BROMOPHOS JMPR 1977
Explanation
Bromophos was evaluated by the Joint Meeting in 1972 (FAO/WHO, 1973),
when a temporary acceptable daily intake was allocated and temporary
tolerances were established. Further work was required to elucidate
renal function and testicular pathology in dogs, and to assess the
carcinogenic potential of bromophos. The additional studies described
below have been received and are discussed in this monograph addendum.
Residue aspects of bromophos were again evaluated in 1975 (FAO/WHO,
1976). The requirements for further information from that Meeting, and
questions arising from the Codex Committee on Pesticide Residues, are
listed in the section "Residues in Food and Their Evaluation".
EVALUATION FOR ACCEPTABLE DAILY INTAKE
BIOCHEMICAL ASPECTS
Biotransformation
Bromophos is subject to the reactions which are common to the
organophosphorothionates: conversion of the thiono sulphur to oxygen;
removal of one or both methyl groups, removal of the
bromodichlorophenyl group (with subsequent conversion of the resulting
phenol to conjugates), and almost any combination of the above.
The administration of bromophos to rats results in the formation of
type I substrate - cytochrome P450 binding (Szutkowski, 1975).
When doses of 20 mg of bromophos were given orally to pregnant rats,
the compound could be detected within 30 min. in maternal liver,
placenta, foetal brain, liver and muscle. The oxygen analogue was also
detected in foetal muscle. (Ackermann, 1975). The degradation of
bromophos in the presence of reduced glutathione occurred at about the
same rate in human and rat hepatic post-mitochondrial fractions, but
at a slower rate in porcine fractions. The primary detoxification
reaction was that of O-demethylation. (Palut, 1974).
The metabolism of 32P-labelled bromophos and its oxygen analogue
in vitro has been studied in post-mitochondrial supernatant
fractions of rat liver; the O-analogue was more rapidly hydrolyzed
than bromophos, and the principal metabolites were the monodesmethyl
derivates (Palut et al., 1970).
After spraying 20 mg of 32P-labelled bromophos on the skin of
lactating cows, labelled phosphate was detectable in blood and milk.
The blood concentration, calculated as bromophos, was about 0.01 mg/kg
and was not in the form of the O-analogue. The principal metabolite,
desmethylbromophos, was identified in blood and milk at concentrations
of 0.4-0.7 mg/kg, but these residues were not toxicologically
significant (Dedek and Schwarz, 1969)*. Bromophos does not act
systemically in tomato plants; rather, it penetrates from the surface
into the interior of the leaf, and from a nutrient solution into the
root. In addition to unchanged bromophos, bromodichlorophenol was
found as a main metabolite, with small amounts of thio-analogue,
monodesmethyl bromophos, dimethyl phosphorothionate and inorganic
phosphate (Stiasni at al., 1969).
50 strains of bacteria were isolated from garden soil degraded
bromophos. Some strains produced dimethyl phosphorothionate and
monomethyl-phosphorothionate. Others produced mainly the bisdesmethyl
compound. Other metabolites, possibly the oxygen analogues of dimethyl
and monomethyl phosphorothionate, were sometimes detected. (Stenersen,
1969).
TOXICOLOGICAL STUDIES
Special studies on mutagenicity
Bromophos has been tested for mutagenicity using
Drosophila melanogaster as a test organism and no mutagenic activity
was seen (Benes and Stram, 1969)
SPECIAL STUDY ON CARCINOGENICITY
Groups of albino mice of the strain Chbb = NMRI (SPF) were fed
bromophos in the diet for 18 months at dose levels of 0, 85, 350 and
1400 ppm to assess a carcinogenic potential in mice. The number of
mice was 100 males and 100 females in the control group and 50 males
and 50 females in each test group.
Body weights of males and females from the high-dose group were
slightly depressed. Food consumption showed no relevant differences
between the groups.
Plasma cholinesterase was markedly inhibited at all dosage levels.
Erythrocyte and brain cholinesterase inhibition was clear at 350 ppm
and 1400 ppm levels.
Tumours (including leukaemias) were detected in the control and the
three dose groups. The percentage of tumours per group was: 23% in the
control group, 13% in the 85 ppm group, 22% in the 350 ppm group, and
18% in the 1400 ppm group. Primary lung tumours were the most
frequent, followed by lymphocytic leukaemia, lymphosarcoma,
hepatocellular adenoma and reticulum cell neoplasms. Tumours observed
only in a few animals were: granulosa cell tumours of the ovaries,
adenoma and pheochromocytoma of the adrenals, cutis fibrosarcoma,
* In this connection, see also Stiasni, Rehbinder and Deckers,
J. Agr. Fd. Chem. 15: 474, 1967, who concluded that the distribution
pattern of bromophos shows no accumulation in the tissues.
urinary bladder carcinoma, intestinal adenocarcinoma and pleomorphic
sarcoma in the abdominal cavity. The tumours were found in the control
group and/or the three dose groups presenting no dose-related
distribution. Tumours occurred no earlier in the dose groups than in
the control group and there was no difference in the intercurrent
mortality rate. (Kreuzer et al., 1976).
Short-term studies
Dog
Groups of beagle dogs (4 males and 4 females/group) were fed bromophos
in the diet at dosage levels of 0, 20, 80, 320 and 1280 ppm for one
year. Bodyweight was comparable to control at levels of 0, 20, 80 and
320 ppm; a marked depression was observed at the 1280 ppm level. Food
consumption was reduced at the 1280 ppm level. No specific clinical
symptoms were observed and no mortality occurred over the course of
the study. Oestrus occurred less frequently than normal in the female
animals at the 1280 ppm level. There were no differences from control
values in the haematology and urine parameters examined. The clinical
chemistry studies revealed a slight reduction of calcium at the
highest dose and a slightly dose-dependent rise in the serum chloride
level at the 80, 320 and 1280 ppm groups. Total serum proteins were
reduced at the highest dose due to the fall in albumin. Plasma
cholinesterase inhibition occurred at 80, 320 and 1280 ppm levels.
Erythrocyte cholinesterase was inhibited at the 320 and 1280 ppm
levels. Brain cholinesterase inhibition was observed at the 1280 ppm
level. No significant dose-related changes were noted on gross and
histological examination of tissues. Pyelitis was observed in isolated
animals of all study groups. Unilateral atrophy of the germinal
epithelium of a group of tubules in the testis occurred in one dog of
the control group, two at the 80 ppm, one at the 320 ppm and one at
the 1280 ppm level. Focal lymphocytic infiltrates in the epididymis
occurred in one male dog at the 80 ppm and 320 ppm levels and in two
male dogs at the high level dose. Ophthalmological examination
revealed no pathological findings, (Herbst et al., 1975).
Acute Toxicity
TABLE 1. Acute Toxicity of bromophos
Route of LD50,
Species Sex Administration mg/kg Reference
Rat Oral 3750-5180 Jones, et al., 1968
Rat Oral 6000 Novozhilov, 1975
Rat M Oral 1600(1322-1936) Gaines, 1969
Rat F Oral 1730(1373-2180) Gaines, 1969
TABLE 1. (continued)
Route of LD50,
Species Sex Administration mg/kg Reference
Rat M Oral 4000 Kinkel et al., 1966
Rat F Oral. 6100 Kinkel et al., 1966
Rat Dermal 1625-3125 Kinkel et al., 1966
Rat M,F Dermal >5000 Gaines, 1969
Rat Subcut. 1460 Pallade et al., 1970
Mouse M Oral 3700-5850 Kinkel et al., 1966
Mouse F Oral 2829 Kinkel et al., 1966
Mouse Dermal 1040 Kinkel et al., 1966
Guinea Pig Oral 1500 Kinkel at al., 1966
Rabbit M Oral 720 Kinkel et al., 1966
Rabbit Dermal >1000 Jones et al., 1968
Rabbit Dermal 2181 Kinkel et al., 1966
OBSERVATIONS IN HUMANS
Bromophos was administered to four groups of four male and four female
volunteers at levels of 0, 0.2, 0.4 and 0.8 mg/kg body weight/day for
28 days. The material was given orally in capsules.
The lowest dosage did not influence the cholinesterase activity in
either erythrocytes or plasma when administered for 28 days. Higher
dosages inhibited the cholinesterase activity in plasma. This
inhibition showed a time dependence; at 14 and 28 days it was more
than 20% at 0.8 mg/kg bw/day.
The cholinesterase activity in erythrocytes did not show any
inhibition even at 0.8 mg/kg/day.
Checks of behaviour, general condition, appetite, stools, blood count,
clinico-biochemical parameters and ECG did not indicate any
intolerance reactions (Anon., 1977).
COMMENTS
Bromophos was previously evaluated and a temporary ADI for humans of
0-0.006 mg/kg was allocated.
An adequate study in dogs previously requested has been provided.
Renal function tests and testicular histology did not confirm previous
concern. Plasma cholinesterase was more susceptible to depression than
erythrocyte cholinesterase; however, the previous study revealed the
opposite. A no-effect level was demonstrated in the 20 ppm group.
A carcinogenic study was also previously requested and has become
available. In this long-term carcinogenicity study in mice, no
compound-related effect was observed.
A 28-day study in human volunteers showed no significant effect of
bromophos at a level of 0.4 mg/kg bw/day based on plasma
cholinesterase activity.
Attention was paid to the 2,5-dichloro-4-bromophenol part of the
bromphos molecule. The available information indicated that the
synthesis of this phenolic compound does not incur any risk of forming
p-dibenzodioxins.
As the required studies were provided and observations in humans were
available, a higher ADI for humans was allocated.
TOXICOLOGICAL EVALUATION
Level causing no toxicological effects
Dog: 20 mg/kg in diet, equivalent to 0.5 mg/kg bw
Humans: 0.4 (mg/kg bw)/day
ESTIMATE OF ACCEPTABLE DAILY INTAKE FOR HUMANS
0-0.04 mg/kg bw
RESIDUES IN FOOD AND THEIR EVALUATION
Residue aspects of bromophos were last evaluated by the 1975 Joint
Meeting (FAO/WHO, 1976). The Meeting then required residue data on fat
of meat of domestic animals other than sheep (including residues in
milk products, poultry and eggs) and on peanuts before additional
maximum residue limits could be recommended. Further data on residues
in stored wheat and on rice following storage and processing under
full-scale commercial conditions were recorded as desirable.
At the 9th Session of the Codex Committee on Pesticide Residues in
1977 (ALINORM 78/24, paras. 54-55):
1. The Netherlands delegation drew attention to the varying maximum
residue limits for the different kinds of currants. The Joint Meeting
was requested to review the proposed maximum residue limits for red
currants in the light of the proposed figures for blackberries and
blackcurrants.
2. The Netherlands delegation stated that the use of bromophos on
sugar beet could not only leave residues in the roots, but also on the
tops of the beet which were used extensively as cattle feed and,
therefore, could give rise to residues in meat and milk. The Joint
Meeting was requested to propose a maximum residue limit for meat in
the light of the use of bromophos on sugar beet.
No information was supplied concerning the requests of the 1975 Joint
Meeting or the 2nd request of the CCPR.
In response to the first request of the CCPR, the maximum residue
limits recommended by the 1972 Meeting were reviewed. The reported
residue data indicate residues of 0.18-0.36 mg/kg for blackcurrants,
0.14-0.19 mg/kg for blackberries and 0.11-0.76 mg/kg for red currants
7 days after the application of comparable dosages of bromophos. In
view of the range of variation in red currants, and since no further
residue data are available, a uniform MRL of 1 mg/kg for these fruits
is recommended.
The question was raised whether bromophos in grain causes taint. Wheat
treated with 8-12 mg bromophos/kg was milled 0-12 months after storage
and processed to bread. No taint was detectable and no differences in
odour or flavour from bread prepared from untreated wheat were
observed (Bisle and Deckers, 1974).
Information on residues in food moving in commerce was supplied by the
Netherlands (Food Inspection Services, 1977; Table 2).
RECOMMENDATIONS
The maximum residue limits for bromophos in blackberries and black
currants are raised to the limit previously recommended for red
currants.
Commodity Limit, mg/kg
Blackberries, currants
(red & black) 1
Table 2A. Residue of bromphos in food moving in commerce; Netherlands, 1976
lettuce celery spinach Brussels white butter-bean leek potato radish summer
sprouts cabbage carrot
SOURCE N N N N N N N N N N
Range,
mg/kg
0 - 0.05 4 4 1 24 1 0 3 0 0 0
0.051 -0.10 3 0 0 0
0.101 -0.50 0 1 2
0.501 -1.00 0
1.001 -1.50 2
>1.50 2
Total 4 4 1 24 1 3 3 4 1 2
Exceeding - - - - - - - - 4 - -
maximum
residue
limit
___ - National maximum residue limit
N - Produced in Netherlands
I - Imported
Table 2B. Residue of bromphos in food moving in commerce; Netherlands, 1976
winter pear currant strawberry mandarin
carrot
SOURCE N N N N I
Range,
mg/kg
0 -0.05 4 0 0 4 0
0.051 -0.10 0 0 0 1 3
0.101 -0.50 1 1 1 2 3
0.501 -1.00
1.001 -1.50
>1.50
Total 5 1 1 7 6
Exceeding - - - - -
maximum
residue
limit
___ - National maximum residue limit
N - Produced in Netherlands
I - Imported
FURTHER WORK OR INFORMATION
Desirable
1. Further information on residues in rice following storage and
processing under full-scale commercial conditions.
2. Information on the level and fate of bromophos residues in products
of animal origin.
REFERENCES*
Anonymous Influence of Bromophos pure on the Cholinoesterase-Activity
in Plasma and Erythrocytes of healthy volunteers when administered
orally. Unpublished report by Boehringer Sohn submitted to WHO by
Celamerck Co. (unpublished report). (1977)
Benes, V. and Stram, R. (1969) Ind. Med. Surg. 38:442.
Bisle and Deckers Organoleptische, Boehringer, Beurteilung von mit
Bromophos behandelten Weizen. Boehringer, Wissenschaftl. Abteilung,
Ingelheim, Federal Republic of Germany 17 May 1974. (1974)
Dedek, W. and Schwarz, H. (1969) Zeits. Naturforsch., B. 24:744.
FAO/WHO (1973) 1972 evaluations of some pesticide residues in food.
AGP:1972/M/9/1; WHO Pesticide Residues Series, No. 2.
FAO/WHO (1976) 1975 evaluations of some pesticide residues in food.
AGP:1975/M/13; WHO Pesticide Residues Series No. 5.
Food Inspection Services of the Netherlands 1976 (1977);
Bromophos-Residues in Food in Commerce, 25 October 1977.
Gaines T.B. (1969) Acute toxicity of pesticides. Toxicol. Appl.
Pharmacol. 14:515.
Jones, K.H., Sanderson, D.M. and Noakes, D.M. (1968)
World Rev. Pest. Control (London), 7:138.
Kinkel J., Mauacevic, G., Sehring, R. and Bodenstein, G. (1966)
Arch. Toxikol 22:36.
Kreuzer, H., Weibe, J., Guénard, J., Knappen, F. and Stötzer, H.
(1976) Carcinogenicity study with the substance bromophos in mice
using oral administration -- 80 weeks. Unpublished report by H.
Boehringer Sohn submitted to the WHO by Celamerck Co. (unpublished
report).
* In cases in which the original paper has appeared in very obscure
journals, references to Chemical Abstracts are given.
Pallade, S., Gabrielescu, E., London, M. and Siminovici, R. (1970)
Toxicity of some organophosphoric pesticides. Chem. Abstr. 73:97847.
Palut, D. (1974) Detoxication mechanisms of organic phosphorus and
carbamate insecticides. Chem. Abstr. 80:116892.
Palut, D., Grzymala, W. and Syrowatka, T. (1970) Metabolism of some
organophosphorus insecticides in animal model systems. II. Hydrolytic
mechanisms. Chem. Abstr. 72:120478.
Stenersen, J. (1969) Demethylation of the insecticide bromophos by a
glutathione-dependent liver enzyme and by alkaline buffers. J. Econ.
Entomol. 62:1043-45.
Stiasni, M., Deckers, W., Schmidt, K. and Simon, H. (1969) J. Agr.
Fd. Chem. 17:1017.
Szutkowski, M.M. (1975) Effect of a carbon tetrachloride on activation
and detoxification of organophosphorus insecticides in the rat.
Toxicol. Appl Pharmacol. 33:350-55.