Drug compounds have included stable heavy isotopes of carbon, hydrogen, and other elements, mostly as tracers that influence measurement during the drug development process. It's possible that the pharmacokinetics and functional range of medications contribute to the concern over mutagenesis [1].

Absorption, Distribution and Excretion
... Diffusion cell experiments /were conducted/ for aniline (ANI), o-toluidine (OT), 4,4'-methylenedianiline (MDA) and N-phenyl-2-naphthylamine (PBNA). Excised human skin was exposed to different AA concentrations in vehicles containing water and solvents. Recovery for ANI in receptor fluid was about 20-38% and for MDA 15% over 24h. PBNA could not be detected in the receptor fluid. Further data for OT and beta-naphthylamine (BNA) were considered from our recent study. A semi-quantitative percutaneous absorption ranking for AA was derived: BNA>OT>ANI>MDA>PBNA. For aqueous ANI solutions up to saturation a linear relationship of exposed dose and penetrated amount was observed. However, a linear extrapolation of the flux of neat compounds, as often recommended for risk assessment policies, underestimates considerably the percutaneous uptake. The in vitro data support ...findings in rubber industry workers that the percutaneous absorption may significantly contribute to overall exposure of AA.
... Diffusion cells were used to investigate the effects of two skin barrier creams (SBC) and one skin care cream (SCC) on percutaneous penetration of neat aniline (ANI) and o-toluidine (OT) as well as of OT from a mixture with a workplace specific lubricant. The experiments were carried out with untreated and with skin creams treated human skin. A considerable percutaneous penetration enhancement of test compounds was observed for treated skin compared with untreated skin; the highest enhancement (mean factors 6.2-12.3) was found for SBC (based on oil in water emulsion) treated skin. The lowest penetration enhancement showed SCC treated skin (mean factors 4.2-9.7). The in vitro data support ... findings in workers that the percutaneous absorption of aromatic amines significantly increases in presence of skin creams. The efficacy of skin creams to protect the percutaneous penetration of aromatic amines is not confirmed...
... Using diffusion cells ... dermal penetration of ...ortho-toluidine (OT) through human skin /was measured/. /OT/ penetrates through human skin fast (lag time: approximately 0.8 hr) and in high percentages (50% of the applied dose within 24 hr). A skin notation is therefore justified...
Distribution of the carcinogenic agent orthotolidine (OT) in the rat's body has been investigated by diazotizing technique. Levels of free amines were 1.5-2 times those in control 3 days after the last administration (total treatment duration-8 months). Zymbal gland, the target tissue, showed the highest concentrations. Also, bound amine concentrations in Zymbal gland soared up (8 times those in control). Similar results were reported 7 days after the last administration of OT: free amine level in the same site was 7 times that in control. Enhanced free and bound amine concentrations may point to its being withdrawn through Zymbal gland ducts. This may also promote carcinogenesis in them.
For more Absorption, Distribution and Excretion (Complete) data for 2-Aminotoluene (21 total), please visit the HSDB record page.

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Metabolism / Metabolites
Male F344 rats were given sc injection of 50 or 400 mg/kg o-[methyl-(14)C]toluidine ... The results shown that N-acetylation and hydroxylation at the 4 position of o-toluidine are major metabolic pathways in rats. Minor pathways include hydroxylation at the 6 position, oxidation of the methyl group and oxidation of the amino group. Sulfate conjugates predominate over glucuronides by a ratio of 6:1. Detected substances in the urine: o-toluidine, azoxytoluene, o-nitrosotoluene, N-acetyl-o-toluidine, N-acetyl-o-aminobenzyl alcohol, 4-amino-m-cresol, N-acety-4-amino-m-cresol, anthranilic acid, N-acetylanthranilic acid, 2-amino-m-cresol, and unidentified substances. Substances which were excreted via feces or via exhaled air were not mentioned.
4 dogs, older than one year, received i.v. injections of o-toluidine hydrochloride, 0.77 mM/kg bw = 111.1 mg/kg bw dissolved in water. The concentration of o-toluidine in blood was observed for a period of 6 hours ... Products from N-oxidation were extracted from blood with carbon tetrachloride ... Hemoglobin was estimated by measuring the increase of the extinction at 550 u that was caused by adding cyanide to a blood solution of pH = 6.8. Half-life time of plasma elimination was approximately half an hour. 7 hr post application approximately 10 ug o-toluidine per mL of blood was found. Hemoglobin content per total blood pigment versus time course in hours increased with increasing time from application, max value was reached 6 hr post application indicating the presence of a reactive oxidation product. Carbon tetrachloride extracts did not contain o-nitrosotoluene.
Male F344 rats (230-260 g b.w.) were s.c. injected with 0.82 mmol/kg bw o-toluidine as corn oil solution. Urines were collected over a time period of 6 hours. All urines were analyzed within 4 hr of collection using high performance liquid chromatography with electrochemical detection. The amounts of N-hxdroxy-o-toluidine found in the 0-6 hr urines of rats, treated with 0.82 mmol/kg bw o-toluidine, ranged from 0.04 to 0.36 umol.
Determination of metabolizing enzymes in rat liver, kidneys and lung. Male Wistar rats, body weight: 200 - 250 g (6 per group), were given 0, 75 mg/kg bw o-toluidine in sunflower oil ip for 3 days. Rats were fasted for 12 hr after the last administration before being killed. Rats were decapitated on the fourth day, livers, kidneys, and lungs were immediately excised, weighed and homogenized. Analysis were performed (method not mentioned); differences were assumed to be significant when p < 0.05. Effects on enzyme activities by organ: LIVER: Cytochrome b5: 0.545 vs 0.447 nmol/mg protein; NADPH cytochrome c reductase: 201.3 vs 165.8 nmol/mg protein/min; /Aryl hydrocarbon hydrolase/ (AHH): 654 vs 295 pmol/mg protein/min; ratio of Glutathione-S-transferase to AHH activities: 1926 vs 3969 nmol/mg protein/min; ratio of Epoxide Hydrolase to AHH activities: 1.85 vs 3.35 nmol/mg protein/min. KIDNEYS: AHH: 70.35 vs 2.91 pmol/mg protein/min; ratio of Glutathione-S-transferase to AHH activities: 2840 vs 63780 nmol/mg protein/min; ratio of Epoxide Hydrolase to AHH activities: 1.24 vs 34.02 nmol/mg protein/min. LUNGS: AHH: 9.49 vs 4.75 pmol/mg protein/min; ratio of Glutathione-S-transferase to AHH activities: 5184 vs 12484 nmol/mg protein/min; ratio of Epoxide Hydrolase to AHH activities: 4.00 vs 9.89 nmol/mg protein/min.
For more Metabolism/Metabolites (Complete) data for 2-Aminotoluene (14 total), please visit the HSDB record page.

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Biological Half-Life
Following oral application of 500 mg toluidine/kg bw to rats (strain not given) a half-life time of plasma elimination of 12 to 15 hours was derived; i.v. application of approx. 111 mg/kg bw to dogs yielded a half-life time of plasma elimination of half an hour.

Toxicity Summary
IDENTIFICATION: ortho-Toluidine is a synthetic chemical that is a light yellow liquid at ambient temperature. It is used primarily in the manufacture of dyestuffs, although it is also used in the production of rubber, chemicals and pesticides and as a curing agent for epoxy resin systems. HUMAN EXPOSURE: In the occupational environment, there is the potential for significant carcinogenic and genotoxic effects. ANIMAL STUDIES: o-Toluidine is of moderate to low acute toxicity and has the potential to produce minimal skin irritation and mild irritation. Information was not available on the skin or respiratory sensitization potential of o-toluidine. The principal signs of toxicity following acute short term exposure to this chemical are methemaglobinemia and related effects on the spleen. These effects have been observed in rats administered o-toluidine at 225 mg/kg bw/day for 5 days. A no observed adverse effect level (NOEL) has not been identified. In several carcinogenicity studies in which o-toluidine wasadministered orally to rats and mice, there was a significant increase in the incidence of benign and malignant tumors in various tissues. o-Toluidine is generally non-mutagenic in standard bacterial mutagenicity tests, but is clastogenic in mammalian cells in vitro. There is uncertainty concerning the genotoxicity of o-toluidine in vivo; however, some positive results have been reported. Based on the wide distribution of tumors in o-toluidine exposed animals, as well as the clastogenic activity observed in mammalian in vitro assays, o-toluidine may be acting as a genotoxic carcinogen. Information relevant to assessing the risks of reproductive or developmental effects of o-toluidine was not identified.

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Toxicity Data
LC50 (rat) = 862 ppm/4h

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Interactions
SC ADMIN OF BENZIDINE & O-TOLUIDINE SIMULTANEOUSLY TO 116 RATS RESULTED IN EARLIER APPEARANCE OF TUMORS. LATENT PERIOD FOR BENZIDINE WAS TWICE AS LONG AS WITH COMBINED USE (548.6 DAYS & 264.1 DAYS).

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Non-Human Toxicity Values
LD50 Rat oral 940 mg/kg
LD50 Rat oral 670 mg/kg
LD50 Rat Wistar oral 0.75 mL/kg bw (750 mg/kg bw)
LD50 Rat Sprague-Dawley male ip 164 mg/kg bw
For more Non-Human Toxicity Values (Complete) data for 2-Aminotoluene (15 total), please visit the HSDB record page.

[1]. Impact of Deuterium Substitution on the Pharmacokinetics of Pharmaceuticals. Ann Pharmacother. 2019 Feb;53(2):211-216.

o-Toluidine can cause cancer according to an independent committee of scientific and health experts.
O-toluidine appears as a clear colorless or light yellow liquid. May become reddish brown on exposure to air and light. Flash point 185 °F. Has about the same density as water and is very slightly soluble in water. Vapors are heavier than air. Confirmed carcinogen.
O-toluidine is an aminotoluene in which the amino substituent is ortho to the methyl group. It has a role as a carcinogenic agent.
o-Toluidine is primarily used in the manufacture of dyes. o-Toluidine is highly toxic to humans when absorbed through the skin, inhaled as vapor, or swallowed. Acute (short-term) exposure of humans to o- toluidine affects the blood (i.e., methemoglobinemia), with clinical signs of central nervous system depression. The chronic (long-term) effects in workers exposed to o-toluidine include anemia, anorexia, weight loss, skin lesions, central nervous system depression, cyanosis, and methemoglobinemia. Animal studies indicate that chronic exposure too-toluidine causes effects on the spleen, liver, urinary bladder, and blood. Occupational exposure to dyestuffs (including o-toluidine) is associated with an increased risk of bladder cancer. 2-Methylaniline hydrochloride (the hydrochloride salt of o-toluidine) was carcinogenic in rats and mice. o-Toluidine has been classified by EPA as a Group B2, probable human carcinogen.
o-Toluidine has been reported in Camellia sinensis with data available.
o-Toluidine is a synthetic, light sensitive, light yellow liquid that is slightly soluble in water and miscible with carbon tetrachloride, diethyl ether and ethanol. The hydrochloride is a synthetic, light sensitive, white crystalline powder that is soluble in dimethylsulfoxide and ethanol. o-Toluidine and o-toluidine hydrochloride are used primarily as intermediates in the manufacture of dyes and pigments. When heated to decomposition, o-toluidine emits toxic fumes of nitrogen oxides while the hydrochloride also produces hydrochloric acid. Four studies of workers exposed to o-toluidine reported an excess of bladder cancers. o-Toluidine and o-toluidine hydrochloride are reasonably anticipated to be human carcinogens. (NCI05)
The chemical properties of the toluidines are quite similar to those of aniline and toluidines have properties in common with other aromatic amines. Due to the amino group bonded to the aromatic ring, the toluidines are weakly basic. None of the toluidines is very soluble in pure water, but will become soluble if the aqueous solution is acidic due to formation of ammonium salts, as usual for organic amines. At room temperature and pressure, ortho- and meta-toluidines are viscous liquids, but para-toluidine is a flaky solid. This can be explained by the fact that the p-toluidine molecules are more symmetrical and fit into a crystalline structure more easily. p-Toluidine can be obtained from reduction of p-nitrotoluene. p-Toluidine reacts with formaldehyde to form Troger's base.

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Mechanism of Action
Mechanisms of DNA damage by metabolites of carcinogenic o-toluidine in the presence of metals were investigated by the DNA sequencing technique using (32)P-labeled human DNA fragments. 4-Amino-3-methylphenol, a major metabolite, caused DNA damage in the presence of Cu(II). Predominant cleavage sites were thymine and cytosine residues. o-Nitrosotoluene, a minor metabolite, did not induce DNA damage even in the presence of Cu(II), but addition of NADH induced DNA damage very efficiently. The DNA cleavage pattern was similar to that in the case of 4-amino-3-methylphenol. Bathocuproine and catalase inhibited DNA damage by these o-toluidine metabolites, indicating the participation of Cu(I) and H(2)O(2) in the DNA damage. Typical free hydroxyl radical scavengers showed no inhibitory effects on the DNA damage. o-Toluidine metabolites increased the formation of 8-oxo-7,8-dihydro-2'-deoxyguanosine in calf thymus DNA in the presence of Cu(II). UV-visible and ESR spectroscopic studies have demonstrated that 4-amino-3-methylphenol is autoxidized to form the aminomethylphenoxyl radical and o-nitrosotoluene is reduced by NADH to the o-toluolhydronitroxide radical in the presence and absence of Cu(II). Consequently, it is considered that these radicals react with O(2) to form O(-)(2) and subsequently H(2)O(2), and that the reactive species generated by the reaction of H(2)O(2) with Cu(I) participate in the DNA damage. Metal-mediated DNA damage by o-toluidine metabolites through H(2)O(2) seems to be relevant for the expression of the carcinogenicity of o-toluidine.
The in vivo covalent binding of o- and p-toluidine to rat hepatic macromolecules was investigated to determine if a relationship exists between the degree of binding for each isomer and its carcinogenic potency. The ortho-isomer has been shown to be a more potent hepatocarcinogen than the para-isomer. In addition to the macromolecular binding, the tissue distribution of each isomer was also measured. The degree of binding to hepatic macromolecules appeared to be at maximum for both at 24-28 hr following dosing. At 24 hr following dosing, the level of DNA binding of o-toluene was approximately 1.2-fold lower than that of p-toluene. The binding to RNA and protein was also lower for o-toluene than p-toluene, although the differences were not as great as that observed for DNA binding. There were subtle differences in tissue distribution for each isomer. However, in contrast to the macromolecular binding data, the area under the plasma concentration curve for o-toluene was approximately 1.8-fold greater than that for p-toluene. Based on the results of these studies, there was no direct correlation between the degree of macromolecular binding and carcinogenic potency.

C7H2D7N

114.20

114.117

68408-22-0

97917-08-3;636-21-5 (hydrochloride)

7242

Colorless to light yellow liquid

2.7 °F (NTP, 1992)
-14.41 °C
Melting point: -23.7 °C (alpha form); -14.7 °C (beta form)
-16.3 °C
-16 °C (beta-form)
2.7 °F
6 °F

2.158

1

1

0

8

70.8

0

[2H]C1=C([2H])C([2H])=C(N)C(C([2H])([2H])[2H])=C1[2H]

RNVCVTLRINQCPJ-UHFFFAOYSA-N

InChI=1S/C7H9N/c1-6-4-2-3-5-7(6)8/h2-5H,8H2,1H3

2-methylaniline

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

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

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

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.)