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    ERYTHROSINE

    First draft prepared by Dr J.C. Larsen,
    Institute of Toxicology, National Food Agency of Denmark

    1.  EXPLANATION

         Erythrosine was evaluated for acceptable daily intake for man
    (ADI) by the Joint FAO/WHO Expert Committee on Food Additives at its
    eighth, thirteenth, eighteenth, twenty-eighth, thirtieth and thirty-
    third meetings (Annex 1, references 8, 19, 35, 66, 73 and 83). 
    Toxicological monographs or monograph addenda were published after
    the thirteenth, eighteenth, twenty-eighth, thirtieth and thirty-
    third meetings (Annex 1, references 20, 36, 67, 74 and 84).  At its
    eighteenth meeting the Committee allocated an ADI of 0-2.5 mg/kg
    body weight.  This ADI was reduced at the twenty-eighth meeting to
    0-1.25 mg/kg body weight and made temporary following observations
    that erythrosine produced effects on thyroid function in short term
    studies in rats and that, in long-term studies, male rats receiving
    4% erythrosine in the diet developed thyroid tumours.  At the
    thirtieth meeting the Committee reduced the temporary ADI to 0-0.6
    mg/kg body weight, based on studies on the biochemical effects of
    erythrosine on thyroid hormone metabolism and regulation, and
    required further data from pharmacokinetic studies relating the
    amount of absorption to the amount ingested, which would enable a
    correlation to be established between blood/tissue levels of
    erythrosine and effects on the thyroid.  At the thirty-third meeting
    the Committee further reduced the temporary ADI to 0-0.05 mg/kg body
    weight, based on a no-observed-effect level with respect to thyroid
    function in human beings ingesting 60 mg per person per day
    (equivalent to 1 mg per kg body weight per day) for 14 days and
    applying a safety factor of 20.  The Committee again requested the
    pharmacokinetic studies required by the previous Committee.

         Since the previous evaluation, additional information have
    become available and are summarized and discussed in the following
    monograph addendum.

    2.  BIOLOGICAL DATA

    2.1  Biochemical aspects

    2.1.1  Absorption, distribution, and excretion

         No new information.

    2.1.2  Biotransformation

         No new information.

    2.1.3  Effects on enzymes and other biochemical parameters

         See 2.2.6.  Special studies on thyroid function.

    2.2  Toxicological studies

    2.2.1  Acute toxicity studies

         No new information.

    2.2.2  Short-term studies

         See 2.2.6.  Special studies on thyroid function.

    2.2.3  Long-term/carcinogenicity studies

    2.2.3.1  Mouse

         No new information.

         The long term feeding study reviewed at the thirtieth meeting
    (Richter  et al., 1981; see Annex 1, reference 74) has now been
    published (Borzelleca & Hallagan, 1987).

    2.2.3.2  Rat

         No new information.

         The results of the two long term feeding studies in rats after
    in utero exposure to erythrosine that were reviewed at the thirtieth
    meeting (Brewer  et al., 1981; Brewer  et al., 1982; see Annex 1,
    reference 74) have now been published (Borzelleca  et al., 1987). 
    In the statistical analyses thyroid follicular cell adenomas and
    carcinomas are treated as separate tumour classes.  The authors'
    conclusion remains that erythrosine at a level of 4% in the diet for
    128 weeks induces an increased incidence in thyroid follicular cell
    adenomas in male rats (15/69 compared to 1/69 in controls).  The
    incidence of thyroid follicular cell carcinomas (3/69) was not

    statistically significantly different from the control value (2/69). 
    In the females at the 4% level the incidence of thyroid follicular
    cell adenomas (5/68) or carcinomas (0/68) were not different than
    the controls (5/66 and 0/66, respectively).  In female rats fed 0.1,
    0.5, or 1% erythrosine in the diet, a numerical increase in adenomas
    was observed (1/68, 3/67 and 5/768, respectively compared to 1/138
    control females), but the increases were not statistically
    significant.  (The incidences of females with carcinomas were 0/68,
    0/67 and 1/68 compared to 0/138).  In the males at the 0.1, 0.5 and
    1.0 levels the incidence of adenomas (0/67, 2/68, and 1/69 compared
    to 0/139) and carcinomas (3/67, 1/68, and 3/69 compared to 0/139)
    were not considered significantly different.

         The microscopic findings in the thyroids from the above-
    mentioned studies and the statistics used have been reviewed (FD&C
    Red No. 3 Review Panel, 1987; Federal Register, 1990).  Slight
    discrepancies in the diagnoses of adenomas/carcinomas were reported. 
    When the combined incidence of adenomas and carcinomas was used in
    the statistical evaluation the following results were obtained:  As
    might be expected an increased incidence of combined adenomas and
    carcinomas was seen in the males fed 4% erythrosine in the diet
    (18/68 compared to 2/68 in control males).  A statistically
    significant increase was also found for combined adenomas and
    carcinomas in male rats fed 0.1, 0.5 or 1.0% erythrosine for 122
    weeks (3+3/64, 7+1/66, 1+3/57, respectively, compared to 0+1/128 in
    control male rats).  In the female rats a significant increase in
    tumour yield was only found in the 1.0% group (5+1/68 compared to
    1+0/138 in controls).

    2.2.4  Reproduction studies

         No new information.

    2.2.5  Special studies on genotoxicity

         Erythrosine was tested for the induction of point mutations in
    the  Salmonella typhimurium plate incorporating assay using strains
    TA1535, TA1537, TA1538, TA98, and TA100.  No mutagenic effects were
    observed.  In a modified assay using the addition of flavin
    mononucleotide to the activation mixture negative results were also
    obtained (Cameron  et al., 1987).

         Erythrosine was non-mutagenic in the Ames test in strains
    TA97a, TA98, TA100, TA102, and TA104 to a concentration of 2
    mg/plate, with or without metabolic activation with rat liver S9 or
    caecal-cell free extracts.  The comutagens harman and norharman (+/-
    S9) did not affect mutagenicity.  A dose dependent suppression in
    spontaneous reversion frequencies was observed.  Toxicity
    (phototoxicity) was observed in the repair-deficient strains (TA97a,
    TA98 and TA100) but not in the repair-proficient strains (TA102 and

    TA104).  Erythrosine was antimutagenic to benzo(a)pyrene and
    mitomycin C but not to 4-nitroquinoline-N-oxide and
    methylmethanesulfonate (Lakdawalla & Netrawali, 1988a).

         Erythrosine did not induce DNA repair in rat hepatocytes  in
     vitro at concentrations up to 1 mM, or  in vivo after an oral
    dose of 200 mg/kg body weight (Kronbrust & Barfknecht, 1985).

         In the mouse lymphoma assay using L5178Y TK+/- cells
    erythrosine was reported positive both with and without the addition
    of S9.  At concentrations exerting high toxicity the response was
    similar to the positive control ethylmethanesulfonate (Cameron  et
     al., 1987).  These results are in contrast to the results obtained
    by Lin & Brusick (1986).

         Erythrosine was reported to increase the yield of multigene
    sporulation minus mutants of  Bacillus subtilis excision repair-
    proficient strain 168 when incubated in the presence of fluorescent
    light.  This effect was not seen in the excision repair-deficient
    strain her-9 (exc).  Erythrosine was highly toxic to both strains
    (Lakdawalla & Netrawali, 1988b).

         Erythrosine was tested for genotoxicity in V79 Chinese hamster
    lung cells.  Reduced colony size was seen at 200 g/ml and more than
    90% lethality was seen at 400 g/ml.  Erythrosine was non-mutagenic
    to V79 cells at the HGPRT and Na+, K+, ATPase gene loci, and did
    not increase the frequency of sister-chromatid exchanges with or
    without rat hepatocyte activation.  At 300 g/ml erythrosine
    produced an increase in micronucleus frequency in the absence of
    hepatocytes.  A dose related increase in the mitotic frequency was
    observed due to an increase in the number of first mitosis.  Thus
    increased genotoxicity was observed only at concentrations well in
    the range of cytoxicity (Rogers  et al., 1988).

         A re-evaluation of an earlier published mouse micronucleus test
    (Lin & Brusick, 1986) revealed a positive response at the low dose
    used (24 mg/kg body weight erythrosine given i.p.), but not at the
    two higher doses (80 and 240 mg/kg body weight) (Brusick, 1989).

    2.2.6  Special studies on thyroid function

         Three groups of 160 male Sprague-Dawley rats were administered
    erythrosine at dose levels of 0.0, 0.25 or 4.0% in the diet
    (corresponding to 0.0, 147.1 or 2514.3 mg per kg body weight per
    day).   Physical observations and body weight and food consumption
    measurements were performed on all animals pretest and at weekly
    intervals during the treatment period.  Necropsy was performed with
    up to 20 animals per test group at days 0, 3, 7, 10, 14, 21, 30 and

    60.  Serum was prepared from blood samples taken from the abdominal
    aorta at each sacrifice interval and analyzed by radioimmunoassays
    for thyrotropin (TSH), thyroxine (T4), 3,5,3'-triiodothyronine
    (T3) and 3,3'5'-triiodothyronine (rT3).  Thyroid and pituitary
    were weighed at each interval and organ/body weight ratios were
    calculated.  Gross postmortem examinations were conducted on the
    thyroid and pituitary only.  Three rats receiving 4.0% erythrosine
    in the diet died spontaneously during the second week of the study. 
    The animals receiving 4% erythrosine in the diet lost weight during
    the first week of the study and the mean body weights were
    significantly lower than control values throughout the study (13% at
    week one and 17% at week 8).  Food consumption of the animals
    receiving 4.0% erythrosine in the diet was significantly lower than
    the control value at week one, but after week 2 it was comparable. 
    This probably reflected a palatability problem during the first two
    weeks.  The absolute pituitary weights of males receiving 4%
    erythrosine were statistically significantly lower than control
    values at days 7, 10, 14, 21 and 60.  The differences were
    considered to reflect the body weight differences between the high-
    dose animals and the controls.  The absolute thyroid/parathyroid
    weights of the rats at the 4% level were generally lower than the
    control values, but the differences were slight and may be due to
    the body weight differences between these groups.  The relative
    weights of these organs were significantly greater at day 21; 
    otherwise relative weights were only slightly greater and not
    significant.  Thyroid/parathyroid absolute and relative weights of
    the rats fed 0.25% erythrosine were significantly lower at day 60,
    otherwise they were comparable to controls.  Gross postmortem
    examinations of thyroid and pituitaries did not show treatment
    related changes (Kelly & Daly, 1988).

         The analysis of serum hormone levels in these rats reveals the
    following:  There was a change (slight increase) in serum TSH levels
    in the control rats during the 60 day experimental period.  The
    baseline (day 0) TSH level was significantly lower than the levels
    on days 21, 30, and 60.  In the 0.25% group serum TSH concentrations
    were significantly increased over baseline (day 0) at days 14, 21,
    30 and 60.  When compared to the TSH levels in control animals a
    significant increase was observed at days 21, 30 and 60.  In the
    4.0% group the TSH levels were significantly increased over the
    baseline (day 0) level and the corresponding control levels at all
    time points.  When compared to the 0.25% group the serum TSH levels
    in the high dose group were significantly greater at days 3, 7, 10,
    and 14.  Serum T4 concentrations were increased over baseline and
    control values at days 10 and 14 in the 0.25% group, while in the
    4.0% group the T4 concentrations were increased at all time points. 
    Furthermore, the high dose animals had significantly greater T4
    concentrations than the low dose animals at days 7,10, 21, 30 and
    60.  Serum T3 concentrations in the low dose rats were comparable
    to the control values except for a decrease at day 30.  In the high

    dose rats serum T3 concentrations were significantly lower than
    baseline (day 0) and control values at all time points.  In
    addition, serum T3 concentrations were decreased compared to those
    of the low dose animals on days 3, 10, 14, 21, 30, and 60.  Serum
    rT3 concentrations were increased above baseline (day 0) in the low
    dose group at days 7, 10, 14, 21, 30 and 60; and increased above
    control values at days 10, 14 and 21.  A marked increase in serum
    rT3 over controls and low dose animals was seen in the high dose
    group at all time points.

         The results indicate that the ingestion of a dietary
    concentration of 4% erythrosine induces a rapid and sustained
    increase in serum TSH, T4, and rT3 and a comparable decrease in
    serum T3 concentrations, and that these changes are also induced,
    but are less pronounced, after 0.25% in the diet.  These findings
    are consistent with an inhibition by erythrosine of the deiodination
    in the 5'-position of T4 and rT3, resulting in a decreased
    production of T3 from T4 and a decreased deiodination of rT3,
    respectively (Braverman & DeVito, 1988).

         Three groups of 80 male Sprague-Dawley rats were administered
    erythrosine at dose levels of 0.0, 0.03, 0.06 and 4.0% in the diet
    for a maximum of 60 days (corresponding to 0.0, 17.5, 35.8, and
    2671.7 mg/kg body weight per day, respectively).  Control animals
    (100 males) received standard laboratory diets.  Physical
    observations, body weight and food consumption measurements were
    performed on all animals pretest and at weekly intervals during the
    study period.  For the determination of baseline data, 20 control
    animals were bled for radioimmunoassays of TSH, T4, T3, and rT3
    and sacrificed on test day 0, prior to the initiation of dosing. 
    Additional necropsy intervals were staggered so that on days 7, 21,
    30 and 60, an additional 20 animals per group at each interval were
    bled for radioimmunoassay samples.  Brain, pituitary and thyroid
    were weighed and organ/body and organ/brain weight ratios were
    calculated for all animals.  Gross postmortem examinations were
    performed on the thyroids, pituitary and brains of all animals.  In
    the animals receiving 4% erythrosine in the diet  a substantial loss
    of body weight and decreased food consumption during week 1 of the
    study, probably due to poor palatability of the diet, resulted in
    statistically significantly lower body weights of the animals
    throughout the study period.  The absolute and relative
    thyroid/parathyroid weights of the animals receiving 4.0%
    erythrosine were increased at days 21 and 30, and at day 60
    (relative organ to body weight ratio).  The absolute and relative
    (organ to brain weight ratio) pituitary weights of animals at the
    4.0% level were lower than control values at day 7.  In the 0.03%
    group absolute and relative thyroid/parathyroid weights were greater
    than corresponding control values at day 21 and 30, but comparable
    to control values at days 7 and 60.  Thus, no consistent and dose
    related changes in organ weight, absolute or relative, were found at

    the lower doses.  Gross postmortem examination of the thyroid,
    pituitaries and brain did not reveal any treatment related effects
    (Kelly & Daly, 1989).

         In the 0.03% and 0.06% groups there were no significant changes
    in serum TSH, T4, T3, and rT3, concentrations during the 60 day
    treatment period.  In the 4.0% group TSH concentrations were
    significantly greater than the corresponding control values at days
    21, 30, and 60.  A 41% increase after 7 days was not statistically
    significant compared to the control value.  Serum TSH concentrations
    were significantly greater than those of the 0.03 group at days 21,
    30, and 60, and the 0.06% group at day 30.  In the 4.0% group serum
    T4 concentrations were slightly elevated above controls during the
    treatment period.  However, the increase was only statistically
    significant on day 30.  In the high dose animals serum T3
    concentrations were significantly lower than controls at all time
    points.  Serum rT3 concentrations were markedly increased in the
    high dose animals compared to controls or animals fed 0.03% and
    0.06% erythrosine at all time points (Braverman & DeVito, 1989).

    2.3  Observations in humans

         No new information.

    3.  COMMENTS

         The Committee considered additional studies on thyroid hormone
    metabolism and regulation in male rats during 60-day feeding trials
    with erythrosine.  The studies showed a rapid onset in the expected
    hormonal changes  of a statistically significant rise in serum
    levels of thyrotropin, thyroxine (T4), and 3,3,5'-triiodothyronine
    (rT3), and a decrease in serum 3,5,3'-triiodothyronine (T3) after
    ingestion of 40 mg/kg erythrosine in the diet.  A no-observed-effect
    level of 0.6 mg/kg erythrosine in the diet corresponding to 30 mg
    per kg of body weight per day was obtained.  The changes seen in
    these studies are consistent with the hypothesis that erythrosine
    inhibits the hepatic conversion of circulating T4 to T3, and the
    resulting decrease in the concentration of T3 stimulates the serial
    release of thyrotropin-releasing hormone from the hypothalamus and
    then thyrotropin from the pituitary.  The sustained increases in the
    levels of thyrotropin produce hyperstimulation of the thyroid, which
    may be associated with the tumorigenic effects noted below.

         The Committee also reconsidered the carcinogenicity data from
    two long-term feeding studies on erythrosine in which an increase in
    the incidence of thyroid follicular-cell adenomas in male rats was
    demonstrated at a level of 40 mg/kg of erythrosine in the diet. 
    When thyroid follicular-cell adenomas and carcinomas were combined
    in the statistical analysis, significant (but not clearly dose-
    related) increases in the incidence of thyroid tumours in male rats
    given 1, 5, 10 and 40 mg/kg of erythrosine in the diet were found. 
    Effects in females were significant only at one dose level.  The
    Committee agreed that it was appropriate to combine thyroid
    follicular-cell adenomas and carcinomas in the statistical analysis,
    in view of evidence that adenomas are an earlier stage of carcinomas
    in the thyroid.

         The Committee reviewed additional data on the mutagenicity of
    erythrosine, and, taking into account extensive data from other
    mutagenicity studies, concluded that the compound is not genotoxic.

    4.  EVALUATION

         While a no-effect-level could not be determined for the
    tumorigenic effect of erythrosine in rats, the Committee considered
    that the occurrence of thyroid tumours in rats was most likely
    secondary to hormonal effects and concluded that it would be
    possible to establish an ADI from the no-effect-level for effects on
    thyroid function.  In view of the differences in thyroid physiology
    between humans and rats the Committee based its evaluation on the
    previously reported no-observed-effect level derived from human
    data.  Therefore the Committee allocated an ADI of 0-0.1 mg/kg of
    body weight for erythrosine, based on the no-effect-level at 60 mg
    per person per day (equivalent to 1 mg per kg body weight per day)
    and a safety factor of 10.

    5.  REFERENCES

    BORZELLECA, J.F., & HALLAGAN, J.B. (1987).  Lifetime
    toxicity/carcinogenicity study of FD&C Red No. 3 in mice.   Fd.
     Chem. Toxicol., 25, 735-737.

    BORZELLECA, J.F., CAPEN, C.C. & HALLAGAN, J.B. (1987).  Lifetime
    toxicity/carcinogenicity study of FD&C Red No. 3 (erythrosine) in
    rats.   Fd. Chem. Toxicol., 25, 723-733.

    BRAVERMAN, L.E. & DEVITO, W.J. (1988).  Effects of FD&C Red No. 3
    (Tetraiodofluorescein) on serum thyroid hormone and TSH
    concentrations in male Sprague-Dawley rats; a 60-day study. 
    Unpublished report dated December 7, 1988.  Submitted to WHO by
    Certified Color Manufacturers' Association, Washington, D.C., USA.

    BRAVERMAN, L.E. & DeVITO, W.J. (1989).  Effects of FD&C Red No. 3 on
    serum TSH and serum thyroid hormone concentrations in male Sprague-
    Dawley rats;  Results of a 60-day study (B/d Project No. 88-3378). 
    Unpublished report dated July 26, 1989.  Submitted to WHO by
    Certified Color Manufacturers' Association, Washington, D.C., USA.

    BRUSICK, D.J. (1989).  Addendum to review the genotoxicity of FD&C
    Red No. 3.  Unpublished report.  Submitted to WHO by Certified Color
    Manufacturers' Association, Washington, D.C., USA.

    CAMERON, T.P., HUGHES, T.J., KIRBY, P.E., FUNG, V.A. & DUNKEL, V.C.
    (1987).  Mutagenic action of 27 dyes and related chemicals in the
     Salmonella/microsome and mouse lymphoma TK+/- assays.   Mutat.
     Res., 189, 223-261.

    FD&C RED NO. 3 REVIEW PANEL (1987).  An inquiry into the mechanism
    of carcinogenic action of FD&C Red No. 3 and its significance for
    risk assessment.  Unpublished report.  Submitted to WHO by Certified
    Color Manufacturers' Association, Washington, D.C., USA.

    FEDERAL REGISTER (1990).  Termination of provisional listing of FD&C
    Red No. 3 for use in cosmetics and externally applied drugs and of
    lakes of FD&C Red NO. 3 for all uses.  Department of Health and
    Human Services, Food and Drug Administration, 21 CFR parts 81 and 82
    (Docket Nos. 76C-0044 and 76N-0366), Thursday, February 1.

    KELLY, C.M. & DALY, I.W. (1988).  A 60-day study to investigate the
    effects of FD&C Red No. 3 on thyroid economy in male Sprague-Dawley
    rats.  Bio/Dynamics Inc. Project No. 88-3378.  Report dated December
    7, 1988.

    KELLY, C.M. & DALY, I.W. (1989).  A 60-day study to investigate the
    effects of FD&C Red No. 3 on thyroid economy in male Sprague-Dawley
    rats.  Bio/Dynamics Inc., Project No. 88-3378.  Report dated August
    2, 1989.  Submitted to WHO by Certified Color Manufacturers'
    Association, Washington, D.C., USA.

    KORNBRUST, D. & BARFKNECHT, T. (1985).  Testing of 24 food, drug,
    cosmetic, and fabric dyes in the  in vitro and the  in vivo/in
     vitro rat hepatocyte primary culture/DNA repair assays.   Environ.
     Mutat., 7, 101-120.

    LAKDAWALLA, A.A. & NETRAWALI, M.S. (1988a).  Mutagenicity,
    comutagenicity, and antimutagenicity of erythrosine (FD&C Red 3), a
    food dye, in the Ames/ Salmonella assay.   Mutat. Res., 204, 131-
    139.

    LAKDAWALLA, A.A. & NETRAWALI, M.S. (1988b).  Erythrosine, a
    permitted food dye, is mutagenic in the  Bacillus subtilis 
    multigene sporulation assay.   Mutat. Res., 206, 171-176.

    LIN, G.H.Y. & BRUSICK, D.J. (1986).  Mutagenicity studies on FD&C
    Red No. 3.   Mutagenesis, 1, 253-259.

    ROGERS, C.G., BOYES, B.G., MATULA, T.I, HEROUX-METCALF, C. &
    CLAYSON, D.B. (1988).  A case report:  A multiple end-point approach
    to evaluation of cytotoxicity and genotoxicity of erythrosine (FD&C
    Red No. 3) in a V79 hepatocyte-mediated mutation assay.   Mutat.
     Res., 2-5, 415-423.


    See Also:
       Toxicological Abbreviations
       Erythrosine  (FAO Nutrition Meetings Report Series 46a)
       Erythrosine (WHO Food Additives Series 6)
       Erythrosine (WHO Food Additives Series 19)
       Erythrosine (WHO Food Additives Series 21)
       Erythrosine (WHO Food Additives Series 24)
       Erythrosine (WHO Food Additives Series 44)
       ERYTHROSINE (JECFA Evaluation)