[1,3-phenylenebis(1-methylethylidene)]bis[tert-butyl] peroxide

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Three studies are available for assessment of genetic toxicity on the registered substance. Theydid not show any mutagenic activity.

In vitro genotoxicity in bacteria

In a Klimisch 2 study (TNO, 1976), the potential of the substance to induce reverse mutation inSalmonella typhimuriu m(strains: TA 1535, TA 1537, TA 98, TA 100 and TA 1538) was evaluated according to Ames et al. (1975), bydirect plate incorporation (the first experiment and the second without S9mix), atfour dose-levels (0.2, 2, 20, 500 g/plate). No mutagenic activity was recorded, with and without activation, for any of the 5 tested strains.

In vitro genotoxicity in mammalian cells

Two other studies were carried out in order to have information on potential of the registered substance to induce mutations or chromosomal aberrations in mammalian cells: In vitro mammalian cell gene mutation test in L5178Y TK+/- mouse lymphoma cells (Harlan, 2014) and an In vitro mammalian chromosome aberration test in cultured human lymphocytes (Harlan, 2015). Both of the studies are are Klimisch 1 since they were performed according to OECD guidelines and are GLP. Both studies were negative.

In conclusion,[1,3-phenylenebis(1-methylethylidene)]bis[tert-butyl] peroxidewas shown to be not genotoxic in vitro.

Short description of key information:

[1,3-phenylenebis(1-methylethylidene)]bis[tert-butyl] peroxide did not induce reverse mutation in Salmonella typhimurium (strains: TA 1535, TA 1537, TA 1538, TA 98 and TA 100). The substance also did not induce chromosomal aberrations in mammalian cells in vitro.

Endpoint Conclusion:No adverse effect observed (negative)

Link to relevant study records

Reference

Reference 1

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.

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test

Deviations:

no

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Target gene:

Thymidine kinase

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

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).

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'.

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

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.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experimental start date: 17th June 2014 Experimental completion date: 20th Novemner 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted to GLP and in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do no effect the quality of the relevant results.

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Target gene:

Not applicable.

Species / strain / cell type:

lymphocytes: human

Details on mammalian cell type (if applicable):

For each experiment, sufficient whole blood was drawn from the peripheral circulation of a non smoking volunteer 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. The cell-cycle time for the lymphocytes from the donors used in this study was determined using BrdU (bromodeoxyuridine) incorporation to assess the number of first, second and third division metaphase cells and so calculate the average generation time (AGT). The mean value of the AGT for the pool of regular donors used in this laboratory has been determined to be approximately 16 hours under typical experimental exposure conditions.

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

phenobarbitone and beta-naphthoflavone induced rat liver, S9

Test concentrations with justification for top dose:

Experiment 1:
4(20)-hour without S9: 3.125, 6.25, 12.5, 25, 50, 100, 200, 400 µg/mL
4(20)-hour with S9 (2%): 12.5, 25, 50, 100, 200, 400 µg/mL

Experiment 2:
24-hour without S9: 3.125, 6.25, 12.5, 25, 50, 100, 200 µg/mL
4(20)-hour with S9 (1%): 6.25, 12.5, 25, 50, 100, 200 µg/mL

Vehicle / solvent:

Acetone

Untreated negative controls:

yes

Remarks:

0 µg/mL

Negative solvent / vehicle controls:

yes

Remarks:

Solvent treatment groups were used as the vehicle control

True negative controls:

no

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

Absence of S9 at 0.4 and 0.2 µg/mL in Experiments 1 and 2 respectively. Dissolved in Minimal Essential Medium.

Untreated negative controls:

yes

Remarks:

0 µg/mL

Negative solvent / vehicle controls:

yes

Remarks:

Solvent treatment groups were used as the vehicle control

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

+S9: 0.5 µg/mL in both experiments. Dissolved in DMSO.

Details on test system and experimental conditions:

Test Procedure
Culture conditions
Duplicate lymphocyte cultures (A and B) were established for each dose level by mixing the following components, giving, when dispensed into sterile plastic flasks for each culture:

9.05 mL MEM, 10% (FBS)
0.1 mL Li-heparin
0.1 mL phytohaemagglutinin
0.75 mL heparinized whole blood

With Metabolic Activation (S9) Treatment
After approximately 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 0.05 mL of the appropriate solution of vehicle control or test item was added to each culture. For the positive control, 0.1 mL of the appropriate solution was added to the cultures. 1mL 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 of Experiment 1.

In Experiment 2, 1 mL of 10% S9-mix (i.e. 1% final concentration of S9 in standard co-factors), was added. All cultures were then returned to the incubator. The nominal final volume of each culture was 10 mL.

After 4 hours at approximately 37 ºC, 5% CO2 in humidified air, 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 original culture medium. The cells were then re-incubated for a further 20 hours at approximately 37 ºC in 5% CO2 in humidified air.

Without Metabolic Activation (S9) Treatment
In Experiment 1, after approximately 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 re-suspended in the required volume of fresh MEM (including serum) and dosed with 0.05 mL of the appropriate vehicle control, test item solution or 0.1 mL of positive control solution. The total volume for each culture was a nominal 10 mL.

After 4 hours at approximately 37 ºC, 5% CO2 in humidified air, 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. The cells were then returned to the incubator for a further 20 hours.

In Experiment 2, in the absence of metabolic activation, the exposure was continuous for 24 hours. Therefore, when the cultures were established the culture volume was a nominal 9.95 mL. After approximately 48 hours incubation the cultures were removed from the incubator and dosed with 0.05 mL of vehicle control, test item dose solution or 0.1 mL of positive control solution. The nominal final volume of each culture was 10 mL. The cultures were then incubated at approximately 37 ºC, 5% CO2 in humidified air for 24 hours.

The preliminary toxicity test was performed using both of the exposure conditions as described for Experiment 1 and for Experiment 2 in the absence of metabolic activation only.


Preliminary Toxicity Test
Three exposure groups were used:

i) 4 hours exposure to the test item without S9-mix, followed by a 20-hour recovery period in treatment-free media, 4(20)-hour exposure.
ii) 4 hours exposure to the test item with S9-mix (2%), followed by a 20-hour recovery period in treatment-free media, 4(20)-hour exposure.
iii) 24-hour continuous exposure to the test item without S9-mix.

The dose range of test item used was 0, 13.22, 26.45, 52.89, 105.78, 211.56, 423.13, 846.25, 1629.5 and 3385 µg/mL.

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 mitotic index evaluation. Mitotic index data was used to estimate test item toxicity and for selection of the dose levels for the main test.

Experiment 1
Two exposure groups were used for Experiment 1:

i) 4-hour exposure to the test item without S9-mix, followed by 20-hour culture in treatment-free media prior to cell harvest. The dose range of test item used was 3.125, 6.25, 12.5, 25, 50, 100, 200 and 400 µg/mL.

ii) 4-hour exposure to the test item with S9-mix (2%), followed by 20-hour culture in treatment-free media prior to cell harvest. The dose range of test item used was 12.5, 25, 50, 100, 200 and 400 µg/mL.


Experiment 2
Two exposure groups were used for Experiment 2:

i) 24-hour continuous exposure to the test item without S9-mix prior to cell harvest. The dose range of test item used was 3.125, 6.25, 12.5, 25, 50 and 100 µg/mL.

ii) 4-hour exposure to the test item with S9-mix (1%) followed by 20-hour culture in treatment-free media prior to cell harvest. The dose range of test item used was 6.25, 6.25, 12.5, 25, 50, 100 and 200 µg/mL.


Cell Harvest
Mitosis was arrested by addition of demecolcine (Colcemid 0.1 µg/mL) two hours before the required harvest time. After incubation with demecolcine, the cells were centrifuged, the culture medium was drawn off and discarded, and the cells re-suspended in 0.075M hypotonic KCl. After approximately fourteen minutes (including centrifugation), most of the hypotonic solution was drawn off and discarded. The cells were re-suspended and then fixed by dropping the KCl cell suspension into fresh methanol/glacial acetic acid (3:1 v/v). The fixative was changed at least three times and the cells stored at approximately 4 ºC to ensure complete fixation prior to slide preparation.

Evaluation criteria:

Evaluation criteria
The following criteria were used to determine a valid assay:

Negative Control
The frequency of cells with chromosome aberrations (excluding gaps) in the vehicle control cultures will normally be within the laboratory historical control data range.

Positive Control
All the positive control chemicals must induce a clear positive response (p≤0.01). Acceptable positive responses demonstrate the validity of the experiment and the integrity of the S9-mix.

Statistics:

The frequency of cells with aberrations excluding gaps and the frequency of polyploid cells was compared, where necessary, with the concurrent vehicle control value using Fisher's Exact test.

Species / strain:

lymphocytes: Human

Metabolic activation:

with and without

Genotoxicity:

not determined

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Preliminary Toxicity Test
The dose range for the Preliminary Toxicity Test was 13.22 to 3385 µg/mL. The maximum dose was the 10 mM concentration.

A precipitate of the test item was observed in the parallel blood-free cultures at the end of the exposure at and above 105.78 µg/mL in the 4(20)-hour exposure group in the absence of S9-mix at and above 846.25 µg/mL in the 4(20)-hour exposure group in the presence of S9-mix and at and above 52.89 µg/mL in the continuous exposure group.

Haemolysis was observed following exposure to the test item at and above 52.89 µg/mL in the 4(20)-hour exposure group in the absence of S9-mix and at 3385 µg/mL only in the 4(20)-hour exposure group in the presence of S9-mix and the 24-hour continuous exposure group. Haemolysis is an indication of a toxic response to the erythrocytes and not indicative of any genotoxic response to the lymphocytes.

Microscopic assessment of the slides prepared from the exposed cultures showed that metaphase cells were present up to 3385 µg/mL in the 4(20)-hour exposures in the presence and absence of metabolic activation. The maximum dose with metaphases present in the 24-hour continuous exposure was 1692.5 µg/mL. The mitotic index data are presented in Table 1. The test item induced some evidence of toxicity in all of the exposure groups.

The selection of the maximum dose levels for the main tests were partially based on toxicity and the onset of the formation of precipitate. This is clearly demonstrated in the 24-hour exposure data where 52% mitotic inhibition was recorded at 52.89 µg/mL and 76% mitotic inhibition was observed at 423.13 µg/mL where the onset of the precipitate was noted. It was considered that maximum test item exposure was occurring at this point.


Chromosome Aberration Test - Experiment 1
The dose levels of the controls and the test item are given in the table below:

Group Final concentration of
[1,3-phenylenebis(1-methylethylidene)]bis[tert-butyl] peroxide CAS# 2212-81-9 (µg/mL)
4(20)-hour without S9 0*, 3.125, 6.25*, 12.5*, 25*, 50, 100*, 200, 400, MMC 0.4*
4(20)-hour with S9 (2%) 0*, 12.5, 25*, 50, 100*, 200*, 400, CP 5*

The qualitative assessment of the slides determined that the toxicity was similar to that observed in the Preliminary Toxicity Test and that there were metaphases suitable for scoring present up to 400 µg/mL both exposure groups.

Precipitate observations were made at the end of exposure and a precipitate was noted at 400 µg/mL in the 4(20)-hour exposure group in the absence of S9-mix. No precipitate was observed the in presence of S9-mix. Haemolysis was observed at and above 50 µg/mL in the absence of S9-mix only.
The mitotic index confirm the qualitative observations in that a dose-related inhibition of mitotic index was observed, and that 37% and 59% mitotic inhibition was achieved at 25 and 100 µg/mL, respectively, in the absence of S9-mix. The toxicity observed at 50 µg/mL in the absence of S9-mix precluded its selection for metaphase analysis. In the presence of S9-mix, a dose-related inhibition of mitotic index was observed and that 35% and 54% mitotic inhibition was achieved at 100 and 200 µg/mL, respectively.

The maximum dose level selected for metaphase analysis in both exposure groups was based on toxicity, and was 100 µg/mL and 200 µg/mL in the absence and presence of S9-mix, respectively.

The chromosome aberration data are given in Table 4 and Table 5. All of the vehicle control cultures had frequencies of cells with chromosome aberrations within the expected range. The positive control items induced statistically significant increases in the frequency of cells with aberrations. The metabolic activation system was therefore shown to be functional and the test method itself was operating as expected.

The test item did not induce any statistically significant increases in the frequency of cells with aberrations either in the absence or presence of metabolic activation.

The polyploid cell frequency data are given in Table 8. The test item did not induce statistically significant increases in the numbers of polyploid cells at any dose level in either of the exposure groups.

Chromosome Aberration Test - Experiment 2
The dose levels of the controls and the test item are given in the table below:

Group Final concentration of
[1,3-phenylenebis(1-methylethylidene)]bis[tert-butyl] peroxide CAS# 2212-81-9 (µg/mL)
24-hour without S9 0*, 3.125, 6.25*, 12.5*, 25*, 50, 100, MMC 0.2*
4(20)-hour with S9 (1%) 0*, 6.25*, 12.5*, 25*, 50, 100, 200, CP 5*

The qualitative assessment of the slides determined that there were metaphases suitable for scoring present at 100 µg/mL and 200 µg/mL in the absence and presence of S9-mix, respectively.

A precipitate of the test item was observed at the end of exposure at 200 µg/mL in the 4(20)-hour exposure group and at 100 µg/mL in the 24 hour continuous exposure group. No haemolysis was observed at the end of exposure in either exposure group.

The mitotic index data are given in Table 3. They confirm the qualitative observations in that a dose-related inhibition of mitotic index was observed in both exposure groups. In the absence of S9-mix, 23% and 86% mitotic inhibition was achieved at 25 and 50 µg/mL, respectively. In the presence of S9-mix, 33% and 50% mitotic inhibition was achieved at 12.5 and 25 µg/mL, respectively.

The maximum dose level selected for metaphase analysis was, therefore, 25 µg/mL in both exposure groups and was based on toxicity. The toxicity observed at 50 µg/mL in both exposure groups precluded its selection for metaphase analysis.

The chromosome aberration data are given in Table 6 and Table 7. All of the vehicle control cultures had frequencies of cells with chromosome aberrations within the expected range. The positive control items induced statistically significant increases in the frequency of cells with aberrations. The metabolic activation system was therefore shown to be functional and the test method itself was operating as expected.

The test item did not induce any statistically significant increases in the frequency of cells with chromosome aberrations either in the absence or presence of metabolic activation.

The polyploid cell frequency data are given in Table 8. The test item did not induce a statistically significant increase in the numbers of polyploid cells at any dose level in either of the exposure groups.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

LIST OF TABLES

 Table 1               Mitotic Index - Preliminary Toxicity Test

Table 2               Mitotic Index - Experiment 1.

Table 3               Mitotic Index - Experiment 2.

Table 4               Results of Chromosome Aberration Test - Experiment 1 Without Metabolic Activation (S9)

Table 5               Results of Chromosome Aberration Test - Experiment 1 With Metabolic Activation (2% S9)

Table 6               Results of Chromosome Aberration Test - Experiment 2 Without Metabolic Activation (S9)

Table 7               Results of Chromosome Aberration Test - Experiment 2 With Metabolic Activation (1% S9)

Table 8               Mean Frequency of Polyploid Cells (%)

LIST OF APPENDICES

 Appendix 1     Chromosome Structural Aberrations: Classification and Historical Control Data

Appendix 2     Copy of the Certificate of Analysis

Appendix 3     Monitoring Authority Statement of GLP Compliance

 

Conclusions:

Interpretation of results (migrated information):
negative

The test item did not induce statistically significant increase in the frequency of cells with chromosome aberrations, in either the absence or presence of a liver enzyme metabolizing system, in either of two separate experiments. The test item was therefore considered to be non clastogenic to human lymphocytes in vitro.

Executive summary:

Introduction

This report describes the results of anin vitrostudy for the detection of structural chromosomal aberrations in cultured mammalian cells. It supplements microbial systems insofar as it identifies potential mutagens that produce chromosomal aberrations rather than gene mutations (Scottet al., 1991). 

 

 

Methods…….

Duplicate cultures of human lymphocytes, treated with the test item, were evaluated for chromosome aberrations at up to four dose levels, together with vehicle and positive controls. Four treatment conditions were used for the study; i.e. in Experiment 1, 4 hours in the presence of an induced rat liver homogenate metabolizing system (S9), at a 2% final concentration with cell harvest after a 20-hour expression period and a 4 hours exposure in the absence of metabolic activation (S9) with a 20-hour expression period. In Experiment 2, the 4 hours exposure with addition of S9 was repeated (using a 1% final S9 concentration), whilst in the absence of metabolic activation the exposure time was increased to 24 hours.

 

The dose levels used in the main experiments were selected using data from the preliminary toxicity test and were as follows:

 

Experiment 1:

Group	Final concentration of test item [1,3-phenylenebis(1-methylethylidene)]bis[tert-butyl] peroxide CAS# 2212-81-9(µg/mL)
4(20)-hour without S9	3.125, 6.25, 12.5, 25, 50, 100, 200, 400
4(20)-hour with S9 (2%)	12.5, 25, 50, 100, 200, 400

 

Experiment 2:

Group	Final concentration of test item [1,3-phenylenebis(1-methylethylidene)]bis[tert-butyl] peroxide CAS# 2212-81-9(µg/mL)
24-hour without S9	3.125, 6.25, 12.5, 25, 50, 100, 200
4(20)-hour with S9 (1%)	6.25, 12.5, 25, 50, 100, 200

 

 

Results…….

All vehicle (acetone) controls had frequencies of cells with aberrations within the range expected for normal human lymphocytes.

 

All the positive control items induced statistically significant increases in the frequency of cells with aberrations. The metabolic activation system was therefore shown to be functional and the test method itself was operating as expected.

 

The test item did not induce any statistically significant increases in the frequency of cells with aberrations, in either of two separate experiments, using a dose range that included a dose level that induced approximately 50% mitotic inhibition.

 

 

Conclusion

The test item,[1,3-phenylenebis(1-methylethylidene)]bis[tert-butyl] peroxide CAS# 2212-81-9was considered to be non-clastogenic to human lymphocytesin vitro.

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1976

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

2d: Study was conducted in accordance with recognized published scientific procedure for examining the genotoxicity potential of a test compound in S. typhimurium bacteria strains. Test method utilized recognized positive controls that gave the expected positive responses, confirming the sensitivity of the method.

Qualifier:

no guideline followed

Principles of method if other than guideline:

According to Ames method: Ames, B.N., McCann, J. and Yamasaki, E. (1975). Methods for detecting carcinogens and mutagens with Salmonella/Mammalian-Microsome Mutagenicity Test. Mutation Research, 31, 347-364.

GLP compliance:

no

Type of assay:

bacterial reverse mutation assay

Target gene:

- The set of Salmonella typhimurium was a gift of Dr B.N. Ames, Berkeley, California, USA, and was checked for histidine requirement, for sensitivity crystal violet, deoxychloate, and UV.

Species / strain / cell type:

other: S. typhimurium TA 1535, TA 1537, TA1538, TA 98 and TA 100

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9: it was prepared from 2 male rats induced with 0.1 % phenobarbitone in their drinking water for 5 days. Activation propertieswere checked with  TA1538 and 4-BP. The optimum activity was produced with 100 ul/plate with 4-aminobiphenyl.

Test concentrations with justification for top dose:

Doses used in Exp. 1 were: 0.2, 2, 20 and 500 ug/plate.
Doses used in Exp. 2 were: 0.02 and  0.2 ug/plate.

Vehicle / solvent:

Solvant used: dimethylsulfoxide

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: methylmethanesulfonate (TA 1535, TA 100); N-Methyl-N-Nitro-N-Nitrosoguanidine (MNNG)(TA 1535, TA 100);  9-Aminoacridine (9-AA) (TA1537), 4-aminobiphenyl (TA100, TA98, TA1538)

Remarks:

with S9

Positive control substance:

other: methylmethanesulfonate (TA 100); N-Methyl-N-Nitro-N-Nitrosoguanidine (MNNG) (TA 1535, TA 100);  9-Aminoacridine (9-AA) (TA1537)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium; in agar (plate incorporation); preincubation; in suspension; as impregnation on paper disk


DURATION
- Preincubation period:
- Exposure duration:
- Expression time (cells in growth medium):
- Selection time (if incubation with a selection agent):
- Fixation time (start of exposure up to fixation or harvest of cells):


SELECTION AGENT (mutation assays):
SPINDLE INHIBITOR (cytogenetic assays):
STAIN (for cytogenetic assays):


NUMBER OF REPLICATIONS:


NUMBER OF CELLS EVALUATED:


DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other:


OTHER EXAMINATIONS:
- Determination of polyploidy:
- Determination of endoreplication:
- Other:


OTHER:

Species / strain:

other: S. typhimurium TA 1535, TA 1537, TA 98, TA1538 and TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

other: The positive controls were valid but for 9-AA the positive response was slight

Remarks on result:

other: other:

Remarks:

Migrated from field 'Test system'.

Conclusions:

Interpretation of results (migrated information):
negative

Under these experimental conditions, Perkadox 14R did not induce reverse mutation in Salmonella typhimurium (strains: TA 1535, TA 1537, TA 98, TA 100 and TA 102).

Executive summary:

The potential of the peroxide to induce reverse mutation in Salmonella typhimurium (strains: TA 1535, TA 1537, TA 98, TA 100 and TA 1538) was evaluated according to Ames et al. (1975): direct plate incorporation (the first experiment and the second without S9mix)

Bacterias were exposed to the substance at four dose-levels (0.2, 2, 20, 500 g/plate). The revertant colonies were scored and reported after substraction of spontaneous revertants. Up to 500 µg Perkadox 14 R per plate did not reveal any mutagenic activity with and without activation.

No increase in the number of revertants was observed for all doses with and without metabolic activation on the 5 tested strains.

Under these experimental conditions, the substance did not show any mutagenic activity in the bacterial reverse mutation test with Salmonella typhimurium R.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

No testing required as all in vitro studies are negative

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / bone marrow chromosome aberration

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

other:

Justification for classification or non-classification

[1,3-phenylenebis(1-methylethylidene)]bis[tert-butyl] peroxide was not genotoxic in vitro. According to the directive 67/548/EEC and according to regulation (EC) No 1272 -2008, the substance is not classified.