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    INTERNATIONAL PROGRAMME ON CHEMICAL SAFETY

    WORLD HEALTH ORGANIZATION





    SAFETY EVALUATION OF CERTAIN 
    FOOD ADDITIVES



    WHO FOOD ADDITIVES SERIES: 42





    Prepared by the Fifty-first meeting of the Joint FAO/WHO
    Expert Committee on Food Additives (JECFA)





    World Health Organization, Geneva, 1999
    IPCS - International Programme on Chemical Safety

    PROCESSED EUCHEUMA SEAWEED (addendum)

    First draft prepared by
    Dr J.B. Greig
    Department of Health, Skipton House, London, United Kingdom

          Explanation 
          Biological data
              Toxicological studies
                   Short-term studies of toxicity
                   Genotoxicity
          Comments
          Evaluation
          References


    1.  EXPLANATION

         Processed  Eucheuma seaweed was previously considered by the
    Committee at its thirtieth, thirty-ninth, forty-first, and
    forty-fourth meetings (Annex 1, references 73, 101, 107, and 116). At
    the thirtieth and thirty-ninth meetings, the Committee was unable to
    evaluate its use in food because no relevant toxicological data were
    available. At its forty-first meeting, the Committee considered a
    90-day feeding study in rats, for which complete details were not
    available, and some studies of genotoxicity. The Committee allocated a
    temporary ADI of 0-20 mg/kg bw to processed  Eucheuma seaweed from
     E. cottonii pending submission of the data on individual animals in
    the 90-day study. At its forty-fourth meeting, the Committee reviewed
    those data and the results of new assays for genotoxicity and
    cytotoxicity. Because of the chemical relationship between processed
     Eucheuma seaweed and traditionally refined carrageenan, the
    Committee considered that toxicological data on the latter were
    relevant to the safety assessment of the carrageenan polysaccharide
    constituents of processed  Eucheuma seaweed, but could not replace
    adequate toxicological studies on processed  Eucheuma seaweed itself.
    Although the data for individual animals in the 90-day feeding study
    in rats confirmed the accuracy of the summary data and the conclusions
    derived from them, the Committee expressed reservations about the
    design, conduct, and documentation of the study. Additionally, none of
    the available studies of genotoxicity was considered to be adequate,
    because of deficiencies in their conduct or reporting. The Committee
    therefore extended the temporary ADI, pending submission of the
    results of a new 90-day study of toxicity in rats and of an
    appropriate battery of genotoxicity studies on processed seaweed from
     E. cottonii, all meeting present-day standards.

         In addition, in response to a request that the specifications
    include  E. spinosum as a source of processed  Eucheuma seaweed, the
    Committee determined at the forty- fourth meeting that the results of
    a separate 90-day feeding study in rodents and of a separate battery
    of genotoxicity assays were required for that inclusion, as
     iota-carrageenan, the major component of  E. spinosum, caused

    ulcerative lesions in the caecum of guinea-pigs. The Committee also
    requested the results of a separate battery of assays of genotoxicity.
    Additionally, the Committee concluded that a complete review of all
    data on carrageenan should be undertaken in 1998  (vide supra).

         At the present meeting, the Committee reviewed the results of a
    new 90-day study in rats.


    2.  BIOLOGICAL DATA

    2.1  Toxicological studies

    2.1.1  Short-term studies of toxicity

         Processed  Eucheuma seaweed derived from two sources,
     E. cottonii or  E. spinosum, was fed to groups of 20 male and 20
    female Sprague-Dawley rats at concentrations of 0.5, 1.5, or 5% in the
    diet for at least 90 days. Groups of 30 male and 30 female rats fed
    basal diet for the same period were used as controls. Positive control
    groups of 10 male and 10 female Sprague-Dawley rats were fed diets
    containing 5% conventionally processed carrageenans derived from the
    same seaweeds over the same period. Additional groups of 10 male and
    10 female rats were fed the 5% processed  Eucheuma seaweed and
    conventionally processed carrageenan diets for 90 days and then basal
    diet for 28 days. The study was carried out to present-day standards;
    the only deviation was a slight inhomogeneity of mixing outside the
    defined range. Ophthalmological examinations of controls and rats fed
    5% test diets were carried out before the start of the study and again
    one week before they were killed. All animals were observed daily, and
    body weights and food intake were measured twice weekly. Urine samples
    were collected on the day before necropsy. The animals were killed by
    exsanguination, and haematological and clinical chemical parameters in
    blood, plasma, and serum were measured. The weights of 10 major organs
    or tissues from each animal were recorded, and a comprehensive set of
    tissues were fixed. Tissues from controls, rats fed 5% processed
     Eucheuma seaweed derived from  E. cotonii or  E. spinosum, and all
    animals that died or were killed during the study were examined by
    light microscopy. The lungs of all animals were examined.

         No ophthalmological abnormalities were found, and the alterations
    in general condition during the study, usually consisting of red
    staining on the face, were considered by the authors not to be due to
    treatment. Statistically significant reductions in body weight of
    about 5% below control values at more than half of the measurement
    times were seen in males fed the 5%  Eucheuma seaweed from both
    sources; these changes did not persist in the corresponding groups
    that were allowed to recover. Males fed 5% conventionally processed
    carrageenans derived from  E. spinosum had statistically
    significantly reduced body weights throughout the study, which were
    10% lower than those of controls at the end of the study. These
    changes persisted during the recovery phase. Of the females, only
    those fed 5% conventionally processed carragee-nans derived from

     E. spinosum had statistically significant reductions in body weight
    at more than half of the measurement times during the treatment and
    recovery phases. The food intakes of treated animals showed no
    consistent changes that could be related to the body-weight changes. 

         No dose-related functional changes were recorded during week 12
    in a subset of 10 animals from each group. Similarly, urinalysis
    provided no evidence of any biologically significant effect.
    Occasional statistically significant alterations in haematological
    parameters were reported, but the decreases in erythrocyte, leukocyte,
    and lymphocyte counts, haemoglobin concentration, and haematocrit were
    related to dose only in females fed 1.5 or 5% processed  Eucheuma 
    seaweed derived from  E. cotonii. Similar changes were not observed
    in rats allowed to recover. Serum analysis revealed no dose-related
    response, except an increase of about 7% in the albumin:globulin ratio
    in male rats fed 5% processed  Eucheuma seaweed derived from
     E. spinosum.

         At necropsy, the weight of the full caecum relative to body
    weight was statistically significantly higher in rats fed 5% processed
     Eucheuma seaweed from either source. The weight of the empty caecum
    was increased only in males. No significant change was seen in rats
    allowed to recover. 

         Isolated histopathological findings in males fed 5% processed
     Eucheuma from either source were of no obvious toxicological
    significance. Significantly increased incidences of perivascular
    cuffing, haemorrhage, interstitial pneumonitis, and foci of foamy
    macrophages were seen in the lungs of some control and treated groups,
    with no indication of a dose-response relationship. The lesions were
    attributed by the study authors to mild infection. Hydrometria of the
    cervix and uterus were seen commonly in females and were considered
    not to be relevant. Observations of follicular hyperplasia of lymphoid
    tissue were attributed to infection (Robbins, 1997a,b).

    2.1.2  Genotoxicity

         The results of assays for genotoxicity were reviewed at the
    forty-fourth meeting of the Committee and were considered to be
    inadequate (Sylianco et al., 1993).

         Assays for reverse mutation in  Salmonella typhimurium strains
    TA98, TA100, TA102, TA1535, and TA1537 both with and without an
    Aroclor 1254-induced rat liver microsome fraction (Ames test) were
    conducted with processed  Eucheuma seaweed derived from  E. cotonii 
    or  E. spinosum, each at 10 concentrations in the range of 0-10
    mg/plate. The results were negative (Jackson, 1997).


    3.  COMMENTS

         The Committee reviewed the results of a new 90-day study in rats
    fed processed  Eucheuma seaweed from two sources,  E. cottonii and
     E. spinosum, at concentrations of 0, 0.5, 1.5, or 5% in the diet, as
    well as conventionally processed carrageenan from these two sources at
    5% in the diet. At the highest concentrations in the diet, the intakes
    of the seaweeds were equal to 4300 and 5000 mg/kg bw per day,
    respectively, for male and female rats fed the material from
     E. cottonii and to 4500 and 5100 mg/kg bw per day, respectively, for
    male and female rats fed the material from  E. spinosum. No adverse
    effects were noted in the study. The changes observed during the
    course of feeding the highest concentration of processed  Eucheuma 
    seaweed from these two sources, most notably an increase in the
    relative weights of the full and empty caecum, were considered to be
    the consequence of accumulation of poorly absorbed material in the
    caecum and to be of no toxicological significance. There was no
    indication that the effects of the processed  Eucheuma seaweeds
    differ from those of conventionally prepared carrageenans from the
    same seaweed species. 

         Processed  Eucheuma seaweed derived from either  E. cottonii or
     E. spinosum was not mutagenic in well-conducted assays for reverse
    mutation in  Salmonella typhimurium strains. The Committee considered
    that no further studies of genotoxicity were required.


    4.  EVALUATION

         In view of the lack of toxicity of processed  Eucheuma seaweeds
    derived from  E. cottonii or  E. spinosum, the Committee determined
    that both species could be included in the specifications for
    processed  Eucheuma seaweeds. Additionally, because of the
    similarities between processed  Eucheuma seaweeds and conventionally
    processed carrageenans and in the effects they caused in the recent
    90-day study of toxicity, the Committee included carrageenans and
    processed  Eucheuma seaweed in a temporary group ADI 'not specified'
    to be reviewed in 2001 (see monograph on carageenans).


    5.  REFERENCES

    Jackson, L.I. (1997)  Salmonella mutation test (Ames test) with a
    semi-refined carrageenan from two sources. Unpublished BIBRA Report
    No. 3160/2/1/97 from BIBRA International, Carshalton, Surrey, United
    Kingdom. Submitted to WHO by Dr H.J. Bixler, Seaweed Industry
    Association of the Philippines, Searsport, Maine, United States. 

    Robbins, M. (1997a) Validation of the determination of carrageenan in
    diet mixtures. Unpublished BIBRA Report No. 3160/3/1/97 from BIBRA
    International, Carshalton, Surrey, United Kingdom. Submitted to WHO by
    Dr H.J. Bixler, Seaweed Industry Association of the Philippines,
    Searsport, Maine, United States. 

    Robbins, M.C. (1997b) A 90-day feeding study in the rat with
    semi-refined carrageenan from two sources, including a recovery phase.
    Unpublished report of Project No. 3160/1/2/97 from BIBRA
    International, Carshalton, Surrey, United Kingdom. Submitted to WHO by
    Dr H.J. Bixler, Seaweed Industry Association of the Philippines,
    Searsport, Maine, United States.

    Sylianco, C.Y.L., Balboa, J., Serrame, E., & Guantes, E. (1993)
    Non-genotoxic and antigenotoxic activity of PNG carrageenan.
     Philipp. J. Sci., 122, 139-153. 
    


    See Also:
       Toxicological Abbreviations
       Processed Eucheuma seaweed (WHO Food Additives Series 32)
       PROCESSED EUCHEUMA SEAWEED (JECFA Evaluation)