Nitrile hydratase recombinant from E.coli

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Genetic toxicity in vitro Ames - key study (Saigo, 2003):

Under the conditions of this study, the test material shows no mutagenicity.

 

In vitro Micronucleus test - Morris (2021):

Under the conditions of this study, the test material did not induce any statistically significant increases in the frequency of binucleate cells with micronuclei in either the absence or presence of a metabolising system. The test material was therefore considered to be non-clastogenic and non aneugenic to human peripheral blood lymphocytes in vitro.

 

In vitro Mammalian gene mutation study - Smith (2021):

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.

Link to relevant study records

Reference

Reference 1

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

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

Target gene:

Thymidine kinase gene

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.

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

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.

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.

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

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.

Reference 2

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

05 January 2021 to 08 March 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

Version / remarks:

29 July 2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell micronucleus test

Specific details on test material used for the study:

A correction was made in the formulations for the water content of 84.2 %

Species / strain / cell type:

lymphocytes: human

Details on mammalian cell type (if applicable):

CELLS USED
For lymphocytes:
- Method: For each experiment, sufficient whole blood was drawn from the peripheral circulation of a non smoking volunteer (18-35) who had been previously screened for suitability.
- The volunteer had not knowingly been exposed to high levels of radiation or hazardous chemicals and had not knowingly recently suffered from a viral infection.
- Based on over 20 years in house data for cell cycle times for lymphocytes using BrdU (bromodeoxyuridine) incorporation to assess the number of first, second and third division metaphase cells to calculate the average generation time (AGT) for human peripheral blood lymphocytes it is considered to be approximately 16 hours. Therefore using this average the in-house exposure time for the experiments for 1.5 x AGT is 24 hours.

- The details of the donors used are:
Preliminary Toxicity Test: male, aged 24 years
Main Experiment: female, aged 29 years


MEDIA USED
- Cells (whole blood cultures) were grown in Eagle's minimal essential medium (MEM) with HEPES buffer, supplemented “in-house” with L-glutamine, penicillin/streptomycin, amphotericin B and 10% fetal bovine serum (FBS), at approximately 37ºC with 5% CO2 in humidified air. The lymphocytes of fresh heparinized whole blood were stimulated to divide by the addition of phytohaemagglutinin (PHA).

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 prior to the dosing of the test cultures and contained the S9 fraction (20 % (v/v)), MgCl2 (8mM), KCl (33mM), sodium orthophosphate buffer pH 7.4 (100mM), glucose-6-phosphate (5mM) and NADP (5mM).
- 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 into culture media, was 2 %.
- 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 high levels of precipitate observed in the solubility check, the concentration levels of test item used were 0, 0.78, 1.56, 3.13, 6.25, 12.5, 25, 50, 100, and 200 µg/mL.

Main test: The dose levels of test item used were 0, 1.56, 3.13, 6.25, 12.5, 25, and 50 µg/mL.
4-hour without S9: 0*, 1.56, 3.13*, 6.25*, 12.5*, 25, 50, MMC 0.2*
4-hour with S9 (2 %): 0*, 1.56, 3.13*, 6.25*, 12.5*, 25, 50, CP 6*
24-hour without S9: 0*, 1.56, 3.13*, 6.25*, 12.5*, 25, 50, DC 0.075*

* = Dose levels selected for analysis of micronucleus frequency in binucleate cells
MMC = Mitomycin C
DC = Demecolcine

Vehicle / solvent:

The test material formed a suspension suitable for dosing in culture medium at 50 mg/mL in MEM 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 MEM medium, and serial dilutions prepared.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

mitomycin C

other: Demecolcine (DC)

Remarks:

Absence of S9-mix

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:

POSITIVE CONTROLS
Demecolcine (DC) is not one of the suggested positive control substances listed in the OECD 487 guideline but the substance is a derivative of Colchicine, one of the recommended substances. There are sufficient laboratory historical control data to demonstrate its effectiveness and suitability as an aneugen.

TEST MATERIAL PREPARATION
The test material was a UVCB and therefore the maximum concentration was initially 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.

The test material was formulated within two hours of it being applied to the test system; it is assumed that the test material formulation was stable for this duration. No analysis was conducted to determine the homogeneity, concentration or stability of the test material formulation because it is not a requirement of the OECD guideline. This is an exception with regard to GLP and has been reflected in the GLP compliance statement.

NUMBER OF REPLICATIONS:
- Number of cultures per concentration (single, duplicate, triplicate): Duplicate lymphocyte cultures (A and B), (quadruplicate for the solvent) were established for each dose level, by mixing the following components, giving, when dispensed into sterile plastic flasks for each culture:

8.3 - 9.4 mL MEM, 10% (FBS)
0.1 mL Li-heparin
0.1 mL phytohaemagglutinin
0.4 - 0.5 mL heparinized whole blood

TREATMENT
- 4-Hour Exposure With Metabolic Activation (S9):
After 44 to 48 hours incubation at approximately 37 ºC, 5 % CO2 in humidified air, the cultures were transferred to tubes and centrifuged. Approximately 9 mL of the culture medium was removed, reserved, and replaced with the required volume of MEM (including serum) and 1 mL of the appropriate solution of solvent control or test material was added to each culture. For the positive control, 0.1 mL of the appropriate solution was added to the cultures. One mL of 20 % S9-mix (i.e. 2 % final concentration of S9 in standard co factors) was added to the cultures of the Preliminary Toxicity Test and the Main Experiment. All cultures were then returned to the incubator. The nominal total volume of each culture was 10 mL.

After 4 hours at approximately 37 ºC, the cultures were centrifuged, the treatment medium removed by suction and replaced with an 8 mL wash of MEM culture medium. After a further centrifugation, the wash medium was removed by suction and replaced with the reserved original culture medium, supplemented with Cytochalasin B at a final concentration of 4.5 µg/mL, and then incubated for a further 24 hours.

- 4-Hour Exposure Without Metabolic Activation (S9):
After 44 to 48 hours incubation at approximately 37 ºC with 5 % CO2 in humidified air, the cultures were decanted into tubes and centrifuged. Approximately 9 mL of the culture medium was removed and reserved. The cells were then resuspended in the required volume of fresh MEM (including serum) and dosed with 1 mL of the appropriate solvent control, test material solution or 0.1 mL of positive control solution. The nominal total volume for each culture was 10 mL.

After 4 hours at approximately 37 ºC, the cultures were centrifuged, the treatment medium was removed by suction and replaced with an 8 mL wash of MEM culture medium. After a further centrifugation the wash medium was removed by suction and replaced with the reserved original culture medium, supplemented with Cytochalasin B, at a final concentration of 4.5 µg/mL, and then incubated for a further 24 hours.

- 24-Hour Exposure Without Metabolic Activation (S9):
The exposure was continuous for 24 hours in the absence of metabolic activation. Therefore, when the cultures were established the culture volume was a nominal 9 mL. After approximately 48 hours incubation the cultures were removed from the incubator and dosed with 1 mL of solvent control, test material dose solution or 0.1 mL of positive control solution. The nominal total volume of each culture was 10 mL. The cultures were then incubated for 24 hours, the tubes and the cells were washed in MEM before resuspension in fresh MEM with serum. At this point Cytochalasin B was added at a final concentration of 4.5 µg/mL, and then the cells were incubated for a further 24 hours.

The extended exposure detailed above is a modification of the suggested cell treatment schedule in the 487 Guideline and is considered to be an acceptable alternative. This is because it avoids any potential interaction between Cytochalasin B and the test material during exposure to the cells and any effect this may have on the activity or response. Additionally, as the stability or reactivity of the test material is unknown prior to the start of the study this modification of the schedule is considered more effective and reproducible due to the in-house observations on human peripheral blood lymphocytes and their particular growth characteristics in this study type and also the significant laboratory historical control data using the above format.

The Preliminary Toxicity Test was performed using the exposure conditions as described for the Main Experiment but using single cultures for the test item dose levels and duplicate cultures for the solvent controls, whereas the Main Experiment used duplicate cultures for the test material and quadruplicate cultures for the solvent controls.

PRELIMINARY TOXICITY TEST
Three exposure groups were used:
i) 4-hour exposure to the test material without S9-mix, followed by a 24-hour incubation period in treatment-free media, in the presence of Cytochalasin B, prior to cell harvest.
ii) 4-hour exposure to the test material with S9-mix (2%), followed by a 24-hour incubation period in treatment-free media, in the presence of Cytochalasin B, prior to cell harvest.
iii) 24-hour continuous exposure to the test material without S9-mix, followed by a 24-hour incubation period in treatment-free media, in the presence of Cytochalasin B, prior to cell harvest.

Parallel flasks, containing culture medium without whole blood, were established for the three exposure conditions so that test item precipitate observations could be made. Precipitate observations were recorded at the beginning and end of the exposure periods.

Using a qualitative microscopic evaluation of the microscope slide preparations from each treatment culture, appropriate dose levels were selected for the evaluation of the frequency of binucleate cells and to calculate the cytokinesis block proliferation index (CBPI). Coded slides were evaluated for the CBPI. The CBPI data were used to estimate test material toxicity and for selection of the dose levels for the exposure groups of the main experiment. The maximum dose level selected for the main experiment was based around the lowest precipitating concentration.

MAIN EXPERIMENT
Three exposure groups were used for the Main Experiment:
i) 4-hour exposure to the test material without S9-mix, followed by a 24-hour incubation period in treatment-free media, in the presence of Cytochalasin B, prior to cell harvest.
ii) 4-hour exposure to the test material with S9-mix (2%), followed by a 24-hour incubation period in treatment-free media, in the presence of Cytochalasin B, prior to cell harvest.
iii) 24-hour continuous exposure to the test material without S9-mix, followed by a 24-hour incubation period in treatment-free media, in the presence of Cytochalasin B, prior to cell harvest.

Parallel flasks, containing culture medium without whole blood, were established for the three exposure conditions so that test material precipitate observations could be made. Precipitate observations were recorded at the beginning and end of the exposure periods

CELL HARVEST
At the end of the Cytochalasin B treatment period the cells were centrifuged, the culture medium was drawn off and discarded, and the cells resuspended in MEM. The cells were then treated with a mild hypotonic solution (0.0375M KCl) before being fixed with fresh methanol/glacial acetic acid (19:1 v/v). The fixative was changed at least three times and the cells stored at approximately 4ºC prior to slide making.

MICRONUCLEUS TEST
- Methods of slide preparation and staining technique used including the stain used (for cytogenetic assays): The peripheral blood lymphocytes were re-suspended in several mL of fresh fixative before centrifugation and re suspension in a small amount of fixative. Several drops of this suspension were dropped onto clean, wet microscope slides and left to dry with gentle warming. Each slide was permanently labeled with the appropriate identification data.

When the slides were dry, they were stained in 5 % Giemsa for 5 minutes, rinsed, dried and a cover slip applied using mounting medium.

- Number of cells spread and analysed per concentration (number of replicate cultures and total number of cells scored): A minimum of approximately 500 cells per culture were scored for the incidence of mononucleate, binucleate and multinucleate cells.

- Criteria for scoring micronucleated cells (selection of analysable cells and micronucleus identification): The micronucleus frequency in 1000 binucleated cells was analysed per culture (2000 binucleated cells per concentration for the test material and positive control and 4000 binucleated cells for the solvent controls). Cells with 1, 2 or more micronuclei were recorded and included in the total. Experiments with human peripheral blood lymphocytes have established a range of micronucleus frequencies acceptable for control cultures in normal volunteer donors.

The criteria for identifying micronuclei were that they were round or oval in shape, non refractile, not linked to the main nuclei and with a diameter that was approximately less than a third of the mean diameter of the main nuclei. Binucleate cells were selected for scoring if they had two nuclei of similar size with intact nuclear membranes situated in the same cytoplasmic boundary. The two nuclei could be attached by a fine nucleoplasmic bridge which was approximately no greater than one quarter of the nuclear diameter.

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: CBPI: the CBPI value was expressed as a percentage of the solvent controls. The CBPI indicates the number of nuclei per cell during the period of exposure to Cytochalasin B. It was used to calculate cytostasis by the following formula:

% Cytostasis = 100 - 100{(CBPIT – 1) / (CBPIC – 1)}

Where:
CBPI= (No.mononucleate cells + (2 x No.binucleate cells) + (3 x No.multinucleate cells)) / Total number of cells

Key:
T = test chemical treatment culture
C = solvent control culture

ACCEPTABILITY CRITERIA
The following criteria were used to determine a valid assay:
• The concurrent negative control was considered acceptable for addition to the laboratory historical negative control data range.
• All the positive control chemicals induced positive responses that were compatible with those in the laboratory historical positive control data range and produced a statistically significant increase when compared with the concurrent negative control. Acceptable positive responses demonstrated the validity of the experiment and the integrity of the S9 mix.
• Cell proliferation criteria in the solvent control were considered to be acceptable.
• The study was performed using all three exposure conditions using a top concentration which meets the requirements of the current testing guideline.
• The required number of cells and concentrations were analyzed

Evaluation criteria:

Providing that all of the acceptability criteria are fulfilled, a test material is considered to be clearly negative if, in all of the experimental conditions examined:
1. None of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control.
2. There is no dose-related increase when evaluated with an appropriate trend test.
3. The results in all evaluated dose groups are within the range of the laboratory historical control data.
The test system is then considered to be unable to induce chromosome breaks and/or gain or loss.
Providing that all of the acceptability criteria are fulfilled, a test material may be considered to be clearly positive, if in any of the experimental conditions examined, there is one or more of the following applicable:
1. At least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control.
2. The increase is dose-related in at least one experimental condition when evaluated with an appropriate trend test.
3. The results are substantially outside the range of the laboratory historical negative control data.
When all the criteria are met, the test item is considered able to induce chromosome breaks and/or gain or loss in this test system.
There is no requirement for verification of a clear positive or negative response.

Statistics:

The frequency of binucleate cells with micronuclei was compared, where necessary, with the concurrent solvent control value using the Chi-squared Test on observed numbers of cells with micronuclei. Other statistical analyses may be used if considered appropriate (Hoffman et al., 2003). A toxicologically significant response was recorded when the p value calculated from the statistical analysis of the frequency of binucleate cells with micronuclei was less than 0.05 and there was a dose-related increase in the frequency of binucleate cells with micronuclei.

The dose-relationship (trend-test) was assessed using a linear regression model. An arcsin square-root transformation was applied to the percentage of binucleated cells containing micronuclei (excluding positive controls). A linear regression model was then applied to these transformed values with dose values fitted as the explanatory variable. The F-value from the model was assessed at the 5 % statistical significance level.

Key result

Species / strain:

lymphocytes: human

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 TOXICITY TEST
Precipitate of the test material was observed at and above 25 µg/mL in all three of the exposure groups.
Microscopic assessment of the slides prepared from the exposed cultures showed that binucleate cells were present at up to 200 µg/mL in all three of the exposure groups. The CBPI data for the 4-hour exposures in the absence and presence of S9 and the 24-hour exposure are presented in Table 'CBPI Data - Preliminary Toxicity Test - 4-hour exposure without Metabolic Activation (S9)', Table 'CBPI Data - Preliminary Toxicity Test - 4-hour exposure with Metabolic Activation (S9)', Table 'CBPI Data - Preliminary Toxicity Test - 24-hour exposure without Metabolic Activation (S9)' respectively. The test material induced no evidence of toxicity in any of the three exposure groups.

The maximum dose level used in the Main Experiment was limited by test material precipitate.

MICRONUCLEUS TEST - MAIN EXPERIMENT
The qualitative assessment of the slides determined that there were binucleate cells suitable for scoring at the maximum dose level of test material of 50 µg/mL in all three of the exposure groups. Precipitate of test material was observed at and above 12.5 µg/mL in all three of the exposure groups.

The CBPI data for the 4-hour exposure groups in the absence and presence of S9 and for the 24-hour exposure group are given in Table 'CBPI Data – Main Experiment - 4-Hour Exposure without Metabolic Activation (S9)', Table 'CBPI Data – Main Experiment - 4-Hour Exposure with Metabolic Activation (S9)' and Table 'CBPI Data – Main Experiment - 24-Hour Exposure without Metabolic Activation (S9) ', respectively. They confirm the qualitative observations in that no marked dose-related test item-induced toxicity was observed.

The maximum concentration selected for analysis of micronuclei in binucleate cells was 12.5 µg/mL in all three exposure groups. This was the lowest precipitating dose level.

A summary of the results for all three exposure groups is presented in Table 'Summary of Results'.

The solvent control cultures had frequencies of cells with micronuclei within the expected range and were considered acceptable for addition to the laboratory historical negative control data range.

The positive control items induced statistically significant increases in the frequency of cells with micronuclei with responses that were compatible with those in the laboratory historical positive control data range. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

The test material was non-toxic and did not induce any statistically significant increases in the frequency of binucleate cells with micronuclei in any of the three exposure groups using a dose range that included the lowest precipitating concentration. The response observed at 3.13 µg/mL in the 4-hour exposure group in the absence of S9, exceeded the upper limit of the current historical control data for a solvent. However, since the response would be considered acceptable for inclusion in the laboratory solvent historical control range and was only slightly above the HCD for a solvent it is considered to be of no biological relevance. There were also no statistically significant concentration-related increases in any of the three exposure groups when evaluated with a trend test. These results fulfilled the criteria for a clearly negative outcome.

Remarks on result:

other: Non-clastogenic and non-aneugenic under study conditions

Summary of Results

Exposure Condition	Treatment/ Concentration (μg/mL)	Mean Cytostasis  (%)	% Binucleated cells containing micronuclei ^(a)
			Mean	p-value ^(b)	Trend test p-value ^(d)
4-hour -S9	Vehicle (MEM)	0	0.50	-
	3.13	0‡	0.85	0.103
	6.25	0‡	0.65	0.460	0.577
	12.5 P	0‡	0.40	-
	MMC 0.2	27	2.60	1.890 E-12***	-
4-hour +S9	Vehicle (MEM)	0	0.35	-
	3.13	0‡	0.40	0.763
	6.25	0‡	0.10	-	0.084
	12.5 P	0‡	0.15	-
	CP 6	43	1.35	9.318 E-06***	-
24-hour -S9	Vehicle (MEM)	0	0.20	-
	3.13	0‡	0.20	-
	6.25	1	0.20	-	0.439
	12.5 P	0‡	0.25	0.695
	DC 0.075	55	6.95	3.175 E-57***	-

(a) The percentage of micronucleated cells determined in a sample of 2000 binucleate cells (4000 for vehicle)

(b) p-values are for comparison with the control using Chi-square test

(d) Trend test p-values using Linear regression model applied to control and test material concentrations

P Precipitate observed at end of exposure period in blood-free cultures

‡ Cytostasis reported as 0 as the CBPI value is equal to or higher than the solvent control

MMC Mitomycin C

CP Cyclophosphamide

DC Demecolcine

MEM Minimal Essential Medium

*** P<0.001

 

 

CBPI Data - Preliminary Toxicity Test - 4-hour exposure without Metabolic Activation (S9)

Treatment/ Concentration (µg/mL)	Mononucleate Cells	Binucleate Cells	Multinucleate Cells	CBPI ^(c)	Cytostasis (%)
Vehicle                    (MEM)	282	188	30	1.46	0
	306	183	11
0.78	-	-	-	-	-
1.56	-	-	-	-	-
3.13	287	190	23	1.47	0‡
6.25	268	197	35	1.53	0‡
12.5	270	205	25	1.51	0‡
25 P	281	197	22	1.48	0‡
50 P	277	197	26	1.50	0‡
100 P	-	-	-	-	-
200 P	-	-	-	-	-

(c)  Mean value for vehicle

-  Not selected for scoring

P   Precipitate observed at end of exposure period in blood-free cultures

‡ Cytostasis reported as 0 as the CBPI value is equal to or higher than the solvent control

MEM Minimal Essential Medium

 

 

CBPI Data - Preliminary Toxicity Test - 4-hour exposure with Metabolic Activation (S9)

Treatment / Concentration  (µg/mL)	Mononucleate Cells	Binucleate Cells	Multinucleate Cells	CBPI ^(c)	Cytostasis (%)
Vehicle                 (MEM)	261	216	23	1.52	0
	261	220	19
0.78	-	-	-	-	-
1.56	-	-	-	-	-
3.13	236	243	21	1.57	0‡
6.25	242	246	12	1.54	0‡
12.5	235	238	27	1.58	0‡
25 P	209	256	35	1.65	0‡
50 P	226	243	31	1.61	0‡
100 P	-	-	-	-	-
200 P	-	-	-	-	-

(c) Mean value for vehicle

-  Not selected for scoring

P   Precipitate observed at end of exposure period in blood-free cultures

‡ Cytostasis reported as 0 as the CBPI value is equal to or higher than the solvent control

MEM Minimal Essential Medium

 

 

CBPI Data - Preliminary Toxicity Test - 24-hour exposure without Metabolic Activation (S9)

Treatment / Concentration  (µg/mL)	Mononucleate Cells	Binucleate Cells	Multinucleate Cells	CBPI ^(c)	Cytostasis (%)
Vehicle                            (MEM)	102	330	68	1.96	0
	85	337	78
0.78	-	-	-	-	-
1.56	-	-	-	-	-
3.13	103	329	68	1.93	3
6.25	84	345	71	1.97	0‡
12.5	111	327	62	1.90	6
25 P	117	321	62	1.89	7
50 P	85	345	70	1.97	0‡
100 P	-	-	-	-	-
200 P	-	-	-	-	-

(c)  Mean value for vehicle

-  Not selected for scoring

P   Precipitate observed at end of exposure period in blood-free cultures

‡ Cytostasis reported as 0 as the CBPI value is equal to or higher than the solvent control

MEM Minimal Essential Medium

 

 

CBPI Data – Main Experiment - 4-Hour Exposure without Metabolic Activation (S9)

Treatment / Concentration (µg/mL)	Replicate	Mononucleate Cells	Binucleate Cells	Multinucleate Cells	CBPI	Mean CBPI	Mean Cytostasis (%)
Vehicle (MEM)	A1	132	325	43	1.82	1.81	0
	A2	139	325	36	1.79
	B1	116	365	19	1.81
	B2	119	354	27	1.82
1.56	A	127	341	32	1.81	1.84	0‡
	B	115	339	46	1.86
3.13	A	94	379	27	1.87	1.9	0‡
	B	59	418	23	1.93
6.25	A	133	339	28	1.79	1.84	0‡
	B	82	389	29	1.89
12.5 P	A	109	369	22	1.83	1.82	0‡
	B	120	353	27	1.81
25 P	A	-	-	-	-	-	-
	B	-	-	-	-
50 P	A	-	-	-	-	-	-
	B	-	-	-	-
MMC  0.2	A	222	269	9	1.57	1.6	27
	B	194	301	5	1.62

MMC Mitomycin C

-  Not selected for scoring

P   Precipitate observed at end of exposure period in blood-free cultures

‡ Cytostasis reported as 0 as the CBPI value is equal to or higher than the solvent control

MEM Minimal Essential Medium

 

 

CBPI Data – Main Experiment - 4-Hour Exposure with Metabolic Activation (S9)

Treatment / Concentration (µg/mL)	Replicate	Mononucleate Cells	Binucleate Cells	Multinucleate Cells	CBPI	Mean CBPI	Mean Cytostasis (%)
Vehicle (MEM)	A1	207	270	23	1.63	1.65	0
	A2	200	281	19	1.64
	B1	177	292	31	1.71
	B2	214	265	21	1.61
1.56	A	201	278	21	1.64	1.64	2
	B	214	257	29	1.63
3.13	A	169	297	34	1.73	1.69	0‡
	B	197	279	24	1.65
6.25	A	204	269	27	1.65	1.66	0‡
	B	200	268	32	1.66
12.5 P	A	195	273	32	1.67	1.7	0‡
	B	176	290	34	1.72
25 P	A	-	-	-	-	-	-
	B	-	-	-	-
50 P	A	-	-	-	-	-	-
	B	-	-	-	-
CP 6	A	320	179	1	1.36	1.37	43
	B	312	185	3	1.38

CP Cyclophosphamide

-  Not selected for scoring

P   Precipitate observed at end of exposure period in blood-free cultures

‡ Cytostasis reported as 0 as the CBPI value is equal to or higher than the solvent control

MEM Minimal Essential Medium

 

 

CBPI Data – Main Experiment - 24-Hour Exposure without Metabolic Activation (S9)

Treatment / Concentration (µg/mL)	Replicate	Mononucleate Cells	Binucleate Cells	Multinucleate Cells	CBPI	Mean CBPI	Mean Cytostasis (%)
Vehicle (MEM)	A1	30	383	87	2.11	2.1	0
	A2	37	386	77	2.08
	B1	46	367	87	2.08
	B2	37	371	92	2.11
1.56	A	23	377	100	2.15	2.15	0‡
	B	29	370	101	2.14
3.13	A	28	376	96	2.14	2.13	0‡
	B	42	360	98	2.11
6.25	A	46	372	82	2.07	2.08	1
	B	45	364	91	2.09
12.5 P	A	47	357	96	2.1	2.14	0‡
	B	21	368	111	2.18
25 P	A	-	-	-	-	-	-
	B	-	-	-	-
50 P	A	-	-	-	-	-	-
	B	-	-	-	-
DC - 0.075	A	276	218	6	1.46	1.49	55
	B	254	233	13	1.52

DC Demecolcine

-  Not selected for scoring

P   Precipitate observed at end of exposure period in blood-free cultures

‡ Cytostasis reported as 0 as the CBPI value is equal to or higher than the solvent control

MEM Minimal Essential Medium

Conclusions:

Under the conditions of this study, the test material did not induce any statistically significant increases in the frequency of binucleate cells with micronuclei in either the absence or presence of a metabolising system. The test material was therefore considered to be non-clastogenic and non aneugenic to human peripheral blood lymphocytes in vitro.

Executive summary:

The clastogenic and aneugenic potential of the test material on the nuclei of normal peripheral blood lymphocytes was investigated according to the standardised guideline OECD 487, under GLP conditions. 

Duplicate cultures of human peripheral blood lymphocytes, treated with the test material, were evaluated for micronuclei in binucleate cells at three dose levels, together with solvent (quadruplicate cultures) and positive controls (duplicate cultures). Three exposure conditions were used for the study using a 4‑hour exposure in the presence and absence of a standard metabolising system (S9) at a 2 % final concentration, and a 24-hour exposure in the absence of metabolic activation. At the end of the exposure period, the cell cultures were washed and then incubated for a further 24 hours in the presence of Cytochalasin B.

The dose levels used in the Main Experiment were selected using data from the Preliminary Toxicity Test where the results indicated that the maximum concentration should be limited by the onset of test material precipitate. The dose levels selected for the Main Experiment were as follows:

Main Experiment

Exposure Group	Final concentration of Test Material (µg/mL)
4-hour without S9	0, 1.56, 3.13, 6.25, 12.5, 25, 50
4-hour with S9 (2%)
24-hour without S9

 All solvent Minimal Essential Medium (MEM) controls had frequencies of cells with micronuclei within the range expected for normal human peripheral blood lymphocytes and were considered acceptable for addition to the laboratory historical negative control data range.

The test material was non-toxic and did not induce any statistically significant increases in the frequency of binucleate cells with micronuclei in any of the three exposure groups using a dose range that included the lowest precipitating concentration. There were also no statistically significant concentration-related increases in any of the three exposure groups when evaluated with a trend test.  These results fulfilled the criteria for a clearly negative outcome.

The positive control items induced statistically significant increases in the frequency of cells with micronuclei with responses that are compatible with those in the laboratory historical positive control data range. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

Under the conditions of this study, the test material did not induce any statistically significant increases in the frequency of binucleate cells with micronuclei in either the absence or presence of a metabolising system. The test material was therefore considered to be non-clastogenic and non aneugenic to human peripheral blood lymphocytes in vitro.

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

09 September 2003 to 26 September 2003

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: See 'Version / remarks'

Version / remarks:

Standards established by the Minister of Labor pursuant to the provisions of Paragraph 2-1 of Article 57 of Industrial Safety and Health Law Test Guidelines (September 1, 1988 Notice No. 77 of the Ministry of Labor), Notice (June 2, 1997 Notice No. 67 of the Ministry of Labor) for amending the standards established by the Minister of Labor pursuant to the provisions of Paragraph 2-1 of Article 57 of the Industrial Safety and Health Act, and Administrative Communication “Concerning Specific Method for Mutagenicity Test Using Microorganisms and Method for Evaluating Test Results” - 08 February 1999

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

- Histidine requirement in the Salmonella typhimurium strains.
- Tryptophan requirement in the Escherichia coli strain.

Species / strain / cell type:

E. coli WP2 uvr A

Details on mammalian cell type (if applicable):

CELLS USED
- Storage: 0.07 mL of DMSO were added to 0.8 mL of bacterial suspension and dispensed and frozen with dry ice/acetone, and identified with a label. After that, it was stored in a super-low-temperature freezer. It was thawed when it was used for the test.
- Amino acid requirements, membrane mutation rfa, chemical-resistant factors, and UV sensitivity were inspected.

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Details on mammalian cell type (if applicable):

CELLS USED
- Storage: 0.07 mL of DMSO were added to 0.8 mL of bacterial suspension and dispensed and frozen with dry ice/acetone, and identified with a label. After that, it was stored in a super-low-temperature freezer. It was thawed when it was used for the test.
- Amino acid requirements, membrane mutation rfa, chemical-resistant factors, and UV sensitivity were inspected.

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system: S9
- source of S9: S9 fraction was prepared from the livers of SD male rats (7 weeks old; weight: 215 ± 8.8 g)
- method of preparation of S9 mix: 9 mL of distilled water were added to 1 vial of Cofactor-I and it was dissolved. After the solution was sterilised by filtration by use of a syringe filter having pore size of 0.20 μm, 1 mL of S9 was added, and S9 mix was obtained
- composition of S9 mix (1 mL):
S9 = 0.1 mL
MgCl2 = 8 μmol
KCl = 33 μmol
G-6-P = 5 μmol
NADPH = 4 μmol
NADH = 4 μmol
Sodium phosphate buffer solution = 100 μmol

Test concentrations with justification for top dose:

A dose setting test was conducted for the purpose of setting the dose levels of the test material used for the test. The bacterial strains doses chosen were 5, 15, 50, 150, 500, 1500 and 5000 μg/plate. The results of the dose setting test indicated no mutagenicity or growth inhibition against the bacterial strains at all of the doses of all bacterial strains, 5000 μg/plate was used as the largest dose in both of the case where the metabolic activation system was used and the case where no metabolic activation system was used.
For the mutagenicity test, the bacterial strain doses chosen were: 156, 313, 625, 1250, 2500 and 5000 μg/plate.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Water
- Justification for choice of solvent/vehicle: The test material readily mixed with water, and was stable in water.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Water

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

sodium azide

other: 2-Aminoanthracene and 2-(2-furyl)-3-(5-nitro-2-furyl) acrylamide

Details on test system and experimental conditions:

METHOD OF APPLICATION:
The preincubation method was adopted for the test. The test was conducted in the case where the metabolic activation system was used and in the case where no metabolic activation system was used.
- 0.5 mL of a 0.1 mol/L sodium phosphate buffer solution (pH 7.4, in the case of not using the metabolic activation system) or S9 mix (in the case of using the metabolic activation system) and 0.1 mL of the bacterial suspension having been subjected to preculture were added to a small test tube (13x100 mm) previously sterilised.
- Next, 0.1 mL of a negative control substance, a prepared test material liquid or a positive control substance solution was added to the test tube, and the test tube was shook (back-and-forth motion type, 120 times/min, amplitude: 3.3 cm) for 20 minutes by means of a constant-temperature shaking culture device set to 37 ℃. After completion of shaking culture, 2 mL of the top agar that had been kept warm were added and mixed. After that, it was poured over a minimal glucose agar plate culture medium and spread uniformly while moving the plate. Further, it was checked if there was precipitation when the prepared test material liquid was added.
- The plate was left to stand on a horizontal plane. After the top agar solidified, the plate was turned upside down and put in a heater-type incubator. The top agar was incubated for 48 hours in the incubator.
- For each group, 2 plates were used and identified by writing a group number on each plate with a marker pen. The plates having the same group number were identified by writing “a” and “b” on them.

EVALUATION OF EXPERIMENTAL DATA
Observation was made visually and by use of a stereoscopic microscope to check if there is growth inhibition against the bacterial in the background and if there was precipitation of the test material. Further, growth inhibition was classified as follows:
(-): The level of the growth of the background bacteria on the plate is about the same as that of the negative control growth.
(*): The growth of the background bacteria on the plate is obviously inhibited in comparison with that of the negative control group.
(**): The growth of the background bacteria on the plate is inhibited by the test substance, and as a result, the background bacteria die out, and there were minute colonies observed visually. Further, this case was regarded as the number of revertant colonies being “0”.
(***): The growth of the background bacteria on the plate is inhibited strongly by the test material, and as a result, the background bacteria die out, and there were no minute colonies observed visually.
The number of the revertant colonies was counted visually. However, in the case of the positive control groups, the number of the revertant colonies was measured twice for each one plate (turned round by approx. 90 degrees) by use of a corona analyzer (with area correction made), and the average of the measurement values was taken as the measurement value.

Evaluation criteria:

The results were judged to be positive when the number of revertant colonies per plate (average of the number of revertant colonies of 2 plates) in the test material treatment group increased two or more times the number of revertant colonies in the negative control, the number of revertant colonies increased with an increase in dose, and it was confirmed that there was reproducibility between dose setting test and mutagenicity test. Any other cases were judged to be negative.

Statistics:

No statistical processing was carried out for the result in this test.

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

DOSE SETTING TEST:
- Precipitation of test material: No precipitation was observed up to 5000 μg/plate, regardless of whether or not the metabolic activation system was used.
- Growth Inhibition: No growth inhibition was observed up to 5000 μg/plate, regardless of whether or not the metabolic activation system was used.
- Mutagenicity: There was no increase in the number of revertant colonies two or more times higher than the negative control.
Since the results of the dose setting test indicated no mutagenicity or growth inhibition against the bacterial strains at all of the doses of all bacterial strains, 5000 μg/plate was used as the largest dose in both of the case where the metabolic activation system was used and the case where no metabolic activation system was used. Further, in the mutagenicity test, the doses of the test bacterial strains, the metabolic activation system and the positive control group were used in the same way as in the dose setting test.

MUTAGENICITY TEST:
- Precipitation of test material: There was no precipitation of the test substance up to 5000 μg/plate or more, regardless of whether or not the metabolic activation system was used.
- Growth Inhibition against Bacterial Strains: There was no growth inhibition against the test substance up to 5000 μg/plate, regardless of whether or not the metabolic activation system was used.
- Measurement of Number of Revertant Colonies: There was no increase in the number of revertant colonies two or more times higher than the negative control.

VALIDITY OF TEST:
The number of the revertant colonies of the negative control and the positive control showed appropriate values within the range (mean3±S.D.) From this, it was judged that the test was conducted under appropriate conditions.

Mutagenicity Test Results

Metabolic Activation System: yes or no	Dose of Test Material (μg/plate)	Number of Revertant Colonies (per plate)
		Base Pair Substitution Type			Frameshift Type
		TA100	TA1535	WP2uvrA	TA98	TA1537
(-)S9 mix	Negative control Water for injection	108 , 117 (113 )	9, 9 (9)	21, 24 (23 )	20, 19 (20 )	9, 7 (8)
	156	120 , 111 (116)	8,7 (8)	23, 27 (25)	20 , 20 (20)	9,7 (8)
	313	127, 125 (126)	10,8 (9)	22, 21 (22)	21 , 22 ( 22)	10,8 (9)
	625	127 , 125 (126)	10,9 (10)	24,21 (23)	19 , 16 (18)	10,10 (10)
	1250	119 , 118 (119)	8,8 (8)	22 , 23 (23)	20 , 23 (22)	9, 9 (9)
	2500	135 ,113 (124)	8,7 (8)	23 ,20 (22)	17,20 (19)	10,9 (10)
	5000	120 ,118 (119)	9,7 (8)	18, 22 (20)	20, 18 (19)	10, 9 (10)
(+)S9 mix	Negative control Water for injection	124 ,114 (119 )	11,12 (12)	24 ,22 (23 )	26 , 33 (30 )	10, 14 (12)
	156	122 ,127 (125)	10,11 (11)	20, 23 ( 22)	28, 29 ( 29)	14, 11 ( 13)
	313	121 ,133 (127)	11 , 12 ( 12)	22 ,23 (23)	25 ,27 (26)	12,14 (13)
	625	127 ,104 (116)	14,10 (12)	23 24 (24)	26 ,32 (29)	10 , 11 ( 11)
	1250	112 ,137 (125)	9,14 (12)	28 , 27 (28)	30 , 33 (32)	11, 15 (13)
	2500	122 ,124 (123)	13,10 (12)	22, 22 (22)	23 , 26 (25)	11, 14 (13)
	5000	129 ,130 (130)	11,9 (10))	24 , 30 (27)	29 , 30 (30)	11, 14 (13)
Positive control: Not requiring S9 mix	Name	AF-2	NaN3	AF-2	AF-2	9AA
	Dose (μg/plate)	0.01	0.5	0.02	0.1	80
	No. of colonies/plate	489, 504 (497)	396, 328 (362)	301, 305 (303)	355, 397 (376)	388, 431 (410)
Positive control: Requiring S9 mix	Name	2AA	2AA	2AA	2AA	2AA
	Dose (μg/plate)	1	2	10	0.5	2
	No. of colonies/plate	1526, 1500 (1513)	335, 370 (353)	206, 238 (222)	843, 832 (838)	313, 314 (314)

 

1. Figures in ( ) show the average of two plates.
2. Names of positive controls
AF-2: 2(2furyl)-3-(5-nitro-2 furyl)acrylamide. NaN3:Sodiuma azide.
9AA:9-Aminoacridine hydrochloride hydrate. 2AA:2-Aminoanthracene

Conclusions:

Under the conditions of this study, the test material shows no mutagenicity.

Executive summary:

The genetic toxicity of the test material was investigated in a study equivalent to OECD 471, under GLP conditions.

The test material was investigated using microorganisms according to the preincubation method using five bacterial strains, namely, Salmonella typhimurium TA98, TA100, TA1535, TA1537 and Escherichia coli WP2uvrA both in the presence and absence of metabolic activation. 

The following doses were used:

Dose setting test (In the case of not using and using a metabolic activation system): All bacterial strains: 5, 15, 50, 150, 500, 1500 and 5000 μg/plate

Mutagenicity test (In the case of not using and using a metabolic activation system): All bacterial strains: 156, 313, 625, 1250, 2500 and 5000 μg/plate

In both dose setting test and mutagenicity test, there was no increase in the number of revertant colonies two or more times higher than the negative control in the cases of all five bacterial strains, regardless of whether or not there was a metabolic activation system. There was no growth inhibition observed against the bacterial strains up to 5000 μg/plate, regardless of whether or not there was a metabolic activation system. There was no precipitation of the test material observed up to 5000 μg/plate, regardless of whether or not there was a metabolic activation system.

Under the conditions of this study, the test material shows no mutagenicity.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Genetic toxicity in vitro Ames - key study (Saigo, 2003):

The genetic toxicity of the test material was investigated in a study equivalent to OECD 471, under GLP conditions. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

The test material was investigated using microorganisms according to the preincubation method using five bacterial strains, namely, Salmonella typhimurium TA98, TA100, TA1535, TA1537 and Escherichia coli WP2uvrA both in the presence and absence of metabolic activation. 

The following doses were used:

Dose setting test (In the case of not using and using a metabolic activation system): All bacterial strains: 5, 15, 50, 150, 500, 1500 and 5000 μg/plate

Mutagenicity test (In the case of not using and using a metabolic activation system): All bacterial strains: 156, 313, 625, 1250, 2500 and 5000 μg/plate

In both dose setting test and mutagenicity test, there was no increase in the number of revertant colonies two or more times higher than the negative control in the cases of all five bacterial strains, regardless of whether or not there was a metabolic activation system. There was no growth inhibition observed against the bacterial strains up to 5000 μg/plate, regardless of whether or not there was a metabolic activation system. There was no precipitation of the test material observed up to 5000 μg/plate, regardless of whether or not there was a metabolic activation system.

Under the conditions of this study, the test material shows no mutagenicity.

 

In vitro Micronucleus test - Morris (2021):

The clastogenic and aneugenic potential of the test material on the nuclei of normal peripheral blood lymphocytes was investigated according to the standardised guideline OECD 487, under GLP conditions. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

Duplicate cultures of human peripheral blood lymphocytes, treated with the test material, were evaluated for micronuclei in binucleate cells at three dose levels, together with solvent (quadruplicate cultures) and positive controls (duplicate cultures). Three exposure conditions were used for the study using a 4‑hour exposure in the presence and absence of a standard metabolising system (S9) at a 2 % final concentration, and a 24-hour exposure in the absence of metabolic activation. At the end of the exposure period, the cell cultures were washed and then incubated for a further 24 hours in the presence of Cytochalasin B.

The dose levels used in the Main Experiment were selected using data from the Preliminary Toxicity Test where the results indicated that the maximum concentration should be limited by the onset of test material precipitate. The dose levels selected for the Main Experiment were as follows:

Main Experiment

Exposure Group	Final Concentration of Test Material (µg/mL)
4-hour without S9	0, 1.56, 3.13, 6.25, 12.5, 25, 50
4-hour with S9 (2%)
24-hour without S9

All solvent Minimal Essential Medium (MEM) controls had frequencies of cells with micronuclei within the range expected for normal human peripheral blood lymphocytes and were considered acceptable for addition to the laboratory historical negative control data range.

The test material was non-toxic and did not induce any statistically significant increases in the frequency of binucleate cells with micronuclei in any of the three exposure groups using a dose range that included the lowest precipitating concentration. There were also no statistically significant concentration-related increases in any of the three exposure groups when evaluated with a trend test. These results fulfilled the criteria for a clearly negative outcome.

The positive control items induced statistically significant increases in the frequency of cells with micronuclei with responses that are compatible with those in the laboratory historical positive control data range. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

Under the conditions of this study, the test material did not induce any statistically significant increases in the frequency of binucleate cells with micronuclei in either the absence or presence of a metabolising system. The test material was therefore considered to be non-clastogenic and non aneugenic to human peripheral blood lymphocytes in vitro.

 

In vitro Mammalian gene mutation study - Smith (2021):

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 item 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.

Justification for classification or non-classification

In accordance with the criteria for classification as defined in Annex I, Regulation (EC) No. 1272/2008, the substance does not require classification with respect to genetic toxicity.