Tony Henwood, Histopathology, the Children’s Hospital at Westmead, NSW, Australia
Most of the ethanols used in Histopathology are Denatured Alcohols. Denatured alcohol, also called methylated spirit (methylated spirits in Australia and New Zealand) or denatured rectified spirit, is ethanol that has additives to make it poisonous, bad tasting, foul smelling or nauseating, to discourage recreational consumption. In some cases, it is also dyed. Pyridine, methanol, or both can be added to make denatured alcohol poisonous, and denatonium can be added to make it bitter (1).
There is a dark side to Denatured Alcohol. In the 1920’s, during the American Prohibition era, frustrated that people continued to consume so much alcohol even after it was banned, federal officials had decided to try a different kind of enforcement. They ordered the poisoning of industrial alcohols manufactured in the United States, products regularly stolen by bootleggers and resold as drinkable spirits. The practice was called “denaturing”. The idea was to scare people into giving up illicit drinking. Instead, by the time Prohibition ended in 1933, the federal poisoning program, by some estimates, had killed at least 10,000 people (2). “They put all kinds of poisonous stuff into the alcohol. There was benzene, there was mercury, there was this list of formulas that's heart-stopping horrible. But in particular they put more wood alcohol, or methanol, because their own tests showed bootleggers couldn't get it out — it's too closely bonded to the drinking alcohol” (2).
Ethanol is the commonly used dehydrant in histology. However, because it is drinkable, pure ethanol is taxed and regulated in many countries. Ethanol is also available with chemicals added to make it undrinkable. These government approved denatured formulations are less regulated and require minimal documentation to purchase. “Reagent Alcohol,” comprised of approximately 90% ethanol, 5% methanol and 5% isopropanol, is one example of a denatured ethanol. Only this blend can be labelled as “Reagent Alcohol.” Alcohols labelled as “denatured ethanol” use other chemical denaturants (e.g., gasoline, ammonia, pine tar) that can possibly cause excess drying of tissue samples; they should only be used after validation (3).
So what are the “Denaturants” added to the ethanols that we are likely to use in Histopathology. Looking at those MSDS available on-line can give us some indications.
Methyl isobutyl ketone - The typical toxicity effects of Methyl isobutyl ketone in humans exposed at 50 to 100 ppm are mucous membrane irritation and weak effects on the central nervous system (CNS) such as headache.
Denatonium Benzoate - Denatonium benzoate gets its name from "denatured alcohol", and that is where it is often used. It is the bitterest tasting substance known.
Heptane – It has been reported that exposure to 1000 ppm of heptane for 6 minutes caused slight dizziness in humans; exposures to higher levels caused vertigo, incoordination, and inappropriate behaviour (4).
Ethyl acetate – has a sweet smell and it is used to impart flavour to any fruit flavoured candy, baked food, gum etc. It is highly flammable, as well as toxic when ingested or inhaled, and this chemical can be seriously damaging to internal organs in the case of repeated or prolonged exposure.
Iso-propanol - acts as central nervous system (CNS) depressants, and around 15 g of isopropyl alcohol can have a toxic effect on a 70-kg human if left untreated. Poisoning can occur from ingestion, inhalation, or skin absorption.
So, we have another issue we need to be aware of in Histotechnology: The appropriate use of “Denatured Alcohol”. It might be prudent to consider possible difficulties that the use of this reagent might cause to our stains and processes.
1. Wikipedia “Denatured Alcohol” https://en.wikipedia.org/wiki/Denatured_alcohol
2. Blum, D., “The Chemist’s War”.
4. Feldman, A. T., & Wolfe, D. (2014). Tissue processing and hematoxylin and eosin staining. In Histopathology (pp. 31-43). Humana Press, New York, NY.
5. NIOSH “Heptane” https://www.cdc.gov/niosh/pel88/142-82.html.