increased incidence or decreased latency of liver tumours in the black strain at any time during the study or in the pseudoagouti strain at the 18-month sacrifice; however, at 18 months, 0/34 control and 12/36 (33%) treated agouti mice had developed hepatocellular adenomas, and one carcinoma was found in each of the treated and control groups. Treated agouti and pseudoagouti mice showed clear increases in the incidences of adenomas and slight increases in the incidences of carcinomas at 24 months (Table 6).

Table 6. Incidences^a of hepatocellular adenoma and carcinoma in (YS × VY) F₁ hybrid mice given diets containing lindane

From Wolff et al. (1987)

^a Mice bearing tumours/mice examined

Increased incidences of Clara cell hyperplasia in the lung were seen at all sacrifice intervals for each strain, and the incidence of lung tumours was increased at later times in the agouti and pseudoagouti strains (Table 7). After recovery, the incidences of Clara cell hyperplasia (agouti and black) and lung tumours (agouti) remained slightly elevated as compared with the controls (Wolff et al., 1987).

Table 7. Incidences^a of Clara cell hyperplasia and lung tumours in (YS × VY) F₁ hybrid mice given diets containing lindane

From Wolff et al. (1987)

^a Mice bearing lesions/mice examined

Groups of 50 Crl:CD-1®(ICR)BR mice of each sex received diets containing lindane (purity, 99.78%) at a concentration of 0, 10, 40 or 160 ppm for 78 weeks. The animals were observed for clinical signs of toxicity and deaths twice daily, and comprehensive examinations (including palpation) were conducted weekly. Body weight and food consumption were measured weekly during the first 14 weeks and monthly thereafter. Haematological evaluations were conducted at the end of the study period. All animals were necropsied grossly, and 43 tissues and any gross lesions from animals in the control group and at the highest dose and those of animals dying intercurrently were examined histologically.

No significant treatment-related effects were seen on clinical signs, mortality rates, body weight, body-weight gain, food consumption or haematological parameters. The relative weight of the uterus and cervix of animals at 160 ppm that died intercurrently were decreased by 50% (p < 0.05), and these animals also had a decreased incidence of uterine cysts (30%) when compared with controls (79%). Given the lack of corroborative histopathological findings, these effects are considered not to be adverse or toxicologically relevant. At the end of the study, males at the highest concentration had a statistically significant increase in the incidences of centrilobular hepatocyte hypertrophy (34%; 5% in controls; p < 0.01) and eosinophilic foci of hepatocellular alteration (21%; 5% in controls; p < 0.05). Although no histopathological changes were seen in the livers of females, a statistically significant (p < 0.05) increase in the incidence of bronchiolar–alveolar adenomas was reported at 160 ppm (22%; 6% in controls) in females only (Table 8). It should be noted, however, that these changes were not dose-dependent, and the tumour response was variable. Furthermore, the incidence of this finding in control animals was at the low end of the range for other controls in the same laboratory (6%). Nonetheless, the incidence in females at the highest dose (19%) exceeded the rate in other controls. When the slides were re-examined, two additional adenomas were identified in both the control and the high-concentration group. While the incidence of carcinomas was not increased when compared with concurrent controls, the combined incidence of adenomas and carcinomas at the highest concentration was increased (29%; 12% in controls; p < 0.05). The NOAEL was 40 ppm, equal to 5.2 mg/kg bw per day, on the basis of an increase incidence of alveolar–bronchiolar adenomas in females at 160 ppm, equal to 21 mg/kg bw per day (Chase, 2000).

Table 8. Incidences of alveolar–bronchiolar tumours in mice given diets containing lindane

From Chase (2000)

* p < 0.05

Rats

Groups of 50 male and 50 female Wistar rats received diets containing lindane (purity, 99.75%) at a concentration of 0, 1, 10, 100 or 400 ppm for 1 year for the study of toxicity. An additional 15 rats of each sex per group were designated for interim sacrifice (followed by gross necropsy and histopathological examination) after 30 days and 26, 52 and 78 weeks (52 weeks of treatment followed by a 26-week recovery period). For the study of carcinogenicity, 55 rats of each sex per dose received the diets containing lindane at the same concentrations for 102 weeks, and five rats of each sex per dose were used to assess the concentrations of lindane in liver, kidney, brain and blood. The animals were necropsied and evaluated histologically at the end of the study. An additional 25 animals of each sex were examined to establish baseline parameters for haematology, clinical chemistry and urine analysis. Ten rats of each sex from this group were killed, and their kidneys, liver and lungs were examined histologically. During both phases of the study, blood and urine were collected in weeks 3, 12, 24, 51, 77 and 103 from 10 rats of each sex per dose.

During the phase of the study in which toxicity was studied, no treatment-related clinical signs of toxicity were reported. Alopecia, swollen limbs and aggressive behaviour were observed sporadically at similar rates in control and treated groups. Mortality rates were not affected. Although statistically significant decreases in body-weight gain were reported sporadically for animals of each sex at concentrations > 100 ppm, only females at the highest concentration had statistically significant decreases at week 52. These decreases in body-weight gain were found in conjunction with a marginal (7%) decrease in food consumption, which was not statistically or biologically significant. Food use efficiency was not affected by treatment. Haematological and clinical chemical parameters were not affected. Statistically significant increases in urine volume, with decreased pH and specific gravity, were reported in males at the highest concentration during the first 25 weeks of the study but were no longer present at week 52. A statistically significant increase in urea and creatinine concentrations was reported in males at dietary concentrations > 100 ppm during week 12 but was apparent only in animals at 400 ppm at week 24 and had resolved by the end of the dosing period (52 weeks), as was the case for the increase in urinary output. No consistent changes in organ weights (absolute or relative to body weight) were reported. Gross necropsy after the 30-day and 26-week interim sacrifices revealed an increased incidence of pale kidneys in males (30 days: 0/10, 5/10 and 6/10 in controls, 100 and 400 ppm, respectively; 26 weeks: 0/10 and 3/10 in controls and at 400 ppm). This finding was not present at necropsy at week 52 or week 78. Periacinar hepatocyte hypertrophy was observed in animals of each sex at concentrations > 100 ppm, and renal lesions suggestive of alpha_2u-globulin-mediated nephrotoxicity were found in males at concentrations > 10 ppm. After a 26-week recovery period, the histopathological renal changes had resolved, and periacinar hepatocyte hypertrophy was observed only in females at 100 ppm with no dose–response relationship.

In the phase of the study designed to investigate carcinogenicity, the clinical signs of toxicity consisted of convulsions in 11 females at the highest concentration. The survival rates at the end of the study were 36%, 36%, 31%, 20% and 16% for males and 49%, 38%, 44%, 35% and 18% for females at 0, 1, 10, 100 and 400 ppm, respectively. The survival rate of males at the highest concentration was similar to that of controls through week 93, but the survival rate of females at this concentration was significantly decreased, 50% survival being reached at week 89, compared with week 104 for the control group. Body-weight gain was significantly (p < 0.01) decreased for males at 100 and 400 ppm during the first few weeks of the study as compared with controls. Because the final body weights of males at 100 ppm were similar to those of controls, the initial reduction in weight gain was considered not biologically significant. The final body weights of males at 400 ppm were significantly (–14%; p < 0.05) lower than those of controls. The body weights and body-weight gains of treated females were similar to those of controls throughout the study. Food consumption by the groups at the highest concentration was decreased by 15% in males and 19% in females during the first week of the study, but the total food consumption over the entire study was similar to control levels.

Platelet counts were significantly (p < 0.05 or 0.01) increased (by ­ 20%) in males at 100 and 400 ppm at week 12 and in males and females at these concentrations at week 24 but not at later times. Males and females at 400 ppm showed significant (p < 0.05 or 0.01) decreases in erythrocyte parameters at week 104 as compared with the controls: the