In order to evaluate the safety of the product we intend to produce, 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 and the cleavage product of the protection group we intend to use and compared the required amounts for the existing solution to ours as well as the dangers both 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 which cannot be filtered out in purification plants expose consumers via their drinking water.

Toxicology

Toxicology of boric acid
dermal:
LD₅₀ rat :  > 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 man [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 :  > 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.

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 had a clear advantage because of the much lower concentration in the product. While there are 0.01617 moles boric acid per kg laundry detergent the concentration of the photocaged amino acid is only 3,6 ∙ 10^-9 moles 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 500,000 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.