Nitrile hydratase recombinant from E.coli

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

30 March 2021 to 27 April 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Report date:

2021

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)

Version / remarks:

29 July 2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell gene mutation tests using the thymidine kinase gene

Test material

Test material information

Constituent 1

Reference substance name:

Nitrile hydratase recombinant from E.coli

EC Number:

617-328-0

Cas Number:

82391-37-5

Molecular formula:

Not available for a UVCB substance

IUPAC Name:

Nitrile hydratase recombinant from E.coli

Test material form:

solid - liquid: suspension

Details on test material:

Appearance: Light yellow suspension

Method

Target gene:

Thymidine kinase gene

Species / strain

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

CELLS USED
- Type and source of cells: L5178Y TK+/- 3.7.2c mouse lymphoma cell line

For cell lines:
- Absence of Mycoplasma contamination: Master stocks of cells were tested and found to be free of mycoplasma.
- Cell cycle length, doubling time or proliferation index: The cells have a generation time of approximately 12 hours and were sub-cultured accordingly.

CELL CLEANSING
The TK +/- heterozygote cells grown in suspension spontaneously mutate at a low but significant rate. Before the stocks of cells were frozen they were cleansed of homozygous (TK -/-) mutants by culturing in THMG medium for 24 hours. This medium contained Thymidine (9 µg/mL), Hypoxanthine (15 µg/mL), Methotrexate (0.3 µg/mL) and Glycine (22.5 µg/mL). For the following 24 hours the cells were cultured in THG medium (i.e. THMG without Methotrexate) before being returned to R10 medium.

MEDIA USED
- The stocks of cells were stored in liquid nitrogen at approximately -196 °C. Cells were routinely cultured in RPMI 1640 medium with Glutamax-1 and HEPES buffer (20 mM) supplemented with Penicillin (100 units/mL), Streptomycin (100 µg/mL), Sodium pyruvate (1 mM), Amphotericin B (2.5 µg/mL) and 10 % donor horse serum (giving R10 media) at 37 °C with 5 % CO2 in air.
- RPMI 1640 with 20 % donor horse serum (R20), 10 % donor horse serum (R10), and without serum (R0), were used during the course of the study. All donor horse serum was purchased heat inactivated from the supplier.

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9: The S9 Microsomal Enzyme Fraction was purchased.
- method of preparation of S9 mix: The S9 mix was prepared by mixing S9 with 100 mM phosphate buffer containing NADP (5 mM), G­6 P (5 mM), KCl (33 mM) and MgCl2 (8 mM) to give a 20 % S9-mix concentration.
- concentration or volume of S9 mix and S9 in the final culture medium: The final concentration of S9 when dosed at a 10 % volume of S9-mix was 2 % for the Preliminary Toxicity Test and the Mutagenicity Test.
- other: The protein content was adjusted to approximately 20 mg/mL prior to use.

Test concentrations with justification for top dose:

Preliminary toxicity test: Due to the precipitate observed in the solubility check, the maximum dose concentration was limited to 50 µg/mL for all three exposure groups and the dose range used in the preliminary toxicity test was 0, 0.20, 0.39, 0.78, 1.56, 3.13, 6.25, 12.5, 25, and 50 µg/mL.

Main test: 0, 0.2, 0.39, 0.78, 1.56, 3.13, 6.25 µg/mL

Results from the preliminary toxicity test were used to set the test item concentrations for the mutagenicity experiments. Maximum concentrations were selected using the following criteria:

i) For non-toxic test materials the upper test material concentrations was 10 mM, 2 mg/mL or 2 µL/mL whichever was the lowest. When the test material is a substance of unknown or variable composition (UVCB) the upper concentration may need to be higher, up to a maximum concentration of 5 mg/mL.
ii) Precipitating concentrations were not tested beyond the onset of precipitation regardless of the presence of toxicity beyond this point.
iii) In the absence of precipitate and if toxicity occurred, the highest concentration should lower the Relative Total Growth (RTG) to approximately 10 to 20 %. This optimum upper level of toxicity was confirmed by an IWGT meeting in New Orleans, USA.

Vehicle / solvent:

The test material formed a suspension suitable for dosing in culture medium at 50 mg/mL in solubility checks performed in house. Prior to each experiment, the test material was thawed in a water bath at 25 °C for no more than 90 minutes, accurately measured, gently agitated in culture medium, and serial dilutions prepared. The solubility of the test material was explored in the in vitro micronucleus study.

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

Absence of S9-mix

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

Presence of S9-mix

Details on test system and experimental conditions:

TEST MATERIAL PREPARATION
The test material was a UVCB and therefore the maximum concentration in the solubility check was set at 5000 µg/mL, the maximum recommended concentration. A correction was made for the water content of the test material (84.2 %) when the formulations were prepared.

There was no significant change in pH when the test material was dosed into media and the osmolality did not increase by more than 50 mOsm.

No analysis was conducted to determine the homogeneity, concentration or stability of the test material formulation. The test material was formulated within two hours of it being applied to the test system; it is assumed that the formulation was stable for this duration. This is an exception with regard to GLP and has been reflected in the GLP compliance statement.

PRELIMINARY TOXICITY TEST
- A preliminary toxicity test was performed on cell cultures at 1 x 10^7 cells/mL, using a 4 hour exposure period both with and without metabolic activation (S9) and a 24-hour exposure.
- Following the exposure periods the cells were washed twice with R10, resuspended in R20 medium, counted and then serially diluted to 2 x 10^5 cells/mL. The cultures were incubated at 37 °C with 5 % CO2 in air and sub-cultured after 24 hours by counting and diluting to 2 x 10^5 cells/mL in R20 medium. After a further 24 hours the cultures were counted and then discarded.
- The cell counts were then used to calculate Suspension Growth (SG) values. The SG values were then adjusted to account for immediate post exposure toxicity, and a comparison of each exposure SG value to the concurrent solvent control performed to give a percentage Relative Suspension Growth (%RSG) value.

MUTAGENICITY TEST
- Several days before starting the experiment, an exponentially growing stock culture of cells was set up so as to provide an excess of cells on the morning of the experiment.
- The cells were counted and processed to give 1 x 10^6 cells/mL in 10 mL aliquots in R10 medium in sterile plastic universals for the 4-hour exposure groups in both the absence and presence of metabolic activation, and 0.3 x 10^6 cells/mL in 10 mL cultures were established in 25 cm^2 tissue culture flasks for the 24-hour exposure group in the absence of metabolic activation.
- The exposures were performed in duplicate (A + B) at eight concentrations of the test material (0, 0.10, 0.20, 0.39, 0.78, 1.56, 3.13, 6.25, and 12.5 µg/mL in all three of the exposure groups), solvent and positive controls. To each universal was added 2 mL of S9 mix if required, 2 mL of the exposure dilutions, (0.2 mL or 0.15 mL for the positive controls), and sufficient R0 medium to bring the total volume to 20 mL.
- The exposure vessels were incubated at 37°C for 4 or 24 hours with continuous shaking using an orbital shaker within an incubated hood.

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: RSG, Cloning efficiency, RTG, MF:
- Calculation of Percentage Relative Suspension Growth (%RSG):

The cell counts obtained immediately post exposure and over the 2-day expression period were used to calculate the Percentage Relative Suspension Growth.
4-Hour Suspension Growth (SG) = (24-hour cell count/2) x (48-hour cell count/2)
24-Hour Suspension Growth (SG) = (0-hour cell count/1.5) x (24-hour cell count/2) x (48 hour cell count/2)
Day 0 Factor = dose 0-hour cell count/solvent control 0-hour cell count
4-Hour %RSG= [(dose SG x dose Day 0 Factor)/solvent control SG] x100
24-Hour %RSG= dose SG/solvent control SG x100

- Calculation of Day 2 Cloning Efficiency (%V):
Since the distribution of colony-forming units over the wells is described by the Poisson distribution, the day 2 cloning efficiency (%V) was calculated using the zero term of the Poisson distribution [P(0)] method.

P(0) = number of negative wells / total wells plated
%V = -ln P(0) x 100 / (number of cells/well)

- Calculation of Relative Total Growth (RTG):
For each culture, the relative cloning efficiency, RCE, was calculated:

RCE = %V / Mean solvent control %V

Finally, for each culture RTG is calculated:
RTG = RCE x RSG

- Calculation of Mutant Frequency (MF)
MF per survivor = [(-ln P(0) selective medium)/cells per well in selective medium)]/surviving fraction in non-selective medium.


ASSESSMENTS
- Measurement of Survival, Cloning Efficiency and Mutant Frequency:
At the end of the exposure periods, the cells were washed twice using R10 medium then resuspended in R20 medium at a cell density of 2 x 10^5 cells/mL. The cultures were incubated at 37 °C with 5 % CO2 in air and sub-cultured every 24 hours for the expression period of two days, by counting and dilution to 2 x 10^5 cells/mL.
On Day 2 of the experiment, the cells were counted, diluted to 10^4 cells/mL and plated for mutant frequency (2000 cells/well) in selective medium containing 4 µg/mL 5 trifluorothymidine (TFT) in 96-well plates. Cells were also diluted to 10 cells/mL and plated (2 cells/well) for cloning efficiency (%V) in non-selective medium.
The daily cell counts were used to obtain a Relative Suspension Growth (%RSG) value that gives an indication of post exposure toxicity during the expression period as a comparison to the solvent control, and when combined with the cloning efficiency (%V) data, a Relative Total Growth (RTG) value.

- Plate scoring:
96 well plates were scored using a magnifying mirror box after ten to twelve days incubation at 37 °C with 5 % CO2 in air. The number of positive wells (wells with colonies) was recorded together with the total number of scorable wells (normally 96 per plate). The numbers of small and large colonies seen in the TFT mutant plates were also recorded as the additional information may contribute to an understanding of the mechanism of action of the test material. Colonies are scored manually by eye using qualitative judgment. Large colonies are defined as those that cover approximately ¼ to ¾ of the surface of the well and are generally no more than one or two cells thick. In general, all colonies less than 25 % of the average area of the large colonies are scored as small colonies. Small colonies are normally observed to be more than two cells thick. To assist the scoring of the TFT mutant colonies 0.025 mL of thiazolyl blue tetrazolium bromide (MTT) solution, 2.5 mg/mL in phosphate buffered saline (PBS), was added to each well of the mutant plates. The plates were incubated for two hours. MTT is a vital stain that is taken up by viable cells and metabolised to give a brown/black color, thus aiding the visualisation of the mutant colonies, particularly the small colonies.

DATA EVALUATION
Dose selection for the mutagenicity experiments was made using data from the preliminary toxicity test in an attempt to obtain the desired levels of toxicity. This optimum toxicity is approximately 20 % survival (80 % toxicity), but no less than 10 % survival (90 % toxicity). Relative Total Growth (RTG) values were the primary factor used to designate the level of toxicity achieved by the test material for any individual concentration. However, under certain circumstances, %RSG values may also be taken into account when designating the level of toxicity achieved. Concentrations that have RTG survival values less than 10 % are excluded from the mutagenicity data analysis, as any response they give would be considered to have no biological or toxicological relevance.

Evaluation criteria:

An approach for defining positive and negative responses is recommended to assure that the increased MF is biologically relevant. In place of statistical analysis generally used for other tests, it relies on the use of a predefined induced mutant frequency (i.e. increase in MF above the concurrent control), designated the Global Evaluation Factor (GEF) of 126 x 10^-6, i.e. the mutant frequency of the concurrent solvent control plus 126, which is based on the analysis of the distribution of the solvent control MF data from participating laboratories.

Providing that all acceptability criteria are fulfilled, a test material is considered to be clearly positive if, in any of the experimental conditions examined, the increase in MF above the concurrent background exceeds the GEF and the increase is concentration related (e.g., using a trend test). The test material is then considered able to induce mutation in this test system.

Providing that all acceptability criteria are fulfilled, a test material is considered to be clearly negative if, in all experimental conditions examined there is no concentration related response or, if there is an increase in MF, it does not exceed the GEF. The test material is then considered unable to induce mutations in this test system.

Results and discussion

Test results

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

PRELIMINARY CYTOTOXICITY TEST
The dose range of the test material used in the preliminary toxicity test was 0.20 to 50 µg/mL. The results for the Relative Suspension Growth (%RSG) are shown in table 'Relative Suspension Growth (%RSG)'.

There was no evidence of any marked dose-related reductions in the Relative Suspension Growth (%RSG) of cells treated with the test material in any of the three exposure groups. Precipitate of the test material was observed at and above 6.25 µg/mL in all three of the exposure groups at the end of the exposure periods. Therefore, following the recommendations of the OECD 490 guideline, the maximum concentrations in the subsequent Mutagenicity Test were limited by the onset of test material precipitate.

MUTAGENICITY TEST
There was no evidence of any toxicity, in any of the three exposure groups, following exposure to the test material, as indicated by the %RSG and RTG values. A summary of the results from the test are shown in Table 'Summary of Results'.
There was also no evidence of any marked reductions in cloning efficiency (%V) in any of the three exposure groups, therefore indicating that residual toxicity had not occurred .
The onset of test material precipitate was observed at 6.25 µg/mL in all three of the exposure groups. Therefore, following the recommendations of the OECD 490 guideline, the 12.5 µg/mL concentrations were not plated for cloning efficiency or 5-TFT resistance due to the presence of test material precipitate. Acceptable levels of toxicity were seen with the positive control substances.

The solvent controls had mutant frequency values that were considered acceptable for the L5178Y cell line at the TK +/- locus. The positive controls produced marked increases in the mutant frequency per viable cell achieving the acceptability criterion recommended by the OECD guideline, indicating that the test system was operating satisfactorily, and that the metabolic activation system was functional.

The study met the criteria for a valid assay in that the highest concentration tested was the lowest precipitating dose level, the total suspension growth of the solvent controls and the mutant frequencies for the solvent were in the expected ranges and the positive controls demonstrated marked increases in mutant frequency.

The test material did not induce any increases in the mutant frequency at any of the concentrations in the main test that exceeded the Global Evaluation Factor (GEF), using a dose range that included the lowest precipitating concentration in all three of the exposure groups, and at least four analysable concentrations in each exposure group, as recommended by the OECD 490 guideline. The results observed were considered to fulfill the criteria for a clearly negative outcome.

Any other information on results incl. tables

Relative Suspension Growth (%RSG)

Dose (mg/mL)	% RSG (-S9) 4-Hour Exposure	% RSG (+S9) 4-Hour Exposure	% RSG (-S9) 24-Hour Exposure
0	100	100	100
0.20	121	99	109
0.39	92	83	92
0.78	122	88	90
1.56	132	99	99
3.13	114	84	81
6.25	129	98	93
12.5	122	104	110
25	137	80	96
50	137	91	70

 

Summary of Results

Concentration (μg/mL)	4 hours -S9			Concentration (μg/mL)	4 hours +S9			Concentration (μg/mL)	24 hours -S9
	%RSG	RTG	MF§		%RSG	RTG	MF§		%RSG	RTG	MF§
0	100	1	126.67	0	100	1	107.43	0	100	1	105.93
0.1	101	NP	NP	0.1	100	NP	NP	0.1	98	NP	NP
0.2	98	0.93	111.39	0.2	108	1.16	107.37	0.2	81	1	113.54
0.39	96	1.02	125.67	0.39	91	1.02	122.98	0.39	89	1.06	98.63
0.78	110	1.03	144.07	0.78	114	1.17	143.95	0.78	87	0.93	105.35
1.56	110	1.36	104.32	1.56	104	1.05	129.91	1.56	89	1.03	78.77
3.13	92	1.07	83.77	3.13	103	1.07	115.04	3.13	101	1.09	110.62
6.25 P	105	1.15	100.03	6.25 P	108	1.17	134.34	6.25 P	111	1.39	64.67
12.5 P	98	NP	NP	12.5 P	108	NP	NP	12.5 P	102	NP	NP
MF threshold for a positive response = 252.67				MF threshold for a positive response = 233.43				MF threshold for a positive response = 231.93
Positive Control				Positive Control				Positive Control
EMS - 400 μg/mL	83	0.7	1139.22	CP - 1.5 μg/mL	85	0.64	1000	EMS - 150 μg/mL	54	0.39	1394.79

%RSG = Relative Suspension Growth, RTG = Relative Total Growth, CP = Cyclophosphamide, EMS = Ethylmethanesulphonate, MF§ = 5-TFT resistant mutants/10^6 viable cells 2 days after exposure, P = Precipitate present at the end of exposure period, NP = Not plated for cloning efficiency or 5-TFT resistance

Applicant's summary and conclusion

Conclusions:

Under the conditions of this study the test material did not induce any increases in the mutant frequency at the TK +/- locus in L5178Y cells that exceeded the Global Evaluation Factor (GEF), consequently it is considered to be non-mutagenic.

Executive summary:

The mutagenicity of the test material on the thymidine kinase, TK ^+/-, locus of the L5178Y mouse lymphoma cell line was investigated in accordance with the standardised guideline OECD Test Guideline 490, under GLP conditions. 

One main Mutagenicity Test was performed. In this main test, L5178Y TK ^+/- 3.7.2c mouse lymphoma cells (heterozygous at the thymidine kinase locus) were treated with the test material at eight concentrations in duplicate, together with solvent (R0 medium), and positive controls using 4 hour exposure groups both in the absence and presence of metabolic activation (2 % S9), and a 24-hour exposure group in the absence of metabolic activation.

The dose range of test material used in the main test was selected following the results of a preliminary toxicity test. The maximum concentrations in the Mutagenicity Test were limited by the onset of test material precipitate in all three of the exposure groups, as recommended by the OECD 490 guideline. The concentrations plated for cloning efficiency and expression of mutant colonies were as follows:

Mutagenicity Test

Group	Concentration of Nitrile hydratase - (µg/mL) plated for cloning efficiency and mutant frequency
4-hour without S9	0, 0.2, 0.39, 0.78, 1.56, 3.13, 6.25
4-hour with S9 (2%)
24-hour without S9

The solvent controls had mutant frequency values that were considered acceptable for the L5178Y cell line at the TK ^+/- locus.  The positive controls produced marked increases in the mutant frequency per viable cell achieving the acceptability criterion recommended by the OECD guideline, indicating that the test system was operating satisfactorily, and that the metabolic activation system was functional.

The test material did not induce any increases in the mutant frequency at any of the concentrations in the main test that exceeded the Global Evaluation Factor (GEF), using a dose range that included the lowest precipitating concentration in all three of the exposure groups, and at least four analysable concentrations in each exposure group, as recommended by the OECD 490 guideline. The results observed in all three of the exposure groups were considered to fulfill the criteria for a clearly negative outcome.

Under the conditions of this study the test material did not induce any increases in the mutant frequency at the TK +/- locus in L5178Y cells that exceeded the Global Evaluation Factor (GEF), consequently it is considered to be non-mutagenic.