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    d-PHENOTHRIN

    EXPLANATION

         Phenothrin was evaluated by the Joint Meeting in 1979 and 1980
    and a temporary ADI was estimated in 1980 (Annex I, FAO/WHO 1980b,
    1981b). It was reviewed in 1982 and 1984 (Annex I, FAO/WHO 1983b,
    1985c). The rodent reproduction, chronic toxicity and oncogenicity
    studies evaluated in 1980 were performed by Industrial Bio-Test
    Laboratories (IBT). Repeat studies were required to be submitted by
    1988. In 1984 a temporary ADI for d-phenothrin was estimated based on
    6-months rat and dog studies. Data were submitted indicating that
    metabolism and toxicity were similar for phenothrin and d-phenothrin
    and that data for phenothrin can be used to support d-phenothrin. The
    required data and some additional data have been submitted and are
    summarized in this monograph addendum.

    EVALUATION FOR ACCEPTABLE INTAKE

    BIOLOGICAL DATA

    Biochemical aspects

    Absorption, distribution and excretion

    Rats

         Studies were done using the 1R,cis- and 1R,trans- isomers
    of phenothrin. Groups of 5 rats/sex were given either 14C-(1R,
    Trans)-phenothrin or 14C-(1R, cis)-phenothrin as:

    (1)  a single oral dose at 4 mg/kg bw;

    (2)  a single oral dose at 200 mg/kg bw; or

    (3)  a single oral dose at 4 mg/kg bw 24 hours after the last of
         14 daily oral doses of unlabelled material at the same
         dose level.

    With single doses of the trans- isomer at 4 or 200 mg/kg bw, 56-69%
    of the administered radioactivity was recovered in faeces with 25-40%
    in urine by 7 days after dosing. Following repeated doses with 4 mg/kg
    bw, faecal excretion within 7 days decreased to 24-29% while urinary
    excretion increased to 70-75%. With the cis- isomer, 80-87% of
    single doses (4 or 200 mg/kg bw) was excreted in faeces and 11-18% in
    urine within 7 days. Following repeated doses the amount excreted in
    faeces decreased slightly to 72%, with 24% in urine. No sex
    differences were apparent with either isomer.

         Tissue levels were generally low under all the dosing regimens of
    this study. The highest tissue residues were in fat. With the trans-
    isomer fat residues were 2-10% lower than those with the cis-
    isomer. Skin with hair, and carcass had the next highest residues -
    possibly due to residual fat. Levels in other tissues were less than
    10 ng phenothrin equivalents/gm tissue with single and repeated low
    doses and less than 0.6 g/gm tissue for the single high dose.

         Fat levels were higher following repeated doses of 4 mg/kg bw
    than following a single dose at this level with both isomers. Females
    showed a larger increase than males, having lower tissue residues
    following a single dose and higher tissue residues than males
    following repeated doses (Isobe et al., 1987).

    Biotransformation

         The metabolism of the 1R,cis- and 1R,trans- isomers of
    phenothrin was studied in the rat. In faeces a major proportion of the
    excreted radioactivity was intact phenothrin. The authors stated that
    this was considered to be unabsorbed material since intact phenothrin
    is not excreted in bile. Phenothrin in faeces accounted for 14-16%
    with the trans- and 17-25% with the cis- isomer of the repeated
    4 mg/kg bw dose; 44-45% (trans) an 41-44% (cis) of the single
    4 mg/kg bw dose; and 44-60% (trans.) and 50-59% (cis) of the
    single 200 mg/kg bw dose.

         Five faecal metabolites were identified for the cis- isomer.
    These retained the ester linkage and were derived from oxidation
    involving the alcohol moiety, the isobutenyl moiety and cyclopropane
    ring of the acid moiety. These metabolites accounted for 0.8-9.2% of
    the 14C dose.

         Most of the urinary metabolites were derived following ester
    cleavage. With both isomers a major metabolite was 4-OH-PB
    acid-sulfate, accounting for 15-55% and 7-18% of the dose of
    14C given as trans- and cis- isomer, respectively, by the three
    dose regimens in both sexes. A metabolic pathway was proposed
    (Isobe et al., 1987). (See Figure 1.)

    Toxicological studies

    Special studies on carcinogenicity

    Mice

         Groups of 50 male and 50 female B6C3F1 SPF mice were fed diets
    containing 0, 300, 1000 or 3000 ppm d-phenothrin for 104 weeks.
    Clinical observations, body weight gain, and food consumption were
    recorded throughout the study. Ophtalmoscopic observations and urinary
    analyses were performed at 6, 12, 18 and 24 months. At 104 weeks,
    blood samples were taken from 10 animals/sex/dose for haematological
    and clinical chemistry examinations. All survivors at 104 weeks were
    sacrificed and organ weights were recorded. Histopathological
    examination of about 40 tissues/organs was performed for all animals
    sacrificed or dying during the study. Additional groups of 10, 10 and
    20 mice/sex/dose were fed the same diets for 26, 53 or 78 weeks,
    respectively. Blood samples were taken from 10 animals/sex/dose at
    each of these time periods for haematological and blood chemistry
    determinations. Organ weights were recorded at necropsy.
    Histopathological examination was performed on about 40 tissues/organs
    from mice sacrificed at 53 weeks.

         Mortality was 2-6% at 78 weeks and 16-28% at termination in the
    main study. The pattern of mortality did not show any relationship to
    treatment. Effects due to treatment were reduced body weight gains in
    males and increased liver/body weight ratios in both sexes in mice
    given diet containing 3000 ppm d-phenothrin. Histopathologically,
    "hepatocytic hypertrophy" (large hepatocytes) was observed in males
    and females at 3000 ppm after 53 weeks of treatment but was not
    observed to a significant degree after 104 weeks' treatment. This
    observation may, therefore, have been of an adaptive effect.
    Paradoxical changes in kidney/body weight ratios were observed at
    3000 ppm: reduced in males, increased in females. There were no
    histopathological changes in kidney to explain these differences. No
    tumour type was observed to be statistically significantly increased
    in any group. There was a slightly higher incidence of hepatocytic
    adenomas and carcinomas in treated groups, but the incidences were not
    strictly dose-related. There was also a slight increase in reported
    metastases of hepatic carcinomas in males and females at 1000 and
    3000 ppm. Most of the observed metastases were located only in lung.
    Although the incidence of tumours did not indicate a carcinogenic
    response, it would be valuable to have the metastatic lesions in lung
    confirmed.

         In this study no effects were observed at the lowest dose level
    of 300 ppm (equal to 40.3 mg/kg bw/day) (Amyes et al., 1987).

    Rats

         Groups of 80 male and 80 female F344 SPF rats/dose level were
    given diet containing d-phenothrin at levels of 0, 300, 1000 or
    3000 ppm for 105 (males) or 118 (females) weeks. The rats were
    separated into replicate groups of 40 animals/sex/dose to minimise
    bias. Five rats/sex/dose/replicate were sacrificed at 52 weeks for
    interim evaluation. Throughout the study clinical observations, body
    weight gains, food consumption and ophthalmoscopic observations were
    monitored. Blood samples were taken from 5 rats/sex/group/replicate on
    weeks 26, 50, 78 and 104 for haematological and clinical chemistry
    examinations. Urine samples were collected from the same animals for
    analysis. Surviving males were sacrificed on week 106 and females on
    week 119. Organ weights were recorded for these animals. All rats
    which died or were sacrified were necropsied. Histopathological
    examinations of about 40 tissues from 50 rats/sex/dose level dying
    prior to or sacrificed at termination and from the 10 rats/sex/dose
    sacrificed at 52 weeks were performed.

    CHEMICAL STRUCTURE 1

         Mortality was 42-56% in males and 14-36% in females at 104 weeks.
    There was no increase in mortality as a result of treatment. There was
    a slight but statistically significant reduction in body weight gain
    during the first year of the study in females given 3000 ppm. This
    group also had reduced alanine aminotransferase (ALT) and aspartate
    aminotransferase (AST) levels at weeks 25, 49 and 77. Males given
    3000 ppm had reduced ALT activity at week 49 only. Liver/body weight
    ratio was increased in females given 3000 ppm at week 52 only and in
    males given 3000 ppm at weeks 52 and 105 (statistically significant
    only at week 105). Histopathologically the only observation of note
    was a slight increase in the incidence of hepatocytic hypertrophy in
    males given 3000 ppm. There were no statistically significant
    increases in the incidences of any tumour type. However, there were
    increases in the incidence of preputial gland adenomas and carcinomas
    in males. The incidence at 0, 300, 1000 and 3000 ppm were 1, 1, 1 and
    4 adenomas and 0, 0, 1 and 3 carcinomas, respectively.

         In this study only the left preputial gland was examined
    routinely for histopathology. The right preputial gland was examined
    only when there were gross observations. However, the three carcinomas
    in the high dose group were apparently not detected grossly and,
    therefore, there may have been additional tumours in the right glands
    that were not detected. Pathologists studying this gland have reported
    that small tumours of the preputial gland cannot be detected when only
    gross lesions are examined (Resnick & Ward, 1981). Incidences of
    preputial tumours in F344 rats which have been published for the NTP
    program in the United States indicate incidences of 2.2% with adenomas
    and 2.7% with carcinomas among 2,320 control males in 2-year studies
    and 0.4% with adenomas and 4.7% with carcinomas in 529 males in a
    lifespan study (Solleveld et al., 1984). In one NTP study a total of
    7 preputial gland tumours were observed in a group of 50 males
    (untreated) but it is not indicated how many of the tumours were
    adenomas or carcinomas (Haseman, 1983). The procedure for examining
    this gland was not given in either of these studies. It appears likely
    that in this gland adenomas progress to carcinomas (Resnick & Ward,
    1981).

         On the basis of this information the observed incidences of these
    tumours do not appear of concern. However, it would be of interest to
    have the preputial glands reexamined to determine the true incidence
    of tumours in this study. In this study there was no apparent effect
    at the 1000 ppm dose level (equivalent to 50 mg/kg bw/day) on any of
    the examined parameters (Martin et al., 1987).

    Special study on mutagenicity

         The ability of d-phenothrin to cause chromosomal aberrations in
    Chinese hamster ovary cells was tested in vitro. Based on the
    results of a cytotoxicity test, 4 concentrations (2  10-5 to
    2  10-4 M without S-9 mix, and 5  10-5 to 5  10-4 with S-9
    mix) were tested. There was no increase above control levels in the
    incidence of chromosomal aberrations at any of the dose levels tested.
    Positive controls significantly increased the number of aberrations
    observed (Kogiso et al., 1986).

         The ability of d-phenothrin to induce unscheduled DNA synthesis
    was tested in human cells (HeLa S3). Because no cytotoxicity was noted
    with dose levels up to 4 mg/ml, concentrations tested were 0.25 to
    4 mg/ml. No significant increases in uptake of tritiated thymidine
    were observed following treatment with d-phenothrin in contrast to the
    results with the positive controls. There was no evidence, under the
    conditions of this test, that d-phenothrin induced unscheduled DNA
    synthesis (Forster et al., 1984).

    Special study on reproduction

    Rats

         In a two-generation reproduction study, groups of 30 male and 30
    female Charles River CD (Sprague-Dawley-derived) rats were given diets
    containing 0, 300, 1000 or 3000 ppm d-phenothrin (92.9% pure) during
    growth, mating, gestation and lactation for 2 litters per generation.
    The rats were mated on a 1:1 basis after 91 days of dietary exposure
    to produce the F1a litter. The rats were subsequently remated to
    produce the F1b litter. From the F1b litter 30 males and 30
    females were selected to be parents to the F2a and F2b litters.
    From the F2b litter 1 male and 1 female/litter/dose level were
    maintained on test for 13 weeks.

         Body weight gains of F0 rats given 3000 ppm were slightly lower
    than controls during the pre-mating period. The difference in body
    weight was maintained throughout the study but did not increase with
    time. In the F1 parents there was a slight reduction in body weight
    gain up to week 13 (pre-mating) at 3000 ppm which was maintained in
    males but females approached control values during the second mating.
    The F2b pups maintained for 13 weeks showed no effect on body weight
    gain. There were no treatment-related effects on food consumption,
    water intake, regularity of oestrus cycle, or reproductive performance
    (fertility, gestation, litter size, viability, pup sex and weight).
    Development of the F1b and F2b pups was normal as indicated by
    auditory and visual responses, pinna unfolding, hair growth, tooth
    eruption and eye opening. Liver/body weight ratios were increased in
    F0 adult females, F1 adult females and F2 weanling males and

    females given diet containing 3000 ppm of d-phenothrin. There was no
    consistent histopathological change suggestive of a treatment-related
    effect. In this study the first mating (F0->F1a) was mainly
    consanguinous. There were no apparent differences in the results of
    these matings and the subsequent non-consanguinous matings. The
    offspring of this mating were not further used in the study so the
    departure from protocol did not invalidate the study as a whole. The
    authors concluded that d-phenothrin had no effect on reproductive
    performance or development of Charles River rats at dietary levels of
    up to 3000 ppm (equivalent to 150 mg/kg bw/day) over 2 generations.
    The NOEL for parental effects was 1000 ppm (equivalent to 50 mg/kg
    bw/day) (Tesh et al., 1987).

    Short-term studies

    Rats

         Groups of 20 Sprague-Dawley weanling rats/sex/dose level were
    given diets containing d-phenothrin (92.9% pure) at levels of 0, 1000,
    3000 or 10000 ppm for 6 months. Additional groups of 10 rats/sex/dose
    level were given the same diets for 3 months. No deaths occurred and
    no clinical signs of toxicity or opthalmoscopic effects were observed.
    A dietary level of 10000 ppm caused depressed body weight gain in the
    first 3 months of feeding in both sexes, slight reduction of RBC
    count, haematocrit and haemoglobin at 3 and 6 months in males, reduced
    serum cholinesterase at 3 and 6 months in females and increased
    liver/bw, kidney/bw and adrenal/bw ratios in both sexes. At 3000 ppm
    liver/bw ratio was increased in both sexes at 3 and 6 months,
    kidney/bw was increased in males at 3 months and adrenal/bw ratio was
    increased in females at 3 months. Water intakes were reduced in males
    at 3000 ppm and females at 10000 ppm and in both sexes serum albumen
    levels and A/G ratios were increased and serum sodium levels were
    reduced at 3 and 6 months at 10000 ppm. The biological relevance of
    these observations was not established. No effects were observed on
    food intake or urinary parameters and there were no treatment-related
    histopathological lesions. The NOEL for this study was 1000 ppm (equal
    to 55 mg/kg bw/day). (Murakami et al., 1981).

    Dogs

         Groups of 6 male and 6 female purebred beagle dogs (24-29 weeks
    old) were given diets containing d-phenothrin at 0, 100, 300 or
    1000 ppm for 6 months. The diets were prepared weekly. Two batches of
    d-phenothrin were used: a 95.5% pure lot was used weeks 1-4 (week 4
    for the 100 ppm diet); and a 91.3% pure lot was used weeks 4-26 (week
    4 for the 300 and 1000 ppm diets). No deaths occurred and there were
    no treatment-related effects or clinical signs of toxicity, food
    consumption, haematology, urinalysis, ophthalmoscopic observations, or

    gross or histopathology. Body weights were slightly (<10%) lower than
    in controls in the 1000 ppm group (both sexes) throughout the study.
    Serum alkaline phosphatase levels were elevated in both males and
    females in the 1000 ppm group. Liver/body weight ratios were increased
    in both males and females at 1000 ppm. The NOEL for this study is
    considered to be 300 ppm (equal to 9.3 mg/kg bw/day based on food
    intake and body weight). (Pence et al., 1981).

         Groups of 4 male and 4 female beagle dogs/dose level were given
    diet containing 0, 100, 300, 1000 or 3000 ppm d-phenothrin for 52
    weeks. Clinical observations, body weight gain and food consumption
    were monitored throughout the study. Blood and urine samples were
    analyzed on weeks 13, 26, 39 and 52. All dogs were given
    ophthalmoscopic examinations prior to initiation and at termination.
    On week 52 all dogs were sacrificed and organ weights were recorded.
    Histopathological examinations of about 40 tissues/organs from each
    dog were performed.

         No deaths occurred in the study. There were no treatment-related
    effects on body weight gain, food consumption, haematology, urinalysis
    or ophthalmoscopy. Among females emesis was slightly more frequent at
    3000 ppm but was not unequivocally treatment-related. Alkaline
    phosphatase levels were slightly increased in both males and females
    at 3000 ppm but was statistically significant only in males on week 13
    and was higher than the expected range only in males. There was a
    tendency to reduced serum albumen levels and albumen/globulin ratios
    at 10.00 and 3000 ppm but the levels were within the expected range at
    the performing laboratory. Liver/body weight ratios were increased in
    males and females at 3000 ppm but were statistically significant only
    in females. Histopathologically there were increased incidences of the
    diagnosis of microcyst in the pituitary in females at 3000 ppm, focal
    degeneration accompanied by the presence of acicular crystalline
    material in adrenal cortex in males at 3000 ppm and hepatocellular
    enlargement in males and females at 3000 ppm. Focal degeneration in
    adrenal cortex without crystalline material was observed in one male
    at 1000 ppm. One male at 1000 ppm had slight diffuse hepatocellular
    enlargement. The authors concluded that the NOEL for this study was
    300 ppm (equal to 8.2 mg/kg bw/day) (Cox et al., 1987).

    COMMENTS

         d-Phenothrin is a mixture of predominantly 1R,cis- and
    1R,trans- isomers (cis:trans ratio of 20:80) of the
    chrysanthemic acid ester of 3-phenoxybenzyl alcohol.

         Following either single or repeated doses of both the 1R,cis-
    and 1R, trans- isomers of phenothrin, excretion was virtually
    complete within 7 days after dosing. After a single dose of either
    isomer the primary route of excretion was via the faeces, being higher
    for the cis- isomer. After repeated doses of the cis- isomer the
    faecal route was still the predominant route, although the proportion
    recovered in faeces was slightly less. Urinary excretion was the
    predominant route of elimination of the trans- isomer. Tissue levels
    were low with residues primarily in the fat. Tissue levels were higher
    following repeated doses than following a single dose. Much of the
    material recovered from the faeces was intact phenothrin, which was
    considered to be unabsorbed material. Faecal metabolites of the cis-
    isomer retained the ester linkage and were derived from oxidations.
    Urinary metabolites were formed from both isomers following ester
    cleavage after single or repeated dosing in both sexes.

         In feeding studies in dogs, mice and rats, liver/body weight
    ratios were increased. After one year in dogs and two years in rats
    and mice large hepatocytes were observed. Histopathological changes
    were also seen in the adrenals of dogs after one year, but not at 6
    months. In mice there were slightly increased incidences of
    hepatocellular adenomas (both sexes) and carcinomas (in females) in
    all treated groups but these were not dose-related. Despite what were
    reported to be pulmonary metastases of hepatic carcinomas in both
    sexes at 1000 and 3000 ppm, the Meeting did not consider the compound
    to be a hepatic carcinogen in this species. However, it was concerned
    that the lung lesions be properly identified.

         In rats there was an increase in the incidence of adenomas and
    carcinomas of the preputial gland in males at 3000 ppm. Since only the
    left gland was examined routinely, the true incidence of these tumours
    is unknown. Although it would be of interest to have additional data
    concerning these tumours, it was considered unlikely that the
    incidences of preputial gland tumours were toxicologically relevant.

         A two-generation reproduction study in rats was flawed by
    consanguinous matings in the first mating period. However, subsequent
    matings were non-consanguinous and the offspring of the consanguinous
    matings were not used. Therefore, the study was considered acceptable.
    No reproductive effects were observed at dose levels up to and
    including 3000 ppm (equivalent to 150 mg/kg bw/day). The NOEL for
    parental effects (body weight gain depression) was 1000 ppm
    (equivalent to 50 mg/kg bw/day).

         There was no increase in chromosomal aberrations in Chinese
    hamster ovary cells or unscheduled DNA synthesis in human HeLa S3
    cells.

         Re-examination of the teratology studies in rabbits and mice
    evaluated previously (1980 JMPR) indicated that in the mouse study
    dosing was on days 7-12 of gestation. This period does not cover the
    complete period of organogenesis. No other teratology study in a
    rodent species was available. A rodent teratology study covering the
    entire period of organogenesis is desirable.

    TOXICOLOGICAL INFORMATION

    LEVELS CAUSING TO TOXICOLOGICAL EFFECT

         Mouse:    300 ppm in the diet, equal to 40.3 mg/kg bw/day
         Rat:      1000 ppm in the diet, equivalent to 50 mg/kg bw/day
         Dog:      300 ppm in the diet, equal to 7.1 mg/kg bw/day

    ESTIMATE OF ACCEPTABLE DAILY INTAKE FOR MAN

         0-0.07 mg/kg bw for "d-phenothrin"

    STUDIES WHICH WILL PROVIDE INFORMATION VALUABLE FOR THE CONTINUED
    EVALUATION OF THE COMPOUND

    1.   Histopathological examination of all preputial glands from
         the rat oncogenicity study.

    2.   Re-examination of the metastatic liver tumours in the lung
         in the mouse oncogenicity study to determine whether they
         represent true metastases.

    3.   A teratogenicity study in a rodent species.

    REFERENCES

    Amyes, S.J., Martin, P.A., Ashby, R., Lee, P., Brown, P.M., Fowler,
    J.S.L. & Finn, J.P. 1987. Sumithrin: Oncogenicity and toxicity study
    in mice. Unpublished report from Life Science Research. Submitted by
    Sumitomo Chemical Co., Ltd.

    Cox, R.H., Sutherland, J.D., Voelker, R.W., Alsaker, R.D., Vargas,
    K.J., Lewis, S.A. & Hagen, W.J. 1987. Chronic Toxicity Study in Dogs
    with Sumithrin -J.G.- Unpublished report from Hazleton Laboratories
    Inc. Submitted by Sumitomo Chemical Co., Ltd.

    Forster, R., Tipins, R.S., De Venezia, V. & Nunziata, A. 1984.
    Unscheduled DNA synthesis in human cells. Cell Line: HeLa S3. Test
    Substance: Sumithrin. Unpublished report from Life Sciences Research,
    Roma Toxicology Center. Submitted by Sumitomo Chemical Co., Ltd.

    Haseman, J.K. 1983. Patterns of Tumor Incidence in Two-Year Cancer
    Bioassay Feeding Studies in Fischer 344 Rats. Fundamental and Applied
    Toxicology 3:1-9.

    Isobe, N., Matsunaga, H., Nakatsuka, I. & Yoshitake, A. 1987.
    Metabolism of (1R, trans) and (1R, cis)-isomers of Phenothrin in
    Rats. Unpublished report from Sumitomo Chemical Co., Ltd.

    Kogiso, S., Hara, M., Ito, K., Iwawaki, H. & Yoshitake, A. 1986.
    In vitro chromosomal aberration test of S-2539F in Chinese hamster
    ovary cells (CHO-K1). Unpublished report from Sumitomo Chemical Co.,
    Ltd.

    Martin, P.A., Amyes, S.J., Ashby, R., Lee, P., Brown, P.M., Fowler,
    J.S.L. & Finn, J.P. 1987. Sumithrin: Combined Toxicity and
    Oncogenicity Study in Rats. Unpublished Report from Life Science
    Research, Submitted by Sumitomo Chemical Co., Ltd.

    Murakami, M., Hiromori, T., Ito, S. & Hosokawa, S. 1981. Six Month
    Oral Toxicity Study of S2539 Forte (SumithrinR) in Rats. Unpublished
    report from Sumitomo Chemical Co., Ltd.

    Pence, D.H., Hagen, W.H., Alsaker, R.D., Hastings, T.F., Dawkins,
    B.G., Tacey, R.L. & Marshall, P.M. 1981. Subchronic Toxicity Study in
    Dogs S2539-F. Unpublished report from Hazleton Laboratories Inc.
    Submitted by Sumitomo Chemical Co., Ltd.

    Resnik, G. & Ward, J.M. 1981. Morphology of Hyperplastic and
    Neoplastic Lesions in the Clitoral and Preputial Gland of the F344
    Rat. Veterinary Pathology 18:228-238.

    Solleveld, H.A., Haseman, J.K. & McConnell, E.E. 1984. Natural History
    of Body Weight Gain, Survival and Neoplasia in the F344 Rat. Journal
    of the National Cancer Institute 72(4):929-940.

    Tesh, J.M., Willoughby, C.R. & Fowler, J.S.L. 1987. Sumithrin: Effects
    upon reproductive performance of rats treated continuously throughout
    two successive generations. Unpublished report from Life Science
    Research. Submitted by Sumitomo Chemical Co., Ltd.
    


    See Also:
       Toxicological Abbreviations
       Phenothrin, d- (EHC 96, 1990)
       Phenothrin, d- (HSG 32, 1989)