Cyclohexanone, peroxide

Genetic toxicity: in vitro

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:

Experimental starting date: 23rd February 2015 Experimental completion date: 7th april 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of relevant results.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Cyclohexanone, peroxide

EC Number:

235-527-7

EC Name:

Cyclohexanone, peroxide

Cas Number:

12262-58-7

Molecular formula:

Multi-constituent substance (equilibrium mixture of a number of cyclohexyl based organic peroxides, hydrogen peroxide, cyclohexanone and water)

IUPAC Name:

Peroxycyclohexanone

Test material form:

other: Liquid

Details on test material:

Identification: Cyclohexanone peroxide (CAS No.012262-58-7)
Batch: 1405426797
Purity: 33.9% Cyclohexanone peroxide (mixture of cyclohexyl peroxy components)
Expiry / Retest Date: 18 February 2016
Storage Conditions: Ambient 10 oC not to exceed 25 oC.

Method

Target gene:

Thymidine kinase, TK +/- locus of the L5178Y mouse lymphoma cell line

Species / strain

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

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 applicable

Metabolic activation:

with and without

Metabolic activation system:

phenobarbital/beta-naphthaflavone

Test concentrations with justification for top dose:

Experiment 1 and 2: 0, 3.75, 7.5, 15, 30, 45, 60, 75 and 90 µg/ml

Vehicle / solvent:

DMSO

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

+S9

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

-S9

Details on test system and experimental conditions:

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 test item had a molecular weight of 148.2. Therefore, the maximum dose level in the solubility test was set at 1482 µg/mL, the maximum recommended dose level, and no correction for the purity of the test item was applied. 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 are in the following table:

Dose level
µg/mL 0 5.79 11.58 23.16 46.31 92.63 185.25 370.5 741 1482

pH 7.24 7.27 7.21 7.22 7.25 7.27 7.24 7.20 7.21 7.19
Osmolality
mOsm 457 461 - - - 473 - - 467 455
- Not required

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.


4.4 Control Preparation
Vehicle and positive controls were used in parallel with the test item. Solvent (DMSO) (CAS No. 67-68-5) treatment groups were used as the vehicle controls. Ethylmethanesulphonate (EMS) (CAS No. 62-50-0) Sigma batch BCBK5968V and BCBN1209V at 400 µg/mL was used as the positive control in the absence of metabolic activation. Cyclophosphamide (CP) (CAS No. 6055-19-2) Sigma-Aldrich batch MKBS0021V at 1.5 µg/mL was used as the positive control in the presence of metabolic activation. The positive controls were formulated in DMSO.


4.5 Microsomal Enzyme Fraction
PB/BNF S9 was prepared in-house on 23 November 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 Experiments 1 and 2.

4.6 Preliminary Toxicity Test
A preliminary toxicity test was performed on cell cultures at 5 x 105 cells/mL, using a 4 hour exposure period both with and without metabolic activation (S9), and at 1.5 x 105 cells/mL using a 24-hour exposure period without S9. The dose range used in the preliminary toxicity test was 5.79 to 1482 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 105 cells/mL, unless the mean cell count was less than 3 x 105 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 105 cells/mL, unless the mean cell count was less than 3 x 105 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).


4.7 Mutagenicity Test
4.7.1 Experiments 1 & 2
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 106 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 (3.75 to 90 µg/mL in the absence of metabolic activation, and 7.5 to 180 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 or 0.15 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 was performed under the same conditions as Experiment 1 to clarify a suspected positive result.

Evaluation criteria:

Please refer to "Any other information on materials and methods"

Statistics:

Please refer to "Any other information on materials and methods"

Results and discussion

Test results

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

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 onset of test item-induced toxicity was sharp in all three of the exposure groups. A precipitate of the test item was observed at and above 741 µg/mL in both 4-hour exposure groups. Based on the %RSG values observed, the maximum dose levels in the subsequent Mutagenicity Test were limited by test item induced toxicity.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

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 (Tables 3 and 6). There was also evidence of reductions in viability (%V), therefore indicating that residual toxicity had occurred in both of the exposure groups (Tables 3 and 6). Based on the RTG and %RSG values observed, optimum levels of toxicity were considered to have been achieved in the presence of metabolic activation (Tables 3 and 6). The toxicity observed at 90 µg/mL in the absence of metabolic activation exceeded the upper acceptable limit of 90%. Therefore, this dose level was excluded from the statistical analysis. Several vital dose levels around optimum toxicity exhibited excessive heterogeneity which was believed to be caused by test item induced cytotoxicity, therefore these dose levels were excluded from statistical analysis. Acceptable levels of toxicity were seen with both positive control substances (Tables 3 and 6).

 

The vehicle controls had mutant frequency values that were considered acceptable for the L5178Y cell line at the TK +/- locus. In the presence of metabolic activation the vehicle control mutant frequency was marginally high but still considered to be acceptable. 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 (Tables 3 and 6).

 

The test item induced both statistically significant and dose related (linear-trend) increases in the mutant frequency x 10^-6per viable cell, in both the absence and presence of metabolic activation. In the absence of metabolic activation, it should be noted that the highest MF values observed were either excluded due to excessive heterogeneity or excessive toxicity, however the GEF value was significantly exceeded in these dose levels giving a cause for concern. In the presence of metabolic activation the highest MF value observed was just outside the acceptable limit for toxicity where the GEF value was markedly exceeded, however the dose level with optimum toxicity had a statistically significant increase in MF and the GEF was exceeded. With the data considered and issues with heterogeneity it was decided to perform an Experiment 2 with the same conditions as Experiment 1 to clarify the result. Precipitate of the test item was not observed at any of the dose levels during the course of the experiment.

 

The numbers of small and large colonies and their analysis are presented in Tables 4 and 7. In both the absence and presence of metabolic activation the colony formation was predominantly due to small colonies, indicating a possible contribution to clastogenic activity.

 

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 (Tables 9 and 12). The toxicity observed was very similar to the values observed in Experiment 1. There was also evidence of a modest reduction in viability (%V) in both the absence and presence of metabolic activation, therefore indicating that residual toxicity had occurred (Tables 9 and 12). Based on the RTG and %RSG values observed, optimum levels of toxicity were considered to have been achieved in both the absence and presence metabolic activation. In the presence of metabolic activation, the 180 µg/mL dose level was plated for viability and 5-TFT resistance as sufficient cells were available at the time of plating. However, this dose level was later excluded from statistical analysis due to excessive toxicity. Acceptable levels of toxicity were seen with both positive control substances (Tables 9 and 12).

 

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 (Tables 9 and 12).

 

The positive response observed in Experiment 1 was reproduced without any issues with heterogeneity in Experiment 2. The test item induced both statistically significant and dose related (linear-trend) increases in the mutant frequency x 10^-6per viable cell in both the absence and presence of metabolic activation (Tables 9 and 12). The GEF value was exceeded at several cytotoxic dose levels including optimum toxicity in both exposure conditions. Therefore, the effect was considered to be toxicologically significant. The test item was now considered to have been adequately tested. Precipitate of the test item was not observed at any of the dose levels during the course of the experiment.

 

The numbers of small and large colonies and their analysis are presented in Tables 10 and 13. In both the absence and presence of metabolic activation the colony formation was predominantly due to small colonies, indicating a possible contribution to clastogenic activity.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
positive

The test item induced toxicologically significant and dose related increases in the mutant frequency at the TK +/- locus in L5178Y cells in both the absence and presence of metabolic activation in both experiments and is therefore considered to be mutagenic under the conditions of the test

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…….

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 (DMSO), and positive controls using 4-hour exposure groups both in the absence and presence of metabolic activation (2% S9). In Experiment 2, the same experimental conditions as Experiment 1 were used to clarify a suspected positive result obtained in Experiment 1.

 

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:

 

Experiments 1 & 2

Group	Concentration of Cyclohexanone peroxide (CAS No. 012262-58-7) (µg/mL) plated for viability and mutant frequency
4-hour without S9	7.5, 15, 30, 45, 60, 75, 90
4-hour with S9 (2%)	30, 45, 90, 120, 150, 180

 

Results…………

The maximum dose levels used in the Mutagenicity Test were limited by test item-induced toxicity. Precipitate of the test item was not observed at any of the dose levels during the course of 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 and dose related increases in the mutant frequency at the TK +/- locus in L5178Y cells in both the absence and presence of metabolic activation in both experiments. The GEF value was markedly exceeded and the mutagenic response was reproducible in both exposure groups. The colony formation was predominantly small colonies indicative of a possible contribution to clastogenic activity.

 

Conclusion

The test item induced toxicologically significant and dose related increases in the mutant frequency at the TK +/- locus in L5178Y cells in both the absence and presence of metabolic activation in both experiments and is therefore considered to be mutagenic under the conditions of the test.