PESTICIDE RESIDUES IN FOOD - 1997
Sponsored jointly by FAO and WHO
with the support of the International Programme
on Chemical Safety (IPCS)
TOXICOLOGICAL AND ENVIRONMENTAL
EVALUATIONS 1994
Joint meeting of the
FAO Panel of Experts on Pesticide Residues
in Food and the Environment
and the
WHO Core Assessment Group
Lyon 22 September - 1 October 1997
The summaries and evaluations contained in this book are, in most
cases, based on unpublished proprietary data submitted for the purpose
of the JMPR assessment. A registration authority should not grant a
registration on the basis of an evaluation unless it has first
received authorization for such use from the owner who submitted the
data for JMPR review or has received the data on which the summaries
are based, either from the owner of the data or from a second party
that has obtained permission from the owner of the data for this
purpose.
LINDANE (addendum)
First draft prepared by
P.H. van Hoeven-Arentzen and M.E. van Apeldoorn
Centre for Substances and Risk Assessment
National Institute of Public Health and the Environment
Bilthoven, The Netherlands
Explanation
Evaluation for acceptable daily intake
Toxicological studies
Short-term toxicity
Long-term toxicity and carcinogenicity
Genotoxicity
Reproductive toxicity
Special studies
Hyaline droplet formation in rat kidneys
Immune responses
Comments
Toxicological evaluation
References
Explanation
Lindane was evaluated toxicologically by the JMPR in 1963, 1965, 1966,
1970, 1973, 1977, and 1989 (Annex 1, references 2, 4, 6, 14, 20, 28,
and 56). An ADI of 0-0.01 mg/kg bw was established in 1977. On the
basis of additional data, the 1989 JMPR allocated an ADI of 0-0.008
mg/kg bw. An Environmental Health Criteria monograph on lindane has
been published (WHO, 1991). Additional short-term studies on toxicity
after dermal exposure, long-term toxicity and carcinogenicity,
genotoxicity, and reproductive toxicity have become available, which
were evaluated at the present Meeting.
Evaluation for acceptable daily intake
1. Toxicological studies
(a) Short-term toxicity
Rats
Groups of 49 male and 49 female Charles River rats (strain Crl:(WI)BR;
5-7 weeks of age) received lindane (purity, 99.5%) formulated in 5%
aqueous carboxymethylcellulose as dermal applications on the clipped
dorsal skin under occlusive conditions for 6 h per day on five
consecutive days per week for 13 weeks at doses of 0, 10, 60, or 400
mg/kg bw per day in a volume of 4 ml/kg bw. In each group, 13 rats of
each sex were selected for an interim kill after six weeks of
treatment, and 13 rats of each sex were allowed to recover from the
13-week treatment for another six weeks and were then killed. At each
kill, three animals of each sex per group were selected for
determination of the concentrations of lindane in brain, liver,
kidney, and fat. The limits of quantification were 10 ng/ml in plasma,
0.1 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 Negativea 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 Negativeb Murli (1990)
ovary cells harvest at 10 h; not toxic
Chomosomal aberration Chinese hamster 25-305 µg/ml in DMSO; 99.7 Negativeb 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 Negativeb; 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 alpha2µ-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 alpha2µ-globulin. The authors concluded that lindane induces
a protein with an antigenic structure corresponding to
alpha2µ-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 alpha2µ-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 alpha2µ-globulin, and staining was scored on a scale
from 0-5, from none to markedly severe. Alpha2µ-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 alpha2µ-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
'alpha2µ-globulin nephropathy'. This type of nephropathy is
considered not to be relevant for humans. Therefore, in dtermining the
NOAELs in studies in rats, these male-specific renal effects were not
taken into account.
In a 13-week study of dermal toxicity, rats were exposed to doses of
0, 10, 60, or 400 mg/kg bw per day. At the highest dose, clinical
signs of neurological effects (convulsions) were observed. Other
targets were the kidneys of male animals and the liver, as
demonstrated by changes in organ weight and histopathological changes.
Since the male-specific renal effects were not taken into account, the
NOAEL for dermal exposure was 10 mg/kg bw per day, on the basis of
increased liver weight and histopathological changes in the liver.
In another 13-week study of dermal toxicity, rabbits were exposed to
doses of 0, 10, 60, or 400 mg/kg bw per day. At the highest dose,
clinical neurological effects (convulsions) were observed. The NOAEL
for dermal exposure was 10 mg/kg bw per day on the basis of increased
liver and adrenal weights and centrilobular hypertrophy of the liver.
In a two-year study of toxicity and carcinogenicity, rats were exposed
to dietary concentrations of lindane at 0, 1, 10, 100, or 400 ppm. At
the highest dose, neurological effects (convulsions), reduced
body-weight gain, decreased survival rates (also in males at 100 ppm),
and changes in erythrocyte parameters were observed. Other changes
seen at 400 ppm, and to a lesser extent at 100 ppm, were changes in
weight and in the histological appearances of the liver and kidneys.
The effects on the kidneys were confined to male rats, with a slight
increase in hyaline droplet formation that was also observed in male
rats at 1 and 10 ppm. Since the male-specific renal effects were not
taken into account, the NOAEL was 10 ppm, equal to 0.47 mg/kg bw per
day, on the basis of a slight increase in mortality and effects on the
liver. There was no evidence of carcinogenicity.
A two-generation study in rats given lindane at dietary concentrations
of 0, 1,20, or 150 ppm did not indicate reproductive toxicity. The
main effects found in progeny at 150 ppm were on weight gain;
decreased viability of pups was seen up to day 4 post partum. In the
pups of the second generation, there was a slight delay in tooth
eruption and hair growth Pups of the F2 generation at 150 ppm that
died before weaning showed increased incidences of hydronephrosis and
hydroureter. The NOAEL for reproductive and developmental toxicity was
20 ppm, equivalent to 1 mg/kg bw per day. Histopathological effects in
the kidneys were observed only in male parents at 20 and 150 ppm.
Since the male-specific renal effects were not taken into account, the
NOAEL for parental toxicity was 20 ppm, equivalent to 1 mg/kg bw per
day, on the basis of effects on body-weight gain and the liver and
increased kidney weights in animals of each sex.
Lindane did not induce chromosomal aberrations or unscheduled DNA
synthesis in vitro.
Functional effects and histological changes in the immune system were
induced by lindane (purity, 97% or unknown) in mice, rats, and
rabbits. In rats and rabbits, effects were seen at doses equivalent to
1 mg/kg bw per day and higher, but they were not seen in rats at 0.25
mg/kg bw per day. In mice exposed to doses of 0.012 mg/kg bw per day
and higher, an initial immunostimulation followed by immunosuppression
was observed. It was noted, however, that the purity of the test
material used in these studies was lower than that specified by
current FAO specifications, namely > 99% gamma-hexachloro-
cyclohexane.
The toxicological effects that are relevant for estimating hazard for
humans are those on the liver and the central nervous system. In
published studies, however, lindane of a purity of 97% or of unknown
purity has been found to affect the immune system. As immunotoxic
effects were observed at doses close to or even lower than the NOAEL
found in the two-year study in rats, the Meeting decided that
additional data should be generated on immunotoxicity. Further, the
Meeting recommended that, when the new results become available, a
full re-evaluation be performed to consider the validity of the
studies that have been reviewed previously and to consider any new
information that becomes available.
The Meeting established a temporary ADI at 0-0.001 mg/kg bw on the
basis of the NOAEL of 0.5 mg/kg bw per day in the two-year study of
toxicity and carcinogenicity in rats, using a safety factor of 500.
Pending clarification of the immunotoxicity of lindane that meets FAO
specifications, this ADI provides a 10-fold margin of safety over the
LOAEL of 0.012 mg/kg bw per day in a study of immunotoxicity in mice.
Toxicological evaluation
Levels that cause no toxic effect
Mouse: 300 ppm, equivalent to 15 mg/kg bw per day (26-week
study of effects on the liver)
50 ppm, equal to 7.8 mg/kg bw per day (80-week study of
carcinogenicity)
30 mg/kg bw per day (maternal and developmental
toxicity in a study of developmental toxicity)
< 0.012 mg/kg bw per day (24-week study of
immunotoxicity with 97% pure lindane)
Rat: 10 ppm, equal to 0.75 mg/kg bw per day (13-week study
of toxicity)
4 ppm, equal to 0.29 mg/kg bw per day (three-month
study of toxicity, LOAEL= 20 ppm)
10 ppm, equal to 0.5 mg/kg bw per day (two-year study
of toxicity and carcinogenicity)
20 ppm, equivalent to 1 mg/kg bw per day
(two-generation study of reproductive toxicity)
5 mg/kg bw per day (maternal toxicity in a study of
developmental toxicity)
Dog: 50 ppm, equal to 1.6 mg/kg bw per day (two-year study
of toxicity)
Rabbit: <5 mg/kg bw per day (maternal toxicity in a study of
developmental toxicity)
Estimate of temporary acceptable daily intake for humans
0-0.001 mg/kg bw
Studies without which the determination of an ADI is impracticable,
to be provided by 2000
Confirmatory study of immunotoxicity in mice with lindane that meets
the current FAO specification (> 99% gamma-hexachlorocyclohexane)
Studies that would provide information useful for continued
evaluation of the compound
Further observations in humans
References
Amyes, S.J. (1990). Lindane: Combined oncogenicity and toxicity study
by dietary administration to Wistar rats for 104 weeks. Unpublished
report LSR No. 90/CIL002/0839 from Life Science Research Ltd, United
Kingdom. Submitted to WHO by Centre International d'Etudes du Lindane,
Brussels, Belgium.
Toxicological criteria for setting guidance values for dietary and non-dietary exposure to lindane
Human exposure Relevant route, study type, species Results, remarks
Short-term Oral, toxicity, dog LD50 = 40 mg/kg bw
(1-7 days) Dermal, toxicity, rabbit LD50 = 900 mg/kg bw
Inhalation, 4 h, toxicity, rat LC50 = 1600 mg/m3
Skin, irritation, rabbit Not irritating
Eye, irritation, rabbit Slightly irritating
Skin, sensitization, galnea-pig Not sensitizing
Medium-term Repeated inhalation, 90 days, toxicity, rat NOAEL = 0.6 mg/m3 per day: clinical signs and
(1-26 weeks) increased cytochrome P450 enzymes
Repeated dermal, 90 days, toxicity, rat/rabbit NOAEL = 10 mg/kg bw per day: hepatic effects
Repeated oral, 90 days, toxicity, rat NOAEL = 0.75 mg/kg bw per day: changes in liver
and kidney weight
Repeated oral, developmental toxicity, rabbit No NOAEL for maternal toxicity; NOAEL =
10 mg/kg bw per day: fetuses with 13 ribs
Repeated oral, reproductive toxicity, rat NOAEL = 1 mg/kg bw per day: developmental
toxicity and hepatic effects
Long-term Repeated oral, 2 years, toxicity/carcinogenicity, rat NOAEL = 0.5 mg/kg bw per day: hepatic changes
(> 1 year) and decreased survival; no carcinogenicity
Andre, F., Gillon, J.,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 alpha2µ-globulin in kidneys of rats treated with
lindane. Unpublished report from Centre International d'Etudes du
Lindane (document No. 464-001). Submitted to WHO by Centre
International d'Etudes du Lindane, Brussels, Belgium.
WHO (1991) Lindane (Environmental Health Criteria 124), Geneva,
International Programme on Chemical Safety.