2

FD&C Green No. 3 is currently approved for use as an inert ingredient in non-food use pesticide 

products. The Joint FAO/WHO Expert Committee on Food Additives (JECFA) has assigned 

maximum acceptable daily intakes (ADIs) of 25 mg/kg body weight for FD&C Green No. 3. It is 

currently used as a biological stain; color additive in food, drugs and cosmetics. 

FD&C Green No. 3 is not acutely toxic via the oral route in rats and dogs given the median lethal 

doses (LD50) were greater than 2000 mg/kg body weight and 200 mg/kg body weight, respectively. 

Groups of 60 male and female Charles-River SC- mice, including 120 control animals, were fed 

diets containing 0, 0.5, 1.5 or 5.0% (equivalent to 0, 750, 2250 and 7500 mg/kg bw/day) Fast 

Green FCF beginning at 43 days of age for approximately 24 months. There were no treatment 

related effects on mortality in this study. The mean body weights for the females in the 5% dose 

group were consistently lower than the controls. Histological examination of all animals did not 

reveal any treatment related lesions. There was also no difference between control and treated 

animals in terms of the incidence of benign and malignant neoplasms. The no observed adverse 

effect level (NOAEL) was 5% in the diet (equivalent of 7500 mg/kg bw/day), the highest dose 

tested. 

Multiple long-term studies in mice, dogs and rats fed diets containing Fast Green FCF were 

conducted. Microscopic examination revealed no treatment related lesions attributable to feeding 

of the color in any of the studies. There were also no treatment related effects on growth or 

mortality. 

A carcinogenicity study with a in utero phase was conducted with Charles-River albino rats. 

Groups of 60 male and female rats of the F0 generation were fed diets containing 0, 1.25, 2.5 or 

5.0% (equivalent to 0, 625m 1250 and 2500 mg/kg bw/day) Fast Green FCF for 2 months prior to 

mating and throughout gestation and lactation. There were also 120 control animals used in this 

study. Following the reproductive phase, a maximum of 4 rats/sex/litter were randomly selected 

from the F1 generation for the long-term carcinogenicity study. Seventy animals of each sex/group 

were fed the test substance at the same concentrations as the parent generation. During the 

premating period, there were no treatment-related effects on mortality or body-weight gain. 

However, there was a dose-related increase in food consumption. 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 day 4-14 of lactation. Mean pup weight was reduced in all 

test groups, most distinctly in the high-dose group. In the F1 generation mortality was slightly 

higher in all treatment groups as compared to control groups. However, it did not occur in a doesrelated manner. At interim sacrifice the mean and relative thyroid weights were elevated in highdose males and relative kidney weights were elevated in high-dose females. At termination, males 

and females showed elevated thyroid weights. Kidney weights were elevated in both sexes of the 

5% group. “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.” It was concluded that the observation of only three neoplasms in the highdose male group, two of which were of questionable neoplastic character, and the absence of 

evidence of a pre-neoplastic process in the observed hyperplasia, established that there is no 

indication of a neoplastic effect on the urinary bladder from administration of Fast Green FCF.” 

The NOAEL for carcinogenicity was 5% in the diet (equivalent of 2500 mg/kg bw/day; the highest 

dose tested). No reproductive toxicity was observed at doses up to 5% in the diet (equivalent to 
3

2500 mg/kg bw/day). The NOAEL for systemic toxicity is parental animals was 2.5% in the diet 

(equivalent to 1250 mg/kg bw/day) based on decreased in food consumption, increased in thyroid 

and kidney weights seen at the LOAEL of 5% in the diet. The NOAEL for off spring toxicity was 

2.5% in the diet (equivalent to 1250 mg/kg bw/day) based on decreased in pup body weight and 

pup mortality seen at the LOAEL of 5% in the diet. 

A 3-generation reproduction study was completed on Fast Green FCF in Long-Evans rats at dose 

levels of 0, 10, 100, 300 or 1000 mg/kg bw/day. No treatment related effects on 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 also no macroscopic or microscopic tissue abnormalities attributable to 

treatment. The NOAEL was 1000 mg/kg bw/day. 

In a Salmonella/microsome assay, Fast Green FCF was determined to be non-mutagenic. Negative 

results were also found in bacterial DNA repair tests. 

Immunotoxicity and neurotoxicity studies were not available for review. However, signs of 

immunotoxicity and neurotoxicity were not observed in any of the studies that were submitted and 

EPA concluded that studies are not required at this time. 

The metabolism potential of Fast Green FCF was tested in rats and dogs. Almost all of the color 

was excreted unchanged in the feces of the rats and no color was found in the urine. The color did 

not exceed 5% of the given dose in the bile of the dogs. After feeding, the color was found in the 

bile of rats, but not in the urine. It was concluded that the amount found in the bile provides an 

estimate of the amount absorbed through the gastrointestinal tract. 

The available toxicity studies indicate that FD&C Green No. 3 has a very low overall toxicity. The 

lowest NOAEL in the database is 1000 mg/kg bw/day. In the carcinogenicity study with an in 

utero phase, the effects on the pups (decreased body weights and pup mortality) and kidney and 

thyroid toxicity in adults were observed at 5% in diet (equivalent to 2500 mg/kg/day). Since these 

effects were observed at 2.5 times the limit dose of 1000 mg/kg/day, there are low concerns for the 

hazard. Since no endpoint of concern was identified for the acute and chronic dietary exposure 

assessments and short- and intermediate-term dermal and inhalation exposure assessments, a 

quantitative risk assessment for FD&C Green No. 3 is not necessary. 

There is no evidence of qualitative or quantitative susceptibility of infants and children in the 

available database. As part of its qualitative assessment, the Agency did not use safety factors for 

assessing risk, and no additional safety factor is needed for assessing risk to infants and children. 

The available toxicity studies suggest low toxicity FD&C Green No. 3. The toxicity database for 

FD&C Green No. 3 contains acute oral toxicity studies and chronic toxicity studies, including 

carcinogenicity and reproductive toxicity studies. The database also contains mutagenicity studies 

and metabolism data. There is no indication, based upon the available data, that FD&C Green No. 

3 is a neurotoxic or immunotoxic chemical or results in increased qualitative or quantitative 

susceptibility in infants or children. Based on this information, there is no concern, at this time, for 

increased sensitivity to infants and children to this chemical when used as inert ingredient in 

pesticides formulations. 
4

Dietary exposure to FD&C green No. 3 can occur from eating food that has come in contact with 

surfaces treated with pesticide formulations containing this inert ingredient. Dietary exposure to 

FD&C Green No. 3 can also occur from eating foods which contain FD&C Green NO. 3 as an 

ingredient. Non-dietary exposure from the proposed use is not expected, however non-dietary 

exposure may occur from other approved uses of this chemical. Occupational exposure is also 

possible. However, since there are no toxicological effects of concern identified in the available 

database, it is not necessary to conduct assessments of residential (non-occupational) exposures 

and risks. There are no dermal or inhalation toxicological endpoints of concern to the Agency; 

therefore quantitative assessments have not been conducted. 

EPA has not found FD&C Green No. 3 to share a common mechanism of toxicity with any other 

substances, and FD&C Green No. 3 does do not appear to produce a toxic metabolite produced by 

other substances. For the purposes of this tolerance action, therefore, EPA has assumed that the 

FD&C Green No. 3 does not have a common mechanism of toxicity with other substances. 

In terms of terrestrial, aquatic and atmospheric fate, FD&C Green No. 3 is expected to be mobile 

in soil as it is expected to exist almost entirely in anion form in the environment. Anions do not 

adsorb more strongly to soils containing organic carbon and clay than their neutral counterparts. 

Volatilization from moist soil and water surfaces is not expected. Due to the ionic nature of this 

chemical, bioconcentration in aquatic organisms is not expected. Ecological modeling indicates 

that FD&C Green No. 3 has potentially low toxicity to aquatic organisms. 

 

Potential areas of environmental justice concerns, to the extent possible, were considered in this 

human health risk assessment, in accordance with U.S. Executive Order 12898, "Federal Actions 

to Address Environmental Justice in Minority Populations and Low-Income Populations," 

http://www.eh.doe.gov/oepa/guidance/justice/eo12898.pdf. 

Based on the lack of toxicological effects of concern and upon the conditions discussed above, the 

EPA concludes that there is a reasonable certainty that no harm will result to the general 

population, or to infants and children from aggregate exposure to residues of the FD&C Green No. 

3 when used as an inert ingredient in pesticide formulations under 40 CFR 180.940(a).

I. BACKGROUND 

This is the decision document for FD&C Green No. 3 (CAS Reg. No. 2353-45-9). The firm 

Exponent (1150 Connecticut Ave., NW; Suite 1100; Washington, DC 20036), on behalf of Ecolab 

(370 N. Wabasha St., St. Paul, MN 55102) has submitted a request to the Environmental 

Protection Agency (EPA or “the Agency”) to exempt from the requirements of a tolerance FD&C 

Green No. 3 when used as an inert ingredient in antimicrobial formulations, for use on food 

contact surfaces in public eating places, dairy processing equipment, and food processing 

equipment and utensils under 40 CFR 180.940(a). The chemical is also referred to as Fast Green 

FCF in this document, as this name is synonymous with FD&C Green No. 3. 

FD&C Green No. 3 was evaluated by the Joint FAO/WHO Expert Committee on Food Additives 

(JECFA) multiple times, most recently in 1987. JECFA established an acceptable daily intake 

(ADI) of 25 mg/kg body weight. 
5

II. Inert Ingredient Profile 

A. Summary of Uses 

1. Proposed Use Pattern 

The proposed use pattern requested in this petition for FD&C Green No. 3 is as an inert 

ingredient in antimicrobial formulations, for use on food contact surfaces in public eating 

places, dairy processing equipment, and food processing equipment and utensils under 40 

CFR 180.940(a), without limitation. 

2. Inert uses 

FD&C Green No. 3 is currently approved for use as an inert ingredient in non-food use 

pesticide products. 

 

3. Other uses 

The Joint FAO/WHO Expert Committee on Food Additives (JECFA) have assigned a 

maximum acceptable daily intake (ADIs) for FD&C Green No. 3. It is currently used as a 

biological stain; color additive in food, drugs and cosmetics. 

 

Table 1. FDA Approved Color Additive Uses 

Chemical 21 CFR 74 Uses 

FD&C Green No. 

3 

74.203 Coloring in food, including 

dietary supplements 

 74.1203 Coloring in drugs 

 74.2203 Coloring in cosmetics 
6

III. Physical Chemical Properties 

Some of the physical and chemical characteristics of FD&C Green No. 3, along with its structure 

and nomenclature, are found in Table 3. 

Table 2. Physical and Chemical Properties of FD&C Green No. 3 

Parameter Value Source 

Structure ChemIDplus Lite, 2013 

CAS Chemical Name 

Benzenemethanaminium, N-ethylN-(4-((4-(ethyl((3- 

sulfophenyl)methyl)amino)phenyl)(

4-hyd- roxy-2- 

sulfophenyl)methylene)-2,5-

cyclohexadien-1-ylidene)-3-sulfo-, hydroxide, inner salt, disodium 

salt 

ChemIDplus Lite, 2013 

Chemical Formula C37-H36-N2-O10-S3 ChemIDplus Lite, 2013 

Synonyms C.I. Food Green 3, disodium salt, 

Fast Green FCF ChemIDplus Lite, 2013 

CAS # 2353-45-9 ChemIDplus Lite, 2013 

SMILES Notation 

C(=C1\C=C\C(=[N+](\Cc2cc(ccc2)

S(=O)(=O)[O-

])CC)C=C1)(/c1c(cc(cc1)O)S(=O)(

=O)O)c1ccc(cc1)N(Cc1cc(ccc1)S(=

O)(=O)O)CC.[Na].[Na] 

ChemIDplus Lite, 2013 

Color/Form 

Dark green powder or granules with 

a metallic luster. Red to brownviolet powder for dark green 

crystals 

PubChem, 2013 

Molecular Weight 808.12 PubChem, 2013 

Water Solubility 20.0 g/100 mL at 25 deg C 

(2.0X10+5 mg/L) PubChem, 2013 

Melting Point (°C) 290 °C PubChem, 2013 

Atmospheric OH Rate 4.18E-10 cm3/molecule-sec (est) ChemIDplus Lite, 2013 
7

Constant 

Partition Coefficient 

(usually the Log P or Kow)

Log P= -5.420 (est) 

Log Kow= -3.22 (est) ChemIDplus Lite, 2013

IV. Hazard Assessment 

A. Summary of Toxicity Data. 

FD&C Green No. 3 is not acutely toxic. Long term studies of the effects of the color administered 

in the diet to rats, mice and dogs did not indicate any significant toxic effects. A long term 

carciongencity study did not show any carcinogenic effects from dietary administration of FD&C 

Green No. 3. A reproductive study did not show any effects on fertility or fetal abnormalities. 

There is no evidence of this chemical being considered a neurotoxic or immunotoxic chemical. 

Studies also show that this chemical is non-mutagenic. Metabolism studies indicate that primarily 

all of the FD&C Green No. 3 that was administered via the diet in rats and mice was excreted 

unchanged in the feces. 

B. Toxicity Profile 

Acute Toxicity 

In one acute oral toxicity study in rats, reported in JECFA, the LD50 was determined to be greater 

than 2000 mg/kg body weight. In a second study with dogs the LD50 was greater than 200 mg/kg. 

No other acute studies were found in the literature. 

Subchronic Toxicity 

No subchronic toxicity studies are available in the database. 

Chronic Toxicity (Oral)

Groups of 60 male and female Charles-River SC- mice, including 120 control animals, were fed 

diets containing 0, 0.5, 1.5 or 5.0% (equivalent to 0, 750, 2250 and 7500 mg/kg bw/day) Fast 

Green FCF beginning at 43 days of age for approximately 24 months. Hematological 

examinations were conducted at 3, 6, 12 and 18 months on 10 animals/sex/group. Post-mortem 

examination was conducted on all animals that died during treatment or were killed in moribund 

condition. Animals that survived to termination were also subject to post-mortem examination. 

All survivors from the control and 5% dose groups were subject to histological examination of 

tissues including adrenals, aorta, bone and marrow (femur), brain (3 sections), eyes (with optic 

nerve), gall bladder, gastrointestinal tract (esophagus, 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, 
8

skin, spinal cord, spleen testes with epididymides, thymus, thyroid/parathyroid, trachea, urinary 

bladder, uterus, and gross lesions and tissue masses. All animals that that died or were killed in 

extremis from these groups were subject to the same histological examinations. All animals in the 

lower dose test groups were examined for signs of gross changes and tissue masses. 

There were no treatment related effects on mortality in this study. The mean body weights for the 

females in the 5% dose group were consistently lower than the controls. Mean body weights for 

males were consistently lower than the controls at weeks 52 and 78. There were no other effects 

on body weight noted in the study. In high-dose males at 18 months, there were slight reductions 

in hemoglobin, hematocrit and erythrocyte counts. However, there were no other consistent or 

dose-related changes observed. Histological examination of all animals did not reveal any 

treatment related lesions. There was also no difference between control and treated animals in 

terms of the incidence of benign and malignant neoplasms. The NOAEL was 5% in the diet 

(equivalent of 7500 mg/kg bw/day), the highest dose tested. (JECFA, 1986) 

In a second study with 50 male and 50 female C3HeB/FeJ mice were fed diets containing 1.0 or 

2.0% Fast Green FCF for 2 years (equivalent to 1500 and 3000 mg/kg bw/day). One hundred mice 

of each sex served as controls. After 78 weeks, 56 controls, 27 animals in the 1.0% test group and 

17 animals in the 2.0% test group still survived. Microscopic examination reveled no treatment 

related lesions attributable to feeding of the color. (JECFA, 1986) 

In a third study in dogs, 4 beagle dogs/ group, equally divided by sex, were fed Fast Green FCF for 

a time period of 2 years at 0, 1.0 and 2.0% in the diet (equivalent to 250 and 500 mg/kg bw/day). 

Histopathology attributable to the color 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. (JECFA, 1986) 

In a fourth study in rats, groups of 50 weanling Osborne-Mendel rats, evenly divided by sex, were 

fed diets containing 0, 0.5, 1.0, 2.0 or 50.% Fast Green FCF for 2 years (equivalent to 250, 500, 

1000 or 2500 mg/kg bw/day). There were no treatment related effects on growth or mortality. 

Microscopic examination revealed no lesions attributed to the feeding of the test substance. 

(JECFA, 1986) 

In a fifth study in rats, the color was fed at a dietary level of 4.0% (equivalent to 2000 mg/kg 

bw/day) for a time period of 18 to 20 months. This resulted in gross staining of the forestomach, 

glandular stomach, small intestine and colon. Granular deposits were also noted in the stomach. 

No tumors were observed. (JECFA, 1986) 

Carcinogenicity (Oral)

A carcinogenicity study with a in utero phase was conducted with Charles-River albino rats. 

Groups of 60 male and female rats of the F0 generation were fed diets containing q, 1.25, 2.5 or 

5.0% (equivalent to 0, 625m 1250 and 2500 mg/kg bw/day) Fast Green FCF for 2 months prior to 

mating and throughout gestation and lactation. There were also 120 control animals used in this 

study. Following the reproductive phase, a maximum of 4 rats/sex/litter were randomly selected 
9

from the F1 generation for the long-term carcinogenicity study. Seventy animals of each sex/group 

were fed the test substance at the same concentrations as the parent generation. After 12 months, 

an interim kill was carried out for 10 rats of each sex per group. The remaining animals continued 

to receive the test diet for 29 months (males) or 31 months (females). Hematology, clinical 

chemistry tests and urinalysis were performed on 10 rats/sex/group at 3, 6, 12, 18 and 24 months. 

Gross autopsies were performed on all animals. Histological examination of tissues was 

conducted on all animals killed at interim sacrifice and all survivors from the control and 5% dose 

groups, as well as all animals that died or were killed in extremis for these dose groups. 

Histological examination of tissues included adrenals, aorta, bone and marrow (femur), brain (3 

sections), eyes (with optic nerve), gall bladder, gastrointestinal tract (esophagus, 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 and tissue masses. All 

animals in the lower dose test groups were examined for signs of gross changes and tissue masses. 

During the premating period, there were no treatment-related effects on mortality or body-weight 

gain. However, there was a dose-related increase in food consumption. There were no treatmentrelated effects on the number of successful pregnancies or pup viability at birth, but pup mortality 

was increased in the 5% dose group during day 4-14 of lactation. Mean pup weight was reduced in 

all test groups, most distinctly in the high-dose group. 

In the F1 generation mortality was slightly higher in all treatment groups as compared to control 

groups. However, it did not occur in a does-related manner. In spite of elevated food intake in 

high-dose males, mean body weights were consistently lower than controls. Fasting blood 

glucose levels were elevated in females in all groups at 3 and 12 months. Females in the 1.25 and 

2.5% groups showed elevated levels at 18 months. Males in all treatment groups showed elevated 

levels at 12 and 18 months. 

At interim sacrifice (12 months), the mean and relative thyroid weights were elevated in high-dose 

males and relative kidney weights were elevated in high-dose females. At termination, males in 

the 2.5 and 5% dose groups and females of the 5% group showed elevated thyroid weights. 

Kidney weights were elevated in both sexes of the 5% group. Females in the 2.5% group also 

showed increased kidney weights. There were no treatment related effects seen in urinalyses, 

hematology determinations, physical observations or opthanlmology. 

“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 1 Control 2 1.25% 2.5% 5% 

Number 

examined 

58 61 58 55 60 

Number with 

neoplasia 

1 2 1 2 5 

Number with 

hyperplasia 

1 4 7 10 3 
10

“Non-statistically-significant increases in testicular Leydig cell tumors and neoplastic nodules in 

the liver were also observed. When time-to-tumor analysis was performed on pathology incidence 

data, the increased incidence of bladder tumors was confirmed and the incidence of several other 

tumor types showed statistically-significant differences related to treatment, including neoplastic 

nodules in the liver (males and females), female mammary adenomas and pituitary adenomas, 

male parathyroid adenomas, male thyroid medullary carcinomas, female uterine leiomyosarcomas, 

and male testicular interstitial/Leydig cell tumors.” 

“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.” (JECFA, 1986) 

The results of the carcinogenicity study were re-evaluated in 1987. “A review of the 

histopathological data from the rat oral carcinogenicity study was performed from which it was 

concluded that inappropriate statistical tests had been performed on some of the data by the testing 

laboratory. Using appropriate statistical procedures the reviewers concluded that there were no 

significant differences in tumour incidence between combined control groups and the high-dose 

group with respect to tumours of the liver, testes, or thyroid; the tissues from the low- and 

intermediate-dose groups were not examined. In a blind review of the slides of the bladders from 

all dose groups, only three proliferative lesions, two benign (papilloma) and one malignant 

(carcinoma), were observed in bladders of male rats in the high-dose group and a treatment-related 

increase in the incidence or severity of transitional cell hyperplasia was not detected. It was 

concluded that the observation of only three neoplasms in the high-dose male group, two of which 

were of questionable neoplastic character, and the absence of evidence of a pre-neoplastic process 

in the observed hyperplasia, established that there is no indication of a neoplastic effect on the 

urinary bladder from administration of Fast Green FCF.” The NOAEL for carcinogenicity was 5% 

in the diet (equivalent of 2500 mg/kg bw/day; the highest dose tested). No reproductive toxicity 

was observed at doses up to 5% in the diet (equivalent to 2500 mg/kg bw/day). The NOAEL for 

systemic toxicity is parental animals was 2.5% in the diet (equivalent to 1250 mg/kg bw/day) 

based on decreased in food consumption, increased in thyroid and kidney weights seen at the 

LOAEL of 5% in the diet. The NOAEL for off spring toxicity was 2.5% in the diet (equivalent to 

1250 mg/kg bw/day) based on decreased in pup body weight and pup mortality seen at the LOAEL 

of 5% in the diet. (JECFA, 1987) 

Reproductive Toxicity

A 3-generation reproduction study was completed on Fast Green FCF in Long-Evans rats at dose 

levels of 0, 10, 100, 300 or 1000 mg/kg bw/day. The first generation parents (10 males and 10 

females) were given the appropriate dose of the test substance 2 weeks prior to mating with dosing 

continuing throughout gestation, lactation, and post-weaning phases for 3 successive generations. 

The F0 generation rats were mated twice. The F 1a litters were necropsied at weaning and 10 

males and 10 females from the F 1b litters were used for breeding. 
11

After and 80-day growth period, animals from the F 1b generation were mated 3 times. And the 

offspring from the F2a and F2b generations were treated in the same manner as the F1a and F1b 

animals. After completion of the third mating, half of the pregnant dams were sacrificed on day 19 

of gestation with the uterine contents being examined for total embryos/resorption sites and 

corpora lutea per ovary. The other half were allowed to deliver normally 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 sacrificed. 

Gross necropsies were performed on all parent animals. Offsping from the F1a, F2a, F2c and F3a 

generations were subject to gross necropsies at weaning. Tissues from 5 animals of each sex/dose 

of the F1b parents and the F3a generation were fixed at necropsy. Tissue examination from the 

control and high dose groups included stomach, ileum, jejunum, colon, liver, spleen, hearth, lungs, 

adrenals, kidneys, urinary bladder, thyroid, ovaries, and uterus or testes. 

No treatment related effects on 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 also no macroscopic or 

microscopic tissue abnormalities attributable to treatment. The NOAEL was 1000 mg/kg bw/day. 

(JECFA, 1986) 

Mutagenicity

In a Salmonella/microsome assay, Fast Green FCF was determined to be non-mutagenic. Negative 

results were also found in bacterial DNA repair tests. In another test, color-induced cell 

transformation occurred in cultured Fisher rat embryo cells at a concentration of 1μg/ml. 

Chromosome damage was also reported in an in vitro test using Chinese hamster ovary cells. 

(JECFA, 1986) 

Neurotoxicity

No neurotoxicity studies were available for review. However, signs of neurotoxicity were not 

observed in any of the studies that were submitted and EPA concluded that a study is not required 

at this time. 

Immunotoxicity

No immunotoxicity studies were available for review. However, signs of immunotoxicity were not 

observed in any of the studies that were submitted and EPA concluded that studies are not required 

at this time. 

Metabolism and Pharmacokinetics

The metabolism potential of Fast Green FCF was tested in rats and dogs. Each species was given 

orally 200 mg of the color. In the rats, the urine and feces were collected for 36 hours. In the 

dogs, a bile fistula was made for bile analysis. Almost all of the color was excreted unchanged in 

the feces of the rats and no color was found in the urine. The color did not exceed 5% of the given 
12

dose in the bile of the dogs. After feeding, the color was found in the bile of rats, but not in the 

urine. It was concluded that the amount found in the bile provides an estimate of the amount 

absorbed through the gastrointestinal tract. (JECFA, 1986) 

C. Toxicity Endpoint Selection 

The available toxicity study indicates that FD&C Green No. 3 has a very low overall toxicity. The 

lowest NOAEL in the database is 1000 mg/kg bw/day. In the carcinogenicity study with an in 

utero phase, the effects on the pups (decreased body weights and pup mortality) and kidney and 

thyroid toxicity in adults were observed at 5% in diet (equivalent to 2500 mg/kg/day). Since these 

effects were observed at 2.5 times the limit dose of 1000 mg/kg/day, there are low concerns for the 

hazard. Since no endpoint of concern was identified for the acute and chronic dietary exposure 

assessments and short- and intermediate-term dermal and inhalation exposure assessments, a 

quantitative risk assessment for FD&C Green No. 3 is not necessary.

V. FQPA Safety Factor Considerations 

Section 408(b)(2)(c) of FFDCA provides that EPA shall apply an additional tenfold (10X) margin 

of safety for infants and children in the case of threshold effects to account for prenatal and 

postnatal toxicity and the completeness of the database on toxicity and exposure unless EPA 

determines based on reliable data that a different margin of safety will be safe for infants and 

children. This additional margin of safety is commonly referred to as the FQPA safety factor (SF). 

In applying this provision, EPA either retains the default value of 10X, or uses a different 

additional safety factor when reliable data available to EPA support the choice of a different factor. 

There is no evidence of qualitative or quantitative susceptibility of infants and children in the 

available database. 

 

As part of its qualitative assessment, the Agency did not use safety factors for assessing risk, and 

no additional safety factor is needed for assessing risk to infants and children. The available 

toxicity studies suggest low toxicity of FD&C Green No. 3. The toxicity database for FD&C Green 

No. 3 contains an acute oral toxicity study and chronic toxicity studies, including carcinogenicity, 

and reproductive toxicity studies. No reproductive or developmental toxicity was observed in the 

3-generation reproduction study. The database also contains mutagenicity studies, and metabolism 

data. There is no indication, based upon the available data, that FD&C Green No. 3 is a neurotoxic 

or immunotoxic chemical or results in increased qualitative or quantitative susceptibility in infants 

or children. Based on this information, there is no concern, at this time, for increased sensitivity to 

infants and children to this chemical when used as inert ingredient in pesticides formulations. 

VI. Exposure 

A. Dietary Exposure 
13

Dietary exposure to FD&C Green No. 3 can occur from eating food that has come in contact with 

surfaces treated with pesticide formulations containing this inert ingredient. Dietary exposure to 

FD&C Green No. 3 can also occur from eating foods which contain FD&C Green NO. 3 as an 

ingredient. However, since an endpoint of concern for risk assessment was not identified, a 

quantitative dietary exposure assessment for FD&C Green No. 3 was not conducted. 

 i. Acute exposure. No adverse effects attributable to a single exposure of FD&C Green No. 3 

were seen in the toxicity databases. Therefore, acute dietary risk assessment for FD&C Green No. 

3 is not required.

 ii. Chronic exposure. A chronic dietary exposure assessment was not conducted for FD&C 

Green No. 3 because no toxicological endpoint of concern was identified for chronic dietary 

exposure. The available studies/literature contained no evidence of a chronic dietary endpoint of 

concern. 

iii. Dietary exposure from drinking water: Dietary exposure from drinking water to FD&C 

Green No. 3 can occur by drinking water that has been contaminated by run-off from a pesticide 

treated area, such as countertops. Since an endpoint for risk assessment was not identified, a 

quantitative dietary exposure assessment from drinking water for FD&C Green No. 3 was not 

conducted. 

iv. Cancer exposure. 

Based on the available toxicity data (carcinogenicity study in rats and mice) and lack of 

mutagenicity concerns, FD&C Green No. 3 is not expected to be carcinogenic. Therefore, a cancer 

dietary exposure assessment is not necessary to assess cancer risk. 

 B. Residential (Non-Occupational) Exposure 

The term “residential exposure” is used in this document to refer to non-occupational, non-dietary 

exposure (e.g., for lawn and garden pest control, indoor pest control, termiticides, and flea and tick 

control on pets). The proposed use of FD&C Green No. 3 as a dye 40 CFR 180.940(a) is not 

expected to result in residential exposure to this chemical. Residential exposure is possible based 

on other currently approved inert uses of this chemical. 

However, since there are no toxicological effects of concern identified in the available database_, 

it is not necessary to conduct assessments of residential (non-occupational) exposures and risks. 

There are no dermal or inhalation toxicological endpoints of concern to the Agency therefore 

quantitative assessments have not been conducted. 

VII. Aggregate Risk 

i. Acute Aggregate Risk. There was no hazard attributable to a single exposure seen the in the 

toxicity database for the FD&C Green No. 3; therefore an acute aggregate risk assessment is not 

required. 
14

ii. Short-term Aggregate risk. Short-term aggregate exposure takes into account short-term 

residential exposure plus chronic exposure to food and water (considered to be a background 

exposure level). The requested use pattern for FD&C Green No. 3 could result in short-term 

residential exposure. 

Since there are no oral, dermal or inhalation toxicological endpoints of concern identified in the 

available database, it is not necessary to aggregate dermal and inhalation exposures with estimated 

dietary exposures. 

Residential Post-application Exposure

Assessments of residential post-application exposure are not necessary since there are no 

toxicological endpoints of concern identified in the available database. 

VIII. Aggregate Risk Assessments and Risk Characterization 

i. Acute Aggregate Risk. There was no hazard attributable to a single exposure seen the in the 

toxicity database for FD&C Green No. 3, therefore, an acute aggregate risk assessment is not 

required. 

ii. Short-Term/Intermediate-Term Aggregate Risk. Short-term and intermediate-term 

aggregate risk assessments for FD&C Green No. 3 were not conducted. The assessments are not 

necessary since there are no dermal or inhalation toxicological effects of concern identified in the 

available database. There are no risks that exceed the Agency’s level of concern. 

IX. Occupational Exposure 

In the case of FD&C Green No. 3, the Agency has reviewed the available toxicological 

information. The data indicate that there are no adverse toxicological effects (endpoints of 

concern) identified in the available database, therefore a quantitative risk assessment is not 

necessary and is not conducted. 

 

X. Cumulative Exposure 

Cumulative effects from substances with a common mechanism of toxicity. Section 408(b)(2)(D)(v) 

of FFDCA requires that, when considering whether to establish, modify, or revoke a tolerance, the 

Agency consider “available information” concerning the cumulative effects of a particular 

pesticide's residues and “other substances that have a common mechanism of toxicity.” 

EPA has not found FD&C Green No. 3 to share a common mechanism of toxicity with any other 

substances, and FD&C Green No. 3 does do not appear to produce a toxic metabolite produced by 

other substances. For the purposes of this tolerance action, therefore, EPA has assumed that the 

FD&C Green No. 3 does not have a common mechanism of toxicity with other substances. For 

information regarding EPA's efforts to determine which chemicals have a common mechanism of 
15

toxicity and to evaluate the cumulative effects of such chemicals, see EPA's website at 

http://www.epa.gov/pesticides/cumulative.

XI. Enviromental Fate 

“Fast Green FCF's production and use as a color additive in foods, drugs and cosmetics 

may result in its release to the environment through various waste streams. If released to 

air, Fast Green FCF will exist solely in the particulate phase in the atmosphere because it is 

a salt and is nonvolatile. Particulate-phase Fast Green FCF will be removed from the 

atmosphere by wet or dry deposition. Fast Green FCF may be susceptible to direct 

photolysis by sunlight; its absorption maximum is 628 nm. If released to soil, Fast Green 

FCF is expected to be mobile; it is expected to exist almost entirely in anion form in the 

environment and anions generally do not adsorb more strongly to soils containing organic 

carbon and clay than their neutral counterparts. Volatilization from moist soil surfaces is 

not expected to be an important fate process because the compound exists as an anion and 

anions do not volatilize. If released into water, Fast Green FCF is not expected to adsorb to 

suspended solids and sediment based upon this compound's ionic nature in the 

environment. The concentrations of azo dyes with a phenolic OH and two sulfonic acid 

groups remain constant throughout activated sludge treatment and are therefore not 

expected to be biodegradable in water and soils. Volatilization from water surfaces is not 

expected to be an important fate process based upon the ionic character of the compound. 

The potential for bioconcentration in aquatic organisms is low based on the ionic character 

of the compound. Hydrolysis is not expected to be an important environmental fate process 

since this compound lacks functional groups that hydrolyze under environmental 

conditions. Occupational exposure to Fast Green FCF may occur through dermal contact 

with this compound at workplaces where Fast Green FCF is produced or used. Use data 

indicate that the general population may be exposed to Fast Green FCF via ingestion of 

food and drug products that contain this color additive, and dermal contact with cosmetics 

that use this compound.” (US NLM, TOXNET search) 

XII. Ecological Effects 

Ecological Structure-Activity Relationship Model (ECOSAR) estimations indicate that FD&C 

Green No. 3 has potentially low toxicity to aquatic organisms (fish and invertebrates) and to algae. 

XIII. Determination of Safety. 

Taking into consideration all available information on FD&C Green No. 3, EPA has determined 

that there is a reasonable certainty that no harm to any population subgroup will result from 

aggregate exposure to FD&C Green No. 3 under reasonable foreseeable circumstances. Therefore, 

the establishment of an exemption from tolerance under 40 CFR 180.940(a) for residues of FD&C 

Green No. 3 when used as an inert ingredient (dye) in antimicrobial formulations, for use on food 

contact surfaces in public eating places, dairy processing equipment, and food processing 

equipment and utensils, is safe under FFDCA section 408. 
16

XIV. Environmental Justice 

Potential areas of environmental justice concerns, to the extent possible, were considered in this 

human health risk assessment, in accordance with U.S. Executive Order 12898, "Federal Actions 

to Address Environmental Justice in Minority Populations and Low-Income Populations," 

http://www.eh.doe.gov/oepa/guidance/justice/eo12898.pdf). 

As a part of every pesticide risk assessment, OPP considers a large variety of consumer subgroups 

according to well-established procedures. In line with OPP policy, HED estimates risks to 

population subgroups from pesticide exposures that are based on patterns of that subgroup’s food 

and water consumption, and activities in and around the home that involve pesticide use in a 

residential setting. Extensive data on food consumption patterns are compiled by the USDA under 

the Continuing Survey of Food Intake by Individuals (CSFII) and are used in pesticide risk 

assessments for all registered food uses of a pesticide. These data are analyzed and categorized by 

subgroups based on age, season of the year, ethnic group, and region of the country. Additionally, 

OPP is able to assess dietary exposure to smaller, specialized subgroups and exposure assessments 

are performed when conditions or circumstances warrant. Whenever appropriate, non-dietary 

xposures based on home use of pesticide products and associated risks for adult applicators and for 

toddlers, youths, and adults entering or playing on treated areas postapplication are evaluated. 

Further considerations are currently in development as OPP has committed resources and expertise 

to the development of specialized software and models that consider exposure to bystanders and 

farm workers as well as lifestyle and traditional dietary patterns among specific subgroups. 

XV. Human Studies 

This assessment does not rely on any studies in which human subjects were intentionally exposed 

to a pesticide. 

XVI. International limits 

In making its tolerance decisions, EPA seeks to harmonize U.S. tolerances with international 

standards whenever possible, consistent with U.S. food safety standards and agricultural practices. 

EPA considers the international maximum residue limits (MRLs) established by the Codex 

Alimentarius Commission (Codex), as required by FFDCA section 408(b)(4). The Codex 

Alimentarius is a joint United Nation Food and Agriculture Organization/World Health 

Organization food standards program, and it is recognized as an international food safety 

standards-setting organization in trade agreements to which the United States is a party. EPA may 

establish a tolerance that is different from a Codex MRL; however, FFDCA section 408(b)(4) 

requires that EPA explain the reasons for departing from the Codex level. The Codex has not 

established a MRL for FD&C Green No. 3. 

 
17

References 

ECOSAR, 2000. Ecological Structure Activity Relationships Version 0.99g. 

Environmental Protection Agency. http://www .epa. gov I oppt!N ewchems/21 ecosar.htm 

ECOTOX. 2002 U.S. Environmental Protection Agency, 2002 ECOTOX User Guide: 

ECOTOXicology Database System. Version 3.0 http://www.epa.gov/ecotox/ Search 

terms: CAS Reg Nos: 2353-45-9. 

FDA, 2011, Code of Federal Regulations, Title 21, Volume 1, 21 CFR 74.203 

HSDB. 2004. Hazardous Substance Data Bank (HSDB). Online Scientific Search Engine, 

National Library of Medicine, National Institutes of Health. http://toxnet.nlm.nih.govIcgibin/sis/htmlgen?HSDB 

Search terms: FD&C Green No. 3 (CAS RN 2353-45-9); August 18, 2013 

WHO Food Additive Series 21, Prepared By: The Joint FAO/WHO Expert Committee on Food 

Additives (JECFA), 1987, FAST GREEN FCF, International Program Chemical Safety (IPCS) 

INCHEM 

 

WHO Food Additive Series 20, Prepared By: The Joint FAO/WHO Expert Committee on Food 

Additives (JECFA), 1986, FAST GREEN FCF, International Program Chemical Safety (IPCS) 

INCHEM 

WHO Food Additive Series 16, Prepared By: The Joint FAO/WHO Expert Committee on Food 

Additives (JECFA), 1982, FAST GREEN FCF, International Program Chemical Safety (IPCS) 

INCHEM 

WHO, 30th Report of the Joint Expert Committee on Food Additives, 1987, Evaluation of certain 

food additives and contaminants, Technical Report Series, 751, Page 24. 

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