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Genetic toxicity: in vitro

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Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
15 August 2014 to 07 October 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Based on OECD guideline and GLP compliant

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2014
Report Date:
2014

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
mammalian cell gene mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Test material form:
other: Solid

Method

Target gene:
thymidine kinase
Species / strain
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
The L5178Y TK+/- 3.7.2c mouse lymphoma cell line was obtained from Dr. J. Cole of the MRC
Cell Mutation Unit at the University of Sussex, Brighton, UK. The cells were originally
obtained from Dr. D. Clive of Burroughs Wellcome (USA) in October 1978 and were frozen in
liquid nitrogen at that time.

- Type and identity of media: 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.
- Periodically checked for Mycoplasma contamination: yes. Master stocks of cells were tested and found to be free of mycoplasma.
Additional strain / cell type characteristics:
not specified
Test concentrations with justification for top dose:
Group Concentration of Diacetate (CAS: 24085-06-1) (µg/mL) plated for mutant frequency
4-hour without S9 2.5, 5, 10, 20, 30, 40
4-hour with S9 (2%) 10, 20, 40, 80, 120, 160
Vehicle / solvent:
The vehicle control was DMSO
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
Solvent (DMSO) treatment groups were used as the vehicle controls.
True negative controls:
yes
Positive controls:
yes
Positive control substance:
cyclophosphamide
ethylmethanesulphonate
Remarks:
Ethylmethanesulphonate Sigma batch BCBK5968V at 400 µg/mL was used as the positive control in the absence of metabolic activation. Cyclophosphamide Acros batch A0302605 at 2 µg/mL was used as the positive control in the presence of metabolic activation.
Details on test system and experimental conditions:
Cell Line
The L5178Y TK+/- 3.7.2c mouse lymphoma cell line was obtained from Dr. J. Cole of the MRC Cell Mutation Unit at the University of Sussex, Brighton, UK. The cells were originally obtained from Dr. D. Clive of Burroughs Wellcome (USA) in October 1978 and were frozen in liquid nitrogen at that time.

Cell Culture
The stocks of cells are 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. The cells have a generation time of approximately 12 hours and were subcultured accordingly. RPMI 1640 with 20% donor horse serum (R20) and without serum (R0) are used during the course of the study. Master stocks of cells were tested and found to be free of mycoplasma.

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.

Test Item Preparation
Following solubility checks performed in-house, the test item was accurately weighed and
formulated in DMSO prior to serial dilutions being prepared. The molecular weight of the test
item was 250.24, therefore the maximum dose level investigated initially was 2502.4 µg/mL,
which was equivalent to 10 mM. The purity of the test item was 98.25% and was therefore
accounted for when formulating the dosing solutions. There was no marked change in pH when
the test item was dosed into media and the osmolality did not increase by more than 50 mOsm
(Scott et al. 1991). The pH and osmolality readings were within the following ranges respectively: 7.20-7.22; 422-454 mOsm
No analysis was carried out to determine the homogeneity, concentration or stability of the test
item formulation. The test item 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.

Control Preparation
Vehicle and positive controls were used in parallel with the test item. Solvent (DMSO)
treatment groups were used as the vehicle controls. Ethylmethanesulphonate (EMS) Sigma
batch BCBK5968V at 400 µg/mL was used as the positive control in the absence of metabolic
activation for Experiment 1. Cyclophosphamide (CP) Acros batch A0302605 at 2 µg/mL was
used as the positive control in the presence of metabolic activation for Experiment 1. The
positive controls were formulated in DMSO.


3.5 Microsomal Enzyme Fraction
PB/BNF S9 was prepared in-house on 27 July 2014 from the livers of male Sprague-Dawley rats
weighing approximately 250g. These had each received, orally, three consecutive daily doses of
phenobarbital/β-naphthoflavone (80/100 mg per kg per day) prior to S9 preparation on the fourth
day. This procedure was designed and conducted to cause the minimum suffering or distress to
the animals consistent with the scientific objectives and in accordance with the Harlan
Laboratories Ltd, Shardlow, UK policy on animal welfare and the requirements of the United
Kingdom’s Animals (Scientific Procedures) Act 1986 Amendment Regulations 2012. The
conduct of the procedure may be reviewed, as part of the Harlan Laboratories Ltd, Shardlow, UK
Ethical Review Process. The S9 was stored at approximately -196 °C in a liquid nitrogen
freezer.


S9-mix was prepared by mixing S9, NADP (5 mM), G-6-P (5 mM), KCl (33 mM) and MgCl2 (8 mM) in R0.

20% S9-mix (i.e. 2% final concentration of S9) was added to the cultures of the Preliminary Toxicity Test and of Experiment 1.

Preliminary Toxicity Test
A preliminary toxicity test was performed on cell cultures at 5 x 10E5 cells/mL, using a 4 hour exposure period both with and without metabolic activation (S9), and at 1.5 x 10E5 cells/mL using a 24-hour exposure period without S9. The dose range used in the preliminary toxicity test was 9.78 to 2502.4 µg/mL for all three of the exposure groups. Following the exposure period the cells were washed twice with R10, resuspended in R20 medium, counted and then serially diluted to 2 x 10E5 cells/mL, unless the mean cell count was less than 3 x 10E5 cells/mL in which case all the cells were maintained.

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 10E5 cells/mL, unless the mean cell count was less than 3 x 10E5 cells/mL in which case all the cells were maintained. 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 treatment toxicity, and a comparison of each treatment SG value to the concurrent vehicle control performed to give a percentage Relative Suspension Growth (%RSG) value.

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

i) Maximum recommended dose level, 5000 µg/mL or 10 mM.

ii) The presence of excessive precipitate where no test item-induced toxicity was observed.

iii) Test item-induced toxicity, where the maximum dose level used should produce 10 to 20% survival (the maximum level of toxicity required). This optimum upper level of toxicity was confirmed by an IWGT meeting in New Orleans, USA (Moore et al 2002).

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 10E6 cells/mL in 10 mL aliquots in R10 medium in sterile plastic universals. The treatments were performed in duplicate (A + B), both with and without metabolic activation (2% S9 final concentration) at eight dose levels of the test item (0.63 to 40 µg/mL in the absence of metabolic activation, and 2.5 to 160 µg/mL in the presence of metabolic activation), vehicle and positive controls. To each universal was added 2 mL of S9-mix if required, 0.2 mL of the treatment dilutions, (0.2 mL for the positive controls) and sufficient R0 medium to bring the total volume to 20 mL.

The treatment vessels were incubated at 37 °C for 4 hours with continuous shaking using an orbital shaker within an incubated hood.

Measurement of Survival, Viability and Mutant Frequency
At the end of the treatment period, the cells were washed twice using R10 medium then resuspended in R20 medium at a cell density of 2 x 10E5 cells/mL. The cultures were incubated at 37 °C with 5% CO2 in air and subcultured every 24 hours for the expression period of two days, by counting and dilution to 2 x 10E5 cells/mL, unless the mean cell count was less than 3 x 10E5 cells/mL in which case all the cells were maintained.

On Day 2 of the experiment, the cells were counted, diluted to 10E4 cells/mL and plated for mutant frequency (2000 cells/well) in selective medium containing 4 µg/mL 5 trifluorothymidine (TFT) in 96-well microtitre plates. Cells were also diluted to 10 cells/mL and plated (2 cells/well) for viability (%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 treatment toxicity during the expression period as a comparison to the vehicle control, and when combined with the Viability (%V) data a Relative Total Growth (RTG) value.

Plate Scoring
Microtitre plates were scored using a magnifying mirror box after ten to fourteen 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 mutation plates were also recorded as the additional information may contribute to an understanding of the mechanism of action of the test item (Cole et al, 1990). Colonies are scored manually by eye using qualitative judgement. 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 mutation plates. The plates were incubated for two hours. MTT is a vital stain that is taken up by viable cells and metabolized to give a brown/black color, thus aiding the visualization of the mutant colonies, particularly the small colonies.
Evaluation criteria:
A mutation assay is considered acceptable if it meets the following criteria:
1. The majority of the plates, for either viability (%V) or TFT resistance are analyzable for each experiment.
2. The absolute viability (%V) at the time of mutant selection of the solvent controls is 65 to 120 %.
3. The total suspension growth of the solvent control following 4 hour treatment, calculated by the day 1 fold-increase in cell number multiplied by the day 2 fold increase in cell number, should be in the range of 8 to 32. Following 24 hour treatment the total suspension growth should be in the range of 32 to 180.
4. The in-house vehicle control mutant frequency is in the range of 50 – 170 x 10E6 cells. Vehicle control results should ideally be within this range, although minor errors in cell counting and dilution, or exposure to the metabolic activation system, may cause this to be slightly elevated. Experiments where the vehicle control values are markedly greater than 200 x 10E6 mutant frequency per survivor are not acceptable and will be repeated.
5. Positive control chemicals (EMS and CP) should induce at least three to five fold increases in mutant frequency greater than the corresponding vehicle control. The positive controls should ideally yield an absolute increase in total MF, that is an increase above spontaneous background MF (an induced MF [IMF]), of at least 300 x 10E6 cells.
6. The upper limit of cytotoxicity observed in the positive control culture should be the same as for the experimental cultures i.e. the relative total growth (RTG) and percentage relative suspension growth (%RSG) should be greater than 10 % of the concurrent selective control group.
7. The highest concentration of the test item should be 10 mM or 5000 µg/mL, unless limited by toxicity or solubility of the test item. If toxicity occurred, the highest concentration should lower the relative total growth (RTG) and/or percentage relative suspension growth (%RSG) to ~ 10 to 20 % of surival.
Statistics:
A) Calculation of Percentage Relative Suspension Growth (%RSG)
The cell counts obtained immediately post treatment 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/vehicle control 0-hour cell count
%RSG = [(dose SG x dose Day 0 Factor)/vehicle control SG] x 100

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

P(0)= number of negative wells/total wells plated
%V= -lnP(0) x100/(number of cells/well)

C) 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 RTGis calculated:
RTG = (RCE x %RSG)/100%


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

The experimental data was analyzed using a dedicated computer program, Mutant 240C by York Electronic Research, which follows the statistical guidelines recommended by the UKEMS (Robinson W D et al., 1989).

Results and discussion

Test results
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Cytotoxicity was observed in the preliminary cytotoxicity test.
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
Positive controls validity:
valid
Additional information on results:
Following consultation with the Sponsor, it was considered unnecessary to perform the scheduled second experiment as an unequivocal positive mutagenic response was obtained in the 4-hour exposure groups in Experiment 1. This is in accordance with the current OECD 476 test guideline.
Remarks on result:
other: other: S9 activation
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Preliminary Cytotoxicity Test

The dose range of the test item used in the preliminary toxicity test was 9.78 to 2502.4 µg/mL.

The results for the Relative Suspension Growth (%RSG) were as follows:

Dose(µg/mL)   % RSG (-S9) 4-Hour Exposure  % RSG (+S9) 4-Hour Exposure  % RSG (-S9) 24-Hour Exposure 
 0  100  100  100
 9.78  85  101  73
 19.55  43  94  31
 39.1  2  76  1
 78.2  1  35  0
 156.4  2  3  1
 312.8  2  2  1
 625.8  2  3  1
 1251.2  2  3  1
 2502.4  5  3  1
       

There was evidence of marked reductions in the Relative Suspension Growth (%RSG) of cells treated with the test item when compared to the concurrent vehicle controls in all three of the exposure groups. The toxicity curve of the test item was very steep in all three exposure groups. Overall, greasy / oily precipitate of the test item was observed at and above 625.6 µg/mL. Based on the %RSG values observed, the maximum dose level in the subsequent Mutagenicity Test was limited by test item-induced toxicity.

Table 1 Summary of Results

Treatment

(µg/mL)

 

4-Hours-S9

Treatment

(µg/mL)

 

4-Hours+S9

%RSG

RTG

MF§

%RSG

RTG

MF§

0

100

1.000

119.96

0

100

1.00

139.54

0.63 Ø

94

 

 

2.5 Ø

97

 

 

1.25 Ø

87

 

 

5    Ø

95

 

 

2.5

88

0.94

126.20

10

96

1.19

118.10

5

78

0.79

121.07

20

92

1.00

126.16

10

73

0.63

166.36

40

77

0.8

150.85

20

45

0.37

311.22 *

80

50

0.49

448.76

30

21

0.13

1136.01 *

120    X

21

0.08

1550.66

40     X

2

0.01

720.52

160   X

4

0.00

1196.14

Linear Trend                                                ***

Linear Trend                                                ***

EMS

400

59

0.42

874.06

CP

2

59

0.35

1097.42

; %RSG = Relative Suspension Growth; RTG = Relative Total Growth; § or # = Positive wells per tray, 96 wells plated unless otherwise stated in parenthesis; CP = Cyclophosphamide; EMS = Ethylmethanesulphonate; MF§ = 5-TFT resistant mutants/106

viable cells 2 days after treatment; Ø = Not plated for viability or 5-TFT resistance; X = Treatment excluded from test statistics due to toxicity; * = p<0.05; *** = p<0.001

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
positive with metabolic activation
positive without metabolic activation

The test item induced toxicologically significant increases in the mutant frequency at the TK +/- locus in L5178Y cells following four hours exposure to the test item and is therefore considered to be mutagenic under the conditions of the test. These increases were observed in both the absence and presence of metabolic activation, and were predominantly due to small colony formation indicating a possible clastogenic response.
Executive summary:

Introduction

The study was conducted according to a method that was designed to assess the potential mutagenicity of the test item on the thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line. The method was designed to be compatible with the OECD Guidelines for Testing of Chemicals No.476 "In Vitro Mammalian Cell Gene Mutation Tests" adopted 21 July 1997, Method B17 of Commission Regulation (EC) No. 440/2008 of 30 May 2008, the US EPA OPPTS 870.5300 Guideline, and in alignment with the Japanese MITI/MHW guidelines for testing of new chemical substances.

Methods

In Experiment 1, L5178Y TK +/- 3.7.2c mouse lymphoma cells (heterozygous at the thymidine kinase locus) were treated with the test item at eight dose levels, using duplicate cultures, together with vehicle (DMSO) and positive controls using 4-Hour exposure groups both in the absence and presence of metabolic activation (2% S9). An unequivocal mutagenic response was obtained in the 4-hour exposure groups in Experiment 1 and therefore, following consultation with the Sponsor, a repeat experiment was not performed. This is in accordance with the current OECD 476 test guideline.

The dose range of test item used in the main test was selected following the results of a preliminary toxicity test. The dose levels plated out for viability and expression of mutant colonies were as follows:

Experiment 1

Group                            Concentration of Diacetate (CAS: 24085-06-1) (µg/mL) plated for mutant frequency

4-hour without S9                                                               2.5, 5, 10, 20, 30, 40

4-hour with S9 (2%)                                                         10, 20, 40, 80, 120, 160

Results

The maximum dose level used in the Mutagenicity Test was limited by test item-induced toxicity, and the presence of precipitate effectively reducing exposure of the test item to the cells. Precipitate of the test item was not observed at any of the dose levels in the Mutagenicity Test. The vehicle controls (DMSO) had mutant frequency values that were considered acceptable for the L5178Y cell line at the TK +/- locus. The positive control treatment induced marked increases in the mutant frequency indicating the satisfactory performance of the test and of the activity of the metabolizing system.

The test item induced toxicologically significant dose-related (linear-trend) increases in the mutant frequency in the 4-hour exposure groups in both the absence and presence of metabolic activation. The increases in mutant frequency observed were predominantly due to small colony formation, indicating clastogenic activity resulting in structural chromosome damage.

Conclusion

The test item was considered to be mutagenic to L5178Y cells under the conditions of the test.