Absorption, Distribution and Excretion
High-performance liquid chromatography (HPLC) was used to determine the presence of benzidine and its monoacetyl and diacetylbenzidine in urine samples from 18 workers at a small Direct Black 38 production facility. Acetylated metabolites were detected in all urine samples, and benzidine was detected in all but two. Two workers showed extremely high excretion rates of benzidine and its metabolites in their urine. This study highlights the potential risk of bladder cancer in such production facilities in developing countries, where benzidine-based dye production is not yet regulated. This study also investigated the absorption timelines of two azo dyes on rat and rabbit skin. The study used the benzidine-derived dye CI Direct Black 38 and a related dye CI Direct Black 19, the latter replacing benzidine with p-phenylenediamine. Male New Zealand white rabbits and male Fischer 344 rats undergoing specific pathogen clearance (SPF) were shaved on the left back, and a 2 cm × 2 cm area was smeared with the radiolabeled dye. …The levels of radioactive material in the urine and feces of rats treated with CI Direct Black 38 were extremely low. After 144 hours, the cumulative excretion of radioactive material in urine was 0.05% of the total skin dose, and in feces, it was 0.16%. The cumulative excretion of radioactive material in rabbits treated with CI Direct Black 38 was higher after 144 hours: 3.12% of the total skin dose in urine and 5.12% in feces. After 144 hours, the excretion of radioactive material in both animals treated with CI Direct Black 38 continued to increase. Under experimental conditions, CI Direct Black 38 was less stable on rabbit skin than CI Direct Black 19, although differences in skin permeability and microbiome may be partly to attributable to this.

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Metabolisms/Metabolites
Urine analysis of hamsters following a single administration of purified Direct Black 38 (100 mg/kg) showed high concentrations of benzidine, monoacetylbenzidine, diacetylbenzidine, 4-aminobenzidine, and alkaline hydrolysates of benzidine and 4-aminobenzidine in the urine within the first 8 hours of excretion.
Studies have shown that Direct Black 38 is metabolized in animals to form benzidine, a human carcinogen.
Preliminary field studies by the National Institute for Occupational Safety and Health (NIOSH) also indicated higher-than-expected levels of benzidine in the urine of individuals exposed to these dyes (Direct Black 38, Direct Brown 95, and Direct Blue 6), warranting increased vigilance. Both laboratory and field studies demonstrate that these benzidine-derived dyes can be metabolized to benzidine, which is present in the urine of both animals and humans. Based on data from a short-term study, scientists at the National Cancer Institute believe that exposure to benzidine derivative dyes may have carcinogenic potential, most likely through the metabolism of the dye into benzidine in animals. In a 13-week subchronic toxicity study of Direct Black 38, researchers detected benzidine and monoacetylbenzidine in the urine of rats and mice. Analysis of 24-hour urine samples at 4 and 12 weeks (rats) and 3 and 11 weeks (mice) showed a significant correlation between the concentration of dye in food and the amount of benzidine excreted. Mice bioconverted the dye into benzidine at a much higher rate than rats. For more complete data on the metabolism/metabolites of CI Direct Black 38 (out of 10), please visit the HSDB records page.

Toxicity Data
LCLo (Rat) = 180,000 mg/m³/1h

[1]. Beije B. Induction of unscheduled DNA synthesis in liver and micronucleus in bone marrow of rats exposed in vivo to the benzidine-derived azo dye, Direct Black 38. Mutat Res. 1987 Apr;187(4):227-34.

According to an independent committee of scientific and health experts, Direct Black 38 (technical grade) may be carcinogenic. Direct Black 38 is a gray-black microcrystalline or black powder. (NTP, 1992) Direct Black 38 is a benzidine-based azo dye that metabolizes in the body to free benzidine. Direct Black 38 is primarily used for dyeing textiles, leather, and paper. Benzidine and its metabolites have been detected in the urine of workers exposed to benzidine-based dyes. Exposure to this dye is strongly associated with the development of bladder cancer. (NCI05)

C34H25NA2O7S2

781.7295

781.111

1937-37-7

Direct Black 38 free acid;22244-14-0

5284349

Light brown to black solid powder

1.61g/cm3

109-110°C

1.768

11.918

4

16

7

54

1460

0

XRPLBRIHZGVJIC-UHFFFAOYSA-L

InChI=1S/C34H27N9O7S2.2Na/c35-22-10-15-27(26(36)18-22)41-38-24-11-6-19(7-12-24)20-8-13-25(14-9-20)40-42-32-28(51(45,46)47)16-21-17-29(52(48,49)50)33(34(44)30(21)31(32)37)43-39-23-4-2-1-3-5-23;;/h1-18,44H,35-37H2,(H,45,46,47)(H,48,49,50);;/q;2*+1/p-2

disodium;4-amino-3-[[4-[4-[(2,4-diaminophenyl)diazenyl]phenyl]phenyl]diazenyl]-5-hydroxy-6-phenyldiazenylnaphthalene-2,7-disulfonate

NSC-47756; NSC 47756; C.I. Direct Black 38

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 (e.g. under nitrogen), avoid exposure to moisture and light.

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

DMSO : ~25 mg/mL (~31.98 mM)
H2O : ~5 mg/mL (~6.40 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (3.20 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (3.20 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
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.

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Solubility in Formulation 3: 3.85 mg/mL (4.92 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with ultrasonication (<60°C).

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