690. Amitraz (Pesticide residues in food: 1984 evaluations)

PESTICIDE RESIDUES IN FOOD - 1984

Sponsored jointly by FAO and WHO

EVALUATIONS 1984

The monographs

Data and recommendations of the joint meeting
of the FAO Panel of Experts on Pesticide Residues
in Food and the Environment and the
WHO Expert Group on Pesticide Residues
Rome, 24 September - 3 October 1984

Food and Agriculture Organization of the United Nations
Rome 1985

AMITRAZ

Explanation

Amitraz was evaluated by the Joint Meeting in 1980 (FAO/WHO,
1981) ^1/ and a temporary ADI of 0-0.0005 mg/kg body weight was
allocated. Further data required included a long-term carcinogenicity
study in mice using Amitraz; a separate long-term carcinogenicity
study using the metabolite N(2,4-dimethyl-phenyl)N-methylformamidine;
and studies on the mutagenic activity of 2,4-xylidine, N,N'-bis
(2,4-dimethylphenyl) formamidine and 2,4-dimethyl-formanilide. Since
the previous evaluation (FAO/WHO, 1981) additional data have become
available and are reviewed in this monograph addendum.

EVALUATION FOR ACCEPTABLE DAILY INTAKE

BIOCHEMICAL ASPECTS

Absorption, distribution and excretion

Male and female B6C3F1 mice, either untreated or pre-dosed for 3
weeks with 400 ppm. Amitraz in the diet, were orally intubated with a
single dose of 10 mg/kg bw ¹⁴C- Amitraz. In the first 24 h, 85.9
percent of the radio-labelled dose was excreted and 62.3 percent was
present in the urine. It was completely excreted by 96 h, 72.7 percent
being in the urine. The route and rate of excretion was similar in
both sexes, as well as in untreated and pre-dosed mice. The highest
¹⁴C- Amitraz concentration was found in liver, adrenals and eyes,
with the least activity being in bone and muscle (Campbell & Needham,
1983).

Following administration of a single oral dose of ¹⁴C- Amitraz
to Sprague-Dawley CD strain rats at 1, 10, 50 and 100 mg/kg body
weight, the compound was rapidly excreted and effectively metabolized,
with no apparent sex differences identified. At all dose levels,
4-formamido-3-methylbenzoic acid and 4-acetamido-3-methylbenzoic acid
were the major metabolites, which together accounted for up to 32
percent of the total urinary excretion. The excretion of the
metabolite N(2,4-dimethylphenyl-N'-methylformamidine) was found to be
dose-dependent. At 1 mg Amitraz/kg bw, approximately 4 percent was
excreted; at 100 mg/kg bw, 23-38 percent of the dose was excreted as
this metabolite. Minor metabolites identified accounted for
approximately 2 percent of the total excretion and included
2,4-dimethylformanilide and 4-amino-3-methylbenzoic acid (Campbell &
Needham, 1984a).

^1/ See Annex 2 for FAO and WHO documentation

In separate studies, beagles were dosed orally (via capsule) or
dermally with ¹⁴C- Amitraz. Peak blood levels of radiolabel were
observed in the first 8 h following oral administration. Approximately
80 percent of the oral dose was excreted in the first 24 h and 100
percent within 72 h, preferentially in the urine. After dermal
treatment, peak blood levels occurred in the 24-72 h post-treatment
interval, and only 25-40 percent of the dermal dose was recovered in
urine and faeces over a 10-day collection period, thus demonstrating
the poor dermal absorption of Amitraz. The metabolism of Amitraz by
either oral or dermal administration was essentially the same with
4-formamido-3-methylbenzoic acid, the predominant residue in both
blood and urine. Parent compound and the first formed hydrolysis
products (N-2,4-di-methylphenyl-N-methylformamidine and
2,4-dimethylformanilide) were never observed at measurable levels in
either blood or urine (Hornish & Nappier, 1983).

Approximately 57-81 percent of the applied radioactivity was
recovered in dermal washings 12 h after application of a diluted E.C.
formulation of ¹⁴C-Amitraz (18 mg a.i.) to the shaved dorsal shin of
pigs. Approximately 6.7 percent of the applied radioactivity was
recovered in the excreta collected over 60 h post-application. Tissue
residues were less than 0.05 ppm (Campbell, 1984a).

¹⁴C-Amitraz, given orally to baboons at 10 mg/kg bw, was rapidly
excreted in both sexes, mainly in urine (75-83 percent of the dose
recovered within the first 24 h). Tissue residues were similar in both
sexes and were highest in the liver and eyes and lowest in muscle
(Campbell & Needham, 1984b).

Two male human volunteers received a single oral dose of
¹⁴C-Amitraz of 0.25 mg/kg bw. Approximately 62.9 percent was
recovered in the urine within the first 24 h, and 87.9 percent within
72 h. Urinary metabolites were the same as in other species examined
(Campbell, 1984b,c).

TOXICOLOGICAL STUDIES

Special Studies on Hormone Levels

The effect of technical Amitraz on the oestrus cycle and hormone
levels was evaluated in groups of female B6C3F1 mice fed diets
containing 0, 25, 100 and 400 ppm Amitraz for 28 weeks. Pro-oestrus
was prolonged and a trend towards reduced duration of dioestrus was
evident at 400 ppm on examination of vaginal smear data. There
were lower blood levels of progesterone and higher levels of
dehydroepiandrosterone sulphate in the 400 ppm groups, and to a lesser
degree in the 100 ppm group, when compared to controls. Liver weights
were increased at 400 and 100 ppm. There were no treatment-related
effects at 25 ppm (Hounsell & Rush, 1984).

Special Study on Carcinogenicity

In a pilot study, groups of 20 male and female B6C3F1 mice were
administered Amitraz in the diet at doses of 0, 100, 200, 400, 600 and
800 ppm for 13 weeks. Aggressive behaviour was observed in males in
the 400 to 800 ppm groups. Overall, body weight gain was reduced in
males at >400 ppm and in females at >200 ppm. There were no
significant differences in food consumption between control and
treated groups. There was no microscopic evaluation of tissues (Colley
et al., 1981).

Groups of 75 male and 75 female B6C3F1 hybrid mice (33 days old)
were administered diets containing 25, 100 and 400 ppm Amitraz for 104
weeks. The untreated controls consisted of 100 male and 100 female
B6C3F1 hybrid mice (33 days old). Clinical signs, food consumption and
body weight were recorded throughout the study. Survivors were killed
at the end of the 2-year dosing period and all animals were subjected
to a complete gross autopsy. A complete collection of tissues was
taken from all animals and preserved for future examination.
Histopathological examination was performed on all tissues from the
high dose group and controls. At the low and intermediate dosage
levels, liver, pancreas, spleen, lung, stomach, pituitary, thyroid,
adrenals, ovaries, uterus, sternum and all macroscopically abnormal
tissues were examined microscopically.

Males at 400 ppm exhibited hyperactivity and aggressive
behaviour. The incidence of cutaneous ulceration and inflammation of
the perigenital and perianal areas was greater at 100 and 400 ppm than
at 25 ppm and in controls. The incidence of urogenital swelling was
greater in all treatment groups than that observed for the controls.
Incidences of gross adverse effects for treated females were
comparable to control incidences. Mortality was increased in high dose
group animals compared to other groups, including the control group.

Mean body weight gain was reduced throughout the study for high
dose animals, with a significant decrease in 100 ppm females over the
last 74 weeks of the study. Food consumption was marginally reduced
initially at 100 and 400 ppm but was comparable to controls from week
25 to termination.

An increase in liver and lymph node involvement was apparent for
males and females fed 400 ppm. The incidence of preputial gland
enlargement of 400 ppm males was greater than that observed for
controls (20/75 vs 12/100). Prominence of the limiting ridge of the
stomach was greater than that of controls for females at all levels
and for males at 100 and 400 ppm. A decrease in the production of
myeloid elements accompanied by an increase in erythroid elements
resulted in a significant decrease in the M/E ratio for males fed
400 ppm and females fed 100 and 400 ppm amitraz. The incidence of
spleen haematopoiesis in males and stomach focal hyperkeratosis in
males and females fed all three dosage levels were greater than that
observed in the control groups.

There is an apparent liver involvement in females with an
increased incidence, compared to controls, in heptocellular tumours at
the 400 ppm level (15/75 vs 2/100 hepatocarcinoma; 16/75 vs 4/100
hepatocellular adenoma). This was accompanied by a dose-response
increase in the incidence of hyperplastic nodules, telangiectatic and
basophilic foci of the liver at 25, 100 and 400 ppm. Males at the
400 ppm level exhibited an apparent increase, compared to controls, in
the incidence of hyperplastic nodules, and telangiectatic and
basophilic foci of liver (Colley et al., 1983).

Special Studies on Mutagenicity

See Tables 1 and 2 for a summary of the mutagenicity studies
evaluated.

COMMENTS

Amitraz was first reviewed by the JMPR in 1980; a temporary ADI
was allocated pending further toxicological information. New studies
on metabolism have shown that after oral administration amitraz is
rapidly metabolized and excreted, mainly in the urine; the pattern of
metabolism is qualitatively similar in mouse, rat, dog, baboon and
human. Mutagenicity studies were all negative, both in the presence
and absence of metabolic activation.

In the carcinogenicity study in B6/C3 F₁ mice the only
significant change in tumour incidence observed after treatment with
amitraz for 104 weeks was the increased incidence of hepatocellular
tumours in female mice at 400 ppm, which was not considered to be
significant to human health (FAO/WHO 1983a).

The request for a long-term carcinogenicity study of
N-(2,4-dimethylphenyl)-N'-methylformamidine was considered, but was
found to be clearly unnecessary as the new metabolic studies have
indicated that this compound has been effectively examined. An ADI was
estimated on the basis of the data submitted.

TOXICOLOGICAL EVALUATION

Level Causing no Toxicological Effect

Mouse: 2.5 mg/kg bw/day

Rat: 60 ppm in the diet equivalent to 3 mg/kg bw

Dog: 0.25 mg/kg bw/day

Estimate of Acceptable Daily Intake for Man

0 - 0.003 mg/kg bw

TABLE 1. Special Studies on Mutagenicity (Amitraz)

Test System Test Concentrations Purity Results Reference
Organism of Amitraz used

Ames' Test S. typhimurium 0, 33, 100, 333, 97-99% Negative. McGregor &
(with and without TA 98 1000, 3300, Positive control Prentice, 1983
metabolic activation) TA 100 10,000 µg/plate gave the expected
TA 1535 response.
TA 1537
TA 1538

Cell Transformation C3H/10 T ´ Activated 97-99% Negative McGregor & Riach,
(with and without clone 8 mouse 12.5, 25, 1983a
metabolic activation) embryo 37.5 µg/ml
fibroblasts Non-activated
5, 10, 15 µ/ml

Unscheduled DNA Human embryonic 20, 60, 100, 140, 97-99% Negative. McGregor & Riach,
Synthesis (with and fibroblasts 180, 220, 260, Positive control 1983b
without metabolic 300 µg/ml gave the expected
activation) response.

Mouse Lymphoma Mouse Activated 97-99% Negative McGregor & Riach,
Forward Mutation L5178Y TK+/- Up to 33 µ/ml 1984
Assay phenotype cells Non-activated
(with and without Up to 20 µg/ml
metabolic activation)

TABLE 2. Special Studies on Mutagenicity (Metabolites of Amitraz)

Test System Test Concentration Test Results Reference
Organism Used Material

Ames' Test S. typhimurium Up to 5000 N-(2,4-dimethyl- Negative. Richold et al.,
(with and with- TA 98 µg/plate phenyl)-N'-methyl- 1983a
out metabolic TA 100 formamidine and
activation) TA 1535 2,4-dimethylformanilide Negative. Richold et al.,
TA 1537 1983b.
TA 1538

Micronucleus Mouse, bone 56.3, 112.5 and 2,4-dimethylaniline Negative. Hounsell &
marrow 225 mg/kg, twice No increase in Walker, 1983
24 h apart polychromatic
erythrocytes
containing
micronuclei

Mouse Lymphoma Mouse 1, 3.3, 10, 2,4-dimethylaniline Positive in McGregor & Riach,
Forward Mutation L5178Y TK +/- 33.3, 100, 200, activated 1983c
Assay phenotype 300, 333.3, systems at
(with and with- 400, 500, 3.3 µg/ml and
out metabolic 600 µg/ml above. Negative
activation) in non-activated
systems up to
300 µg/ml.

Cell Transformation C3H/10 T ´ Activated 2,4-dimethylaniline Negative. McGregor et al.,
(with and clone 8 mouse 5, 10 and 20 µg/ml 1984.
without metabolic embyro Non-activated
activation) fibroblasts 100, 200 and
400 µg/ml

FURTHER WORK OR INFORMATION

Desirable

Further observations in humans.

REFERENCES

Campbell, J.K. Dermal absorption of ¹⁴C-amitraz in E.C. formulation by
1984a male and female pigs given a single topical application of
18 mg A.I. Report submitted by FBC Limited to WHO.
(Unpublished)

Campbell, J.K. Urinary excretion of ¹⁴C-amitraz by two human males
1984b following a single oral dose of 0.25 mg/kg body weight.
Report submitted by FBC Limited to WHO. (Unpublished)

Campbell, J.K. A comparison of the metabolism of ¹⁴C-amitraz in rat,
1984c mouse, baboon and human. Report submitted by FBC Limited to
WHO. (Unpublished)

Campbell, J.K. & Needham, D. Excretion and tissue residues of
1983 ¹⁴C-amitraz in male and female mice given a single oral
dose of 10 mg 14C-amitraz per kg body weight. Report
submitted by FBC Limited to WHO. (Unpublished)

Campbell, J.K. & Needham, D. The metabolism of ¹⁴C-amitraz by male and
1984a female rats. Report submitted by FBC Limited to WHO.
(Unpublished)

Campbell, J.K. & Needham, D. Excretion and tissue residues of
1984b ¹⁴C-amitraz in a male and female baboon given a single oral
dose of 10 mg 14C-amitraz per kg body weight. Report
submitted by FBC Limited to WHO. (Unpublished)

Colley, J., Dawe, S., Heywood, R., Prentice, D.E. & Gibson, W.A.
1981 Amitraz toxicity by mice by dietary administration for 13
weeks. Report from Huntingdon Research Center, Huntingdon,
U.K., submitted by FBC Limited to WHO. (Unpublished)

Colley, J., Dawe, S., Heywood, R., Street, A.E. & Gibson, W.A.
1983 Amitraz: 104 week tumorigenicity in mice. Report from
Huntingdon Research Centre, Huntingdon, U.K., submitted by
FBC Limited to WHO. (Unpublished)

Hornish, R.E. & Nappier, J.M. The absorption, metabolism of mitaban
1983 (amitraz) in the dog from oral and dermal exposure. Report
from Agricultural Research and Development Laboratories, The
Upjohn Company, U.S., submitted by FBC Limited to WHO.
(Unpublished)

Hounsell, I.A.G. & Walker, A.K. A micronucleus study in mice using BTS
1983 24868 (2,4-dimethylaniline). Report submitted by FBC Limited
to WHO. (Unpublished)

McGregor, D.B. & Prentice, R.D. Technical amitraz: Ames bacterial
1983 mutagenicity test. Report submitted by FBC Limited to WHO.
(Unpublished)

McGregor, D.B. & Riach, G.G. Technical amitraz: Induction of
1983a morphological transformation in C3H10T¹/2 cells. Report
submitted by FBC Limited to WHO. (Unpublished)

McGregor, D.B. & Riach, G.G. Technical amitraz: Unscheduled DNA
1983b synthesis in human embryonic cells. Report submitted by FBC
Limited to WHO. (Unpublished)

McGregor, D.B. & Riach, G.G. Technical BTS 24868: Mouse lymphoma
1983c mutation assay. Report submitted by FBC Limited to WHO.
(Unpublished)

McGregor, D.B. & Riach, G.G. Technical amitraz. Mouse lymphoma
1984 mutation assay. Report submitted by FBC Limited to WHO.
(Unpublished)

McGregor, D.B., Brown, A.G. & Riach, G.G. Technical BTS 24868:
1984 Induction of cell transformation in C3H10T¹/2 cells. Report
submitted by FBC Limited to WHO. (Unpublished)

Richold, M., Jones, E. & Fenner, L.A. Technical BTS 27271: Ames
1983a bacterial mutagenicity test. Report submitted by FBC Limited
to WHO. (Unpublished)

Richold, M., Jones, E. & Fenner, L.A. Technical BTS 27919: Ames
1983b bacterial mutagenicity test. Report submitted by FBC Limited
to WHO. (Unpublished)

    See Also:
       Toxicological Abbreviations
       Amitraz (ICSC)
       Amitraz (Pesticide residues in food: 1980 evaluations)
       Amitraz (Pesticide residues in food: 1983 evaluations)
       Amitraz (Pesticide residues in food: 1984 evaluations)
       Amitraz (JMPR Evaluations 1998 Part II Toxicological)