Cyclohexanone, peroxide

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The test substance is positive in two in vitro mutagenicity assays, Ames and MLA.

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:

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.

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

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Target gene:

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

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

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

+S9

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"

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'.

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.

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.

Reference 2

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:

February 1997 to March 1997

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Apparently well conducted GLP study.

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine operon for Salmonella

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254 induced rat liver S9

Test concentrations with justification for top dose:

In the direct plate assay CYCLONOX LE·50 was tested up to 2000 ug/plate in the absence and presence of S9·mix.
In the preincubation assay, CYCLONOX LE-50 was tested up to 666 ug/plate in the absence and presence of S9-mix.

Vehicle / solvent:

dimethylsulphoxide

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rate

Negative solvent / vehicle controls:

yes

Remarks:

dimethylsulphoxide

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

sodium azide

methylmethanesulfonate

other: daunomycine

Remarks:

-S9

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rate

Negative solvent / vehicle controls:

yes

Remarks:

dimethylsulphoxide

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

+S9

Details on test system and experimental conditions:

Test system: Salmonella typhimurium bacteria
Source: Dr. Bruce N. Ames, University of California at
Berkeley, U.S.A. (TA1535 and TA1537: 1994, TA98: 1991, TA100: 1993)

The characteristics of the individual Salmonella typhimurium strains were as
follows:

Strain Histidine mutation
TA1537 hisC3076
TA9S hisD3052/R-factor*
TA1535 *hisG46
TA100 hisG46/R-factor

*: R-factor = plasmid pKM101 (increases error-prone DNA repair)

Each tester strain contained the following additional mutations:
rfa: deep rough (defective lipopolysaccharide cellcoat)
gal : mutation in the galactose metabolism
chl : mutation in nitrate reductase
bio : defective biotin synthesis
uvr8: loss of the excision repair system (deletion of the ultraviolet repair B gene)

The Salmonella typhimurium strains were regularly checked to confirm their
histidine-requirement, crystal violet sensitivity, ampicillin resistance
(TA98 and TA100), UV-sensitivity and the number of spontaneous revertants.
Stock CUltures of the four strains were stored in liquid nitrogen (-196°C) .

Dose range finding test
Selection of a adequate range of doses for the direct plate mutation assay
and the preincubation assay was based on a dose range finding test with
strain TA 100. both with and without S9-mix. Eight concentrations were
tested in triplicate. The highest concentration of test article used in the
subsequent mutagenesis assays was the level at which the test substance
inhibited bacterial growth.

Direct plate mutation assay
Five different doses (increasing with approximately half-log steps) of the
test substance were tested in triplicate in each strain in a direct plate
assay.
The test substance was tested both in the absence and presence of S9-mix in
each strain.
Top agar in top agar tubes was molten and heated to 45°C. The following
solutions were successively added to 3 ml molten top agar: 0.1 ml of a
fresh bacterial culture (10 x EE9 cells/ml) of one of the tester strains, 0.1 ml
of a dilution of the test SUbstance in dimethylsulphoxide and either 0.5 ml
S9-mix (in case of activation assays) or 0.5 ml 0.1 M phosphate buffer (in
case of non-activation assays). The ingredients were mixed on a Vortex and
the content of the top agar tube was poured onto a selective agar plate.
After solidification of the top agar, the plates were turned and incubated
in the dark at 37°C for 48 h. After this period revertant (histidine
independent) colonies were counted.

Preincubation assay
Five different doses (increasing with approximately half-log steps) of the
test substance were tested in triplicate in each strain in a preincubation
assay.
The test substance was tested both in the absence and presence of 59~mix in
each strain.
Top agar in top agar tubes was molten and heated to 45°C. The following
solutions were preincubated for 30 minutes by 70 rpm at 37°C, either 0.5 ml
0.1 M phosphate buffer (in case of non-activation assays) or 0.5 ml S9-mix
(in case of activation assays), 0.1 ml of a fresh bacterial culture (10 x EE9
cells/ml) of one of the tester strains and 0.1 ml of a dilution of the test
substance in dimethylsulphoxide. After the preincubation period the
solutions were added to 3 ml molten top agar. The ingredients were mixed on
a Vortex and the content of the top agar tube was poured onto a selective
agar plate. After solidification of the top agar, the plates were turned
and incubated in the dark at 37°C for 48 h. After this period revertant
colonies (histidine independent) were counted.

Colony counting
The revertant colonies (histidine independent) were counted automatically
with a Protos model 50000 colony counter or manually, if less than 40
colonies per plate were present.

Evaluation criteria:

No formal hypothesis testing was done.
A test substance is considered negative (not mutagenic) in the test if:
a) The total number of revertants in any tester strain at any concentration
is not greater than two times the solvent control value, with or
without metabolic activation.
b) The negative response should be reproducible in at least one
independently repeated experiment.
A test substance is considered positive (mutagenic) in the test if:
a) It induces at least a 2-fold, dose related increase in the number of
revertants with respect to the number induced by the solvent control in
any of the tester strains, either with or without metabolic activation.
However, any mean plate count of less than 20 is considered to be not
significant.
b) The positive response should be reproducible in at least one
independently repeated experiment.

The preceding criteria were not absolute and other modifying factors might
enter into the final evaluation decision.

Key result

Species / strain:

S. typhimurium, other: 100, 1535, 1537, 98

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

DIRECT PLATE MUTATION ASSAY
In the absence of S9-mix, strain TA1537 showed a 2.6-fold, dose-related,
increase in the number of (His+) revertants compared to the number of
revertants in the solvent control. In the presence of S9-mix, strain TA1537
showed a 3.0-fold, dose-related, increase in the number of revertants.
In the absence of S9-mix, strain TA98 showed a 2.3-fold, dose-related,
increase in the number of revertants compared to the number of revertants in
the solvent control.
In the absence of S9-mix. strain TA100 showed a 1.7-fold, dose-related,
increase in the number of revertants compared to the number of revertants in
the solvent control.
The strains TA98 and TA100, in the presence of S9-mix, and strain TA1535,
showed negative responses over the entire dose range, i.e. no dose-related,
two-fold. increase in the number of revertants.

The negative and strain-specific positive control values were within our
laboratory background historical control data ranges indicating that the
test conditions were adequate and that the metabolic activation system
functioned properly.

PREINCUBATION ASSAY
In the dose range finding test, in the absence of S9-mix, strain TA100
showed negative responses over the entire dose range, i.e. no dose-related,
two-fold, increase in the number of revertants. In the presence of S9-mix,
strain TA100 showed a 2.0-fold increase in the number of revertants compared
to the number of revertants in the solvent control.
In the preincubation assay, in the absence of S9-mix, strain TA1537 showed a
5.6-fold increase in the number of (His+) revertants compared to the number
of revertants in the solvent control. In the presence of S9-mix, strain
TA1537 showed a 3.3-fold increase in the number of revertants.
In the absence of S9-mix, strain TA98 showed a 1.9-fold increase in the
number of revertants compared to the number of revertants in the solvent
control.
Strain TA98, in the presence of S9-mix, and the strains TA1535 and TA100,
showed negative responses over the entire dose range, i.e. no dose-related,
two-fold, increase in the number of revertants.

The negative and strain-specific positive control values were within our
laboratory background historical control data ranges indicating that the
test conditions were adequate and that the metabolic activation system
functioned properly.

Remarks on result:

other: see details in text and tables

DIRECT PLATE ASSAY- DOSE RANGE FINDING TEST

The test article was tested in strain TA100 with concentrations of 3, 10, 33, 100, 333, 1000, 3330 and 5000 ug/plate in the absence and presence of S9mix. The dose range finding test is reported as a part of the first experiment of the mutation test.

Precipitate

The test article did not precipitate in the top agar. Precipitation of the test article was not observed on the plates at the start or at the end of the incubation period in tester strain TA100.

Toxicity

To determine the toxicity of test article, the reduction of the bacterial background lawn, the increase in the size of the microcolonies and the reduction of the His+ revertant colonies were observed. Both in the absence and presence of S9-mix, a complete lack of any microcolony background lawn was observed at test substance concentrations of 3330 and 5000 ug/plate. Based on these data, the test article was tested in the direct plate mutation assay up to a concentration of 2000 ug/plate in the absence and the presence of S9-mix.

PREINCUBATION ASSAY - DOSE RANGE FINDING TEST

The test article was tested in strain100, 333, 1000, 3330 and 5000 ug/plate in the absence and presence of S9mix.

Precipitate

The test article did not precipitate in the top agar. Precipitation of the test article was not observed on the plates at the start or at the end of the incubation period in tester strain TA100.

Toxicity

In the absence of S9-mix, a complete lack of any microcolony background lawn was observed at test substance concentrations of 1000 ug/plate and upwards. In the presence of S9-mix, an extreme reduction of the bacterial background lawn and an increase in the size of the microcolonies was observed at the test substance concentration of 1000 ug/plate. A complete lack of any microcolony background lawn was observed at test substance concentrations of 3330 and 5000 ug/plate. Based on these data, the test article was tested in the preincubation assay up to a concentration of 666 ug/p1ate in the absence and the presence of S9mix.

Conclusions:

Based on the results of this study it is concluded that the test article is mutagenic in the Salmonella typhimurium reverse mutation assay.

Executive summary:

The test article was tested in the Salmonella typhimurium reverse mutation assay with four histidine-requiring strains of Salmonella typhimurium (TA1535, TA1537, TA100 and TA98) in a direct plate assay and a preincubation assay.

In the direct plate assay the test article was tested up to 2000 ug/plate in the absence and presence of S9·mix. In the preincubation assay, the test article was tested up to 666 ug/plate in the absence and presence of S9-mix.

In the absence of S9-mix, strain TA1537 showed 2.6- and 5.6-fold increases in the number of revertant (His+) colonies in the direct plate assay and the preincubation assay respectively. In the presence of S9-mix, strain TA1537 showed 3.0 and 3.3-fold increases in the number of revertant (His+) colonies in the the direct plate assay and the preincubation assay respectively. In the absence of S9-mix, strain TA98 showed 2.3- and 1.9-fold increases in the number of revertant (His+) colonies in the direct plate assay and the preincubation assay respectively. In the absence of S9-mix, strain TA 100, showed a 1.7-fold increase in the number of revertant (His+) colonies in the direct plate assay. In the presence of S9-mix, strain TA100 showed a 2.0-fold increase in the number of revertants in the dose range finding study of the preincubation assay only. In the presence of S9-mix, strain TA98 and in the absence and presence of S9-mix, strain TA1535 showed negative responses over the entire dose range i.e. no dose-related two-fold increase in the number of revertants in both assays. In the presence of S9-mix in the direct plate assay and in the absence and presence of S9-mix, in the preincubation assay, TA100 showed negative responses over the entire dose range. Based on the results of this study it is concluded that test article is mutagenic in the Salmonella typhimurium reverse mutation assay.

Reference 3

Endpoint:

in vitro cytogenicity / micronucleus study

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

an in vitro cytogenicity study in mammalian cells or in vitro micronucleus study does not need to be conducted because adequate data from an in vivo cytogenicity test are available

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

In an in vivo micronucleus assay the test substance is negative.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2008

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Apparently well conducted GLP study.

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

micronucleus assay

Species:

mouse

Strain:

Swiss

Sex:

male/female

Details on test animals or test system and environmental conditions:

Breeder: Charles River Laboratories, l'Arbresle, France.
Age: on the day of treatment, the animals were approximately 6 weeks old.
Weight: at the beginning of treatment the mean body weight was 32 g for males (ranging from
29 to 34 g) and 24 g for females (ranging from 22 to 27 g).
Veterinary care at CIT: upon their arrival at CIT, the animals were given a complete
examination to ensure that they were in good clinical conditions.
Acclimation: at least 5 days before the day of treatment.
Constitution of groups: upon arrival, the animals were randomly allocated to the groups by sex.
Subsequently, each group was assigned to a different treatment group.
Identification: individual tail marking upon treatment.

Environmental conditions
Upon their arrival at CIT, the animals were housed in an animal room, with the following
environmental conditions:
⋅ temperature: 22 ± 2°C,
⋅ relative humidity: 30 to 70%,
⋅ light/dark cycle: 12 h/12 h (07:00 – 19:00),
⋅ ventilation: at least 12 cycles/hour of filtered non-recycled fresh air.
The temperature and relative humidity were under continuous control and recording. The
housing conditions (temperature, relative humidity and ventilation) and corresponding
instrumentation and equipment were verified and calibrated at regular intervals.
The animals were housed by groups in polycarbonate cages. Each cage contained autoclaved
sawdust (SICSA, Alfortville, France).
Sawdust is analyzed by the supplier for composition and contaminant levels.

Food and water
All animals had free access to SsniffR/M-H pelleted maintenance diet (SSNIFF Spezialdiäten
GmbH, Soest, Germany).
Each batch of food is analysed by the supplier for composition and contaminant levels.
Drinking water filtered by a FG Millipore membrane (0.22 micron) was provided ad libitum.
Bacteriological and chemical analysis of water are performed regularly by external laboratories,
These analyses include the detection of possible contaminants (pesticides, heavy metals and
nitrosamines).
No contaminants were known to have been present in the diet, drinking water or bedding
material at levels which may be expected to interfere with or prejudice the outcome of the study.

Route of administration:

intraperitoneal

Vehicle:

Corn oil

Details on exposure:

For the main test, the test item was dissolved in the vehicle in order to achieve the
concentrations of 1.875, 3.75, 7.5 and 15 mg/mL and then homogenized using a magnetic
stirrer. Using a treatment volume of 10 mL/kg, the target dose-levels were 18.75, 37.5, 75 and
150 mg/kg/day.
The preparations were made immediately before use.

Duration of treatment / exposure:

two treatments

Frequency of treatment:

two treatments separated by 24 hours

Remarks:

Doses / Concentrations:
18.75, 37.5, 75 and 150 mg/kg/day
Basis:
nominal conc.

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Positive control(s):

Cyclophosphamide

Tissues and cell types examined:

Bone marrow

Details of tissue and slide preparation:

Preparation of the bone marrow smears
At the time of sacrifice, all the animals were killed by CO2 inhalation in excess. The femurs of
the animals were removed and the bone marrow was flushed out using fetal calf serum. After
centrifugation, the supernatant was removed and the cells in the sediment were resuspended by
shaking. A drop of this cell suspension was placed and spread on a slide. The slides were
air-dried and stained with Giemsa. The slides were coded so that the scorer is unaware of the
treatment group of the slide under evaluation ("blind" scoring).

Microscopic examination of the slides
For each animal, the number of the micronucleated polychromatic erythrocytes (MPE) was
counted in 2000 polychromatic erythrocytes; the polychromatic (PE) and normochromatic (NE)
erythrocyte ratio was established by scoring a total of 1000 erythrocytes (PE + NE).
The analysis of the slides was performed at Microptic, cytogenetic services (2 Langland Close
Mumbles, Swansea SA3 4LY, UK), in compliance with GLP, and the Principal Investigator was
Natalie Danford.

Evaluation criteria:

For a result to be considered positive, a statistically significant increase in the frequency of MPE
must be demonstrated when compared to the concurrent vehicle control group. Reference to
historical data (appendix 3), or other considerations of biological relevance was also taken into
account in the evaluation of data obtained.

Statistics:

Normality and homogeneity of variances will be tested using a Kolmogorov Smirnov test and a
Bartlett test.
If normality and homogeneity of variances were demonstrated, the statistical comparisons was
performed using a Student t-test (two groups) or a one-way analysis of variance (≥ 3 groups)
followed by a Dunnett test (if necessary).
If normality or homogeneity of variances was not demonstrated, a Mann/Whitney test
(two groups) or a Kruskall Wallis test (≥ 3 groups) was performed followed by a Dunn test
(if necessary).

All these analyses were performed using the software SAS Enterprise Guide V2 (2.0.0.417, SAS
Institute Inc), with a level of significance of 0.05 for all tests.

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

PRELIMINARY TOXICITY TEST
In order to select the top dose-level for the cytogenetic study, 50, 75, 100, 150, 200, 300 and
2000 mg/kg were administered to three males and three females.
At 2000 mg/kg/day, the three females treated and one male out of three died following the
first treatment. The two surviving males were sacrificed before the second administration of the
test item.
At 300 mg/kg/day, two females out of three and two males out of three died following the
first treatment. The two surviving animals (one male and one female) were sacrificed before the
second administration of the test item.
At 200 mg/kg/day (administered to females only), two females out of three died following the
second treatment.
At 150 mg/kg/day (administered to females only), no mortality was induced. Piloerection and in
addition hypoactivity for one female, were observed.
At 100 mg/kg/day, one male out of three died following the second treatment and piloerection
was noted in the two surviving males. No clinical signs and no mortality were noted in the
three females treated.
At 75 mg/kg/day (administered to males only), piloerection was noted in the treated animals and
no mortality was induced. No clinical signs and no mortality were noted in the three females.
At 50 mg/kg/day, no mortality was induced. Piloerection and sometimes half-closed eyes were
noted in males. No clinical signs and no mortality were noted in the three females treated.
The top dose-level for the cytogenetic test was selected according to the criteria specified in the
international guidelines; since toxic effects were observed, the choice of the top dose-level was
based on the level of toxicity, such that a higher dose-level was expected to induce lethality.
Consequently, 75 mg/kg/day for males or 150 mg/kg/day for females were selected as the top
dose-level for the main test. The two other selected dose-levels were 18.75 and 37.5 mg/kg/day,
for males, and 37.5 and 75 mg/kg/day, for females.

Except for piloerection regularly observed in males (solvent control and the three dose-levels
treated groups) and sometimes noted in females (highest dose group only), no clinical signs and
no mortality were observed in treated animals.
For either males or females, the mean values of MPE as well as the PE/NE ratio in the groups
treated with the test item, were equivalent to those of the vehicle control or solvent groups.
The mean values of MPE as well as the PE/NE ratio for the vehicle and positive controls were
consistent with our historical data.
Cyclophosphamide induced a significant increase in the frequency of MPE, indicating the
sensitivity of the test system under our experimental conditions. The study was therefore
considered valid.

Conclusions:

Interpretation of results (migrated information): negative
Under experimental conditions, the test item (35% of cyclohexanone peroxide in a solvent melange) did
not induce damage to the chromosomes or the mitotic apparatus of mice bone marrow cells after
two intraperitoneal administrations, at a 24-hour interval, at the dose-levels of 18.75, 37.5 and
75 mg/kg/day and 37.5, 75 and 150 mg/kg/day for males and females respectively.

Executive summary:

The objective of this study was to evaluate the potential of the test item (35% of cyclohexanone peroxide in a solvent melange) to induce structural or numerical damage in bone marrow cells of mice. The study was performed according to the international guidelines (OECD 474, Commission Directive No. B12) and in compliance with the Principles of Good Laboratory Practice Regulations.

In the main study, three groups of five male and five female Swiss Ico: OF1 (IOPS Caw) mice were given intraperitoneal administrations of CYCLOHEXANONE PEROXIDE at dose-levels of 18.75, 37.5 and 75 mg/kg/day, for males, and of 37.5, 75 and 150 mg/kg/day for females, over a 2-day period. One group of five males and five females received the solvent control (Mixture diacetonealcohol/Diisobutylphtalate/Cyclohexanone) at 65% of the highest dose-level of the test item (i.e. 48.75 mg/kg/day for males and 97.5 mg/kg/day for females), under the same experimental conditions. One group of five males and five females received the vehicle (corn oil) under the same experimental conditions, and acted as control group. One group of five males and five females received the positive control test item (Cyclophosphamide) once by oral route at the dose-level of 50 mg/kg. The animals of the treated, solvent and vehicle control groups were killed 24 hours after the last treatment and the animals of the positive control group were killed 24 hours after the single treatment. Bone marrow smears were then prepared. For each animal, the number of the micronucleated polychromatic erythrocytes (MPE) was counted in 2000 polychromatic erythrocytes. The polychromatic (PE) and normochromatic (NE) erythrocyte ratio was established by scoring a total of 1000 erythrocytes (PE + NE).

The top dose-level for the cytogenetic test was selected according to the criteria specified in the international guidelines; since toxic effects were observed, the choice of the top dose-level was based on the level of toxicity, such that a higher dose-level was expected to induce lethality. Consequently, 75 mg/kg/day for males or 150 mg/kg/day for females were selected as the top dose-level for the main test. The two other selected dose-levels were 18.75 and 37.5 mg/kg/day, for males, and 37.5 and 75 mg/kg/day, for females. Except for piloerection regularly observed in males (solvent control and the three dose-levels treated groups) and sometimes noted in females (highest dose group only), no clinical signs and no mortality were observed in treated animals. For either males or females, the mean values of MPE as well as the PE/NE ratio in the groups treated with the test item, were equivalent to those of the vehicle control or solvent groups.

The mean values of MPE as well as the PE/NE ratio for the vehicle and positive controls were consistent with our historical data. Cyclophosphamide induced a significant increase in the frequency of MPE, indicating the sensitivity of the test system under our experimental conditions. The study was therefore considered valid.

Reference 2

Endpoint:

in vivo mammalian somatic cell study: gene mutation

Data waiving:

other justification

Justification for data waiving:

other:

Justification for type of information:

TESTING PROPOSAL ON VERTEBRATE ANIMALS
[Please provide information for all of the points below. The information should be specific to the endpoint for which testing is proposed. Note that for testing proposals addressing testing on vertebrate animals under the REACH Regulation this document will be published on the ECHA website along with the third party consultation on the testing proposal(s).]

NON-CONFIDENTIAL NAME OF SUBSTANCE:
- Name of the substance on which testing is proposed to be carried out: cyclohexanone peroxide
- Name of the substance for which the testing proposal will be used [if different from tested substance]: same as above

CONSIDERATIONS THAT THE GENERAL ADAPTATION POSSIBILITIES OF ANNEX XI OF THE REACH REGULATION ARE NOT ADEQUATE TO GENERATE THE NECESSARY INFORMATION [please address all points below]:
- Available GLP studies: No GLP Comet study is available
- Available non-GLP studies: No non-GLP Comet study is available
- Historical human data: Not available
- (Q)SAR: Not suitable for this endpoint
- In vitro methods: No in vitro test to address this endpoint is available
- Weight of evidence: No relevant structural analogs
- Grouping and read-across: No relevant structural analogs
- Substance-tailored exposure driven testing [if applicable]: Not applicable
- Approaches in addition to above [if applicable]: None
- Other reasons [if applicable]: The substance has positive OECD 471 and OECD 476 test results, and as such, an in vivo study must be proposed to properly address the mutagenicity of the substance.

CONSIDERATIONS THAT THE SPECIFIC ADAPTATION POSSIBILITIES OF ANNEXES VI TO X (AND COLUMN 2 THEREOF) OF THE REACH REGULATION ARE NOT ADEQUATE TO GENERATE THE NECESSARY INFORMATION:
- Specific adaptations described in column 2 are not applicable

FURTHER INFORMATION ON TESTING PROPOSAL IN ADDITION TO INFORMATION PROVIDED IN THE MATERIALS AND METHODS SECTION:
- Details on study design / methodology proposed [if relevant]: Comet assay

Qualifier:

according to guideline

Guideline:

OECD Guideline 489 (In vivo Mammalian Alkaline Comet Assay)

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Justification for classification or non-classification

Based on the available information the test substance is not classified for mutagenicity.