FAST GREEN FCF
EXPLANATION
Fast Green FCF was evaluated at the twenty-fifth meeting of the
Committee (Annex 1, reference 56) when inadequacies were identified in
earlier long-term feeding studies in rats and mice. The previously-
allocated ADI was converted to a temporary ADI of 12.5 mg/kg b.w.
pending the results of adequate long-term feeding studies and
multigeneration reproduction/teratogenicity studies.
Since the previous evaluation, additional data have become
available and are summarised and discussed in the following monograph.
The previously-published monograph has been expanded and is reproduced
in its entirety below.
BIOLOGICAL DATA
Biochemical aspects
Rats and dogs were given orally 200 mg of the colour. In the rats
the urine and faeces were collected for 36 hours. In the dogs, a bile
fistula was made for bile analysis. Almost all the administered colour
was excreted unchanged in the faeces of rats. No colour was found in
the urine. In the bile of the dogs, the amount of colour never
exceeded 5% of the given dose. After feeding, the colour was found in
the bile of rats and rabbits, but not in their urine. It was concluded
that the quantity found in the bile provides a reasonable estimate of
the amount absorbed from the gastrointestinal tract (Hess & Fitzhugh,
1953; 1954; 1955).
Following i.v. injection in rats, over 90% of the colour was
excreted in the bile within 4 hours (Iga et al., 1971).
Fast Green FCF was found to have a high binding affinity for
plasma protein (Gangolli et al., 1967; 1972; Iga et al., 1971).
Toxicological studies
Special studies on carcinogenicity (see also long-term studies)
Mice
Groups of 60 (120 controls) male and female Charles-River CD-1
mice were fed diets containing 0, 0.5, 1.5, or 5.0% Fast Green FCF
from 43 days of age for approximately 24 months. Ten animals/sex/group
were subjected to haematological examination at 3, 6, 12, and 18
months. All animals dying or killed in a moribund condition and all
survivors to termination were subjected to detailed post-mortem
examination. The following tissues were examined histologically from
all survivors from the control and 5%-dose groups as well as all
animals dying or killed in extremis from these groups: adrenals,
aorta, bone and marrow (femur), brain (3 sections), eyes (with optic
nerve), gall bladder, gastrointestinal tract (oesophagus, stomach,
duodenum, ileum, caecum, colon), heart, kidneys, liver, lung, lymph
nodes (mesenteric and mediastinal), mammary gland, nerve (sciatic),
ovaries, pancreas, pituitary, prostate, salivary gland, seminal
vesicles, skeletal muscle, skin, spinal cord, spleen, testes with
epididymides, thymus, thyroid/parathyroid, trachea, urinary bladder,
uterus, and gross lesions/tissue masses. In addition, gross
changes/tissue masses were examined histologically from all animals in
the lower-dose groups.
No treatment-related effects on mortality were observed. The mean
body weights of females in the 5%-dose group were consistently lower
than controls and the mean body weights of males in the 5%-dose group
were lower than controls at weeks 52 and 78. No other consistent
differences in body weight were noted. Slight reductions in
haemoglobin, haematocrit, and erythrocyte counts were noted in the
high-dose males at 18 months but no other consistent or dose-related
haematological changes were observed. Histological examination did not
reveal any treatment-related lesions and the incidence, origins,
and histology of benign and malignant neoplasms did not differ
significantly between controls and treated animals (Hogan & Knezevich,
1981).
Rats
Eighteen weanling Osborne-Mendel rats of both sexes received
weekly s.c. injections of approximately 30 mg (1 ml of a 3% aqueous
solution) of Fast Green FCF for 94-99 weeks. Subcutaneous
fibrosarcomas appeared at the site of injection in 15 animals (Nelson
& Hagan, 1953; Hansen et al., 1966).
Two groups of 16 female rats (control groups of 10 rats) were
given s.c. injections of 0.5 ml of a 3% or 6% solution (the rats
received with each injection 15 or 30 mg, respectively). The colour
used in the experiment was certified as 92% pure and was supplied as
the disodium sulfonate salt. The 10 control rats were given distilled
water injections. At first, injections of 6% were given 3 times a
week; after 17 weeks it became necessary to reduce the dose to 3%.
Thereafter, both groups were given injections of 3% twice weekly for
9 weeks. The rest of the time, 22 weeks, both groups were injected
usually once a week, while occasionally 2 injections were tolerated.
Growth inhibition was observed. Thirteen out of 16 animals receiving
6% of the colour had fibrosarcomas. The animals given 3% also showed
fibrosarcomas (10 out of 12). The controls did not show neoplastic
tissue at the site of injection (Hasselbach & O'Gara, 1960).
Subcutaneous injection of 1 ml of an 0.8% solution twice weekly
produced histological changes suggestive of subsequent sarcoma
formation unassociated with chemical carcinogenic potential (Grasso &
Golberg, 1966).
No tumours were produced in 11 hamsters injected with 1 mg of the
dye in 0.1 ml water (Price et al., 1978).
A carcinogenicity study with an in utero phase was carried out
in Charles-River albino rats. Groups of 60 (120 controls) male and
female rats of the F0 generation were fed diets containing 0, 1.25,
2.5, or 5.0% Fast Green FCF for 2 months prior to mating and
throughout gestation and lactation. Following the reproductive phase,
a maximum of 4 animals of each sex/litter were randomly selected from
the F1 generation for the long-term carcinogenicity study. Groups of
70 animals of each sex/group were given Fast Green FCF in the diet at
the same concentrations as the parent generation. An interim kill of
10 animals of each sex per group was carried out after 12 months; the
remaining animals continued to receive the test diets for 29 months
(males) or 31 months (females). Haematology, clinical chemistry tests,
and urinalysis were performed on 10 rats of each sex/group at 3, 6,
12, 18, and 24 months. Gross autopsies were performed on all animals
that died on test or were killed in a moribund condition and on all
F1 generation animals at interim and terminal sacrifice. The
following tissues were examined histologically from all animals killed
at interim sacrifice and all survivors from the control and 5%-dose
groups, as well as all animals dying or killed in extremis from
these groups: adrenals, aorta, bone and marrow (femur), brain
(3 sections), eyes (with optic nerve), gastrointestinal tract
(oesophagus, stomach, duodenum, ileum, caecum, colon), heart, kidneys,
liver, lung, lymph nodes (mesenteric, mediastinal) mammary gland,
nerve (sciatic), ovaries, pancreas, pituitary, prostate, salivary
gland, seminal vesicles, skeletal muscle, skin, spinal cord, spleen,
testes with epididymides, thymus, thyroid/parathyroid, trachea,
urinary bladder, uterus, and gross lesions/tissue masses. In addition,
gross changes/tissue masses were examined histologically from all
animals in the lower-dose groups. Subsequently, the urinary bladder
from males of the 1.25- and 2.5%-dose groups were also examined
histologically.
During the premating period, no treatment-related effects were
seen on mortality or body-weight gain but there was a dose-related
increase in food consumption. After mating there were no treatment-
related effects on the number of successful pregnancies or pup
viability at birth, but pup mortality was increased in the 5%-dose
group during the period 4-14 days of lactation. Mean pup weight was
reduced in all treated groups, most markedly in the high-dose group.
In the F1 generation, mortality was slightly higher in all
treated groups than in controls, but it did not vary in a dose-related
manner. Mean body weights of the high-dose males were consistently
lower than controls, even though their food intake was elevated.
Fasting blood glucose levels were elevated in females in all treated
groups at 3 and 12 months, females in the 1.25 and 2.5% groups at 18
months, and males in all treated groups at 12 and 18 months.
At interim (12 months) sacrifice, the mean absolute and relative
thyroid weights were elevated in the high-dose males while the
relative kidney weights were elevated in the high-dose females. At
termination, the thyroid weights were elevated in males of the
2.5- and 5%-dose groups and females of the 5% group; kidney weights
were elevated in both sexes of the 5%-dose group and females of the
2.5% group. No treatment-related effects were seen in urinalyses,
haematology determinations, physical observations, or ophthalmology.
Histopathological examination revealed an increased incidence of
urothelial hyperplasia in treated males and of urinary bladder
transitional cell/urothelial neoplasms in males of the 5%-dose group.
The overall incidences in males are summarized below:
Group Control Control 1.25% 2.5% 5%
1 2
Number examined 58 61 58 55 60
Number with neoplasia 1 2 1 2 5
Number with hyperplasia 1 4 7 10 3
Non-statistically-significant increases in testicular Leydig cell
tumours and neoplastic nodules in the liver were also observed. When
time-to-tumour analysis was performed on pathology incidence data, the
increased incidence of bladder tumours was confirmed and the incidence
of several other tumour types showed statistically-significant
differences related to treatment, including neoplastic nodules in the
liver (males and females), female mammary adenomas and pituitary
adenomas, male parthyroid adenomas, male thyroid medullary carcinomas,
female uterine leiomyosarcomas, and male testicular
interstitial/Leydig cell tumours.
Of the non-neoplastic pathology, chronic nephropathy was a common
finding in all groups but the severity was greater in females in the
5%-dose group. Other lesions did not appear to be related to treatment
(Knezevich & Hogan, 1981).
Special studies mutagenicity
Fast Green FCF was non-mutagenic in the Salmonella/microsome
assay (Brown et al., 1978) and negative results were also obtained
in bacterial DNA repair tests (Kada et al., 1972; Rosenkranz &
Leifer, 1980). The colour was inactive in a gene conversion assay in
diploid yeast (Sankaranarayanan & Murthy, 1979).
In one of 2 experiments, colour-induced cell transformation
occurred in cultured Fisher rat embryo cells at a concentration of
1 µg/ml (Price et al., 1978) and chromosome damage was reported in
an in vitro test using Chinese hamster ovary cells (Au & Hsu, 1979).
Special studies on reproduction
Rats
A 3-generation reproduction study was carried out on Fast Green
FCF in Long-Evans rats at dose levels of 0, 10, 100, 300, or
1,000 mg/kg b.w./day. The first generation parents (10 males, 20
females) were given the appropriate dose of Fast Green FCF in the diet
2 weeks before the first mating, and dosing continued throughout the
gestation, lactation, and post-weaning phases for three successive
generations. The F0 generation rats were mated twice, the F1a
litters being necropsied at weaning, and selected animals (10 males,
20 females) from the F1b litters were used for breeding.
Following an 80-day growth period, animals from the F1b
generation were mated 3 times and the offspring of the F2a and F2b
generations were treated identically to the F1a and F1b generations.
Following the third mating, half of the pregnant dams were sacrificed
on day 19 of gestation, the uterine contents were examined for total
embryos/resorption sites, and the corpora lutea per ovary were
recorded. The other half were allowed to deliver normally (F2c) and
were sacrificed at weaning.
The F2b animals were mated once and allowed to raise their
offspring to weaning when both parents and offspring were culled.
Gross necropsies were performed on all parent animals and on
F1a, F2a, F2c, and F3a offspring at weaning. Selected tissues from
5 animals of each sex/dose from the F1b parents and the F3a
generation at weaning were fixed at necropsy, and the following
tissues examined histologically from the control and high-dose group:
stomach, ileum, jejunum, colon, liver, spleen, heart, lungs, adrenals,
kidneys, urinary bladder, thyroid, ovaries, and uterus or testes.
No effects attributable to treatment were observed with respect
to food consumption, body weight, adult mortality, mating performance,
pregnancy and fertility rates, gestation length, offspring survival,
weights and sex, litter survival, resorption rates, or necropsy
findings. There were no macroscopic or microscopic tissue
abnormalities of either F1b- or F3a-generation animals considered to
be attributable to treatment (Smith, 1973).
Acute toxicity
LD50
Species Route (mg/kg b.w.) Reference
Rat Oral > 2,000 Lu & Lavallee,
1964
Dog Oral > 200 Radomski &
mg/dog Deichman, 1956
Short-term studies
Dogs
Four beagles/group, equally divided by sex, were fed Fast Green
FCF at 0, 1.0, or 2.0% of the diet for 2 years. Histopathology
attributable to the colour was limited to green blobs of pigment in
the renal cortical tubular epithelial cytoplasm of a male dog at the
high-dose level; a female dog at the high-dose level showed slight
interstitial nephritis and slight bone marrow hyperplasia (Hansen
et al., 1966).
Long-term studies
Mice
Groups of 50 male and 50 female C3HeB/FeJ mice were fed diets
containing 1.0 or 2.0% Fast Green FCF for 2 years and 100 mice of each
sex served as controls. After 78 weeks, 56 controls, 27 animals in the
1.0%-treatment group, and 17 animals in the 2.0%-treatment group still
survived. Microscopic examination revealed no lesions that were
attributed to feeding of the colour (Hansen et al., 1966).
Rats
Groups of 50 weanling Osborne-Mendel rats, evenly divided by sex,
were fed diets containing 0, 0.5, 1.0, 2.0, or 5.0% colour for 2
years. No effects on growth or mortality were observed. Microscopic
examination revealed no lesions that were attributed to the feeding of
the colour (Hansen et al., 1966).
The colour was fed at a dietary level of 4.0% to 5 male and 5
female rats for periods from 18 to 20 months. This procedure resulted
in gross staining of the forestomach, glandular stomach, small
intestine, and colon. Granular deposits were noted in the stomach. No
tumours were observed (Willheim & Ivy, 1953).
Observations in man
No data available.
Comments
The production of local sarcomata at the site of s.c. injection
in rats is not considered to constitute evidence of carcinogenicity by
the oral route. The mouse oral carcinogenicity study was negative but
in the rat study, an increased incidence of urothelial hyperplasia
and/or neoplasia of the bladder was observed. The biological
significance of observed differences in benign and malignant tumours
at other sites is questionable since, in some cases, statistically-
significant differences were observed between the 2 control groups
and, apart from the bladder, complete histological examination was not
performed on the low- and intermediate-dose groups.
Biochemical studies have shown that the colour is poorly absorbed
and the 3-generation reproduction/teratogenicity study was uneventful.
In view of the equivocal results of the most recent
carcinogenicity study in rats, the evaluation is based on the earlier
study, pending complete histological examination of all groups of rats
and biometric examination of the data.
EVALUATION
Level causing no toxicological effect
Mouse: 5% in the diet equal to 18,600 mg/kg b.w./day falling to
8,000 mg/kg b.w./day.
Rat: 5% in the diet equivalent to 2,500 mg/kg b.w./day.
Estimate of temporary acceptable daily intake for man
0-12.5 mg/kg b.w.
Further work or information
Required by 1986
Complete histological examination of all dose-groups in the long-
term carcinogenicity feeding-study in the rat and biometric
examination of the data.
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See Also:
Toxicological Abbreviations
Fast green FCF (FAO Nutrition Meetings Report Series 46a)
Fast Green FCF (WHO Food Additives Series 16)
Fast Green FCF (WHO Food Additives Series 21)
FAST GREEN FCF (JECFA Evaluation)
Fast Green FCF (IARC Summary & Evaluation, Volume 16, 1978)