HPRT Assay

HPRT-Test in vitro

This in vitro experiment was performed to assess the potential of the test item to induce gene mutations by means of a HPRT (hypoxanthine-guanine-phosphoribosyl-transferase) assay using the Chinese Hamster V79 cell line. The HPRT system detects base pair mutations, frameshift mutations and small deletions and insertions.

The test is performed in accordance with the OECD Guideline for Testing of Chemicals, Section 4, No. 476 at Eurofins BioPharma Product Testing Munich GmbH ¹⁾ with chemicals and pharmaceuticals.

Other tests for mutagenicity are the Reverse Mutation Assay (using Bacteria) and the Mouse Lymphoma Assay (using mammalian cells).

Experimental Performance

Protocol
Cell line	Chinese Hamster V79 cell
Endpoint	Detection of gene mutations Cell viability determination by relative survival
Concentrations	At least 4 test item concentrations evaluated in single culture Maximum concentrations: · Chemicals: 2 mg/mL, 2 µL/mL or 10 mM, whichever is the lowest (5 mg/mL for not defined components/ UVCB) · Pharmaceuticals: 0.5 mg/mL or 1 mM, whichever is the lowest
Exposure time	short-term incubation for 4 h
Metabolic activation	5% Rat liver homogenate S9
Quality controls	Negative control: cell culture medium Positive controls: ethylmethanesulfonate, 7,12-dimethylbenz(a)anthracene
Vehicles of test chemicals	Cell culture medium, 1% dimethylsulfoxid, 10% physiological saline, 0.5% tetrahydrofuran, 0.5% ethanol
Data capture	Colony counting

Test principal

In order to estimate the mutagenic effect of a substance by the HPRT assay, the colony formation ability of V79 cells is investigated by adding 6-thioguanine (TG) to the culture medium. Mutagenic substances cause a forward mutation in the gene coding for the enzyme hypoxanthine-guanine-phosphoribosyltransferase (HPRT). Mutations in this gene lead to an incorrect expression of the enzyme sequence, causing its loss of function, which is necessary for the nucleotide synthesis through the Salvage pathway.

Cells can either obtain nucleotides through de-novo biosynthesis or via energy-saving recycling pathways (Salvage Pathway) using free bases (e.g. adenine, guanine).

V79 cells with an intact hprt-gene incorporate the toxic purine base analogue TG into their DNA via the Salvage pathway resulting in inhibition of cellular metabolism and cytotoxicity (figure 1)

The loss of HPRT enzyme function due to mutations leads to increased de-novo synthesis as the Salvage pathway is excluded. Affected V79 cells are therefore unable to incorporate toxic TG into the DNA. Thus mutant cells are able to proliferate in the presence of TG, whereas normal cells, which contain HPRT, are not.

Figure 1: Scheme of the Test principal

Test procedure

First Experiment (short-term incubation)
Approx. 2 - 6 x 10⁶ cells per treatment group will be seeded in complete culture medium
Approx. 24 h after seeding, the cells will be exposed to designated concentrations of the test item either in the presence or absence of metabolic activation. After 4 h the cultures will be checked for precipitation and the treatment medium containing the test item will be removed. The cells will be washed twice with PBS, trypsinised and counted with a cell counter.
Expression:		Survival:
One cell culture flask will be seeded with at least 2 x 10⁶ cells per treatment group in complete culture medium.		Two flasks will be seeded with approx. 200 cells in complete culture medium for each treatment group.
Cells will be subcultured within the following (7 – 9) days after treatment in complete culture medium in a sufficient number of cells (at least 2 x 10⁶ cells per treatment group).		After incubation for an appropriate time (6 - 7 days) colonies will be fixed with methanol, stained with Giemsa and counted.
Mutant frequency:	Cloning efficiency:
At the end of the expression period (after 7 - 9 days) about 4 x 10⁵ cells for each treatment group will be seeded in 5 cell culture petri dishes with selective medium containing 6-thioguanine for further incubation.	At the end of the expression period (after 7 - 9 days), two flasks will be seeded with approx. 200 cells in complete culture medium for each treatment group.
After incubation for an appropriate time (9 - 11 days) colonies will be fixed with methanol, stained with Giemsa and counted.	After incubation for an appropriate time (6 - 8 days) colonies will be fixed with methanol, stained with Giemsa and counted.

HPRT_Mutagenic Concentration

Figure 2: left side: non mutagenic concentration / right side: mutagenic concentration

Historical Laboratory Control Data of the Negative Control
	Negative Control Number of Mutants/10⁶ cells
Mean	-S9	+S9
Min	26	27
Max	5	8
SD	43	54
RSD [%]	8.43	9.02
n =	32.94	33.08
LCL	86	92
UCL	8.7	9.2

Mean: mean of mutants/10⁶ cells

Min.: minimum of mutants/10⁶ cells

Max.: maximum of mutants/10⁶ cells

SD: standard deviation

RSD: relative standard deviation

n: number of control values

LCL: lower control limit

UCL: upper control limit

Evaluation of Mutagenicity

A test chemical is considered to be clearly positive if, in any of the experimental conditions examined

at least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
the increase is concentration-related when evaluated with an appropriate trend test, and
any of the results are outside the distribution of the historical negative control data.
if there is by chance a low spontaneous mutant frequency in the corresponding negative and solvent controls a concentration-related increase of the mutations within their range has to be discussed.

Significance

The HPRT test can substitute the Mouse Lymphoma Assay as part of a test battery in case of precipitation of the test item observed after treatment.

References

OECD Guideline for Testing of Chemicals, Section 4, No. 476, "In vitro Mammalian Cell Gene Mutation Tests using the Hprt and xprt genes" adopted July 29, 2016.
Bradley, M.O., B. Bhuyan, M.C. Francis, R. Langenbach, A. Peterson and E. Huberman (1981). Mutagenesis by chemical agents in V79 Chinese hamster cells: a review and analysis of the literature. A report of the gene-tox program. Mutat. Res. 87, 81-142