Hazardous Substances Data Bank (HSDB) - 1405 - PubChem

10/08/2021 Hazardous Substances Data Bank (HSDB) : 1405 - PubChem
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DATA SOURCES HAZARDOUS SUBSTANCES DATA BANK (HSDB) ANNOTATION RECORD
Methylene blue
Hazardous Substances
1405
DataBank Number
Related CIDs
Related PubChem Records
6099
https://pubchem.ncbi.nlm.nih.gov/source/hsdb/1405#section=Absorption-Distribution-and-Excretion-(Complete) 1/51
1 Human Health Effects
1.1 Toxicity Summary

IDENTIFICATION AND USE: Methylene blue is a solid. Water solutions are deep blue. It is used as a stain in bacteriology, as
mixed indicator, a dye, a redox colorimetric agent, and a targeting agent for melanoma. It is also used as a medication to
treat drug-induced methemoglobinemia. HUMAN STUDIES: Methemoglobinemia is usually treated with methylene blue.
However, in patients with glucose-6-phosphate dehydrogenase deficiency, methylene blue can induce
methemoglobinemia. Preclinical studies have shown that low-dose methylene blue increases mitochondrial cytochrome
oxidase activity in the brain and improves memory retention after learning tasks, including fear extinction. The intracameral
use of 1% methylene blue has a cytotoxic effect on the corneal endothelium and iris epithelium. Several cases of suspected
serotonin syndrome have been reported in patients who received methylene blue in combination with serotonin active
agents. Allergic hypersensitivity reaction to methylene blue-treated plasma transfusion has also been reported. A preterm
infant had methemoglobulinemia and hemolytic anemia after enteral administration of methylene blue. There is
epidemiologic evidence that methylene blue is a teratogen, and the drug can cause fetal harm if administered during
pregnancy. Use of methylene blue in amniocentesis has been associated with atresia of the ileum and jejunum, ileal
occlusion, and other adverse effects in neonates. Use of methylene blue during pregnancy has resulted in hemolytic
anemia, hyperbilirubinemia, methemoglobinemia, respiratory distress, skin staining, and phototoxicity in neonates. ANIMAL
STUDIES: Methylene blue treatment resulted in methemoglobin formation and oxidative damage to red blood cells, leading
to a regenerative anemia and a variety of tissue and biochemical changes secondary to erythrocyte injury. An early change
was a dose-related increase in methemoglobin, where the response of rats and mice was similar in magnitude. Mice
appeared to be more sensitive than rats to the formation of Heinz bodies and the development of anemia that was
characterized by a decrease in hemoglobin, hematocrit, and erythrocyte count. Splenomegaly was apparent in all treated
mice and in the 100 mg/kg (males only) and 200 mg/kg rats at necropsy. Methylene blue was embryotoxic in the rat.
Methylene blue caused the mice to deliver before gestation day 18 (term gestation). This response was observed in 45%,
50% and 83% of animals receiving methylene blue at 50, 60 or 85 mg/kg, respectively. Under cell-free conditions
methylene blue induced DNA damage. It is characterized by a high number of base modifications sensitive to the repair
endonuclease FPG protein (formamidopyrimidine-DNA glycosylase). Methylene blue was mutagenic in cultured
mammalian cells. In contrast, results from the mouse micronucleus assay suggest that the genotoxicity is not expressed in
vivo. The greatest concerns with methylene blue therapy in veterinary use are the development of Heinz body anemia or
other red cell morphological changes, methemoglobinemia, and decreased red cell life spans. Cats tent to be very sensitive
to these effects and some consider it contraindicated in feline patients, but dogs and horses can also develop hematologic
adverse effects at relatively low dosages. ECOTOXICITY STUDIES: Methylene blue had a teratogenic effect in angelfish.
1.2 Evidence for Carcinogenicity (Complete)

Evaluation: No data were available to the Working Group for humans. There is limited evidence for the carcinogenicity of
methylene blue in experimental animals. Overall evaluation: Methylene blue is not classifiable as to its carcinogenicity in
humans (Group 3).
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. V108 179 (2016)
1.3 Human Toxicity Excerpts (Complete)

/HUMAN EXPOSURE STUDIES/ OBJECTIVE: Preclinical studies have shown that low-dose methylene blue increases
mitochondrial cytochrome oxidase activity in the brain and improves memory retention after learning tasks, including fear
extinction. The authors report on the first controlled experiment to examine the memory-enhancing effects of posttraining
methylene blue administration on retention of fear extinction and contextual memory following fear extinction training.
METHOD: Adult participants displaying marked claustrophobic fear were randomly assigned to double-blind administration
of 260 mg of methylene blue (N=23) or administration of placebo (N=19) immediately following six 5-minute extinction
trials in an enclosed chamber. Retesting occurred 1 month later to assess fear renewal as indexed by peak fear during
exposure to a nontraining chamber, with the prediction that the effects of methylene blue would vary as a function of fear
reduction achieved during extinction training. Incidental contextual memory was assessed 1 and 30 days after training to
assess the cognitive-enhancing effects of methylene blue independent of its effects on fear attenuation. RESULTS:
Consistent with predictions, participants displaying low end fear posttraining showed significantly less fear at the 1-month
follow-up if they received methylene blue posttraining compared with placebo. In contrast, participants displaying
moderate to high levels of posttraining fear tended to fare worse at the follow-up if they received methylene blue
posttraining. Methylene blue's enhancement of contextual memory was unrelated to initial or posttraining claustrophobic
fear. CONCLUSIONS: Methylene blue enhances memory and the retention of fear extinction when administered after a
successful exposure session but may have a deleterious effect on extinction when administered after an unsuccessful
exposure session.
PMID:25018057
Full text: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4467026
Telch MJ et al; Am J Psychiatry 171 (10): 1091-8 (2014)
/HUMAN EXPOSURE STUDIES/ Renal blood flow (RBF) and arterial blood pressure (BP) were monitored in 12 patients
undergoing nephrolithotomy in the lateral flexed position. All patients were preoxygenated and were anesthetized with
sodium thiopental, N2O, O2, and fentanyl. Maintenance relaxation was obtained with pancuronium bromide. Arterial
pressure was monitored by percutaneous arterial catheter. Following exposure of the kidney and renal pedicle, an
electromagnetic flow probe was attached to the renal artery and baseline flows recorded. Following baseline measurements,
20 mL of 1 percent methylene blue was given intravenously. All patients studied showed an immediate rise in BP, and 11/12
showed a simultaneous decrease in RBF. The average fall in RBF was 35 percent at one minute. Both parameters returned to
normal values on the average in 177 seconds. The decreased RBF appeared to be part of a generalized vasoconstrition
caused either by sympathetic reflexes or by the direct action of methylene blue.
PMID:788550
Birch AA, Boyce WH; Anesth Analg 55 (5): 674-6 (1976)
/SIGNS AND SYMPTOMS/ Large IV doses of methylene blue may produce nausea, vomiting, abdominal pain, precordial
pain, dizziness, headache, profuse sweating, dyspnea, hypertension, and mental confusion. Urinary tract irritation may occur.
High IV dosage or high local concentrations of methylene blue may cause formation of methemoglobin and cyanosis.
American Society of Health-System Pharmacists; Drug Information 2018. Bethesda, MD. 2018
/CASE REPORTS/ We report the case of a 70-year-old female patient who developed corneal edema and iris discoloration
following the inadvertent use of 1% methylene blue instead of 0.025% trypan blue to stain the anterior capsule during
cataract phacoemulsification surgery. Copious irrigation was performed upon realization of incorrect dye use. Corneal
edema and iris discoloration developed during the early postoperative period and persisted at 24-months follow-up.
However, keratoplasty was not required. The intracameral use of 1% methylene blue has a cytotoxic effect on the corneal
endothelium and iris epithelium. Copious irrigation for at least 30 min using an anterior chamber maintainer may improve
outcomes.
PMID:27224079
Timucin OB et al; Arq Bras Oftalmol 79 (2): 121-2 (2016)
/CASE REPORTS/ OBJECTIVES: Methylene blue commonly is used as a dye or an antidote, but also can be used off label as a
vasopressor. Serotonin toxicity is a potentially lethal and often misdiagnosed condition that can result from drug
interaction. Mild serotonin toxicity previously was reported in settings in which methylene blue was used as a dye. The
authors report 3 cases of life-threatening serotonin toxicity in patients undergoing chronic selective serotonin reuptake
inhibitor (SSRI) therapy who also underwent cardiac surgery and received methylene blue to treat vasoplegic syndrome.
DESIGN: An observational study. SETTING: A cardiothoracic intensive care unit (ICU) in a teaching hospital. PARTICIPANTS:
Three patients who received methylene blue after cardiac surgery, later discovered to be undergoing chronic SSRI therapy.
INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: All 3 patients received high doses of fentanyl during general
anesthesia. They all developed vasoplegic syndrome and consequently were given methylene blue in the ICU. All 3 patients
developed serotonin toxicity, including coma, after this administration and diagnostic tests were negative for acute
intracranial pathology. Coma lasted between 1 and 5 days. Two patients were discharged from the ICU shortly after
awakening, whereas the third patient experienced a complicated postoperative course for concomitant refractory low-
cardiac-output syndrome. CONCLUSIONS: Patients undergoing chronic SSRI therapy should not be administered methylene
blue to treat vasoplegic syndrome.
PMID:26703972
Martino EA et al; J Cardiothorac Vasc Anesth 30 (2): 423-7 (2016)
/CASE REPORTS/ INTRODUCTION: Despite its favorable safety profile, there have been reports of methylene blue-induced
encephalopathy and serotonin syndrome in patients undergoing parathyroidectomy. We report a case of serotonin
syndrome following methylene blue administration in a cardiothoracic surgery patient. CASE REPORT: A 59-year-old woman
taking preoperative venlafaxine and trazodone was given a single dose of 2 mg/kg methylene blue (167 mg) during a
planned coronary artery bypass and mitral valve repair. Postoperatively, she was febrile to 38.7 °C and developed full-body
tremors, rhythmic twitching of the perioral muscles, slow conjugate roving eye movements, and spontaneous movements of
the upper extremities. Electroencephalography revealed generalized diffuse slowing consistent with toxic encephalopathy,
and a computed tomography scan showed no acute process. The patient's symptoms were most consistent with a
methylene blue-induced serotonin syndrome. Her motor symptoms resolved within 48 hours and she was eventually
discharged home. DISCUSSION: Only 2 cases of methylene blue-induced serotonin syndrome during cardiothoracic surgery
have been described in the literature, with this report representing the third case. Methylene blue and its metabolite, azure
B, are potent, reversible inhibitors of monoamine oxidase A which is responsible for serotonin metabolism. Concomitant
administration of methylene blue with serotonin-modulating agents may precipitate serotonin syndrome.
PMID:25613051
Smith CJ et al; J Pharm Pract 28 (2): 207-11 (2015)
/CASE REPORTS/ Methylene blue has been used not only as a diagnostic agent, but also as an agent in the treatment of
ifosfamide-induced encephalopathy (IIE) for several years. Recently, several cases of suspected serotonin syndrome have
been reported in patients who received methylene blue in combination with serotonin active agents. Rodent models have
revealed that methylene blue is a potent, reversible inhibitor of monoamine oxidase A. It is well known that serotonin active
drugs, in combination with monoamine oxidase inhibitors can produce profound serotonin syndrome. To date, cases of
serotonin syndrome, which resulted from concurrent methylene blue and serotonin active agents, have been published in
the anesthesia literature. We report the first known case of serotonin syndrome in a patient receiving methylene blue for
IIE.
PMID:22235061
McDonnell AM et al; J Oncol Pharm Pract 18 (4): 436-9 (2012)
/CASE REPORTS/ Methylene blue-treated fresh-frozen plasma (MB-FFP) is mainly used in Europe. The advantage of the
methylene blue system is that units can be treated individually. The combined action of methylene blue and illumination is
a photodynamic process preventing viral RNA and DNA replication. We report the first immediate allergic hypersensitivity
reaction to methylene blue-treated plasma transfusion. The clinical course and subsequent assessment of the allergic
reaction, including skin tests and basophil activation test, confirmed methylene blue-induced IgE-mediated anaphylaxis. All
immediate reactions after MB-FFP transfusion should be investigated to document the underlying mechanism.
PMID:21310720
Dewachter P et al; Br J Anaesth 106 (5): 687-9 (2011)
/CASE REPORTS/ BACKGROUND: Methylene blue allergy is a well-known entity associated with food or lymph node
location. Inactivation of viruses by methylene blue in fresh-frozen plasma (FFP-MB) has been recently introduced in France
after many years of use in other parts of Europe. CASE REPORT: We describe here two anaphylactic shock reactions
occurring during FFP-MB infusion in patients after cardiac surgery. The follow-up was favorable using epinephrine infusion
in one patient and extracorporeal circulatory assistance in the other. RESULTS: The allergy was suspected based on the
following arguments: chronology of the event, absence of other allergen infused to patients, and an associated symptom
(generalized rash). Methylene blue allergy was documented by prick tests and/or intradermal reactions with methylene
blue and patent blue. Additional allergic tests with basophil activation tests confirm the role of the dye in the anaphylactic
reaction. CONCLUSION: These two cases questioned the safety of FFP-MB.
PMID:20667044
Nubret K et al; Transfusion 51 (1): 125-8 (2011)
/CASE REPORTS/ Acute generalized exanthematous pustulosis is a rare severe pustular cutaneous adverse reaction
characterized by a rapid clinical course with typical histological findings. It is accompanied by fever and acute eruption of
non-follicular pustules overlying erythrodermic skin. The causative agents are most frequently antibacterial drugs. We
present a patient with acute generalized exanthematous pustulosis caused by methylene blue and indigotin dyes.
PMID:22016599
Sener O et al; Korean J Intern Med 26 (3): 360-3 (2011)
/CASE REPORTS/ BACKGROUND: Intraoperative staining of the parathyroid glands with intravenously administered
methylene blue is well described and has been demonstrated as an effective and safe method to facilitate
parathyroidectomy. However, there have been several /cases/ of the development of postoperative neurological toxicity in
patients who received methylene blue infusion during parathyroidectomy. We report the method of methylene blue
infusion during parathyroidectomy at our institution. METHODS: Seven adult patients who had undergone
parathyroidectomy for secondary hyperparathyroidism associated with chronic renal failure were included in this study.
Methylene blue was administered at a constant rate of 4 mg/kg/hr with a 1% solution just before the start of operation. The
infusion was stopped after the first parathyroid gland was identified. RESULTS: The mean dose of methylene blue used was
2.2 +/- 0.8 mg/kg. Consequently, the dose of methylene blue by this method could be decreased to less than half of the
previously administered dose (6 mg/kg) at our institution. CONCLUSIONS: The dose of methylene blue used should be kept
to the minimum required to identify the parathyroid glands in each case.
PMID:25199318
Kadoya T et al; Masui 63 (8): 862-5 (2014)
/CASE REPORTS/ This is the first report of a fatal outcome from serotonin toxicity, precipitated by an interaction between
methylene blue and venlafaxine. Methylene blue-associated serotonin toxicity has been described before but usually as
mild toxicity. Its presentation after general anesthesia may be atypical and therefore more difficult to diagnose. However,
the syndrome is completely preventable if serotonin re-uptake inhibiting agents are stopped beforehand.
PMID:24846936
Top WM et al; Neth J Med 72 (3): 179-81 (2014)
/CASE REPORTS/ A preterm infant had methemoglobulinemia and hemolytic anemia after enteral administration of
methylene blue. The dye was administered to exclude a tracheoesophageal fistula. Methylene blue is a noxious product,
especially in neonates. It should be considered a potential cause of acquired methemoglobulinemia, even after enteral
administration.
PMID:14694406
Allegaert K et al; J Pediatr Surg 39 (1): E35-7 (2004)
/CASE REPORTS/ ... Three premature neonates exposed to methylene blue ... experienced severe hemolytic reactions
requiring exchange transfusions. Two neonates were subsequently diagnosed with G6PD deficiency.
PMID:11048840
Gauthier TW; J Matern Fetal Med 9 (4): 252-4 (2000)
/CASE REPORTS/ A case is presented in which methylene blue dye was injected intraamniotically to confirm rupture of the
membranes. Significant neonatal morbidity occurred as a direct result of the methylene blue dye injection. A syndrome of
toxic side effects is described including hemolytic anemia, hyperbilirubinemia, and methemoglobinemia. The metabolic
process is discussed.
PMID:6823393
McEnerney JK, McEnerney LN; Obstet Gynecol 61 (3 Suppl): 35S-37S (1983)
/CASE REPORTS/ ... Two infants, one a neonate with trisomy 21 exposed to methylene blue as an intraoperative diagnostic
marker and the other a neonate treated with methylene blue for type II glutaric acidemia. ... Within hours after exposure to
methylene blue, the infants voided green-blue urine, followed by hyperbilirubinemia, recurrent anemia requiring
transfusions, and red blood cell dysmorphology, including the appearance of blister cells and Heinz bodies visible in both
Wright's- and supravital-stained peripheral blood smears. After the initiation of phototherapy, both infants exhibited
cutaneous bullae followed by desquamation. ... Significant neonatal morbidity may occur following postpartum
administration of methylene blue. Toxic manifestations include hyperbilirubinemia, Heinz body hemolytic anemia, and
possibly desquamation of the skin.
PMID:8130867
Sills MR, Zinkham WH; Arch Pediatr Adolesc Med 148 (3): 306-10 (1994)
/CASE REPORTS/ A documented severe immunoglobulin E-mediated hypersensitivity reaction associated with use of 1%
methylene blue for detection of tubal permeability occurring during general anesthesia /was reported/. Clinical symptoms,
biological assessment results, and cutaneous test positivity confirmed an anaphylactic reaction to methylene blue. This case
report confirms the need for systematic allergological investigation of all drugs and substances administered during the
perioperative period in the event of a hypersensitivity reaction occurring during anesthesia. ...
PMID:15976222
Dewachter P et al; Anesth Analg 101 (1): 149-50 (2005)
/CASE REPORTS/ The case of a neonate who developed Heinz-body anemia and hyperbilirubinemia following intra-amniotic
injection of less than 50 mg of methylene blue to diagnose premature rupture of fetal membranes is reported. Total serum
level of bilirubin rose to a maximum of 16.1 mg/dL by 58 hr after birth and then gradually decreased over the next 2 wk.
The infant was treated with phototherapy from 2 to 12 days of age. At 3 and 15 days of age, the infant received packed
erythrocyte transfusions for anemia. ...
Vincer MJ et al; Can Med Assoc J 136: 503-4 (1987)
/CASE REPORTS/ Methylene blue is used to check tubal patency during laparoscopy. A case of methemoglobinemia which
was induced by methylene blue is presented. Methemoglobinemia is usually treated with methylene blue; however, in
patients with glucose-6-phosphate dehydrogenase deficiency, methylene blue can induce methemoglobinemia.
PMID:9605379
Bilgin H et al; Acta Anaesthesiol Scand 42 (5): 594-5 (1998)
/CASE REPORTS/ A 65-year-old man underwent parathyroidectomy for hyperparathyroidism secondary to renal failure.
Intra-operatively he received methylene blue infusion (7.5 mg/kg, a total of 650 mg in 500 mL 0.9% sodium chloride) for
visualization of parathyroid glands. At the end of surgery, following extubation he developed agitation, intense shivering
and hyperpyrexia, and his level of consciousness decreased to a Glasgow Coma score of 7. The differential diagnoses
included methylene blue toxicity or malignant hyperpyrexia. His lungs were ventilated, and intravenous dantrolene was
administered to control hyperpyrexia. Hemodialysis was started to remove the methylene blue dye.
PMID:16704594
Mathew S et al; Anaesthesia 61 (6): 580-3 (2006)
/CASE REPORTS/ Methylene blue is utilized as the main treatment of methemoglobinemia conventionally, but it may be
ineffective in individuals with glucose-6-phosphate dehydrogenase (G6PD) deficiency. ... A G6PD-deficient patient /is
reported/ who suffered from aniline-induced methemoglobinemia with initial good response Heinz body but hemolytic
anemia appeared later 3 d after methylene blue therapy. G6PD deficiency was identified. He recovered uneventfully with
hydration, packed blood transfusion and adjuvant luvela-N(dl-alpha-tocopheryl nicotinate) medication. Caution should be
taken in using methylene blue as antidote of acute methemoglobinemia, especially when a history of G6PD deficiency is
obscure.
PMID:11824767
Liao YP et al; Vet Hum Toxicol 44 (1): 19-21 (2002)
/EPIDEMIOLOGY STUDIES/ OBJECTIVES: The purpose of this study was to investigate whether patients who received
methylene blue as treatment for vasoplegia during cardiac surgery with cardiopulmonary bypass had decreased morbidity
and mortality. DESIGN: Retrospective analysis. SETTING: Single tertiary care university hospital. PARTICIPANTS: Adult patients
who suffered from vasoplegia and underwent all types of cardiac surgery with cardiopulmonary bypass at this institution
between 2007 and 2008. INTERVENTIONS: With IRB approval, the authors reviewed the charts of the identified patients and
divided them into 2 groups based on whether they had received methylene blue. Two hundred twenty-six patients were
identified who met the inclusion criteria for the study. Fifty-seven of these patients had received methylene blue for
vasoplegia. The authors collected data on preoperative and intraoperative variables as well as outcomes. MEASUREMENTS
AND MAIN RESULTS: The patients who received methylene blue had higher rates of in-hospital mortality, a compilation of
morbidities, as well as renal failure and hyperbilirubinemia. A multiple logistic regression model demonstrated that receiving
methylene blue was an independent predictor of in-hospital mortality (p value: 0.007, OR 4.26, 95% CI: 1.49-12.12),
compilation of morbidities (p value: 0.001, OR 4.80, 95% CI: 1.85-12.43), and hyperbilirubinemia (p value:<0.001, OR 6.58,
95% CI: 2.91-14.89). Using propensity score matching, the association with morbidity was again seen but the association
with mortality was not found. CONCLUSIONS: The current study identified the use of methylene blue as treatment for
vasoplegia to be independently associated with poor outcomes. While further studies are required, a thorough risk-benefit
analysis should be applied before using methylene blue and, perhaps, it should be relegated to rescue use and not as first-
line therapy.
PMID:23972738
Weiner MM et al; J Cardiothorac Vasc Anesth 27 (6): 1233-8 (2013)
/PREGNANCY AND HUMAN REPRODUCTION/ Because methylene blue exhibits germicidal, oxidation, and reduction
properties, /it was/ asked whether this agent causes adverse effects on gametes, embryos, and/or secretions of the
reproductive tract. Time- and dose-dependent inhibition of human sperm motility by methylene blue was observed, ...
Furthermore, the presence of methylene blue in human uterine, fallopian tube, and peritoneal fluids altered protein mobility
in polyacrylalamide gels, and yielded apparent values of follicle-stimulating hormone and estradiol up to 260% of actual
values (P less than 0.05). ...
PMID:2493404
Coddington CC et al; Fertil Steril 51 (3): 480-5 (1989)
/PREGNANCY AND HUMAN REPRODUCTION/ ... To determine if methylene blue has potential detrimental effects on
ovarian tissue, /the investigators/ compared the effect of methylene blue and indigo carmine on human granulosa luteal
cell functon in vitro. Human oocyte-cumulus complexes were obtained during in vitro fertilization cycles and one to three
were placed in an organ culture dish. After insemination with sperm, oocytes were removed the day after retrieval and the
attached granulosa luteal cell were washed daily for 3 more days by changing 2 mL of culture medium. All the dishes were
treated with human chorionic gonadotropin for the next 24 hr and progesterone production during this interval was taken
as baseline. Test chemicals were added with human chorionic gonadotropin for the next 48 hr with daily media changes. The
P production during the last 24 hr of chemical treatment was expressed as a percentage of the baseline. Methylene blue
significantly reduced P production whereas indigo carmine did not appear to have any effect. Moreover, under inverted
microscopy more than 90% of the granulosa luteal cells contained several sma11 bluish intracellular granules when exposed
to 0.01% methylene blue but not 0.01% indigo carmine. These results indicate that methylene blue may be taken up and
processed by granulosa luteal cells and inhibits P production. ...
PMID:8118115
Mahadevan MM et al; Reprod Toxicol 7 (6): 631-3 (1993)
/PREGNANCY AND HUMAN REPRODUCTION/ Methylene blue is contraindicated in women who are or may become
pregnant. There is epidemiologic evidence that methylene blue is a teratogen, and the drug can cause fetal harm if
administered during pregnancy. Use of methylene blue in amniocentesis has been associated with atresia of the ileum and
jejunum, ileal occlusion, and other adverse effects in neonates. Use of methylene blue during pregnancy has resulted in
hemolytic anemia, hyperbilirubinemia, methemoglobinemia, respiratory distress, skin staining, and phototoxicity in
neonates. If methylene blue is used during pregnancy or if the patient becomes pregnant, the patient should be apprised of
potential fetal hazard.
/ALTERNATIVE and IN VITRO TESTS/ ... The use of methylene blue as a means of suppressing the production of superoxide
radicals O2- by acting as an alternative electron acceptor for xanthine oxidase /is investigated/. Previous work has indicated
that methylene blue accepts electrons from xanthine oxidase at the iron-sulfur center. Initial experiments ... demonstrated
that (pairs of electrons from each enzymatic oxidation are transferred to methylene blue; the reduction of methylene blue
can be achieved by model Iron-sulfur centers, similar to the iron-sulfur center of xanthine oxidase; reduced methylene blue
auto-oxidizes to produce hydrogen peroxide directly, rather than O2-. and methylene blue is effective at non-toxic levels
(2-5 mg/kg) in preventing free radical damage to liver and kidney tissue in an in vitro model of ischemia and reoxygenation.
...
PMID:1650213
Salaris SC et al; Biochem Pharmacol 42 (3): 499-506 (1991)
/ALTERNATIVE and IN VITRO TESTS/ BACKGROUND AND PURPOSE: Methylene blue (MB) is commonly employed as a
treatment for methemoglobinemia, malaria and vasoplegic shock. An increasing number of studies indicate that MB can
cause 5-HT toxicity when administered with a 5-HT reuptake inhibitor. MB is a potent inhibitor of monoamine oxidases, but
other targets that may contribute to MB toxicity have not been identified. Given the role of the 5-HT transporter (SERT) in
the regulation of extracellular 5-HT concentrations, the present study aimed to characterize the effect of MB on SERT.
EXPERIMENTAL APPROACH: Live cell imaging, in conjunction with the fluorescent SERT substrate 4-(4-(dimethylamino)-
styryl)-N-methylpyridinium (ASP(+) ), [(3) H]5-HT uptake and whole-cell patch-clamp techniques were employed to examine
the effects of MB on SERT function. KEY RESULTS: In EM4 cells expressing GFP-tagged human SERT (hSERT), MB
concentration-dependently inhibited ASP(+) accumulation (IC(50) : 1.4 +/- 0.3 uM). A similar effect was observed in N2A
cells. Uptake of [(3) H]5-HT was decreased by MB pretreatment. Furthermore, patch-clamp studies in hSERT expressing cells
indicated that MB significantly inhibited 5-HT-evoked ion currents. Pretreatment with 8-Br-cGMP did not alter the inhibitory
effect of MB on hSERT activity, and intracellular Ca(2+) levels remained unchanged during MB application. Further
experiments revealed that ASP(+) binding to cell surface hSERT was reduced after MB treatment. In whole-cell radioligand
experiments, exposure to MB (10 uM; 10 min) did not alter surface binding of the SERT ligand [(125) I]RTI-55.
CONCLUSIONS AND IMPLICATIONS: MB modulated SERT function and suggested that SERT may be an additional target
upon which MB acts to produce 5-HT toxicity.
PMID:21542830
Oz M et al; Br J Pharmacol 166 (1): 168-76 (2012)
/ALTERNATIVE and IN VITRO TESTS/ Methylene blue, currently in phase 3 clinical trials against Alzheimer Disease,
disaggregates the Tau protein of neurofibrillary tangles by oxidizing specific cysteine residues. Here, we investigated if
methylene blue can inhibit caspases via the oxidation of their active site cysteine. Methylene blue, and derivatives, azure A
and azure B competitively inhibited recombinant Caspase-6 (Casp6), and inhibited Casp6 activity in transfected human
colon carcinoma cells and in serum-deprived primary human neuron cultures. Methylene blue also inhibited recombinant
Casp1 and Casp3. Furthermore, methylene blue inhibited Casp3 activity in an acute mouse model of liver toxicity. Mass
spectrometry confirmed methylene blue and azure B oxidation of the catalytic Cys163 cysteine of Casp6. Together, these
results show a novel inhibitory mechanism of caspases via sulfenation of the active site cysteine. These results indicate that
methylene blue or its derivatives could (1) have an additional effect against Alzheimer Disease by inhibiting brain caspase
activity, (2) be used as a drug to prevent caspase activation in other conditions, and (3) predispose chronically treated
individuals to cancer via the inhibition of caspases.
PMID:26400108
Pakavathkumar P et al; Sci Rep 5: 13730 (2015)
/ALTERNATIVE and IN VITRO TESTS/ Human skin fibroblasts isolated in vitro from subjects carrying the Mediterranean
variant of glucose 6-phosphate dehydrogenase exhibit an 85% decrease of this enzymic activity. There is a 26% and 94%
decr of the hexose monophosphate shunt of the NADPH/NADP ratio, respectively. Incubation with 0.1 mM methylene blue
activates the hexose monophosphate shunt 7 times that of normal fibroblasts and only 2.2 times that of glucose 6-
phosphate-deficient cells. ...
PMID:6331645
Feo F et al; Cancer Res 44 (8): 3419-25 (1984)
/ALTERNATIVE and IN VITRO TESTS/ Methylene blue chloride has been used previously as a supravital stain to facilitate the
excision and subsequent transplantation of mammary epithelial structures by delineating them from surrounding connective
tissue and fat, which stain less intensely. This study was undertaken to determine why methylene blue chloride selectively
stains epithelium and if it has any long-term toxicity to epithelial cells. Light microscopy indicated that methylene blue
chloride was taken up into cell cytoplasm, but neither thin-section nor scanning electron microscopy indicated the
mechanism of uptake or cytoplasmic localization. Spectrophotometry indicated that relative methylene blue chloride
uptake was greatest by macrophage and mammary epithelial cell lines and least by endothelial and fibroblast cell lines,
offering an explanation for the more intense staining of epithelium. Toxicity of methylene blue chloride to the cell lines was
dose dependent and corresponded roughly to the methylene blue chloride uptake capacity of the cell lines. Low
concentrations were stimulatory to growth of some cell lines in serum-free medium. Comparisons of primary epithelial
cultures from 28 pairs of methylene blue chloride-treated and untreated human breast tissues indicated no significant
effect of methylene blue chloride on the growth of cells of any classification (normal, nonmalignant atypical, and
malignant). These results confirm that supravital methylene blue chloride staining of mammary tissues may be used with
confidence that it will not bias the outcome of subsequent experiments requiring healthy, proliferating mammary
epithelium.
PMID:6640545
Buehring GC, Jensen HM; Cancer Res 43 (12): 6039-44 (1983)
/OTHER TOXICITY INFORMATION/ ... In the first part of this study, the in vitro vascular reactivity of methylene blue labeled
saphenous veins was compared with that of veins that were not marked with methylene blue. The vasoactive agents tested
were designed to examine multiple pathways. They included potassium chloride, prostaglandin F2 alpha, phenylephrine,
serotonin, angioteinsin II, BHT-933 (alpha 2-adrenergic agonist), sodium nitroprusside, acetylcholine, isoproterenol, and
verapamil. Compared with unmarked veins, those marked with methylene blue demonstrated a significant impairment of
both vasoconstrictor and vasodilator function. These observations were made on a relatively small number of patients and
could therefore be attributed to inherent differences between patients or surgical procedures. In the second part of this
study, these variables were eliminated by dividing a single vein from one patient into three segments for a 45-minute
exposure to external only methylene blue, internal and external methylene blue, or no methylene blue. The segments were
then evaluated for vasoreactivity in vitro. Externally applied methylene blue reduced vasoconstriction regardless of the
agonist. Further, both endothelium dependent and independent vasodilation was diminished by external methylene blue
exposure. In veins exposed to methylene blue both internally and externally the results were similar but the magnitude of
impairment greater. ...
PMID:7815805
Barber DB et al; J Thorac Cardiovasc Surg 109 (1): 21-9 (1995)
1.4 Populations at Special Risk (Complete)

A preterm infant had methemoglobulinemia and hemolytic anemia after enteral administration of methylene blue. The dye
was administered to exclude a tracheoesophageal fistula. Methylene blue is a noxious product, especially in neonates. It
should be considered a potential cause of acquired methemoglobulinemia, even after enteral administration.
PMID:14694406
Allegaert K et al; J Pediatr Surg 39 (1): E35-7 (2004)
Hemolysis and hemolytic anemia may occur, especially in young infants and patients with glucose-6-phosphate
dehydrogenase (G-6-PD) deficiency.
Methylene blue is utilized as the main treatment of methemoglobinemia conventionally, but it may be ineffective in
individuals with glucose-6-phosphate dehydrogenase (G6PD) deficiency. ... A G6PD-deficient patient /is reported/ who
suffered from aniline-induced methemoglobinemia with initial good response Heinz body but hemolytic anemia appeared
later 3 d after methylene blue therapy. G6PD deficiency was identified. He recovered uneventfully with hydration, packed
blood transfusion and adjuvant luvela-N(dl-alpha-tocopheryl nicotinate) medication. Caution should be taken in using
methylene blue as antidote of acute methemoglobinemia, especially when a history of G6PD deficiency is obscure.
PMID:11824767
Liao YP et al; Vet Hum Toxicol 44 (1): 19-21 (2002)
... Methemoglobinemia is usually treated with methylene blue; however, in patients with glucose-6-phosphate
dehydrogenase deficiency, methylene blue can induce methemoglobinemia.
PMID:9605379
Bilgin H et al; Acta Anaesthesiol Scand 42 (5): 594-5 (1998)
2 Emergency Medical Treatment
2.1 Antidote and Emergency Treatment (Complete)

/SRP:/ 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 if 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. /Poisons A and B/
Currance, P.L. Clements, B., Bronstein, A.C. (Eds).; Emergency Care For Hazardous Materials Exposure. 3rd revised edition, Elsevier Mosby, St.
Louis, MO 2007, p. 160
/SRP:/ 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 needed. Administer oxygen by nonrebreather mask at
10 to 15 L/min. Monitor for pulmonary edema and treat if necessary ... . 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 ... . Cover
skin burns with dry sterile dressings after decontamination ... . /Poisons A and B/
Louis, MO 2007, p. 160
/SRP:/ Advanced treatment: Consider orotracheal or nasotracheal intubation for airway control in the patient who is
unconscious, has severe pulmonary edema, or is in severe respiratory distress. Positive-pressure ventilation techniques with
a bag valve mask device may be beneficial. Consider drug therapy for pulmonary edema ... . Consider administering a beta
agonist such as albuterol for severe bronchospasm ... . Monitor cardiac rhythm and treat arrhythmias as necessary ... . Start
IV administration of D5W TKO /SRP: "To keep open", minimal flow rate/. Use 0.9% saline (NS) or lactated Ringer's (LR) if
signs of hypovolemia are present. For hypotension with signs of hypovolemia, administer fluid cautiously. Watch for signs of
fluid overload ... . Treat seizures with diazepam (Valium) or lorazepam (Ativan) ... . Use proparacaine hydrochloride to assist
eye irrigation ... . /Poisons A and B/
Louis, MO 2007, p. 160-1
/EXPL ANTR/ Treatment of a basic dye, methylene blue, by electrochemical oxidation, fly ash adsorption, and combined
electrochemical oxidation-fly ash adsorption was compared. Methylene blue at 100 mg/L was used in this study. The
toxicity was also monitored by the Vibrio fischeri light inhibition test. When electrochemical oxidation was used, 99% color
and 84% COD were removed from the methylene blue solution in 20 min at a current density of 428 Am(-2), NaCl of 1000
mg/L, and pH(0) of 7. However, the decolorized solution showed high toxicity (100% light inhibition). For fly ash adsorption,
a high dose of fly ash (>20,000 mg/L) was needed to remove methylene blue, and the Freundlich isotherm described the
adsorption behavior well. In the combined electrochemical oxidation-fly ash adsorption treatment, the addition of 4000
mg/L fly ash effectively reduced intermediate toxicity and decreased the COD of the electrochemical oxidation-treated
methylene blue solution. The results indicated that the combined process effectively removed color, COD, and intermediate
toxicity of the methylene blue solution.
PMID:20399000
Wang KS et al; J Environ Manage 91 (8): 1778-84 (2010)
3 Animal Toxicity Studies
3.1 Non-Human Toxicity Excerpts (Complete)

/LABORATORY ANIMALS: Subchronic or Prechronic Exposure/ Methylene blue was administered by gavage to 30 animals
(rats and mice)/sex/dose group in a 0.5% aqueous methylcellulose suspension at doses of 0, 25, 50, 100, and 200 mg/kg.
Blood samples from 10 animals/sex/dose group were collected at the end of study weeks 1, 6, and 13. Methylene blue
treatment resulted in methemoglobin formation and oxidative damage to red blood cells, leading to a regenerative anemia
and a variety of tissue and biochemical changes secondary to erythrocyte injury. An early change was a dose-related
increase in methemoglobin, where the response of rats and mice was similar in magnitude. Mice appeared to be more
sensitive than rats to the formation of Heinz bodies and the development of anemia that was characterized by a decrease in
hemoglobin, hematocrit, and erythrocyte count. Splenomegaly was apparent in all treated mice and in the 100 mg/kg
(males only) and 200 mg/kg rats at necropsy. There was a dose-related increase in absolute and relative spleen weight for
both species. Microscopic examination revealed increased splenic hematopoiesis in all mice treatment groups and in rats at
the 50 mg/kg dose level and above. Splenic congestion and bone marrow hyperplasia were also observed in these rat-dose
groups. Mice at the higher doses showed hematopoiesis in the liver and accumulation of hemosiderin in Kupffer cells. These
gross and microscopic findings are consistent with the development of hemolytic anemia. A dose-related increase in the
reticulocyte count during study weeks 6 and 13 suggested a compensatory response to anemia.
PMID:11752692
Hejtmancik MR et al; Toxicol Sci 65 (1): 126-34 (2002)
/LABORATORY ANIMALS: Developmental or Reproductive Toxicity/ ... Pregnant Riv: TOX rats were treated with methylene
blue on day 16 pc by intraamniotic injection of 5 uL of 1% to 4% in water. The results showed no intestinal atresias at day
20 pc. However, increased incidences of dead conceptuses and of cleft palate and digit malformations were caused by the
operation procedure. In addition high frequencies of dead conceptuses were observed at 3% and 4% methylene blue.
Clinically, 10 mL 1% methylene blue in water is injected in a total amniotic volume of around 200 ml. /In this study/ 5 uL
solution /was injected/ in 0.5 mL total amniotic volume in the rat. Therefore, concentrations in the order of the clinically
used dosage appear embryotoxic in the rat. ...
Piersma AH et al; Reprod Toxicol 6 (2): 185 (1992)
/LABORATORY ANIMALS: Developmental or Reproductive Toxicity/ The vital dye methylene blue has been shown to be
teratogenic when injected into the amnion in the second trimester. On the other hand, the teratogenic potential of
transplacental exposure to methylene blue has not been determined. Methylene blue was administered subcutaneously to
ICR (CD-1) mice at 0, 35, 50, 60, or 70 mg/kg on gestation day 8 (plug day = day 0). Teratological assessments were carried
out at term gestation, on gestation day 18. Since methylene blue inhibits soluble guanylate cyclase enzyme activity,
zaprinast (ZPN), a selective cGMP-phosphodiesterase type V inhibitor, was administered to prevent developmental
disorders initiated by methylene blue at 50 mg/kg. There was a dose-dependent increment of embryolethality. Methylene
blue treatment also produced axial skeleton and neural tube defects. Coadministration of ZPN (20 mg/kg per three times)
abolished completely methylene blue-induced neural tube defects and reduced by one-half the incidence of fetuses
exhibiting axial skeletal defects. ZPN did not provide protection against the embryocidal effects of methylene blue. This
study showed that transplacental exposure to methylene blue is teratogenic in the mouse. Coadministration of ZPN
prevented partly methylene blue-induced teratogenesis, which supports the hypothesis that imbalance of cGMP pathway
accounts, in part, for the teratogenicity of methylene blue.
PMID:11598927
Tiboni GM et al; Teratology 64 (4): 213-20 (2001)
/LABORATORY ANIMALS: Developmental or Reproductive Toxicity/ The present study was undertaken to test whether
methylene blue, a soluble guanylate cyclase inhibitor, can initiate delivery in the mouse. The potential adverse effects of
methylene blue on the fetal growth process were also investigated. ICR (CD-I) mice were injected subcutaneously with
methylene blue at 0 (vehicle), 5, 30, 50, 60 or 85 mg/kg on gestation days 15.5 and 16 (plug day = gestation day 0).
Methylene blue caused the mice to deliver before gestation day 18 (term gestation). This response was observed in 45%,
50% and 83% of animals receiving methylene blue at 50, 60 or 85 mg/kg, respectively (p < 0.05 vs. controls). In a dose-
dependent fashion, methylene blue induced a statistically significant (p < 0.05) derangement of the fetal growth process.
The present study provides the first evidence that exposure to methylene blue during late gestation induces preterm
delivery and fetal growth restriction. ...
PMID:11695226
Tiboni GM et al; In Vivo 15 (4): 333-7 (2001)
/LABORATORY ANIMALS: Neurotoxicity/ ... The aim of the current study was to investigate the potentially neurotoxic effects
of methylene blue using a combination of in vivo and in vitro experimental approaches. Isoflurane-anesthetized adult rats
were used to evaluate the impact of a single bolus intravascular administration of methylene blue on systemic
hemodynamic responses and on the minimum alveolar concentration of isoflurane using the tail clamp test. In vivo,
methylene blue-induced cell death was evaluated 24 hr after methylene blue administration using Fluoro-Jade B staining
and activated caspase-3 immunohistochemistry. In vitro, neurotoxic effects of methylene blue were examined in
hippocampal slice cultures by measuring excitatory field potentials as well as propidium iodide incorporation after
methylene blue exposure. The impact of methylene blue on dendritic arbor was evaluated in differentiated single cell
neuronal cultures. Bolus injections of methylene blue significantly reduced isoflurane minimum alveolar concentration and
initiated widespread neuronal apoptosis. Electrophysiologic recordings in hippocampal slices revealed a rapid suppression
of evoked excitatory field potentials by methylene blue, and this was associated with a dose-dependent effect of this drug
on cell death. Dose-response experiments in single cell neuronal cultures revealed that a 2-hr long exposure to methylene
blue at non-cell-death-inducing concentrations could still induce significant retraction of dendritic arbor. ...
PMID:18362601
Vutskits L et al; Anesthesiology 108 (4): 684-92 (2008)
/GENOTOXICITY/ Acridine orange and methylene blue in the dark were shown to be weak to moderate mutagens
(induction of resistance to T5 phage) in repair-deficient strains of Escherichia coli B/r. Methylene blue and acridine orange
likely act through different mechanisms since methylene blue is only a moderate mutagen in strain WP6 and the other
repair deficient strains tested.
PMID:6379434
Webb RB et al; Mutat Res 137 (1): 1-6 (1984)
/GENOTOXICITY/ The specific recognition of DNA modifications by repair endonucleases was used to characterize the DNA
damage induced by photosensitizers in the presence of visible light. Under cell-free conditions chemically unrelated
photosensitizers (methylene blue, acridine orange, proflavin, riboflavin, hematoporphyrin) induce the same type of DNA
damage. It is characterized by a high number of base modifications sensitive to the repair endonuclease FPG protein
(formamidopyrimidine-DNA glycosylase), while both the number of DNA strand breaks and the number of sites of base
loss (sensitive to exonuclease III or endonuclease IV) is low. Therefore, the damage is markedly different from that induced
by hydroxyl radicals. Mechanistically the generation of the base modifications sensitive to FPG protein involves singlet
oxygen in some but possibly not all cases as substituting D20 for water increases the reaction yield six-fold in the case of
methylene blue but only 1.4-fold in the case of acridine orange. In plasmids from Salmonella typhimurium strains treated
with methylene blue or acridine orange plus light ... the same type of damage was observed as under cell-free conditions. ...
PMID:7683082
Epe B et al; Mutat Res 299 (3-4): 135-45 (1993)
/GENOTOXICITY/ ... Under cell-free conditions, two types of DNA damage profiles are obtained. The profiles induced by
chemically generated singlet oxygen and by various photosensitizers (acridine orange, methylene blue, riboflavin,
hematoporphyrin) plus light are dominated by base modifications sensitive to formamidopyrimidine-DNA glycosylase (FPG
protein), while 5,6-dihydropyrimidines (recognized by endonuclease III), sites of base loss (AP sites, recognized by
endonuclease IV and exonuclease III) and strand breaks are minor lesions. In contrast, the DNA damage profile induced by
hydroxyl radicals (gamma-rays) consists of approx. equal levels of base modifications. AP sites and strand breaks. The
damage profiles induced by Fe(III)-EDTA in the presence of superoxide and by Fe(III)-nitrilotriacetate in the presence of
hydrogen peroxide do not differ from that by hydroxyl radicals. ...
PMID:8383892
Epe B et al; Toxicol Lett 67 (1-3): 57-72 (1993)
/GENOTOXICITY/ ... This study investigates the potential for methylene blue genotoxicity in two mammalian test systems.
Different concentrations of methylene blue were prepared in plasma (heat-treated at 56 degrees C for 1 hour to reduce
cytotoxicity) and used, without illumination, in an in vitro mouse lymphoma cell assay designed to detect forward mutations
in the gene encoding thymidine kinase. The assay was performed in the presence or absence of rat liver S9 microsomal
fraction. Similarly prepared samples of methylene blue in heat-treated plasma were used in an in vivo mouse micronucleus
assay. Each system included a negative vehicle control (heat-treated plasma without methylene blue) and a positive control
consisting of a known genotoxic agent. Intravenous administration to mice of 62 mg/kg of methy1ene blue did not increase
the frequency of micronuclei in polychromatic red cells harvested from bone marrow. However, methy1ene blue
concentrations of 10 ug/mL (with S9 activation) and 30 ug/mL without S9 activation) significantly increased the thymidine
kinase mutation frequency of mouse lymphoma cells to approximately 110 x 10(-6), from a spontaneous frequency of 28 x
10(-6). Methylene blue is mutagenic in cultured mammalian cells. In contrast, results from the mouse micronucleus assay
suggest that the genotoxicity is not expressed in vivo. ...
PMID:7740612
Wagner SJ et al; Transfusion 35 (5): 407-13 (1995)
/ALTERNATIVE and IN VITRO TESTS/ The rate of oxidation of glutathione by direct reaction with methylene blue was
equivalent in normal and glucose 6-phosphate dehydrogenase deficient erythrocytes, supporting a non-NADPH-
dependent mechanism of glutathione oxidation in cells exposed to the dye. These results may explain the clinical finding of
hemolysis in glucose 6-phosphate dehydrogenase deficient patients receiving methylene blue. Although leukomethylene
blue formed from the reaction of the dye with glutathione also reduces methemoglobin the rate is slower than the NADPH
reduction catalyzed by diaphorase II. Conversion of oxyhemoglobin to methemoglobin by methylene blue occurred to the
same extent in normal and glucose 6-phosphate dehydrogenase deficient cells.
Kelner MJ, Alexander NM; Res Commun Chem Pathol Pharmacol 52 (2): 265-8 (1986)
/IMMUNOTOXICITY/ Inducible nitric oxide synthase (iNOS) critically contributes to the development of endotoxin-mediated
inflammation. It can be induced by cytokines or endotoxins via distinct signaling pathways. Lipopolysaccharide (LPS)
triggers iNOS expression through activation of the inhibitor of kappaB-alpha (IkappaB-alpha)-nuclear factor kappaB (NF-
kappaB) cascade, whereas interferon-gamma (IFN-gamma) acts primarily through Janus kinase (JAK)-signal transducer and
activator of transcription 1 (STAT1). Methylene blue (MB), an agent used clinically to treat numerous ailments, has been
shown to reduce NO accumulation through suppression of iNOS activity. But it remains unclear whether MB affects iNOS
induction. This knowledge gap is addressed in the present study using cultured cells and endotoxemic mice. With mouse
macrophages, MB treatment prevented the LPS- and/or IFN-gamma-stimulated iNOS protein expression. Real-time PCR
experiments showed that iNOS mRNA transcription was robustly blocked by MB treatment. The inhibitory effect of MB on
iNOS expression was confirmed in vivo in endotoxemic mice. Further analysis showed that MB had no significant effect on
IkappaB-alpha degradation and NF-kappaB or STAT1 phosphorylation in LPS/IFN-gamma-stimulated cells. The nuclear
transport of active NF-kappaB or STAT1 was also not affected by MB treatment. But MB treatment markedly reduced the
binding of NF-kappaB and STAT1 to their DNA elements. Chromatin immunoprecipitation assays confirmed that MB
reduced NF-kappaB and STAT1 bindings to iNOS promoter inside the cell. These studies show that MB attenuates
transcriptional factor binding amid iNOS mRNA transcription, providing further insight into the molecular mechanism of MB
in disease therapy.
PMID:25736558
Huang C et al; J Cell Biochem 116 (8): 1730-40 (2015)
3.2 Ecotoxicity Excerpts (Complete)

/AQUATIC SPECIES/ Failure to inflate the swim bladder is regarded a major obstacle in the rearing of many fish species. A
study of swim bladder non-inflation (SBN) in angelfish, Pterophyllum scalare. A normal developing primordial swim bladder
was first discernable at the end of the first day post-hatch (p.h.) as a cluster of epithelial cells with a central lumen,
surrounded by presumably mesenchymal cells. Initial inflation occurred on the fourth day p.h. Prior to inflation the swim
bladder epithelium consisted of an outer squamous and inner columnar layer. Cells of the inner layer were filled at their
basal region with an amorphous material, which disappeared upon inflation. A pneumatic duct was absent, and larvae
presented no need to reach the water surface for inflation, suggesting that angelfish are pure physoclists. A model for the
role of the amorphous material in normal initial inflation is proposed. Abnormal swim bladders were apparent from the
fourth day p.h., and methylene blue (MB) at a concentration of 5 ppm significantly increased the prevalence of SBN.
Histologically, abnormal swim bladders in larvae hatched in 5 ppm MB could not be distinguished from those in fish raised
under routine conditions (0.5 ppm MB). ...MB may have a teratogenic effect in angelfish.
PMID:18261035
Perlberg ST et al; J Fish Dis 31 (3): 215-28 (2008)
/AQUATIC SPECIES/ The acute and 7-day subchronic toxicity of methylene blue were determined for fathead minnow larvae
using U.S. Environmental Protection Agency methods. The 96-hr lethal concentration (LC50) was 45 and 15 mg/L at 20 and
25 °C, respectively. The 7-day chronic value for survival under laboratory light and low light was the same (2.1 mg/L).
However, under laboratory light, larval growth was impaired at lower concentrations (chronic value = 0.6 mg/L) than under
low light (chronic value = 2.1 mg/L). To assess prior methylene blue treatment on larval sensitivity, embryos, larvae <24 hr
old, and embryos followed by larvae were treated with 3 mg/L methylene blue. Cadmium LC50 values were similar between
methylene blue-treated and untreated control groups. When larvae treated with methylene blue as embryos were
subchronically exposed to an effluent, survival was similar to that of the untreated group. However, dry weight of the
treated larvae was significantly lower. Differences in dry weight between treated and untreated groups were reduced as the
age of the larvae at test initiation increased. Treatment of incubating embryos using 3 mg/L methylene blue does not
appear to affect larvae sensitivity to cadmium but may affect the growth of larvae used in subchronic testing.
Rifici LM et al; Environ Toxicol Chem 15 (8): 1304-8 (1996)
/OTHER TOXICITY INFORMATION/ Methylene blue, a heterocyclic aromatic chemical compound used to treat fish diseases
in the ornamental fish aquaculture industry, is believed to impair nitrification as a side effect. However, very little is known
about the toxicity of methylene blue to nitrifying micro-organisms. Here, /is reported/ the susceptibility of six bacterial and
one archaeal ammonia-oxidizing micro-organisms to methylene blue within the range of 10 ppb to 10 ppm. Remarkably
high susceptibility was observed in the archaeal species Nitrosopumilus maritimus compared to the bacterial species.
Ammonia oxidation by Nitrosopumilus maritimus was inhibited 65% by 10 ppb of methylene blue. Of the bacterial species
examined, Nitrosococcus oceani was the most resistant to methylene blue toxicity. For similar inhibition of Nitrosococcus
oceani (75% inhibition), one thousand times more methylene blue (10 ppm) was needed. The examination of single cell
viability on Nitrosomonas marina demonstrated that methylene blue is lethal to the cells rather than reducing their single
cell ammonia oxidation activity. The level of susceptibility to methylene blue was related to the cell volume,
intracytoplasmic membrane arrangement and the evolutionary lineage of nitrifying micro-organisms. Our findings are
relevant for effectively using methylene blue in various aquaculture settings by helping minimize its impact on nitrifiers
during the treatment of fish diseases. In the future, resistant nitrifiers such as Nitrosococcus oceani may be purposely added
to aquaculture systems to maintain nitrification activity during treatments with methylene blue. The susceptibility of six
bacterial and one archaeal nitrifying micro-organisms to methylene blue was tested. Remarkably high susceptibility was
observed in the archaeal species compared to the bacterial species. The level of resistance to methylene blue was related to
the cell volume, cytomembrane system and the taxonomic position of the nitrifying micro-organisms. This may be
significant in the design and management of engineered nitrification systems and the stability of the nitrification process in
various ecosystems if these systems are exposed to harmful chemicals or toxins.
PMID:26661751
Sipos AJ, Urakawa H; Lett Appl Microbiol 62 (2): 199-206 (2016)
3.3 National Toxicology Program Studies (Complete)

Groups of 10 male and 10 female core study rats and groups of 10 male and 10 female clinical pathology study rats were
administered methylene blue trihydrate in 0.5% aqueous methylcellulose solution by gavage at doses of 0, 125, 250, 500,
1,000, or 2,000; mg/kg, 5 days per week for 5 weeks. In the 500 mg/kg groups, one male died the first week of the study and
one male and four females died the second week of the study. All rats in the 1,000 mg/kg group died by study day 10, and
all rats in the 2,000 mg/kg group died by study day 6. Final mean body weights of male and female rats in the 250 and 500
mg/kg groups were significantly less than those of the vehicle controls. Dosed rats developed methemoglobinemia and a
regenerative Heinz body anemia. Significant increases in spleen weights occurred in all surviving dosed groups. There were
also significant decreases in the thymus weights of 250 and 500 mg/kg males and 125 and 250 mg/kg females. Spleen
lesions associated with methylene blue trihydrate administration included hematopoietic cell proliferation, pigmentation,
lymphoid depletion of the lymphoid follicles, and capsular fibrosis. Hyperplasia of the bone marrow occurred in all dosed
groups of rats. Liver lesions associated with methylene blue exposure included centrilobular necrosis in rats dying early,
hematopoietic cell proliferation, and Kupffer cell pigmentation with erythrophagocytosis. /Methylene blue trihydrate/
DHHS/NTP; Toxicology and Carcinogenesis Studies of Methylene Blue Trihydrate) in F344/N Rats and B6C3F1 Mice (Gavage Study) (2008)
Technical Rpt Series No. 540 NIH Pub No. 08-4429. Available from, as of September 5, 2018:
https://ntp.niehs.nih.gov/results/pubs/longterm/reports/longterm/tr500580/listedreports/tr540/index.html
Groups of 10 male and 10 female core study mice were administered methylene blue trihydrate in 0.5% aqueous
methylcellulose solution by gavage at doses of 0, 125, 250, 500, 1,000, or 2,000 mg/kg, 5 days per week for 5 weeks. None
of the mice in the 500, 1,000, and 2,000 mg/kg groups survived to the end of the study. In the 250 mg/kg groups, two
females died on days 16 and 18 and two males died on days 6 and 13. Mean body weights of surviving dosed mice were
similar to those of the vehicle controls. Thinness, abnormal respiration, hypothermia, lethargy, ataxia, and ruffled fur were
observed in a few surviving animals in the 250 mg/kg groups. Hypothermia and abnormal posture were observed in mice in
the 500, 1,000, and 2,000 mg/kg groups. Dosed mice developed methemoglobinemia and a regenerative Heinz body
anemia. Significant increases in spleen weights occurred in all surviving dosed groups of mice compared to vehicle controls.
Significant decreases occurred in the thymus weights of 250 mg/kg males and females. The heart weights of 125 and 250
mg/kg females were significantly increased. Lesions in the spleen associated with methylene blue trihydrate administration
included hematopoietic cell proliferation, pigmentation, and congestion. Liver lesions associated with methylene blue
trihydrate administration included periportal degeneration, hematopoietic cell proliferation, and Kupffer cell pigmentation
with erythrophagocytosis. The incidences of bone marrow pigmentation were significantly increased in all dosed groups of
mice. Forestomach lesions that were related to methylene blue trihydrate administration included focal ulcer, inflammation,
and squamous hyperplasia. /Methylene blue trihydrate/
Groups of 10 male and 10 female core study rats and groups of 20 male and 20 female clinical pathology study rats were
administered methylene blue trihydrate in 0.5% aqueous methylcellulose solution by gavage at doses of 0, 25, 50, 100 or
200 mg/kg, 5 days per week for 14 weeks. Mean body weights of males in the 200 mg/kg group were significantly less than
those of the vehicle controls. Dosed rats developed methemoglobinemia and a regenerative Heinz body anemia. Significant
increases in spleen weights occurred in males and females administered 50 mg/kg or greater. Thymus and lung weights of
50, 100, and 200 mg/kg males (except relative lung weight at 100 mg/kg) were significantly less than those of the vehicle
controls. Spleen lesions in dosed rats included hematopoietic cell proliferation, congestion, lymphoid depletion of the
lymphoid follicles, and capsular fibrosis. The incidences of bone marrow hyperplasia were significantly increased in groups
administered 50 mg/kg or greater. There were no consistent effects of methylene blue trihydrate administration on
reproductive system measures in male or female rats. /Methylene blue trihydrate/
Groups of 10 male and 10 female core study mice and groups of 20 male and 20 female clinical pathology study mice were
administered methylene blue trihydrate in 0.5% aqueous methylcellulose solution by gavage at doses of 0, 25, 50, 100, or
200 mg/kg, 5 days per week for 14 weeks. Mean body weights of all dosed groups were similar to or only slightly less than
those of the vehicle control groups. Dosed mice developed methemoglobinemia and a regenerative Heinz body anemia.
Spleen weights of 100 and 200 mg/kg males and 50 mg/kg or greater females were significantly greater than those of the
vehicle control groups. Heart weights were significantly increased in 200 mg/kg males. In females, there were significant
decreases in thymus weights at 50 mg/kg or greater. Males had decreased sperm motility and increased epididymal sperm
counts at 200 mg/kg. In all dosed groups, the incidences of hematopoietic cell proliferation and pigmentation in the spleen
were significantly greater than those in the vehicle controls. In the liver, the incidences of hematopoietic cell proliferation
were significantly increased in males and females in the 100 and 200 mg/kg groups, and the incidences of Kupffer cell
pigmentation were significantly increased in groups administered 50 mg/kg or greater. The incidences of bone marrow
pigmentation were significantly increased in all dosed groups of mice except 25 mg/kg females. /Methylene blue
trihydrate/
Groups of 50 male and 50 female rats were administered methylene blue trihydrate in 0.5% aqueous methylcellulose
solution by gavage at doses of 0, 5, 25, or 50 mg/kg, 5 days per week for 2 years. Additional groups of 10 male and 10
female rats were administered the same doses for up to 18 months and were evaluated at 2 weeks and 3, 12, and 18 months
for hematology. Survival of all dosed groups of rats was similar to that of the vehicle controls. Mean body weights of 25 and
50 mg/kg male rats were less than those of the vehicle controls after weeks 29 and 21, respectively. In the 25 and 50 mg/kg
females, mean body weights were less after weeks 73 and 53. Dosed male and female rats developed methemoglobinemia,
and females developed a regenerative Heinz body anemia. The incidences of pancreatic islet cell adenoma and adenoma or
carcinoma (combined) were increased in all dosed groups of males, were significantly increased in 25 mg/kg males, and
exceeded the historical range in controls (all routes). The incidence of pancreatic islet cell hyperplasia was significantly
increased in the 50 mg/kg males. In the spleen, the incidence of hematopoietic cell proliferation in 50 mg/kg males was
significantly increased; the incidences of capsular fibrosis were significantly increased in all dosed groups of males and in 5
and 50 mg/kg females. /Methylene blue trihydrate/
Groups of 50 male and 50 female mice were administered methylene blue trihydrate in a 0.5% aqueous methylcellulose
solution by gavage at doses of 0, 2.5, 12.5, or 25 mg/kg, 5 days per week for 2 years. Additional groups of 30 male and 30
female mice were administered the same doses for up to 18 months and were evaluated at 2 weeks and 3, 12, or 18 months
for hematology. Survival of dosed male and female groups exceeded that of the vehicle controls in a generally dose-related
manner. Mean body weights of dosed female mice began to increase after weeks 29, 61, and 85, reaching final values that
were 113%, 111%, and 106% of vehicle controls for the 2.5, 12.5, and 25 mg/kg groups, respectively. Dosed mice developed
methemoglobinemia and a regenerative Heinz body anemia. The incidences of carcinoma and of adenoma or carcinoma
(combined) of the small intestine occurred with a positive trend in males. The incidences of malignant lymphoma occurred
with a positive trend in females, and the incidence in 25 mg/kg males exceeded the historical control range. The incidences
of hematopoietic cell proliferation of the spleen were significantly increased in 12.5 and 25 mg/kg males and in 25 mg/kg
females. The incidences of inflammation of the nose were significantly increased in 12.5 and 25 mg/kg females. /Methylene
blue trihydrate/
Methylene blue trihydrate was mutagenic in Salmonella typhimurium strains TA98 and TA100 with and without rat or
hamster liver S9 activation enzymes; mutagenicity was also observed in Escherichia coli strain WP2 uvrA/pKM101 with and
without rat liver S9. In cytogenetic tests with cultured Chinese hamster ovary cells, methylene blue trihydrate induced sister
chromatid exchanges and chromosomal aberrations with and without S9. However, in contrast to the positive results in the
in vitro tests, no increase in the frequency of micronucleated erythrocytes was observed in bone marrow or blood samples
collected from male mice and analyzed 48 hours after a single intraperitoneal injection of methylene blue trihydrate or in
peripheral blood of male and female mice administered methylene blue trihydrate by gavage for 3 months. In the 3-month
micronucleus tests, a dose-related increase in the percentage of reticulocytes among the total erythrocyte population was
observed in both male and female mice. Adjunct studies were conducted with three metabolites of methylene blue
trihydrate: Azure A, Azure B, and Azure C. All three compounds were tested in the Ames assay, and all were positive, with
and without rat liver S9 activation enzymes, in Salmonella typhimurium strains TA98 and TA100 and Escherichia coli strain
WP2 uvrA/pKM101. /Methylene blue trihydrate/
3.4 Non-Human Toxicity Values (Complete)

LD50 Mouse iv 77 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.
485
LD50 Mouse ip 150 mg/kg

485
LD50 Mouse oral 3500 mg/kg

484
LD50 Rat iv 1250 mg/kg

484
LD50 Rat ip 180 mg/kg

484
LD50 Rat oral 1180 mg/kg

484
LD50 Mouse ip 67 mg/kg

Gosselin, R.E., R.P. Smith, H.C. Hodge. Clinical Toxicology of Commercial Products. 5th ed. Baltimore: Williams and Wilkins, 1984., p. II-385
3.5 Ecotoxicity Values (Complete)

LC50; Species: Penaeus californiensis (Shrimp) age 6 months, weight 2.54 g; Conditions: saltwater, static, 27 °C, salinity 24
ppt; Concentration: 100,000 ug/L for 1 hr
Hanks KS; Aquaculture 7: 293-4 (1976) as cited in the ECOTOX database. Available from, as of August 28, 2018
LC50; Species: Heteropneustes fossilis (Indian catfish) adult, weight 22.2 g, length 18.3 cm; Conditions: freshwater, renewal,
18-22 °C; Concentration: 188500 ug/L for 24 hr (95% confidence interval: 184260-192840 ug/L) /formulated product/
Ahmad G, Srivastava G; Sci Environ 5 (1-2): 25-32 (1983) as cited in the ECOTOX database. Available from, as of August 28, 2018
LC50; Species: Ictalurus punctatus (Channel catfish); Conditions: freshwater, static, 17 °C, hardness 42 mg/L CaCO3;
Concentration: 120000 ug/L for 24 hr (95% confidence interval: 110000-131000 ug/L)
Willford WA; USDOI, FWS, Bur Sport Fish Wildl; Invest Fish Control No.18, Resourc Publ No.35: 10 (1966) as cited in the ECOTOX database.
Available from, as of August 28, 2018
LC50; Species: Ictalurus punctatus (Channel catfish); Conditions: freshwater, static, 17 °C, hardness 42 mg/L CaCO3;
LC50; Species: Lepomis macrochirus (Bluegill); Conditions: freshwater, static, 12 °C, hardness 42 mg/L CaCO3; Concentration:
51000 ug/L for 24 hr (95% confidence interval: 40200-64800 ug/L)
LC50; Species: Lepomis macrochirus (Bluegill); Conditions: freshwater, static, 12 °C, hardness 42 mg/L CaCO3; Concentration:
LC50; Species: Morone saxatilis (Striped bass) larvae; Conditions: freshwater, static, 21.1 °C, dissolved oxygen >4 mg/L;
Concentration: 1000 ug/L for 24, 48, 72, 96 hr /formulated product/
Hughes JS; La Dep Wildl Fish 318-43, 2417: 15 (1973) as cited in the ECOTOX database. Available from, as of August 28, 2018
LC50; Species: Morone saxatilis (Striped bass) fingerling; Conditions: freshwater, static, 21.1 °C, dissolved oxygen >4 mg/L;
Concentration: 15000 ug/L for 24 hr /formulated product/
LC50; Species: Morone saxatilis (Striped bass) fingerling; Conditions: freshwater, static, 21.1 °C, dissolved oxygen >4 mg/L;
Concentration: 12000 ug/L for 48, 72, 96 hr /formulated product/
LC50; Species: Mystus vittatus (Striped catfish) adult, weight 12.6 g, length 10.2 cm; Conditions: freshwater, renewal, 18-22
°C; Concentration: 27500 ug/L for 24 hr (95% confidence interval: 24020-31490 ug/L) /formulated product/
Ahmad G, Srivastava G; Sci Environ 5 (1-2): 25-32 (1983) as cited in the ECOTOX database. Available from, as of September 4, 2018
LC50; Species: Oncorhynchus mykiss (Rainbow trout); Conditions: freshwater, static, 12 °C, hardness 42 mg/L CaCO3;
Available from, as of September 4, 2018
LC50; Species: Oncorhynchus mykiss (Rainbow trout); Conditions: freshwater, static, 12 °C, hardness 42 mg/L CaCO3;
LC50; Species: Oryzias latipes (Medaka) length 2 cm, weight 0.2 g; Conditions: freshwater, static, 25 °C; Concentration: 18000
ug/L for 24 hr
Tonogai Y et al; J Toxicol Sci 7 (3): 193-203 (1982) as cited in the ECOTOX database. Available from, as of September 4, 2018
LC50; Species: Oryzias latipes (Medaka, length 2 cm, weight 0.2 g); Conditions: freshwater, static, 25 °C; Concentration: 13000
ug/L for 48 hr
Tonogai Y et al; J Toxicol Sci 7 (3): 193-203 (1982) as cited in the ECOTOX database. Available from, as of September 4, 2018
LC50; Species: Pimephales promelas (Fathead minnow) age 24-48 hr, larvae; Conditions: freshwater, static, 25 °C, hardness
60 mg/L CaCO3, alkalinity 53 mg/L CaCO3, conductivity 140 umhos/cm; Concentration: >50000 ug/L for 24 hr /formulated
product/
Rifici LM et al; Environ Toxicol Chem 15 (8): 1304-8 (1996) as cited in the ECOTOX database. Available from, as of September 4, 2018
60 mg/L CaCO3, alkalinity 53 mg/L CaCO3, conductivity 140 umhos/cm; Concentration: 75000 ug/L for 24 hr (95%
confidence interval: 69000-81000 ug/L) /formulated product/
LC50; Species: Pimephales promelas (Fathead minnow, age 24-48 hr, larvae); Conditions: freshwater, renewal, 25 °C, pH 7.6-
7.9, soft water, conductivity 132-140 umhos/cm, dissolved oxygen 6.9-8.2 mg/L; Concentration: 2800 ug/L for 7 days (95%
confidence interval: 2500-3300 ug/L) /formulated prodcut/
LC50; Species: Salmo trutta (Brown trout); Conditions: freshwater, static, 12 °C, hardness 42 mg/L CaCO3; Concentration:
LC50; Species: Salmo trutta (Brown trout); Conditions: freshwater, static, 12 °C, hardness 42 mg/L CaCO3; Concentration:
LC50; Species: Salvelinus fontinalis (Brook trout); Conditions: freshwater, static, 12 °C, hardness 42 mg/L CaCO3;
LC50; Species: Salvelinus fontinalis (Brook trout); Conditions: freshwater, static, 12 °C, hardness 42 mg/L CaCO3;
LC50; Species: Salvelinus namaycush (Lake trout); Conditions: freshwater, static, 12 °C, hardness 42 mg/L CaCO3;
LC50; Species: Salvelinus namaycush (Lake trout); Conditions: freshwater, static, 12 °C, hardness 42 mg/L CaCO3;
3.6 Ongoing Test Status

The following link will take the user to the National Toxicology Program (NTP) Test Status of Agents Search page, which
tabulates the results and current status of tests such as "Short-Term Toxicity Studies", "Long-term Carcinogenicity Studies",
"Developmental Studies", "Genetic Toxicology Studies", etc., performed with this chemical. Testing status for methylene blue
trihydrate is available. /Methylene blue trihydrate/[Available from, as of February 1, 2019: https://ntpsearch.niehs.nih.gov/?
e=True&ContentType=Testing+Status]
EPA has released the Interactive Chemical Safety for Sustainability (iCSS) Dashboard. The iCSS Dashboard provides an
interactive tool to explore rapid, automated (or in vitro high-throughput) chemical screening data generated by the Toxicity
Forecaster (ToxCast) project and the federal Toxicity Testing in the 21st century (Tox21) collaboration. /The title compound
was tested by ToxCast and/or Tox21 assays/[USEPA; ICSS Dashboard Application; Available from, as of February 4, 2019:
http://actor.epa.gov/dashboard/]
3.7 FIFRA Requirements (Complete)

Residues of the following chemical substances are exempted from the requirement of a tolerance when used in accordance
with good manufacturing practice as ingredients in an antimicrobial pesticide formulation, provided that the substance is
applied on a semi-permanent or permanent food-contact surface (other than being applied on food packaging) with
adequate draining before contact with food. (a) The following chemical substances when used as ingredients in an
antimicrobial pesticide formulation may be applied to: Food-contact surfaces in public eating places, dairy-processing
equipment, and food-processing equipment and utensils. Methylene blue is included on this list. Limit: When ready for use,
the end-use concentration is not to exceed 0.4 ppm.
40 CFR 180.940(a) (USEPA); U.S. National Archives and Records Administration's Electronic Code of Federal Regulations. Available from, as of
August 29, 2018: https://www.ecfr.gov
adequate draining before contact with food. ... (c) The following chemical substances when used as ingredients in an
antimicrobial pesticide formulation may be applied to: Food-processing equipment and utensils. Methylene blue is included
on this list. Limit: When ready for use, the end-use concentration is not to exceed 0.4 ppm.
40 CFR 180.940(c) (USFDA); U.S. National Archives and Records Administration's Electronic Code of Federal Regulations. Available from, as of
4 Metabolism/ Pharmacokinetics
4.1 Metabolism/Metabolites (Complete)

Methylene blue can be reduced to a colorless form, leukomethylene blue; together, these compounds form a reversible
oxidation-reduction system. In low concentrations, methylene blue accelerates conversion of methemoglobin to
hemoglobin. In patients with methemoglobinemia, methylene blue is reduced to leukomethylene blue by methemoglobin
reductases in erythrocytes; leukomethylene blue then reduces methemoglobin to hemoglobin. In high concentrations,
methylene blue oxidizes the ferrous iron of reduced hemoglobin to the ferric state, thereby changing hemoglobin to
methemoglobin.
Following distribution into tissues, methylene blue is rapidly reduced to leukomethylene blue (leucomethylthioninium
chloride). Metabolism to leucomethylene blue may be less efficient in neonates than in older individuals.
4.2 Absorption, Distribution and Excretion (Complete)

... The concentration of methylene blue in whole blood was measured using high-performance liquid chromatography in
seven volunteers after IV and oral administration of 100 mg methylene blue with and without mesna. The distribution of
methylene blue in different tissues was measured in rats after intraduodenal and IV application. The time course of
methylene blue in whole blood after IV administration showed a multiphasic time course with an estimated terminal half-
life of 5.25 hr. Following oral administration, the area under the concentration-time curve was much lower (9 nmol/min/mL
vs 137 nmol/min/mL). Co-administration of mesna, which could influence distribution by ion-pairing, did not alter the
pharmacokinetics. The urinary excretion of methylene blue and its leukoform was only moderately higher after IV
administration (18% vs 28% dose). Intraduodenal administration to rats resulted in higher concentrations in intestinal wall
and liver but lower concentrations in whole blood and brain than IV methylene blue. Differences in organ distribution of
methylene blue are mainly responsible for the different pharmacokinetics after oral and IV administration. ...
PMID:10952480
Peter C et al; Eur J Clin Pharmacol 56 (3): 247-50 (2000)
Methylene blue is well absorbed from the GI tract, and peak plasma concentrations occur approximately 1-2 hours after an
oral dose. ... Following distribution into tissues, methylene blue is rapidly reduced to leukomethylene blue
(leucomethylthioninium chloride). Metabolism to leucomethylene blue may be less efficient in neonates than in older
individuals. Methylene blue is excreted in urine and bile. About 75% of an oral dose of methylene blue is excreted in urine,
mostly as stabilized colorless leukomethylene blue. On exposure to air, the urine turns green or blue, due to the presence
of the oxidation product methylene azure (methylene blue sulfone). Some unchanged drug is also excreted in urine.
BACKGROUND: Although blue dye is routinely used for lymphatic mapping, it is not used for lymphatic mapping in
pregnancy-associated breast cancer, because of concern of fetal risk. METHODS: To investigate the safety of blue dye for
lymphatic mapping in pregnant women, the pharmacokinetics of methylene blue dye were examined in 10 nonpregnant
women, and the results were extrapolated to estimate maximal fetal exposure to the dye. RESULTS: Plasma and urine
measurements indicated that the dye quickly distributed from the breast injection site to the circulation, with 32% of the
total dose excreted in urine within 48 hours. Combined with existing data on organ distribution of methylene blue, the
estimated maximal dose to the fetus is 0.25 mg (5% of the administered dose), likely further reduced by other physiologic
factors related to pregnancy. CONCLUSIONS: The analysis suggests that methylene blue dye can be used for lymphatic
mapping in pregnancy-associated breast cancer with minimal fetal risk.
PMID:21167367
Pruthi S et al; Am J Surg 201 (1): 70-5 (2011)
The disposition and urinary excretion pharmacokinetics of methylene blue were determined after its intravenous
administration at 15 mg/kg to mature female sheep. Comparisons were made between methylene blue administered alone
or subsequent to 50 mg/kg sodium nitrite. The overall elimination rate constant (beta) of methylene blue, 0.0076 +/-
0.0016 min-1, was not influenced by prior administration of sodium nitrite. However, the distribution rate was significantly
altered by sodium nitrite. Very little of the methylene blue was eliminated in the urine either intact or as leukomethylene
blue in spite of its relatively short half life. ...
PMID:6492250
Burrows GE; J Vet Pharmacol Ther 7 (3): 225-31(1984)
4.3 Biological Half-Life (Complete)

Following IV administration, the estimated half-life of methylene blue is 5-6.5 hours.
... The concentration of methylene blue in whole blood was measured using high-performance liquid chromatography in
seven volunteers after IV and oral administration of 100 mg methylene blue with and without mesna. The distribution of
methylene blue in different tissues was measured in rats after intraduodenal and IV application. The time course of
methylene blue in whole blood after IV administration showed a multiphasic time course with an estimated terminal half-
life of 5.25 hr. ...
PMID:10952480
Peter C et al; Eur J Clin Pharmacol 56 (3): 247-50 (2000)
4.4 Mechanism of Action (Complete)

The mechanism of modulation of cyclic guanosine monophosphate accumulation by methylene blue, a putative inhibitor of
soluble guanylate cyclase, was investigated in cultured rabbit pulmonary arterial smooth muscle cells. Control or methylene
blue pretreated rabbit pulmonary arterial smooth muscle were stimulated with sodium nitroprusside, nitrosothiols or
endothelium derived relaxing factor released basally from bovine pulmonary arterial endothelial cells, in short term
cocultures. The putative endothelium-derived relaxing factor, S-nitroso-L-cysteine, a stab1e deaminated analog of S-
nitroso-L-cysteine, S-nitroso-3-mercaptoproprionic acid and sodium nitroprusside produced concentration-dependent (1-
100 uM) increase (1.5- to 12-fold) in rabbit pulmonary arterial smooth muscle cells cyclic guanosine monophospate levels.
Methylene blue pretreatment inhibited S-nitroso-L-cysteine and sodium nitroprusside induced cyclic guanosine
monophosphate accumulation by 51% to 100%, but S-nitroso-3-mercaptoproprionic acid mediated responses were not
altered by methylene blue. The inhibition profile of methylene blue on nitrovasodilator induced cyclic guanosine
monophosphate accumulation was quantitatively reproduced by extracellular generation of superoxide anion with xanthine
(100 uM) and xanthine oxidase (5 mU). Similarly to methylene blue pretreatment, superoxide anion generation had no
effects on base-line cyclic guanosine phosphate levels or cyclic guanosine phosphate responses elicited by S-nitroso-3-
mercaptoproprionic acid. Furthermore, methylene blue induced a dose and time dependent generation of superoxide
anion from rabbit pulmonary arterial smooth muscle cells, as evidenced from spectrophotometric determination of
cytochrome c reduction. Inhibition of cyclic guanosine monophosphate accumulation in response to S-nitroso-L-cysteine
and sodium nitroprusside by methylene blue was completely prevented by superoxide dismutase but not catalase.
Selective pretreatment of endothelial cells with methylene blue before co-culture with untreated rabbit pulmonary arterial
smooth muscle produced a reduction in rabbit pulmonary arterial smooth muscle cyclic guanosine monophosphate levels of
a magnitude comparable with that seen in cocultures of methylene blue pretreated rabbit pulmonary arterial smooth
muscle with untreated endothelial cells, and which was partially prevented by superoxide dismutase.
PMID:1328604
Marczin N et al; J Pharmacol Exp Ther 263 (1): 170-9 (1992)
5 Pharmacology
5.1 Therapeutic Uses (Complete)

Enzyme Inhibitors
National Library of Medicine's Medical Subject Headings. Methylene Blue. Online file (MeSH, 2018). Available from, as of August 29, 2018:
https://meshb.nlm.nih.gov/search
/CLINICAL TRIALS/ ClinicalTrials.gov is a registry and results database of publicly and privately supported clinical studies of
human participants conducted around the world. The Web site is maintained by the National Library of Medicine (NLM) and
the National Institutes of Health (NIH). Each ClinicalTrials.gov record presents summary information about a study protocol
and includes the following: Disease or condition; Intervention (for example, the medical product, behavior, or procedure
being studied); Title, description, and design of the study; Requirements for participation (eligibility criteria); Locations where
the study is being conducted; Contact information for the study locations; and Links to relevant information on other health
Web sites, such as NLM's MedlinePlus for patient health information and PubMed for citations and abstracts for scholarly
articles in the field of medicine. Methylene blue is included in the database.
NIH/NLM; ClinicalTrials.Gov. Available from, as of August 29, 2018: https://clinicaltrials.gov/
Drug-induced methemoglobinemia. /Included in US product label/

NIH; DailyMed. Current Medication Information for Methylene blue injection (Updated: November 20, 2017). Available from, as of September 5,
2018: https://dailymed.nlm.nih.gov/dailymed/drugInfo.cfm?setid=fde64824-2be5-4d85-8d57-5098ca6890bb
Methylene blue posses weak urinary antiseptic properties. Methylene blue directly inhibits calcium binding by oxalate and
by organic stone matrix. The drug also acts as a crystal poison at the interface, reducing the tendency of calcium oxalate
particles to aggregate. In addition, it reverses intracellular acidosis (such as that in renal tubule acidosis), apparently by
competing with diphosphopyridine nucleotide as a hydrogen receptor.
Methylene blue has been used in the management of ifosfamide-induced encephalopathy. Although methylene blue has
resulted in rapid improvement in CNS function in some patients, beneficial effects have not been consistently reported and
further clinical studies are needed to establish the role of the drug in the management of ifosfamide-induced
encephalopathy. Methylene blue does not appear to be effective when used prophylactically in an attempt to prevent
ifosfamide-associated encephalopathy. /NOT included in US product label/
Methylene blue is used as a bacteriologic stain, as an indicator dye, and for surgical and medical marking. Methylene blue
has been administered by local injection or instillation as a diagnostic (visualizing) dye in a variety of procedures, including
sentinel lymph node biopsy in cancer patients (e.g., breast cancer patients), endoscopic evaluation of lesions in patients with
gastroesophageal reflux disease (GERD) or Barrett's esophagus, urologic evaluation in patients with ureteral or renal pelvis
injury, and thoroscopic procedures in patients with pulmonary nodules. /NOT included in US product label/
Although methylene blue was used in the past as an antidote for cyanide poisoning, it is no longer recommended for this
use. Cyanide poisoning usually is treated with an antidote regimen consisting of amyl nitrite, sodium nitrite, and sodium
thiosulfate or with hydroxocobalamin. When sodium nitrite is used for cyanide poisoning, methylene blue should not be
used in an attempt to treat excessive methemoglobinemia induced by the antidote because reduced cyanide binding and
increased toxicity occurs. Methylene blue is of no value in the treatment of carbon monoxide poisoning. /NOT included in
US product label/
Although further study is needed to evaluate safety and efficacy, methylene blue has been used topically as a
photosensitizer for photodynamic therapy (PDT) in the topical treatment of dermatologic or mucocutaneous infections (e.g.,
herpes labialis, eczema herpeticum, oral candidiasis, cutaneous leishmaniasis, chromoblastomycosis) or chronic
dermatologic or mucocutaneous conditions (e.g., plaque psoriasis, oral lichen planus). /NOT included in US product label/
Methylene blue was used in the past as a mild urinary antiseptic and stimulant to mucous surfaces in the treatment of
cystitis and urethritis, but is no longer recommended for this use. /NOT included in US product label/
Methylene blue has been used alone and in combination with ascorbic acid for the management of chronic urolithiasis.
Although methylene blue appears to have some inhibitory effect on the formation of calcium oxalate and calcium
phosphate crystals in some patients, the drug is not currently recommended for this use and is ineffective in dissolving
previously formed stones. /NOT included in US product label/
OBJECTIVE: To assess the efficacy of methylene blue (MB) monotherapy in semi-immune adults with uncomplicated malaria
in Burkina Faso. METHODS: In an open-label controlled phase II study with 60 semi-immune adults with uncomplicated
falciparum malaria in Nouna, north-western Burkina Faso, MB monotherapy (390 mg twice daily) was given sequentially to
groups of 20 adults for 7 days (MB7), 5 days (MB5) and 3 days (MB3), respectively. The primary outcome was the rate of
adequate clinical and parasitological response (ACPR) on day 28 of follow-up. RESULTS: Of the study population, 27/58
(47%) and 5/51 (10%) patients still had parasites on days 2 and 3, respectively, of follow-up resulting in 9/58 (16%) early
treatment failures. By day 14, no recrudescence was observed but in 4/19 (MB5) and 2/20 (MB3) individuals by day 28. The
PCR-corrected rate of ACPR was 72%, 58% and 85% in groups 7, 5 and 3, respectively, by per protocol analysis. Self-limiting
dysuria was the most frequent adverse event. CONCLUSIONS: MB acts slowly against the blood stages of P. falciparum. MB
alone needs to be given for at least 7 days to be efficacious in the treatment of falciparum malaria but should be used in
combination with a fast acting antimalarial.
PMID:20374561
Bountogo Met al; Trop Med Int Health 15 (6): 713-7 (2010)
VET: Intra-operative methylene blue can be used to preferentially stain islet-cell tumors of the pancreas in dogs in order to
aid in their surgical removal or in determining the animal's prognosis. However, IV methylene blue is rarely used for this
purpose due to its adverse effects and delayed effect colorizing pancreatic cells.
Plumb D.C. Veterinary Drug Handbook. 8th ed. (pocket). Ames, IA: Wiley-Blackwell, 2015., p. 955
VET: In dogs, methylene blue may be considered for treating methemoglobinemia secondary to drugs or toxins (eg, phenol,
mothballs, hydroxyurea, phenazopyridine).
VET: Methylene blue is used primarily for treating methemoglobinemia secondary to oxidative agent (nitrates, chlorates) in
ruminants. It is also employed occasionally a adjunctive or alternative therapy for cyanide toxicity.
/EXPL THER/ Sirtuins maintain energy balance. Particularly, sirtuin 1 (SIRT1) activation mimics calorie restriction and nutrient
utilization. However, no medications are available for the up-regulation of SIRT1. Methylene blue (MB) had been in clinical
trials for the treatment of neurological diseases. This study investigated the effect of MB on sirtuin expression in association
with the treatment of steatosis and steatohepatitis, and explored the underlying basis. The effects of MB on mitochondrial
function, molecular markers, pharmacokinetics, and histopathology were assessed using hepatocyte and/or mouse models.
Immunoblotting, PCR and reporter assays were done for molecular experiments. After oral administration, MB was well
distributed in the liver. MB treatment increased NAD(+)/NADH ratio in hepatocytes. Of the major forms, MB treatment up-
regulated SIRT1, and thereby decreased PGC-1alpha acetylation. Consistently, hepatic mitochondrial DNA contents and
oxygen consumption rates were enhanced. MB treatment also notably activated AMPK, CPT-1 and PPARalpha: the AMPK
activation relied on SIRT1. Activation of LXRalpha and the induction of SREBP-1c and its target genes by T0901317 were
diminished by MB. In addition, MB treatment antagonized the ability of palmitate to acetylate PGC-1alpha, and increase
SERBP-1c, FAS, and ACC levels. In mice fed on a high-fat diet for 8 weeks, MB treatment inhibited excessive hepatic fat
accumulation and steatohepatitis. The ability of MB to activate SIRT1 promotes mitochondrial biogenesis and oxygen
consumption and activates AMPK, contributing to anti-lipogenesis in the liver. Our results provide new information on the
potential use of MB for the treatment of steatosis and steatohepatitis.
PMID:24486702
Shin SY et al; Eur J Pharmacol 727: 115-24 (2014)
/EXPL THER/ BACKGROUND: Low back pain (LBP) is a common health problem and a substantial part of LBP is presumed to
be attributable to degeneration of the intervertebral disc. For patients suffering from intractable discogenic LBP, there are
few evidence-based effective interventional treatment options available. In 2010, the results of a randomized controlled trial
(RCT) were published concerning "intradiscal methylene blue injection" (IMBI), in which this intervention appeared to be
very successful in relieving discogenic pain. Therefore, we decided to repeat this study to investigate whether we could
replicate the published results. The results of our preliminary feasibility study gave reason to set up an RCT. The aim of this
RCT is to evaluate if IMBI is a more effective treatment of discogenic low back pain as an intradiscal placebo intervention,
and furthermore, to assess the cost-effectiveness of this intervention. METHODS/DESIGN: Consecutive discogenic low back
pain patients referred to four specialized pain treatment facilities are being screened for eligibility. After a positive
standardized provocation discography and informed consent, patients are randomized into two groups. The treatment
group receives an intradiscal injection with methylene blue, lidocaine, and contrast, and the control group receives
intradiscal isotonic saline with lidocaine and contrast. Main outcome measures are pain at the 6-month follow-up, patient's
global impression of change, cost-effectiveness, quality of life, disability, and analgesic intake. DISCUSSION: The importance
of this study is emphasized by the fact that for intractable discogenic low back pain patients, evidence-based effective pain
treatments are rare. If this study establishes clinical success and cost-effectiveness, IMBI could become the "pain treatment
of choice" for a selected group of patients with chronic discogenic low back pain for whom noninvasive treatment options
have failed.
PMID:26590962
Geurts JW et al; Trials 16: 532 (2015)
/EXPL THER/ PURPOSE: Glaucoma is a progressive optic neuropathy characterized by loss of retinal ganglion cells (RGCs)
and optic nerve degradation. Existing treatments focus on lowering intraocular pressure (IOP); however, vision loss may still
progress. Neuroprotective drugs may be useful as an adjunct approach to prevent further loss of RGCs, although efficacious
drugs are lacking. One agent, methylene blue, protects neurons during several neurodegenerative models. Methylene blue
potentiates the electron transport chain by shuttling elections from NADH and FADH2 to coenzyme Q (CoQ) and
cytochrome c. The purpose of this study was to determine if methylene blue could protect RGCs from noxious stimuli.
METHODS: Primary rat RGCs were isolated and cultured following a sequential immunopanning technique using P3-P7
Sprague-Dawley rats. Approximately 25,000 RGCs were seeded per coverslip and cultured for 3 days before testing. The
RGCs were treated for 24 hours with rotenone or staurosporine or for 72 hours of hypoxia. Methylene blue was then
assessed for protection of RGCs during each of these insults. Cell viability was measured using calcein Am and ethidium
homodimer-1. Cytochrome c oxidase activity was measured using a cytochrome c oxidase assay kit to monitor the health of
mitochondria. RESULTS: Methylene blue (1 uM and 10 uM) significantly protected RGCs against 24 hours of 1 uM rotenone.
Methylene blue (1 uM and 10 uM) significantly protected RGCs against 24 hours of treatment with 1 uM staurosporine and
protected RGCs against 72 hours of hypoxia. Methylene blue increased cytochrome c oxidase activity in the presence of
hydrogen peroxide. CONCLUSIONS: Methylene blue is a neuroprotective compound that can protect RGCs from toxic
insults. Methylene blue's ability to increase cytochrome c oxidase and protect RGCs against these noxious stimuli supports
its suggested mechanism of action, which is to preserve the electron transport chain. Further testing is needed to determine
if methylene blue would be an efficacious treatment for the protection of neurodegeneration that occurs during optic
neuropathy.
PMID:22661471
Daudt DR 3rd et al; Invest Ophthalmol Vis Sci 53 (8): 4657-67 (2012)
/EXPL THER/ STUDY OBJECTIVE: Calcium channel blocker poisonings account for a substantial number of reported deaths
from cardiovascular drugs. Although supportive care is the mainstay of treatment, experimental therapies such as high-dose
insulin-euglycemia and lipid emulsion have been studied in animal models and used in humans. In the most severe cases,
even aggressive care is inadequate and deaths occur. In both experimental models and clinical cases of vasodilatory shock,
methylene blue improves hemodynamic measures. It acts as a nitric oxide scavenger and inhibits guanylate cyclase that is
responsible for the production of cyclic guanosine monophosphate (cGMP). Excessive cGMP production is associated with
refractory vasodilatory shock in sepsis and anaphylaxis. The aim of this study is to determine the efficacy of methylene blue
in an animal model of amlodipine-induced shock. METHODS: Sprague-Dawley rats were anesthetized, ventilated, and
instrumented for continuous blood pressure and pulse rate monitoring. The dose of amlodipine that produced death within
60 minutes was 17 mg/kg per hour (LD50). Rats were divided into 2 groups: amlodipine followed by methylene blue or
amlodipine followed by normal saline solution, with 15 rats in each group. Rats received methylene blue at 2 mg/kg during
5 minutes or an equivalent amount of normal saline solution in 3 intervals from the start of the protocol: minutes 5, 30, and
60. The animals were observed for a total of 2 hours after the start of the protocol. Mortality risk and survival time were
analyzed with Fisher's exact test and Kaplan-Meier survival analysis with the log rank test. RESULTS: Overall, 1 of 15 rats (7%)
in the saline solution-treated group survived to 120 minutes compared with 5 of 15 (33%) in the methylene blue-treated
group (difference -26%; 95% confidence interval [CI] -54% to 0.3%). The median survival time for the normal saline solution
group was 42 minutes (95% CI 28.1 to 55.9 minutes); for the methylene blue group, 109 minutes (95% CI 93.9 to 124.1
minutes). Pulse rate and mean arterial pressure (MAP) differences between groups were analyzed until 60 minutes. Pulse
rate was significantly higher in the methylene blue-treated group beginning 25 minutes after the start of the amlodipine
infusion (95% CI 30 to 113 minutes) that was analyzed until 60 minutes. MAP was significantly higher in the methylene
blue-treated group starting 25 minutes after the amlodipine infusion (95% CI 2 to 30 minutes) that was analyzed until 60
minutes. CONCLUSION: Methylene blue did not result in a significant difference in mortality risk. There was an increased
pulse rate, MAP, and median survival time in the methylene blue group.
PMID:25441767
Jang DH et al; Ann Emerg Med 65 (4): 410-5 (2015)
/EXPL THER/ INTRODUCTION: The purpose of this study is to describe a case where methylene blue improved
hemodynamics in a poisoned patient. CASE REPORT: This is a single case report where a poisoned patient developed
vasodilatory shock following ingestion of atenolol, amlodipine, and valsartan. Shock persisted after multiple therapies
including vasopressors, high-dose insulin, hemodialysis, and 20% intravenous fat emulsion. Methylene blue (2 mg/kg IV
over 30 min) was administered in the ICU with temporal improvement as measured by pulmonary artery catheter
hemodynamic data pre- and post-methylene blue administration. Within 1 hr of methylene blue administration, systemic
vascular resistance improved (240 dyn s/cm5 increased to 1204 dyn s/cm5), and vasopressor requirements decreased with
maintenance of mean arterial pressure 60 mmHg. DISCUSSION: Methylene blue may improve hemodynamics in drug-
induced vasodilatory shock and should be considered in critically ill patients poisoned with vasodilatory medications
refractory to standard therapies.
PMID:26310944
Laes JR et al; J Med Toxicol 11 (4): 460-3 (2015)
/EXPL THER/ OBJECTIVE: To examine if methylene blue (MB) can counteract or prevent protamine (P) cardiovascular effects.
METHODS: The protocol included five heparinized pig groups: Group Sham - without any drug; Group MB - MB 3 mg/kg
infusion; Group P - protamine; Group P/MB - MB after protamine; Group MB/P - MB before protamine. Nitric oxide levels
were obtained by the nitric oxide/ozone chemiluminescence method, performed using the Nitric Oxide Analizer 280i
(Sievers, Boulder, CO, USA). Malondialdehyde plasma levels were estimated using the thiobarbiturate technique. RESULTS:
1) Groups Sham and MB presented unchanged parameters; 2) Group P - a) Intravenous protamine infusion caused mean
arterial pressure decrease and recovery trend after 25-30 minutes, b) Cardiac output decreased and remained stable until
the end of protamine injection, and c) Sustained systemic vascular resistance increased until the end of protamine injection;
3) Methylene blueinfusion after protamine (Group P/MB) - a) Marked mean arterial pressure decreased after protamine, but
recovery after methylene blue injection, b) Cardiac output decreased after protamine infusion, recovering after methylene
blue infusion, and c) Sustained systemic vascular resistance increased after protamine infusion and methylene blue
injections; 4) Methylene blue infusion before protamine (Group MB/P) - a) Mean arterial pressure decrease was less severe
with rapid recovery, b) After methylene blue, there was a progressive cardiac output increase up to protamine injection,
when cardiac output decreased, and c) Sustained systemic vascular resistance decreased after protamine, followed by
immediate Sustained systemic vascular resistance increase; 5) Plasma nitrite/nitrate and malondialdehyde values did not
differ among the experimental groups. CONCLUSION: Reviewing these experimental results and our clinical experience, we
suggest methylene blue safely prevents and treats hemodynamic protamine complications, from the endothelium function
point of view.
PMID:27737405
Albuquerque AA et al; Braz J Cardiovasc Surg 31 (3): 226-231 (2016)
/EXPL THER/ Protamine sulfate is used during coronary artery bypass graft surgery to reverse the anticoagulating effects of
heparin. Vasoplegic syndrome is a state of endothelial dysregulation that produces profound vasodilatation that is
refractory to vasopressors. This syndrome leads to systemic hypoperfusion and may progress to death. Up to 27% of
patients after cardiac bypass may experience vasoplegia. Symptoms of vasoplegia may also be present in many different
clinical settings. This case report describes a 57-year-old woman who after cardiac bypass experienced a severe protamine
reaction with profound hypotension, which was unresponsive to volume resuscitation and vasopressor therapy. A dramatic
increase in blood pressure resulted almost immediately after administration of methylene blue. This patient had no prior
risk factors for a protamine reaction other than her current cardiac surgery. A review of the pathophysiologic characteristics
associated with vasoplegia and the pharmacodynamics of methylene blue will potentially enable anesthesia providers to
utilize this lifesaving drug when needed.
PMID:22848976
Lutjen DL, Arndt KL; AANA J 80 (3): 170-3 (2012)
/EXPL THER/ BACKGROUND: Huntington's disease (HD) is a Polyglutamine disease caused by the presence of cytosine-
adenine-guanine (CAG) repeats in the first exon of Huntingtin (Htt), a large protein with multiple functions. In addition to
neurodegeneration of specific brain regions, notably the striatum, HD also shows alterations in peripheral tissues, such as
the heart, skeletal muscles or peripheral endocrine glands. Mutant Huntingtin (mHtt)-driven mitochondrial impairment may
underlie some of the CNS and peripheral tissues dysfunctions, especially in tissues with high energy demand such as the
heart. OBJECTIVE: The aim of this study is to characterize two new inducible Drosophila HD heart models and to assay the
therapeutic potential of methylene blue in these HD models. METHODS: We report the construction of inducible Drosophila
HD heart models, expressing two Nter fragments of the protein encompassing either exon 1 or the first 171 amino acids and
the characterization of heart phenotypes in vivo. RESULTS: We show that both mHtt fragments are able to impair fly cardiac
function with different characteristics. Additionally, expression of mHtt, which was limited to adulthood only, leads to mild
heart impairment, as opposed to a strong and age-dependent phenotype observed when mHtt expression was driven
during both developmental and adult stages. We report that treatment with methylene blue (MB), a protective compound
in mitochondria-related diseases, partially protects the fly's heart against mHtt-induced toxicity, but does not rescue
neuronal or glial phenotypes in other fly models of HD. This may be linked to its low penetration through the fly's blood-
brain barrier. CONCLUSIONS: Our data suggest that improvement of mitochondrial function by MB, or related compounds,
could be an efficient therapeutic strategy to prevent cardiac failure in HD patients.
PMID:26397898
Heidari R et al; J Huntingtons Dis 4 (2): 173-86 (2015)
/EXPL THER/ Methylene blue (MB), the first lead chemical structure of phenothiazine and other derivatives, is commonly
used in diagnostic procedures and as a treatment for methemoglobinemia. We have previously demonstrated that MB
could function as an alternative mitochondrial electron transfer carrier, enhance cellular oxygen consumption, and provide
protection in vitro and in rodent models of Parkinson's disease and stroke. In the present study, we investigated the
structure-activity relationships of MB in vitro using MB and six structurally related compounds. MB reduces mitochondrial
superoxide production via alternative electron transfer that bypasses mitochondrial complexes I-III. MB mitigates reactive
free radical production and provides neuroprotection in HT-22 cells against glutamate, IAA and rotenone toxicity. Distinctly,
MB provides no protection against direct oxidative stress induced by glucose oxidase. Substitution of a side chain at MB's
10-nitrogen rendered a 1000-fold reduction of the protective potency against glutamate neurototoxicity. Compounds
without side chains at positions 3 and 7, chlorophenothiazine and phenothiazine, have distinct redox potentials compared
to MB and are incapable of enhancing mitochondrial electron transfer, while obtaining direct antioxidant actions against
glutamate, IAA, and rotenone insults. Chlorophenothiazine exhibited direct antioxidant actions in mitochondria lysate assay
compared to MB, which required reduction by NADH and mitochondria. MB increased complex IV expression and activity,
while 2-chlorphenothiazine had no effect. Our study indicated that MB could attenuate superoxide production by
functioning as an alternative mitochondrial electron transfer carrier and as a regenerable anti-oxidant in mitochondria.
PMID:23118969
Poteet E et al; PLoS One 7 (10): e48279 (2012)
/EXPL THER/ Methylene blue (MB) is an FDA-grandfathered drug with memory-enhancing effects at low doses, but
opposite effects at high doses. We investigated the effects of four MB doses (0.1, 0.5, 5.0, or 10.0 uM) on zebrafish memory
retention in the T-maze task. After training fish to swim into a certain arm of the T-maze, the fish were placed into a tank
containing one of the four MB doses or a control tank containing blue food dye. Subsequently, fish were placed into the T-
maze for memory retention testing. Results indicated that MB produced hormetic dose-response effects on memory. Fish
that received the 0.5 uM dose performed significantly better at the T-maze than those that received higher doses. Fish who
received 5.0 uM did not exhibit a significant difference in performance from control fish, and the fish that received the 10.0
uM dose performed significantly worse than lower doses. These findings support the utility of zebrafish in comparative
research and their potential value for testing of MB and other neuropsychopharmacological treatments in animal models of
memory disorders.
PMID:27482828
Echevarria DJ et al; Zebrafish 13 (6): 489-494 (2016)
/EXPL THER/ Perinatal asphyxia induces retinal lesions, generating ischemic proliferative retinopathy, which may result in
blindness. Previously, we showed that the nitrergic system was involved in the physiopathology of perinatal asphyxia. Here
we analyze the application of methylene blue, a well-known soluble guanylate cyclase inhibitor, as a therapeutic strategy to
prevent retinopathy. Male rats (n = 28 per group) were treated in different ways: 1) control group comprised born-to-term
animals; 2) methylene blue group comprised animals born from pregnant rats treated with methylene blue (2 mg/kg) 30
and 5 min before delivery; 3) perinatal asphyxia (PA) group comprised rats exposed to perinatal asphyxia (20 min at 37 °C);
and 4) methylene blue-PA group comprised animals born from pregnant rats treated with methylene blue (2 mg/kg) 30
and 5 min before delivery, and then the pups were subjected to PA as above. For molecular studies, mRNA was obtained at
different times after asphyxia, and tissue was collected at 30 days for morphological and biochemical analysis. Perinatal
asphyxia produced significant gliosis, angiogenesis, and thickening of the inner retina. Methylene blue treatment reduced
these parameters. Perinatal asphyxia resulted in a significant elevation of the nitrergic system as shown by NO synthase
(NOS) activity assays, Western blotting, and (immuno)histochemistry for the neuronal isoform of NOS and NADPH-
diaphorase activity. All these parameters were also normalized by the treatment. In addition, methylene blue induced the
upregulation of the anti-angiogenic peptide, pigment epithelium-derived factor. Application of methylene blue reduced
morphological and biochemical parameters of retinopathy. This finding suggests the use of methylene blue as a new
treatment to prevent or decrease retinal damage in the context of ischemic proliferative retinopathy.
PMID:26984891
Rey-Funes M et al; Am J Physiol Regul Integr Comp Physiol 310 (11): R1011-9 (2016)
/EXPL THER/ Paraquat (PQ) poisoning often leads to severe oxidative liver injury. Recent studies have reported that
methylene blue (MB) can prevent oxidative stress-induced diseases. This study tested the hypothesis that MB treatment
reduced acute liver injury induced by PQ in rats. Adult male Sprague-Dawley (SD) rats were randomly divided into four
groups: (1) normal group, (2) MB group (2 mg/kg i.p.), (3) PQ group (35 mg/kg i.p.) and (4) PQ+MB group (MB 2 mg/kg i.p.
administrated 2 hr after PQ). We evaluated the changes of liver histopathology, serum liver enzymatic activities, oxidative
stress, heme oxygenase-1 expression, and mitochondrial permeability transition. The rats were injected with PQ produced
liver injury, evidenced by histological changes and elevated serum alkaline phosphatase and alanine transaminase levels; PQ
also led to oxidative stress, an increase of malondialdehyde content and mitochondrial permeability transition pore
opening. Pathological damage and all of the above mentioned markers were reversed in the animals treated with MB than
in those who received PQ alone. Meanwhile, MB significantly increased the contents of superoxide dismutase, adenosine
triphosphate and the expression of heme oxygenase-1. In conclusion, MB had a protective effect against PQ-induced
hepatic damage in rats. The mechanisms of the protection seem to be the inhibition of mitochondrial permeability transition
opening and the increase of heme oxygenase-1 expression.
PMID:25943564
Chen JL et al; Int Immunopharmacol 28 (1): 808-12 (2015)
/EXPL THER/ INTRODUCTION: Chemotherapy still is the most effective way to control malaria, a major public health problem
in sub-Saharan Africa. The large-scale use of the combination therapy artemether-lumefantrine for malaria treatment in
Africa predisposes lumefantrine to emergence of resistance. There is need to identify drugs that can be used as substitutes
to lumefantrine for use in combination therapy. Methylene blue, a synthetic anti-methemoglobinemia drug, has been
shown to contain antimalarial properties, making it a candidate for drug repurposing. The present study sought to
determine antiplasmodial effects of methylene blue against lumefantrine- and pyrimethamine-resistant strains of P.
berghei. METHODOLOGY: Activity of methylene blue was assessed using the classical four-day test on mice infected with
lumefantrine-resistant and pyrimethamine-resistant Plasmodium berghei. A dose of 45 mg/kg/day was effective for testing
ED90. Parasitemia and mice survival was determined. RESULTS: At 45 mg/kg/day, methylene blue sustained significant
parasite inhibition, over 99%, for at least 6 days post-treatment against lumefantrine-resistant and pyrimethamine-resistant
P. berghei (p = 0.0086 and p = 0.0191, respectively). No serious adverse effects were observed. CONCLUSIONS: Our results
indicate that methylene blue at a concentration of 45 mg/kg/day confers over 99% inhibition against lumefantrine- and
pyrimethamine-resistant P. berghei for six days. This shows the potential use methylene blue in the development of
antimalarials against lumefantrine- and pyrimethamine-resistant parasites.
PMID:27367013
Mwangi VI et al; J Infect Dev Ctries 10 (6): 635-42 (2016)
/EXPL THER/ BACKGROUND: Low back pain is a leading cause of pain and disability. The intervertebral disk has been
identified as the most common source of chronic low back pain. Although prior treatments directed at intervertebral disks
have been disappointing, recent studies show promising improvement of pain and function after a single intradiskal
injection of methylene blue. OBJECTIVE: To assess changes in pain and function in patients with diskogenic low back pain,
diagnosed by diskography, after an intradiskal injection of methylene blue. STUDY DESIGN: Prospective trial. METHODS:
Patients diagnosed with diskogenic pain by diskography underwent a single treatment of intradiskal injection of methylene
blue, determined by prior provocation diskography. MAIN OUTCOME MEASURES: Pain and function measurements were
completed at baseline and 1, 2, and 6 months after treatment. Treatment was considered a categorical success based on a
30% improvement in pain according to a visual analog scale (VAS) and function on the Oswestry Disability Index (ODI).
Treatment was considered a categorical failure if less than 30% improvement in pain and function was achieved or if the
patient pursued other invasive treatment options during the trial period. RESULTS: Sixteen patients received the intradiskal
methylene blue injection. Eleven patients received a single-level injection, 4 patients received a 2-level injection, and one
patient received injections at 3 levels. For the VAS, at 1, 2, and 6 months after the injection, the categorical success rates
were 25%, 21%, and 25%, respectively. For the ODI, at 1, 2 and 6 months after the injection, the categorical success rates
were 25%, 21%, and 33%, respectively. The overall categorical success rates at 1, 2, and 6 months after the injection were
19%, 21%, and 25%, respectively. CONCLUSION: This small trial did not demonstrate overall clinical success of intradiskal
methylene blue injection for patients diagnosed with diskogenic pain by diskography.
PMID:24780850
Levi DS et al; PM R 6 (11): 1030-7 (2014)
/EXPL THER/ Methylene blue is an FDA approved compound with a variety of pharmacologic activities. It inhibits
aggregation of several amyloidogenic proteins known to be deposited in neurodegenerative diseases. Recently, it has been
proposed that methylene blue shows significant beneficial effects in a phase 2 clinical trial by slowing cognitive decline in
Alzheimer's disease patients. To analyze its therapeutic potential, we investigated the effect of methylene blue on
neurotoxicity in a zebrafish model for tauopathies. Transgenic expression of the frontotemporal dementia associated Tau-
P301L mutation recapitulates a number of the pathological features observed in humans including abnormal
phosphorylation and folding of Tau, tangle formation and Tau dependent neuronal loss. Upon incubation of zebrafish larvae
with methylene blue, neither abnormal phosphorylation nor neuronal cell loss, reduced neurite outgrowth or a swimming
defect were rescued. Methylene blue is biologically active in zebrafish since it reduced aggregation of a huntingtin variant
containing a stretch of 102 glutamine residues. However, although huntingtin aggregation was largely prevented by
methylene blue, huntingtin-dependent toxicity was unaffected. Our findings are consistent with the hypothesis that toxicity
is not necessarily associated with deposition of insoluble amyloid proteins.
PMID:20381619
van Bebber F et al; Neurobiol Dis 39 (3): 265-71 (2010)
/EXPL THER/ OBJECTIVES: Methylene blue, once discarded due to its unsettling yet mild side effects, has now found a
renewed place in the pharmacopoeia of modern medicine. The continued spread of drug-resistant Plasmodium vivax and
Plasmodium falciparum has also led to a recent re-examination of methylene blue's potent antimalarial properties. Here we
examine the ex vivo susceptibility profile of Plasmodium spp. isolates to methylene blue; the isolates were from a region on
the Thai-Myanmar border where there are increasing rates of failure when treating vivax malaria with chloroquine.
METHODS: To do this we used a newly developed ex vivo susceptibility assay utilizing flow cytometry and a portable flow
cytometer with a near-UV laser. RESULTS: P. vivax (median methylene blue IC50 3.1 nM, IQR 1.7-4.3 nM) and P. falciparum
(median methylene blue IC50 1.8 nM, IQR 1.6-2.3 nM) are susceptible to methylene blue treatment at physiologically
relevant levels. Unfortunately, the addition of chloroquine to combination treatments with methylene blue significantly
reduces the ex vivo effectiveness of this molecule. CONCLUSIONS: Our data support further efforts to employ methylene
blue as a safe, low-cost antimalarial to treat drug-resistant malaria.
PMID:25150147
Suwanarusk R et al; J Antimicrob Chemother 70 (1): 124-9 (2015)
/EXPL THER/ Methylene blue (MB, methylthioninium chloride) is a phenothiazine that crosses the blood brain barrier and
acts as a redox cycler. Among its beneficial properties are its abilities to act as an antioxidant, to reduce tau protein
aggregation and to improve energy metabolism. These actions are of particular interest for the treatment of
neurodegenerative diseases with tau protein aggregates known as tauopathies. The present study examined the effects of
MB in the P301S mouse model of tauopathy. Both 4 mg/kg MB (low dose) and 40 mg/kg MB (high dose) were administered
in the diet ad libitum from 1 to 10 months of age. We assessed behavior, tau pathology, oxidative damage, inflammation
and numbers of mitochondria. MB improved the behavioral abnormalities and reduced tau pathology, inflammation and
oxidative damage in the P301S mice. These beneficial effects were associated with increased expression of genes regulated
by NF-E2-related factor 2 (Nrf2)/antioxidant response element (ARE), which play an important role in antioxidant defenses,
preventing protein aggregation, and reducing inflammation. The activation of Nrf2/ARE genes is neuroprotective in other
transgenic mouse models of neurodegenerative diseases and it appears to be an important mediator of the neuroprotective
effects of MB in P301S mice. Moreover, we used Nrf2 knock out fibroblasts to show that the upregulation of Nrf2/ARE genes
by MB is Nrf2 dependent and not due to secondary effects of the compound. These findings provide further evidence that
MB has important neuroprotective effects that may be beneficial in the treatment of human neurodegenerative diseases
with tau pathology.
PMID:24556215
Stack C et al; Hum Mol Genet 23 (14): 3716-32 (2014)
/EXPL THER/ The overproduction of reactive oxygen species (ROS) and reactive nitrogen species (RNS) has been known to
contribute to the pathogenesis of noise-induced hearing loss. In this study, we discovered that in BALB/c mice pretreatment
with methylene blue (MB) for 4 consecutive days significantly protected against cochlear injury by intense broad-band
noise for 3 hr. It decreased both compound threshold shift and permanent threshold shift and, further, reduced outer hair
cell death in the cochlea. MB also reduced ROS and RNS formation after noise exposure. Furthermore, it protected against
rotenone- and antimycin A-induced cell death and also reversed ATP generation in the in vitro UB-OC1 cell system.
Likewise, MB effectively attenuated the noise-induced impairment of complex IV activity in the cochlea. In addition, it
increased the neurotrophin-3 (NT-3) level, which could affect the synaptic connections between hair cells and spiral
ganglion neurons in the noise-exposed cochlea, and also promoted the conservation of both efferent and afferent nerve
terminals on the outer and inner hair cells. These findings suggest that the amelioration of impaired mitochondrial electron
transport and the potentiation of NT-3 expression by treatment with MB have a significant therapeutic value in preventing
ROS-mediated sensorineural hearing loss.
PMID:24763057
Park JS et al; Cell Death Dis 5: e1200 (2014)
5.2 Drug Warnings (Complete)

/BOXED WARNING/ WARNING: SEROTONIN SYNDROME WITH CONCOMITANT USE OF SEROTONERGIC DRUGS. Methylene
Blue Injection may cause serious or fatal serotonergic syndrome when used in combination with serotonergic drugs. Avoid
concomitant use of Methylene blue injection with selective serotonin reuptake inhibitors (SSRIs), serotonin and
norepinephrine reuptake inhibitors (SNRIs), and monoamine oxidase inhibitors.
Large IV doses of methylene blue may produce nausea, vomiting, abdominal pain, precordial pain, dizziness, headache,
profuse sweating, dyspnea, hypertension, and mental confusion. Urinary tract irritation may occur. High IV dosage or high
local concentrations of methylene blue may cause formation of methemoglobin and cyanosis.
Hemolysis and hemolytic anemia may occur, especially in young infants and patients with glucose-6-phosphate
dehydrogenase (G-6-PD) deficiency.
Hypersensitivity, manifested as wheal and flare reactions at the injection site, has been reported. Severe hypersensitivity
reactions, including anaphylaxis, generalized urticaria, and hypotension, tachycardia, and bronchospasm, have been
reported following local instillation or injection of methylene blue.
Methylene blue imparts a blue-green color to saliva, urine, feces, and skin. Bluish skin discoloration from excessive
methylene blue dosage can be mistaken for methemoglobinemia.
IV administration of methylene blue has caused adverse local effects, including pain, burning sensation, rash, necrosis,
abscess, ulceration, and thrombophlebitis; extravasation has caused tissue necrosis. Oral administration of methylene blue
has caused adverse GI effects and dysuria. Topical application of methylene blue may stain the skin; skin stains may be
removed by hypochlorite solution. Subcutaneous or intradermal injection of methylene blue has caused adverse skin and
tissue reactions (e.g., erythematous macular lesions, superficial ulcers, abscess formation, skin and fat necrosis) at the
injection site. Intrathecal injection of the drug has caused neural damage.
Methylene blue is contraindicated in patients with known hypersensitivity to the drug. Epinephrine and other appropriate
agents and equipment should be available for immediate use in case an anaphylactic reaction occurs.
Methylene blue is contraindicated in women who are or may become pregnant.

Subcutaneous, intrathecal, and intraspinal injection of methylene blue are contraindicated.

To prevent local high concentrations of methylene blue and production of additional methemoglobin, IV injections must be
made slowly and recommended dosage should not be exceeded.
Long-term administration of methylene blue may result in marked anemia due to accelerated destruction of erythrocytes;
hemoglobin concentrations should be checked frequently.
Because of the risk of paradoxical methemoglobinemia and hemolysis, methylene blue should be avoided in patients with
known or suspected G-6-PD deficiency.
Methylene blue should be used with caution in patients with severe renal impairment. Some clinicians suggest that initial
dosage may not need to be reduced when methylene blue is used for the treatment of methemoglobinemia in patients
with renal impairment, but creatinine clearance should be considered if the drug is given by continuous IV infusion.
Because of the risk of serotonin syndrome, methylene blue generally should not be used in patients receiving serotonergic
drugs.
Use of methylene blue in neonates and young infants has caused hemolysis, hemolytic anemia, hyperbilirubinemia, and
phototoxicity. Fatalities have been reported.
Methylene blue is contraindicated in women who are or may become pregnant. There is epidemiologic evidence that
methylene blue is a teratogen, and the drug can cause fetal harm if administered during pregnancy. Use of methylene blue
in amniocentesis has been associated with atresia of the ileum and jejunum, ileal occlusion, and other adverse effects in
neonates. Use of methylene blue during pregnancy has resulted in hemolytic anemia, hyperbilirubinemia,
methemoglobinemia, respiratory distress, skin staining, and phototoxicity in neonates. If methylene blue is used during
pregnancy or if the patient becomes pregnant, the patient should be apprised of potential fetal hazard.
Very serious complications are reported following intrathecal injection of methylene blue. The clinical indication was to
assess spontaneous /rhinorrhea/. It is now generally agreed that intrathecal application of dyestuff solutions, especially of
methylene blue, is no longer permissible.
Steiner HH, Steiner-Milz HG; Laryngol Rhinol Otol 65 (12): 699-701 (1986)
Common adverse effects of methylene blue include dyspnea, precordial pain, restlessness, and apprehension. Rare effects
include tremors, hemolytic anemia, pulmonary edema, and even death. Giving too large a dose saturates the nicotinamide
adenine dinucleotide-dependent enzyme system, resulting in an excess of the oxidant methylene blue. This additional
oxidant stress may, paradoxically, increase methemoglobin and possible cause hemolysis. Patients with significant G6PD
deficiency will not be able to reduce methylene blue to leukomethylene blue, and it should be avoided.
Dart, R.C. (ed). Medical Toxicology. Third Edition, Lippincott Williams & Wilkins. Philadelphia, PA. 2004., p. 222
VET: In cats and dogs, administration of methylene blue to treat methemoglobinemia can cause oxidative damage to
erythrocytes, including Heinz bodies, limiting the dose that can be used therapeutically. ... Heinz bodies can increase in cats
from methylene blue treatment without producing anemia.
Papich, M.G. Saunders Handbook of Veterinary Drugs Small and Large Animal. 3rd ed. St. Louis, MO: Elsevier Saunders, 2011, p. 496
VET: The greatest concerns with methylene blue therapy are the development of Heinz body anemia or other red cell
morphological changes, methemoglobinemia, and decreased red cell life spans. Cats tent to be very sensitive to these
effects and some consider it contraindicated in feline patients, but dogs and horses can also develop hematologic adverse
effects at relatively low dosages. Methylene blue has the potential to cause serotonin syndrome. Necrotic abscesses may
develop if injected SC or extravasation occurs during IV administration.
5.3 Interactions (Complete)

Although the clinical importance is unclear, in vitro studies indicate that the antimalarial effects of methylene blue and
artemisinin, artemether, or artesunate are synergistic and the antimalarial effects of methylene blue and mefloquine or
quinine are additive against Plasmodium falciparum. Antagonism occurred when methylene blue was used with
chloroquine or pyrimethamine in vitro.
Methylene Blue Injection may cause serious or fatal serotonergic syndrome when used in combination with serotonergic
drugs. Avoid concomitant use of Methylene blue injection with selective serotonin reuptake inhibitors (SSRIs), serotonin
and norepinephrine reuptake inhibitors (SNRIs), and monoamine oxidase inhibitors.
This is the first report of a fatal outcome from serotonin toxicity, precipitated by an interaction between methylene blue
and venlafaxine. Methylene blue-associated serotonin toxicity has been described before but usually as mild toxicity. Its
presentation after general anesthesia may be atypical and therefore more difficult to diagnose. However, the syndrome is
completely preventable if serotonin re-uptake inhibiting agents are stopped beforehand.
PMID:24846936
Top WM et al; Neth J Med 72 (3): 179-81 (2014)
Sodium azide (NaN(3)) is widely employed to quench singlet oxygen during photodynamic therapy (PDT), especially when
PDT is used to kill bacteria in suspension. We observed that addition of NaN(3) (100 uM or 10 mM) to gram-positive
Staphylococcus aureus and gram-negative Escherichia coli incubated with methylene blue (MB) and illuminated with red
light gave significantly increased bacterial killing (1-3 logs), rather than the expected protection from killing. A different
antibacterial photosensitizer, the conjugate between polyethylenimine and chlorin(e6) (PEI-ce6), showed reduced PDT
killing (1-2 logs) after addition of 10 mM NaN(3). Azide (0.5 mM) potentiated bacterial killing by Fenton reagent (hydrogen
peroxide and ferrous sulfate) by up to 3 logs, but protected against killing mediated by sodium hypochlorite and
hydrogen peroxide (considered to be a chemical source of singlet oxygen). The intermediacy of N(3)() was confirmed by
spin-trapping and electron spin resonance studies in both MB-photosensitized reactions and Fenton reagent with addition
of NaN(3). We found that N(3)() was formed and bacteria were killed even in the absence of oxygen, suggesting the direct
one-electron oxidation of azide anion by photoexcited MB. This observation suggests a possible mechanism to carry out
oxygen-independent PDT.
PMID:23044264
Huang L et al; Free Radic Biol Med 53 (11): 2062-71 (2012)
The sonodynamic damage to protein in the presence of methylene blue (MB) and the various influencing factors including
ultrasonic irradiation time and MB concentration on the damage of protein were studied by fluorescence and absorption
spectra. In addition, the mechanisms of the synergistic effects of ultrasound and MB were studied by oxidation-extraction
photometry with several reactive oxygen species (ROS) scavengers. The results indicated that the damage of protein
induced by the synergistic effects of ultrasound and MB were more serious than those that ultrasound or MB alone was
applied. The damage of protein could be mainly due to the generation of ROS. The damage degree of protein increased
with the increase of ultrasonic irradiation time and MB concentration because of the increased quantities of ROS generation.
Both (1)O2 and *OH were the important mediators of the ultrasound-inducing protein damage in the presence of MB.
PMID:25025307
He LL et al; Spectrochim Acta A Mol Biomol Spectrosc 134: 361-6 (2015)
The therapeutic index of very toxic, oxidative drugs could be improved by concurrent treatment with other agents, such as
methylene blue, which affect the concentration of intracellular reducing agents. In support of this hypothesis, methylene
blue was found to protect mice against the toxic effects of doxorubicin without reducing doxorubicin's antineoplastic
activity.
Hrushesky WJ et al; Larcet 1 (8428): 565-7 (1985)
The effect of sodium taurodeoxycholate infused iv on the pharmacokinetics of methylene blue was studied in the rat to
investigate the role of ion pair complexation in the body on drug elimination and disposition. Distribution volume of
methylene blue was increased by sodium taurodeoxycholate infusion. Apparent partition coeff of methylene blue between
phosphate buffer (pH 7.4) and n-octanol was increased markedly by sodium taurodeoxycholate; the increase in distribution
volume seemed to be the result of decreased polarity of methylene blue by ion-pair formation with sodium
taurodeoxycholate. But total body clearance and biliary excretion clearance of methylene blue were not increased by
sodium taurodeoxycholate.
Shim CK; Arch Pharm Res 9 (1): 49-54 (1986)
The disposition and urinary excretion pharmacokinetics of methylene blue were determined after its iv administration at 15
mg/kg to mature female sheep. Comparisons were made between methylene blue administered alone or subsequent to 50
mg/kg sodium nitrite. The overall elimination rate constant (beta) of methylene blue, 0.0076 +/- 0.0016 min/L, was not
influenced by prior administration of sodium nitrite. However, the distribution rate was significantly altered by sodium
nitrite. Very little of the methylene blue was eliminated in the urine either intact or as leucomethylene blue in spite of its
relatively short half-life.
Burrows GE; J Vet Pharmacol 7 (3): 225-31 (1984)
... Methylene blue was administered to a 58-yr-old woman undergoing a parathyroidectomy under general anesthesia. The
patient had a background of obsessive compulsive disorder treated with paroxetine. Postoperatively, she demonstrated
symptoms and signs of serotonin syndrome; specifically tachycardia, agitation, dystonia and abnormal eye movements.
These clinical findings spontaneously resolved themselves over the subsequent 48 hr. An interaction between methylene
blue and serotonergic agents may give rise to the serotonin syndrome. ...
PMID:18166746
Ng BK et al; Can J Anaesth 55 (1): 36-41 (2008)
6 Environmental Fate & Exposure
6.1 Environmental Fate/Exposure Summary

Methylene blue's production and use as a paper dye, in silk dyeing and microscopy stain, chemical intermediate, medicinal
agent, and cosmetic dye may result in its release to the environment through various waste streams. If released to air, an
estimated vapor pressure of 7.0X10-7 mm Hg at 25 °C indicates methylene blue will exist in both the vapor and particulate
phases in the atmosphere. Vapor-phase methylene blue will be degraded in the atmosphere by reaction with
photochemically-produced hydroxyl radicals; the half-life for this reaction in air is estimated to be 2 hrs. Particulate-phase
methylene blue will be removed from the atmosphere by wet and dry deposition. The trihydrate of methylene blue has UV
absorption maxima 688 and 609 nm and, therefore, methylene blue may be susceptible to direct photolysis by sunlight. If
released to soil, methylene blue is expected to be immobile based upon an estimated Koc of 8000. The estimated pKa of
methylene blue is 3.14, indicating that this compound will exist almost entirely in the cation form in the environment and
cations generally adsorb more strongly to soils containing organic carbon and clay than their neutral counterparts.
Methylene blue is not expected to volatilize from dry soil surfaces based upon its vapor pressure. More than 90% of color
removal efficiency was observed when using an anaerobic sludge reactor and a low methylene blue concentration,
suggesting that biodegradation may occur in soil or water under specific conditions. If released into water, methylene blue
is expected to adsorb to suspended solids and sediment based upon the estimated Koc. The pKa indicates methylene blue
will exist almost entirely in the cation form at pH values of 5 to 9 and, therefore, volatilization from water surfaces is not
expected to be an important fate process. An estimated BCF of 3 suggests the potential for bioconcentration in aquatic
organisms is low. Hydrolysis is not expected to be an important environmental fate process since this compound lacks
functional groups that hydrolyze under environmental conditions (pH 5 to 9). Occupational exposure to methylene blue
may occur through inhalation and dermal contact with this compound at workplaces where methylene blue is produced or
used. The general public is not likely to be exposed to methylene blue unless by direct medical treatment. (SRC)
6.2 Probable Routes of Human Exposure (Complete)

NIOSH (NOES Survey 1981-1983) has statistically estimated that 74,208 workers (43,305 of these were female) were
potentially exposed to methylene blue in the US(1). Occupational exposure to methylene blue may occur through
inhalation and dermal contact with this compound at workplaces where methylene blue is produced or used. The general
public is not likely to be exposed to methylene blue unless by direct medical treatment(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 Sept 5, 2018:
https://web.archive.org/web/20110717195709/https://www.cdc.gov:80/noes/noes1/agtindex.html
6.3 Artificial Pollution Sources (Complete)

Methylene blue's production and use as a paper dye, in silk dyeing and microscopy stain, chemical intermediate, medicinal
agent, and cosmetic dye(1,2) and photosensitizer(3) may result in its release to the environment through various waste
streams(SRC). Methylene blue is a cationic dye(4). The compound is used as an antidote to methemglobinemia(5).
Methylene Blue-curcumin is used in the treatment of Alzheimer's disease(2).
(1) Berneth H; Azine Dyes. Ullmann's Encyclopedia of Industrial Chemistry. (1999-2018). New York, NY: John Wiley & Sons. Online Posting
Date: 15 Apr 2008 (2) Gregory P; Azine Dyes. Kirk-Othmer Encyclopedia of Chemical Technology. (1999-2018). New York, NY: John Wiley &
Sons. Online Posting Date: 18 Jan 2013. (3) Smith RE; Azine Dyes. Kirk-Othmer Encyclopedia of Chemical Technology. (1999-2018). New York,
NY: John Wiley & Sons. Online Posting Date: 18 Jan 2013. (4) O'Neil MJ, ed; The Merck Index. 15th ed. Cambridge, UK: Royal Soceity of
Chemistry, p. 1124 (2013) (5) Brunton L et al, eds; Goodman & Gilman's The Pharmacological Basis of Therapeutics. 12th ed., New York, NY:
McGraw Hill Medical. p. 86 (2001)
6.4 Environmental Fate (Complete)

TERRESTRIAL FATE: Based on a classification scheme(1), an estimated Koc value of 8000(SRC), determined from a structure
estimation method(2), indicates that methylene blue is expected to be immobile in soil(SRC). The estimated pKa of
methylene blue is 3.14(3), indicating that this compound will exist almost entirely in the cation form in the environment and
cations generally adsorb more strongly to soils containing organic carbon and clay than their neutral counterparts(4).
Volatilization from moist soil is not expected because the compound exists as a cation and cations do not volatilize.
Methylene blue is not expected to volatilize from dry soil surfaces(SRC) based upon an estimated vapor pressure of 7.0X10-
7 mm Hg at 25 °C(SRC), determined from a fragment constant method(2). More than 90% of color removal efficiency was
observed when using an anaerobic sludge reactor and a low methylene blue concentration(5), suggesting that
biodegradation may occur in soil under specific conditions(SRC).
(1) Swann RL et al; Res Rev 85: 17-28 (1983) (2) US EPA; Estimation Program Interface (EPI) Suite. Ver. 4.11. Nov, 2012. Available from, as of
Sept 6, 2018: https://www2.epa.gov/tsca-screening-tools (3) Wishart DS et al; DrugBank: a knowledgebase for drugs, drug actions and drug
targets. Nucleic Acids Res. 2008 Jan;36(Database issue):D901-6. PMID:18048412.Available from, as of Sept 5, 2018: https://www.drugbank.ca
(4) Doucette WJ; pp. 141-188 in Handbook of Property Estimation Methods for Chemicals. Boethling RS, Mackay D, eds. Boca Raton, FL: Lewis
Publ (2000) (5) Ong S-A et al; J Hazard Mat 124(1-3): 88-94 (2005). Available from, as of Sept 10, 2018:
https://www.ncbi.nlm.nih.gov/pubmed/16002211?dopt=Abstract
AQUATIC FATE: Based on a classification scheme(1), an estimated Koc value of 8000(SRC), determined from a structure
estimation method(2), indicates that methylene blue is expected to adsorb to suspended solids and sediment(SRC). An
estimated pKa of 3.14(3) indicates methylene blue will exist almost entirely in the cation form at pH values of 5 to 9 and,
therefore, volatilization from water surfaces is not expected to be an important fate process(SRC). According to a
classification scheme(4), an estimated BCF of 3(SRC), from an estimated log Kow of 0.75(2) and a regression-derived
equation(2), suggests the potential for bioconcentration in aquatic organisms is low(SRC). More than 90% of color removal
efficiency was observed when using an anaerobic sludge reactor and a low methylene blue concentration(5), suggesting
that biodegradation may occur in water under specific conditions(SRC).
(1) Swann RL et al; Res Rev 85: 17-28 (1983) (2) US EPA; Estimation Program Interface (EPI) Suite. Ver. 4.11. Nov, 2012. Available from, as of
Sept 5, 2018: https://www2.epa.gov/tsca-screening-tools (3) Wishart DS et al; DrugBank: a knowledgebase for drugs, drug actions and drug
targets. Nucleic Acids Res. 2008 Jan;36(Database issue):D901-6. PMID:18048412.Available from, as of Sept 5, 2018: https://www.drugbank.ca
(4) Franke C et al; Chemosphere 29: 1501-14 (1994) (5) Ong S-A et al; J Hazard Mat 124(1-3): 88-94 (2005). Available from, as of Sept 10,
2018: https://www.ncbi.nlm.nih.gov/pubmed/16002211?dopt=Abstract
ATMOSPHERIC FATE: According to a model of gas/particle partitioning of semivolatile organic compounds in the
atmosphere(1), methylene blue, which has an estimated vapor pressure of 7.0X10-7 mm Hg at 25 °C(SRC), determined from
a fragment constant method(2), will exist in both the vapor and particulate phases in the ambient atmosphere. Vapor-phase
methylene blue is degraded in the atmosphere by reaction with photochemically-produced hydroxyl radicals(SRC); the half-
life for this reaction in air is estimated to be 2 hrs(SRC), calculated from its rate constant of 2.1X10-10 cu cm/molecule-sec at
25 °C(SRC) that was derived using a structure estimation method(2). Particulate-phase methylene blue may be removed
from the air by wet and dry deposition(SRC). The trihydrate of methylene blue has UV absorption maxima at 688 and 609
nm(3) which suggests that methylene blue may be susceptible to direct photolysis by sunlight(SRC).
(1) Bidleman TF; Environ Sci Technol 22: 361-367 (1988) (2) US EPA; Estimation Program Interface (EPI) Suite. Ver. 4.11. Nov, 2012. Available
from, as of Sept 5, 2018: https://www2.epa.gov/tsca-screening-tools (3) O'Neil MJ, ed; The Merck Index. 15th ed. Cambridge, UK: Royal
Society of Chemistry, p. 1124 (2013)
6.5 Environmental Biodegradation (Complete)

ANAEROBIC: The objective of this study is to evaluate the decolorization of Methylene Blue (MB) by an up-flow anaerobic
sludge blanket (UASB) reactor. The UASB reactor was operated under batch condition with total treatment volume of 3 L
and operation time of 24 hrs per batch. It was found that the color of MB disappeared within a few minutes after entering
into the UASB reactor due to reduction by anaerobic biomass. However, the reduced MB was re-oxidized again by air after
discharged from the reactor and thus caused low color removal efficiency. The presence of suitable amount of organic
content (sucrose and peptone) as an electron donor played an important factor for color removal. It was observed that
more than 90% of color removal efficiency was achieved in the UASB reactor with 0.627 mmol/L of MB concentration and
the presence of low amount of organic content (< 0.45 g COD/(L d)). Biological dye reduction kinetics depends on the
concentration of dye and reducing equivalents. The kinetic behavior of MB biodegradation by microbes was also
investigated to determine the model involved in the process.
PMID:16002211
Ong S-A et al; J Hazard Mat 124(1-3): 88-94 (2005)
6.6 Environmental Abiotic Degradation (Complete)

The rate constant for the vapor-phase reaction of methylene blue with photochemically-produced hydroxyl radicals has
been estimated as 2.1X10-10 cu cm/molecule-sec at 25 °C(SRC) using a structure estimation method(1). This corresponds to
an atmospheric half-life of about 2 hours at an atmospheric concentration of 5X10+5 hydroxyl radicals per cu cm(2).
Methylene blue is not expected to undergo hydrolysis in the environment due to the lack of functional groups that
hydrolyze under environmental conditions(2). The trihydrate of methylene blue has UV absorption maxima at 668 and 609
nm(3,4) and, therefore, methylene blue may be susceptible to direct photolysis by sunlight(SRC). Methylene blue is reduced
to a colorless form then oxidized back to the dye, imparting good fastness to oxidation. The compound is also used as a
photosensitizer(5).
(1) US EPA; Estimation Program Interface (EPI) Suite. Ver. 4.11. Nov, 2012. Available from, as of Sept 6, 2018: https://www2.epa.gov/tsca-
screening-tools (2) Lyman WJ et al; Handbook of Chemical Property Estimation Methods. Washington, DC: Amer Chem Soc pp. 7-4, 7-5, 8-12
(1990) (3) O'Neil MJ, ed; The Merck Index. 15th ed. Cambridge, UK: Royal Society of Chemistry, p. 1124 (2013) (4) Green FJ; The Sigma-Aldrich
Handbook of Stains, Dyes, and Indicators. Milwaukee, WI: Aldrich Chem Co. p. 448-49 (1990) (5) Smith RE; Azine Dyes. Kirk-Othmer
Encyclopedia of Chemical Technology. (1999-2018). New York, NY: John Wiley & Sons. Online Posting Date: 18 Jan 2013.
6.7 Environmental Bioconcentration (Complete)

An estimated BCF of 3 was calculated in fish for methylene blue(SRC), using an estimated log Kow of 0.75(1) and a
regression-derived equation(1). According to a classification scheme(2), this BCF suggests the potential for bioconcentration
in aquatic organisms is low(SRC).
(1) US EPA; Estimation Program Interface (EPI) Suite. Ver. 4.11. Nov, 2012. Available from, as of DATE: https://www2.epa.gov/tsca-screening-
tools (2) Franke C et al; Chemosphere 29: 1501-14 (1994)
The aquatic plant, Hydrilla verticillata was shown to remove methylene blue from aqueous solution rapidly; 100, 500, and
1000 ppm methylene blue were 95, 95 and 42% absorbed, respectively, in 180 minutes using 0.5 g Hydrilla in 100 mL
solution(1).
(1) Low KS et al; Environ Technol 14: 115-24 (1994)
6.8 Soil Adsorption/Mobility (Complete)

Using a structure estimation method based on molecular connectivity indices(1), the Koc of methylene blue can be
estimated to be 8000(SRC). According to a classification scheme(2), this estimated Koc value suggests that methylene blue
is expected to be immobile in soil. The estimated pKa of methylene blue is 3.14(3), indicating that this compound will exist
almost entirely in the cation form in the environment and cations generally adsorb more strongly to soils containing organic
carbon and clay than their neutral counterparts(4).
(1) US EPA; Estimation Program Interface (EPI) Suite. Ver. 4.11. Nov, 2012. Available from, as of Sept 5, 2018: https://www2.epa.gov/tsca-
screening-tools (2) Swann RL et al; Res Rev 85: 17-28 (1983) (3) Wishart DS et al; DrugBank: a knowledgebase for drugs, drug actions and
drug targets. Nucleic Acids Res. 2008 Jan;36(Database issue):D901-6. PMID:18048412.Available from, as of Sept 5, 2018:
https://www.drugbank.ca (4) Doucette WJ; pp. 141-188 in Handbook of Property Estimation Methods for Chemicals. Boethling RS, Mackay D,
eds. Boca Raton, FL: Lewis Publ (2000)
Laboratory adsorption studies determined that methylene blue was strongly adsorbed to soil organic matter, sodium
montmorillonite, and sodium kaolinite(1); desorption studies using three different extraction methods found that only 2% of
adsorbed methylene blue was extractable(2).
(1) Baughman GL et al; Dye Solubilities. In: Advances in Color Chemistry. Freeman M, Peters MT, eds., Elsevier (1993) (2) Weber JB; Advan
Chem Ser 111: 55-120 (1972)
6.9 Volatilization from Water/Soil (Complete)

Methylene blue is a cationic thiazine dye(1). An estimated pKa of 3.14(2) indicates methylene blue will exist almost entirely
in the cation form at pH values of 5 to 9 and, therefore, volatilization from water or moist soil surfaces is not expected to be
an important fate process(SRC). Methylene blue is not expected to volatilize from dry soil surfaces(SRC) based upon an
estimated vapor pressure of 7.0X10-7 mm Hg(SRC), determined from a fragment constant method(3).
(1) Green FJ; The Sigma-Aldrich Handbook of Stains, Dyes, and Indicators. Milwaukee, WI: Aldrich Chem Co. p. 448-49 (1990) (2) Wishart DS et
al; DrugBank: a knowledgebase for drugs, drug actions and drug targets. Nucleic Acids Res. 2008 Jan;36(Database issue):D901-6.
PMID:18048412.Available from, as of Sept 5, 2018: https://www.drugbank.ca (3) US EPA; Estimation Program Interface (EPI) Suite. Ver. 4.11.
Nov, 2012. Available from, as of Sept 5, 2018: https://www2.epa.gov/tsca-screening-tools
6.10 Environmental Water Concentrations (Complete)

GROUNDWATER: Methylene blue was identified as one of several organic waste water contaminants in groundwater
affected by a residential 1989 septic system on Cape Cod, MA. Concentrations ranged from 0.25-3 ug/L. The compound was
detected to a depth of 3.5 meters below land surface at 0.3, 0.9 and 1.5 ug/L in November 2003, July 2003, and August
2004, respectively(1).
(1) Swartz CH et al; Environ Sci Technol 40: 4894-4902 (2006)
6.11 Effluent Concentrations (Complete)
Aqueous waste effluents are generated at manufacturing sites where azine and thioazine dyes (such as methylene blue) are
produced(1). Losses of dyes (in general) to wastewater effluents during manufacture have been estimated to be 1-2%(2). For
the organic dye industry in general, it has been estimated that as much as 10% of the dye is lost to wastewater effluents
during dyeing operations(2,3).
(1) Steadman TR et al; Industrial Process Profiles for Environmental Use: Chapter 7. Organic Dyes and Pigments Industry. USEPA-600/2-77-
023g p. 106-13 (1977) (2) Brown D et al; Chemosphere 10: 245-61 (1981) (3) Porter JJ; Text Res J 43: 735-44 (1973)
6.12 Atmospheric Concentrations (Complete)

RURAL/REMOTE: Mean concentrations of 103 and 887 ng/cu methylene blue active substances (MBAS) in PM10 coastal
aerosols from the northwest Mediterranean basin. Values correspond to samples from the remote Porquerolles Island-Var,
France and more populated site of San Rossore National Park - Tuscany, Italy, respectively(1).
(1) Becagli S et al; Atmos Environ 45: 6788-6801 (2011)
7 Environmental Standards & Regulations
7.1 Allowable Tolerances (Complete)

adequate draining before contact with food. (a) The following chemical substances when used as ingredients in an
antimicrobial pesticide formulation may be applied to: Food-contact surfaces in public eating places, dairy-processing
equipment, and food-processing equipment and utensils. Methylene blue is included on this list. Limit: When ready for use,
the end-use concentration is not to exceed 0.4 ppm.
40 CFR 180.940(a) (USEPA); U.S. National Archives and Records Administration's Electronic Code of Federal Regulations. Available from, as of
adequate draining before contact with food. ... (c) The following chemical substances when used as ingredients in an
antimicrobial pesticide formulation may be applied to: Food-processing equipment and utensils. Methylene blue is included
on this list. Limit: When ready for use, the end-use concentration is not to exceed 0.4 ppm.
40 CFR 180.940(c) (USFDA); U.S. National Archives and Records Administration's Electronic Code of Federal Regulations. Available from, as of
7.2 FDA Requirements (Complete)

The Approved Drug Products with Therapeutic Equivalence Evaluations identifies currently marketed prescription drug
products, including methylene 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 August 29, 2018:
https://www.fda.gov/cder/ob/
Drug products containing certain active ingredients offered over-the-counter (OTC) for certain uses. A number of active
ingredients have been present in OTC drug products for various uses, as described below. However, based on evidence
currently available, there are inadequate data to establish general recognition of the safety and effectiveness of these
ingredients for the specified uses: Methylene blue is included in orally administered menstrual drug products.
21 CFR 310.545(a)(24) (USFDA); U.S. National Archives and Records Administration's Electronic Code of Federal Regulations. Available from, as
of August 29, 2018: https://www.ecfr.gov
8 Chemical/Physical Properties
8.1 Molecular Formula

C16-H18-Cl-N3-S
O'Neil, M.J. (ed.). The Merck Index - An Encyclopedia of Chemicals, Drugs, and Biologicals. Cambridge, UK: Royal Society of Chemistry, 2013., p.
1124
8.2 Molecular Weight

319.852
Haynes, W.M. (ed.). CRC Handbook of Chemistry and Physics. 95th Edition. CRC Press LLC, Boca Raton: FL 2014-2015, p. 3-370
8.3 Color/Form (Complete)

Dark green crystals or powder from chloroform-ethyl ether
Solutions have a deep blue color /Methylene blue zinc-free dye/

Osol, A. (ed.). Remington's Pharmaceutical Sciences. 16th ed. Easton, Pennsylvania: Mack Publishing Co., 1980., p. 781
8.4 Odor
Slight odor
ASHP. American Hospital Formulary Service -Drug Information 87. Bethesda, MD: American Society of Hospital Pharmacists, 1987. (Plus
Supplements, 1987), p. 2045
8.5 Melting Point

100-110 °C (decomposes)
Kirk-Othmer Encyclopedia of Chemical Technology. 3rd ed., Volumes 1-26. New York, NY: John Wiley and Sons, 1978-1984., p. V3: 379 (1978)
8.6 LogP
log Kow = 0.75 (est)
US EPA; Estimation Program Interface (EPI) Suite. Ver. 4.1. Nov, 2012. Available from, as of Sept 4, 2018: https://www2.epa.gov/tsca-
screening-tools
8.7 Solubility (Complete)

In water, 43,600 mg/L at 25 °C.
Baughman GL et al; Dye Solubilities. In: Advances in Color Chemistry. Freeman M, Peters MT, eds., Elsevier (1993)
Soluble in water
Soluble in ethanol, chloroform; slightly soluble in pyridine; insoluble in ethyl ether

Soluble in glacial acetic acid and glycerol; insoluble in xylene and oleic acid, in ethanol 2%, and in acetone 0.5%.
8.8 Vapor Pressure

7.0X10-7 mm Hg at 25 °C (est)
screening-tools
8.9 Other Experimental Properties (Complete)

Dark green crystals or crystalline powder with bronze luster. Odorless; stable in air. Absorption max: 668, 609 nm. Soluble in
water and chloroform; sparingly soluble in alcohol. Deep blue solution in water or alcohol. Forms double salts. /Methylene
blue trihydrate/
1124
Dark green crystals or powder with bronze-like luster; odorless or slight odor. Stable in air. Soluble in water, alcohol,
chloroform. Water solutions are deep blue. /Methylene blue trihydrate/
Larranaga, M.D., Lewis, R.J. Sr., Lewis, R.A.; Hawley's Condensed Chemical Dictionary 16th Edition. John Wiley & Sons, Inc. Hoboken, NJ 2016.,
p. 904
Crystalline powder ... bronze-like luster /zinc salt/

ASHP. American Hospital Formulary Service -Drug Information 87. Bethesda, MD: American Society of Hospital Pharmacists, 1987. (Plus
Supplements, 1987), p. 2045
Hydroxyl radical reaction rate constant = 2.05X10-10 cu cm/molec-sec at 25 °C (est)

screening-tools
9 Spectral Information
9.1 UV Spectra
UV: 6-680 (Organic Electronic Spectral Data, Phillips et al, John Wiley & Sons, New York)
Lide, D.R., G.W.A. Milne (eds.). Handbook of Data on Organic Compounds. Volume I. 3rd ed. CRC Press, Inc. Boca Raton ,FL. 1994., p. V4: 4124
9.2 IR Spectra
IR Spectra IR: 1372 (Coblentz Society Spectral Collection)
10 Chemical Safety & Handling
10.1 Fire Fighting Procedures (Complete)

Suitable extinguishing media: Use water spray, alcohol-resistant foam, dry chemical or carbon dioxide.
Sigma-Aldrich; Safety Data Sheet for Methylene Blue solution, Product Number: 03978, Version 5.4 (Revision Date 08/11/2015). Available from,
as of September 5, 2018: https://www.sigmaaldrich.com/safety-center.html
Advice for firefighters: Wear self-contained breathing apparatus for firefighting if necessary.
10.2 Personal Protective Equipment (PPE) (Complete)

Eye/face protection: Use equipment for eye protection tested and approved under appropriate government standards such
as NIOSH (US) or EN 166(EU).
Skin protection: Handle with gloves.

Body Protection: Impervious clothing. The type of protective equipment must be selected according to the concentration
and amount of the dangerous substance at the specific workplace.
Respiratory protection: Respiratory protection not required. For nuisance exposures use type OV/AG (US) or type ABEK (EU
EN 14387) respirator cartridges. Use respirators and components tested and approved under appropriate government
standards such as NIOSH (US) or CEN (EU).
10.3 Preventive Measures (Complete)

ACCIDENTAL RELEASE MEASURES: Personal precautions, protective equipment and emergency procedures: Avoid breathing
vapors, mist or gas. Environmental precautions: No special environmental precautions required.
Appropriate engineering controls: General industrial hygiene practice.

Gloves must be inspected prior to use. Use proper glove removal technique (without touching glove's outer surface) to
avoid skin contact with this product. Dispose of contaminated gloves after use in accordance with applicable laws and good
laboratory practices. Wash and dry hands.
10.4 Stability/Shelf Life (Complete)

Stable under recommended storage conditions.
10.5 Storage Conditions (Complete)
Keep container tightly closed in a dry and well-ventilated place. Storage class (TRGS 510): Non Combustible Liquids.
Commercially available methylene blue 10-mg/mL solution for IV use should be stored at 20-25 °C, but may be exposed to
temperatures ranging from 15-30 °C.
10.6 Cleanup Methods (Complete)

ACCIDENTAL RELEASE MEASURES: Personal precautions, protective equipment and emergency procedures: Avoid breathing
vapors, mist or gas. Environmental precautions: No special environmental precautions required. Methods and materials for
containment and cleaning up Keep in suitable, closed containers for disposal.
10.7 Disposal Methods (Complete)

SRP: Expired or waste pharmaceuticals shall carefully take into consideration applicable DEA, EPA, and FDA regulations. It is
not appropriate to dispose by flushing the pharmaceutical down the toilet or discarding to trash. If possible return the
pharmaceutical to the manufacturer for proper disposal being careful to properly label and securely package the material.
Alternatively, the waste pharmaceutical shall be labeled, securely packaged and transported by a state licensed medical
waste contractor to dispose by burial in a licensed hazardous or toxic waste landfill or incinerator.
Product: Offer surplus and non-recyclable solutions to a licensed disposal company; Contaminated packaging: Dispose of as
unused product.
11 Manufacturing/Use Information
11.1 Uses (Complete)

For methylene blue (USEPA/OPP Pesticide Code: 039505) there are 0 labels match. /SRP: Not registered for current use in
the U.S., but approved pesticide uses may change periodically and so federal, state and local authorities must be consulted
for currently approved uses./
National Pesticide Information Retrieval System's Database Database on Methylene Blue (61-73-4). Available from, as of October 17, 2018:
https://npirspublic.ceris.purdue.edu/ppis/
Stain in bacteriology; reagent for several chemicals; as mixed indicator; as redox colorimetric agent. Targeting agent for
melanoma.
1124
Dyeing cotton and wool, biological and bacteriological stains, reagent in oxidation-reduction titrations in volumetric
analysis, indicator
p. 904
Methylene blue is mainly used to dye paper and office supplies, but also to tone up silk colors. ... as a stain in microscopy,
as a hydrogen acceptor in enzymatic dehydrogenations, as a redox indicator in iodometry and titanometry, and as a
cosmetic dye.
Berneth H; Azine Dyes. Ullmann's Encyclopedia of Industrial Chemistry. 7th ed. (1999-2018). NY, NY: John Wiley & Sons. Online Posting Date:
Apr 15, 2008.
Basic (cationic) dyes, eg, methylene blue, ... have also been used as temporary hair colorants.
Kirk-Othmer Encyclopedia of Chemical Technology. 3rd ed., Volumes 1-26. New York, NY: John Wiley and Sons, 1978-1984., p. V12 106 (1980)
Enzyme Inhibitors
National Library of Medicine's Medical Subject Headings. Methylene Blue. Online file (MeSH, 2018). Available from, as of August 29, 2018:
https://meshb.nlm.nih.gov/search
MEDICATION
MEDICATION (VET)
11.2 Manufacturers
Methylene blue Formulators
Table: 5 of 35 Formulators listed
Company Location
GE Healthcare Oslo, Norway
Akorn Inc Lake Forest IL 60045
Prodibio 13013 Marseille France
Aquarium Muenster Pahlsmeier GmbH D-48291 Telgte Germany
Guna Inc Whitehall PA 18052
US NLM; DailyMed. Current Medical Information. US Natl Lib Med, Natl Inst Health, Health Human Serv. Available from, as of Sept 4, 2018:
https://dailymed.nlm.nih.gov/dailymed/about.cfm
Sigma-Aldrich Fine Chemicals, 3050 Spruce St., St. Louis, MO 63103, (314) 534-4900
Sigma-Aldrich; Product Search Methylene blue (61-73-4). Available from, as of Sept 7, 2018: https://www.sigmaaldrich.com/united-
states.html
11.3 Methods of Manufacturing (Complete)
Methylene blue ... is obtained by oxidation of 4-aminodimethylaniline in the presence of sodium thiosulfate to give the
quinonediiminothiosulfonic acid, reaction with dimethylaniline, oxidation to the indamine, and cyclization to give the
thiazine. The dye is commercially available as the hydrochloride and as the zinc chloride double salt.
Berneth H; Azine Dyes. Ullmann's Encyclopedia of Industrial Chemistry. 7th ed. (1999-2018). NY, NY: John Wiley & Sons. Online Posting Date:
Apr 15, 2008.
By treating a solution of N,N-dimethyl-p-phenylenediamine and N,N,-dimethylaniline hydrochlorides with H2S and FeCl3
or another suitable oxidizing agent.
Troy, D.B. (Ed); Remmington The Science and Practice of Pharmacy. 21 st Edition. Lippincott Williams & Williams, Philadelphia, PA 2005, p.
1348
By oxidation of p-aminodimethylaniline with ferric chloride in the presence of hydrogen sulfide. The dye is the zinc
chloride double salt of the chloride.
p. 904
N,N-Dimethyl-p-phenylene diamine ... reacts with sodium thiosulfate ... to form the thiosulfonic acid which condenses with
N,N-dimethylaniline ... in the presence of sodium dichromate ... to the indamine, then with copper sulfate ... and sodium
dichromate to methylene blue.
Smith, RE; Azine Dyes. Kirk-Othmer Encyclopedia of Chemical Technology (1999-2018). John Wiley & Sons, Inc. Online Posting Date: December
4, 2000
11.4 General Manufacturing Information (Complete)

C16H18N3SCl.3H2O (medicinal); (C16H18N3SCl)2.ZnCl2H20 (dye)
p. 904
Methylene blue has also been described as a "photosensitizer" which means that it can absorb light and transfer the
absorbed energy to another compound.
Methylene blue was first isolated in 1876. Its use covers many areas, including as an antiseptic, disinfectant, and cyanide-
antidote. Methylene blue is also an antimicrobial agent in plastic materials such as food packaging wraps, surgical drapes,
face masks, and other film materials. Light exposure of the dye dispersed in plastic generates reactive singlet oxygen, which
is the active antimicrobial species. Methylene blue is approved for topical, intravenous, and oral administration. Its cellular
adsorption selectivity, subcellular localization, reduction to leuko-dye by mitochondria, and photoreaction with DNA have
been of interest for decades. Recent applications of this technology to HIV phototherapy have reviewed previous methylene
blue literature .
Sturmer DM; Kirk-Othmer Encyclopedia of Chemical Technology. (2001). NY, NY: John Wiley & Sons; Dyes, Senstizing. Online Posting Date:
July 13, 2001.
Thiazine dyes are generally of minor importance in the textile field due to what is presently considered as inferior fastness
Berneth H; Azine Dyes. Ullmann's Encyclopedia of Industrial Chemistry. (1999-2018). New York, NY: John Wiley & Sons. Online Posting Date:
15 Apr 2008
11.5 Formulations/Preparations (Complete)

Provided technically as the zinc double chloride; used medically in a zinc free form.
1124
Parenteral: Injection, for IV use, 10 mg/mL (Available from one or more manufacturer, distributor, and/or repackager by
generic (nonproprietary) name)
Grade: USP, technical.

p. 904
11.6 Impurities (Complete)
3-(dimethylamino)-7-(methylamino)phenothiazin-5-ylium
Council of Europe, European Directorate for the Quality of Medicines. European Pharmacopoeia, 5th Ed., Supplement 5.4; Strasbourg, France,
p.3977 (2005)
11.7 U.S. Production (Complete)

Production volumes for non-confidential chemicals reported under the Inventory Update Rule.
Year Production Range (pounds)
1986 No Reports
1990 No Reports
1994 No Reports
1998 10 thousand - 500 thousand
2002 No Reports
US EPA; Non-confidential Production Volume Information Submitted by Companies for Chemicals Under the 1986-2002 Inventory Update Rule
(IUR). Phenothiazin-5-ium, 3,7-bis(dimethylamino)-, chloride (61-73-4). Available from, as of October 30, 2008:
https://www.epa.gov/oppt/iur/tools/data/2002-vol.html
12 Laboratory Methods
12.1 Clinical Laboratory Methods (Complete)

Analyte: methylene blue; matrix: tissue (muscle); procedure: high-performance liquid chromatography with mass
spectrometry detection; limit of detection: 1.3 ppm
Voyksner RD et al; Biomed Environ Mass Spectrom 19: 523-534 (1990). As cited in: Lunn G; HPLC and CE Methods for Pharmaceutical Analysis.
CD-ROM. New York, NY: John Wiley & Sons (2000)
12.2 Analytic Laboratory Methods (Complete)

The following method is applicable for the examination of ground and surface waters, domestic and industrial waste
effluents, and treatment process samples. Method 425.1 for the determination of methylene blue employs active
substances colormetric technique. The method is applicable over the range of 0.025 to 100 mg/l. On a sample of filtered
river water, spiked with 2.94 mg linear alkyl sulfonate/liter, 110 analysts obtained a mean of 2.98 mg/l with a standard
deviation of 0.272. On a sample of tap water spiked with 0.48 mg linear alkyl sulfonate/liter, 110 analysts obtained a mean
of 0.49 mg/l with a standard deviation of 0.048. On a sample of distilled water spiked with 0.27 mg linear alkyl
sulfonate/liter, 110 analysts obtained a mean of 0.24 mg/l with a standard deviation of 0.036.
USEPA; Methods for Chemical Analysis of Water and Wastes p.425.1-1 (1983) EPA-600/4-79-020
Method: AOAC 923.11; Procedure: titrimetric method; Analyte: methylene blue; Matrix: drugs; Detection Limit: not provided.
Official Methods of Analysis of AOAC International, 18th Edition Online. Methylene Blue (61-73-4). Available from, as of November 5, 2008:
https://www.aoac.org
Analyte: methylene blue; matrix: chemical identification; procedure: infrared absorption spectrophotometry with
comparison to standards
U.S. Pharmacopeia. The United States Pharmacopeia, USP 31/The National Formulary, NF 26; Rockville, MD: U.S. Pharmacopeial Convention,
Inc., p.2678 (2008)
Analyte: methylene blue; matrix: chemical purity; procedure: visible absorption spectrophotometry at 663 nm with
comparison to standards
Inc., p.2678 (2008)
Analyte: methylene blue; matrix: pharmaceutical preparation (injection solution); procedure: visible absorption
spectrophotometry at 663 nm with comparison to standards
Inc., p.2679 (2008)
Analyte: methylene blue; matrix: pharmaceutical preparation (injection solution); procedure: thin-layer chromatography with
comparison to standards (chemical identification)
Inc., p.2679 (2008)
Analyte: methylene blue; matrix: pharmaceutical preparation (injection solution); procedure: visible absorption
spectrophotometry at 663 nm with comparison to standards (chemical purity)
Inc., p.2679 (2008)
Analyte: methylene blue; matrix: chemical identification; procedure: ultraviolet and visible absorption spectrophotometry
with maxima at 255-260 nm, 285-290 nm, 765-685 nm, and 740-750 nm with comparison to standards
p.3977 (2005)
Analyte: methylene blue; matrix: chemical identification; procedure: thin-layer chromatography with comparison to
standards
p.3977 (2005)
Analyte: methylene blue; matrix: chemical purity; procedure: dissolution in water; addition of potassium chromate solution
and dilution with water; addition of dilute sulfuric acid and potassium iodide solution; addition of water; titration with
sodium thiosulfate with starch solution as indicator

p.3977 (2005)
13 Synonyms and Identifiers

Synonyms
61-73-4
Methylene blue
3,7-Bis(dimethylamino)phenazathionium chloride
3,7-Bis(dimethylamino)phenothiazin-5-ium chloride
Aizen Methylene Blue BH
Aizen Methylene Blue FZ
Basic Blue 9
C.I. Basic Blue 9
C.I. Solvent Blue 8
3H-Phenothiazine, 7-(dimethylamino)-3-(methylimino)-, 3-methochloride
Phenothiazin-5-ium, 3,7-bis(dimethylamino)-, chloride

Phenothiazin-5-ium, 3,7-bis(dimethylamino)-, chloride (1:1)
Chlorure de methylthioninium (French)
Cloruro de metiltioninio (Spanish)
Methylene Blue A
Methylene Blue B
Methylene Blue BB
Methylene Blue D
Methylene Blue G
Methylene Blue N
Methylene Blue 2B
Methylene Blue 2BF
Methylene Blue 2BN
Methylene Blue 2BP
Methylene Blue BBA
Methylene Blue BD
Methylene Blue BP
Methylene Blue BPC
Methylene Blue BX
Methylene Blue BZ
Methylene Blue chloride
Methylene Blue chloride (biological stain)
Methylene Blue FZ
Methylene Blue GZ
Methylene Blue HGG
Methylene Blue I (medicinal)
Methylene Blue IAD
Methylene Blue JFA
Methylene Blue NF (medicinal)
Methylene Blue NZ
Methylene Blue Polychrome
Methylene Blue SG
Methylene Blue SP
Methylene Blue USP (medicinal)
Methylene Blue ZF
Methylene Blue Zinc Free

Methylene Blue ZX
Methylenium ceruleum
Methylthioninium chloride
Methylthioninii chloridum (Latin)
Modr Methylenova (Czech)
Modr Rozpoustedlova 8 (Czech)
Modr Zasadita 9 (Czech)

Solvent Blue 8
Swiss Blue
Tetramethylthionine chloride
Urolene Blue
USEPA/OPP Pesticide Code: 039505.
13.1 Substance Title

Methylene blue
13.2 Associated Chemicals (Complete)

Methylene blue trihydrate; 7220-79-3
Leucomethylene blue; 613-11-6
14 Administrative Information
14.1 Hazardous Substances DataBank Number

1405
14.2 Last Revision Date

20190531
14.3 Last Review Date

Reviewed by SRP on 3/28/2019
14.4 Update History

Complete Update on 2019-05-31, 56 fields added/edited/deleted
Field Update on 2008-10-01, 1 fields added/edited/deleted
Field Update on 2008-08-25, 1 fields added/edited/deleted
Complete Update on 03/05/2003, 2 fields added/edited/deleted.

Field Update on 02/14/2003, 1 field added/edited/deleted.
Complete Update on 02/13/2002, 1 field added/edited/deleted.


Field update on 12/20/1992, 1 field added/edited/deleted.

Field update on 12/29/1989, 1 field added/edited/deleted.
Complete Update on 12/25/1984
Created 19830401 by GCF

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DATA SOURCES HAZARDOUS SUBSTANCES DATA BANK (HSDB) ANNOTATION RECORD

1 Human Health Effects

1.1 Toxicity Summary

1.2 Evidence for Carcinogenicity (Complete)

1.3 Human Toxicity Excerpts (Complete)

1.4 Populations at Special Risk (Complete)

2 Emergency Medical Treatment

2.1 Antidote and Emergency Treatment (Complete)

3 Animal Toxicity Studies

3.1 Non-Human Toxicity Excerpts (Complete)

3.2 Ecotoxicity Excerpts (Complete)

3.3 National Toxicology Program Studies (Complete)

3.4 Non-Human Toxicity Values (Complete)

LD50 Mouse ip 150 mg/kg

LD50 Mouse oral 3500 mg/kg

LD50 Rat iv 1250 mg/kg

LD50 Rat ip 180 mg/kg

LD50 Rat oral 1180 mg/kg

LD50 Mouse ip 67 mg/kg

3.5 Ecotoxicity Values (Complete)

3.6 Ongoing Test Status

3.7 FIFRA Requirements (Complete)

4.1 Metabolism/Metabolites (Complete)

4.2 Absorption, Distribution and Excretion (Complete)

4.3 Biological Half-Life (Complete)

4.4 Mechanism of Action (Complete)

5.1 Therapeutic Uses (Complete)

Drug-induced methemoglobinemia. /Included in US product label/

5.2 Drug Warnings (Complete)

Methylene blue is contraindicated in women who are or may become pregnant.

Subcutaneous, intrathecal, and intraspinal injection of methylene blue are contraindicated.

5.3 Interactions (Complete)

6 Environmental Fate & Exposure

6.1 Environmental Fate/Exposure Summary

6.2 Probable Routes of Human Exposure (Complete)

6.3 Artificial Pollution Sources (Complete)

6.4 Environmental Fate (Complete)

6.5 Environmental Biodegradation (Complete)

6.6 Environmental Abiotic Degradation (Complete)

6.7 Environmental Bioconcentration (Complete)

6.8 Soil Adsorption/Mobility (Complete)

6.9 Volatilization from Water/Soil (Complete)

6.10 Environmental Water Concentrations (Complete)

6.11 Effluent Concentrations (Complete)

6.12 Atmospheric Concentrations (Complete)

7 Environmental Standards & Regulations

7.1 Allowable Tolerances (Complete)

7.2 FDA Requirements (Complete)

8.1 Molecular Formula

8.2 Molecular Weight

8.3 Color/Form (Complete)

Solutions have a deep blue color /Methylene blue zinc-free dye/

8.5 Melting Point

8.7 Solubility (Complete)

Soluble in ethanol, chloroform; slightly soluble in pyridine; insoluble in ethyl ether

8.8 Vapor Pressure

8.9 Other Experimental Properties (Complete)