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Testing Status of Agents at NTP
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Home » Testing Information » Testing Status of Agents at NTP » Methylene Blue » Executive Summary Methylene Blue: Exposure and Toxic. Effects
Consumer exposure to methylene blue results from the medicinal
and therapeutic uses of this compound. In addition, consumer exposure
may occur from the use of methylene blue in food and
(See note) and
hair colorants.
No quantitative data were found on consumer exposure to this compound.
Occupational exposure to methylene blue may occur in industry,
laboratories and health care facilities. Data from the National
Occupational Exposure Survey (NOES), conducted by the National
Institute for Occupational Safety and Health (NIOSH) between 1981
and 1983, indicate that 69, 563 workers, including 42,026 female
employees, were potentially exposed to methylene blue in the workplace.
The NOES data base does not contain information on the frequency,
level or duration of exposure to workers of any chemicals listed
therein [NIOSH, 1990].
No data were found on environmental exposure to methylene blue.
In addition, methylene blue was administered by intravenous drip
for 2-3 hours to six other lactating goats and infused into the
mammary glands of six additional lactating goats as a 10% aqueous
solution at 10 mg/kg.
Methylene blue was detected in the milk of goats 5 minutes after
beginning the IV drip. At equilibrium, drug concentrations in
the milk were 5 to 7 times higher than in the blood. Methylene
blue was detected in the blood of the goats 30 minutes to 12 hours
following intramammary infusion.
Urine and fecal recovery of methylene blue in cows and steers
was less than 2% and 1%, respectively. Low concentrations (quantity
not specified) of methylene blue were found in the kidneys of
these animals by day three post-treatment, and no residual levels
were detected in any body organ or fluid by day six [Ziv, et al.,
1982].
Urine collected from the female mongrel dog after administration
of the 15 mg/kg and the 10 mg/kg doses of methylene blue contained
2.4% and 3.8% of the administered dose, respectively. Blood samples
in the female yielded no methylene blue 2.5 and 3.5 hours after
administration. Blood concentrations from the male beagle dog
indicated values below the assay sensitivity level (0.02 mg/ml)
0-6.9 hours after dosing. The authors report that these results
indicate poor absorption of methylene blue in the dog [DiSanto
and Wagner, 1972].
After 120 minutes, the amount of methylene blue absorbed
in rats, guinea pigs, and rabbits was 54%, 36%, and 47% respectively.
The surface areas of the small intestines of the three animals
were calculated, and absorption ratios per unit surface area of
the small intestine were determined to be 1.0:0.77:1.2 (rabbit:guinea
pig:rat).
Eight percent, 46%, and 30% of the original methylene blue was
found to be metabolized to leucomethylene blue in rats, guinea
pigs and rabbits respectively. The remaining ratio of leucomethylene
blue per unit surface area of the small intestine was determined
to be 1.0:0.25:1.5 (rabbit:rat:guinea pig).
Urinary and biliary excretion of total methylene blue (methylene
blue + leucothylene blue) was determined in rabbits. After 180
minutes, 12.8% of the methylene blue was excreted in urine and
8.0% was excreted in the bile.
Rat and guinea pig small intestines were incubated with methylene
blue solution (quantity not specified) in vitro. Methylene
blue in the mucosal fluid was found to be reduced to leucomethylene
blue and nearly all of the methylene blue disappeared in approximately
90 minutes in both rats and guinea pigs. Leucomethylene blue was
detected in the serosal fluid in rats, guinea pigs and rabbits,
and the amount increased with time. The authors report that leucomethylene
blue was able to penetrate through the small intestine barrier
[Watanabe and Mori, 1977].
Six percent of the administered dose of methylene blue was excreted
after four hours. Most of the methylene blue was eliminated as
leucomethylene blue. There was a significant increase (p=0.05)
in urinary excretion of methylene blue at 1, 2, and 3 hours in
the group receiving sodium nitrate, although for both groups,
excretion rates were low. The half-life of methylene blue was
101.6 ± 16.2 minutes and was not influenced by the presence
of sodium nitrate.
The overall elimination rate constant of methylene blue (0.0076
+ 0.0016) was not found to be influenced by the administration
of sodium nitrite in sheep given 15 mg/kg of methylene blue. Only
the kinetics of the distribution phase (distribution rate, alpha)
were significantly changed (p=0.05) by the administration of sodium
nitrate.
The authors suggest that methylene blue is eliminated by metabolism
and/or biliary excretion, or is sequestered in tissues with slow
release into the peripheral circulation. Elimination in urine
occurs in very low concentrations over a long period, and the
presence of sodium nitrate has little influence on either the
rate of elimination or the metabolic pathway [Burrows, 1984].
From non-linear (one fluid-one tissue) model analysis, it was
determined that the average volume of distribution of the five
sets of blood samples was 0.222 L/kg, or 22.2% of body weight.
The authors suggested that the linear volume of distribution obtained
by the non-linear model may have physiological significance. Using
this non-linear model, no systemic trends in the estimated parameters
in relation to dose were found. The concentration data correlated
well with the classical linear, two-compartment open model, systemic
trends in the estimated parameters in relation to dose were observed.
By this method, the average volume of distribution was found to
be 87.6% of the body weight.
In order to investigate the uptake of methylene blue by the tissues,
and to obtain data on the amount of methylene blue bound as a
fraction of dose, methylene blue at doses of 2.0, 5.0, 7.5, 10.0,
15.0, and 25.0 mg/kg was administered intravenously into the penis
vein of adult male Sprague-Dawley rats. The rats were sacrificed
3 minutes post-injection, blood was collected, and the heart,
lungs, liver, and kidneys were examined for tissue uptake of methylene
blue.
The amount of methylene blue found in the blood was negligible
compared to the amount detected in the tissues studied. These
organs accounted for an average of 29.8% of the administered dose
of methylene blue (See Table 1). The authors reported that uptake
of methylene blue by rat tissue was rapid supporting the non-linear
heterogenous, one-compartment, one tissue-type model observed
in the beagle dog study [DiSanto and Wagner, 1972].
Methylene blue uptake was significantly higher (p<0.001)
in human erythrocytes than in cat or dog erythrocytes. In humans,
virtually all of methylene blue uptake occurred within 20 minutes.
Feline methylene blue uptake was significantly greater (p value
not reported) than uptake by the dog. However, the authors report
that this result may not be significant due to varying degrees
of hemolysis in feline blood samples [Harvey and Kaneko, 1974].
Gosselin et al. have given methylene blue a toxicity
rating of 4 [Gosselin, et al., 1984]. Acute exposure to methylene
blue by intravenous injection has been found to cause hypertension,
sweating, chest pain, confusion, nausea, vomiting, dizziness,
and cyanosis, as well as urine and stool discoloration [Arena,
1986; Gennaro, 1985].
Large, oral doses of methylene blue may cause fever
[McEvoy, 1989]. Methylene blue has also been reported to cause
quadriplegia after intrathecal injection [Driesbach, 1980]. Subcutaneous
injection of methylene blue may cause necrotic abscesses [Reynolds
and Prasad, 1989].
Methylene blue induces hemolytic anemia in individuals
with glucose-6-phosphate dehydrogenase (G-6-PD) enzyme deficiency.
Dark-skinned races have a higher incidence of methylene blue-induced
hemolytic anemia than lighter skinned races, but the disease has
not been noted in North American Indians or Eskimos [Thienes and
Haley, 1972].
Methylene blue is a severe eye irritant [Lenga, 1988].
High concentrations of methylene blue have been found to induce
corneal and conjunctival injury [Grant, 1986].
Two of the patients experienced a blue staining of
the conjunctiva, either in one or both eyes, which was believed
to be caused by methylene blue. Four patients were diagnosed with
follicular conjunctivitis, with three of the four showing symptoms
in both eyes. One patient with follicular conjunctivitis in both
eyes also demonstrated a concurrent blue discoloration of the
limbus in one eye. It was reported that the follicular conjunctivitis
observed may have been caused by the naphazoline or methylene
blue [Brownstein, et al, 1989].
Eight days later both feet became swollen, and five
weeks following the injection of methylene blue, ulcers formed
on the left foot at the injection sites. Three days later, additional
ulcers appeared on the patient's right foot at the sites of methylene
blue injection. The ulcers were necrotic and extended to the deep
fascial layer of the skin, which later required grafting [Perry
and Meinhard, 1974].
Ninety minutes after injection of methylene blue,
a zone of skin pallor with surrounding traces of blue color was
noted at the injection site. During the following 48 hours, necrosis
of the skin developed and became progressively larger during the
next five days. In addition, an incomplete left median nerve palsy
developed. A debridement procedure revealed complete necrosis
of the skin, subcutaneous tissue and underlying fatty tissue of
the left antecubital fossa. When the superficial fascia of the
arm was excised, "dark blue coloration oozed from the wound."
It was determined that the patient had suffered an
accidental injection of methylene blue into the subcutaneous tissues
of the left antecubital fossa. The authors report that although
the patient made a complete recovery from his presenting life-threatening
acute illness, his course was complicated by severe tissue necrosis
of the arm which added substantially to his morbidity and resulted
in diminished function of the arm 18-months post-injury [Ruhlen,
1982].
Four months after the injection, paraparesis had
progressed, urinary retention persisted, and sensory loss had
occurred. Eighteen months after the injection, there was complete
loss of strength in the right lower limb. Three and one half years
after the injection, total paraplegia was observed, which persisted
until the patient died five years later.
The authors reported three phases of spinal cord
and nerve damage which may have resulted following the injection
of methylene blue. The first phase occurred shortly after injection
and resulted in direct damage to the surface of the cord and its
surface blood vessels, probably accentuated by poor perfusion
during a period of hypotension. The second stage was a temporary
recovery possibly related to a regression of edema around the
areas of infarction in the cord, and third was progression to
total paraplegia. This last phase may have resulted from progressive
ischemia of the cord accompanying fibrosis and scarring of the
meninges [Sharr, et al.,1978].
The other twin and mother voided bluish-tinged urine.
However, no other signs of methylene blue toxicity were observed.
[Spahr, et al., 1980].
Low Apgar scores and blue tracheal secretions were
reported after delivery. In addition, a chest x-ray revealed mild
hyaline membrane disease. The primary toxic effect observed in
the infant was hyperbilirubinemia secondary to hemolysis. On day
three of life, the infant's methemoglobin level was elevated at
4.1 g/dl, (normal value < 1 g/dl), and a blood transfusion
was required. The direct fraction of the bilirubin was elevated
after day 7, secondary to inspissated bile syndrome caused by
hepatic cellular injury from the hemolysis. Additional tests revealed
low thyroid function and blood in the urine. The infant was discharged
from the hospital on day 48 of life and was reportedly "neurologically
and developmentally" healthy [McEnerney, 1983].
At birth, the infant's skin, oral mucosa and urine
were stained a deep blue. The infant became jaundiced and developed
hemolytic anemia and hyperbilirubinemia. The infant was treated
with phototherapy and by three weeks of age, symptoms of methylene
blue toxicity had subsided [Serota, et al., 1979].
Following 2 exchange transfusions and phototherapy,
his symptoms gradually diminished. The infant's skin color was
noted to gradually lighten, and the bluish discoloration completely
disappeared 72 hours after delivery [Cowett, et al., 1976].
Adverse effects resulting from acute toxicity in
animals exposed to methylene blue by parenteral administration
include hemoconcentration, hypothermia, acidosis, hypercapnia,
hypoxia, increased blood pressure and increased frequency and
amplitude of respiration [Gosselin, et al, 1984].
Methylene blue has been found to cause corneal and conjunctival injury in rabbits. Intracameral injection of methylene blue has reportedly caused loss of cells from the anterior polar region of the lens, with temporary opacity. This condition reportedly cleared as repopulation of cells from the periphery occurred [Grant, 1986].
LD50 animal studies on methylene blue found in the literature are summarized in Table 2.
The LC50 of methylene blue in Heteropneustes fossilis
was 188.5, 181.5, 172.0. and 165.5 ppm for 24, 48, 72, and 96
hours, respectively. LC50 values for Mystus vittatus were
27.5, 24.0, 22.0, and 18.5 ppm for 24, 48, 72, and 96 hours, respectively.
The no-effect concentration of methylene blue for Herteropneustes
fossilis was 50.48 ppm and 5.48 ppm for Mystus vittatus.
Toxic effects observed for both species of catfish included irritation
as evidenced by erratic movements and violent actions of the pelvic
fins. At high concentrations, signs of restlessness, muscle spasm,
body torsion and massive mucous secretion from the general body
surface of the fish were observed, and the fish became sluggish
and lethargic [Ahmed, et al., 1983].
Numerous reports have indicated an association between methylene blue and the formation of Heinz bodies in animals, caused by the irreversible oxidative denaturation of hemoglobin [Christiansen, 1980; Rentsch and Wittekind, 1967; Ridder and Oehme, 1974]. Studies concerning the effects of methylene blue on Heinz body formation and other hematological parameters are summarized in Table 3.
In the erythrocytes of the cat, small Heinz bodies
began to appear after two hours of incubation and were present
at high concentrations after four hours. In contrast, dog and
human erythrocytes did not contain Heinz bodies until six hours
after incubation, and the Heinz bodies were smaller than those
seen in the feline erythrocytes. Feline erythrocytes incubated
under identical conditions without methylene blue did not develop
Heinz bodies [Harvey and Kaneko, 1974].
No data were found on the prechronic effects of methylene
blue in humans.
No data were found on the prechronic effects of methylene
blue in animals.
Chronic application of methylene blue-containing
eye drops has been found to result in strong staining of bulbar
and palpebral conjunctiva, the lid margins and slight staining
of the corneal epithelium [Grant, 1986]. No other data were found
on the chronic effects of methylene blue in humans. In addition,
no data were found on the carcinogenic effects of methylene blue
in humans.
After 28 days of exposure to methylene blue, hypertrophy
and hyperplasia in the mucous cells of the skin had led to distortion
of these cells and to massive mucous secretion. The maximum degree
of severity was observed after 84 days of exposure, at which time
complete loss of cellular organization was observed [Ahmed and
Shrivastava, 1985].
Methylene blue induced alterations in the electrophoretic
profile of proteins or polypeptides in all reproductive tract
fluids examined. Artificially high levels of follicle-stimulating
hormone (FSH) and estradiol (E2), directly measured by radioimmunoassay,
were identified. Methylene blue at concentrations of 0.005%, 0.01%,
and 0.05% increased levels of FSH and E2 by 20% (p<0.05), 60%
(p<0.05), and 160% (p<0.05), respectively.
In this same study, the effect of methylene blue
on sperm motility was examined. Semen from men believed to have
normal fertility (sperm motility > 85%, sperm concentration
at least 40 x 106) was used to prepare aliquots. The aliquots
were incubated, assessed for initial motility, and added to a
medium containing methylene blue at concentrations described above.
Sperm motility was measured after 5, 15, 30, 45, and 60 minutes.
Sperm motility was found to be significantly inhibited
by methylene blue in a time- and dose-dependent manner (p<0.0001).
At the lowest concentration of methylene blue (0.0005%), less
than 50% were motile after 15 minutes, less than 25% were motile
after 30 minutes and less than 15% were motile after 45 minutes.
Results of sperm motility measurements were similar at concentrations
of 0.001%, while at higher concentrations of methylene blue, inhibition
of sperm motility was increased. At concentrations of 0.01% methylene
blue, less than 5% of the sperm were motile after 5 minutes. After
45 and 60 minutes, sperm were unable to recover motility after
exposure to methylene blue at concentrations greater than 0.005%
[Coddington, et al., 1989].
Methylene blue was found to cause maternal toxicity,
as poor pregnancy weight gain was observed. No other information
on maternal effects, including reference to a maximum tolerated
dose, was provided.
From the 10 litters born to rats exposed to methylene
blue, 100 implantations were identified. Sixty-two normal fetuses
were delivered. Ninety percent of the litters had one or more
resorptions and 38.0% of all recognizable implantations had terminated
in resorptions. In the control group, 40.8% of the litters had
one or more resorptions and 10.6 % of all implantations reportedly
terminated in resorptions (p values not reported) [Telford, et
al., 1962].
No data were found on the genetic effects of methylene
blue in humans.
Methylene blue was found to be mutagenic under all
test conditions. However, the mutagenicity of methylene blue was
not enhanced by the addition of S9 or EDTA-Fe. Further, the addition
of superoxide dismutase (100 µg/ml) inhibited the mutagenicity
of methylene blue by 40%, although complete inhibition was not
obtained. 2,5-Dimethyl furan (1x10-3 M), D-alpha-tocopherol (5x10-4
M), and hydroquinone (5x10-5 M) markedly inhibited the mutagenicity
of methylene blue at a concentration of 8 µg/plate, while
ethanol (0.1 M), D-mannitol (10 mM), formic acid (2 mM), and 1,1-dimethoxyethane
(0.5 M) did not significantly affect the mutagenicity of methylene
blue. The authors reported that singlet oxygen recoverers inhibited
the mutagenicity of methylene blue. However, hydroxy radical scavengers
did not significantly affect the mutagenicity of this compound
[Yamaguchi, 1981].
Conflicting results were found in an abstract describing
a Japanese study which reported that methylene blue was mutagenic
to Bacillus subtilis (strain not specified) in the rec-assay.
In addition, these authors report that methylene blue was also
mutagenic to Escherichia coli (strain not specified) and
Salmonella typhimurium (strain not specified). These latter
studies were conducted both in the light and in the dark [Fujita
et al., 1976].
Methylene blue was found to be an "effective"
sensitizing agent for mutagenesis. Methylene blue mutation rates
appeared to be proportional to fluence rate and dye concentration.
In continuous cultures, rates of photomutagenesis of methylene
blue were independent of growth rate [Webb, et al., 1979].
Three strains of Salmonella typhimurium containing
the base pair substitution mutation his G46 (DG2657, DG2678, TA1530)
were exposed to a visible light source for various times (20 to
90 seconds) following sensitization for 30 minutes with 30 µg/ml
of methylene blue. Methylene blue was found to cause photodynamic-induced
base-pair substitution in the three strains tested. The frequency
of induced revertants was significantly higher in strain TA1530
(uvrB) than in DG2657 (wild-type), and was significantly lower
in strain DG2678 (rec) than in either of the other two strains
(p values not reported).
Three strains of S. typhimurium containing
the frameshift mutation hisD3052 were exposed to a visible light
source for 30 to 120 seconds, following sensitization for 30 minutes
with 30 µg/ml of methylene blue. Frameshift mutations were
induced in all of the strains tested. The frequency of induced
revertants was significantly higher in strain DG2659 (wild type),
than in both DG2666 (rec) and DG2594 (uvrB) [Imray and MacPhee,
1975].
The combined treatment of methylene blue and visible
light was found to induce DNA damage (primarily base modifications)
in all strains tested. Salmonella typhimurium strains treated
with methylene blue in the absence of visible light were not mutated.
In addition, visible light in the absence of methylene blue reportedly
caused insignificant (TA100, TA 2638) or very low (TA104) mutation
rates. The authors reported that singlet oxygen is the reactive
species in S. typhimurium DNA damage induced by methylene
blue/visible light [Epe, et al., 1989].
No data were found on the immunotoxic effects of
methylene blue.
Gross examination of the spinal cords of all five
cats revealed blue discoloration of the dura, leptomeninges and
spinal cord. Microscopic examination of the spinal cord showed
acute inflammation of the leptomeninges and nerve roots, deposition
of fibrin, localized necrosis of the dura, swelling and inflammation
of myelin sheaths and axonal swelling in the anterior and posterior
roots of the grey matter.
Eight cats in this same study that received epidural
injections of either physiologic saline or prilocaine solution
exhibited no significant gross or microscopic changes [Poppers,
et al., 1970].
Methylene blue acts as an electron acceptor in the transfer of electrons from reduced pyridine nucleotides (NADPH and NATPH) to methemoglobin, facilitating reduction of ferric to ferrous iron. Methylene blue is readily reduced to leucomethylene blue, which is readily reoxidized to methylene blue [Gennaro, 1985].
After the administration of methylene blue in
the treatment of methemo-globinemia, methylene blue is converted
to a leuco base by the coenzyme diphosphopyridine nucleotide (DPN).
This leuco base rapidly reduces ferric iron (Fe3+) to ferrous
iron (Fe2+) [Dreisbach, 1980]. If the dose of methylene blue is
high, the oxidation potential favors the formation of methemoglobin
from hemoglobin [Gennaro, 1985].
The leucomethylene blue formed reduces oxygen to
hydrogen peroxide. The hydrogen peroxide formed is initially detoxified
through the hexose monophosphate shunt. When the capacity of the
shunt is exhausted, reduced glutathione is depleted. The excess
hydrogen peroxide then oxidizes both membrane lipid components
and hemoglobin. The oxidation of the hemoglobin leads to the formation
of sulfhemoglobin and consequently Heinz bodies. Hemolysis is
thought to be secondary to the damage to the lipid membrane and
the pitting of the Heinz bodies by the reticuloendothelial cells
(See Figure 1, Heinz Body Formation From Excess Methylene Blue)
[Kirsch and Cohen, 1980].
In the absence of methylene blue, there was a
significant decrease in GSH (p<0.01) in feline erythrocytes
incubated with glucose. Percent of preincubation glucose remaining
was 85% in feline erythrocytes and 99%-100% in dog and human erythrocytes.
When methylene blue was included in the media, there
was a significant decrease in GSH in the red blood cells of the
dog and cat (p<0.01 and <0.001, respectively). This decrease
was not seen in the human red blood cells. Ninety-five percent
of the GSH was still present at the end of the incubation period
in the human red blood cells, compared to 64% in the cat and 76%
in the dog. When glucose was not included in the media, there
was no significant difference between any of the species in the
percent of GSH remaining in the red blood cells. This was true
whether methylene blue was present or not [Harvey and Kaneko 1974].
Web page last updated on August 15, 2005
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