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Genetic toxicity in vitro

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Reference
Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: The study is well conducted and documented, however no OECD guideline was followed and the study is not GLP
Principles of method if other than guideline:
19 amino acids were tested for mutagenicity in E. coli K-12 uvrB DNA repair-deficient strain. The specific goal was finding a type of mutagenesis that is regulated by DNA repair genes.
GLP compliance:
no
Type of assay:
other: DNA repair assay in bacteria
Target gene:
uvrB gene, which functions in the error-free excision repair of many bulky mutagenic lesions
Species / strain / cell type:
other: K-12 strain
Details on mammalian cell type (if applicable):
DNA repair-deficient strains: SR250, SR 251, SR256 and SR1034.
Wild type strain: SR248
Test concentrations with justification for top dose:
2 mM
Untreated negative controls:
yes
Details on test system and experimental conditions:
19 amino acids were individually tested for their ability to revert lacZ53 cells to Lac+
Logarithmic-phase cells were prepared by diluting overnight cultures 1:50 and shaking the cultures at 37°C until a concentration of 4 x 10e8 colony-forming units (CFU) per mL was reached. The cells were pelleted and resuspende at 1.5 CFU/mL.
Cells were spread on Glu-600 plates (600 µg/mL glucose) or Glu-0 plates. On the GLu-600 plates 2 mM of the test compound was added.
Plates were incubated for 3 days at 37°C, after which the Lac+ mutants were scored.
The mean number of mutants was determined and the ration test/control was calculated.
Mean number of preexisting mutants per plate were: 1 (wild type), 15 (uvrB and 0 (uvrB umuC).
Mean number of plate mutants per control plate were: 60 (wild-type), 355 (uvrB), 21 (uvrB umuC).
Evaluation criteria:
2 criteria: The mean mutant frequency +/- 1 SD (range) for the amnio-acid-supplemented plates did not overlap the range for the control plates, and the mean relative (test/control) range for the uvrB strain did not overlap the mean relative range for the uvrB umuC strain.
Statistics:
The mutagenesis was normalized for constant growth.
Species / strain:
other: K-12 strain
Metabolic activation:
not applicable
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Untreated negative controls validity:
valid
Remarks on result:
other: all strains/cell types tested

Effect of amino acids on spontaneous mutagenesis in E. coli

Values are the means of relative mutants per total cells data (mutant frequency on test plates divided by mutant frequency on control plates) from triplicate plate-assay experiments for LacZ53 strains.

Amino acid tested

Relative Lac+mutagenesis in the presence of 2 mM

uvrB

uvrB umuC

Histidine

0.93

1.04

Values did not indicate a significant effect on mutagenesis by either of two criteria.

Conclusions:
Interpretation of results: negative

L-histidine did not show a significant effect on Lac reversion in the uvrB strain, and thus, was found not to bu mutagenic.
Executive summary:

In this study, 19 amino acids were tested to see if their catabolism was mutagenic, using uvrB cells. More specifically, the goal was to find a type of mutagenesis that is regulated by DNA repair genes. The study was not GLP and no OECD guideline was followed. A plate assay for mutagenesis was performed in which E. coli strains were incubated with the test substance for 3 days at 37°C with or without glucose present. Hereafter, Lac+ mutants were scored and the ratio test/control was determined, taking into account the growth-normalization factors.

L-histidine did not show a significant effect on Lac reversion in the uvrB strain, and thus, was found not to be mutagenic.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vitro:

The chromosome aberration assay of Zhang (1992) investigated the effect ot L-histidine on sister-chromatid exchange (SEC) frequency in human peripheral blood lymphocytes. The chromosome preparations were made according to the Giemsa method. No guideline was followed to conduct this study, however, it is similar to the OECD 479. The concentrations tested were 10, 50 and 100 µg/mL.

An increase in the number of SCE was seen at the three tested concentrations, when compared to control cells. A statistical significant dose-related increase in the mean number of the SCEs was not investigated. Nevertheless, it was concluded that L-histidine can induce SCE in human peripheral blood lymphocytes at any concentration.

However, as L-histidine is an essential substance for the growth of the peripheral blood lymphocytes it is very unexpected that it can induce SCE. The current hypothesis is that the exogenous addition of amino acids to the cells can result in an imbalance of the amino acids in the medium, causing metabolic disturbances in the cells. And after influencing the activity of various enzymes this might induce SCE.

In the gene mutation assay of Sargentini and Smith (1986) 19 amino acids, including L-histidine, were tested to see if their catabolism was mutagenic, using uvrB cells. More specifically, the goal was to find a type of mutagenesis that is regulated by DNA repair genes. The study was not GLP and no OECD guideline was followed, however, the study was well conducted and documented. A plate assay for mutagenesis was performed in which E. coli strains were incubated with the test substance for 3 days at 37°C with or without the presence of glucose. Hereafter, Lac+ mutants were scored and the ratio test/control was determined, taking into account the growth-normalization factors.

L-histidine did not show a significant effect on Lac reversion in the uvrB strain, and thus, L-histidine was found not to be mutagenic.

Justification for classification or non-classification

The criteria used for the classification of genotoxicity are Table 3.5.1 and section 3.5.2.3. of the CLP regulation No 1272/2008. According to these criteria L-histidine does not require classification for germ cell mutagenicity. Moreover, the genotoxic/mutagenic potential of L-histidine can be excluded for various reasons. L-histidine is a naturally occuring essential amino acid and a normal constituent in living cells. It is ingested daily in significant amounts and a basic metabolite and building block of all living organisms. Aditionally, L-histidine was shown not be a carcinogen (Ikezaki, 1996).

In conclusion, we can state that L-histidine does not require to be classified according to the criteria of the CLP regulation for this endpoint.