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Toxicological information

Genetic toxicity: in vitro

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Administrative data

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:
The study was conducted between 24 June 2014 and 23 September 2014.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study.
Cross-reference
Reason / purpose:
reference to other study

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
Qualifier:
according to
Guideline:
EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test
Deviations:
no
Qualifier:
according to
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
mammalian cell gene mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: flakes
Details on test material:
Identification: [1,3-phenylenebis(1-methylethylidene)]bis[tert-butyl] peroxide CAS# 2212-81-9
Physical State / Appearance: White flakes
Chemical name: [1,3-phenylenebis(1-methylethylidene)]bis[tert-butyl] peroxide
Molecular weight: 338.48 g/mol
CAS number: 2212-81-9
Batch: 0909144588
Purity: >99% (not accounted for in formulations)
Expiry / Retest Date: 01 October 2019
Storage Conditions: Approximately -20o C, in the dark

Method

Target gene:
Thymidine kinase
Species / strain
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
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.
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Test concentrations with justification for top dose:
Experiment 1
4-hour without S9: 1.65, 3.31, 6.63, 13.25, 26.5 µg/mL.
4-hour with S9: 0.83, 1.65, 3.31, 6.63, 13.25, 26.5 µg/mL.

Experiment 2
24-hour without S9: 3.38, 6.75, 13.5, 18, 22.5, 27 µg/mL.
4-hour with S9: 1.69, 3.38, 6.75, 13.5, 27, 36 µg/mL.

Experiment 3
4-hour with S9 (2%): 15, 20, 25, 30, 35, 40 µg/mL.
Vehicle / solvent:
Acetone
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
ethylmethanesulphonate
Details on test system and experimental conditions:
Test Item Preparation
Following solubility checks performed in-house for the Chromosome Aberration Test performed on the same test item (Harlan Laboratories Ltd. Project No. 41401119), the test item was accurately weighed and formulated in acetone prior to serial dilutions being prepared. The test item had a molecular weight of 338.48 therefore the maximum proposed dose level in the solubility test was set at 3385 µg/mL, the 10mM limit dose level and no correction for the purity of >99% was applied. However, acetone is toxic to L5178Y cells at dose volumes greater than 0.5% of the total culture volume. Therefore, the test item was formulated at 677 mg/ml and dosed at 0.5% to achieve the 10 mM limit dose level of 3385 µg/mL. 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) in the Chromosome Aberration Test performed on the same test item, the solubility data from which was considered acceptable for use in this study.
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 (Acetone) treatment groups were used as the vehicle controls. Ethylmethanesulphonate (EMS) Sigma batch BCBK5968V at 400 µg/mL and 150 µg/mL for Experiment 1 and Experiment 2, respectively, was used as the positive control in the absence of metabolic activation. Cyclophosphamide (CP) Acros batch A0302605 at 2 µg/mL was used as the positive control in the presence of metabolic activation. The positive controls were formulated in DMSO.

Preliminary Toxicity Test
A preliminary toxicity test was performed on cell cultures at 5E+05 cells/mL, using a 4 hour exposure period both with and without metabolic activation (S9), and at 1.5E+05 cells/mL using a 24-hour exposure period without S9. The dose range used in the preliminary toxicity test was 13.22 to 3385 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 2E+05 cells/mL, unless the mean cell count was less than 3E+05 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 2E+05 cells/mL, unless the mean cell count was less than 3E+05 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
Experiment 1
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 1E+06 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 (1.65 to 212 µg/mL in the absence of metabolic activation, and 0.83 to 106 µg/mL in the presence of metabolic activation), vehicle and positive controls. To each universal was added 2 mL of S9 mix if required, 0.1 mL of the treatment dilutions, (0.2 mL for the positive control) 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.

Experiment 2
As in Experiment 1, an exponentially growing stock culture of cells was established. The cells were counted and processed to give 1E+06 cells/mL in 10 mL cultures in R10 medium for the 4 hour treatment with metabolic activation cultures. In the absence of metabolic activation the exposure period was extended to 24 hours therefore 0.3E+06 cells/mL in 10 mL cultures were established in 25 cm2 tissue culture flasks. 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 (0.84 to 27 µg/mL in the absence of metabolic activation, and 1.69 to 54 µg/mL in the presence of metabolic activation), vehicle and positive controls. To each culture vessel was added 2 mL of S9 mix if required, 0.1 mL of the treatment dilutions, (0.2 mL for the positive controls) and sufficient R0 medium to give a final volume of 20 mL (R10 was used for the 24 hour exposure group).

The treatment vessels were incubated at 37 °C with continuous shaking using an orbital shaker within an incubated hood for 24 hours in the absence of metabolic activation and 4 hours in the presence of metabolic activation.

Experiment 3
As in the previous experiments, an exponentially growing stock culture of cells was established. The cells were counted and processed to give 1E+06 cells/mL in 10 mL cultures in R10 medium for the 4 hour treatment with metabolic activation cultures only. The treatments were performed in duplicate (A + B), with metabolic activation (2% S9 final concentration) at eight dose levels of the test item (15 to 50 µg/mL), vehicle and positive control. To each culture vessel was added 2 mL of S9 mix, 0.1 mL of the treatment dilutions, (0.2 mL for the positive control) and sufficient R0 medium to give a final volume of 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, for each experiment, the cells were washed twice using R10 medium then resuspended in R20 medium at a cell density of 2E+05 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 2E+05 cells/mL, unless the mean cell count was less than 3E+05 cells/mL in which case all the cells were maintained.

On Day 2 of the experiment, the cells were counted, diluted to 1E+04 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.

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

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 = (-1n P(0) x 100) / number of cells per 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)/100%

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:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Marked reductions in the Relative Suspension Growth (%) of cells treated with the test item when compared to the concurrent vehicle controls.
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
Mutagenicity Test
Experiment 1
There was evidence of marked toxicity following exposure to the test item in both the absence and presence of metabolic activation, as indicated by the RTG and %RSG values. There was also evidence of reductions in viability (%V) in the absence of metabolic activation, therefore indicating that residual toxicity had occurred in this exposure group. Based on the RTG and %RSG values, it was considered that optimum levels of toxicity were not achieved in either the absence or presence of metabolic activation, despite using a very narrow dose interval, due to the very steep toxicity curve of the test item. Whilst optimum levels of toxicity were not achieved, a dose level in the absence of metabolic activation that exceeded the upper limit of toxicity was plated as sufficient cells were available at the time of plating. The excessive toxicity observed at and above 53 µg/mL, in both the absence and presence of metabolic activation, resulted in these dose levels not being plated for viability or 5-TFT resistance. Acceptable levels of toxicity were seen with both positive control substances.

The vehicle controls had mutant frequency values that were considered acceptable for the L5178Y cell line at the TK +/- locus. Both of the positive controls produced marked increases in the mutant frequency per viable cell indicating that the test system was operating satisfactorily and that the metabolic activation system was functional.

The test item did not induce any statistically significant or dose related (linear-trend) increases in the mutant frequency x 10-6 per viable cell, at any of the dose levels (including the dose level that exceeded the upper limit of acceptable toxicity in the absence of metabolic activation), in either the absence or presence of metabolic activation. Precipitate of the test item was observed at and above 53 µg/mL immediately upon dosing.

Experiment 2
As was seen previously, there was evidence of marked toxicity in both the absence and presence of metabolic activation, as indicated by the RTG and %RSG values. There was evidence of modest reductions in viability (%V) in the presence of metabolic activation, therefore indicating that residual toxicity had occurred in this exposure group. Based on the %RSG and RTG values observed, optimum levels of toxicity were considered to have been achieved in both the absence and presence of metabolic activation. The excessive toxicity observed at and above 45 µg/mL in the presence of metabolic activation, resulted in these dose levels not being plated for viability or 5-TFT resistance. Acceptable levels of toxicity were seen with both positive control substances.

The 24-hour exposure without metabolic activation (S9) treatment, demonstrated that the extended time point had no marked effect on the toxicity of the test item.

The vehicle (solvent) controls had mutant frequency values that were considered acceptable for the L5178Y cell line at the TK +/- locus. Both of the positive controls produced marked increases in the mutant frequency per viable cell indicating that the test system was operating satisfactorily and that the metabolic activation system was functional.

The test item did not induce any statistically significant dose related (linear-trend) increases in the mutant frequency x 1E-06 per viable cell in the absence of metabolic activation. A statistically significant increase in mutant frequency was observed at 27 µg/mL. However, the GEF was not exceeded, there was no evidence of any increases in absolute numbers of mutant colonies, and the mutant frequency value observed would have been considered acceptable for vehicle controls. The response was therefore considered to be artefactual and of no toxicological significance.

The test item induced a marked statistically significant dose related (linear-trend) increase in the mutant frequency x 1E-06 per viable cell in the presence of metabolic activation. A statistically significant increase in mutant frequency was also observed at 36 µg/mL, the upper surviving dose level. The GEF was exceeded at this dose level, there were increases in mutant colony numbers, there was evidence of modest increases in mutant frequency, and the mutant frequency value observed exceeded the upper limit for vehicle controls. However, there was no marked shift towards small colony formation that would have indicated a clastogenic response. It was considered that the response may have been due to a cytotoxic mechanism and not a true genotoxic response. It was therefore considered appropriate, following consultation with the Sponsor, to perform a third experiment using a 4 hour exposure group in the presence of metabolic activation to confirm or disprove the response observed. Precipitate of the test item was not observed at any of the dose levels in either the absence or presence of metabolic activation.

Experiment 3
As was seen previously, there was once again evidence of marked toxicity, as indicated by the RTG and %RSG values. There was also once again evidence of a modest reduction in viability (%V), therefore indicating that residual toxicity had occurred. Based on the RTG and / or %RSG values observed, optimum levels of toxicity were considered to have been achieved. The excessive toxicity observed at and above 45 µg/mL, resulted in these dose levels not being plated for viability or 5 TFT resistance. The toxicity observed at 40 µg/mL markedly exceeded the upper acceptable limit of 90%, therefore, this dose was excluded from the statistical analysis. Acceptable levels of toxicity were seen with the positive control substance.

On this occasion the test item did not induce a statistically significant dose related (linear-trend) increase in the mutant frequency x 1E-06 per viable cell in the presence of metabolic activation. Statistically significant increases in mutant frequency were also not observed at any of the individual dose levels. A modest increase in mutant frequency was observed at 30 µg/mL. However, the GEF was not exceeded and there was also evidence of heterogeneity (poor correlation between A and B plates) with modest increases in mutant frequency only observed in the A plates, despite the A and B viability plates for that dose level having very similar values. There was also no evidence of a shift towards small colony formation that would have indicated a clastogenic response. It should also be noted that there was no evidence of any increases in mutant frequency at 35 µg/mL, a dose level that achieved optimum levels of toxicity with very similar viability plate counts as those for the 30 µg/mL dose level. It was therefore considered that, with no evidence of a consistent and reproducible increase in mutant frequency that exceeded the GEF, the increases in mutant frequency may be due to a cytotoxic mechanism and not a true genotoxic response. The response observed in the presence of metabolic activation in Experiment 2 was therefore considered not to be reproducible and of no toxicological significance. Precipitate of the test item was not observed at any of the dose levels.
Remarks on result:
other: all strains/cell types tested
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 13.22 to 3385 µg/mL in all three of the exposure groups. The results for the Relative Suspension Growth (%RSG) were as follows:

Dose

(mg/mL)

% RSG (-S9)

4-Hour Exposure

% RSG (+S9)

4-Hour Exposure

% RSG (-S9)

24-Hour Exposure

0

100

100

100

13.22

91

88

70

26.45

52

37

4

52.89

39

31

0

105.78

46

13

0

211.56

14

33

0

423.13

12

12

0

846.25

28

43

0

1692.5

41

38

0

3385

18

32

0

 

There was evidence of marked reductions in the Relative Suspension Growth (%) of cells treated with the test item when compared to the concurrent vehicle controls in all three of the exposure groups, with the most marked reductions observed in the 24-hour exposure group in the absence of metabolic activation. A precipitate of the test item was observed immediately upon dosing at and above 105.78 µg/mL in all three of the exposure groups. The precipitate of the test item became greasy / oily in appearance at 846.25 and 1692.5 µg/mL and aggregated at 3385 µg/mL. Based on the precipitate observations and the %RSG values observed, maximum exposure of the test item to the cells was considered to occur at 211.56 µg/mL in the 4-hour exposure group in the absence of metabolic activation, and at 105.78 µg/mL in the 4-hour exposure group in the presence of metabolic activation, due to the presence of precipitate effectively reducing exposure of the test item to the cells. These dose levels were therefore selected as the maximum dose levels in the subsequent Experiment 1. For the 24-hour exposure group in the absence of metabolic activation in Experiment 2, the maximum dose level was limited by test item-induced toxicity only.

Summary of results

Experiment 1

Treatment

(µg/mL)

4-Hours-S9

Treatment

(µg/mL)

4-Hours+S9

 

%RSG

RTG

MF§

 

%RSG

RTG

MF§

0

 

100

1.00

148.63

 

0

 

100

1.00

124.14

 

1.65

 

89

0.89

168.85

 

0.83

 

100

1.06

130.16

 

3.31

 

83

0.95

152.72

 

1.65

 

102

1.09

101.31

 

6.63

 

89

0.99

144.21

 

3.31

 

95

0.97

114.24

 

13.25

 

51

0.48

161.51

 

6.63

 

89

0.95

90.94

 

26.5

X

4

0.01

201.16

 

13.25

 

80

0.86

126.10

 

53

Ø

1

 

 

 

26.5

 

54

0.51

129.99

 

106

Ø

2

 

 

 

53

Ø

1

 

 

 

212

Ø

3

 

 

 

106

Ø

1

 

 

 

Linear trend

 

NS

Linear trend

 

NS

EMS

 

 

 

 

 

CP

 

 

 

 

 

400

 

69

0.47

1036.04

 

2

 

60

0.33

1220.40

 

 

 

 

 

 

 

 

 

 

 

 

 

Experiment 2

Treatment

(µg/mL)

24-Hours-S9

Treatment

(µg/mL)

4-Hours+S9

 

%RSG

RTG

MF§

 

%RSG

RTG

MF§

0

 

100

1.00

132.58

 

0

 

100

1.00

126.54

 

0.84

Ø

84

 

 

 

1.69

 

108

0.93

170.37

 

1.69

Ø

91

 

 

 

3.38

 

102

0.95

151.16

 

3.38

 

88

1.01

136.33

 

6.75

 

106

0.97

155.71

 

6.75

 

92

1.05

144.95

 

13.5

 

94

0.83

127.28

 

13.5

 

73

0.93

141.54

 

27

 

80

0.70

164.62

 

18

 

44

0.70

145.89

 

36

 

20

0.15

329.81

*

22.5

 

27

0.47

136.47

 

45

Ø

3

 

 

 

27

 

9

0.14

187.35

*

54

Ø

1

 

 

 

Linear trend

 

NS

Linear trend

 

***

EMS

 

 

 

 

 

CP

 

 

 

 

 

150

 

60

0.50

1139.47

 

2

 

66

0.38

1267.39

 

Experiment 3

Treatment

(µg/mL)

4-Hours+S9

 

%RSG

RTG

MF§

0

 

100

1.00

143.62

 

15

 

93

1.05

144.28

 

20

 

80

0.86

138.15

 

25

 

70

0.68

167.86

 

30

$$

29

0.30

268.90

 

35

 

10

0.09

172.60

 

40

X

2

0.02

182.70

 

45

$

1

 

 

 

50

$

1

 

 

 

Linear trend

 

NS

CP

 

 

 

 

 

2

 

69

0.43

1028.79

 

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

The test item did not induce any toxicologically significant increases in the mutant frequency at the TK +/- locus in L5178Y cells.
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 VitroMammalian 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….

Initially, two independent experiments were performed. 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 in duplicate, together with vehicle, (acetone), and positive controls using 4-hour exposure groups both in the absence and presence of metabolic activation (2% S9). In Experiment 2, the cells were treated with the test item at eight dose levels using a 4‑hour exposure group in the presence of metabolic activation (2% S9) and a 24-hour exposure group in the absence of metabolic activation. However, due to the response observed in the presence of metabolic activation in Experiment 2, a third experiment was performed, after consultation with the Sponsor, to confirm or disprove the response using a 4‑hour exposure group in the presence of metabolic activation (2% S9) only.

 

The dose ranges of test item used in the main test were 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[1,3-phenylenebis(1-methylethylidene)]bis[tert-butyl] peroxide CAS# 2212-81-9(µg/mL) plated for mutant frequency

4-hour without S9

1.65, 3.31, 6.63, 13.25, 26.5

4-hour with S9 (2%)

0.83, 1.65, 3.31, 6.63, 13.25, 26.5

 

Experiment 2

Group

Concentration of[1,3-phenylenebis(1-methylethylidene)]bis[tert-butyl] peroxide CAS# 2212-81-9(µg/mL) plated for mutant frequency

24-hour without S9

3.38, 6.75, 13.5, 18, 22.5, 27

4-hour with S9 (2%)

1.69, 3.38, 6.75, 13.5, 27, 36

Experiment 3

Group

Concentration of[1,3-phenylenebis(1-methylethylidene)]bis[tert-butyl] peroxide CAS# 2212-81-9(µg/mL) plated for mutant frequency

4-hour with S9 (2%)

15, 20, 25, 30, 35, 40

 

Results……..

The maximum dose levels used in the Mutagenicity Test were limited by a combination of 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 observed at and above 53 µg/mL immediately upon dosing in Experiment 1. 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 did not induce any toxicologically significant increases in the mutant frequency at any of the dose levels, either with or without metabolic activation, in either the first, second, or confirmatory third experiment.

 

Conclusion

The test item did not induce any toxicologically significant increases in the mutant frequency at the TK +/- locus in L5178Y cells.