Mercury-containing vaccine preservative

The viability of all examined cell lines was suppressed entirely in the presence of 4.6 μg/ml (12.5 μM) of Thimerosal. The MTD for HepG2, C2C12, PBMC, and Vero cells was 2, 1.6, 1, and 0.29 μg/ml (5.5, 4.3, 2.7 and 0.8 μM), respectively. The IC50 of Thimerosal exposure for HepG2, C2C12, PBMC, and Vero cells was 2.62, 3.17, 1.27, and 0.86 μg/ml (7.1, 8.5, 3.5 and 2.4 μM), respectively. As for antimicrobial effectiveness, the growth capability of Candida albicans and Staphylococcus aureus was suppressed entirely in the presence of 6.25 µg/ml (17 μM) Thimerosal. The complete growth inhibition of Pseudomonas aeruginosa in culture media was achieved in 100 µg/ml (250 µM) Thimerosal concentration. This value was 12.5 µg/ml (30 μM) for Aspergillus brasiliensis.[3]

In the periaqueductal gray (PAG), the injection of Thimerosal (THIM) results in a dose-dependent increase in the density of μ-opioid receptors (MORs). MOR density in the lateral periaqueductal gray (LPAG) and dorsomedial periaqueductal gray (DMPAG) areas is statistically significantly increased by treatment with increasing doses of Thimerosal. In the caudate putamen (CPU), Thimerosal likewise increases MOR density at a dosage of 3,000 μg Hg/kg. On the other hand, MOR density in the dentate gyrus (DG) is decreased when Thimerosal is administered at both higher doses [1]. Measuring 10 to 14 weeks after the injections, glutamate and aspartate levels rise but glycine and alanine levels fall due to the long-lasting effects of immerosal treatment (4 injections, im, 240 μg Hg/kg on postnatal days 7, 9, 11, and 15). Glutamate and aspartate concentrations at microdialysis time are unaffected by four injections of Thimerosal at a dose of 12.5 μg Hg/kg. When Thimerosal is applied to the perfusion fluid of the prefrontal cortex (PFC), glutamate overflow increases quickly. The Thimerosal impact on glutamate and aspartate is inhibited by coadministration of the neurosteroid dehydroepiandrosterone sulfate (DHEAS; 80 mg/kg; ip); the steroid does not affect these amino acids on its own. Thimerosal's acute effect on glutamate is likewise inhibited by co-application of dehydroepiandrosterone sulfate (DHEAS) in perfusion fluid[2].

The safety of Thimerosal exposure on cells was analyzed through an MTT cell toxicity assay. The viability of four cell types, including HepG2, C2C12, Vero Cells, and Peripheral blood mononuclear cells (PBMCs), was examined in the presence of different Thimerosal concentrations and the maximum tolerable dose (MTD) and the half maximal inhibitory concentration (IC50) values for each cell line were determined. The antimicrobial effectiveness of Thimerosal was evaluated on four control strains, including Pseudomonas aeruginosa, Staphylococcus aureus, Candida albicans, and Aspergillus brasiliensis, to obtain the minimum inhibitory concentration (MIC) of Thimerosal. The MIC test was performed in culture media and under optimal growth conditions of microorganisms in the presence of different Thimerosal concentrations.[3]

Thimerosal, a mercury-containing vaccine preservative, is a suspected factor in the etiology of neurodevelopmental disorders. We previously showed that its administration to infant rats causes behavioral, neurochemical and neuropathological abnormalities similar to those present in autism. Here we examined, using microdialysis, the effect of thimerosal on extracellular levels of neuroactive amino acids in the rat prefrontal cortex (PFC). Thimerosal administration (4 injections, i.m., 240 μg Hg/kg on postnatal days 7, 9, 11, 15) induced lasting changes in amino acid overflow: an increase of glutamate and aspartate accompanied by a decrease of glycine and alanine; measured 10-14 weeks after the injections. Four injections of thimerosal at a dose of 12.5 μg Hg/kg did not alter glutamate and aspartate concentrations at microdialysis time (but based on thimerosal pharmacokinetics, could have been effective soon after its injection). Application of thimerosal to the PFC in perfusion fluid evoked a rapid increase of glutamate overflow. Coadministration of the neurosteroid, dehydroepiandrosterone sulfate (DHEAS; 80 mg/kg; i.p.) prevented the thimerosal effect on glutamate and aspartate; the steroid alone had no influence on these amino acids. Coapplication of DHEAS with thimerosal in perfusion fluid also blocked the acute action of thimerosal on glutamate. In contrast, DHEAS alone reduced overflow of glycine and alanine, somewhat potentiating the thimerosal effect on these amino acids. Since excessive accumulation of extracellular glutamate is linked with excitotoxicity, our data imply that neonatal exposure to thimerosal-containing vaccines might induce excitotoxic brain injuries, leading to neurodevelopmental disorders. DHEAS may partially protect against mercurials-induced neurotoxicity.[2]

Absorption, Distribution and Excretion
Less than 0.01% of the ingested dose is absorbed by the gastrointestinal tract (rat study). Gastrointestinal tract. 266 liters in one study. High concentrations of mercury detected in fecal samples suggest that ethylmercury may be excreted via the gastrointestinal tract. Mercury concentrations in the aqueous humor and excised corneal flaps of nine patients who underwent corneal transplantation were measured. A contact lens that had been stored for several weeks in a solution containing thimerosal was worn in one eye for 4 hours. After 4 hours, the contact lens was removed, and mercury concentrations in the aqueous humor, corneal flap, and the contact lens itself were measured. Compared to the control group, the subjects showed significantly higher mercury levels in both the aqueous humor and corneal flap; however, almost no mercury residue remained on the contact lens after 4 hours. Mercury levels in the corneal tissue of the subjects ranged from 0.6 to 14 nanograms per tissue. Mercury levels in the aqueous humor samples of the subjects ranged from 20 to 46 nanograms per milliliter. Of the 13 infants who received topical treatment with 0.1% thimerosal tincture for omphalocele, 10 died. The total number of administrations ranged from 9 to 48. Mercury concentrations were measured in various tissues from 6 of these infants. The mean mercury concentrations in fresh liver, kidney, spleen, and heart tissue samples ranged from 5152 to 11330 ppb, suggesting that repeated topical application may lead to percutaneous absorption. Urinary mercury levels were measured in 26 patients with hypogammaglobulinemia receiving weekly intramuscular injections of IgG replacement therapy (containing 0.01% thimerosal). The IgG dose ranged from 25 mg/kg to 50 mg/kg, with each dose containing 0.6–1.2 mg of mercury. The estimated total mercury dose ranged from 4 to 734 mg, with treatment durations from 6 months to 17 years. Urinary mercury levels were elevated in 19 patients; however, no clinical evidence of chronic mercury poisoning was found in any of them. Forty full-term infants aged 6 months and under received a thimerosal-containing vaccine (diphtheria-tetanus-acellular pertussis vaccine, hepatitis B vaccine, and some children also received a Haemophilus influenzae type b vaccine). Twenty-one infants in the control group received a thimerosal-free vaccine. Blood, urine, and stool samples were collected 3–28 days post-vaccination. The total mercury content (organic and inorganic mercury) in the samples was determined using cold atomic absorption spectrometry. The average mercury intake of infants exposed to thimerosal was: 45.6 μg (range 37.5–62.5 μg) for 2-month-old infants and 111.3 μg (range 87.5–175.0 μg) for 6-month-old infants. The blood mercury levels in 2-month-old infants exposed to thimerosal ranged from less than 3.75 to 20.55 nmol/L (parts per billion); the blood mercury levels in 6-month-old infants were all less than 7.50 nmol/L. Quantifiable mercury was detected in only 1 of 15 blood samples from the control group. Following vaccination, urinary mercury concentrations were low, but fecal mercury concentrations were higher in 2-month-old infants (mean 82 ng/g dry weight) and 6-month-old infants (mean 58 ng/g dry weight) exposed to thimerosal. The estimated blood half-life of ethylmercury is 7 days (95% CI 4–10 days). Vaccination with thimerosal-containing vaccines does not appear to raise blood mercury concentrations in infants above safe levels. Ethimerosal appears to be rapidly excreted from the blood via feces after thimerosal vaccination. Metabolites/Metabolites Ethimerosal (etHg) is derived from the metabolism of thimerosal (sodium o-carboxyphenyl thioethyl), the most widely used form of organic mercury. Organic mercury is primarily absorbed through the gastrointestinal tract and then distributed throughout the body via the bloodstream. Organic mercury forms complexes with free cysteine and cysteine and sulfhydryl groups on proteins such as hemoglobin. These complexes mimic methionine, allowing them to be transported throughout the body, including across the blood-brain barrier and placenta. Organic mercury is metabolized into inorganic mercury, which is eventually excreted in urine and feces. (T11)

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Biological Half-Life
A study aimed to investigate the pharmacokinetics of thimerosal in mice. The estimated half-lives (in days) were: blood 8.8 days, brain tissue 10.7 days, heart 7.8 days, liver 7.7 days, and kidney 45.2 days. Ethylmercury has a longer half-life (averaging about 50 days in humans), which can lead to its accumulation and may be harmful to the developing fetal brain, as the fetal brain is more susceptible to the effects of organic mercury compounds than the adult brain.

Protein Binding
95% to 99% of mercury in blood plasma (depending on animal species and experimental conditions) is bound to albumin (and other plasma proteins). A significant portion of albumin is filtered out at the glomeruli.

[1]. Olczak M, et al. Neonatal administration of thimerosal causes persistent changes in mu opioid receptors in the ratbrain. Neurochem Res. 2010 Nov;35(11):1840-7.
[2]. Duszczyk-Budhathoki M, et al. Administration of thimerosal to infant rats increases overflow of glutamate and aspartate in the prefrontal cortex: protective role of dehydroepiandrosterone sulfate. Neurochem Res. 2012 Feb;37(2):436-47.
[3]. J Trace Elem Med Biol. 2023 May:77:127129. doi: 10.1016/j.jtemb.2023.127129. Epub 2023 Jan 4.

Sodium thiomercurate of o-carboxyphenylthioethylmercury is a light milky white crystalline powder with a slight odor: pH (1% aqueous solution) 6.7. (NTP, 1992)
Thimerosal is an alkylmercury compound (containing approximately 49% mercury by weight) used as a preservative and antifungal agent. It has disinfectant, antifungal, preservative, and sensitizing effects. It contains ethylmercuric thiosalicylate.
Thimerosal (INN), commonly known as thimerosal in the United States, is an organomercury compound. This compound is a mature and widely used preservative and antifungal agent. Developed in 1927, thimerosal has long been used as a preservative in certain cosmetics, topical medicines, and biological agents (including vaccines). The safety and toxicity of thimerosal have been a major concern for decades. Although thimerosal has been banned in some countries, it is still used as a preservative in some vaccines in the United States and many vaccines in developing countries. Thimerrosal is a standardized chemical allergen. Its physiological effects are achieved through increased histamine release and cell-mediated immunity. Thimerosal is an organomercury compound, a derivative of thiosalicylic acid, with antibacterial and antifungal properties. Although its mechanism of action is not fully elucidated, thimerosal can inhibit the sulfhydryl-containing active sites of various enzymes and bind to sulfhydryl compounds such as glutathione, cysteine, and the sulfhydryl groups of proteins. Furthermore, thimerosal can activate InsP3 calcium channels on the endoplasmic reticulum membrane, triggering the release of calcium ions from intracellular calcium stores, leading to an influx of extracellular calcium ions. Therefore, thimerosal may induce or inhibit calcium-dependent cellular functions. Thimerosal is an organomercury compound primarily used as a preservative and antifungal agent. Developed by the pharmaceutical company Eli Lilly and Company in 1928 and registered under the trade name Merthiolate, thimerosal has been used as a preservative in vaccines, immunoglobulin preparations, skin test antigens, antivenom, ophthalmic and nasal products, and tattoo inks. Mercury is a heavy metal, silvery-white in color, belonging to the d-block elements, and is one of the six elements that are liquid at room temperature or near room temperature and atmospheric pressure. It is a naturally occurring substance that can combine with other elements (such as chlorine, sulfur, or oxygen) to form inorganic mercury compounds (salts). Mercury can also combine with carbon to form organomercury compounds. (L1, L267)
Ethylmercuric sulfonylbenzoate has been used as a preservative in vaccines, antivenom, and ointments. It was previously used as a topical disinfectant. It degrades into ethylmercuric acid and thiosalicylate.

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Pharmaceutical Indications
Used as a preservative in certain cosmetics, topical medicines, and biological agents (including vaccines).

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Mechanism of Action
Although its mechanism of action is not fully elucidated, thimerosal can inhibit the thiol-containing active sites of various enzymes and bind to thiol compounds, including glutathione, cysteine, and the thiol groups of proteins. Furthermore, thimerosal can activate InsP3 calcium channels on the endoplasmic reticulum membrane, thereby triggering the release of intracellular calcium, leading to calcium-induced influx of extracellular calcium. Therefore, thimerosal may induce or inhibit a variety of calcium signaling-dependent cellular functions. Ethylmercury is metabolized to inorganic mercury more rapidly than methylmercury. This metabolic difference may explain the nephropathy caused by toxic doses of ethylmercury. Furthermore, in vitro experiments have shown that high doses (405 μg/L to 101 mg/L) of thimerosal can increase oxidative stress and induce apoptosis, which may explain its damaging effects on the nervous system. Currently, the mechanism of action of low-dose ethylmercury is not fully understood. However, the short half-life of ethylmercury (a metabolite of thimerosal) limits its application in vaccines. Ethylmercury is a lipophilic cation capable of crossing the blood-brain barrier. Under intracellular pH and [Cl⁻] conditions, the octanol/water partition coefficients of methylmercury and ethylmercury are 1.4 to 1.8; therefore, these two organomercury compounds are primarily present intracellularly as lipophilic cations. Previous studies have shown that lipophilic cations accumulate in mitochondria in a manner driven by the Nernst equation and steady-state membrane potential. Since the typical mitochondrial membrane potential of astrocytes and neurons is between 140 and 170 mV, it can be expected that the concentration of these organomercury compounds in the mitochondria is about 1,000 times that in the cytoplasm.

C11H15HGNAO2S

404.81

405.992

C, 26.70; H, 2.24; Hg, 49.55; Na, 5.68; O, 7.90; S, 7.92

54-64-8

16684434

Cream colored, crystalline powder

298.6ºC at 760mmHg

234-237 °C (dec.)(lit.)

250 °C

1.577

0

3

3

14

180

0

C1=CC=C(C(=C1)C(=O)[O-])[S-].[CH2]C.[Hg+].[Na+]

RTKIYNMVFMVABJ-UHFFFAOYSA-L

InChI=1S/C7H6O2S.C2H5.Hg.Na/c8-7(9)5-3-1-2-4-6(5)10;1-2;;/h1-4,10H,(H,8,9);1H2,2H3;;/q;;2*+1/p-2

sodium;(2-carboxylatophenyl)sulfanyl-ethylmercury

HSDB7151; HSDB-7151; HSDB 7151

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment, avoid exposure to moisture.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

H2O : 100 mg/mL (229.95 mM )

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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 (Please use freshly prepared in vivo formulations for optimal results.)