IPCS INCHEM Home


    PESTICIDE RESIDUES IN FOOD - 1983


    Sponsored jointly by FAO and WHO






    EVALUATIONS 1983





    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
    Geneva, 5 - 14 December 1983

    Food and Agriculture Organization of the United Nations
    Rome 1985

    NITROFEN

    TOXICOLOGY 

    IDENTITY

    Chemical Name

    2,4-dichlorophenyl 4-nitrophenyl ether (IUPAC)

    2,4-dichloro-p-nitrophenyl ether

    Synonyms

    TOKR Herbicide, NIPR Herbicide, NIPDIAR Herbicide, NIPDINR
    Herbicide, NIPQ-PR Herbicide, FW925

    Common Names

    nitrofen (ISO), niclofen (Canada), nitrofene (France).

    Structural Formula

    STRUCTURAL FORMULA

    Molecular Formula

    C12H9O3Cl2N

    Other Information on Identify and Properties

    Molecular mass                         284. 11

    Physical state                         dark brown to black semi-solid
                                           (technical compound)

    Melting point                          70-71°C (pure compound)

    Specific density                       1.80 g/ml at 83°C
                                           (technical product)

    Vapour pressure                        2 x 10-6 mm Hg at 250°C

    Solubility (25°C) in

    - water                                about 1 mg/l
    - acetone                              about 25%
    - ethyl acetate                        50-60%
    - methanol                             about 25%
    - methylene chloride                   58-68%
    - xylene                               about 25%

    Stability                              hydrolitically stable but rapidly
                                           photodegraded in solution and on
                                           surfaces

    Purity of Technical Product

    nitrofen technical contains > 95% pure compound

    Formulations

    Nitrofen is primarily formulated as a liquid emulsifiable concentrate
    containing 25 percent active ingredient and a wettable powder
    containing 50 percent active ingredient. It is also available in
    combination with other herbicides and has been available as a granular
    formulation.

    EVALUATION FOR ACCEPTABLE DAILY INTAKE

    BIOCHEMICAL ASPECTS

    Absorption, Distribution, Excretion and Metabolism

    Rat

    Samples of urine and faeces from two experiments in which rats had
    been fed nitrophenyl ring - and dichlorophenyl ring-14C-labelled
    nitrofen, respectively, were used to determine the metabolic rate of
    nitrofen. Faeces contained approximately 75 percent and urine
    approximately 20 percent of the administered dose. Unmetabolized
    nitrofen was excreted in the faeces but not in the urine and
    represened ca. 36 percent of the total excretion. Nitrofen and 
    non-hydroxylated metabolites accounted for ca. 61 percent of the total
    of the excreted dose, with relatively higher levels in urine (28
    percent) than faecesœ

    Hydroxylation occurred in the dichlorophenyl ring. Unidentified polar
    metabolites (conjugates) were found in the urine. Cleavage of the
    diphenyl ether moiety was not observed (Roser et al. 1971).

    One male and one female albino rat were each given (by stomach tube)
    nine daily doses of 125 mg/kg b.w. 14C-nitrofen uniformly labelled
    in the nitrophenyl ring. After the dosing period, the rats were
    maintained on a nitrofen-free diet for two days, and then sacrificed.

    Recovery of total administered dose was 81.8 percent in the female
    rate and 94.2 percent in the male rat, with 54.1 percent and 75.6
    percent, respectively, appearing in the faeces and 18.0 percent and
    13.7 percent in the urine. Metabolism to 14CO2 occurred to a minimal
    extent (0.01 percent). A small portion (1.12 percent) of the
    administered dose of nitrofen was found in all tissues and organs, the
    highest concentrations of nitrofen and/or its metabolites were found

    in the fat (211.9 ppm), spleen (54.3 ppm), stomach (42.5 ppm, blood
    (40.7 ppm), kidneys and adrenals (58.1 ppm), liver (29.4 ppm) and skin
    and hair (26.2 ppm). (Adler et al. 1970a)

    Albino rats (one female and one male) were each given (by stomach
    tube) daily doses of nitrofen (uniformly 14C-labelled in the
    dichlorophenyl rins) at a level of 115 mg/kg b.w. for eight days.
    After the dosing period, the rats were maintained on a nitrofen-free
    diet for three days and were then sacrificed.

    Throughout the experiment, urine, faeces and exhaled CO2 were
    collected separately and were assayed for radioactivity, as were
    tissues removed from the male rat at sacrificeœ

    Recovery of total administered dose was 104 percent in the female rat
    and 106 percent in the male rat, with 73.9 percent and 80.0 percent,
    respectively, appearing in the faeces and 18.0 percent and 13.7
    percent in the urine. Metabolism to 14CO2 occurs to a minimal extens
    (0œ01 percent). Some radioactivity (0.89 percent) is found in all
    tissues, with the highest concentrations being found in the blood
    (21.9 ppm), fat (18.4 ppm), large intestine (13.5 ppm), liver (12.9
    ppm), kidney and adrenals (11.0 ppm) and skin and hair (10œ6 ppm).
    (Adler et al. 197Ob)

    Groups of weanling albino Wistar rats (25 males and 25 females per
    group) were fed diets containing technical grade (95 percent nitrofen
    at levels of 0, 10, 100 and 1 000 ppm) for 97 weeks.

    Tissues (fat, muscle, liver, kidney, blood) collected at week 97 and
    excreta (urine, faeces) collected at week 65, were pooled and analysed
    for nitrofen content by sas-chromatography.

    The concentration of nitrofen was highest in fat (20.6, 166.0 and
    814.0 ppm, respectively, at the three dietary levels tested), followed
    by muscle (0.92, 8.2 and 18.0 ppm), kidney (0.15, 3.2 and 9.0), blood
    (0.18, 0.92 and 3.4 ppm) and liver (0.04, 0.44 and <0.01). Excretion
    of unchanged nitrofen occurs almost exclusively in the faeces (35.0
    ppm at the highest level). (Ambrose et al. 1971) (Full experimental
    data were not reported.)

    In a comparative study of the fate of 14C-nitrofen, sets of two
    female and two male Osborne-Mendel rats and two female Fischer-344
    rats were fed diets containing technical nitrofen at levels of 60 or 
    1 300 ppm. After two weeks, the animals received a single oral pulse
    dose of 3 and 65 mg/kg b.w. uniformly dichlorophenyl-rins-labelled
    14C-nitrofen, respectively. The same treatment was repeated after
    four weeks of dietary nitrofen administration. Urine and faeces were
    collected daily after the first pulse dose until sacrifice. One animal
    of each strain from each dose level was sacrificed at 24 and 96h after
    the second 14C-nitrofen dose. Blood, liver, pancreas, fat and skin
    samples were analysed for radioactivity and metabolites. By 92h after
    the second pulse dose, the animals in all groups had eliminated
    between 3 and 22 percent of the 14C in the urine and between 30 and

    100 percent in the faeces. After the two-week exposure, the female
    Osborne-Mendel rats excreted 14C in the urine more rapidly than the
    males; both sexes of Osborne-Mendel rat excreted 14C more rapidly in
    the faeces than did the female Fischer rats. There were no obvious
    differences related to sex or strain in tissue concentrations, in
    tissue binding or in the metabolite profiles in urine, faeces or
    liver; some differences were noted in the pancreas. At 24h after
    dosing, concentrations in the blood, fat, liver, pancreas and skin of
    14C (calculated as nitrofen) were 0.15 to 0.25, 4.8 to 7.6, 0.5 to
    0.8, 1.1 to 1.4 and 0.5 to 0.9 ppm, respectively, for the 3 mg/kg dose
    groups and 2.7 to 4.1, 39 to 80, 6.2 to 14, 3.3 to 5.3 and 2.9 to 5.0
    ppm, respectively, for the 65 mg/kg dose groups. By 92h after dosing,
    the concentrations in the same tissues had declined to 0.06 to 0.09,
    3.2 to 4.2, 0.21 to 0.22, 0.3 to 1.4 and 0.4 to 0.8 ppm, respectively,
    for the 3 mg/kg groups and 1.6 to 2.2, 1.0 to 12.6, 1.3 to 2.5, 0.6 to
    2.1 and 0.3 to 0.9 ppm, respectively, for the 64 mg/kg groups. About
    14 to 29 percent and 22 to 42 percent of the 14C were bound in the
    liver of low-dose and high-dose animals, respectively, while 1 to 6
    percent and 16 to 36 percent were bound in the pancreas. The urinary
    metabolites were generally very hydrophillic. Traces of
    hydroxyaminonitrofen, acetamidonitrofen and aminonitrofen were
    observed but urinary metabolites were not identified further. No
    parent compound was detected; 33 percent of the total applied dose was
    excreted in the faeces as the parent compound, 14.4 percent as a polar
    unknown, 5 percent as acetamidonitrofen, 2.8 percent as 3'-hydroxy
    acetamidonitrofen, 2.8 percent as 4-hydroxyaminonitrofen, 2.4 percent
    as aminonitrofen and 0.2 percent as 5-hydroxynitrofen. With time, the
    concentrations of nitrofen, aminonitrofen and hydroxyaminonitrofen
    decreased, and the concentrations of the acetylated compounds
    increased. While the metabolic profile in the liver paralleled that in
    the urine, the pancreatic metabolites were more lipophilic and
    consisted exclusively of the parent compound in the 3 mg/kg groups,
    while the 65 ms/ks groups exhibited a variety of metabolites,
    including aminonitrofen, hydroxyaminonitrofen, hydroxynitrofen and
    2,4-dichloropheno) in addition to the 20 to 74 percent parent
    compound. Nitrofen appeared to accumulate to a greater extent in the
    pancreas of high-dose female Osborne-Mendel rats than in the pancreas
    of the males of this strain or the Osborne-Mendel female pancreas
    contained 2.23 ppm nitrofen while the pancreas of each of the other
    two groups averaged 0.83 and 1.08 ppm. This strain and sex difference
    was not seen in the 3 mg/kg animals which were also killed at 24h
    (1.07, 1.19 and 1.36 ppm nitrofen) (Steiserwalt et al. 1980a).

    FIGURE 1

    Groups of three female Sprague-Dawley rats received a single dermal
    application of 14C-nitrofen (uniformly labelled in the dichlorophenyl
    ring) as an undiluted emulsifiable concentrate at a dose level of 125
    or 375 mg/kg or as an aqueous dilution (ca. 1.8 percent a.i.) at
    levels of 9 or 27 mg/kg a.i.

    Urine and faeces were collected twice daily. One rat per group was
    sacrificed at 24, 48 and 96 h after dosing. Blood, liver and kidney
    were assayed for radioactivity.

    Less than 2 percent of the applied doses remained at the application
    sites after 96 h while only 30-40 percent of the dose was excreted in
    the urine (5-9 percent) and faeces (26-33 percent) during that time.
    Blood, liver and kidneys also contained radioactivity at all sacrifice
    times (Deckert & Steiserwalt 1979a).

    14C-nitrofen (uniformly labelled in the dichlorophenyl ring) was
    dermally applied as undiluted emulsifiable concentrate at a level of
    120 mg/kg to a female Sprague-Dawley rat. Urine, faeces and volatile
    materials were collected daily at 24 h intervals. Total 14C recovery
    in this study was 87 percent. At 96 h post-dosing, the rat was
    sacrificed and blood, organs and tissue specimens were removed for
    14C analysis. After 96 h 8.7 percent of the dose was excreted in
    urine and 36 percent in faeces. Only 0.02 percent of the dose was
    found as 14CO2, while other volatile materials accounted for 0.001
    percent. 14C was found in every tissue analysed. The highest
    concentrations were found in skin (17.6 percent of the dose), fat
    (484.5 ppm = 2.1 percent of the dose) and muscle at the application
    site (158.3 ppm = 0.9 percent of the dose), whereas only 0.4 percent
    of the dose remained unabsorbed. A total of 38 percent of the applied
    dose was found in tissues. This figure, when compared with the lower
    values found in oral studies, suggests that nitrofen may be better
    absorbed dermally than by the oral route (Deckert & Steiserwalt
    1979b).

    Groups of 12 pregnant Sprague-Dawley rats were treated dermally with
    14C nitrofen (uniformly labelled in the dichlorophenyl-ring) as
    diluted emulsifiable formulation at levels of 0.3 or 27 mg/kg b.w.
    a.i. for 6 h on day 11 of gestation. Another group received a single
    dose intravenously at a level of 0.3 mg/kg b.w.

    At the end of the six-hour application period, only 16 percent of the
    dermally applied dose could be removed by washing. Low-dose dermal
    application produced blood levels similar to those produced by i.v.
    administration, except at the one hour sampling. Urinary 14C
    excretion patterns were similar for both dermal application (low and
    high dose) and i.v. administration. However, 11 days after dosing, 15
    and 20 percent of the dermal (both levels) and i.v. doses,
    respectively, were excreted in urine. Foetal 14C excretion patterns
    were also similar for both routes of administration, with 45-48
    percent and 75 percent of the dose being excreted by 11 days after
    dermal and i.v.  admin-istration, respectively. After 11 days, 61-65
    percent and 97 percent of the dermal and i.v. doses, respectively, had

    been excreted. For the higher dermal dose, most maternal tissue 14C
    concentrations peaked one to two days after dosing (day 12-13 of
    gestation) with the highest concentrations occurring in the fat and
    gonads. At 0.3 mg/kg (both routes) heart, thryroid, adrenals,
    Harderian glands and pancreas approached maximum levels after six
    hours.

    Peak 14C levels of most maternal tissues increased with increasing
    dose increment and reached a higher level following i.v.
    administration than after a dermal application of 0.3 mg/kg.

    Placental and foetal 14-C concentrations also peaked within one to
    two days after dosing. At 0.3 mg/kg, mean placental 14-C levels were
    similar by either route, while the foetal 14-C level two days after
    dermal application was five-fold higher than those after i.v.
    administration, ten-fold higher than that found in maternal whole
    blood at the same time, but only three-fold lower than after the high
    dermal dose. At the low dermal dose, the peak or plateau placental
    and foetal 14C levels were comparable with those in maternal tissues
    (other than the fat and gonads). After i.v. administration and the
    high dermal dose, tissue levels in the mother were much higher than in
    the placenta or foetus.

    In the amniotic fluid, both 0.3 mg/kg dose groups showed the highest
    14C levels at 11 days after dosing (day 22 of gestation); the 
    high-dose dermal group had a steady high 14C level throughout this
    period.

    Eleven days after dermal application (27 mg/kg), 14C concentrations
    in maternal organs were generally higher than in foetal organs, except
    for the heart, where levels were nearly the same. Radioactivity was
    detected in foetal organs in descending order from kidney to heart,
    lung, liver and brain. Metabolic profiles in the placenta and the
    embryo/foetus showed the presence of nitrofen, aminonitrofen and other
    unidentified metabolites with peak or plateau concentrations occurring
    within one to two days. Concentrations of nitrofen and minor
    metabolites decreased with time, while aminonitrofen appeared to reach
    a plateau and then decrease at a slower rate. More than 90 percent of
    14 in the urine and faeces was readily extractable (Steiserwalt
    et al. 1982).

    Dog

    Groups of six-month-old beagles (two males and two females/group) were
    fed diets containing technical grade 95 percent pure) nitrofen at
    levels of 0, 20, 200 or 2 000 ppm for two years. Tissues (fat, muscle,
    bone, liver, kidney, spleen and blood) pooled at week 104 and excreta
    (urine and faeces) at week 103 were analysed for unchanged nitrofen by
    sas-chromatography. The nitrofen concentration was highest in body fat
    (38, 175, 515 ppm, respectively, at the three dietary levels tested),
    with lower levels found in bone (3.9, 19.2, 21.8 ppm), muscle (1.26,
    4.76, 20.0 ppm), kidney (1.4, 4.6, 13.6 ppm), liver (1.0, 2.0, 3.95
    ppm) and blood (0.1, 0.2, 0.4 ppm). Unchanged nitrofen was excreted

    almost exclusively in the faeces (39.0 ppm at the highest level)
    (Ambrose et al. 1971). (Full experimental data were not provided.)

    In vitro dermal absorption

    Skin sections from an adult female Sprague-Dawley rat and an adult
    woman (postmortem) were mounted in Franz Diffusion Cells and 5 µl of
    diluted 14C-nitrofen emulsifiable formulation (ca. 1.8 percent a.i.)
    was applied to each. After six hours of exposure, the rat skin
    absorbed from 24.7 to 61.7 percent of the 14C while the human skin
    absorbed from 13.8 to 25.3 percent of the label. The cell-by-cell
    average rat: human absorption ratio of 1.99+/-0.34 indicated that
    nitrofen was absorbed by human skin at approximately half the rate
    that it passed into rat skin (Steiserwalt et al. 1980b).

    TOXICOLOGICAL STUDIES

    Special Studies on Reproduction

    Rat

    In a three-generation, two-litter per generation, reproduction study
    groups of 28-day-old Wistar rats (25 males and 25 females/group) were
    fed diets containing technical grade (95 percent pure) nitrofen at
    levels of 0, 10, 100 or 1 000 ppm for 11 weeks. Body weights for
    parental rats before mating and at weaning of respective litters were
    not adversely affected for Fo and F1b generations. Body weight of
    F2b parent generation rats was slightly decreased for rats at 100
    ppm, in breeding the F3a litter and in breeding the F3b litter fed 10
    ppm. This decrease was due to lower initial body weights of rats used
    for succeeding generations. Although there was considerable
    inconsistency within and between matings, average data reported did
    not support a dose-related effect on fertility and gestation indices.
    Viability and lactation indices were not adversely affected at 100
    ppm. However, the viability index for rats at 1 000 ppm was seriously
    affected, as stillborn pups outnumbered live pups; indeed no pups were
    available for continuance beyond the Fo generation. In all
    generations, the number of stillborn pups from the 100 ppm group
    numbered 64 compared with 30 in the control.

    No structural or microscopic cellular abnormalities were detected in
    any stillborn pups in F1b litters. Weaning weights indicated no 
    diet-related trends. Overall, only the 10 ppm dietary level of
    nitrofen exhibited no adverse effects on reproduction. Examination of
    a variety of tissues from 10 F3b generation rats of each sex from the
    0, 10 and 100 ppm dietary levels showed no histopathological lesions
    (Ambrose et al. 1971). (Full experimental data were not provided.)

    Groups of Sherman rats (10 males and 20 females/group) were fed
    dietary levels of 0, 20, 100 and 500 ppm technical nitrofen (89
    percent) pure). Breeding and F1a and F1b litters commenced at 68 and
    200 days, respectively. Offspring were observed through weaning. At
    the time of weaning of the F1b litters, 20 female and ten male

    weanlings were selected from the 0, 20 and 100 ppm groups, continued
    on the parental diet and pair-mated when 112 days old to produce the
    F2a generation. Neither the food consumption nor the body weight gain
    were greatly affected in any of the nitrofen-treated groups. At the
    500 ppm dietary level no offspring of the F1a and F1b generation
    survived the neonatal period. The number of pups born alive and the 
    survivors-to-weaning were reduced at the 100 ppm dietary level but not
    at 20 ppm (equal to 1.1 and 1.8 mg/kg b.w. in F1b and F1a,
    respectively) (Kimbrough et al. 1974). (Full experimental data were
    not provided.)

    Groups of 25 male Sprague-Dawley rats were fed diets containing 0,
    100, 500 or 2 500 ppm of nitrofen technical (95.7 percent pure) for 95
    days. At that time, male and untreated female rats were cohabited
    (1:1) for a maximum of 10 days. During co-habitation, males were fed
    an untreated diet and daily doses of 0, 6, 30 or 155 mg/kg of nitrofen
    were administered by gavage. The nitrofen dosage was based on the
    dietary intake of the compound (mg/kg/day) during weeks 10-13 of the
    study.

    No significant effects were detected on the reproductive performance
    of male as indicated by the average number of days for mating to
    occur, percentage of females impregnated or number of mated females
    that delivered. Furthermore, no differences were noted in the length
    of gestation, litter size, percentage of live or dead births or sex
    ratio of the offspring. No compound-related signs of toxicity were
    evident in any of the progeny from birth to day 35. Necropsies of
    offspring that died and of female parents which were killed after
    lactation revealed no compound-related gross abnormalities. Neither
    viability nor mean weights of offspring were affected at any dose
    level. Male reproductive performance was not affected at dietary
    concentrations up to and including 2 500 ppm, equal to 186 mg/kg/day
    (O'Hara et al. 1983).

    Special Studies on Teratogenicity and Postnatal Effects

    Mouse

    Two studies on postnatal dose response and standard teratology are
    summarized together.

    Purified nitrofen (99.6 percent pure) in maize oil was administered to
    CD-1 mice by gavage. In the postnatal study, 24 animals were dosed
    (50, 100, 150 and 200 mg/kg/day) on days 7-17 of gestation and allowed
    to give birth. In the teratology study animals were dosed 50, 100 and
    200 mg/kg/day) on days 7-17 and sacrificed on day 18. Foetuses were
    removed and analysed for visceral and skeletal malformations.
    Concomitant control groups were utilized in all the studies.

    In the postnatal study there was a significant dose-related delay in
    time of birth. Significant dose-related reductions in growth and
    viability were noted at all dose levels. There was considerable litter
    mortality in the 100 mg/kg/day groups between days 3 and 17
    postpartum. Only 23 percent of the pups alive on day 3 were still
    alive by day 17. Mortality in the 50 mg/kg/day dose group was not
    different from control values during this period. Microphthalmia or
    anophthalmia was seen in a high percentage of the animals in the 100
    mg/kg/day dose group. A dose-related growth retardation was noted in
    pups until 60 days of age at 50 mg/kg and above. There were delays in
    the percent of female offspring showing vaginal opening at 30 days and
    in the percentage of animals breeding by 60 days at 50 mg/kg and
    above. Litter size was unaffected by treatment.

    In the teratology study dose-related increases in maternal weight gain
    and liver/ body weight ratios were recorded. Nitrofen significantly
    increased the supraoccipital scores, indicating retarded skeletal
    development of the skull. It was teratogenic at 100 and 200 mg/kg/day,
    the most common defects noted being cleft palate and undescended
    testes (Chernoff et al. 1980). (Full experimental data were not
    available.)

    After being dosed at levels of 100 mg/kg b.w. nitrofen on days 7 to 17
    of gestation, pregnant CD-1 mice showed reduced mean litter size.
    Surviving pups had delayed eye opening and absence of the Harderian
    gland (Gray et al. 1982a).

    Groups of pregnant CD-1 mice were dosed by gavage with nitrofen (99.6
    percent pure) in maize oil at levels of 0, 6.25, 12.5, 25 and 50
    mg/kg/day. There were 23, 19, 27, 21 and 17 litters, respectively, on
    day 1. The animals were observed until necropsy at 110-130 days of
    age.

    The administration of nitrofen did not cause any adverse effects in
    the dams, as measured by maternal viability or weight gain from days 7
    to 19 of gestation. By day 3 the number of pups was reduced
    significantly at 50 mg/kg (20 percent). Body weights were initially
    unaffected after birth at 50 mg/kg but subsequent body weight was
    reduced in the pups from the 12.5 mg/kg treatment group. There was a
    dose-related increase in the frequency of lethal defects in the pups,
    i.e. cleft palate, diaphragmatic hernia and extreme abdominal
    distention. Lung weight was significantly reduced by nitrofen
    treatment at all doses, including the lowest dose of 6.25 mg/kg.
    Seminal vesicle weights were significantly reduced at 6.25, 12.5 and
    50 mg/kg. Obvious intraorbital defects were present in some offspring
    in the 25 mg/kg group and above. Prenatal nitrofen treatment
    significantly reduced the weight of the Harderian glands at all doses,
    including the lowest dose of 6.25 mg/kg. Prenatal nitrofen treatment
    of 50 mg/kg significantly delayed puberty in the female mice, as
    indicated by the percentage of females with patent vasina on day 30.
    In addition, the 50 mg/kg females paired with treated males produced
    significantly fewer pups in the second litter.

    A cross-fostering experiment with 100 mg/kg demonstrated that the
    defects noted in the present study were produced solely by prenatal
    exposure; pups exposed to nitrofen in the milk alone as a consequence
    of any accumulation of nitrofen in the dam during gestation were
    unaffected (Gray et al. 1983).

    Rat

    Groups of 26 pregnant FDRL rats were administered nitrofen by gavage
    on days 6-16 of gestation. The control animals received maize oil (1
    ml/kg b.w.) whereas the test animals received 100 and 200 ppm of
    nitrofen in the same volume of vehicle. Thus the dosage corresponds to
    5 and 10 mg/kg b.w./day, respectively. On day 22, the animals were
    sacrificed and Caesarean section performed. There were no differences
    between control and treated groups with respect to mean dam weight,
    implantation sites, foetal viability or mean live foetal weight.
    Resorptions were increased in the high-dose group. No malformed
    foetuses were seen at the time of Caesarean sections, nor was there
    evidence of soft tissue anomalies. Numbers of incomplete cranial bone
    ossification and scoliosis were slightly increased in both treated
    groups. These variations were considered "consistent with changes
    previously observed in the colony of rats" and, thus, not 
    treatment-related (Vosin 1971).

    Groups of seven pregnant Wistar rats were administered by gavage daily
    doses of nitrofen technical (96.2 percent pure) at levels of 68 mg/kg
    b.w. on days 6 through 15 of gestation. Five pregnant control rats
    received only peanut oil. Parameters evaluated were neonatal mortality
    and histomorphology of the lungs of dead and moribund newborns in the
    nitrofen-treated group and of four-day-old survivors from the control
    and treated groups. All the offspring from the control group were
    alive at birth. Most (85 percent) of the offspring from the treated
    group were born dead or died within the first few hours after delivery
    and exhibited generalized cyanosis. Histological examinations of the
    lungs of moribund offspring from the treated group revealed a large
    number of alveoli bordered by a cubical epithelium (Siou 1979b).

    Groups of 9-12 pregnant Sherman rats were dosed by oral intubation on
    days 7 through 15 of gestation. Dosage levels were 0 (peanut oil), 10,
    20 or 50 mg/kg/day for technical nitrofen (89 percent pure); 0, 20, or
    50 mg/kg/day for purified nitrofen (99 percent pure); and 0 or 0.04
    mg/kg/day for 2,7-dichlorodibenzo-p-dioxins (DCDD) (this dose is
    equivalent to a contamination level of 2 000 ppm (0.2 percent) in the
    technical nitrofen for a 20 mg/kg dose). The pups were observed to
    weaning. At the dosage levels of 20 and 50 mg/kg/day a dose-related
    decrease of live-born rats was observed for both the technical and
    purified nitrofen groups. None of the pups survived at the 50 mg/kg
    dose level and survival-to-weaning was severely reduced (50 percent)
    in both the technical and purified nitrofen groups. No effect of
    technical nitrofen on the number of live pups born and their 
    survival-to-weaning was observed at 10 mg/kg dose level. Extensive
    inflammation, fibrosis and epithelial proliferation of the lung were

    seen in sucklings that died at 11 to 15 days. No effect on 
    survival-to-weaning was observed in DCDD-treated rats.

    Additional groups of ten female and ten male rats were fed technical
    nitrofen at dietary levels of 0 and 500 ppm. The rats were started on
    the diet at weaning and pair-mated when they were 90 to 100 days old.
    On day 20 of pregnancy, foetuses were removed by Caesarean section and
    examined for malformations. The live foetuses were kept in an 
    incubator for six hours and closely checked for viability. Most of the
    foetuses of the controls were pink and survived until they were killed
    after six hours. The pups from nitrofen-treated dams were cyanotic and
    died one to three hours following the Caesarean section.

    Two more pups of pregnant rats were given by oral intubation technical
    nitrofen at levels of 0 or 50 mg/kg/day on days 7 to 18 of gestation.
    The foetuses from these rats were removed by Caesarean section on day
    21 of pregnancy. Lungs from 18 exposed and 16 control foetuses from
    four litters in each group were removed one to three hours following
    the Caesarean section and studied with light- and electron-microscopy.
    Pups from treated dams showed cyanosis at birth. The cyanosis
    increased during the next 45 to 60 minutes and most of the pups from
    treated dams died, while the control rats developed pink colour soon
    after birth and survived. Lungs of treated foetuses were poorly
    expanded and alveolar epithelium was cuboidal and resembled cells of
    earlier gestational age (Kimbrough et al. 1974).

    When purified nitrofen (containing only trace amounts (ppm) of 
    4-aminonitrofen) was administered to pregnant Long-Evans rats at
    different days of gestation, gestation day 11 was demonstrated as
    critical: a single dose of 150 mg/kg on day 11 killed 56 percent of
    the newborn pups withing 48 h.

    Groups of 4-7 Long-Evans dams were given a single dose of purified
    nitrofen at levels of 0, 75, 115, 150, 200 or 250 mg/kg on day 11 of
    gestation. Survival of neonates to five days was monitored. A 
    dose-related increase in neonatal mortality was observed. The LD50
    for the neonates was estimated at 116 mg/kg of maternal body weight.
    Initial experiments demonstrated that animals surviving the first five
    days usually lived to maturity. Thus, the mortality is most accurately
    described by the fraction dying within the first five postnatal days.
    Surviving offspring were autopsied at 35 days of age and examined for
    gross lesions. The predominant lesion observed in all treated groups
    was hydronephrosis. The incidence of hydronephrosis in survivors
    paralleled the neonatal mortality and the lesion was most frequent
    among animals exposed on day 11. The incidence of hydronephrosis was
    also dose-related among animals exposed only on day 11. 
    Nitrofen-induced hydronephrosis was not considered incompatible with
    life.

    In a further teratology study purified nitrofen was administered to
    pregnant Long-Evans rats at dose levels of 0, 70, 115, 150, 250, 265
    and 400 mg/kg b.w. on day 11 of gestation and foetuses collected on
    day 22. Foetuses exposed to nitrofen and examined at term (day 22),

    showed delayed skeletal ossification. There was a dose-related
    depression in foetal body weight. Nitrofen exposure on day 11 induced
    hydronephrosis, which was dose-related in incidence and severity.
    Diaphragmatic hernias were also observed in the treated groups. The
    overall frequency of soft tissue anomalies was also dose-dependent.
    Mean litter frequencies of these anomalies ranged from 45 percent at
    LD15 (70 mg/kg) to 91 percent at LD99+ (400 mg/kg). Nitrofen exposure
    produced a dose-related increase in the frequency of cardiac
    malformations, which included in order of decreasing frequency:
    ventricular septal defects (VSD), double outlet right ventricules
    (DORV), and transposition of great vessels. Incidence of lethal
    cardiac malformations was 38 percent and 53 percent at 150 mg/kg and
    250 mg/kg, respectively. No anomalies were seen in the control
    litters. None of the cardiac or diaphragmatic defects in the term
    foetuses were present in survivors, suggesting that the heart and
    diaphragm are the target organs in nitrofen-induced neonatal deaths
    (Costlow & Manson 1981).

    Groups of five pregnant Sprague-Dawley rats were treated with nitrofen
    (98 percent pure) at 0, 20, 31.2 or 50 mg/kg/day by gavage on days 
    8-18 of gestation. Significant differences in birth weights were
    apparent in pups from the 31.2 and 50 mg/kg/day groups. A decrease in
    the lung to body weight ratio of foetuses exposed to nitrofen was also
    observed. Most, if not all, pups were born alive and lacked
    externally-observable malformations. Within minutes after birth,
    treated pups exhibited laboured breathing and became cyanotic. Death
    usually ensued 30 min. to 4 h postpartum. Survival was significantly
    depressed in all treatment groups and all time intervals (1 h, 24 h,
    25 days) measured after birth. On examination, symptomatic 
    nitrofen-exposed pups suffered massive atelectasis at autopsy, with
    few expanded air sacs in the 31.2 and 50 mg/kg groups. In contrast,
    lungs from newborn controls exhibited normal alveolar expansion. The
    level of glucocorticoids and capacity of the foetal lung to respond to
    glucocorticoids by synthesizing and releasing surfactant were not
    affected by prenatal exposure to nitrofen (Stone & Manson 1981).

    Missing and/or malfunctioning Harderian glands were observed when
    pregnant Sprague-Dawley CD rats were given daily doses of nitrofen at
    a level of 12.5 mg/kg by gavage on days 8 to 16 of gestation (Gray 
    et al. 1982a).

    A postnatal study, using rats (strain unknown) dosed on days 7-16 at
    five doses from 0 to 25 mg/kg, demonstrated that nitrofen caused
    diaphragmatic hernias at 1.39 mg/kg and above and affected the
    haemoglobin level at 12.5 mg/kg (Gray et al. 1982b). (This
    information was provided in abstract form and cannot be further
    interpreted.)

    Groups of five pregnant CD rats were given by gavage daily doses of
    nitrofen at levels of 0, 12.5 or 25 mg/kg b.w. on days 8 to 16 of
    gestation. Dams were sacrificed on day 21 of gestation and foetuses
    removed for examinations. Dose-related increase in foetal mortality
    and dose-related decrease of mean foetal weight were determined.
    Diaphragmatic hernias occurred in both dose groups in a dose-related
    fashion. Brain weight and brain DNA were statistically significantly
    reduced at 25 mg/kg; lung weight and lung surfactants were reduced at
    12.5 and 25.0 mg/kg in a dose-related fashion as were liver weight,
    liver glycogen content, kidney weight and kidney alkaline phosphatase
    levels. However, statistical analysis demonstrated that only effects
    on lung and liver represented specific organ toxicity, while brain and
    kidney effects were correlated to the foetal growth retardation
    (Kavlock et al. 1982).

    When pregnant Sprague-Dawley rats (35/group) were exposed dermally to
    nitrofen emulsion at 0, 0.3, 3.0 or 30.0 mg/kg/day on days 6 to 15 of
    gestation, foetal weight and foetal length were significantly lower in
    the 30.0 mg/kg group compared to the control group. Soft tissue
    anomalies (hydronephrosis, ectopic testes, liver and/or stomach and/or
    intestine protruding into thorax, diaphragmatic hernia, right-sided
    aorta) were observed almost exclusively in the foetuses of the 30.0
    mg/kg group. The foetal viability (live at birth/implantations) in all
    the treated groups was statistically significantly lower than the
    control litters. Neonatal survival index (live at day 4/live at birth)
    was significantly lower in the 30.0 mg/kg group than in the control
    group (Weatherholtz 1979).

    Groups of five to seven pregnant Charles River rats were treated
    dermally with nitrofen (as a water-diluted emulsifiable formulation)
    at levels of 0, 0.6, 4.8 or 19.2 mg/kg/day on days 6 to 15 of
    gestation. A further group received dermally 4.8 mg/kg on day 11 only.
    Dams were sacrificed on day 21 postpartum. Surviving offspring were
    sacrificed on day 27. All maternal rats survived to sacrifice.
    Treatment of pregnant rats with 19.2 mg/kg/day of nitrofen on days 6
    to 15 of gestation resulted in slightly decreased weight gain during
    the treatment interval. The no-observable effect level (NOEL) was 4.8
    mg/kg/day. The treatment caused decreased viability of offspring,
    decreased neonatal survival to day 4 and decreased live offspring of
    pregnant rats treated dermally with 19.2 mg/kg/day of nitrofen on days
    6 to 15 of gestation. Nitrofen did not affect neonatal viability,
    neonatal survival, survival to weaning, growth of offspring or eye
    opening in offspring of dams treated with 0.6 or 4.8 mg/kg/day on days
    6 to 15 of gestation or 4.8 mg/kg/day on day 11 of gestation
    (Hirsekorn & Kane 1981).

    Groups of 25 pregnant Sprague-Dawley rats were treated dermally with
    nitrofen in aqueous emulsion (0, 0.3, 0.6, 1.2 or 12.0 mg/kg b.w./day)
    on days 6 to 15 of gestation. No maternal toxicity occurred. At 12
    mg/kg neonatal survival was reduced. Necropsies of the dead neonates
    showed a high incidence of diaphragmatic hernias. The number of pups
    per litter and viability over days 4-41 of age were unaffected by
    treatment. About half (randomly selected) of the pups from each litter

    and group were necropsied on day 42. Chromo-dacryorrhea, reduced or
    absent Harderian glands and diaphragmatic hernias occurred at 12.0
    mg/kg. The frequency and the severity of dilation of the renal
    pelvices increased with the dose; slight dilation was significantly
    elevated at 0.3 mg/kg and moderate and severe dilation became
    prominent at 0.6 and 12.0 mg/kg, respectively. In the 12.0 mg/kg group
    nitrofen reduced the body weight of the survivors and also the
    relative weights of the Harderian gland, the thyroid gland of both
    sexes; lungs of females were significantly depressed with respect to
    solvent and water control groups. The findings from necropsies
    performed on the remaining offspring on days 146 to 149 postpartum
    were similar to those at day 42. Body weight was unaffected by
    nitrofen exposure but dilated kidneys and diaphragmatic hernias were
    observed. Relative thyroid weight was significantly depressed only in
    the 12 mg/kg dose group. A NOEL was not demonstrated (Costlow et al.
    1983).

    Rabbit

    Groups of 15 pregnant New Zealand rabbits were given encapsulated
    technical nitrofen (96.2 percent pure) at levels of 0, 7, 27 or 108
    mg/kg on days 6 through 18 of gestation. Ten rabbits in each group
    were sacrificed on day 28 of gestation to assess embryotoxic and
    teratogenic effects. The average number of live foetuses and
    implantation sites per pregnant female was slightly decreased in the
    108 mg/kg group sacrificed on day 28 of gestation. Percentage of dead
    foetuses with respect to implantation sites was increased in all
    treated groups compared with the control group, but not in a 
    dose-related manner. The increased incidence of foetuses, with 13
    pairs of ribs was dose-related. No other anomalies were observed. The
    remaining five rabbits in each group were allowed to deliver, and they
    and the newborns were sacrificed 48 h after delivery. The average
    number of live offspring was slightly lower in the 108 mg/kg group.
    Average weights of live foetuses after 48 h were slightly lower in the
    treated groups, but not in a dose-related manner. No significant
    differences were observed in survival rate to 48 h or in resorption
    rates. An increased incidence of 13 ribs was observed in all treated
    groups. Histological examinations of the lungs removed from between 5
    and 17 of the 28-day foetuses and the 48-hour newborns in each group
    did not indicate retardation of lung maturation (Siou 1979a).

    Special Studies on Mutagenicity

    Microbial systems

    Nitrofen, when tested with Salmonella typhimurium strains TA1535,
    TA1538, TA100 and TA98, both with and without S-9 activation, was
    found only moderately mutagenic in TA100 with microsomal enzyme
    activation (Byeon et al. 1976).

    Purified nitrofen (99+ percent) and several samples of nitrofen
    technical (90-98 percent pure) were tested at concentrations of 
    0.001-10.0 µg/plate with S. typhimurium strains TA1535, TA1537 TA98
    and TA100. Tests were carried out both with and without microsomal
    activation. Positive controls were used: 2-aminofluorene and 
    2-acetamidofluorene for TA98 and 2-anthramine for all the strains
    tested. Only one sample of nitrofen technical (90-92 percent pure) was
    completely negative in the test. The other samples of technical
    nitrofen gave statistically significant increases of mutants/plate
    compared with negative control in TA100 strain, both with and without
    activation, but only one was positive in TA98 with activation.
    However, a dose-response relationship was observed only with three
    samples (97, 97 and 98 percent pure), but not with another three (93,
    92.8 and 97.7 percent pure), generally at concentrations of 10-10 000
    µg/plate. Purified nitrofen (99+ percent pure) gave a statistically
    significant increase of revertants/plate in TA100, both with and
    without activation; no dose-response relationship was observed.

    Thus purified nitrofen and three technical samples gave an indication
    of mutagenicity, but the other three technical samples were positive
    (O'Neill & Scribner 1979).

    Nitrofen was found mutagenic in a reversion test using 
    Saccharomyces cereviseae strain 632/4 (auxotrophic for methionine)
    (Guerzoni et al. 1976).

    Mammalian systems

    The mutagenicity of nitrofen was assayed in the murine lymphoma cell
    line L5178Y at concentrations of 0.1 x 10 (E-4), or 2 x 10 (E-4) M.
    Doses were selected as those killing approximately 40 and 80 percent
    of cells. Ethylmethanesulphonate was used as a positive control.
    Induction of methotrexate-resistant mutants of L5178Y cells treated
    in vitro with nitrofen was not significantly different from the
    negative control (Paik & Lee 1977b).

    Nitrofen was tested in the unscheduled DNA repair synthesis assay with
    human lymphocytes (concentrations not clear). Nitromin was used as a
    positive control. 3H-thymidine incorporation in the nitrofen-treated
    cells was not significantly different from the negative control (Paik
    & Lee 1977b).

    Nitrofen was also tested in mouse cells in a BALB/3T3 in vitro
    transformation assay. The compound did not induce a significant
    increase in transformed foci over the applied concentration range of
    0.1 to 60 micrograms/ml. This range corresponding to approximately 50
    to 90 percent survival in the cytotoxicity tests. A negative control
    consisting of assays performed on untreated cells was included. A
    positive control with 3-methyl cholanthrene (MCA), used at level of 5
    µg/ml for each assay, gave a marked increase of the average
    number/flask of foci of transformed cells. Therefore, nitrofen was
    considered to be inactive in the BALB/3T3 in vitro transformation
    assay (Myhr & Brusick 1979).

    Groups of three Swiss Webster albino mice were given intraperitoneally
    two doses, separated by 24 h, of nitrofen (purity not specified) at
    levels of 0, 500 or 1 000 mg/kg b.w. Six hours after the last dose,
    three bone marrow smears from each mouse were prepared and about 2 000
    polychromatic erythrocytes per mouse were analysed for the presence of
    micronuclei. For both dose levels tested, the incidence of micronuclei
    was not statistically significantly different from the control. A
    positive control of cyclophosphamide gave the expected increase of the
    incidence of micronuclei (Paik & Lee 1977a).

    Groups of 5-10 male Swiss mice were given by gavage two doses of
    nitrofen technical (96.2 percent pure) at levels of 0, 0.25, 1.00 and
    1.25 ml/kg, spaced 24 h apart. The animals were sacrificed 6 h after
    the second dose and bone marrow smears were prepared. The incidence of
    polychromatic erythrocytes carrying "micronuclei" in the treated
    groups was not significantly different from the control group (Siou
    1978).

    Groups of 24 male Charles-River CD-1 mice were given by gavage a
    single oral dose of nitrofen technical (95.7 percent pure) at levels
    of 0, 0.39, 0.79 and 1.58 g/kg, representing 1/8, 1/4 and 1/2 of the
    acute oral LD50. Additional groups of 8 male mice were given once
    daily five doses of nitrofen at the same levels. A positive control
    group received triethylamine (TEM) 0.3 mg/kg i.p. Bone marrow slides
    were prepared from 8 animals/group sacrificed at 6, 24 and 48 h after
    the acute dose and 6 h after the last sub-acute dose and TEM dose. All
    animals received colchicine at 2 mg/kg i.m. two hours prior to
    sacrifice. Fifty metaphase cells per animal were scored when possible.
    Owing to poor survival, the high dose subacute group was sacrificed on
    day 4. No statistically significant increase in chromosomal
    aberrations was observed in treated animals compared with controls,
    both in the acute and subacute regimen (Reustle & Scribner 1980).

    Nitrofen technical (95.7 percent pure) was administered in m-xylene
    (99 percent) by dermal application to groups of eight male Charles
    River CD rats at doses of 0, 0.05, 0.125 and 0.50 g/kg, to assess the
    potential of nitrofen to induce chromosomal aberrations in mammalian
    bone marrow cells. A positive control group received a single 0.3
    mg/kg i.p. dose of triethylenemelamine. The doses were administered
    according to both a single and repeated (daily × five days) regimen.
    Animals were killed and bone marrow slides prepared at approximately
    6, 24 and 48 h after the single dose, 24 h after the positive control
    dose and 6 h after the last multiple dose. All animals received
    colchicine (1 mg/kg, i.p.) three hours prior to being killed.
    Metaphase cells (50 per animal) were examined for chromosomal
    aberrations. Nitrofen at 0.5 g/kg and below did not induce a
    significant increase in chromosomal aberrations in bone marrow cells
    at either 6, 24 or 48 h after single or repeated exposure. Chromosomal
    aberrations were consistently elevated at 24 h after treatment with
    triethylmelamine (McLeod & McCarthy 1982).

    Special Studies on Mutagenicity of Aminonitrofen

    Aminonitrofen (99+ percent pure), a metabolite of nitrofen, was tested
    in concentrations of 0.1-1 000 µg/plate with S. typhimurium strains
    TA1535, TA1537, TA98 and TA100 with and without a microsomal enzyme
    preparation. Statistically significant increases of revertants/plate
    were observed with strains TA1535, TA98 and TA100 using a microsomal
    enzyme preparation and a dose-response relationship was exhibited.
    Positive controls were 2-anthramine, 2-aminofluorene and 
    2-acetamidofluorene (O'Neill & Scribner 1979).

    Special Studies on Mutagenicity of 4,4'-Dichloroazobenzene

    4,4'-Dichloroazobenzene (DCAB) (99+ percent technical), a low level
    impurity in technical nitrofen, was tested with the S. typhimurium
    strains TA1535, TA1537, TA98 and TA100 at concentrations of 1.0-1 000
    µg/plate, with and without microsomal activation. Saline buffer
    controls were used with each strain. Statistically significant
    increases of revertants/plate were observed with strain TA100 at 
    10-1 000 µg/plate with metabolic activation. DCAB gave a greater
    response with strain TA98 than with TA100. Positive metabolic
    activation; and 2-anthramine (10 µg/plate), positive with TA1535,
    TA1537 and TA100 with metabolic activation; and 2-acetamidofluorene
    (50 µg/plate), positive with TA98 with metabolic activation (O'Neill &
    Lohse 1980).

    Special Studies on Carcinogenicity

    Mouse

    Groups of 50 male and 50 female young B6C3F1 mice (age not specified)
    were fed diets containing technical nitrofen (stated to be >80
    percent pure; impurities unspecified) dissolved in maize oil for 78
    weeks at levels of 1 775-2 500 mg/kg (low-dose) or 3 550-5 000 mg/kg 
    (high-dose), to give time-weighted average concentrations of 2 348 and
    4 696 mg/kg in the diet. A group of 20 male and 20 female controls
    received the basal diet containing 2 percent maize oil. All mice were
    observed for a further 12 weeks after treatment. Weight gain was
    depressed in both groups of treated females and in the males exposed
    to the high dose.  Beginning at week 54, pronounced abdominal
    distension was displayed by an increasing number of treated mice.  By
    the end of the study, only 10 percent of the control male mice, 34
    percent of the high-dose group and 54 percent of the low-dose group
    were still alive; and, of the female mice, 62 percent were still alive
    in the high-dose group, 54 percent in the low-dose group and 85
    percent of the controls. For neither sex could a positive association
    be established between dosage of nitrofen and mortality. A high
    incidence of hepatocellular carcinomas was found in exposed male and
    female mice: in 4/20 control males, 36/49 low-dose males and 46/48
    high-dose males, in none of the control female mice (both matched and
    pooled controls), in 36/41 low-dose females and in 43/44 high-dose
    females. A few of these tumours metastasized to other sites. The

    difference in incidence between control and exposed male and female
    mice was statistically significant at both dose levels.
    Haemangiosarcomas of the spleen were found in 2/47 of high-dose males
    and haemangiosarcomas of the liver in 1/49 low-dose males and 2/48
    high-dose males. In female mice, haemangiosarcomas were seen in the
    spleen in 1/18 controls, in the liver in 4/44 high-dose animals and in
    the abdominal cavity in 1/43 of the high-dose group.

    The incidence of haemangiosarcomas in the high-dose male mice, but not
    females or low-dose males, was statistically higher than controls
    (National Cancer Institute 1978).

    Groups of 50 male and 50 female six-week-old B6C3F1 mice were fed
    diets containing 3 000 or 6 000 mg/kg technical-grade nitrofen (purity
    not specified) for 78 weeks. The 20 male and 20 female controls
    received the basal diet. All animals received acidified (pH 2.5) water
    ad libitum. After exposure to nitrofen, animals were observed for a
    further 13 weeks. Consistent dose-related depressions in mean body
    weight were noted in male and female mice, reaching about 25 percent
    in high-dose males by the end of the study. At that time, 40/50 of the
    high-dose males, 48/50 of the low-dose males and 19/20 of the control
    males were still alive; of the females, 48/50 of the high-dose and
    43/50 of the low-dose group and 12/20 off the controls survived until
    the end of the study. Although a variety of tumours were noted, only
    the incidence of liver tumours seemed to be related to nitrofen
    exposure. Hepatocellular adenomas were seen in 1/20 control males,
    18/49 low-dose males and 20/48 high-dose males, and hepatocellular
    carcinomas in 0/20 controls, 13/49 low-dose males and 20/48 of the
    high-dose group. In addition, none of the controls, but 3/49 of the
    low-dose group and 4/48 of the high-dose group had hepatoblastoma, a
    very rare tumour in this strain of mice. Of the females, 0/18
    controls, 9/48 low-dose and 17/5 high-dose mice had hepatocellular
    adenomas; hepatocellular carcinomas were observed in 0/18 control,
    5/48 low-dose and 13/50 high-dose female mice. In addition, a
    hepatoblastoma was found in 1/48 females in the low-dose group.

    For both the low- and high-dose groups there was a statistically
    significant increase from the control group in the incidences of
    hepatocellular adenomas and hepatocellular carcinomas (National Cancer
    Institute 1979).

    Rat

    Groups of 50 male and 50 female Osborne-Mendel rats (age not
    specified) were fed diets containing technical grade nitrofen (stated
    to be >80 percent pure; impurities unspecified) in 2 percent maize
    oil for 78 weeks. The dietary concentration for males was 2 300 mg/kg
    (low-dose) or 2 300-4 600 (high-dose), to give a time-weighted average
    of 3 627 mg/kg. The dietary concentrations for female rate were 1 300
    mg/kg (low-dose) or 2 600 mg/kg (high-dose). The low-dose males, the
    control animals and the high- and low-dose females were observed for
    an additional 32 weeks, during which they were maintained on a basal
    laboratory diet plus maize oil; the high-dose males were observed for

    an additional five weeks after dosing was stopped. Groups of 20 male
    and 20 female controls received the basal diet plus 2 percent maize
    oil. A dose-related decrease in body weight was noted; by 75 weeks
    after onset of dosing, the high-dose females weighed roughly one-third
    less than the controls. Fifty percent of the high-dose males were dead
    by week 45 and only 15 survived to week 83; 60 percent of the low-dose
    and 45 percent of the control group survived until the end of the
    study. Of the females, 58 percent at the high-dose, 74 percent at the
    low-dose and 80 percent of the controls survivied until the end of the
    study. Adenocarcinomas of the exocrine pancreas were seen in 2/50
    females at the low dose and in 7/50 at the high dose. All tumours
    showed local invasion and had metastasized to the lungs. No such
    tumour was seen in the 20 matched controls or in the 110 pooled
    controls. The difference was statistically significant for high-dose
    animals. No other tumour showed a statistically significant difference
    in incidence between control and exposed rats. Poor survival precluded
    the evaluation of carcinogenicity in male rats (National Cancer
    Institute 1978).

    Groups of 50 male and 50 female six-week-old Fischer 344 rate were fed
    diets containing 3 000 or 6 000 mg/kg technical-grade nitrofen (purity
    not specified) for 78 weeks. A group of 20 male and 20 female rats
    were given the control diet. All animals received acidified water (pH
    2.5). The animals were followed for an additional 26 weeks, when all
    surviving rats were killed. A dose-related depression in mean body
    weight was noted in animals of both sexes, reaching 15 percent for
    those given the high dose for 60 to 75 weeks. Of the male rats, 45/50
    given the high dose, 42/50 given the low dose and 17/20 of the
    controls survived until termination of the study; of the females,
    38/50, 42/50 and 17/20, respectively, survived. No statistically
    significant difference in the incidence of tumours was observed
    between the exposed and control groups (National Cancer Institute
    1979). However, subsequent detailed histological and morphological
    studies distinguished the nitrofen-induced neoplasm from those
    occurring naturally in the controls (Hoover et al. 1980; Stinson
    et al. 1981).

    Special Studies on Enzyme Induction

    Groups of B6C3F1 mice (six animals per sex) were fed diets containing
    nitrofen (technical, 92 percent pure) at levels of 0, 10, 100 or 1000
    ppm or two weeks prior to determination of individual liver to body
    weight (L:BW) rations and in vitro hepatic p-nitroanisole (pNA) 
    O-demethylase activity. The dose levels were chosen after acute
    studies (three-day gavage dosing), using CD-1 mice, showed a 
    sex-dependent minimum effect level for hepatic pNA-O-demethylase
    activity between 10 and 50 mg/kg/day. The 100 and 1000 ppm dietary
    nitrofen levels resulted in elevated liver mixed function oxidase
    (MFO) activity in both sexes, with females showing a greater response
    (170 percent and 236 percent of control, respectively, for the two
    diet levels). L:BW ratios were increased 20 percent and 30 percent in
    males and females, respectively, but only at the 1000 ppm level. No

    significant changes were observed in either sex fed the 10 ppm
    nitrofen diet. The no-effect level found for MFO induction was much
    lower than that for oncogenicity (2 348 ppm in the National Cancer
    Institute 1978 oncogenicity study). (Deckert & Steigerwalt 1979c)

    Special Studies on Dermal Irritation

    No evidence of irritation developed when both technical grade (95
    percent pure) and pure nitrofen were applied either as a 10 percent
    solution in maize oil or as moistened powder to the skin of albino
    rabbits (Ambrose et al. 1971).

    Special Studies on Cutaneous Sensitization

    There was no visible evidence of erythema, swelling, or discolouration
    of the test sites after each sensitizing injection or after the
    challenging dose of aqueous suspension of technical grade (95 percent)
    nitrofen. Under the conditions of the test, these finding indicate
    that nitrofen is not a sensitizing agent (Ambrose et al. 1971).

    Special Studies on Cataractogenic Effect

    Groups of 30 two-week-old Peking ducklings were fed diets containing
    nitrofen at levels of 0, 0.05 and 0.1 percent for 13 weeks. Distinct
    growth depression occurred at the 0.1 percent nitrofen dose level and
    slight retardation at 0.05 percent. The mortality rate in the highest
    dose group was distinctly increased. No clinical or histologic signs
    of cataract were observed in any of the treated or control animals,
    nor were other eye abnormalities found (Ensel & Seinen 1970).

    Special Studies on Methaemoglobin Formation by Aminonitrofen

    Methaemoglobin formation in vitro by nitroso and amino derivatives
    of nitrofen and other chlorinated biphenyl ethers was investigated in
    suspensions of human erythrocytes. Ability to induce methaemoglobin
    formation among the nitroso and amino derivatives decreased with
    increasing chlorine substitution of the biphenyl ether. However, amino
    derivatives from no methaemoglobin in suspensions of erythrocytes
    alone, but do so after activation by liver homogenate. Methaemoglobin
    formation after intraperitoneal and oral administration to male Wistar
    rats of chlorinated p-aminobiphenyl ether at levels of 0.77 mg/kg b.w.
    gave almost the same result (ca. 40 percent methaemoglobin within 
    60-120 min.) after either route was used (Miyauchi et al. 1981).

    Special Studies on the Stability of the Metabolite Azoxynitrofen

    Azoxynitrofen (a possible vegetable metabolite of nitrofen) was added
    to simulated gastric fluid and simulated intestinal fluid at level of
    7 ppm. There is a slight degradation of azoxynitrofen in gastric fluid
    during 24 h. The intestinal fluid completely decomposes azoxynitrofen
    within 24 h. After 14 days, there is evidence of azoxynitrofen in
    either digestive fluid, and it appears that aminonitrofen is a major
    product of the decomposition (Warso 1972).

    Acute Toxicity

    The acute toxicity of nitrofen in animals is summarized in Table 1.

    Most deaths occurred two to eight days after dosing and were preceded
    by progressive depression. There was no evidence of diarrhoea.
    Postmortem findings were negative in rats that died and in survivors
    autopsied at 14 days (Ambrose et al. 1971).

    Short-Term Studies

    Rat

    Groups of albino Wistar rats (10 males and 10 females per group) were
    fed diets containing technical grade nitrofen (95 percent pure) at
    levels of 0, 100, 500, 2 500, 12 500 and 50 000 ppm for 13 weeks.

    Rats on the 50 000 ppm diet failed to survive beyond the first week
    and survival of rats on the 12 500 ppm diet was adversely affected.
    Growth was depressed in both sexes at 12 500 ppm and in males at 2
    500. In rats on the lower dietary concentrations of nitrofen, no
    adverse effects were noted in growth, food consumption and mortality.
    Haematologic values appeared to be within normal range and urinary
    tests for reducing substances and protein showed no marked differences
    from the controls. Significantly higher liver-to-body weight ratios
    were found in rats at all dietary levels of nitrofen, except for male
    rats at 100 ppm, and appeared to be dose-related. Kidney body weight
    ratios were significantly elevated for females at 12 500 ppm and for
    males at 2 500 and 12 500 ppm. Testes body weight ratios were
    significantly elevated for rats at 2 500 and 12 500 ppm. Lesser
    effects were noted for heart and spleen. Histopathologic findings
    related to treatment appeared to be confined to rats receiving diets
    containing 12 500 and 50 00 ppm nitrofen. The principal findings
    consisted of a hepatitis characterized by oedema, peripheral
    localization of glycogen granules, swelling of the cytoplasm and liver
    nuclei with prominent nucleoli (Ambrose et al. 1971). (Full
    experimental data were not available.)

    Rabbit

    Groups of five male and five female rabbits received dermal
    applications of nitrofen (as a water-diluted emulsifiable formulation)
    at levels of 0, 250 or 1 250 mg/kg b.w., 7 h/day, 5 days/week for
    three weeks. Additional groups received applications on abraded skin.
    There was no mortality and no evidence of any skin irritation. No
    histological changes were noted in the skin of exposed animals. There
    was no evidence of subacute systemic damage to liver, kidney, lung or
    spleen on histological examination (Brown 1964).


        Table 1   Acute Toxicity of Nitrofen in Animals

                                                                                                                      
    Animal          Sex            Route         Vehicle         LD50             a.i.        Reference
                                                                 g/kg b.w.        purity
                                                                                                                      

    Rat             M              oral          maize oil       2.63             95%         Ambrose et al. 1971
                    M              oral          maize oil       3.58             100%        Ambrose et al. 1971
                    M              oral          peanut oil      2.84             99%         Kimbrough et al. 1974
                    F              oral          peanat oil      2.40             99%         Kimbrough et al. 1974

                                   oral          undiluted       5.00                         Swann 1973
                                                 formulation     (as 25%
                                                                 formulation)

                    M+F            dermal        xylene          5.00             99%         Kimbrough et al. 1974

                                   inhalation    undiluted       205 mg/l                     Swann 1973
                                   (1h exper.)   formulation     (as 25%
                                                                 formulation)

                    M+F            inhalation    undiluted       >271 mg/cu.m.                Brown 1965
                                   (1h exper.)   formation       (as a.i.)

    Rabbit                         dermal        undiluted       >2.00                        Swann 1973
                                   (24 h)        fomulation      (as 25%
                                                                 formulation)
                                                                                                                      
    
    Dog

    Groups of six-month-old beagles (two males and two females/group) were
    fed diets containing technical grade (95 percent pure) nitrofen at
    levels of 0, 20, 200 and 2 000 ppm for two years. The dogs on the
    various dietary levels of nitrofen survived the two-year feeding
    period. No toxic signs attributable to nitrofen were seen at any time.
    The general health, appearance, behaviourial pattern, body weight gain
    and cumulative food consumption were not different than those of the
    respective controls. Haematologic values and urinary tests for
    reducing substances and protein showed no effect of treatment at any
    of the test periods. BSP, SGOT, SAP and BUN tests made during the
    month 24 showed no adverse trends. Significantly higher liver-to-body
    weight ratios appeared only in dogs on 2 000 ppm nitrofen.
    Histopathologic examination of major organs revealed no abnormalities
    or lesions resulting from the ingestion of nitrofen for two years, in
    kind or incidence, that were not found in control dogs. In general,
    only the 20 and 200 ppm diet levels of nitrofen exhibited no adverse
    effects (Ambrose et al. 1971).

    Long-Term Studies

    Rat

    Groups of weanling Wistar rats (25 males and 25 females/group)
    received diets containing technical grade (95 percents pure) nitrofen
    at levels of 0, 1, 10, 100 and 1 000 ppm for 97 weeks. The study was
    terminated at 97 weeks, owing to poor survival (50 percent) in all
    groups, including the controls, beyond week 65. Statistically 
    significant decreases in mean body weight were observed in the males
    at 100 and 1000 ppm after the week 52 and in the females at the same
    dose levels until week 26.

    Food consumption, haematological and urinalysis parameters were not
    affected. Significantly higher kidney/body weight and liver/body
    weight ratios in male rats on 1 000 ppm were notable and appeared to
    be attributable to nitrofen. Histopathologic findings revealed no
    lesions in kind or incidence that were not found in the respective
    controls. Overall, the 10 ppm and below diet level of nitrofen was
    reported to cause no adverse effects (Ambrose et al. 1971). (Lack of
    individual animal data precludes further interpretation of this
    study.)

    COMMENTS

    Nitrofen has low acute oral toxicity. Following oral administration,
    nitrofen is partially absorbed and rapidly excreted. A small
    proportion of the administered dose is distributed to all tissues and
    organs. Nitrofen and its metabolites are found primarily in faeces
    (accounting for about 75 percent of the administered dose), while
    urine contains metabolites accounting for about 15 percent of the
    dose. The metabolism of nitrofen involves reduction, hydroxylation and
    conjugation.

    Two reproduction studies were presented. Because of inadequacies in
    the reports the studies were not considered.

    Oral exposures to nitrofen during the period of organogenesis produced
    neonatal mortality, decreases in the time of survival to weaning, and
    a series of abnormalities in rodent offspring. In a mouse
    teratology/post-natal study, the lowest dose tested (6.25 mg/kg b.w.)
    produced increased incidence of lethal defects at birth and resulted
    in decreased lung and seminal vesicle weights at maturity. In a rat
    teratology study, diaphragmatic hernias were observed at and above
    1.39 mg/kg b.w., but full details were not provided.

    Nitrofen in some cases was found mutagenic in the
    Salmonella/microsome assay but it did not display any mutagenic
    potential in several mammalian systems either in vitro or in vivo.

    Two carcinogenicity studies on B6C3F1 mice indicated that nitrofen is
    a liver carcinogen, causing hepatocellular carcinomas and
    hepatocellular adenomas in both sexes, and haemangiosarcomas in male
    mice. In addition, the compound is carcinogenic to female 
    Osborne-Mendel rats, causing adenocarcinomas of the pancreas. Nitrofen
    was not found to be carcinogenic in Fischer 344 rats. Detailed
    histological examinations suggest that naturally occurring and
    nitrofen-induced liver tumours in mice are morphologically different.
    Both studies were of short duration. In the study on Osborne-Mendel
    rats, poor survival precluded the evaluation of carcinogenicity in
    males.

    In a long-term rat-feeding study, a NOEL of 10 ppm was observed.
    However, the duration of the study (97 weeks), the poor survival in
    all groups (50 percent beyond week 65), and the fact that the study
    was not available in extenso, precluded its full evaluation.

    An acceptable daily intake could not be estimated, owing to the
    evidence of carcinogenicity, the lack of a NOEL for the teratology and
    post-natal effects and the inadequacies of several studies, including
    reproduction and long-term studies.

    REFERENCES - TOXICOLOGY

    Adler, I.L. et al.           A material balance study in rats using 
    1970a                            14C- TOK labelled in the nitrophenyl
                                     ring. Rohm and Haas Report No. 23-24.
                                     (Unpublished)

    Adler, I.L. et al.             A material balance study in rats using
    1970b                            14C - TOK labelled in the dichlorophenyl
                                     ring. Rohm and Haas Report No. 23-25.
                                     (Unpublished)

    Ambrose, A.M., Larson,           Toxicologic studies on 
    P.S., Borzelleca, J.F.,          2,4-dichlorophenyl-p-nitrophenyl
    Blackwell Smith, R. &            ether (TOK). Toxicol. Appl. Pharmacol.,
    Hennigar, G.R.                   19:263-275.

    Brown, J.R.                      A study on the acute inhalation toxicity
    1965                             of TOK EC-25. Report from the University
                                     of Toronto, Canada submitted to WHO by
                                     Rohm and Haas. (Unpublished)

    Brown, J.R. & Mastromateo, E.    Subacute percutaneous toxicity of TOK
    1964                             E-25 in the rabbit. Report from the
                                     University of Toronto, Canada,
                                     submitted to WHO by Rohm and Haas.
                                     (Unpublished)

    Byeon, W.H., Hyun, H.H. &        Mutagenicity of pesticides in the
    Lee, S.Y.                        Salmonella/microsome system. Korean J.
    1976                             Microbiol., 14:128-134.

    Chernoff, N., Kavlock, R.J.,     Preliminary report on the perinatal
    Gray, L.E.                       toxicity of nitrofen administered to
    1980                             mice. Rohm and Haas Report.
                                     (Unpublished)

    Costlow, R.D. & Manson, J.M.     The heart and diaphragm: target organs
    1981                             in the neonatal death induced by
                                     nitrofen (2,4-dichlorophenyl-p-
                                     nitrophenyl ether). Toxicology,
                                     20:209-227.

    Costlow, R.D.,                   The effect on rat pups when nitrofen
    Hirsekorn, J.M.,                 (4-(2,4-dichlorophenoxy) nitrobenzene)
    Stiratelli, R.G.,                was applied dermally to the dam
    O'Hara G.P., Black, D.L.,        during organogenesis. Toxicology
    Kane, W.K., Burke, S.S.,         (in press). Submitted to WHO by
    Smith, J.M. & Hayes, A.W.        Rohm and Haas.

    Decker, F.W. &                   Percutaneous absorption of 14C-TOK in
    Steigerwalt, R.B.                rats. Rohm and Haas Report TD77P-59.
    1979a                            (Unpublished)

    Deckert, F.W. &                  Disposition of 14C-TOK after
    Steigerwalt, R.B.                percutaneous application to a rat.
    1979b                            Rohm and Haas Report TD78P-53.
                                     (Unpublished)

    Deckert, F.W. &                  Fourteen day dietary TOK enzyme
    Steigerwalt, R.B.                induction study in mice. Rohm
    1979c                            and Haas Report TD78P-58.
                                     (Unpublished)

    Ensel, A.B. & Seinen, W.         Investigation of TOK on a possible
    1970                             cataractogenic effect in ducklings.
                                     Central Institut Voor Voedingsonderzock.
                                     Report no. R-3306 submitted to WHO by
                                     Rohm and Haas. (Unpublished)

    Gray, L.E., Kavlock, R.J.,       Prenatal exposure to the herbicide
    Chernoff, N., Ferrell, J.,       2,4-dichlorophenyl-p-nitrophenyl
    McLamb, J. & Ostby, J.           ether destroys the rodent Harderian
    1982a                            gland. Science, 215:293-294.

    Gray, L.E., Kavlock, R.J.,       The effects of the prenatal exposure
    Chernoff, N. & Ferrell, J.       to the herbicide TOK on the postnatal
    1982b                            development of the Harderian gland of
                                     the mouse, rat and hamster. Presented
                                     at Soc. Teratology Meeting 1982.
                                     Submitted to WHO by Rohm and Haas.

    Gray, L.E., Kavlock, R.J.,       Postnatal development alterations
    Chernoff,N., Ostby, J. &         following prenatal exposure to the
    Ferrell, J.                      herbicide 2,4-dichlorophenyl-p-
    1983                             nitrophenyl ether. A dose response
                                     evaluation in the mouse. Toxicol.
                                     Appl. Pharmacol. 67:1-14.

    Guerzoni, M.E. &                 Mutagenic activity of pesticides.
    Del Cupolo, L.                   Riv. Sci. Technol. Alimenti Nutr.
    1976                             Um., 6:161-168.

    Hirsekorn, J.M. & Kane, W.W.     TOK E-25 percutaneous range-findings
    1981                             teratology study with postpartum
                                     evaluation. Rohm and Haas Report No.
                                     81R-63. (Unpublished)

    Hoover, K.L., Ward, J.M.  &      Histopathological differences between
    Stinson, S.F.                    nitrofen-induced and naturally occurring
    1980                             hepatocellular carcinomas in the B6C3F1
                                     mouse. J. Nat. Cancer Inst., 65:937-948.

    Kavlock, R.J., Chernoff, N.,     An analysis of fetotoxicity using
    Rogers, E., Whitehouse, D.,      biochemical end-points of organ
    Carver, B., Gray, J. &           differentiation. Teratology,
    Robinson, K.                     26:183-194.
    1982

    Kimbrough, R.D.,                 2,4-dichlorophenyl-p-nitrophenyl ether
    Gaines, T.B. & Linder, R.B.      (TOK). Effect on the lung maturation of
    1974                             rat fetus. Arch. Environ. Health,
                                     28:316-320

    McLeod, P.L. & McCarthy, K.L.    TOK dermal cytogenetic study in rats.
    1982                             Rohm and Haas Report No. 81R-268.
                                     (Unpublished)

    Miyauchi, M., Koizumi, M. &      Studies on the toxicity of chlorinated
    Uematsu, T.                      p-nitro-biphenyl ether.
    1981                             I -  Methemoglobin formation in vitro
                                     and in vivo induced by nitroso and
                                     amino derivatives of chlorinated
                                     biphenyl ether. Biochem. Pharmacol.,
                                     30:3341-3346.

    Myhr, B.C. & Brusik, G.          Evaluation of 78-232 in the in vitro
    1979                             transformation of BALB/3T3 cells assay,
                                     Litton Bionetics Report No. 20992
                                     submitted to WHO by Rohm and Haas.
                                     (Unpublished)

    National Cancer Institute.       Bioassay of nitrofen for possible
    1978                             carcinogenicity. Carcinogenesis
                                     Technical Report Series No. 26,
                                     DHEW Publ. No. (NIH) 78-826.

    National Cancer Institute.       Bioassay of nitrofen for possible
    1979                             carcinogenicity. Carcinogenesis
                                     Technical Report Series No. 184,
                                     DHEW Publ. No. (NIH) 79-1740,1.

    O'Hara, G.P., Can, P.K.,         The effect of nitrofen
    Harris, J.C., Burke, S.S.,       4-(2,4-dichlorophenoxy) nitrobenzene
    Smith, J.M. & Hayes, A.W.        on the reproductive performance
    1983                             of male rats.Rohm and Haas Report
                                     (Unpublished)

    O'Neill, P.J. & Lohse, E.        4,4-DCAB microbial mutagen test. Rohm
    1980                             and Haas Report No. 80R-4. (Unpublished)

    O'Neill, P.J. &                  TOK: Microbial mutagen tests. Rohm and
    Scribner, H.E.                   Haas Report No. TD79M-307. (Unpublished)

    Paik, S.G. & Lee, S.Y.           Genetic effects of pesticides in the
    1977a                            mammalian cells. I. Induction of
                                     micronucleous. Kor. J. Zool.,
                                     20:19-28.

    Paik, S.G. & Lee, S.Y.           Genetic effects of pesticides in the
    1977b                            mammalian cells. II. Mutagenesis in
                                     L5178Y cells and DNA repair induction.
                                     Kor. J. Zool., 20:159-168.

    Reustle, J.A. &                  TOK cytogenetic study in mice. Rohm and
    Scribner, H.E.                   Haas Report No. 79R-173. (Unpublished)
    1980

    Roser, R.L., Adler, I.L. &       A study of the metabolism of 14-C TOK in
    Allen, S.S.                      rats. Rohm and Haas Report No. 23-33.
    1971                             (Unpublished)

    Siou, G.                         Study of the potential mutagenic
    1978                             activity of TOK by the Howell-Jolly
                                     body technique. Cabinet d'Etudes et
                                     de Recherches en Toxicologie
                                     Industrielle, Report No. MBL-2199
                                     submitted to WHO by Rohm and Haas.
                                     (Unpublished)

    Siou, G.                         Study of the effect of TOK on the
    1979a                            prenatal and postnatal development
                                     of the rabbit. Cabinet d'Etudes et
                                     de Recherches en Toxicologie
                                     Industrielle, Report No. MBL-2212
                                     and Addendum submitted to WHO by
                                     Rohm and Haas. (Unpublished)

    Siou, G.                         Study of the effect of TOK on the
    1979b                            postnatal development of the rat.
                                     Cabinet d'Etudes et de Recherches
                                     en Toxicologie Industrielle Report
                                     No. MBL-2248 submitted to WHO by
                                     Rohm and Haas. (Unpublished)

    Steigerwalt, R.B.,               TOK comparative metabolism study.
    Godfrey, W.J. & Deckert,         Rohm and Haas Report No. 79R-169.
    F.W. 1980a                       (Unpublished)

    Steigerwalt, R.B., Lisk, D.C.    Range-finding study on 14C-TOK. Dermal
    & Deckert, F.W.                  absorption by rat and human skin in
                                     vitro. Rohm and Haas Report No. 
                                     80R-110. (Unpublished)

    Steigerwalt, R.B.,               TOK: disposition after percutaneous
    Udinsky, J.R. & Deckert,         applications to pregnant rats. Rohm
    F.W. 1982                        and Haas Report No. 81R-65.
                                     (Unpublished)

    Stinson, S.F., Hoover, K.L.      Quantitation of differences between
    & Ward, J.M.                     spontaneous and induced liver tumors in
    1981                             mice with an automated image analyzer.
                                     Cancer Letters, 14:143-150.

    Stone, L.C. & Manson, J.M.       Effects of the herbicide
    1981                             2,4-dichlorophenyl-p-nitrophenyl
                                     ether (nitrofen) on fetal lung
                                     development in rats. Toxicology,
                                     20:195-207.

    Swann, H.E.                      Acute toxicity report on Rohm and Haas
    1973                             TOK E-25, dark liquid. Food Drug
                                     Res. Labs. Report No. 1-3784-851
                                     submitted to WHO by Rohm and Haas.
                                     (Unpublished)

    Vogin, E.E.                      Effects of FW925 on the fetal
    1971                             development in rats. Food and Drug
                                     Research Laboratories Report,
                                     August 10 submitted to WHO by
                                     Rohm and Haas. (Unpublished)

    Warso, J.P.                      Degradation of azoxynitrofen in
    1972                             simulated digestion fluids. Rohm
                                     and Haas Report No. 23-72-27.
                                     (Unpublished)

    Weatherholtz, W.M.,              Teratogenicity study in rats, TOK
    Kapp, R.W. &                     E-25, TOK E-25 solvent control.
    Greenspun, K.S.                  Report from Hazleton Laboratories,
    1979                             America Inc. submitted to WHO by
                                     Rohm and Haas. (Unpublished)

    RESIDUES

    RESIDUES IN FOOD AND THEIR EVALUATION

    USE PATTERN

    Nitrofen is a selective pre- and early post-emergence herbicide for
    the control of annual grasses and various broad-leaved weeds in cereal
    grains and vegetables.

    Nitrofen is used in France and some other countries under labels that
    prohibit women from handling the product. It is primarily used to
    control backgrass (Alopecurus agrestis), Veronica hederaefolia,
    Galium aparine, Viola tricolor and other weeds that in winter
    wheat and other cereal grains are not adequately controlled by other
    commercially available herbicides.

    The effective use rate is generally between 1.5 and 2 kg/ha applied to
    the soil surface; activity is lost on soil incorporation. Adequate
    soil moisture is necessary for efficacy, but excessive moisture may
    result in crop injury.

    RESIDUES RESULTING FROM SUPERVISED TRIALS

    Residue data have been obtained in several countries where nitrofen
    was applied to various crops at or about the recommended rates. Two to
    four samples were taken at random from the fields and were transported
    to the laboratory within two days. The preparation of the sample and
    the portion of sample to be analysed were in agreement with the
    recommended Codex procedure with two exceptions: only the cloves of
    garlic were sent to the laboratory after removing the outer leaves and
    all the samples were washed before chopping and deep-freezing.

    The analytical results from the United States were obtained by a
    method (Adler and Wargo 1975) that measures the parent compound and
    the majority of metabolites in the form of the derivative of amine
    moiety. The data from other countries indicate the residue level of
    the parent compound only. The results of supervised trials are
    summarized in Table 1.

    FATE OF RESIDUES

    In general, nitrofen undergoes metabolic degradation in all systems
    (animals, plants, soil and Water) studied. Metabolic pathways include
    reduction of the nitro group, conjugation of the resulting amino
    group, hydroxylation of the dichlorophenyl ring and formation of
    naturally occurring materials, as well as total conversion to CO2.
    The structural formula and the occurrence of the important identified
    metabolites are shown in Table 2.

    In Animals

    Cattle milk, urine and faeces collected during the dosing period from
    a cow fed a daily ration of 26.2 kg containing 5 mg/kg purified
    nitrofen for four days did not contain detectable levels of nitrofen
    or aminonitrofen (sensitivity of the method was approximately 0.3
    mg/kg). In vitro studies demonstrated that nitrofen is rapidly
    reduced to aminonitrofen in fresh rumen fluid (T1/2 is less than or
    equal to 10 min). Aminonitrofen was stable in the rumen fluid for 24 h
    (Gutemann & List 1967).

    Similarly, only trace levels of radioactivity (0.02 to 0.07 mg/kg,
    calculated as nitrofen) were found in samples of urine, faeces and
    milk from a cow dosed at 5 mg/kg with radiolabelled nitrofen. The
    levels of total radioactivity were 1.4 and 1.7 mg/kg in the faeces and
    urine, respectively, and 0.14 and 0.16 mg/kg in two samples of milk. A
    fat sample contained 1.06 mg/kg 14C, of which 0.25 mg/kg was parent
    compound (Roser & Adler 1971).


        Table 1  Residues of Nitrofen Resulting from Supervised Trials

                                                                                                                         

    Crop and variety              Country                   Application             Proharvest     Residue        (mg/kg)
                                                               rate                 interval                      Mean
                                                                           
                                                      (kg/a.i./ha)   No.            (days)
                                                                                                                         

    Broccoli (head)               United States1      3.7            1              74             <0.01          <0.015
    Futura                                            4.5            2              63             <0.01          <0.01
    Gem                                               4.5            1              85             <0.01          <0.01
    Green Duke                                        4.75           2              63             <0.01          <0.01
                                                      4.75           2              69             <0.01          <0.01
                                                      5.0            1              70             <0.01          <0.01
                                                      5.3            2              70             <0.01          <0.01
                                                      5.4            1              77             <0.01          <0,01

    Cabbage (head)                United States1
    Head Start                                        4.5            1              69             <0.01          <0.01
                                                      4.5            1              84             <0.01          <0.01
    Green                                             4.5            1              89             <0.01          <0.01
                                                      6.7            1              76             <0.01          <0.01
                                                      6.7            1              90             <0.01          <0.016

    Cauliflower (head)            United States1
     Snoball                                          4.5            1              90             <0.01          <0.01
                                                      4.5            1              93             <0.01          <0.01
                                                      4.6            1              88             <0.01          <0.016
                                                      5.6            1              83             <0.01          <0.01
                                                      6.7            1              59             <0.01          <0.01
                                                      6.7            1              107            <0.01          <0.016

    Celery (stalk)                United States1
    5270 RIMP                                         4.5            1              109            <0.01          <0.01
                                                      5.4            1              127            <0.01          <0.01
    5270                                              5.6            1              52             <0.01          <0.01
    5270R                                             5.6            1              165            <0.01          <0.01
                                                                                                                         

    Table 1 (continued)

                                                                                                                         

    Crop and variety              Country                   Application             Proharvest     Residue        (mg/kg)
                                                               rate                 interval                      Mean
                                                                           
                                                      (kg/a.i./ha)   No.            (days)
                                                                                                                         

    Garlic (bulb)                 United States1
     Cal Late                                         5.6            1              234            <0.01          <0.01
                                                      5.6            1              250            <0.01          <0.01
                                                      6.7            1              229            <0.01          <0.01
                                                      7.8            1              241            <0.01          <0.01

    Rape (grain)                  Denmark2            1.2            1              100            -              <0.01
                                                      1.2            1              101            -              <0.01
                                                      1.2            1              105            -              <0.01
                                                      1.2            1              100            -              <0.01
                                                      1.2            1              101            -              <0.01
                                                      1.2            1              105            -              <0.01

    Rioe (grain)                  United States1
     Early Rose                                       2.2            1              145            <0.01          <0.01
                                                      3.4            1              145            <0.01          <0.01
                                                      3.4            1              160            <0.01          <0.01
                                                      4.5            1              160            <0.01          <0.01
                                                      6.7            1              160            <0.01          <0.01
                                                      9.0            1              166            <0.01          <0.01

    Rice (straw)
     Early Rose                                       2.2            1              145            <0.01          <0.01
                                                      3.4            1              145            <0.01          <0.01
                                                      4.5            1              166            <0.01          <0.01
                                                      9.0            1              166            <0.01          <0.01

                                                                                                                         

    Table 1 (continued)

                                                                                                                         

    Crop and variety              Country                   Application             Proharvest     Residue        (mg/kg)
                                                               rate                 interval                      Mean
                                                                           
                                                      (kg/a.i./ha)   No.            (days)
                                                                                                                         

    Wheat (grain)                 France3
     Chamlein
                                                      4.0            1              239            <0.01          <0.01
                                                      4.0            1              264            <0.01          <0.01
     Joss                                             4.5            1              266            <0.01          <0.01
     Hardy                                            8.0            1              236            <0.01          <0.01
     Chamlein                                         8.0            1              264            <0.01          <0.01
     Hardy                                            8.0            1              265            <0.01          <0,01

    Wheat (grain)                 Federal
                                  Republic
    Joss                          of                  1.6            1              248            -              <0.002
    Carstacht                     Germany4            1.6            1              280            -              <0.002
    Kranich                                           1.6            1              284            -              <0.002
    Caribo                                            1.6            1              286            -              <0.002
                                                      2.0            1              270            -              <0.002
    Benno                                             2.0            1              279            -              <0.002
    Carito                                            2.0            1              286            -              <0.002

    Hyslop                        United States1      1.7            1              294            <0.01          <0.01
    Druchamp                                          2.2            1              163            0.00-0.02      0.01
    Yamhill                                           2.2            1              250            <0.01          <0.01
                                                      2.2            1              267            <0.01          <0.01
                                                      2.2            1              269            <0.01          <0.01
                                                      2.2            1              272            0.01-0.09      0.04
    Nugaines                                          2.2            1              272            0.02-0.03      0.02
    Hyslop                                            2.2            1              281            <0.01          <0.01
    Yamhill                                           2.2            1              284            <0.01          <0.01
                                                      2.2            1              289            <0.01          <0.01
                                                                                                                         

    Table 1 (continued)

                                                                                                                         

    Crop and variety              Country                   Application             Proharvest     Residue        (mg/kg)
                                                               rate                 interval                      Mean
                                                                           
                                                      (kg/a.i./ha)   No.            (days)
                                                                                                                         

    Cajame                                            3.4            1              146            <0.01-0.01     <0.01
    Tehame                                            3.4            1              151            <0.01          <0.01
    Hyslop                                            3.4            1              294            <0.01          <0.01
    Cajame                                            4.5            1              146            <0.01          <0.01
    Tehame                                            4.5            1              151            <0.01-0.01     <0.01

    Druchamp                      United States1      4.5            1              163            <0.01-0.06     0.03
    Yamhill                                           4.5            1              250            <0.01-0.01     <0.01
                                                      4.5            1              267            <0.01          <0.01
                                                      4.5            1              269            <0.01          <0.01
                                                      4.5            1              272            0.01-0.12      0.06
    Nugaines                                          4.5            1              272            0.02-0.06      0.04
    Hylop                                             4.5            1              281            <0.01          <0.01
    Yamhill                                           4.5            1              284            <0.01          <0.01
                                                      4.5            1              289            <0.01          <0.01
    Cajame                                            5.6            1              146            <0.01-0.02     0.01
                                                      6.7            1              146            <0.01-0.01     <0.01
    Tehame                                            6.7            1              151            <0.01-0.01     <0.01
    Yamhill                                           6.7            1              267            <0.01          <0.01

    Wheat (straw)                 Federal Republic
                                  of Germany4
    Joss                                              1.6            1              248            -              <0.002
    Carstacht                                         1.6            1              280            -              0.020
    Kranich                                           1.6            1              284            -              0.007
    Caribo                                            1.6            1              286            -              <0.004
                                                      2.0            1              270            -              0.004
    Benno                                             2.0            1              279            -              0.010
    Carito                                            2.0            1              286            -              0.005

                                                                                                                         

    Table 1 (continued)

                                                                                                                         

    Crop and variety              Country                   Application             Proharvest     Residue        (mg/kg)
                                                               rate                 interval                      Mean
                                                                           
                                                      (kg/a.i./ha)   No.            (days)
                                                                                                                         

    Hyslop                        United States1      1.7            1              294            <0.01          <0.01
    Yamhill                                           2.2            1              252            <0.01          <0.01
                                                      2.2            1              267            <0.01          <0.01
                                                      2.2            1              269            <0.01          <0.01
    Nugaines                                          2.2            1              272            0.02-0.11      0.06
    Yamhill                                           2.2            1              273            <0.01-0.02     0.01
    Druchamp                                          2.2            1              276            0.01-0.04      0.02

    Hyslop                                            2.2            1              281            <0.01          <0.01
    Yamhill                                           2.2            1              284            <0.01          <0.01
                                                      2.2            1              291            <0.01          <0.01
    Hyslop                                            3.4            1              294            <0.01          <0.01
    Yamhill                                           4.5            1              252            <0.01          <0.01
                                                      4.5            1              267            <0.01-0.01     <0.01
                                                      4.5            1              269            <0.01          <0.01
    Nagaines                                          4.5            1              272            0.02-0.05      0.03
    Yamhill                                           4.5            1              273            <0.01-0.03     0.01
    Druchamp                                          4.5            1              276            0.01-0.02      0.01
    Hyslop                                            4.5            1              281            <0.01          <0.01
    Yamhill                                           4.5            1              284            <0.01          <0.01
                                                      4.5            1              291            <0.01-0.02     0.01
                                                      6.7            1              267            <0.01          <0.01
                                                                                                                         

    Sources:
    1 Rohm and Haas 1969-1980;
    2 Institute National de Recherche Agronomique 1975b;
    3 Institute National de Recherche Agronomique 1975a;
    4 Institute National de Recherche Agroncmique 1978;
    5 Samples taken from four experimental fields;
    6 Samples taken from three experimental fields.
    
        Table 2                  Nitrofen Metabolites

                                                                          
    Metabolite                                           Occurrence
                                                                          
                                                 Aminals   Plants   Soil
                                                                          

    CHEMICAL STRUCTURE 1

    azoxy-nitrofen 4,4'-bis 
    (2,4-dichlorophenoxy) azoxy benzene                    x        x

    CHEMICAL STRUCTURE 2

    hydroxy nitrofen chloro, 
    hydroxy-4 '- nitrodiphenylether1             x

    CHEMICAL STRUCTURE 3

    aminonitrofen 
    2,4-dichloro-4'-aminodiphenylether           x         x        x

    CHEMICAL STRUCTURE 4

    formamidonitrofen 
    2,4-dichloro-4'-formamido-diphenylether      x         x        x

                                                                          

    Table 2 (Continued)

                                                                          
    Metabolite                                           Occurrence
                                                                          
                                                 Aminals   Plants   Soil
                                                                          

    CHEMICAL STRUCTURE 5

    acetamidonitrofen 
    2,4-dichloro-4'-acetamidodiphenyletherx      x         x        x

    CHEMICAL STRUCTURE 6

    5-hydroxyaminonitrofen 
    2,4-dichloro, 5-hydroxy 
    4'-aminodiphenyl-ether                       x         x

    CHEMICAL STRUCTURE 7

    hydroxy-acetamido-nitrofen                   x         x        x

    chloro,hydroxy 4'acetamido
    diphenylether1                               x

    CHEMICAL STRUCTURE 9

    monochloronitrofen 2 chloro-4' 
    nitrodiphenyl ether                          x
                                                                          

    Table 2 (Continued)

                                                                          
    Metabolite                                           Occurrence
                                                                          
                                                 Aminals   Plants   Soil
                                                                          

    CHEMICAL STRUCTURE 10

    hydroxy-propionamido-nitrofen1               x

    CHEMICAL STRUCTURE 11

    2,5-dichlorophenol                           x
                                                                          

    1 The substituted carbon atoms have not been identified.
    

    Cattle feeding studies were conducted twice at feeding levels of 0.05,
    0.5 and 5 mg/kg administered in gelatin capsules. Combined residues in
    meat and milk were determined once in four cows and a second time with
    14C-labelled nitrofen. In the multiple cow feeding study, no residues
    of nitrofen were detected in milk or tissues of animals fed 0.05 or
    0.5 mg/kg, based on the total daily diet. Barely detectable residues,
    ranging from 0.002 to 0.004 mg/kg, were found in the milk of a cow fed
    5 mg/kg. At this feeding level, no residue was found in any of the
    tissues except fat (0.01 mg/kg). The sensitivity of the method was
    0.01 mg/kg for tissue, and 0.002 mg/kg for milk (Rohm & Haas 1967b).

    In the radioactive feeding study a cow was dosed orally twice a day
    with 14C-labelled nitrofen. Nitrofen residues were not found in the
    milk from the lowest feeding level (0.05 mg/kg) and only 0.003 and
    0.08 mg/kg were found in milk from the two higher feeding levels, i.e.
    0.5 and 5 mg/kg. The residue in milk was 0.3 percent and 0.8 percent
    of the applied dosage, respectively. Residues were found in edible
    tissues taken from the cow dosed at the 5 mg/kg feeding level.
    Calculated as nitrofen, these residues ranged from 0.05 mg/kg (trips)
    and 0.24 mg/kg (muscle) to 1.15 mg/kg (fat) (Rohm & Haas 1971). The
    parent compound constituted approximately 47, 32 and 23 percent of the
    total residue in milk, kidney and fat, respectively.

    The lowest feeding level (0.05 mg/kg) of radioactive nitrofen is
    almost ten times greater than the calculated maximum level of residue
    that might be eaten by livestock kept on feed containing rice bran,
    rice milling fractions or any other plant by-products that had been
    treated with nitrofen. Consequently, no detectable residues are likely
    to be found in milk and edible tissues.

    Poultry

    Three groups of 15 laying hens each were fed dichlorophenyl 
    ring-labelled 14C nitrofen incorporated in their feed for ten weeks
    at levels of 0.04, 0.12 and 0.47 mg/kg, respectively. Residues in
    tissues and eggs were studied and analysed by liquid scintillation
    counting. Residues in eggs reached plateaux at levels of 0.02, 0.05
    and 0.17 mg/kg, respectively, by week 6 of feeding for the X, 3X and
    10X test group. The residue was located almost exclusively in the egg
    yolk. The excreta collected for one 24 h period in week 3 contained
    98.9 and 80 percent of the total radioactivity of feeding levels of
    0.04 and 0.47 mg/kg, respectively. Hens from each group were
    sacrificed after weeks 3, 5 and 10 of test feeding. Selected tissues
    were removed and analysed by combustion and liquid scintillation
    counting. Average residue levels in tissues from the 1X test group
    ranged from non-detectable (white meat) to 0.18 mg/kg (fat).
    Prolonged feeding of nitrofen to laying hens at a level of 0.04 mg/kg
    daily resulted in total residue of approximately 0.01-0.02 mg/kg in
    eggs. The residue was found almost exclusively in the egg yolk (Adler
    1972).

    Sheep

    A female sheep was given a single oral, 40 mg/kg dose of 14C-labelled
    nitrofen. After 99 h, 76.2 percent of the 14C had been recovered in
    the excreta, with 39 percent appearing in the urine and 37.2 percent
    in the faeces. The residue in the blood peaked at 19 h with a plateau
    from 11 to 31 h; the concentrations were between 3 and 4 mg/kg. When
    killed at 100 h, the highest tissue concentrations were found in the
    fat (23 to 24 mg/kg total radioactivity expressed as nitrofen); the
    gut, liver, thyroid and mammary glands contained from 2 to 4 mg/kg and
    14 other organs had residues of 0.5 to 2 mg/kg (expressed as
    nitrofen). The highest urinary excretion of nitrofen occurred during
    the 11 to 19 h post-treatment collection period; the peak in the
    faeces was during the 39 to 47 h post-treatment collection. Only 2 to
    5.5 percent of the radioactivity in the urine was extractable with
    chloroform. Aminoitrofen and 5-hydroxynitrofen were the principal
    metabolites, with the parent compound accounting for less than 0.3
    percent of the total radioactivity. Small amounts of phenol,
    acetamidonitrofen and the 2-chloro ether, as well as several unknown
    compounds, were found. The bulk of the radioactivity consisted of
    sulphates, glucuronides and glycine conjugates. In contrast, 47 to 86
    percent of the total radioactivity in the faeces could be extracted
    with chloroform. Approximately 30 percent of the total radiocarbon
    excreted in the faeces was the parent nitrofen (11 percent of the
    total dose administered). Aminonitrofen accounted for an average of 21
    percent of the 14C in the faeces, with the concentration increasing
    over time from 14 percent at 23 h to 25 percent at 33 h. In blood
    samples, the parent nitrofen averaged 33.9 percent of the total
    radioactivity, aminonitrofen 20 percent and an unknown labelled
    compound 13.4 percent. The amount of parent compound peaked at 43
    percent at 19 h post-treatment and declined to 28 percent of the total
    by 31 h (Hunt et al. 1977).

    Fish

    The accumulation of residue in fish was studied in a system consisting
    of two tanks. The first tank contained soil treated with 14C-labelled
    nitrofen, while the second one contained water only. The tanks were
    connected and the water circulated. During the first six days, the
    residue in the soil and water declined from 6.29 mg/kg and 0.016 mg/kg
    to 4.31 mg/kg and 0.004 mg/kg, respectively. From day 10, the water
    did not contain detectable residue, while the total residue in soil
    was 3.62 mg/kg at day 28. The residue in the tail (edible portion) of
    crayfish placed in the tank containing soil was 0.48 mg/kg on the
    first day and varied in the range of 0.13-1.04 mg/kg thereafter. In
    the tissue of catfish, kept in the second tank, the highest residue
    (7.42 mg/kg) was measured at day 2. The residue level declined to 0.17
    mg/kg at day 28. Approximately 55 percent of total 14C residue was
    nitrofen (Adler 1971).

    Cultures of intestinal micro-organisms from carp were capable of
    reducing nitrofen to aminonitrofen under both aerobic and anaerobic
    conditions (Miyauchi et al. 1980).

    In Plants

    Soil in which paddy rice was grown was treated pre-emergence with
    dichlorophenyl ring-labelled nitrofen. The rice plants were sprayed
    with nitrophenyl ring-labelled nitrofen in one experiment and with
    dichlorophenyl ring-labelled nitrofen in another. The post-emergence
    spray application was carried out to provide samples with sufficiently
    large residues for metabolic identification and to study metabolic
    routes that might correlate with post-emergence applications of the
    compound to other commodities. Metabolites identified and their
    percentage ratio in the extractable residue are shown in Table 3
    (Roser & Adler 1971).

    The parent compound amounted to 72 percent of the organic extractable
    residue (73.9 percent of total residue) in rice plants treated 10 days
    before sampling. The parent compound (13.7 percent) hydroxy
    acetamidonitrofen (15 percent) and the amino derivative (11.8 percent)
    were the major components of the organic extractable residue (29.1
    percent of total residue) in rice straw 90 days after treatment. The
    bound residues in rice and wheat grain and straw from a 14C 
    pre-emergence application were characterized by Honeycutt and Adler
    (1975).

    64 to 96 percent of the radioactive residues in wheat and rice grain
    were in the starch 110 and 147 days, respectively, after planting and
    treatment. The authors conclude that nitrofen must undergo
    diphenylether bond cleavage followed by ring opening and incorporation
    of the ring fragments into glucose and starch. Similar
    characterization of the wheat and rice straw reaveled approximately 30
    percent of total 14C residues identifiable in the lignin fraction.
    Negligible (5 percent or less) 14C residues were associated with the
    cellulose fraction, except for rice treated pre-emergence (25
    percent).

    Two experimental plots were treated with nitrofen labelled either on
    the dichlorophenyl ring or on the nitrophenyl ring. Each treated plot
    and the control were planted with two varieties, Anza and Era, of
    wheat. Samples were taken at various intervals and analysed with
    methods measuring the parent nitrofen only and all of the residues
    containing an amino group or capable of being converted to amino
    nitrofen. The results of analyses are shown in Table 4 (Wargo 1974).
    Only about half of the total radioactivity could be extracted from the
    earlier samples and only about 20 percent from straw at harvest. The
    method for measuring aminonitrofen, which includes several
    metabolites, resulted in substantially higher residues in the earlier
    samples than the method for the parent compound only. No residues were
    detected by either method in grain and straw at harvest. Most of the
    radioactivity present in grain and straw at harvest was in the form of
    non-toxic natural substances.

        Table 3  Metabolites of Nitrofen Identified in Rice Grain and Straw

                                                                             

    Compound1                                    Extracted 14C(%)
                                                                             

                                       10 days         90 days       90 days
                                       plant           straw         grain

    Nitrofen                           72.3            13.7          2
    Azoxynitrofen                      4.6             2.5
    Aminonitrofen                      3.3             11.8          4
    Formamidonitrofen                  4.8       )
    Acetamidonitrofen                  0.5       )     19
    5-Hydroxy aminonitrofen            1.9       )
    Hydroxy acetamidonitrofen          2.8             15
    Unknowns and polar conjugates      9.8             38            94
                                                                             

    1 Structural formulas of compounds are shown in Table 2.
    

        Table 4  Residues of 14C Nitrofen in Wheat

                                                                                                                                      
                                                             Residues (mg/kg calculated as nitrofen)
                                                                                                                                      

    Label          Variety                   Radioassay                        Nitrofen                 Aminonitrofen
                                                                               (parent compound)        Method
                                  Sample                   Extract             Method
                                                                                                                                      
                                  Immature Plants (at 26 days)

    Control        Era            0.105                    -                   0.04                     0.04
    Control        Anza           0.134                    -                   0.04                     0.02
    NO2            Era            4.04                     2.26                0.99                     1.57
    NO2            Anza           2.42                     1.08                0.63                     0.77
    Cl2            Era            2.80                     -                   0.47                     1.02
    Cl2            Anza           1.12                     0.61                0.25                     0.32

                                  Immatare Plants (at 44 days)

    Control        Era            0.035                    -                   NDR1                     NDR
    Control        Anza           0.053                    -                   NDR                      NDR
    NO2            Era            2.19                     1.17                0.55                     0.87
    NO2            Anza           1.18                     0.68                0.44                     0.51
    Cl2            Era            2.55                     1.34                0.41                     1.12
    Cl2            Anza           1.44                     1.00                0.48                     0.54
                                                                                                                                      

    Table 4 (continued)

                                                                                                                                      
                                                             Residues (mg/kg calculated as nitrofen)
                                                                                                                                      

    Label          Variety                   Radioassay                        Nitrofen                 Aminonitrofen
                                                                               (parent compound)        Method
                                  Sample                   Extract             Method
                                                                                                                                      
                                  Final Harvest (at 110 days)

                                  Grain       Straw        Straw2             Grain     Straw       Grain     Straw

    Control        Era            0.025       0.087        -                  NDR       NDR         NDR       NDR
    Control        Anza           0.034       0.028        -                  NDR       NDR         NDR       NDR
    NO2            Era            0.173       0.992        0.19               NDR       NDR         NDR       NDR
    NO2            Anza           0.148       0.460        0.10               NDR       NDR         NDR       NDR
    Cl2            Era            0.192       1.123        0.22               NDR       NDR         NDR       NDR
    Cl2            Anza           0.122       0.624        0.14               NDR       NDR         NDR       NDR
                                                                                                                                      

    1 NDR = no detectable residue; sensitivity = <0.01 mg/kg
    2 No detectable 14C activity was present in the extract from any sample of grain
    
    In Soil and Water

    Roser and Adler (1971) identified the soil metabolites resulting from
    a preplant/prefood application of dichlorophenyl ring 14C-labelled
    nitrofen to simulated field rice paddies. Characterization of residues
    in soil samples taken 147 days after treatment revealed that the
    parent compound represented 23 percent of the extractable residue,
    which amounted to 36 percent of total 14C activity.  The major
    metabolites were the aminonitrofen (35.4 percent) and
    hydroxyacetamidonitrofen (10.7 percent), while the levels of
    azoxynitrofen, formamidonitrofen and acetamidonitrofen were below 10
    percent of extractable activity. Following a laboratory leaching
    study, Adler and Roser (1971) characterized the 14C products in the
    top 2.5 cm from soil columns. The extracts (6396 percent of total
    activity) contained primarily parent compound (>90 percent), but a
    qualitatively similar spectrum of products was observed as those
    identified in the previous experiment. Niki and Kuwatsuka (1976)
    studied several diphenyl ether herbicides in two rice paddy soils and
    found rapid metabolism of nitrofen occurring in both soils. Under
    flooded conditions, the half-life was less than 10 days while the
    dissipation rate was slower under upland conditions. The primary
    degradation product was the corresponding 4'-amino derivative.
    Watanabe (1973) reported similar rapid dissipation under field
    conditions.

    In sealed metabolism flasks (biometer flasks), the metabolism of 14C
    nitrofen under aerobic conditions were followed through approximately
    100 days (Fischer 1971). Evolution of 14CO2 was detectable from both
    dichlorophenyl and nitrophenyl ring-labelled preparations (ca. 3 and
    1.5 percent of applied 14C, respectively). In order to characterize
    accurately the residue decline patterns of nitrofen from field soil, a
    plot was surface-treated with dichlorophenyl - 14C compound and left
    fallow through the growing season (Fischer 1976). Radioassay of soil
    samples collected through 316 days demonstrated a very rapid initial
    decline of 0-15 cm residues (50 percent dissipation within 18 days )
    followed by a slower decline. In contrast to field conditions nitrofen
    did not undergo dissipation and metabolism under either aerobic or
    anaerobic conditions in the greenhouse (Fischer 1975).

    Fadayomi and Warren (1977) reported essentially no downward movement
    of 14C nitrofen (nitrophenyl ring) that was spiked onto columns of a
    silt loam and a fine sand soil and subjected to 5.4 cm of simulated
    rainfall. Likewise, Abernathy (1972) found essentially no movement of
    nitrofen on thin-layer plates coated with 12 different soil types. The
    leaching tendencies for aged nitrofen residues were also determined
    (Fischer 1974). After aging for 30 days under greenhouse conditions, a
    14C nitrofen (both ring labelled) fortified sandy loam soil was
    placed in 30 cm columns and subjected to daily simulated rainfalls of
    12 mm/day for 45 days. Radioassay of the leachates as well as the
    dissected columns revealed 83 to 102 percent of the applied 14C in
    the top 5 cm. No significant 14C was detectable in the leachates.
    Using nitrophenyl 14C-labelled nitrofen, a total of five soils of
    different types plus river silt were compared as to the extent of

    nitrofen adsorption from an aqueous solution (Adler & Allen 1971). The
    average amount of 14C adsorbed was greater than 90 percent in every
    case from 0.1 ms/l solutions and in all but one (high kaolinite clay
    soil) from 1.0 ms/l solutions. These findings are consistent with
    those of Fadayomi & Warren (1977), in which most or complete
    adsorption of 14C nitrofen was observed by muck soil and bentonite
    clays but not by kaolinite clays. Nitrofen was desorbed slowly when
    the adsorbents were resuspended in distilled water four successive
    times.

    The observed immobility of nitrofen is consistent with its low water
    solubility and relatively high adsorptive properties. The findings of
    laboratory experiments are supported by the results of field
    experiments in which water from wells surrounded by or near to treated
    fields were sampled. No residue of nitrofen (<0.001 mg/l) was
    detectable in 28 samples taken from depths of 3 to 27 m (Zagorski &
    Rogerson 1980).

    Nitrofen has not demonstrated any tendency to hydrolyse in aqueous
    media either in the environment or in the laboratory (Adler 1973).

    Photodecomposition

    Nakagawa & Crosby (1974a) reported that nitrofen exposed in aqueous
    suspensions to sunlight or simulated sunlight underwent rapid ether
    cleavage to form 2,4-dichlorophenol and p-nitrophenol, along with
    other numerous degradation products. In sunlight, more than 50 percent
    of the initial nitrofen degraded after one week of outside exposure.
    In a related publication, Nakagawa & Crosby (1974b) reported that the
    sunlight photolysis of nitrofen in aqueous methanol represented a
    photonucleophilic displacement of nitrophenol by the hydroxide ion of
    water.

    Both nitrophenyl ring-labelled nitrofen and dichlorophenyl 
    ring-labelled nitrofen were placed on glass plates as a thin film and
    irradiated with simulated sunlight. Samples were takes after 48, 165,
    239, 359 and 498 h of illumination. Some of the resulting
    photoproducts were identified by thin-layer chromatography and mass
    spectrometry. Nitrofen containing both ring labels gave rise to at
    least 13 14C-photoproducts. Eleven of the radioactive components of
    the nitrophenyl ring-labelled photoproduct mixture were identical (by
    TLC Rf values) to corresponding 14C-components of the dichlorophenyl 
    ring-labelled photoproduct mixture. One unique component of the
    nitrophenyl ring-labelled 14C nitrofen photolysis mixture was
    identified as 14C-p-nitrophenol. 14C-2,4-dichlorophenol was also
    detected among the photoproducts of the dichlorophenyl ring-labelled
    mixtures. This indicates that diphenyl ether cleavage had occurred to
    some extent during photolysis.

    Most photoproducts of nitrofen were identified as diphenyl ethers,
    such as amino-nitrofen, hydroxyacetamido nitrofen, isocyano nitrofen,
    4-hydroxy-2,5-dichloro-1 (4-nitrophenoxy) benzene, 5-hydroxy-nitrofen,
    acetamidonitrofen, formamido nitrofen, nitroso-nitrofen and 
    5-hydroxy-amino-nitrofen. Total 14C from both labelled compounds
    declined to less than 50 percent within 100 h and it was below 20
    percent after 498 h (Honeycutt 1975).

    METHODS OF RESIDUE ANALYSIS

    Methods are available for determining either the parent compound only,
    or all of the residues containing amino group or those that can be
    converted to aminonitrofen. The parent compound is extracted with
    dichloromethane from crops and with isopropanol-benzene 1:2 (v/v) from
    soil. The extract is cleaned on a Florisil column and the residue is
    determined by gas-liquid chromatography (GLC) using an electron
    capture detector (ECD). The limit of determination is 0.01 mg/kg (Rohm
    & Haas 1967a). Nitrofen residue can also be analysed with 
    multi-residue procedures. The limit of determination was reported to
    be 0.01-0.02 mg/kg depending on the type of sample (Ambrus et al.
    1981).

    Either method is suitable for regulatory purposes. The majority of
    metabolites can be determined with the method of Adler & Wargo (1975),
    which is based on the reduction of the residues to a common amine
    intermediate. The latter compound is further derivatized and
    quantitatively measured by ECD.

    NATIONAL MAXIMUM RESIDUE LIMITS

    National maximum residue limits (MRLs) reported to the Meeting are
    summarized in Table 6.

    APPRAISAL

    Nitrofen is a selective pre- and early post-emergence herbicide for
    the control of animal grasses and various broad-leaved weeds in cereal
    grains and vegetables. Its recommendations for use either prohibit
    women from handling the product or demand stringent handling
    precaution that prevent exposure during application. It has a limited
    use, which is concentrated mainly on wheat. The technical product,
    containing a minimum of 95 percent nitrofen, is formulated as a 25
    percent EC, 50 percent WP or in combination with other compounds. It
    is applied at rates of 2 to 6 kg a.i./ha to the soil surface or to
    weeds at the two to four leaf growth stage.

    Table 6  National Maximum Residue Limits for Nitrofen

                                                                      

    Crop                             Country              MRL
                                                          (mg/kg)
                                                                      

    Barley                           Greece               0.5
    Broccoli                         United States        0.75
    Brussels sprouts                                      0.75
    Cabbage                                               0.75
    Cauliflower                                           0.75
    Carrots                                               0.75
    Celery                                                0.75
    Eggs                                                  0.75
    Fat (poultry)                                         0.2
    Fruits                           Japan                0.1
    Horseradish                      United States        0.05
    Kohlrabi                                              0.75
    Legumes                          Japan                0.1
    Meat, meat by-products and
    fat (except poultry) in
    cattle, goats, pigs, horses,
    sheep                            United States        0.05
    Milk (fat)                                            0.5
    Milk (whole)                                          0.02
    Onion (dry bulb)                 Austria              0.02
                                     United States        0.75
    Parsley                          United States        0.75
    Rice (grain)                     Japan                0.1
    Rice (grain and straw)           United States        0.1
    Roots                            Japan                0.1
    Rye                              The Netherlands      0.01
    Sugarbeet                        United States        0.05
    Taro                                                  0.02
    Vegetables                       Japan                0.1
    Wheat                            Greece               0.5
                                     The Netherlands      0.01
                                     Yugoslavia           0.01
                                                                      

    Supervised trials were carried out in several countries on various
    crops at or about the recommended rates and at twice these rates.
    Broccoli, cabbage, cauliflower, celery, garlic, oilseed rape and rice
    contained no measurable residues (<0.01 mg/kg) regardless of dosage,
    pre-harvest interval or analytical method. No parent compound was
    detectable in many of the wheat samples; in others treated at the
    recommended rate the total residue, measured as the amino derivative,
    ranged, up to 0.09 mg/kg. In contrast to the grain, wheat straw
    contained the parent compound at levels of <0.002-0.02 mg/kg while
    the sum of metabolites and parent compound ranged from 0.01 mg/kg to
    0.11 mg/kg.

    The metabolic pathways of nitrofen include reduction of the nitro
    group, conjugation of the resulting amino group, hydroxylation of the
    dichlorophenyl ring and incorporation into naturally occurring
    materials. The major metabolites identified in plants were largely
    similar to those in animals. In rice plants, the organo-extractable
    residue amounted to 73.9 percent and 29.1 percent of the total at 10
    and 90 days after application. The parent compound accounted for 72
    percent of the extractable residue at day 10, while
    hydroxyacetamidonitrofen (15 percent), the parent compound (13.7
    percent) and the amino derivative (11.8 percent) were the major
    components after 90 days. A similar pattern of metabolite formation
    was observed in wheat. Neither the grain nor the straw contained
    extractable residues at harvest. In studies with the radio-labelled
    compound, 64 to 96 percent of the radioactive carbon was found in the
    starch of rice and wheat grains. Approximately 50 percent of the
    unextractable activity was found in the lignin fraction of the rice
    straw.

    No residue was detectable in milk or tissues of cattle given nitrofen
    by capsule at rates equivalent to 0.05 and 0.5 mg/kg of total daily
    diet. In cattle dosed at 5 mg/kg, the total residue ranged from 0.002
    to 0.004 mg/kg in milk, while the only tissue containing a measurable
    residue was fat (0.01 mg/kg). Further studies with the labelled
    compound administered by capsule resulted in similar levels of
    residues. In milk, the total radioactivity expressed as the parent
    compound was 0.003 mg/kg and 0.08 mg/kg at feeding levels of 0.5 mg/kg
    and 5 mg/kg in the diet, respectively. At the highest dose, the muscle
    and fat contained 0.24 mg/kg and 1.15 mg/kg of radioactive residue,
    respectively. However, no residue was detectable in the milk, urine or
    faeces of cattle when nitrofen was mixed with the ration at a level of
    5 mg/kg, owing to the rapid degradation of the parent compound and
    aminonitrofen in rumen fluid.

    Metabolites were identified in the urine, faeces and blood of sheep
    dosed at the extreme rate of 40 mg/kg. In urine, the bulk of the
    radioactivity consisted of sulphate, glucuronide and glycine
    conjugates. The organo-soluble fraction (2 to 5.5 percent of the total
    residue) consisted mainly of aminonitrofen and hydroxynitrofen, while
    the parent compound amounted to only 0.3 percent. In blood, the parent

    compound averaged 33.9 percent of the total activity, while
    aminonitrofen and unknown compounds amounted to 20 percent and 13.4
    percent, respectively.

    Eggs and tissues of laying hens kept on a diet containing 0.04 mg/kg,
    0.12 mg/kg and 0.48 mg/kg radio-labelled nitrofen were analysed. After
    a 6-week feeding period the residue in eggs reached plateaus at levels
    of 0.02, 0.05 and 0.17 mg/kg, respectively. Almost all of the residue
    in eggs was found in the yolk. Residues ranged from non-detectable in
    white meat to 0.18 mg/kg in fat at the 0.04 mg/kg feeding level.

    As the lowest feeding level is approximately 10 times the calculated
    maximum residue resulting from treated feed components, no detectable
    residue can be expected in the milk and tissues of cattle or in eggs
    and meat of poultry.

    Fish take up nitrofen residues from water. The residue in fish tissues
    is of about the same magnitude as that in the water shortly after the
    beginning of exposure but declines more slowly.

    Under field conditions, the nitrofen concentration in or on soil
    declined rapidly at first (50 percent dissipation within 18 days) but
    much more slowly thereafter. The major metabolites identified in the
    soil were aminonitrofen and hydroxyacetamidonitrofen. Both nitrofen
    and its aged derivates were immobile in soil, which is in keeping with
    their high adsorption coefficient on different soils and the low water
    solubility of parent compound.

    Analytical methods are available for the determination of the parent
    compound alone or the majority of the organo-extractable residue after
    derivatization of aminonitrofen. The former method is recommended for
    regulatory purposes.

    RECOMMENDATIONS

    The Meeting examined the results of supervised trials and metabolite
    studies and concluded that the data available were sufficient for
    estimating MRLs. As no acceptable daily intake was allocated, the MRLs
    were recorded as guideline levels. They refer to the parent compound
    only.

          Commodity          Guideline levels       Preharvest interval
                             (mg/kg)                on which levels are
                                                    (based months)

          Wheat                  0.01*                      5

          Wheat straw            0.05                       5

          *Residue at or about the limit of determination

    FURTHER WORK OR INFORMATION

    Desirable

    Determination of nitrofen residue in wheat bran.

    REFERENCES - RESIDUES

    Abernathy, J.R.                  Linuron, chlorbromuron, nitrofen and
    1972                             fluorodifen absorption and movement in
                                     12 selected Illinois soils. PhD. Thesis,
                                     University of Illinois, Champaign-
                                     Urbana. (Unpublished)

    Adler, I.L.                      A fish residues study using 14C-labelled
    1971                             TOK. Rohm and Haas Research Report No.
                                     23-41. (Unpublished)

    Adler, I.L.                      A study to determine radioactive residue
    1972                             levels in eggs, tissues and excreta of
                                     laying hens fed 14C TOK. Rohm & Haas
                                     Report No. 23-47. (Unpublished)

    Adler, I.L.                      Study of the hydrolysis of the herbicide
    1973                             TOK in water. Rohm & Haas. Report No.
                                     3923-73-3. (Unpublished)

    Adler, I.L, & Allen,             Soil absorption studies with 14C TOK.
    S.S. Jr. 1971                    Rohm and Haas Report No. 23-77-11.
                                     (Unpublished)

    Adler, I.L. & Roser, R.L.        A study of the leaching and metabolism
    1971                             of 14C TOK in soils. Rohm and Haas
                                     Report No. 23-22. (Unpublished)

    Adler, I.L. & Wargo, J.P.        Determination of residues from the
    1975                             herbicide 2,4-dichloro-1-(4-
                                     nitrophenoxy)-benzene in rice and wheat
                                     by electron capture gas-liquid
                                     chromatography. J. Assoc Off. Anal.
                                     Chem., 58:551.

    Ambrus, A., Csatlos, I.,         General method for determination of
    Hargitai, E., Lautos, J.,        pesticide residues in samples of
    Szabó, L., Visi, E. &            plant origin, soil and water. Part
    Zakar, F.                        I-III, H, Assoc. Off. Anal. Chem.,
                                     64:733.

    Fadayomi, O. & Warren, G.F.      Absorption, desorption and leaching of
    1977                             nitrofen and oxyfluorten. Weed Sci.,
                                     25:97.

    Fischer, J.D.                    Dissipation study of TOK in soil and its
    1971                             effects on microbial activity. Rohm and
                                     Haas Report. (Unpublished)

    Fischer, J.D.                    Laboratory leaching study with aged TOK
                                     soil. Rohm & Haas Report No. 3923-74-78.
                                     (Unpublished)

    Fischer, J.D.                    TOK greenhouse soil metabolism study.
    1975                             Rohm & Haas Report No. 3923-75-13.
                                     (Unpublished)

    Fischer, J.D.                    TOK fallow field study. Rohm & Haas
    1976                             Report No. 34H-76-16. (Unpublished)

    Gutemann, W.H. & List, D.J.      Metabolism of TOK herbicide in the dairy
    1967                             cow. J. Dairy Sci. 50:1516.

    Honeycutt, R.C.                  Photolysis of 14C nitrofen by simulated
    1975                             sunlight. Rohm & Haas Report No. 3923-
                                     75-6. (Unpublished)

    Honeycutt, R.C. & Adler,         Characterization of bound residues of
    I.L. 1975                        nitrofen in rice and wheat straw.
                                     J. Agric. Food Chem. 23:1097-1101.

    Hunt, L.M.,                      Absorption, excretion, and metabolism
    Chamberlain, W.F.,               of nitrofen by a sheep. J. Agric. Food
    Gilbert, B.N.,                   Chem., 25:1062-1065.
    Hopkins, D.E. &
    Gingrich, A.R.
    1977

    Institut National de la          Report on residues of nitrofen on
    Recherche Agronomique.           wheat. Laboratorie de Phytopharmacie,
    1975a                            Versailles. (Unpublished)

    Institut National de la          Reports on residues of nitrofen in rape.
    Recherche Agronomique.           (Unpublished)
    1975b

    Institut National de la          Reports on residues of nitrofen in
    Recherche Agronomique.           wheat. (Unpublished)
    1978

    Miyauchi, M., Takagi, M. &       Studies on the toxicity of chlorinated
    Takayoshi, M.                    nitrobiphenyl ethers on fish-III. Bull.
    1980                             Japanese Soc. Sci. Fish., 46(7):837-844.

    Nakagawa, M. & Crosby, D.G.      Photodecomposition of nitrofen. J.
    1974a                            Agric. Food. Chem. 22:849-853.

    Nakagawa, M. & Crosby, D.G.      Photonucleophilic reactions of nitrofen.
    1974b                            J. Agric. Food Chem., 22:930-933.

    Niki, Y. & Kuwatsuka, S.         Degradation of diphenyl ether herbicides
    1976                             in soils. Soil Sci. Plant Nutr. (Tokyo),
                                     22:223.

    Rohm & Haas.                     Determination of TOK residues in crops
    1967a                            and soil. RAR Memorandum No. 514.
                                     (Unpublished)

    Rohm & Haas.                     A study to determine residues levels in
    1967b                            milk and tissues from a cow fed TOK.
                                     Rohm and Haas Report No. 23-6.
                                     (Unpublished)

    Rohm & Haas.                     A study to determine residue levels in
    1971                             milk, tissues and excreta of a cow fed
                                     14 C-labelled TOK. Rohm and Haas Report
                                     No. 23-30. (Unpublished)

    Rohm & Haas.                     Reports on residues of nitrofen: In
    1969-1980                        broccoli, Report Nos. 78-0172,
                                     78-0173, 78-0174, 78-0175, 78-0176,
                                     78-0177, 80-0249, 80-0250, 80-0279,
                                     80-0280, 80-0290, 80-0291; in cabbage,
                                     Report Nos. 78-0336, 78-0337, 80-0245,
                                     80-0246, 80-0286, 0-0287; in
                                     cauliflower, report nos. 78-0166,
                                     78-0167, 78-0168, 78-0169, 78-0170,
                                     78-0171, 80-0247, 80-0248, 80-288,
                                     80-0289; in celery, Report Nos. 80-0238,
                                     80-0251, 80-0292, 80-0293; garlic,
                                     Report Nos. 80-0252, 80-0253, 80-0254,
                                     80-0255; in rice, Report Nos. 2-69-227,
                                     2-70-173, 2-70-190; in wheat, Report
                                     Nos. 72-121-02, 72-122-02, 72-123-02,
                                     72-292-02, 72-293-02, 72-294-02,
                                     73-266-02, 73-267-02, 73-268-02,
                                     73-269-02, 73-270-02, 73-271-02,
                                     73-272-02, 73-309-02, 73-301-02,
                                     73-311-02, 73-312-02, 73-313-02,
                                     73-314-02, 73-315-02, 74-075-02,
                                     74-076-02. (Unpublished)

    Roser, R.L. & Adler, I.L.        The nature of aged residues of TOK.
    1971                             Rohm & Haas Report No. 23-34.
                                     (Unpublished)

    Wargo, J.P.                      TOK and amino-TOK analysis of 14C-TOK
    1974                             treated wheat. Rohm and Haas Report No.
                                     3923-74-8. (Unpublished)

    Watanabe, I.                     Decomposition of pesticides, by soil
    1973                             micro-organisms - Special emphasis on
                                     the flooded soil condition. Jap. Agric.
                                     Res. Quar., 7:15.

    Zorgorski, W.J. &                Analysis of ground water samples for
    Rogerson, T.D.                   residues of KELTHANE and TOK. Rohm
                                     and Haas Report No. 34H-80-1.
                                     (Unpublished)


    See Also:
       Toxicological Abbreviations
       Nitrofen (ICSC)
       Nitrofen (IARC Summary & Evaluation, Volume 30, 1983)