myo-inositol-hexakisphosphate 3-phosphohydrolase

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Bacterial reverse mutation assay (Ames test on the source substance 3 -Phytase, OECD 471, GLP): not mutagenic.
in-vitro mammalian chromosome aberration test RA from the source substance 3 -Phytase (OECD 473, GLP): not clastogenic

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Additional information

Data on the genetic toxicity of 3 -Phytase (IUBMB 3.1.3.8 / CAS 37288 -11 -2) are available.

The substance 3 -Phytase was tested in the S. typhimurium reverse mutation assay with four histidine-requiring strains of S. typhimurium (TA1535, TA1537, TA100 and TA 98) and in the E. coli reverse mutation assay with a tryptophan-requiring strain of E. coli WP2 uvrA. The test was performed in two independent experiments in the presence and absence of S9-mix (Aroclor-1254 induced rat liver S9-mix). The maximum concentrations tested were 3330 µg/plate. At concentrations of 1000 and 3330 µg/plate the protein precipitated but toxicity for the test organisms was not observed. 3-Phytase did not induce a dose related two-fold increase in the number of revertant (His⁺) colonies in each of the 4 tester strains (TA1535, TA1537, TA100 and TA 98) and in the number of revertant (Trp⁺) colonies in tester strain WP2 uvrA both in the absence and presence of S9-metabolic activation at any of the tested concentrations. These results were confirmed in an independently repeated experiment. Based on the results of this study it is concluded that 3 -Phytase is not mutagenic in the S. typhimurium reverse mutation assay and in the E. coli reverse mutation assay.

The potential of 3 -Phytase to induce chromosome aberrations in cultured human peripheral lymphocytes has been investigated in a study conducted according to OECD 473 and in compliance with GLP (Buskens, 2004). 3 -Phytase was tested up to 5000 µg/mL for a 3 h exposure time with a 24 h and 48 h fixation time in the absence and presence of 1.8% (v/v) S9-fraction, respectively. Furthermore, 3 -Phytase was tested up to 3330 µg/mL for a 24 h continuous exposure time with a 24 h fixation time and up to 5000 µg/mL for a 48 h continuous exposure time with a 48 h fixation time in the absence of S9-mix, respectively. No evidence of a test substance related increase in the number of cells with aberrations was observed with or without metabolic activation in short- and long-term treatments in the main experiments. Also in the absence of S9-mix, at the 48 h continuous exposure time, 3 -Phytase did not induce statistically significant, dose dependent increase in the number of cells with chromosome aberrations, both when gaps were included and excluded. The results of the solvent and positive controls were within the range of the historical control data. In conclusion, 3 -Phytase was found to be not-clastogenic to human lymphocytes under the experimental conditions described in this report.

Microbial enzyme preparations are used extensively in food processing and therefore their hazard to human health has been studied thoroughly. There is no data to suggest that enzymes are in any way DNA-reactive. Up to now no enzyme preparations have been demonstrated to be mutagenic in in vitro test systems (Pariza and Johnson et al., 2001). In fact, 102 bacterial mutagenicity tests (according to OECD 471 and 472) and 63 chromosome aberration/mutagenicity tests (OECD 473, 474 and 476) with enzymes preparations are available to date (Pariza and Johnson et al., 2001). Out of these tests, seven of the Ames and six of the chromosomal aberration tests were found to be false positive. The remaining 152 tests (95 Ames and 57 chromosome aberration) were all negative. The false positive results observed in the Ames tests were demonstrated to be due to the growth-enhancing effects of histidine in the enzyme preparations. The false positive results observed in the chromosome aberration tests were attributed to the following: 

 

-         Additional studies on the same endpoint are available, in different in vitro test systems, which all gave negative results.

-         The production of hydrogen peroxide by some enzymatic reactions, which is known to act as a clastogen.

-         No in vivo tests are available to confirm the positive in vitro tests. The main reason for this is because enzymes are metabolised in the body to amino-acids and ceases to exist.

 

These findings underscore the conclusion that testing enzymes preparations in traditional and genetically modified micro-organisms for genotoxicity is unnecessary for safety evaluation. Furthermore, mutagenicity in an in vivo test is a systemic effect, as the agent has to be transported to the target organ. Enzymes are hydrolysed like any protein before absorption in the body, therefore even if enzymes are mutagenic; its mutagenic activity will be lost before it can reach the appropriate cells.  

 

Reference:

Pariza, M. W., and Johnson, E. A. (2001). Evaluating the Safety of Microbial Enzyme Preparations Used in Food Processing: Update for a New Century. Regulatory Toxicology and Pharmacology, 33: 173-186.