Team:Aachen/Safety

Welcome to iGEM Aachen 2016

Toxicological Assessment

In order to evaluate the safety of our LIPs, we made a toxicological assessment, considering how humans and the environment could be affected when getting in touch with it and how probable an exposure is. We collected all data we could on the substance we want to replace. Furthermore, we examined the cleavage product of the protection group we intended to use. We compared the required amounts of chemicals with the existing solution to the amount that would be needed for ours as well as the dangers those substances pose.

Exposure


In our daily lives we get in touch with the contents of liquid washing detergents in several situations:

A. Production
During production of washing detergents, the factory workers are potentially exposed to high doses of the inhibited protease. There is a risk of dermal contact and inhaling the compounds.

B. Home usage
When washing at home the washing detergent can be spilled onto the skin of user. Thereafter residues of the washing liquid can remain in the clothes because of incomplete rinsing and cause dermal exposure.

C. Drinking water
Waterborne substances like boric acid, which cannot be filtered out in purification plants expose consumers via their drinking water.

Toxicology


Toxicology of boric acid
dermal:
LD50 rabbit :  > 2,000 mg kg [1]

oral:
LD50 rat :  2,660 mg kg [2]

Boric acid reacts with the polyhydroxyl ribitol side chain of riboflavin. This increases its water solubility and reduces the amount of available riboflavin in animals and humans [3]

fatal dose human: [4]

  1. 2,000 - 3,000 mg for infants
  2. 5,000 - 6,000 mg for children
  3. 15,000 - 20,000 mg for adults

inhaling:
LC50 rat :  > 0.16 mg L ∙ hr [5]

reprotox:
Boric acid is classified as substance of very high concern [6] and is toxic for fertility. [7-8]


Toxicology of photocaged amino acids and their cleavage products
ONBY (Ortho-nitrobenzyl-tyrosine) and DMNBS (dimethyl-nitrobenzyl-serine) and their cleavage products ONB (2-nitrosobenzaldelyde) and DMNB (Dimethyl-nitrosobenzaldehyde) are not yet characterized in toxicology. Approximate predictions can be made through known data about similar chemicals as other nitroso compounds.

Figure 1: ONB-tyrosine cleavage reaction
Figure 2: DMNB-serine cleavage reaction


Nitrosobenzene
Nitrosobenzene reacts in the human blood circulation with hemoglobin and minimizes its ability of O2 uptake.[9] Acute and chronic toxicity data are unavailable.


Other protection groups
To avoid the risks of the nitrosobenzyl derivatives there are a lot of different photo protection groups, which could be used instead of the ONB and DMNB.[10]


Comparative toxicological risk assessment


In absence of solid toxicity data predominantly for the cleavage products of the photo protection group of the enzyme it is difficult to impossible to conclude on the comparative risk assessment.

Assuming that both boric acid and the photo cleavage degradation product have similar toxicities the project approach has a clear advantage because of the much lower concentration in the product. While there are 16 mmol boric acid per kg laundry detergent the concentration of the photocaged amino acid is only 3,6 nmoles per kg. For every photocaged molecule of amino acid one molecule of protection group exits.

The common liquid laundry detergent contains 0.5 - 1% (w/w) of boric acid and 0.00005 - 0.0001% (w/w) active protease. [11]


Molecules of subtilisin in 100 g of liquid laundry detergent
For the molecular weight of subtilisin E we pasted our known amino acid sequence of 275 AAs into a calculation tool, the DNA sequence can be seen in part BBa_K2020023 [12]

weight of 1 molecule subtilisin E =  27.02 kDa

M (subtilisin E)  =  27701.06 g mol
m (subtilisin E) =  0.0001% ∙ 100 g = 0.0001 g

n (subtilisin E) = 0.0001 g 27701.06 g mol = 3.6 ∙ 10-9 mol

molecules of boric acid in 100 g liquid laundry detergent

M (boric acid) =  61.83 g mol
m (boric acid) =  1% ∙ 100 g = 1 g

n (boric acid) =  1 g 61.83 g mol = 0.01617 mol

molecules of boric acid per molecule of subtilisin E

n (boric acid) n (subtilisin E) =  448300

Conclusion


As the concentration of cleaved photo protection groups is almost 480,000 times lower than the concentration of boric acid its toxicity is allowed to be 448,000 fold higher for the same toxicity of the liquid washing detergent. If the toxicity of cleavage products is less than 448,000 fold higher than the toxicity of boric acid, washing detergents with replaced boric acid were less toxic than the actual.


Example for one washing cycle (100 g liquid laundry detergent)
For this calculation we assume that 1% of the liquid detergent stays in the laundry after washing and the composition is like in the calculation above.


DMNB and ONB group
n (cleaved group) = n (subtilisin E) =  1% ∙ 0.0001 g 27701.06 g mol = 3.6  ∙ 10-11 mol
M (DMNB) = 195 g mol
M (ONB) = 135 g mol
m (DMNB) =  7 ∙ 10-9 g

m (ONB) =  4.9 ∙ 10-9 g


Boric acid
1% ∙ 1 g = 0.01 g

In this scenario 0.1 g of boric acid or 7 ng of DMNB or 4.9 ng of ONB remain in the laundry.


Comparative environmental risk assessment


The diluted compounds of washing detergents end up in the waste water and consequently in the waste water treatment plant.

Boric acid is highly water soluble and can be neither eliminated in the waste water treatment plant nor in the drinking water purification plant. [13]

The LIPs products are organic products, which could be eliminated by a biological waste water treatment plant but this remains to be verified as there is no related data available. As organic compounds they could most likely be filtered by active charcoal and others whereby human exposure by the drinking water can be excluded. Also, organic compounds will likely be degraded over time.


Laboratory Satefy Aspects

Used organism strains


  1. Escherichia coli DH5α
  2. Escherichia coli BL21 DE3
  3. Saccharomyces cerevisiae
  4. Bacillus subtilis

Potential risks


Our project poses the typical risks of working in a biology lab. So we reduce those risks by fulfilling safety level 1 procedures as seen below.


Waste treatment
All biological materials (including genetically modified organism (abbreviation GMO)) or equipment, that was used for handling, is collected separately and autoclaved.


Transportation:
Closed boxes are used for transportation between labs but if possible, transportation between labs should be avoided.


Emergency reaction:

  1. If lab coats or clothes start burning, use emergency showers.
  2. If acids or other harmful liquids get into the eyes, use eye showers.
  3. If injuries occurred, treat them with first aid-kits, report the injury, and go to the hospital if necessary.
  4. If solutions with GMO is running down the bench (or other kinds of contamination), swap it and disinfect the place with Bacillol.

Protective equipment:

  1. Lab coats are mandatory.
  2. Safety glasses are mandatory in special areas of the lab and should be used in other areas if necessary.
  3. Usage of safety gloves are mandatory in special areas of the lab and should be used depending on the working materials.

Other rules:

  1. Do not work alone. There should be at least one person from the lab, who is not part of the iGEM team.
  2. Do not pipette with your mouth.
  3. Disinfect hands after contamination with GMOs.
  4. Disinfect hands and wash them after working in the lab.

Furthermore our project includes the handling of non-canonical amino acids, which we treat according to their special requirements.

Laws and regulations regarding biosafety in germany


  1. Gesetz zur Regelung der Gentechnik: Bundesgesetzblatt (year 2010, page 1934, in german)
  2. Biostoffverordnung: Bundesgesetzblatt (year 2013, page 2514, in german)


References

[1]R. Krieger, “Handbook of Pesticide Toxicology”, Academic Press, San Diego California, volume 2, no. 2, p. 1414, 2001. [2]R. J. Sr. Lewis, “Sax's Dangerous Properties of Industrial Materials”, Wiley-Interscience, 11th Edition, p. 536, 2004. [3]J. T. Pinto, R. S. Rivlin, “Drug-Nutrient Interactions”, vol. 5, pp. 143-51, 1987. [4]R. Krieger, “Handbook of Pesticide Toxicology”, Academic Press, San Diego California, volume 2, no. 2, p. 1430, 2001. [5]European Chemicals Bureau, “IUCLID Dataset for Boric Acid”, 2000 CD-ROM edition, p.26, Oct. 2011. [6]European Chemicals Agency, “Comments and Response to Comments on Annex XV SVHC: Proposal and Justification”, 2010. [Online]. Available: https://echa.europa.eu. [Accessed: 19-Oct-2016]. [7]Human Health and Environment Task Force, “Human and Environmental Risk Assessment on ingredients of Household Cleaning Products”, 2015. [Online]. Available: http://www.heraproject.com. [Accessed: 19-Oct-2016]. [8]R. E. Chapin and W. W. Ku, “The reproductive toxicity of boric acid.,” Environ. Health Perspect., vol. 102, no. Suppl 7, pp. 87–91, Nov. 1994. [9]H. U. Käfferlein et al., “Bildung von Methämoglobin durch Anilin”, IPA-Journal, pp.26, Jan. 2014. [10]P. Klán et al., “Photoremovable Protecting Groups in Chemistry and Biology: Reaction Mechanisms and Efficacy,” Chem. Rev., vol. 113, no. 1, pp. 119–191, Jan. 2013. [11]Marktforschung Dalli-Werke Stolberg GmbH & Co. KG [12]Science Gateway, “Tools: Protein Molecular Weight Calculator”, [Online]. Available: http://www.sciencegateway.org. [Accessed: 19-Oct-2016]. [13]Barufke et al., “Bor: Ableitung einer Geringfügigkeitsschwelle zur Beurteilung von Grundwasserverunreinigungen“, LUBW. Landesanstalt für Umwelt, Messungen und Naturschutz Baden-Württemberg, Feb. 2012

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