Number
208-EN
Section
General Section
Use
Sector
Health services
Other
Function
Dye
Cleaning agent
Process
Manual activities involving hand contact
Product category
laboratory chemicals
washing ad cleaning products
Application
Substitution Trypan Blue
Abstract
SSI have managed to substitute boric acid, ethidium bromide and ether with acetic acid, a non-mutagenic DNA dye and sodium hydroxide. They are currently working on validation of an alternative to Trypan Blue in cell staining.
Substituted substances
Boric acid
CAS No. 10043-35-3 EC No. 233-139-2 Index No. 005-007-00-2
Chemical group
Boron compounds; inorganic acids
Classification: hazard statements
H360FD May damage fertility. May damage the unborn child
Other adverse effects
The substance is: endocrine disruptor cat. 1 (EU EDC database), as listed in the Substance Database according to SUBSPORT Screening Criteria (SDSC).
Ethidium bromide
CAS No. 1239-45-8 EC No. 214-984-6 Index No. 612-278-00-6
Chemical group
Bromine compounds; nitrogen heterocycles
Classification: hazard statements
H341 Suspected of causing genetic defects
H330 Fatal if inhaled
H302 Harmful if swallowed
Diethylether
CAS No. 60-29-7 EC No. 200-467-2 Index No. 603-022-00-4
Chemical group
Ether
Classification: hazard statements
H224 Extremely flammable liquid and vapour
H302 Harmful if swallowed
H336 May cause drowsiness or dizziness
Trypan blue
CAS No. 72-57-1 EC No. 200-786-7 Index No.
Chemical group
Azo compound
Other adverse effects
The substance is: 2B carcinogen (IARC) as listed in the Substance Database according to SUBSPORT Screening Criteria (SDSC).
Alternative Substances
Acetic acid
CAS No. 64-19-7 EC No. 200-580-7 Index No. 607-002-00-6
Chemical group
Organic acid
Classification: hazard statements
H226 Flammable liquid and vapour
H314 Causes severe skin burns and eye damage
Sodium hydroxide
CAS No. 1310-73-2 EC No. 215-185-5 Index No. 011-002-00-6
Chemical group
Sodium compounds; hydroxides
Classification: hazard statements
H314 Causes severe skin burns and eye damage
Nigrosin (acid black 2)
CAS No. 8005-03-6 EC No. Index No.
Chemical group
Dyes
Reliability of information
Evidence of assessment: there is evidence of an official (positive) assessment of the substitution
Evidence of implementation: there is evidence that the solution was implemented and in use at time of publication
Reason substitution
CMR
ED
neurotoxicant
Hazard Assessment
Substances to be substituted: Ethidium bromide is not listed in the SUBSPORTplus Database and has no harmonised classification according to Annex VI of Regulation (EC) No 1272/2008 (CLP Regulation). Boric acid may is a Substance of very high concern and is included on the REACH candidate list for authorization, according to Article 59 of Regulation (EG) No. 1907/2006. (REACH Regulation). Diethyl ether is not listed in the SUBSPORTplus Database and has no harmonised classification according to Annex VI of Regulation (EC) No 1272/2008 (CLP Regulation). Trypan Blue is a Substance of very high concern and is included on the REACH restriction list, according to Article 73 of Regulation (EG) No. 1907/2006. (REACH Regulation). Alternative substances: Acetic acid is not listed in the SUBSPORTplus Database. Sodium hydroxide is not listed in the SUBSPORTplus Database. Nigrosine (acid black 2) is not listed in the SUBSPORTplus Database and has no harmonised classification according to Annex VI of Regulation (EC) No 1272/2008 (CLP Regulation).
Description of Substitution
SSI is performing advanced diagnostics, health data analysis and vaccine production. Being an international reference laboratory, they have to validate every substitution they make according to GMP (Good Manufacturing Practice), GLP (Good Laboratory Practice) and various ISO-standards. Handling of communicable disease agents require strict disinfection procedures able to inactivate all viruses, bacterial- and fungal spores, as do vaccine production where all protein residues have to be removed between batches; this means that they are still dependent on chlorine based cleaning agents, but they have, through improved management, been able to continuously reduce the amounts. Since the majority proportion of the laboratory staffs are women, many of them between age 20 and 45, substituting CMR-chemicals has been a high priority. The staffs have been open minded regarding implementation of substitutions and novel procedures, and are in general keen on continuing the work on safer chemical agents and technological solutions. A substantial number of diagnostics involves gel electrophoresis of DNA, RNA and proteins, which means handling of gel- and running buffers containing boric acid. TBE-buffer (tris(hydroxymethyl)aminomethane, boric acid, EDTA) used to be the standard, but TAE-buffer (tris(hydroxymethyl)aminomethane, acetic acid, EDTA) works equally well except in rare cases with very reduced band resolution where TBE has to be used. This means that the use of boric acid has dropped to a minimum. Ethidium bromide used for visualization of DNA on gels has been phased out in a number of SSI laboratories, substituted by the much less hazardous EZ-vision™ Three DNA Dye. Degreasing of glass ware and steel beads in vaccine production is now done with sodium hydroxide solution instead of diethyl ether with excellent result. Comments to substituted chemicals: TBE-buffers have been substituted with TAE-buffers in almost all analyses involving gel electrophoresis. There are a few exceptions where the resolutions of DNA, RNA or protein bands are hard to retain, and in those specific cases TBE is still in use. Nigrosine (Acid Black 2) is on trial for substituting Trypan Blue in cell staining and visualization. The substitution needs full validation before it is put to use in standard diagnostics. EZ-vision™ Three DNA Dye has substituted Ethidium Bromide in a number of laboratories.According to the producer this is a non-toxic and non- mutagenic DNA dye.
Case/substitution evaluation
SSI is an international reference laboratory and have to follow strict regulations according to diagnostics and vaccine production. Therefore substitutions are time demanding processes that have to pass several cycles of validation. The beneficial part is that all substitutions perform according to some of the strictest level of regulations (Good Manufacturing Practice, Good Laboratory Practice and several ISO standards). MDSD on EZ-vision is provided by VWR / Bie & Berntsen. The full content could not be evaluated by SUBSPORTplus. The alternatives are all less hazardous and can therefore be considered as positive substitutes.
State of implementation
In use
Enterprise using the alternative
SSI National Institute for Health Data and Disease Control www.ssi.dk">http://www.ssi.dk">www.ssi.dk
Availability of Alternative
On the market.
Type of information supplier
Authority
Research
User
Contact
Date, reviewed
November 26, 2021