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Trypan blue

Hazardous Substances DataBank Number
2945
Related PubChem Records

1 Human Health Effects

1.1 Evidence for Carcinogenicity (Complete)

No data are available in humans. Sufficient evidence of carcinogenicity in animals. OVERALL EVALUATION: Group 2B: The agent is possibly carcinogenic to humans.
IARC. Monographs on the Evaluation of the Carcinogenic Risk of Chemicals to Humans. Geneva: World Health Organization, International Agency for Research on Cancer, 1972-PRESENT. (Multivolume work). Available at: https://monographs.iarc.fr/ENG/Classification/index.php, p. S7 73 (1987)

1.2 Human Toxicity Excerpts (Complete)

/HUMAN EXPOSURE STUDIES/ Trypan blue ... Tested by injection of 0.2 ml of 1% soln subconjunctivally in patients caused no injury. A small drop at the same concn applied to the surface of the eye in patients caused practically no irritation, & though it stained superficial tissues, especially degenerating epithelial cells & mucous, it caused no damage.
Grant, W.M. Toxicology of the Eye. 3rd ed. Springfield, IL: Charles C. Thomas Publisher, 1986., p. 382
/SIGNS AND SYMPTOMS/ Short term exposure: Inhalation: no symptoms reported. Skin: may stain skin. Eyes: application of 0.2 mL (0.007 oz) of a 1% solution caused no eye irritation. Ingestion: moderately toxic. Probable lethal dose between 1 oz and 1 pound for a 150 pound person. Long term exposure: A potential occupational carcinogen, mutagen, and teratogen. Has been shown to cause birth defects, cancer, and liver injury in laboratory animals.
Pohanish, R.P. (ed). Sittig's Handbook of Toxic and Hazardous Chemical Carcinogens 5th Edition Volume 1: A-H,Volume 2: I-Z. William Andrew, Norwich, NY 2008, p. 2528
/ALTERNATIVE and IN VITRO TESTS/ Three in vitro experiments were performed. (1) cultured human corneal fibroblasts /were exposed/ to trypan blue (0.0001% to 0.1%) in Eagle modified minimum essential medium (EMEM) or phosphate-buffered saline (PBS) for 15 minutes to 24 hours. Cytotoxicity was evaluated by Mosmann's colorimetric 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MMT) assay. (2) human corneas /were exposed/ in EMEM for 24 hours to trypan blue (0.001% to 0.1%). Fellow donor corneas served as controls. Endothelial survival was evaluated morphologically and by cell density assessment. (3) the endothelial viability of human donor corneas /was morphologically compared/ after exposure to 0.1% trypan blue for 5 to 30 minutes with control corneas. RESULTS: In experiment 1, trypan blue in EMEM was not significantly toxic at concentrations of 0.005% or lower. Higher concentrations were toxic only after exposure to trypan blue for at least 6 hours. In PBS, significant toxicity was found after exposure to 0.1% trypan blue for 30 minutes or longer. Lower concentrations were toxic after longer exposures. In experiment 2, exposure to 0.01% and 0.1% trypan blue for 24 hours resulted in significant loss in cell density. At lower concentrations, the endothelium was affected only morphologically. In experiment 3, endothelial morphology changed in control corneas and after exposure to 0.1% trypan blue for as little as 5 minutes. After 30-minute exposure, morphologic deterioration was more pronounced. CONCLUSIONS: Trypan blue was toxic in vitro to corneal endothelium and corneal fibroblasts at higher concentrations and notably longer exposure times. Toxicity was less in EMEM than in PBS. Clinical Relevance: At commonly used concentrations, both during cataract surgery and in the cornea bank, trypan blue is safe for corneal cells. At higher concentrations or longer exposures, however, caution is warranted.
van Dooren BT et al; Arch Ophthalmol 122 (5): 736-42 (2004)
/ALTERNATIVE and IN VITRO TESTS/ ... Three concentrations (0.06 mg/mL, 0.6 mg/mL, and 4 mg/mL) of trypan blue were applied to human ARPE19 cells for 1 minute. Cell viability was measured using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. RPE cells were sampled daily for 6 consecutive days to assess the effects of trypan blue on cell viability. The effects of trypan blue on the expression of apoptosis related and cell cycle arrest gene expressions including c-fos, c-jun, p53, and p21 were performed using reverse transcription-polymerase chain reaction and immunostaining. RESULTS: The MTT assay showed a concentration dependent suppression effect of trypan blue on cell viability, with higher reduction in the 0.6 mg/ml and 4 mg/mL trypan blue treated groups. No significant change in the expression of c-fos and c-jun was found with all three concentrations of trypan blue. An increase in p53 expression was found in the 4 mg/mL trypan blue treated group at 10-30 minutes after trypan blue application. Immunostaining showed a mild, albeit insignificant, increase of p53 expression in the RPE cells. No significant increase in p21 expression was observed in the 0.06 mg/mL trypan blue treated group but there were significant increases in p21 expression in both the 0.6 mg/mL (p = 0.032) and the 4 mg/mL (p = 0.025) treated groups. CONCLUSIONS: Trypan blue may lead to toxicity on cultured RPE cells as indicated by the reduction in cell viability and changes in the expression of apoptosis related and cell cycle arrest genes at higher concentrations. The application of 0.06 mg/mL trypan blue for 1 minute appeared to have no significant effect on cultured RPE.
Kwok AK et al; Br J Ophthalmol 88 (12): 1590-4 (2004)
/ALTERNATIVE and IN VITRO TESTS/ Indocyanine green, infracyanine green, and trypan blue are frequently used as aids to visualize structures removed during vitreoretinal surgery. But they may have toxic effects on the retina. ... The acute and chronic toxicities of these stains on cultured human retinal pigmented epithelial (RPE) cells /were compared/ using clinically relevant concentrations and an identical experimental setup for each agent. METHODS: Monolayers of RPE cells were incubated with various concentrations of indocyanine green, infracyanine green (each at 0.005%, 0.05%, and 0.5%) or trypan blue (0.05%, 0.06%, 0.1%, 0.15%, and 0.5%) for 5 min (acute exposure) or 6 days (chronic exposure). Using the propidium iodide assay, acute cytotoxicity was monitored at 15-min intervals for up to 3 hr. Chronic cytotoxicity was assessed by monitoring cell calcein esterase activity, cell proliferation, and cell morphology (viability) after 6 days of exposure. RESULTS: Indocyanine and infracyanine green induced acute and chronic toxicities at a concentration above 0.05%. Trypan blue evoked no acute toxicity, but it was chronically cytotoxic at all tested concentrations. CONCLUSIONS: Despite thorough rinsing after application, significant amounts of the not sufficiently water soluble indocyanine and infracyanine green are retained after surgery by the eye. Trypan blue, being more water-soluble than ICG, is probably retained to the least degree. This circumstance is fortunate given that trypan blue exhibits a chronic cytotoxicity comparable to ICG at all clinically relevant concentrations. During vitrectomy, surgeons should aim to expose retinal tissue to only low concentrations of these stains and for as short a period as possible.
Kodjikian L et al; Graefes Arch Clin Exp Ophthalmol 243 (9): 917-25 (2005)

1.3 Skin, Eye, and Respiratory Irritations

May cause eye irritation. May cause skin irritation. May cause respiratory tract irritation.
Sigma-Aldrich Corp; Safety Data Sheet for Trypan blue (Product Number: 302643) Version 3.0 (January 2, 2009). Available from, as of June 15, 2010: https://www.sigmaaldrich.com

1.4 Medical Surveillance (Complete)

PRECAUTIONS FOR "CARCINOGENS": Whenever medical surveillance is indicated, in particular when exposure to a carcinogen has occurred, ad hoc decisions should be taken concerning ... /cytogenetic and/or other/ tests that might become useful or mandatory. /Chemical Carcinogens/
Montesano, R., H. Bartsch, E.Boyland, G. Della Porta, L. Fishbein, R. A. Griesemer, A.B. Swan, L. Tomatis, and W. Davis (eds.). Handling Chemical Carcinogens in the Laboratory: Problems of Safety. IARC Scientific Publications No. 33. Lyon, France: International Agency for Research on Cancer, 1979., p. 23

2 Emergency Medical Treatment

2.1 Antidote and Emergency Treatment (Complete)

Immediate first aid: Ensure that adequate decontamination has been carried out. If patient is not breathing, start artificial respiration, preferably with a demand-valve resuscitator, bag-valve-mask device, or pocket mask, as trained. Perform CPR as necessary. Immediately flush contaminated eyes with gently flowing water. Do not induce vomiting. If vomiting occurs, lean patient forward or place on left side (head-down position, if possible) to maintain an open airway and prevent aspiration. Keep patient quiet and maintain normal body temperature. Obtain medical attention. /Naphthalene and Related Compounds/
Currance, P.L. Clements, B., Bronstein, A.C. (Eds).; Emergency Care For Hazardous Materials Exposure. 3Rd edition, Elsevier Mosby, St. Louis, MO 2005, p. 273
Basic treatment: Establish a patent airway (oropharyngeal or nasopharyngeal airway, if needed). Suction if necessary. Watch for signs of respiratory insufficiency and assist ventilations if necessary. Administer oxygen by nonrebreather mask at 10 to 15 L/min. Monitor for shock and treat if necessary ... . Anticipate seizures and treat if necessary ... . For eye contamination, flush eyes immediately with water. Irrigate each eye continuously with 0.9% saline (NS) during transport ... . Do not use emetics. For ingestion, rinse mouth and administer 5 ml/kg up to 200 ml of water for dilution if the patient can swallow, has a strong gag reflex, and does not drool. Administer activated charcoal ... . /Naphthalene and Related Compounds/
Currance, P.L. Clements, B., Bronstein, A.C. (Eds).; Emergency Care For Hazardous Materials Exposure. 3Rd edition, Elsevier Mosby, St. Louis, MO 2005, p. 273-4
Advanced treatment: Consider orotracheal or nasotracheal intubation for airway control in the patient who is unconscious or is in severe respiratory distress. Start IV administration of 0.9% saline (NS) or lactated Ringer's (LR). Adequate hydration must be maintained to prevent renal failure secondary to myoglobinuria unless signs of cerebral or pulmonary edema are present. For hypotension with signs of hypovolemia, administer fluid cautiously. Watch for signs of fluid overload ... . Administer 1% solution methylene blue if patient is symptomatic with severe hypoxia, cyanosis, and cardiac compromise not responding to oxygen. ... . Treat seizures with diazepam or lorazepam ... . Use proparacaine hydrochloride to assist eye irrigation ... . /Naphthalene and related compounds/
Currance, P.L. Clements, B., Bronstein, A.C. (Eds).; Emergency Care For Hazardous Materials Exposure. 3Rd edition, Elsevier Mosby, St. Louis, MO 2005, p. 274

3 Animal Toxicity Studies

3.1 Non-Human Toxicity Excerpts (Complete)

/LABORATORY ANIMALS: Acute Exposure/ ... Dutch belted rabbits (n=8) were vitrectomized followed by subretinal injection of 2.4 mg/mL PB (285 mOsm) and 1.5 mg/mL TB (312 mOsm); balanced salt solution (BSS) (300 mOsm) served as the control. Animals were examined 6, 12, and 24 hr and 14 days after the procedure by fluorescein angiography (FA) and indirect ophthalmoscopy; for retinal toxicity, histologic evaluation studies were performed by light and transmission electron microscopy. RESULTS: FA examination demonstrated window defects suggestive of retinal pigment epithelium (RPE) atrophy in positions of subretinal TB injection, but this was not observed after subretinal injection of PB or BSS. Histologic evaluation disclosed only minimal abnormalities on the photoreceptor outer segment (POS) after subretinal injection of BSS during all follow-up. Subretinal injection of PB caused POS and photoreceptor inner segment (PIS) abnormalities 12 and 24 hr after surgery as well as outer nuclear layer (ONL) damage 14 days after surgery. Subretinal TB injection resulted in POS and PIS damage at 12 hr follow-up. The ONL damage was observed 24 hr after surgery; additionally, POS, PIS, ONL, and RPE abnormalities were observed 14 days after surgery after TB injection. CONCLUSIONS: Subretinal injection of TB induced more significant clinical and histologic damage of neurosensory retina/RPE than did PB or BSS.
Maia M et al; Curr Eye Res 32 (4): 309-17 (2007)
/LABORATORY ANIMALS: Acute Exposure/ PURPOSE: To evaluate the effects of subretinal injections of indocyanine green (ICG), trypan blue, glucose (GL), and balanced salt solution (BSS) in rabbits. DESIGN: Experimental study. PARTICIPANTS: Twenty Dutch-belted rabbits. METHODS: Ten animals underwent vitrectomy and subretinal injection of 0.02 mL of either 0.05% ICG (279 milliosmoles [mOsm]), 0.15% trypan blue (312 mOsm), 5% GL (280 mOsm), or BSS (300 mOsm), which was tested as a control. Ten additional animals underwent subretinal injection of 0.02 mL of 0.046% ICG (251 mOsm), 0.13% trypan blue (260 mOsm), 4.6% GL (253 mOsm), or BSS (300 mOsm). Animals were examined 6, 12, and 24 hours and 14 days after the procedure by fluorescein angiography and fundus evaluation; histologic studies were performed by light and transmission electron microscopy. ... RESULTS: All subretinal blebs were flat 24 hours after the procedure. Fluorescein angiography showed window defects where ICG and trypan blue had been injected. Subretinal BSS and GL resulted in minimal abnormalities of the photoreceptor outer segments (POS) during follow-up. Hypo-osmolar GL caused edema in all retinal layers; pyknosis of the outer nuclear layer (ONL) was observed 24 hours after injection. Subretinal injection of trypan blue resulted in histologic abnormalities 24 hours and 14 days after surgery. Hypo-osmolar trypan blue caused edema of the POS and the photoreceptor inner segments and pyknosis of the ONL 6 and 12 hours after surgery; the retinal pigment epithelium also was affected 24 hours and 14 days after surgery. Subretinal injection of iso-osmolar and hypo-osmolar ICG caused severe damage of all retinal layers during the entire follow-up. CONCLUSIONS: Subretinal injection of 0.05% ICG results in more substantial retinal damage than that associated with the 0.15% trypan blue subretinal injection. The damage induced by hypo-osmolar solutions was more important than that caused by the iso-osmolar solutions.
Penha FM et al; Ophthalmology 114 (5): 899-909 (2007)
/LABORATORY ANIMALS: Subchronic or Prechronic Exposure/ Injected sc into rats @ 2-wk intervals @ levels of 200-400 mg/kg body wt, trypan blue induced anemia with leukopenia & thrombocytopenia.
IARC. Monographs on the Evaluation of the Carcinogenic Risk of Chemicals to Humans. Geneva: World Health Organization, International Agency for Research on Cancer, 1972-PRESENT. (Multivolume work). Available at: https://monographs.iarc.fr/ENG/Classification/index.php, p. V8 272 (1975)
/LABORATORY ANIMALS: Chronic Exposure or Carcinogenicity/ ... 193 rats of both sexes each received sc injections of 1 mL fortnightly or 0.5 mL weekly of 1% soln ... For 14-410 days; 75/133 surviving 70-410 days developed tumors of reticulo-endothelial system ... Of hepatic lymph nodes ... As histiocytomas, plasmacytomas, paramonocytomas, endotheliomas, hemohistioblastomas.
IARC. Monographs on the Evaluation of the Carcinogenic Risk of Chemicals to Humans. Geneva: World Health Organization, International Agency for Research on Cancer, 1972-PRESENT. (Multivolume work). Available at: https://monographs.iarc.fr/ENG/Classification/index.php, p. V8 270 (1975)
/LABORATORY ANIMALS: Chronic Exposure or Carcinogenicity/ /Rats of different strains, 4-8 wk old given sc injections of 1 mL 1% soln every 2nd wk, total 60-120 mg/animal showed not only tumors in liver & hepatic lymph nodes, but also/ in mesenteric lymph nodes, thymus, thymic lymph nodes, non-abdominal lymph nodes & kidney.
IARC. Monographs on the Evaluation of the Carcinogenic Risk of Chemicals to Humans. Geneva: World Health Organization, International Agency for Research on Cancer, 1972-PRESENT. (Multivolume work). Available at: https://monographs.iarc.fr/ENG/Classification/index.php, p. V8 270 (1975)
/LABORATORY ANIMALS: Chronic Exposure or Carcinogenicity/ Of 35 Wistar rats weighing 200 g & given sc injections of 1 mL of soln of commercial trypan blue every 15 days until death (2-15 mo) or until appearance of sc tumors (7-18 mo), sc tumors (fibrosarcomas) occurred @ injection site in 22 rats; 4 other rats had liver tumors.
IARC. Monographs on the Evaluation of the Carcinogenic Risk of Chemicals to Humans. Geneva: World Health Organization, International Agency for Research on Cancer, 1972-PRESENT. (Multivolume work). Available at: https://monographs.iarc.fr/ENG/Classification/index.php, p. V8 271 (1975)
/LABORATORY ANIMALS: Chronic Exposure or Carcinogenicity/ In Wistar rats chronically exposed, reticuloendothelial neoplasms, particularly in liver, were induced. Studies suggest that the tumor is composed of a macrophage-like cell that retains some characteristics of normal macrophages...
FORD RJ, BECKER FF; AM J PATHOL 106 (3): 326 (1982)
/LABORATORY ANIMALS: Chronic Exposure or Carcinogenicity/ Trypan blue has been identified as a mutagen and a carcinogen. In some strains of rats, particular Wistar rats, chronic exposure induces a reticuloendothelial neoplasm, predominantly in the liver. These tumors were studied with the use of immunologic cell membrane markers, electron microscopy, and histochemistry to characterize tumor cell type. The authors studied this tumor in two inbred lines of Wistar rats to compare the efficacy of two dye regimens on tumor incidence and to ascertain whether a short, intense exposure was as effective as chronic protracted exposure. No significant difference in tumor incidence was observed between the two regimens. These studies suggest that the tumor is composed of a macrophage-like cell that retains some characteristics of normal macrophages and that is a reproducible model for carcinogen-induced lymphoreticular human lymphomas.
Ford RJ, Becker FF; Am J Pathol 106 (3): 326-31 (1982)
/LABORATORY ANIMALS: Developmental or Reproductive Toxicity/ In the monkey /trypan blue/ has caused abortions with doses of 50 mg/kg with single or two daily doses between the 20th to 25th day.
Shepard, T.H. Catalog of Teratogenic Agents. 5th ed. Baltimore, MD: The Johns Hopkins University Press, 1986., p. 584
/LABORATORY ANIMALS: Developmental or Reproductive Toxicity/ Similar malformations /brain hydrocephalus, eyes, vertebral column and cardiovascular system/ are produced in rabbits and chicks.
Doull, J., C.D.Klassen, and M.D. Amdur (eds.). Casarett and Doull's Toxicology. 3rd ed., New York: Macmillan Co., Inc., 1986., p. 168
/LABORATORY ANIMALS: Developmental or Reproductive Toxicity/ /In rats/ two main types of spina bifida were found in the offspring of trypan blue treated dams: open and occult spina bifida. Vertebral abnormalities in open spina bifida fetuses were most frequently present in the lower thoracic region & were accompanied by rib malformations. In occult spina bifida the malformations in the vertebral arches were mainly restricted to the sacral region. Both types of spina bifida were accompanied by hypoplastic tails.
PETERS PW ET AL; ACTA MORPHOL NEERL SCAND 19 (1): 21 (1981)
/LABORATORY ANIMALS: Developmental or Reproductive Toxicity/ /Trypan blue was identified as an agent that/ caused malformations in mammalian species.
Doull, J., C.D.Klassen, and M.D. Amdur (eds.). Casarett and Doull's Toxicology. 3rd ed., New York: Macmillan Co., Inc., 1986., p. 195
/LABORATORY ANIMALS: Developmental or Reproductive Toxicity/ The study compared the fertility of nulliparous Jcl:SD rats, young mature (12 week old) versus middle aged (47 week old) or old animals (64 week old), both groups near the termination of their reproductive capability. Trypan blue was given sc at 0 (control), 10 or 50 mg/kg on days 7 through 9 of gestation to increase the malformation rate. However, no clear teratogenic action of trypan blue was observed. The dose of 10 mg/kg trypan blue was well tolerated, while 50 mg/kg was of severe maternal toxicity and 14 of 53 females died. In comparison with the young control group, the average per litter from middle aged or old dams was significantly decreased for implantation sites, live fetuses and fetal weight, while the percentages of pre-implantation loss and dead implants were extremely high. No increase of the malformation rats occurred with maternal aging. Histologically, middle aged and old rats exhibited age-related lesions of the pituitary, adrenals and reproductive organs with pituitary adenomas, cystic endometrium and sclerotic atrophy of uterine glands being significantly increased in non-pregnant, old rats.
Iida H, Kast A; Z Versuchstierkd 33 (2): 100-6 (1990)
/LABORATORY ANIMALS: Developmental or Reproductive Toxicity/ On day 8 of gestation, Donryu rats were given an ip injection of trypan blue (40 mg/kg), and the offspring were raised. Sixty (23.1%) out of 260 offspring showed abnormalities in the lower half of the body, tail of hind limbs, and the six week survival rate of the rats with these abnormalities was 38.3%. The grown malformed rats showed complete or incomplete lumbosacral agenesis. In these rats, the spinal level of motor paralysis corresponded to the vertebral level of malformation, and the average spinal level of sensory disturbance was 4.1 segments lower than that of motor paralysis. In the lumbar enlargement of the spinal cord, the ventral horns and the ventral roots were more hypoplastic than the dorsal horns and the dorsal roots, which seemed to be the cause of the difference in the levels of motor paralysis and sensory disturbance. Rats displayed mirror movements of their hind limbs induced by a similar method, which might have resulted from dysplasia of the ventral region of the lumbar spinal cord.
Iwai K; Nippon Seikeigeka Gakkai Zasshi 63 (1): 116-30 (1989)
/LABORATORY ANIMALS: Developmental or Reproductive Toxicity/ ... For a teratological study to obtain a model of sacral anomalies, Donryu rats were used. Trypan blue was injected as the teratogen ip on the 9th day of gestation. Of the 198 live fetuses, 5 rats had tail defect and sacral anomalies. Two of the 5 anomalous rats also had imperforate anus. Four of 5 rats had vertebrae intact above the first sacral segment. In these 4 rats the pelvic floor muscle had developed to some degree. All the vertebrae were agenetic below the thoracic segments in the other rat fetus. This rat had no pelvic floor muscles. ...
Inomata Y et al; Nippon Geka Hokan 58 (2): 217-30 (1989)
/LABORATORY ANIMALS: Developmental or Reproductive Toxicity/ /In CD-1 mice/ following exposure to bromodeoxyuridine, acetazolamide, trypan blue, cortisone, or diphenylhydantoin, alizarin stained, cleared fetuses were examined at 18 days postcoitus for unossified cervical vertebral centra; number of ossified caudal vertebrae; number of ribs; and ossification of sternebrae, metatarsals, metacarpals, and phalangeal rows. At all teratogenic doses, in no vehicle-treated groups, and rarely in lower-dose groups, there were significant increases in frequency of unossified cervical centra, the first vertebra being most often affected, and ... /seventh vertebra/ least often affected. In the high-dose cortisone group, there was a significant correlation between unossified first vertebra and cleft palate. No association between abnormality and reduced ossification of cervical vertebrae was seen in other series examined, nor was there any correlation between litter size and abnormality. With minor complications, the number of ossified caudal vertebrae was significantly reduced after exposure at teratogenic dose levels to all compounds except diphenylhydantoin. Although caudal and cervical ossification were correlated with each other in those series examined, neither was correlated with abnormality. Frequency of 14 ribs was increased in bromodeoxyuridine, acetazolamide, and trypan blue but not cortisone or diphenylhydantoin. Other parameters were essentially unaffected. Significantly increased frequency of abnormality, when contrasted with untreated or vehicle-treated groups, was seen at high dose levels in all but diphenylhydantoin treatments, and mortality was increased in acetazolamide, trypan blue, and cortisone. Reduced ossification of cervical centra is an excellent indicator of prenatal exposure to noxious substances, and caudal vertebrae appear to be useful as well. Increased frequency of 14 ribs occurred for all strong teratogens utilized if they were administered on day 7 or day 8 postcoitus.
Beck SL; Teratology 40 (4): 365-74 (1989)
/LABORATORY ANIMALS: Developmental or Reproductive Toxicity/ Using scanning and transmission electron microscopy, the histological and cytological alterations induced in the developing eye by trypan blue were examined. Microphthalmic and anophthalmic eyes were analyzed from 16 day rat fetuses whose dams had received a teratogenic dose of trypan blue. Cell and tissue damage within cornea and mesenchyme were assessed. Corneas from the treated groups were undulating and uneven owing to abnormal lens placement and corneal cell injury. Extensive extracellular spacing, few cell-to-cell contacts, patchy glycocalyces, and abnormal surface morphology characterized the corneal cells of treated tissues. Mesenchyme of severely malformed eyes typically filled the eye field in regions normally occupied by retina and lens. As a tissue, mesenchyme lacked organization and those surface features normally present in component cells were altered. Mesenchymal cells were packed so closely that extracellular spaces were nearly obliterated. Microvilli, filopodia, and lamellipodia, cell processes observed in normal mesenchyme, were altered in both number and structure. The cytoplasm of mesenchymal cells was condensed and decreased in volume. These data suggested that 1) in this trypan blue ocular model, virtually all major tissue types were morphologically altered; 2) because healthy mesenchyme and cornea are required for normal development of lens, optic vesicle and ocular adnexa, ... abnormal cornea and mesenchyme also will compromise these tissues; and 3) since necrotic reminants were not present, this trypan blue model probably causes developmental arrest. The most likely teratogenic pathway is ... on the cell surface as probable site of dye action.
Schmidt KL et al; Teratology 29 (1): 105-16 (1984)
/LABORATORY ANIMALS: Developmental or Reproductive Toxicity/ Neuropathological and electrophysiological studies were designed to clarify the pathomechanism of mirror movements with sacral agenesis. Model animals were obtained from mother rats which had been treated trypan blue on day 8 of gestation. Mirror movements in the hind limbs were observed in 7 out of 56 offspring with lumbosacral agenesis, remarkably when the rats were frightened. The terminal part of the spinal cord was hypoplastic and dysplastic particularly in the ventral half, with sporadical anterior horn cells and no apparent median fissure. On the other hand, no abnormality was found in the brain, brain stem, and upper part of the spinal cord. ...
Inage A et al; Teratology 40 (6): 667 (1989)
/LABORATORY ANIMALS: Developmental or Reproductive Toxicity/ Trypan blue is a potent teratogen in vivo and in vitro in the rat. Many of the abnormalities produced by trypan blue including swollen neural tube and pericardium, subectodermal blisters, hematomas, and generalized edema may result from altered fluid balance in and around the embryo. ... Rat embryos were exposed in utero or in vitro to trypan blue during the early period of organogenesis. Both exposures resulted in defects that are typical of trypan blue treatment. Osmolality of exocoelomic fluid was measured on gestation day 10 in vivo and day 12 in vitro, both after 48 hr of exposure to trypan blue. In both cases exocoelomic fluid osmolality was significantly lower than controls. This was correlated with the presence of edema-related anomalies in the embryo. On gestation day 11 in vivo, three days after maternal injection of trypan blue, exocoelomic fluid osmolalities were significantly higher than controls; however, there was tremendous variability in this parameter in day 11 treated embryos, and some had exocoelomic fluid osmolalities below the control range. Increased frequency of abnormalities was correlated with abnormal exocoelomic fluid osmolality, below and above the control range. Trypan blue probably exerts its teratogenic effects by disturbing the function of the visceral yolk sac. The movements of an amino acid and a monosaccharide across the visceral yolk sac ... on gestation day 12 embryos in vitro ... was not altered by trypan blue exposure. ... Endodermal cells in trypan blue treated yolk sacs contained fewer large, electron dense lysosomes than controls. These were replaced by numerous small vacuoles, which may contain trypan blue. Trypan blue causes osmotic changes in the rat embryo in vivo and in vitro. These changes are correlated with embryonic malformations. ...
Rogers JM et al; Teratology 31 (3): 389-99 (1985)
/LABORATORY ANIMALS: Developmental or Reproductive Toxicity/ Female mice were injected on day 6 of gestation with the teratogenic dye trypan blue. Within 6, 24, and 48 hr of exposure to the teratogen, egg cylinders were ... prepared for electron microscopic analysis. ... On the ultrastructural level, exposure to trypan blue lead within 6 hr to fragmentation of the endoplasmic reticulum and a depletion of free polysomes in the endoderm of the egg cylinders. In ectodermal cells only the distribution of polysomes was disturbed following exposure to trypan blue. Egg cylinders harvested 24 hr after injection of trypan blue had partially recovered. Their endoplasmic reticulum and polysomes appeared closer to controls. The cells of both germ layers of most egg cylinders obtained 48 hr after injection were indistinguishable from controls when viewed with the electron microscope. No consistent changes were found in mitochondria or Golgi apparatus following trypan blue treatment. ... Mouse embryos appear to be able to correct damage sustained during the egg cylinder stage, and ... in spite of earlier injury affecting both germ layers such egg cylinders can develop normally as revealed by microscopic examination.
Hamburgh M et al; Teratology 29 (3): 393-403 (1984)
/LABORATORY ANIMALS: Developmental or Reproductive Toxicity/ Female rats of WM (Wistar-Mishima)/Nem strain were mated with WM/Nem (group W) or BDIX/Nem (group B) or WM/Nem males group BW). On day 8 gestation, pregnant females were treated ip with 1% aqueous solution of trypan blue at a dose of between 20 and 120 mg/kg of body weight. On day 20 of gestation, fetuses were examined for ... malformation. In group W, fetal mortality increased dose dependently at doses higher than 20 mg/kg, and incidences of external, visceral, and skeletal malformations were significantly higher than control at doses of 30 mg/kg and more. In group B, fetal mortality and the incidence of external malformations were significantly higher than control only in the group treated with 120 mg/kg, and no significant increase of visceral and skeletal malformations was shown. It was confirmed that BDIX strain is much more resistant to trypan blue teratogenicity than WM strain. In group BW, nearly the same teratogenic effects were shown as in group W in terms of fetal mortality and incidence of malformation. However, in group WB, teratogenic effects were not so remarkable as in group BW, suggesting patroclinous effects in teratogenic susceptibility to trypan blue. In group BW, sex differences in teratogenic susceptibility were found; male fetuses were more susceptible to trypan blue than females.
Hoshino K et al; Teratology 37 (1): 43-50 (1988)
/LABORATORY ANIMALS: Developmental or Reproductive Toxicity/ The teratogenicity of trypan blue and its related compounds was studied in Wistar rats ... 1) o-Tolidine, 1-amino-8-naphthol-3,6-disulfonic acid or 1-nitronaphthalene-3, 6-disulfonic acid was injected sc on day 7 of pregnancy (sperm = day 0). No fetotoxicity was observed in any group. 2) The main fractions, blue fraction and red fraction, were separated from commercial trypan blue by silica gel ... chromatography. Commercial trypan blue, blue fraction, or red fraction was injected into pregnant rats sc on day 7 of pregnancy. The incidence of malformed fetuses after injection of blue fraction was higher than that of commercial trypan blue, and the types of malformations induced by commercial trypan blue and blue fraction were similar. However, no fetotoxicity was detected after injection of red fraction, 3) blue fraction or red fraction was injected into the exocoelom on day 11 of pregnancy. The incidence of malformed fetuses in the group injected with blue fraction (2.5 ug/embryo) was 39% and the types of malformations were abnormal tail and vertebrae ... .
Ema M et al; Nippon Yakurigaku Zasshi 83 (5): 459-65 (1984)
/LABORATORY ANIMALS: Developmental or Reproductive Toxicity/ Heterozygotes for the tail-short mutant gene in the Balb/c strain have minor skeletal defects and a short, kinky tail. If heterozygote tail-short-/+ mothers are mated with normal- tail +/+ males and are treated with teratogenic doses of trypan blue on the eighth day of pregnancy, the mutant F1 heterozygotes develop exencephaly, folded neural tube and spina bifida significantly more often than non-mutants. This is indicative of gene-teratogen interaction, with the tail-short gene increasing the embryo's susceptibility to trypan blue-induced neural tube defects.
Matta CA; Folia Morphol 38 (1): 12-18 (1990)
/LABORATORY ANIMALS: Developmental or Reproductive Toxicity/ Malformations of fetuses obtained from Wistar rat dams treated with trypan blue during gestation were studied. Fetuses were examined on day 20 gestation. 127 fetuses showed abnormalities of the external features, skeleton and internal organs, separately or in combination. External malformations were found in 108 fetuses. The most frequent external malformation was anomaly of tail. Spina bifida, club foot, exencephaly and anal atresia were also observed frequently. Skeletal malformations were detected in 48 fetuses. Deformity of vertebrae in the lumbar, sacral and/or caudal regions was found in 46 fetuses. Internal malformations were observed in 27 fetuses. Anomaly of heart and/or great vessels hydrocephaly and micro- or anophthalmia were observed frequently. About 90% of the fetuses with skeletal malformations also showed some external malformations. In contrast, about 48% of the fetuses with internal malformations also had some external malformations.
Ema M et al; Jikken Dobutsu 36 (3): 261-5 (1987)
/LABORATORY ANIMALS: Developmental or Reproductive Toxicity/ The teratogenicity of trypan blue after both maternal and intrauterine administrations was studied in Wistar rats, ... 1) The teratogenic dose of trypan blue by maternal sc injection was found to be greater than 50 mg/kg, and the critical period was until day 10 of pregnancy (sperm = day 0). No teratogenicity was detected by oral administration of 250 mg/kg trypan blue. 2) Trypan blue (250 ug/uterine horn) was injected into the uterine cavity on day 4 or 6 of pregnancy. An increase of intrauterine death without malformations was observed in both trypan blue-treated groups. 3) Trypan blue was injected into the exocoelom. The incidence of malformed fetuses were 53% in the group injected with 2.5 ug/embryo trypan blue on day 10 and 32% and 57% in the groups injected with trypan blue at 1.0 and 2.5 ug/embryo on day 11, respectively. An increase of intrauterine death was observed in these groups. Types of malformations observed in these groups were abnormal tail, spina bifida and deformity of vertebrae, and were almost similar to those observed by maternal subcutaneous injection of trypan blue. 4) A trace of trypan blue was found microscopically in the frozen section of embryo after both subcutaneous and intraexocoelom injections of trypan blue. Trypan blue acts directly on embryos to produce malformations.
Ema M, Kanoh S; Nippon Yakurigaku Zasshi 83 (5): 433-40 (1984)
/LABORATORY ANIMALS: Developmental or Reproductive Toxicity/ Yolk-sacs from untreated Sprague-Dawley rat conceptuses were removed on gestational days 9 to 18 and examined for gamma-glutamyl transferase, alkaline phosphatase, lactate dehydrogenase, and glutamic-oxaloacetic transaminase activities. All enzyme activities were found to increase through gestation in total activity as well as in specific activity. Protein and urea nitrogen were also found to increase through gestation whereas triglyceride increased steadily from gestational day 9 to 13 and then appeared to plateau through gestational day 19. Additional rats were treated on gestational day 8 with 75 mg trypan blue (TB)/kg body weight and yolk-sacs taken on gestational day 9 to 18. Yolk-sac gamma-glutamyl transferase and glutamic-oxaloacetic transaminase activities from trypan blue-treated rats were significantly higher than the respective controls during early gestation but recovered to or were lower than control levels during midgestation. Gamma-glutamyl transferase activity in treated rats was significantly higher than the controls on gestational day 15 and 16, and both gamma-glutamyl transferase and glutamic-oxaloacetic transaminase were significantly lower than controls on gestational days 17 and 18. Alkaline phosphatase activity in the trypan blue-treated yolk-sacs was significantly lower than that in controls during early and midgestation but was not significantly different from the control values late in gestation. Triglyceride concentration was not affected early in gestation but significantly decreased on gestational days 16 and 18. Thus, the yolk-sac enzymes monitored, which are associated with nutrition and normal growth, increased in activity through gestational day 18. The yolk-sac toxicant, trypan blue, resulted in significant changes in yolk-sac protein and triglyceride content.
Andrews JE et al; Reprod Toxicol 8 (5): 405-10 (1994)
/LABORATORY ANIMALS: Developmental or Reproductive Toxicity/ The effect of trypan blue on the 11.5-day rat conceptus after intravitelline vessel administration described. Trypan blue significantly retarded the growth and development of conceptuses after 6 hours incubation in vitro. The SEM revealed rounded ectodermal cells, some of which appeared disrupted. These cells seemed to cause some of the intersomitic grooves to disappear, making a number of the somites indistinct from the outside. Unlike cells of uninjected embryos, the surfaces of the affected ectodermal cells lacked microvilli and their perimeters were lined with microvilli-like structures which appeared matted together. It was concluded that trypan blue affected the embryo directly probably by disturbing its fluid and ionic balance.
Mensah-Brown E PK; Acta Anatomica 151 (3): 159-64 (1994)
/LABORATORY ANIMALS: Developmental or Reproductive Toxicity/ In mice and rats, prenatal exposure to the dye Congo red permanently reduces the number of germ cells in male and female offspring. Nine other dyes structurally related to Congo red were examined for developmental testicular toxicity. Only benzidine-based dyes altered testicular development and caused hypospermatogenesis in mice during adulthood. Dimethyl- and dimethoxy-benzidine-based dyes were without effect. Pregnant mice were dosed orally on Days 8-12 of gestation with a benzidine-, dimethylbenzidine-, or a dimethoxybenzidine-based dye and the testes of 45- to 50-day-old male offspring were examined. The testes of postpubertal male offspring exposed to the benzidine based dyes, Congo red, diamine blue, and Chlorazol Black E, were small and contained some tubules completely devoid of germ cells, but the dimethylbenzidine-based dyes, trypan blue, Evans blue, and benzopurpurin 4B, and the dimethoxybenzidine-based dye, Chicago sky blue, did not alter testicular development in this manner. The structure-activity relationship of the dyes for developmental toxicity following oral administration differs considerably from that produced by maternal ip administration.
Gray L E JR, Ostby JS; Fundam Appl Toxicol 20 (2): 177-83 (1993)
/LABORATORY ANIMALS: Developmental or Reproductive Toxicity/ Mouse and rat whole embryo cultures are widely used in teratogenicity studies. Rabbit embryos of the Japanese White strain were explanted on day 9, 10 or 11 of gestation and cultured for 24 or 48 hr. The development of embryos cultured for 48 hr from day 9 or day 10 or for 24 hr from day 11 was nearly the same as that of embryos that had developed in vivo. Slc:SD rat embryos on day 9.5 of gestation were explanted and cultured in rat serum exposed to trypan blue (300-2,700 ug/mL) for 48 hr. Rabbit embryos on day 9 or 10 of gestation were explanted and cultured in rabbit serum containing trypan blue (300-2,700 ug/mL) for 48 or 24 hr. Cultured rat embryos exposed to trypan blue showed neural tube abnormalities, and all growth parameters were suppressed with increasing concentrations of trypan blue. However, trypan blue had no effect on cultured rabbit embryos.
Ninomiya H et al; Toxicol In Vitro 7 (6): 707-17 (1993)
/LABORATORY ANIMALS: Developmental or Reproductive Toxicity/ Techniques, based on computer-assisted analyses of ensembled ECG waveforms serve as a screening procedure for potentially cardiotoxic substances. This procedure was tested on fetal and neonatal rats exposed in utero to two levels of trypan blue, a known cardiovascular teratogen. The offspring of the treated animals exhibited decreased viability, as well as significant levels of both cardiac and noncardiac malformations. Qualitative assessment of ECG irregularities demonstrated an increased number of abnormalities in the treated groups. Correlations were also obtained between observed morphological defects and ECG irregularities in the trypan blue treated groups.
Watkinson WP et al; Clin Biol Res 140 207-22 (1983)
/LABORATORY ANIMALS: Developmental or Reproductive Toxicity/ Trypan blue was administered to Wistar rats at various doses and at different stages of gestation. Teratogenicity and embryolethality were observed by a single (50 and 250 mg/kg) subcutaneous injection and three (25 and 50 mg/kg) subcutaneous injections of trypan blue daily from day 7 of pregnancy. The incidences of malformations in groups given 50 and 250 mg/kg on day 7 were 12 and 59%, respectively. Most of the fetuses with external malformations were accompanied with skeletal and/or internal anomalies. The types of frequently occurring malformations were as follows: exencephaly, spina bifida, tail anomaly, vertebral deformity, hydrocephaly and heart anomaly. Fetal toxicity was decreased after treatment with the mixture of trypan blue and normal rat serum. The serum level of trypan blue in pregnant rats increased to 308 ug/ml at one hour after subcutaneous treatment with trypan blue 50 mg/kg and decreased rapidly, but remained at 58 ug/ml 72 hours later. Lower serum levels of trypan blue were observed in pregnant rats given trypan blue with serum. No fetotoxic effects of serum from pregnant rats treated with trypan blue were observed in recipient rats given the serum on day 7, 8 and 9 of pregnancy.
Ema M, Kanoh S; Nippon Yakurigaku Zasshi 79 (5): 369-81 ()1982
/ALTERNATIVE and IN VITRO TESTS/ To facilitate the peeling of internal limiting membrane or epiretinal membrane in vitreoretinal surgery, trypan blue (TB) and indocyanine green (ICG) have been used. However, the cytotoxicity of these dyes have been /of concern/. The aim of this study was to investigate the neurotoxic effects of TB on rat retinal ganglion cells (RGCs) and compare the effect of TB with ICG. Rat RGCs were purified by a two-step immunopanning procedure. In short-time exposure experiments, purified RGCs cultured for 3 days were exposed to 600 mg/L TB, 1500 mg/L TB and 1500 mg/L ICG for 10 sec to 30 min. The number of viable RGCs was counted after 12 hr in culture. In long-time exposure experiments, purified RGCs were cultured for 3 days in TB solutions ranging from 2 to 800 mg/L or in ICG solutions at concentrations from 2 to 250 mg/L. Then the number of viable cells was counted. Exposure to 600 mg/L TB, 1500 mg/L TB and 1500 mg/L ICG for short time from 10 sec to 30 min caused a time-dependent damage to RGCs. There was no significant difference in cytotoxicity to RGCs between TB and ICG in short-time exposure. In long-time exposure, TB as well as ICG showed neurotoxic effect on RGCs in a dose-dependent manner. Fifty percentage inhibitory concentration (IC50) of TB to RGCs was calculated as 115 mg/L, while that of ICG was 33 mg/L. In conclusion, TB induced neurotoxic effect on RGCs in a dose- and time-dependent manner. During the short-time exposure in surgery, there is likely no difference in neurotoxic effect on RGCs between TB and ICG. However, once the dyes are left in the eyes after surgery, TB may show less toxicity on RGCs than ICG.
Jin Y et al; Exp Eye Res 81 (4): 395-400 (2005)
/ALTERNATIVE and IN VITRO TESTS/ The effect of four azo dyes, Congo Red, trypan blue, benzopurpurine-4B, and Direct Blue 15, and their derivatives, benzidine, o-tolidine, and o-dianisidine on viral interferon induction, following enzymatic dye activation by rat liver S9 fraction, was tested in Rhesus monkey kidney cell monolayers. The agents were added to cell cultures ... and incubated for 24 hr, followed by interferon induction by influenza virus. Three of the four azo dyes tested inhibited the induction of viral interferon by 35 to 60% while trypan blue and the azo dye derivatives induced negligible inhibition. All chemicals tested caused significant inhibition of interferon induction when rat S9 was used for enzymatic activation. Uninduced hamster liver S9 fraction and Aroclor-1254 induced hamster and rat liver S9 fractions demonstrated comparable metabolic activation of the agents tested. ... /Results indicate/ that potential mutagens and carcinogens which require metabolic activation may be identified according to their ability to inhibit the induction of interferon in mammalian cell cultures.
Hahon N; Environmental Research 37 (1): 228-38 (1985)
/OTHER TOXICITY INFORMATION/ Trypan blue was found to ... inhibit respiration and glycolysis of tumor tissue.
IARC. Monographs on the Evaluation of the Carcinogenic Risk of Chemicals to Humans. Geneva: World Health Organization, International Agency for Research on Cancer, 1972-PRESENT. (Multivolume work). Available at: https://monographs.iarc.fr/ENG/Classification/index.php, p. V2 272 (1973)
/OTHER TOXICITY INFORMATION/ Spleen size and sequestering activity increased in all rats receiving trypan blue. Histological studies demonstrated than an increase in mechanical filtration was responsible for the dysfunction.
DUMONT AE ET AL; BR J EXP PATHOL 62 (4): 333 (1981)
/OTHER TOXICITY INFORMATION/ Pregnant Sprague-Dawley rats were dosed ip with 75 mg/kg trypan blue or water on day 8 of gestation. ... Gamma glutamyl transferase activity/mg protein in yolk sacs increased throughout gestation in controls, in contrast to the pattern previously reported for rat placenta. ... Activity in trypan blue treated yolk sacs was similar to controls on days 9 and 10, and tended to be higher than controls on days 11-16, significantly on days 11, 13, and 15. Gamma glutamyl transferase activity/mg protein increased by 18% from days 16-18 in controls, but decreased by 44% in trypan blue treated yolk sacs. Trypan blue has been shown to inhibit pinocytosis in the yolk sac, and the increased specific activity of gamma glutamyl-transferase seen ... beginning on day 11 may be a compensatory response. ...
Andrews JE, Rogers JM; Teratology 41 (5): 536 (1990)
/OTHER TOXICITY INFORMATION/ Retinal projections, particularly the ipsilateral projections, to the medial terminal nucleus of the accessory optic system were investigated by autoradiography in adult rats after being reared in one of three different conditions: (1) normal visual experiences, (2) an application of trypan blue for intrauterine induction of congenital unilateral anophthalmia; and (3) the procedures for intrauterine production of congenital microphthalmia in which both eyes are reduced in size. In congenital monocular rats, there was an increase of uncrossed retinal projections to the medial terminal nucleus which does not normally exist. The expansion of the ipsilateral projection was markedly greater in the monocular rats than those inflicted with microphthalmia.
Chem ST et al; Taiwan I Hsueh Hui Tsa Chlh 88 (1): 27-31 (1989)
/OTHER TOXICITY INFORMATION/ /During/ the characterization of species differences in the effects of chemical carcinogens, several studies demonstrated that hamster hepatocytes are more effective than rat hepatocytes in mediating the metabolic activation of certain chemicals to their genotoxic (ie, mutagenic) derivatives. In /this/ investigation, a comparison of the amount of DNA repair induced in rat and hamster hepatocytes by 7 azo dyes and 17 aromatic amine azo reduction products of the dyes was performed using the primary hepatocyte culture/DNA repair (HPC/DR) assay. Congo Red and its azo reduction product, benzidine, were more potent inducers of DNA repair in hamster than in rat hepatocytes, whereas trypan blue and its reduction product, o-tolidine were equipotent in the 2 hepatocyte systems. ...
Kornbrust DJ, Barfknecht TR; Mutat Res 136 (3): 255-66 (1984)
/OTHER TOXICITY INFORMATION/ The accumulation of iron in the lymph nodes of trypan blue treated rats was examined as a possible experimental counterpart of the lymph node siderosis which occurs in patients with Hodgkin's disease. Lymph nodes removed from the hilus of the liver, retrosternal area, axilla and root of the small bowel mesentery were examined histologically for iron in rats receiving 6-20 sc injections of trypan blue at biweekly intervals and in control rats. An increase in erythrophagocytosis accompanied by a progressive increase in the amount of stainable iron was found in the RE cells of nodes located in the lymphatic outflow tract of the liver. As in patients with Hodgkin's disease, an increase in erythrophagocytosis together with the prolonged retention of iron by RE cells appears to account for the accumulation of iron in the lymph nodes of trypan blue treated rats.
Dumont AE; Lymphology 21 (4): 234-8 (1988)
/OTHER TOXICITY INFORMATION/ The structure-activity relationship of 40 azo compounds in their ability to induce cytochrome P448 and associated monooxygenase activities, as well as UDP-glucuronyltransferase activity, was investigated (in rats). Regardless of their structure, hydrophilic azo dyes and lipophilic azobenzene derivatives were not able to induce these enzyme activities. Only those lipophilic azo dyes with l-azo-2-naphthol or 1-azo-2-naphthylamine moieties were able to induce cytochrome P448 and related monooxygenase activities, as well as UDP-glucuronyltransferase activity. The extent of induction is comparable to or greater than that caused by 3-methylcholanthrene. Those azo dyes capable of inducing P450 type cytochromes can form coplanar structures with 3 fused, 6-membered rings through intramolecular hydrogen bonding. These structures are analogous to polycyclic aromatic hydrocarbons that can also induce.
Fujita S et al; Chem-Biol Interact 52 (1): 15-38 (1984 (Recd 1985)

3.2 Ecotoxicity Excerpts (Complete)

/BIRDS and MAMMALS/ Ovaries of adult Japanese quails were exposed in vivo to ... trypan blue (TB) which binds to plasma albumin, the plasma precursor of the yolk protein alpha-livetin. ... Trypan blue alters vitellogenesis in the non-disc region of follicles in rapid growth in a reversible and dose-dependent way. Less yolk is produced over 24 hr and its morphology is different when compared to controls... . /It was proposed/ that the inhibitory action of TB on vitellogenesis can be explained by a defective receptor-ligand dissociation in endosomes.
D'Herde K, Roels F; Reprod Nutr Dev, 33 (6): 597-608 (1993)

3.3 Non-Human Toxicity Values (Complete)

LD50 Rat oral 6200 mg/kg
Lewis, R.J. Sr. (ed) Sax's Dangerous Properties of Industrial Materials. 11th Edition. Wiley-Interscience, Wiley & Sons, Inc. Hoboken, NJ. 2004., p. 935
LD50 Mouse sc 267 mg/kg
Lewis, R.J. Sr. (ed) Sax's Dangerous Properties of Industrial Materials. 11th Edition. Wiley-Interscience, Wiley & Sons, Inc. Hoboken, NJ. 2004., p. 935
LD50 Mouse iv 328 mg/kg
Lewis, R.J. Sr. (ed) Sax's Dangerous Properties of Industrial Materials. 11th Edition. Wiley-Interscience, Wiley & Sons, Inc. Hoboken, NJ. 2004., p. 935

3.4 Ecotoxicity Values (Complete)

LC50; Species: Oryzias latipes (medaka); Concentration: >1000 mg/L for 48 hr /Conditions of bioassay not specified/
Sigma-Aldrich Corp; Safety Data Sheet for Trypan blue (Product Number: 302643) Version 3.0 (January 2, 2009). Available from, as of June 15, 2010: https://www.sigmaaldrich.com
LC50; Species: Oryzias latipes (medaka, high-eyes); Conditions: freshwater, static, 10 °C; Concentration: >100000 ug/L for 24 hr /formulated product/
Tsuji S et al; J Hyg Chem (Eisei Kagaku) 32 (1): 46-53 (1986) as cited in the ECOTOX database. Available from, as of June 18, 2010
LC50; Species: Oryzias latipes (medaka, high-eyes); Conditions: freshwater, static, 20 °C; Concentration: >100000 ug/L for 24 hr /formulated product/
Tsuji S et al; J Hyg Chem (Eisei Kagaku) 32 (1): 46-53 (1986) as cited in the ECOTOX database. Available from, as of June 18, 2010
LC50; Species: Oryzias latipes (medaka, high-eyes); Conditions: freshwater, static, 30 °C; Concentration: >100000 ug/L for 24 hr /formulated product/
Tsuji S et al; J Hyg Chem (Eisei Kagaku) 32 (1): 46-53 (1986) as cited in the ECOTOX database. Available from, as of June 18, 2010
LC50; Species: Oryzias latipes (medaka, high-eyes); Conditions: freshwater, static, 10 °C; Concentration: >100000 ug/L for 48 hr /formulated product/
Tsuji S et al; J Hyg Chem (Eisei Kagaku) 32 (1): 46-53 (1986) as cited in the ECOTOX database. Available from, as of June 18, 2010
LC50; Species: Oryzias latipes (medaka, high-eyes); Conditions: freshwater, static, 20 °C; Concentration: >100000 ug/L for 48 hr /formulated product/
Tsuji S et al; J Hyg Chem (Eisei Kagaku) 32 (1): 46-53 (1986) as cited in the ECOTOX database. Available from, as of June 18, 2010
LC50; Species: Oryzias latipes (medaka, high-eyes); Conditions: freshwater, static, 30 °C; Concentration: >100000 ug/L for 48 hr /formulated product/
Tsuji S et al; J Hyg Chem (Eisei Kagaku) 32 (1): 46-53 (1986) as cited in the ECOTOX database. Available from, as of June 18, 2010

3.5 TSCA Test Submissions (Complete)

Acute oral toxicity was evaluated in groups of 5 Sherman-Wistar albino rats (2 females & 3 males/group) administered single doses of direct blue 14 as a 25% w/v suspension in water by oral gavage at levels of 4.0, 5.0, 6.25 and 7.9 g/kg of body weight. Mortality was observed in 3 animals in the 6.25 g/kg dose group and in all animals in the highest dose group. The LD50 value was calculated to be 6.2 g/kg (5.5 - 6.9 g/kg 95% confidence limits) by the Thompson moving average method. Clinical observation and gross necropsy results were not reported.
Food and Drug Research Laboratories Incorporated; Acute Oral Toxicity Study. (1972), EPA Document No. 878221036, Fiche No. OTS0215154
Trypan blue (CAS # 72-57-1) was evaluated for acute inhalation toxicity. The test substance was administered at a concentration of 473.5 mg/m3 to 5 male and 5 female rats for 1-hour. No mortality occurred and rats gained weight normally for the 14-day observation period.
CROMPTON & KNOWLES CORP; Acute Dust Inhalation Toxicity in Osborne - Mendel Rats; 02/22/77; EPA Doc No. 878211045; Fiche No. OTS0206237
Trypan blue (CAS # 72-57-1) was evaluated for primary dermal irritation. The test substance was administered to the clipped back of rabbits (sex not reported) at a level of 2 ml of a 40% solution applied to the intact skin (3 rabbits) and abraded skin (3 rabbits) for 10 applications over a period of 12-14 days. The test substance did not produce any signs of skin irritation and all animals gained body weight normally.
CROMPTON & KNOWLES CORP; Skin Irritation Tests in Rabbits; 11/22/82; EPA Doc No. 878212125; Fiche No. OTS0206237

4 Metabolism / Pharmacokinetics

4.1 Metabolism / Metabolites (Complete)

/Trypan blue/ is reduced in vitro by a rat liver enzyme to ortho-tolidine & 2,8-diamino-1-naphthol-3,6-disulfonic acid.
IARC. Monographs on the Evaluation of the Carcinogenic Risk of Chemicals to Humans. Geneva: World Health Organization, International Agency for Research on Cancer, 1972-PRESENT. (Multivolume work). Available at: https://monographs.iarc.fr/ENG/Classification/index.php, p. V8 273 (1975)
Six azo dyes, including trypan blue, were reduced, N-acetylated, and N-conjugated. No N,N'-diacetylated metabolites were detected in rat urine.
KENNALLY JC ET AL; CARCINOGENESIS (LONDON) 3 (8): 947 (1982)
Azo dyes based on beta-diketone coupling components exist preferentially as the tautomeric hydrazones. A series of hydrazone dyes based on benzidine and benzidine congeners ... was prepared. The hydrazone dyes were resistant to enzymatic reduction by FMN supplemented hamster liver post-mitochondrial supernatant (S-9); under identical conditions, azo dyes such as trypan blue were rapidly reduced.
De France BF et al; Food Chem Toxicol 24 (2): 165-9 (1986)
Metabolism experiments were conducted with rats dosed with 9 azo dyes based on dimethyl-, dimethoxy-, or dichlorobenzidine to determine whether the free amine congeners, their monoacetyl or diacetyl metabolites, or alkaline hydrolyzable conjugates were excreted in the urine. 2-mg doses were administered and urine samples were collected at intervals up to 96 hr. Peak levels of metabolites were excreted either 0-12 or 12-24 hr after administration and, in 7 of 9 instances, no metabolites persisted in the urine after 48 hr. Minimum detectable levels of all metabolites were 12 ppb or less. All 9 dyes were converted to measurable levels of their benzidine-congener-based metabolites in rats.
Bowman MC et al; J Anal Toxicol 7 (l): 55-60 (1983)
The ability of rat liver microsomes from phenobarbitone pretreated animals to reduce the azo groups of amaranth, sunset yellow, congo red, trypan blue, chloramine sky blue FF and direct black 38 was measured in vitro. The dyes amaranth and sunset yellow acted as positive controls. Of the dyes derived from (the carcinogen) benzidine or its congeners, only direct black 38 was reduced to an appreciable extent; the rate of reduction was 10% of that for amaranth. The dyes were tested for mutagenicity in the Salmonella/microsome assay, the only active compound being direct black 38. Mutagenicity of this dye may be due in part to the mutagen 1,2,4-triaminobenzene. Mutagenic activity and azo-reduction of direct black 38 was independent of the presence of oxygen. Mammalian liver may play only a minor or negligible role in the azo-reduction of dyes derived from benzidine or its congeners.
Martin CN, Kennelly JC; Carcinogenesis (Lond) 2 (4): 307-12 (1981)

4.2 Absorption, Distribution and Excretion (Complete)

Trypan blue ... was observed in animals to pass from bloodstream through walls of vessels of iris & choroid, but not through walls of retinal vessels.
Grant, W.M. Toxicology of the Eye. 3rd ed. Springfield, IL: Charles C. Thomas Publisher, 1986., p. 959
Following sc or ip injections into mice or rats, trypan blue is rapidly absorbed & widely distributed throughout the body. Max serum concentrations are ... within 2 hr; it appears bound to serum proteins with ... rapid excretion in urine & uptake by the reticulo-endothelial system.
IARC. Monographs on the Evaluation of the Carcinogenic Risk of Chemicals to Humans. Geneva: World Health Organization, International Agency for Research on Cancer, 1972-PRESENT. (Multivolume work). Available at: https://monographs.iarc.fr/ENG/Classification/index.php, p. V8 272 (1973)
Trypan blue never reaches the rat embryo, but accumulates in maternal reticuloendothelial system and in the placenta.
Doull, J., C.D. Klaassen, and M. D. Amdur (eds.). Casarett and Doull's Toxicology. 2nd ed. New York: Macmillan Publishing Co., 1980., p. 168
Experiments with ring-labelled radioactive trypan blue did not give evidence of any embryonic incorporation of the (14)C. The absence of teratogenic action after the initiation of chorio-allantoic placentation also indicated that yolk sac function was important in pathogenesis. The dye can be visualized in the cells of the visceral yolk sac.
Shepard, T.H. Catalog of Teratogenic Agents. 5th ed. Baltimore, MD: The Johns Hopkins University Press, 1986., p. 584
The serum level of trypan blue in pregnant rats increased to 308 ug/mL at one hour after subcutaneous treatment with trypan blue at 50 mg/kg and decreased rapidly, but remained at 58 ug/mL 72 hours later.
EMA M, KANOH S; NIPPON YAKURIGAKU ZASSHI 79 (5): 369 (1981)
Administration of trypan blue to pregnant Sprague-Dawley or Long-Evans rats, during an identical stage of embryogenesis, has been associated with malformation in 97 and 17% of the offspring, respectively. ... The comparative pharmacokinetics of trypan blue in the two strains were investigated. Female Sprague-Dawley and Long-Evans rats were injected sc with 10 mg trypan blue/rat (0.5 ml 2% (w/v) trypan blue in distilled water). ... The peak serum concentration of trypan blue was greater in Sprague-Dawley rats. Pharmacokinetic analysis of concentrations of trypan blue in serum resulted in fitting a two-compartment open model, with first order absorption, for both strains. Elimination of trypan blue from the central compartment was faster in Sprague-Dawley rats. ... The reported differences between the two strains in the teratogenic effects of trypan blue could be attributable, in part, to these observed pharmacokinetic dissimilarities.
Taylor MJ et al; Food Chem Toxicol 22 (11): 875-8 (1984)

4.3 Mechanism of Action (Complete)

A dose of 50 mg per kg /of trypan blue/ appears to be the optimum teratogenic dose. A characteristic observation with trypan blue is that with treatment after the 9th day of gestation defects are rare. This fact has supported other evidence that the mechanism of action was dependent on disruption of yolk sac nutrition. ... Studies have produced evidence indicating a possible action of trypan blue on a nutritive function of the visceral yolk sac. The failure of the trypan blue to act directly upon the embryo is generally held ... .
Shepard, T.H. Catalog of Teratogenic Agents. 5th ed. Baltimore, MD: The Johns Hopkins University Press, 1986., p. 584
The absence of teratogenic action after the initiation of chorio-allantoic placentation also indicated that yolk sac function was important in pathogenesis. The dye can be visualized in the cells of the visceral yolk sac. ... . The protein-trypan blue complex is concentrated in lysosomes. Through disruption of the enzymatic digestive process in the yolk sac lysosome, trypan blue may interfere with normal embryonic nutritive processes.
Shepard, T.H. Catalog of Teratogenic Agents. 5th ed. Baltimore, MD: The Johns Hopkins University Press, 1986., p. 584

5 Pharmacology

5.1 Therapeutic Uses (Complete)

A diazo-naphthalene sulfonate that is widely used as a stain.
NLM; Medical Subject Headings (2010 MeSH) for trypan blue, RN 72-57-1. Available from, as of June 17, 2010: https://www.nlm.nih.gov/cgi/mesh/2010/MB_cgi?term=72-57-1&rn=1
A therapeutic agent in the treatment of sleeping sickness. /Former use/
IARC. Monographs on the Evaluation of the Carcinogenic Risk of Chemicals to Humans. Geneva: World Health Organization, International Agency for Research on Cancer, 1972-PRESENT. (Multivolume work). Available at: https://monographs.iarc.fr/ENG/Classification/index.php, p. V8 269 (1975)
MembraneBlue 0.15% is indicated for use as an aid in ophthalmic surgery by staining the epiretinal membranes during ophthalmic surgical vitrectomy procedures, facilitating removal of the tissue.
US Natl Inst Health; DailyMed. Current Medication Information for MEMBRANEBLUE (trypan blue) injection, solution (March 2010). Available from, as of June 28, 2010: https://dailymed.nlm.nih.gov/dailymed/drugInfo.cfm?id=15868
/Experimental Therapy/ Trypan blue can be used to stain the superior oblique tendon for easy identification and delineation of it at its insertion, making the current surgical technique less difficult.
Saxena R et al; J Pediatr Opthalmol Strabusmus 44 (1): 45-6 (2007)
VisionBlue is indicated for use as an aid in ophthalmic surgery by staining the anterior capsule of the lens. /Included in US product label/
US Natl Inst Health; DailyMed. Current Medication Information for VISIONBLUE (trypan blue) injection, solution (March 2006). Available from, as of June 28, 2010: https://dailymed.nlm.nih.gov/dailymed/drugInfo.cfm?id=15897
Forty patients with stage II to IV idiopathic macular holes were randomly assigned (1:1) in a 2-arm, single-center, randomized controlled study. Internal limiting membrane delamination was performed using indocyanine green (ICG) solution (n = 20) or trypan blue (TB) (n = 20). Two patients did not complete the study, for a total of 19 in each group. ... Visual acuity did not show a significant difference between study groups (95% confidence interval [CI], -2 to 1 lines). The rate of macular hole closures was identical (84%; 95% CI, 60% to 97%). Within-group visual recovery was significant only in the TB group. Central scotomata despite hole closure persisted in 8 patients (42%) in the ICG group and in 5 (26%) in the TB group...
Beutel J et al; Arch Ophthalmol 125 (3): 326-32 (2007)

5.2 Drug Warnings (Complete)

Adverse reactions reported following use of VisionBlue include discoloration of high water content hydrogen intraocular lenses (see Contraindications) and inadvertent staining of the posterior lens capsule and vitreous face. Staining of the posterior lens capsule or staining of the vitreous face is generally self limited, lasting up to one week.
US Natl Inst Health; DailyMed. Current Medication Information for VISIONBLUE (trypan blue) injection, solution (March 2006). Available from, as of June 28, 2010: https://dailymed.nlm.nih.gov/dailymed/drugInfo.cfm?id=15897
It is not known whether this drug is excreted in human milk. Because many drugs are excreted in human milk, caution should be exercised when trypan blue is administered to a nursing woman.
US Natl Inst Health; DailyMed. Current Medication Information for VISIONBLUE (trypan blue) injection, solution (March 2006). Available from, as of June 28, 2010: https://dailymed.nlm.nih.gov/dailymed/drugInfo.cfm?id=15897
VisionBlue is contraindicated when a non-hydrated (dry state), hydrophilic acrylic intraocular lens is planned to be inserted into the eye because the dye may be absorbed by the intraocular lens and stain theintraocular lens.
US Natl Inst Health; DailyMed. Current Medication Information for VISIONBLUE (trypan blue) injection, solution (March 2006). Available from, as of June 28, 2010: https://dailymed.nlm.nih.gov/dailymed/drugInfo.cfm?id=15897
It is recommended that after injection all excess VisionBlue be immediately removed from the eye by thorough irrigation of the anterior chamber.
US Natl Inst Health; DailyMed. Current Medication Information for VISIONBLUE (trypan blue) injection, solution (March 2006). Available from, as of June 28, 2010: https://dailymed.nlm.nih.gov/dailymed/drugInfo.cfm?id=15897
FDA Pregnancy Risk Category: C /RISK CANNOT BE RULED OUT. Adequate, well controlled human studies are lacking, and animal studies have shown risk to the fetus or are lacking as well. There is a chance of fetal harm if the drug is given during pregnancy; but the potential benefits may outweigh the potential risk./
US Natl Inst Health; DailyMed. Current Medication Information for VISIONBLUE (trypan blue) injection, solution (March 2006). Available from, as of June 28, 2010: https://dailymed.nlm.nih.gov/dailymed/drugInfo.cfm?id=15897

5.3 Interactions (Complete)

The effect of L-glutamic acid on the embryolethal and teratogenic action of trypan blue was investigated in Wistar albino rats. L-glutamic acid was either incorporated into the diet, from gestation day 2 to day 20, or suspended in sesame oil and administered by gavage, from gestation day 6 to day 10. A teratogenic dose of trypan blue was injected at day 8 of pregnancy, either intraperitoneally (14 mg/kg maternal body weight) or subcutaneously (160 mg/kg). The amount of glutamic acid consumed, after the injection of trypan blue, ranged from 600 to 1,500 mg/rat/day. At day 20, the fetuses were examined. Glutamic acid failed consistently to protect the rat embryo against the lethal and teratogenic action of trypan blue. These results are in contrast to those obtained in mice. The administration of sesame oil alone was found to cause embryonic death but not malformations.
Beaudoin AR; Teratology 23 (1): 95-9 (1981)

6 Environmental Fate & Exposure

6.1 Environmental Fate / Exposure Summary

Trypan Blue's production and use as a dye for dyeing of textiles, leather, paper, a discharge-print for cotton, a vital stain for most living cells, a stain for collagen and amyloid, in chlamydospore agar for fungi, and in off-line sample measurements of cell viability in fermentation processes may result in its release to the environment through various waste streams. Trypan Blue's former use as a diagnostic agent for ocular diseases and in the treatment of sleeping sickness may have resulted in its release to the environment. Trypan Blue is an ionic compound that will be essentially non-volatile in the environment. Particulate-phase Trypan Blue will be removed from the atmosphere by wet or dry deposition. Trypan Blue absorbs light at 607 nm and therefore will be susceptible to direct photolysis by sunlight. Trypan Blue may be mobile in soils and sediments based on its solubility in water; however, biodegradation studies have observed adsorption to activated sludge indicating that Trypan Blue may be immobile in certain environmental conditions. Volatilization from moist soil, dry soil, or water surfaces is not expected to be an important fate process since it is an ionic compound. Trypan Blue was 75% removed from the aerobic activated sludge inoculum based on DOC and 87% removed based on color removal after 48 days, although adsorption may have been the main process of removal. It degraded 90% after 7 days in anaerobic sludge, indicating Trypan Blue may biodegrade in anaerobic environments. Based on its solubility in water, Trypan Blue may have low potential for bioconcentration in aquatic organisms. 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 Trypan Blue may occur through inhalation of dust and dermal contact with this compound at workplaces where Trypan Blue is produced or used. Use data indicate that the general population may be exposed to Trypan Blue via dermal contact with this compound or other consumer products containing Trypan Blue. (SRC)

6.2 Probable Routes of Human Exposure (Complete)

NIOSH (NOES Survey 1981-1983) has statistically estimated that 813 workers (623 of these were female) were potentially exposed to Trypan Blue in the US(1). Occupational exposure to Trypan Blue may occur through inhalation of dust and dermal contact with this compound at workplaces where Trypan Blue is produced or used. Use data indicate that the general population may be exposed to Trypan Blue via dermal contact with this compound or other consumer products containing Trypan Blue(SRC).
(1) NIOSH; NOES. National Occupational Exposure Survey conducted from 1981-1983. Estimated numbers of employees potentially exposed to specific agents by 2-digit standard industrial classification (SIC). Available from, as of April 29, 2010: https://www.cdc.gov/noes/

6.3 Natural Pollution Sources (Complete)

There are no known natural sources of Trypan Blue(1).
(1) IARC; Monographs on the Evaluation of the Carcinogenic Risk of Chemicals to Man. Geneva: World Health Organization, International Agency for Research on Cancer, 1972-PRESENT. (Multivolume work). Some Aromatic azo Compounds 8: 267-9 (1975)

6.4 Artificial Pollution Sources (Complete)

Trypan Blue's production and use as a dye for dyeing of textiles, leather, paper(1), a discharge-print for cotton(2), a vital stain for most living cells, a stain for collagen and amyloid, in chlamydospore agar for fungi(3), and in off-line sample measurements of cell viability in fermentation processes(4) may result in its release to the environment through various waste streams(SRC). Trypan Blue's former use as a diagnostic agent for ocular diseases(5) and in the treatment of sleeping sickness(1) may have resulted in its release to the environment(SRC); as of 1975, it was no longer used in foods, drugs or cosmetics(1).
(1) IARC; Monographs on the Evaluation of the Carcinogenic Risk of Chemicals to Man. Geneva: World Health Organization, International Agency for Research on Cancer, 1972-PRESENT. (Multivolume work). Some Aromatic Azo Compounds 8: 267-9 (1975)
(2) Green FJ; The Sigma-Aldrich Handbook of Stains, Dyes, and Indicators. Aldrich Chemical Company, Inc: Milwaukee, WI p. 721 (1990)
(3) Sigma-Aldrich; Product Search Trypan Blue (CAS NO. 72-57-1). Available from, as of April 27, 2010: https://www.sigmaaldrich.com/united-states.html
(4) Junker B; Kirk-Othmer Encyclopedia of Chemical Technology. (2001). NY, NY: John Wiley & Sons; Fermentation. Online Posting Date: January 16, 2004.
(5) Dolak TM et al; Ullmann's Encyclopedia of Industrial Chemistry. 7th ed. (2008). NY, NY: John Wiley & Sons; Ophthalmological Preparations. Online Posting Date: June 15, 2000.

6.5 Environmental Fate (Complete)

TERRESTRIAL FATE: Trypan Blue is considered soluble in the water(1) which suggests it may not adsorb to soil(SRC). However, adsorption to activated sludge may have been the main source of removal during a biodegradation screening test(2), indicating that this compound may adsorb to particulate matter under some environmental conditions(SRC). It is not expected to undergo volatilization from water surfaces(3,4) since it is an ionic compound(SRC). Trypan Blue was 75% removed from the aerobic activated sludge inoculum based on DOC and 87% removed based on color removal after 48 days, although the adsorption may have been the main process of removal(5). It degraded 90% after 7 days in anaerobic sludge, indicating Trypan Blue may biodegrade in anaerobic environments(6).
(1) O'Neil MJ, ed; The Merck Index. 15th ed. Whitehouse Station, NJ: Merck and Co., Inc. p. 1681 (2006)
(2) Pagga U, Brown D; Chemosphere 15: 479-91 (1986)
(3) Doucette WJ; pp. 141-188 in Handbook of Property Estimation Methods for Chemicals. Boethling RS, Mackay D, eds. Boca Raton, FL: Lewis Publ (2000)
(4) Baughman GL, Perenich TA; Amer Dyestuff Reporter p. 19-22. February (1988)
(5) Pagga U, Brown D; Chemosphere 15: 479-91 (1986)
(6) Brown D, Hamburger B; Chemosphere 16: 1539-53 (1987)
AQUATIC FATE: Trypan Blue is considered soluble in the water(1) which suggests it may not adsorb to aquatic soils and sediments(SRC). However, adsorption to activated sludge may have been the main source of removal during a biodegradation screening test(2), indicating that this compound may adsorb to particulate matter under some environmental conditions(SRC). It is not expected to undergo volatilization from water surfaces(3,4) since it is an ionic compound(SRC). It's solubility in water(1) suggests bioconcentration in aquatic organisms may be low(5). Trypan Blue was 75% removed from the aerobic activated sludge inoculum based on DOC and 87% based on color removal after 48 days, although the adsorption may have been the main process of removal(6). It degraded 90% after 7 days in anaerobic sludge, indicating Trypan Blue may biodegrade in anaerobic environments(7).
(1) O'Neil MJ, ed; The Merck Index. 15th ed. Whitehouse Station, NJ: Merck and Co., Inc. p. 1681 (2006)
(2) Pagga U, Brown D; Chemosphere 15: 479-91 (1986)
(3) Doucette WJ; pp. 141-188 in Handbook of Property Estimation Methods for Chemicals. Boethling RS, Mackay D, eds. Boca Raton, FL: Lewis Publ (2000)
(4) Baughman GL, Perenich TA; Amer Dyestuff Reporter p. 19-22. February (1988)
(5) Lyman WJ et al; Handbook of Chemical Property Estimation Methods. New York: McGraw Hill p. 5-10 (1982)
(6) Pagga U, Brown D; Chemosphere 15: 479-91 (1986)
(7) Brown D, Hamburger B; Chemosphere 16: 1539-53 (1987)
ATMOSPHERIC FATE: Trypan Blue is an ionic compound that will be essentially non-volatile in the environment(1,2). Therefore it is expected to exist solely in the particulate phase in the ambient atmosphere(SRC). Particulate-phase Trypan Blue may be removed from the air by wet or dry deposition(SRC). Trypan Blue absorbs light at 607 nm(3) and therefore may be susceptible to direct photolysis by sunlight(SRC).
(1) Doucette WJ; pp. 141-188 in Handbook of Property Estimation Methods for Chemicals. Boethling RS, Mackay D, eds. Boca Raton, FL: Lewis Publ (2000)
(2) Baughman GL, Perenich TA; Amer Dyestuff Reporter p. 19-22. February (1988)
(3) Sigma-Aldrich; Product Search Trypan Blue (CAS NO. 72-57-1). Available from, as of April 27, 2010: https://www.sigmaaldrich.com/united-states.html

6.6 Environmental Biodegradation (Complete)

AEROBIC: The dissolved organic carbon and color removals for Trypan Blue were observed during an OECD Static Test with activated sludge inoculua were analyzed. Trypan Blue was 75% removed from the aerobic activated sludge inoculum based on DOC and 87% removed based on color removal after 48 days(1). Most of the removal of Trypan Blue probably was due to adsorption onto the sludge since adsorption equilibrium was obtained after 1 day(1).
(1) Pagga U, Brown D; Chemosphere 15: 479-91 (1986)
ANAEROBIC: Greater than 90% degradation of Trypan Blue was observed within 7 days in aqueous anaerobic biodegradation screening tests with anaerobic sludge inoculua; 4,4'-diamino-3,3'-dimethylbiphenyl was identified as a metabolite in these tests(1).
(1) Brown D, Hamburger B; Chemosphere 16: 1539-53 (1987)

6.7 Environmental Abiotic Degradation (Complete)

Trypan Blue is not expected to undergo hydrolysis in the environment due to the lack of functional groups that hydrolyze under environmental conditions(1). Trypan Blue absorbs light at 607 nm(2) and therefore may be susceptible to direct photolysis by sunlight(SRC).
(1) Lyman WJ et al; Handbook of Chemical Property Estimation Methods. Washington, DC: Amer Chem Soc pp. 7-4, 7-5 (1990)
(2) Sigma-Aldrich; Product Search Trypan Blue (CAS NO. 72-57-1). Available from, as of April 27, 2010: https://www.sigmaaldrich.com/united-states.html

6.8 Environmental Bioconcentration (Complete)

Trypan Blue is soluble in water(1) which suggests bioconcentration in aquatic organisms may be low(2).
(1) O'Neil MJ, ed; The Merck Index. 15th ed. Whitehouse Station, NJ: Merck and Co., Inc. p. 1681 (2006)
(2) Lyman WJ et al; Handbook of Chemical Property Estimation Methods. New York: McGraw Hill p. 5-10 (1982)

6.9 Soil Adsorption / Mobility (Complete)

Trypan Blue is considered soluble in the water(1) which suggests it may not adsorb to soils and sediments(SRC). However, in an activated sludge biodegradation screening test, a COD removal of 84% was attributed to sludge adsorption(2), indicating that this compound may adsorb to particulate matter under some environmental conditions(SRC).
(1) O'Neil MJ, ed; The Merck Index. 15th ed. Whitehouse Station, NJ: Merck and Co., Inc. p. 1681 (2006)
(2) Pagga U, Brown D; Chemosphere 15: 479-91 (1986)

6.10 Volatilization from Water / Soil (Complete)

Trypan Blue is an ionic compound(SRC). Therefore, it is not expected to undergo volatilization(1,2).
(1) Doucette WJ; pp. 141-188 in Handbook of Property Estimation Methods for Chemicals. Boethling RS, Mackay D, eds. Boca Raton, FL: Lewis Publ (2000)
(2) Baughman GL, Perenich TA; Amer Dyestuff Reporter p. 19-22. February (1988)

7 Environmental Standards & Regulations

7.1 CERCLA Reportable Quantities (Complete)

Persons in charge of vessels or facilities are required to notify the National Response Center (NRC) immediately, when there is a release of this designated hazardous substance, in an amount equal to or greater than its reportable quantity of 10 lb or 4.54 kg. The toll free number of the NRC is (800) 424-8802. The rule for determining when notification is required is stated in 40 CFR 302.4 (section IV. D.3.b).
40 CFR 302.4 (USEPA); U.S. National Archives and Records Administration's Electronic Code of Federal Regulations. Available from, as of June 2, 2010: https://www.ecfr.gov

7.2 RCRA Requirements (Complete)

U236; As stipulated in 40 CFR 261.33, when trypan blue, as a commercial chemical product or manufacturing chemical intermediate or an off-specification commercial chemical product or a manufacturing chemical intermediate, becomes a waste, it must be managed according to Federal and/or State hazardous waste regulations. Also defined as a hazardous waste is any residue, contaminated soil, water, or other debris resulting from the cleanup of a spill, into water or on dry land, of this waste. Generators of small quantities of this waste may qualify for partial exclusion from hazardous waste regulations (40 CFR 261.5).
40 CFR 261.33 (USEPA); U.S. National Archives and Records Administration's Electronic Code of Federal Regulations. Available from, as of June 2, 2010: https://www.ecfr.gov

7.3 FDA Requirements (Complete)

The Approved Drug Products with Therapeutic Equivalence Evaluations List identifies currently marketed prescription drug products, including trypan blue, approved on the basis of safety and effectiveness by FDA under sections 505 of the Federal Food, Drug, and Cosmetic Act.
DHHS/FDA; Electronic Orange Book-Approved Drug Products with Therapeutic Equivalence Evaluations. Available from, as of June 17, 2010: https://www.accessdata.fda.gov/scripts/cder/ob/docs/queryai.cfm

8 Chemical / Physical Properties

8.1 Molecular Formula

C34-H28-N6-O14-S4.4Na

8.2 Molecular Weight

960.81
O'Neil, M.J. (ed.). The Merck Index - An Encyclopedia of Chemicals, Drugs, and Biologicals. Whitehouse Station, NJ: Merck and Co., Inc., 2006., p. 1681

8.3 Color / Form (Complete)

Bluish-grey powder
O'Neil, M.J. (ed.). The Merck Index - An Encyclopedia of Chemicals, Drugs, and Biologicals. Whitehouse Station, NJ: Merck and Co., Inc., 2006., p. 1681
Dark greenish-brown powder.
Green FJ; The Sigma-Aldrich Handbook of Stains, Dyes, and Indicators. Aldrich Chemical Company, Inc: Milwaukee, WI p. 721 (1990)
Aqueous solution deep blue with violet tinge
O'Neil, M.J. (ed.). The Merck Index - An Encyclopedia of Chemicals, Drugs, and Biologicals. Whitehouse Station, NJ: Merck and Co., Inc., 2006., p. 1681

8.4 Melting Point

300 °C
Lide, D.R. CRC Handbook of Chemistry and Physics 88TH Edition 2007-2008. CRC Press, Taylor & Francis, Boca Raton, FL 2007, p. 3-514

8.5 Solubility (Complete)

20 mg/mL in methyl Cellosolve, and 0.6 mg/mL in ethanol
Green FJ; The Sigma-Aldrich Handbook of Stains, Dyes, and Indicators. Aldrich Chemical Company, Inc: Milwaukee, WI p. 721 (1990)
Soluble in water
O'Neil, M.J. (ed.). The Merck Index - An Encyclopedia of Chemicals, Drugs, and Biologicals. Whitehouse Station, NJ: Merck and Co., Inc., 2006., p. 1681
In water, <0.1 mg/mL
Green FJ; The Sigma-Aldrich Handbook of Stains, Dyes, and Indicators. Aldrich Chemical Company, Inc: Milwaukee, WI p. 721 (1990)

9 Spectral Information

9.1 UV Spectra

Max absorption (water): 584-617 nm
IARC. Monographs on the Evaluation of the Carcinogenic Risk of Chemicals to Humans. Geneva: World Health Organization, International Agency for Research on Cancer, 1972-PRESENT. (Multivolume work). Available at: https://monographs.iarc.fr/ENG/Classification/index.php, p. V8: 267 (1975)
UV max absorption = 607 nm /60% dye content/
Sigma-Aldrich; Product Search Trypan Blue (CAS NO. 72-57-1). Available from, as of April 27, 2010: https://www.sigmaaldrich.com/united-states.html

10 Chemical Safety & Handling

10.1 NFPA Hazard Classification

NFPA 704 Diamond
0-1-0
NFPA Health Rating
0 - Materials that, under emergency conditions, would offer no hazard beyond that of ordinary combustible materials.
NFPA Fire Rating
1 - Materials that must be preheated before ignition can occur. Materials require considerable preheating, under all ambient temperature conditions, before ignition and combustion can occur.
NFPA Instability Rating
0 - Materials that in themselves are normally stable, even under fire conditions.

10.2 Fire Fighting Procedures (Complete)

This chemical is a noncombustible solid. Use extinguisher appropriate for burning material ... If material or contaminated runoff enters waterways, notify downstream users of potentially contaminated waters. Notify local health and fire officials and pollution control agencies. From a secure, explosion-proof location, use water spray to cool exposed containers. If cooling streams are ineffective (venting sound increases in volume and pitch, tank discolors or shows any signs of deforming), withdraw immediately to a secure position ... The only respirators recommended for fire fighting are self-contained breathing apparatuses that have full facepieces and are operated in a pressure-demand or other positive-pressure mode.
Pohanish, R.P. (ed). Sittig's Handbook of Toxic and Hazardous Chemical Carcinogens 5th Edition Volume 1: A-H,Volume 2: I-Z. William Andrew, Norwich, NY 2008, p. 2529

10.3 Hazardous Reactivities and Incompatibilities (Complete)

Strong oxidizers
Pohanish, R.P. (ed). Sittig's Handbook of Toxic and Hazardous Chemical Carcinogens 5th Edition Volume 1: A-H,Volume 2: I-Z. William Andrew, Norwich, NY 2008, p. 2528

10.4 Personal Protective Equipment (PPE) (Complete)

Safety glasses. Handle with gloves. Choose body protection according to the amount and concentration of the dangerous substance at the work place. Where risk assessment shows air-purifying respirators are appropriate use a full-face particle respirator type N99 (US) or type P2 (EN 143) respirator cartridges as a backup to engineering controls. If the respirator is the sole means of protection, use a full-face supplied air respirator. Use respirators and components tested and approved under appropriate government standards such as NIOSH (US) or CEN (EU).
Sigma-Aldrich Corp; Safety Data Sheet for Trypan blue (Product Number: 302643) Version 3.0 (January 2, 2009). Available from, as of June 15, 2010: https://www.sigmaaldrich.com
PRECAUTIONS FOR "CARCINOGENS": ... Dispensers of liq detergent /should be available./ ... Safety pipettes should be used for all pipetting. ... In animal laboratory, personnel should ... wear protective suits (preferably disposable, one-piece & close-fitting at ankles & wrists), gloves, hair covering & overshoes. ... In chemical laboratory, gloves & gowns should always be worn ... however, gloves should not be assumed to provide full protection. Carefully fitted masks or respirators may be necessary when working with particulates or gases, & disposable plastic aprons might provide addnl protection. ... Gowns ... /should be/ of distinctive color, this is a reminder that they are not to be worn outside the laboratory. /Chemical Carcinogens/
Montesano, R., H. Bartsch, E.Boyland, G. Della Porta, L. Fishbein, R. A. Griesemer, A.B. Swan, L. Tomatis, and W. Davis (eds.). Handling Chemical Carcinogens in the Laboratory: Problems of Safety. IARC Scientific Publications No. 33. Lyon, France: International Agency for Research on Cancer, 1979., p. 8

10.5 Preventive Measures (Complete)

SRP: The scientific literature for the use of contact lenses by industrial workers is inconsistent. The benefits or detrimental effects of wearing contact lenses depend not only upon the substance, but also on factors including the form of the substance, characteristics and duration of the exposure, the uses of other eye protection equipment, and the hygiene of the lenses. However, there may be individual substances whose irritating or corrosive properties are such that the wearing of contact lenses would be harmful to the eye. In those specific cases, contact lenses should not be worn. In any event, the usual eye protection equipment should be worn even when contact lenses are in place.
SRP: Local exhaust ventilation should be applied wherever there is an incidence of point source emissions or dispersion of regulated contaminants in the work area. Ventilation control of the contaminant as close to its point of generation is both the most economical and safest method to minimize personnel exposure to airborne contaminants. Ensure that the local ventilation moves the contaminant away from the worker.
Wear protective gloves and clothing to prevent any reasonable probability of skin contact ... Wear dust-proof chemical goggles and face shield unless full facepiece respiratory protection is worn.
Pohanish, R.P. (ed). Sittig's Handbook of Toxic and Hazardous Chemical Carcinogens 5th Edition Volume 1: A-H,Volume 2: I-Z. William Andrew, Norwich, NY 2008, p. 2529
PRECAUTIONS FOR "CARCINOGENS": Smoking, drinking, eating, storage of food or of food & beverage containers or utensils, & the application of cosmetics should be prohibited in any laboratory. All personnel should remove gloves, if worn, after completion of procedures in which carcinogens have been used. They should ... wash ... hands, preferably using dispensers of liq detergent, & rinse ... thoroughly. Consideration should be given to appropriate methods for cleaning the skin, depending on nature of the contaminant. No standard procedure can be recommended, but the use of organic solvents should be avoided. Safety pipettes should be used for all pipetting. /Chemical Carcinogens/
Montesano, R., H. Bartsch, E.Boyland, G. Della Porta, L. Fishbein, R. A. Griesemer, A.B. Swan, L. Tomatis, and W. Davis (eds.). Handling Chemical Carcinogens in the Laboratory: Problems of Safety. IARC Scientific Publications No. 33. Lyon, France: International Agency for Research on Cancer, 1979., p. 8
PRECAUTIONS FOR "CARCINOGENS": In animal laboratory, personnel should remove their outdoor clothes & wear protective suits (preferably disposable, one-piece & close-fitting at ankles & wrists), gloves, hair covering & overshoes. ... Clothing should be changed daily but ... discarded immediately if obvious contamination occurs ... /also,/ workers should shower immediately. In chemical laboratory, gloves & gowns should always be worn ... however, gloves should not be assumed to provide full protection. Carefully fitted masks or respirators may be necessary when working with particulates or gases, & disposable plastic aprons might provide addnl protection. If gowns are of distinctive color, this is a reminder that they should not be worn outside of lab. /Chemical Carcinogens/
Montesano, R., H. Bartsch, E.Boyland, G. Della Porta, L. Fishbein, R. A. Griesemer, A.B. Swan, L. Tomatis, and W. Davis (eds.). Handling Chemical Carcinogens in the Laboratory: Problems of Safety. IARC Scientific Publications No. 33. Lyon, France: International Agency for Research on Cancer, 1979., p. 8
PRECAUTIONS FOR "CARCINOGENS": ... Operations connected with synth & purification ... should be carried out under well-ventilated hood. Analytical procedures ... should be carried out with care & vapors evolved during ... procedures should be removed. ... Expert advice should be obtained before existing fume cupboards are used ... & when new fume cupboards are installed. It is desirable that there be means for decreasing the rate of air extraction, so that carcinogenic powders can be handled without ... powder being blown around the hood. Glove boxes should be kept under negative air pressure. Air changes should be adequate, so that concn of vapors of volatile carcinogens will not occur. /Chemical Carcinogens/
Montesano, R., H. Bartsch, E.Boyland, G. Della Porta, L. Fishbein, R. A. Griesemer, A.B. Swan, L. Tomatis, and W. Davis (eds.). Handling Chemical Carcinogens in the Laboratory: Problems of Safety. IARC Scientific Publications No. 33. Lyon, France: International Agency for Research on Cancer, 1979., p. 8
PRECAUTIONS FOR "CARCINOGENS": Vertical laminar-flow biological safety cabinets may be used for containment of in vitro procedures ... provided that the exhaust air flow is sufficient to provide an inward air flow at the face opening of the cabinet, & contaminated air plenums that are under positive pressure are leak-tight. Horizontal laminar-flow hoods or safety cabinets, where filtered air is blown across the working area towards the operator, should never be used ... . Each cabinet or fume cupboard to be used ... should be tested before work is begun (eg, with fume bomb) & label fixed to it, giving date of test & avg air-flow measured. This test should be repeated periodically & after any structural changes. /Chemical Carcinogens/
Montesano, R., H. Bartsch, E.Boyland, G. Della Porta, L. Fishbein, R. A. Griesemer, A.B. Swan, L. Tomatis, and W. Davis (eds.). Handling Chemical Carcinogens in the Laboratory: Problems of Safety. IARC Scientific Publications No. 33. Lyon, France: International Agency for Research on Cancer, 1979., p. 9
PRECAUTIONS FOR "CARCINOGENS": Principles that apply to chem or biochem lab also apply to microbiological & cell-culture labs ... . Special consideration should be given to route of admin. ... Safest method of administering volatile carcinogen is by injection of a soln. Admin by topical application, gavage, or intratracheal instillation should be performed under hood. If chem will be exhaled, animals should be kept under hood during this period. Inhalation exposure requires special equipment. ... Unless specifically required, routes of admin other than in the diet should be used. Mixing of carcinogen in diet should be carried out in sealed mixers under fume hood, from which the exhaust is fitted with an efficient particulate filter. Techniques for cleaning mixer & hood should be devised before expt begun. When mixing diets, special protective clothing &, possibly, respirators may be required. /Chemical Carcinogens/
Montesano, R., H. Bartsch, E.Boyland, G. Della Porta, L. Fishbein, R. A. Griesemer, A.B. Swan, L. Tomatis, and W. Davis (eds.). Handling Chemical Carcinogens in the Laboratory: Problems of Safety. IARC Scientific Publications No. 33. Lyon, France: International Agency for Research on Cancer, 1979., p. 9
PRECAUTIONS FOR "CARCINOGENS": When ... admin in diet or applied to skin, animals should be kept in cages with solid bottoms & sides & fitted with a filter top. When volatile carcinogens are given, filter tops should not be used. Cages which have been used to house animals that received carcinogens should be decontaminated. Cage-cleaning facilities should be installed in area in which carcinogens are being used, to avoid moving of ... contaminated /cages/. It is difficult to ensure that cages are decontaminated, & monitoring methods are necessary. Situations may exist in which the use of disposable cages should be recommended, depending on type & amt of carcinogen & efficiency with which it can be removed. /Chemical Carcinogens/
Montesano, R., H. Bartsch, E.Boyland, G. Della Porta, L. Fishbein, R. A. Griesemer, A.B. Swan, L. Tomatis, and W. Davis (eds.). Handling Chemical Carcinogens in the Laboratory: Problems of Safety. IARC Scientific Publications No. 33. Lyon, France: International Agency for Research on Cancer, 1979., p. 10
PRECAUTIONS FOR "CARCINOGENS": To eliminate risk that ... contamination in lab could build up during conduct of expt, periodic checks should be carried out on lab atmospheres, surfaces, such as walls, floors & benches, & ... interior of fume hoods & airducts. As well as regular monitoring, check must be carried out after cleaning-up of spillage. Sensitive methods are required when testing lab atmospheres. ... Methods ... should ... where possible, be simple & sensitive. /Chemical Carcinogens/
Montesano, R., H. Bartsch, E.Boyland, G. Della Porta, L. Fishbein, R. A. Griesemer, A.B. Swan, L. Tomatis, and W. Davis (eds.). Handling Chemical Carcinogens in the Laboratory: Problems of Safety. IARC Scientific Publications No. 33. Lyon, France: International Agency for Research on Cancer, 1979., p. 10
PRECAUTIONS FOR "CARCINOGENS": Rooms in which obvious contamination has occurred, such as spillage, should be decontaminated by lab personnel engaged in expt. Design of expt should ... avoid contamination of permanent equipment. ... Procedures should ensure that maintenance workers are not exposed to carcinogens. ... Particular care should be taken to avoid contamination of drains or ventilation ducts. In cleaning labs, procedures should be used which do not produce aerosols or dispersal of dust, ie, wet mop or vacuum cleaner equipped with high-efficiency particulate filter on exhaust, which are avail commercially, should be used. Sweeping, brushing & use of dry dusters or mops should be prohibited. Grossly contaminated cleaning materials should not be re-used. ... If gowns or towels are contaminated, they should not be sent to laundry, but ... decontaminated or burnt, to avoid any hazard to laundry personnel. /Chemical Carcinogens/
Montesano, R., H. Bartsch, E.Boyland, G. Della Porta, L. Fishbein, R. A. Griesemer, A.B. Swan, L. Tomatis, and W. Davis (eds.). Handling Chemical Carcinogens in the Laboratory: Problems of Safety. IARC Scientific Publications No. 33. Lyon, France: International Agency for Research on Cancer, 1979., p. 10
PRECAUTIONS FOR "CARCINOGENS": Doors leading into areas where carcinogens are used ... should be marked distinctively with appropriate labels. Access ... limited to persons involved in expt. ... A prominently displayed notice should give the name of the Scientific Investigator or other person who can advise in an emergency & who can inform others (such as firemen) on the handling of carcinogenic substances. /Chemical Carcinogens/
Montesano, R., H. Bartsch, E.Boyland, G. Della Porta, L. Fishbein, R. A. Griesemer, A.B. Swan, L. Tomatis, and W. Davis (eds.). Handling Chemical Carcinogens in the Laboratory: Problems of Safety. IARC Scientific Publications No. 33. Lyon, France: International Agency for Research on Cancer, 1979., p. 11

10.6 Shipment Methods and Regulations (Complete)

PRECAUTIONS FOR "CARCINOGENS": Procurement ... of unduly large amt ... should be avoided. To avoid spilling, carcinogens should be transported in securely sealed glass bottles or ampoules, which should themselves be placed inside strong screw-cap or snap-top container that will not open when dropped & will resist attack from the carcinogen. Both bottle & the outside container should be appropriately labelled. ... National post offices, railway companies, road haulage companies & airlines have regulations governing transport of hazardous materials. These authorities should be consulted before ... material is shipped. /Chemical Carcinogens/
Montesano, R., H. Bartsch, E.Boyland, G. Della Porta, L. Fishbein, R. A. Griesemer, A.B. Swan, L. Tomatis, and W. Davis (eds.). Handling Chemical Carcinogens in the Laboratory: Problems of Safety. IARC Scientific Publications No. 33. Lyon, France: International Agency for Research on Cancer, 1979., p. 13
PRECAUTIONS FOR "CARCINOGENS": When no regulations exist, the following procedure must be adopted. The carcinogen should be enclosed in a securely sealed, watertight container (primary container), which should be enclosed in a second, unbreakable, leakproof container that will withstand chem attack from the carcinogen (secondary container). The space between primary & secondary container should be filled with absorbent material, which would withstand chem attack from the carcinogen & is sufficient to absorb the entire contents of the primary container in the event of breakage or leakage. Each secondary container should then be enclosed in a strong outer box. The space between the secondary container & the outer box should be filled with an appropriate quantity of shock-absorbent material. Sender should use fastest & most secure form of transport & notify recipient of its departure. If parcel is not received when expected, carrier should be informed so that immediate effort can be made to find it. Traffic schedules should be consulted to avoid ... arrival on weekend or holiday ... /Chemical Carcinogens/
Montesano, R., H. Bartsch, E.Boyland, G. Della Porta, L. Fishbein, R. A. Griesemer, A.B. Swan, L. Tomatis, and W. Davis (eds.). Handling Chemical Carcinogens in the Laboratory: Problems of Safety. IARC Scientific Publications No. 33. Lyon, France: International Agency for Research on Cancer, 1979., p. 13

10.7 Storage Conditions (Complete)

Avoid creating dust. Where possible, automatically pump material from storage containers to process containers. Sources of ignition, such as smoking and open flames, are prohibited where this chemical is handled, used, or stored. A regulated, marked area should be established where this chemical is handled, used, or stored ...
Pohanish, R.P. (ed). Sittig's Handbook of Toxic and Hazardous Chemical Carcinogens 5th Edition Volume 1: A-H,Volume 2: I-Z. William Andrew, Norwich, NY 2008, p. 2529
PRECAUTIONS FOR "CARCINOGENS": Storage site should be as close as practical to lab in which carcinogens are to be used, so that only small quantities required for ... expt need to be carried. Carcinogens should be kept in only one section of cupboard, an explosion-proof refrigerator or freezer (depending on chemicophysical properties ...) that bears appropriate label. An inventory ... should be kept, showing quantity of carcinogen & date it was acquired ... . Facilities for dispensing ... should be contiguous to storage area. /Chemical Carcinogens/
Montesano, R., H. Bartsch, E.Boyland, G. Della Porta, L. Fishbein, R. A. Griesemer, A.B. Swan, L. Tomatis, and W. Davis (eds.). Handling Chemical Carcinogens in the Laboratory: Problems of Safety. IARC Scientific Publications No. 33. Lyon, France: International Agency for Research on Cancer, 1979., p. 13
VisionBlue is stored at 15-25 °C (59-77 °F). Protect from direct sunlight.
US Natl Inst Health; DailyMed. Current Medication Information for VISIONBLUE (trypan blue) injection, solution (March 2006). Available from, as of June 28, 2010: https://dailymed.nlm.nih.gov/dailymed/drugInfo.cfm?id=15897

10.8 Cleanup Methods (Complete)

Spill handling: evacuate persons not wearing protective equipment from area of spill or leak until cleanup is complete. Remove all ignition sources. Collect powdered material in the most convenient and safe manner and deposit in sealed containers. Ventilate area after clean-up is complete. It may be necessary to contain and dispose of this chemical as a hazardous waste. If material or contaminated runoff enters waterways, notify downstream users of potentially contaminated waters. Contact your Department of Environmental Protection or your regional office of the federal EPA for specific recommendations.
Pohanish, R.P. (ed). Sittig's Handbook of Toxic and Hazardous Chemical Carcinogens 5th Edition Volume 1: A-H,Volume 2: I-Z. William Andrew, Norwich, NY 2008, p. 2529
PRECAUTIONS FOR "CARCINOGENS": A high-efficiency particulate arrestor (HEPA) or charcoal filters can be used to minimize amt of carcinogen in exhausted air ventilated safety cabinets, lab hoods, glove boxes or animal rooms ... . Filter housing that is designed so that used filters can be transferred into plastic bags without contaminating maintenance staff is avail commercially. Filters should be placed in plastic bags immediately after removal ... . The plastic bag should be sealed immediately ... . The sealed bag should be labelled properly ... . Waste liquids ... should be placed or collected in proper containers for disposal. The lid should be secured & the bottles properly labelled. Once filled, bottles should be placed in plastic bags, so that outer surface ... is not contaminated ... . The plastic bag should also be sealed & labelled. ... Broken glassware ... should be decontaminated by solvent extraction, by chemical destruction, or in specially designed incinerators. /Chemical Carcinogens/
Montesano, R., H. Bartsch, E.Boyland, G. Della Porta, L. Fishbein, R. A. Griesemer, A.B. Swan, L. Tomatis, and W. Davis (eds.). Handling Chemical Carcinogens in the Laboratory: Problems of Safety. IARC Scientific Publications No. 33. Lyon, France: International Agency for Research on Cancer, 1979., p. 15

10.9 Disposal Methods (Complete)

Generators of waste (equal to or greater than 100 kg/mo) containing this contaminant, EPA hazardous waste number U236, must conform with USEPA regulations in storage, transportation, treatment and disposal of waste.
40 CFR 240-280, 300-306, 702-799 (7/1/2008)
A potential candidate for rotary kiln incineration at a temperature range of 820 to 1,600 °C and residence times of seconds for liquids and gases, and hours for solids. A potential candidate for fluidized bed incineration at a temperature range of 450 to 980 °C and residence times of seconds for liquids and gases, and longer for solids.
USEPA; Engineering Handbook for Hazardous Waste Incineration p.3-16 (1981) EPA 68-03-3025
PRECAUTIONS FOR "CARCINOGENS": There is no universal method of disposal that has been proved satisfactory for all carcinogenic compounds & specific methods of chem destruction ... published have not been tested on all kinds of carcinogen-containing waste. ... Summary of avail methods & recommendations ... /given/ must be treated as guide only. /Chemical Carcinogens/
Montesano, R., H. Bartsch, E.Boyland, G. Della Porta, L. Fishbein, R. A. Griesemer, A.B. Swan, L. Tomatis, and W. Davis (eds.). Handling Chemical Carcinogens in the Laboratory: Problems of Safety. IARC Scientific Publications No. 33. Lyon, France: International Agency for Research on Cancer, 1979., p. 14
PRECAUTIONS FOR "CARCINOGENS": ... Incineration may be only feasible method for disposal of contaminated laboratory waste from biological expt. However, not all incinerators are suitable for this purpose. The most efficient type ... is probably the gas-fired type, in which a first-stage combustion with a less than stoichiometric air:fuel ratio is followed by a second stage with excess air. Some ... are designed to accept ... aqueous & organic-solvent solutions, otherwise it is necessary ... to absorb soln onto suitable combustible material, such as sawdust. Alternatively, chem destruction may be used, esp when small quantities ... are to be destroyed in laboratory. /Chemical Carcinogens/
Montesano, R., H. Bartsch, E.Boyland, G. Della Porta, L. Fishbein, R. A. Griesemer, A.B. Swan, L. Tomatis, and W. Davis (eds.). Handling Chemical Carcinogens in the Laboratory: Problems of Safety. IARC Scientific Publications No. 33. Lyon, France: International Agency for Research on Cancer, 1979., p. 15
PRECAUTIONS FOR "CARCINOGENS": HEPA (high-efficiency particulate arrestor) filters ... can be disposed of by incineration. For spent charcoal filters, the adsorbed material can be stripped off at high temp & carcinogenic wastes generated by this treatment conducted to & burned in an incinerator. ... LIQUID WASTE: ... Disposal should be carried out by incineration at temp that ... ensure complete combustion. SOLID WASTE: Carcasses of lab animals, cage litter & misc solid wastes ... should be disposed of by incineration at temp high enough to ensure destruction of chem carcinogens or their metabolites. /Chemical Carcinogens/
Montesano, R., H. Bartsch, E.Boyland, G. Della Porta, L. Fishbein, R. A. Griesemer, A.B. Swan, L. Tomatis, and W. Davis (eds.). Handling Chemical Carcinogens in the Laboratory: Problems of Safety. IARC Scientific Publications No. 33. Lyon, France: International Agency for Research on Cancer, 1979., p. 15
PRECAUTIONS FOR "CARCINOGENS": ... Small quantities of ... some carcinogens can be destroyed using chem reactions ... but no general rules can be given. ... As a general technique ... treatment with sodium dichromate in strong sulfuric acid can be used. The time necessary for destruction ... is seldom known ... but 1-2 days is generally considered sufficient when freshly prepd reagent is used. ... Carcinogens that are easily oxidizable can be destroyed with milder oxidative agents, such as saturated soln of potassium permanganate in acetone, which appears to be a suitable agent for destruction of hydrazines or of compounds containing isolated carbon-carbon double bonds. Concn or 50% aqueous sodium hypochlorite can also be used as an oxidizing agent. /Chemical Carcinogens/
Montesano, R., H. Bartsch, E.Boyland, G. Della Porta, L. Fishbein, R. A. Griesemer, A.B. Swan, L. Tomatis, and W. Davis (eds.). Handling Chemical Carcinogens in the Laboratory: Problems of Safety. IARC Scientific Publications No. 33. Lyon, France: International Agency for Research on Cancer, 1979., p. 16
PRECAUTIONS FOR "CARCINOGENS": Carcinogens that are alkylating, arylating or acylating agents per se can be destroyed by reaction with appropriate nucleophiles, such as water, hydroxyl ions, ammonia, thiols & thiosulfate. The reactivity of various alkylating agents varies greatly ... & is also influenced by sol of agent in the reaction medium. To facilitate the complete reaction, it is suggested that the agents be dissolved in ethanol or similar solvents. ... No method should be applied ... until it has been thoroughly tested for its effectiveness & safety on material to be inactivated. For example, in case of destruction of alkylating agents, it is possible to detect residual compounds by reaction with 4(4-nitrobenzyl)-pyridine. /Chemical Carcinogens/
Montesano, R., H. Bartsch, E.Boyland, G. Della Porta, L. Fishbein, R. A. Griesemer, A.B. Swan, L. Tomatis, and W. Davis (eds.). Handling Chemical Carcinogens in the Laboratory: Problems of Safety. IARC Scientific Publications No. 33. Lyon, France: International Agency for Research on Cancer, 1979., p. 17

11 Manufacturing / Use Information

11.1 Uses (Complete)

Trypan Blue has been used as a vital stain as it is excluded by most living cells. It has been used to stain collagen and amyloid. It is also used in chlamydospore agar for fungi.
Sigma-Aldrich Corp; Safety Data Sheet for Trypan blue (Product Number: 302643) Version 3.0 (January 2, 2009). Available from, as of June 15, 2010: https://www.sigmaaldrich.com/united-states.html
As a diagnostic agent for the diagnosis of ocular systemic disease and to diagnose ocular abnormalities. /Not commercially available/
Dolak TM et al; Ullmann's Encyclopedia of Industrial Chemistry. 7th ed. (2008). NY, NY: John Wiley & Sons; Ophthalmological Preparations. Online Posting Date: June 15, 2000
Vital staining of cytoplasm /In table/
Schmidt U, Schulte EKW; Ullmann's Encyclopedia of Industrial Chemistry. 7th ed. (2008). NY, NY: John Wiley & Sons; Stains, Microscopic. Online Posting Date: June 15, 2000
Trypan Blue is used in off-line sample measurements of cell viability in fermentation processes.
Junker B; Kirk-Othmer Encyclopedia of Chemical Technology. (2001). NY, NY: John Wiley & Sons; Fermentation. Online Posting Date: January 16, 2004
For dyeing textiles, leather & paper, as a stain in biological investigations.
IARC. Monographs on the Evaluation of the Carcinogenic Risk of Chemicals to Humans. Geneva: World Health Organization, International Agency for Research on Cancer, 1972-PRESENT. (Multivolume work). Available at: https://monographs.iarc.fr/ENG/Classification/index.php, p. V8: 269 (1975)
Commercially, it is used to dye and, to a lesser degree, discharge-print cotton. In addition, Trypan Blue dyes fully chromed leather an intense blue.
Green FJ; The Sigma-Aldrich Handbook of Stains, Dyes, and Indicators. Aldrich Chemical Company, Inc: Milwaukee, WI p. 721 (1990)
Trypan Blue has been used in the past in the treatment of sleeping sickness, but as of 1975, it was no longer used in foods, drugs or cosmetics.
IARC. Monographs on the Evaluation of the Carcinogenic Risk of Chemicals to Humans. Geneva: World Health Organization, International Agency for Research on Cancer, 1972-PRESENT. (Multivolume work). Available at: https://monographs.iarc.fr/ENG/Classification/index.php, p. V8: 267 (1975)
MEDICATION

11.2 Manufacturers

Sigma-Aldrich
Sigma-Aldrich Corp; Safety Data Sheet for Trypan blue (Product Number: 302643) Version 3.0 (January 2, 2009). Available from, as of June 15, 2010: https://www.sigmaaldrich.com/united-states.html
Trypan Blue Producers 2009.
Company
Shanghai SSS Reagent Co., Ltd.
Address
2588 Jungong Road
City, State, Country
Yangpu District Shanghai 200438, China
Company
Dyestuffs &amp; Chemicals Co.
Address
P.O. Box 22621
City, State, Country
Behera, Egypt
Company
Reactifs RAL, S.A.
Address
Site Montesquieu
City, State, Country
33650 Martillac, France
Company
Simpsons UK Ltd.
Address
6B Lodgeway, Severnbridge Industrial Estate Caldicot
City, State, Country
Gwent NP26 5PT, Great Britain
Company
Appex Dyestuff Industries
Address
C-1/258, Phase II G.I.D.C.
City, State, Country
Vatva Ahmedabad 382.445, India
Company
Belami Fine Chemicals Pvt. Ltd.
Address
29 Gopal Bhuvan, 2/F, 199 Princess Street
City, State, Country
Mumbai 400.002, India
Company
Esjay Chemicals
Address
B/303, Dharam Palace, Shantivan, Borivali (East)
City, State, Country
Mumbai 400.066, India
Company
Karsandas Mavji
Address
203 Rangwala Apartments, Opp. Bank of India, M.G. Road Ghatkopar (West)
City, State, Country
Mumbai 400.086, India
Company
Neha-Chem
Address
317/2534 Motilal Nagar, 2 M.G. Road, Goregaon (West)
City, State, Country
Mumbai 400.090, India
Company
Unisource Chemicals Private Limited
Address
7 Devkaran Mansion, 24/B Vithaldas Road, Princess Street
City, State, Country
Mumbai 400.002, India
Directory of World Chemical Producers, Chemical Information Services, 9101 LBJ Frwy., Suite 310, Dallas, TX 75243, (214) 349-6200. Date downloaded: September 2009. Available from, as of Dec 3, 2010: https://www.chemicalinfo.com/dwcp
Fabricolor Manufacturing Corp, PO Box 2398, 24 1/2 Van Houten Street, Paterson, NJ 07509 (201) 742-3900. Production Site: Paterson, NJ 07509
United States International Trade Commission. Synthetic Organic Chemicals - United States Production and Sales, 1993. USITC Publication 2810, Nov. 1994. Washington, D.C., p. 3-217

11.3 Methods of Manufacturing (Complete)

Prepared by coupling diazotized o-tolidine with 5-amino-4-hydroxy-2,7-naphthalenedisulfonic acid in sodium carbonate solution
O'Neil, M.J. (ed.). The Merck Index - An Encyclopedia of Chemicals, Drugs, and Biologicals. Whitehouse Station, NJ: Merck and Co., Inc., 2006., p. 1681
Prepared by the alkaline coupling of one mole of tetraazotized o-tolidine to two moles of H acid (5-amino-4-hydroxy-2,7-naphthalenedisulfonic acid) and converting the product to the tetrasodium salt.
Green FJ; The Sigma-Aldrich Handbook of Stains, Dyes, and Indicators. Aldrich Chemical Company, Inc: Milwaukee, WI p. 721 (1990)

11.4 General Manufacturing Information (Complete)

USA producton of Trypan Blue was first reported in 1921.
IARC. Monographs on the Evaluation of the Carcinogenic Risk of Chemicals to Humans. Geneva: World Health Organization, International Agency for Research on Cancer, 1972-PRESENT. (Multivolume work). Available at: https://monographs.iarc.fr/ENG/Classification/index.php, p. V8: 267 (1975)
According to USA industry sources, Trypan Blue is not used in foods, drugs or cosmetics. ... its composition varies ... to meet customer shade and intensity requirement.
IARC. Monographs on the Evaluation of the Carcinogenic Risk of Chemicals to Humans. Geneva: World Health Organization, International Agency for Research on Cancer, 1972-PRESENT. (Multivolume work). Available at: https://monographs.iarc.fr/ENG/Classification/index.php, p. V8: 267 (1975)
Trypan Blue, like other polysulfonic acids, is difficult to purify and commercial product is often mixture; thus, some of biological effects described as being caused by Trypan Blue may have been produced by impurities.
IARC. Monographs on the Evaluation of the Carcinogenic Risk of Chemicals to Humans. Geneva: World Health Organization, International Agency for Research on Cancer, 1972-PRESENT. (Multivolume work). Available at: https://monographs.iarc.fr/ENG/Classification/index.php, p. V8: 269 (1975)

11.5 Formulations / Preparations (Complete)

Each mL of VisionBlue 0.06% contains: 0.6 mg trypan blue; 1.9 mg sodium mono-hydrogen orthophosphate (Na2HPO4.2H2O); 0.3 mg sodium di-hydrogen orthophosphate (NaH2PO4.2H2O); 8.2 mg sodium chloride (NaCl); and water for injection.
US Natl Inst Health; DailyMed. Current Medication Information for VISIONBLUE (trypan blue) injection, solution (March 2006). Available from, as of June 28, 2010: https://dailymed.nlm.nih.gov/dailymed/drugInfo.cfm?id=15897
Each mL of MembraneBlue 0.15% contains: 1.5 mg trypan blue; 1.9 mg sodium mono-hydrogen orthophosphate (Na2HPO4.2H2O); 0.3 mg sodium di-hydrogen orthophosphate (NaH2PO4.2H2O); 8.2 mg sodium chloride (NaCl); and water for injection.
US Natl Inst Health; DailyMed. Current Medication Information for MEMBRANEBLUE (trypan blue) injection, solution (March 2010). Available from, as of June 28, 2010: https://dailymed.nlm.nih.gov/dailymed/drugInfo.cfm?id=15868
HPLC analysis reveals that the product of commerce has a total dye content of approximately 25%. Of this, Trypan Blue constitutes 30-40%, or a net Trypan Blue content of approximately 7-10%. A laboratory grade of the dye, with a net Trypan Blue content in excess of 80%, is available.
Green FJ; The Sigma-Aldrich Handbook of Stains, Dyes, and Indicators. Aldrich Chemical Company, Inc: Milwaukee, WI p. 721 (1990)

11.6 Impurities (Complete)

... Substances identified in various samples /are/ 8-amino-2-[4'-(3,3' -dimethyl-biphenylazo)]-1-naphthol-3,6-disulfonic acid; and 8-amino-2-[4'-(3,3' -dimethyl-4-hydroxybiphenylazo)]-1-napthol-3,6-disulfonic acid; and 8-amino-2 -[4'-(3,3'-dimethyl-4-aminobiphenylazo)]-1-napthol-3,6-disulfonic acid ... and in addition ... can contain varying amt of its starting materials, including ortho-tolidine ... and inorganic salts.
IARC. Monographs on the Evaluation of the Carcinogenic Risk of Chemicals to Humans. Geneva: World Health Organization, International Agency for Research on Cancer, 1972-PRESENT. (Multivolume work). Available at: https://monographs.iarc.fr/ENG/Classification/index.php, p. V8: 267 (1975)

11.7 U.S. Production (Complete)

(1972) PROBABLY GREATER THAN 1.82X10+6 GRAMS
SRI
(1975) PROBABLY GREATER THAN 9.08X10+5 GRAMS
SRI

Production volumes for non-confidential chemicals reported under the Inventory Update Rule.

Year
1986
Production Range (pounds)
No Reports
Year
1990
Production Range (pounds)
10 thousand - 500 thousand
Year
1994
Production Range (pounds)
No Reports
Year
1998
Production Range (pounds)
No Reports
Year
2002
Production Range (pounds)
No Reports

[US EPA; Non-confidential Production Volume Information Submitted by Companies for Chemicals Under the 1986-2002 Inventory Update Rule (IUR). 2,7-Naphthalenedisulfonic acid, 3,3'-[(3,3'-dimethyl[1,1'-biphenyl]-4,4'-diyl)bis(azo)]bis

5-amino-4-hydroxy-, tetrasodium salt (72-57-1). Available from, as of June 7, 2010: https://www.epa.gov/oppt/iur/tools/data/2002-vol.html

11.8 U.S. Imports (Complete)

(1983) 4.00X10+5 g /Direct Blue 14/
USITC. IMPORTS OF BENZENOID CHEM & PROD 1983 p.51

12 Laboratory Methods

12.1 Clinical Laboratory Methods (Complete)

PROCEDURE DESCRIBED FOR RESIDUES IN FECES ASSAYS BASED ON REDUCTION OF THE DYES WITH TIN CHLORIDE FOLLOWED BY GAS CHROMATOGRAPHY OF THE RELEASED FREE AMINE. PURITIES RANGED FROM 26.4-83.4%.
LEVINE RA ET AL; J ANAL TOXICOL 6 (4): 157 (1982)

13 Synonyms and Identifiers

Synonyms

72-57-1

Trypan blue

Amanil sky blue

Amidine blue 4B

Azidinblau 3B

Azidine blue 3B

Azirdinblau 3B

Azzurro Diretto 3B

Bencidal blue 3B

Benzaminblau 3B

Benzamine blue

Benzamine blue 3B

Benzanil blue 3BN

Benzanil blue R

Benzoblau 3B

Benzo blue

Benzo blue 3B

Benzo blue 3BS

Bleu diamine

Bleu directe 3B

Bleue diretto 3B

Bleu trypane N

Blue 3B

Blue EMB

Brasilamina blue 3B

Brasilazina blue 3B

Centraline blue 3B

Chloramiblau 3B

Chloramine blue

Chloramine blue 3B

Chlorazol blue 3B

Chrome leather blue 3B

C.I. Direct blue 14

C.I. Direct blue 14, tetrasodium salt

C.I. 23850

Congoblau 3B

Congo blue

Congo blue 3B

Cresotine blue 3B

Diaminblau 3B

Diamine blue

Diamine blue 3B

Diaminineblue

Dianilblau

Dianilblau H3G

Dianil blue

Dianil blue H3G

Diaphthamine blue TH

Diazine blue 3B

Diazol blue 3B

3,3'-[(3,3'-Dimethyl(1,1'-biphenyl)-4,4'-diyl]bis(azo)bis(4-amino-4-hydroxynaphthalene-2,7-disulphonate)

Diphenyl blue 3B

Directakol blue 3BL

Directblau 3B

Direct Blue 14

Direct blue 3B

Direct blue D3B

Direct blue FFN

Direct blue H3G

Direct blue M3B

Hispamin blue 3BX

2,7-Naphthalenedisulfonic acid, 2-57-13,3'-([13-3'-dimethyl(1,1'-biphenyl)-4,4'-diyl]bis(azo))bis(4-amino-4-hydroxy-, tetrasodium salt

2,7-Naphthalenedisulfonic acid, 3,3'-((3,3'-dimethyl(1,1'-biphenyl)-4,4'-diyl)bis(azo))bis(5-amino-4-hydroxy-, tetrasodium salt

2,7-Naphthalenedisulfonic acid, 3,3'-[(3-3'-dimethyl-4,4'-biphenylylene)bis(azo)]bis(5-amino-4-hydroxy-), tetrasodium salt

Naphthaminblau 3B

Naphthaminblau 3BX

Naphthamine blue 2B

Naphthamine blue 3B

Naphthyl amine blue

Niagara blue

Niagara blue 3B

Orion blue 3B

Paramine blue 3B

Parkibleu

Parkipan

Pontamine blue 3BX

Pyrazol blue 3B

Pyrotrop blau

Renolblau 3B

Sodium ditolyldiazobis-8-amino-1-naphthol-3,6-disulfonate

Sodium ditolyldiazobis-8-amino-1-naphthol-3,6-disulphonate

TB

Tetrasodium 3,3'-[(3,3'-dimethyl[1,1'-biphenyl]-4,4'-diyl)bis(azo)]bis[5-amino-4-hydroxynaphthalene-2,7-disulphonate]

Trianol direct blue 3B

Triazolblau 3BX

Tripan blue

Trypanblau (German)

Trypan blue BPC

Trypan blue sodium salt

Trypane blue

13.1 Substance Title

Trypan blue

13.2 Shipping Name / Number DOT/UN/NA/IMO (Complete)

UN 3077; Environmentally hazardous substances, solid, n.o.s. (Tetrasodium 3,3'-[(3,3'-dimethyl[1,1'- biphenyl]-4,4'-diyl)bis(azo)]bis[5-amino-4-hydroxynaphthalene-2,7-disulphonate])[Sigma-Aldrich Corp; Safety Data Sheet for Trypan blue (Product Number: 302643) Version 3.0 (January 2, 2009). Available from, as of June 15, 2010: http://www.sigmaaldrich.com]

13.3 EPA Hazardous Waste Number

U236; A toxic waste when a discarded commercial chemical product or manufacturing chemical intermediate or an off-specification commercial chemical product or manufacturing chemical intermediate.

14 Administrative Information

14.1 Hazardous Substances DataBank Number

2945

14.2 Last Revision Date

20110503

14.3 Last Review Date

Reviewed by SRP on 9/23/2010

14.4 Update History

Complete Update on 2011-05-03, 50 fields added/edited/deleted

Complete Update on 02/14/2003, 1 field added/edited/deleted.

Complete Update on 11/08/2002, 1 field added/edited/deleted.

Complete Update on 08/06/2002, 1 field added/edited/deleted.

Complete Update on 01/14/2002, 1 field added/edited/deleted.

Complete Update on 08/09/2001, 1 field added/edited/deleted.

Complete Update on 05/15/2001, 1 field added/edited/deleted.

Complete Update on 06/12/2000, 1 field added/edited/deleted.

Complete Update on 02/08/2000, 1 field added/edited/deleted.

Complete Update on 02/02/2000, 1 field added/edited/deleted.

Complete Update on 09/21/1999, 1 field added/edited/deleted.

Complete Update on 08/26/1999, 1 field added/edited/deleted.

Complete Update on 06/02/1998, 1 field added/edited/deleted.

Complete Update on 02/27/1998, 1 field added/edited/deleted.

Complete Update on 10/26/1997, 1 field added/edited/deleted.

Complete Update on 04/23/1997, 1 field added/edited/deleted.

Complete Update on 03/17/1997, 2 fields added/edited/deleted.

Complete Update on 02/20/1997, 35 fields added/edited/deleted.

Field Update on 01/27/1997, 1 field added/edited/deleted.

Field Update on 05/11/1996, 1 field added/edited/deleted.

Complete Update on 01/26/1996, 1 field added/edited/deleted.

Complete Update on 05/08/1995, 1 field added/edited/deleted.

Complete Update on 12/30/1994, 1 field added/edited/deleted.

Complete Update on 03/25/1994, 1 field added/edited/deleted.

Complete Update on 04/27/1993, 1 field added/edited/deleted.

Complete Update on 04/26/1993, 1 field added/edited/deleted.

Complete Update on 02/28/1993, 2 fields added/edited/deleted.

Field update on 12/27/1992, 1 field added/edited/deleted.

Complete Update on 11/05/1992, 2 fields added/edited/deleted.

Complete Update on 09/10/1991, 47 fields added/edited/deleted.

Field Update on 03/01/1989, 1 field added/edited/deleted.

Complete Update on 10/14/1986

Created 19830401 by DS

CONTENTS