Bis(4-methylbenzoyl)peroxide

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

A bacterial gene mutation study (Ames Test) was was performed by Poth (1991) to investigate the potential of D1- (4-METHYLBENZOYL)-PEROX1D (INTEROX-PMBP) to induce gene mutations using the Salmonella typhimurium strains TA 1535, TA 1537, TA 1538, TA 98 and TA 100 and in addition Escherichia coli strain WP2. Up to the highest investigated dose, neither a significant and reproducible increase of the number of revertants was found in any strain as compared to the solvent control nor a concentration-dependent enhancement of the revertant number exists. The presence of liver microsomal activation did not influence these findings. Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies. It was concluded that during the described mutagenicity test and under the experimental conditions reported, the test article did not induce point mutations by base pair changes or frameshifts in the genome of the strains used. Therefore, DI-(4 -METHYLBENZOYL)-PEROXID (INTEROX-PMBP) is considered to be non-mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.

In a recent in vitro mammalian cell mutagenicity study (Bis(4-methylbenzoyl)peroxide, 75% in water CAS No. 895-85-2: L5178Y TK +/- Mouse Lymphoma Assay, Harlan Laboratories, 2014), the test item showed negative results. The maximum dose levels used in the Mutagenicity Test were limited by test item-induced toxicity. A precipitate of the test item was observed at 160 µg/mL in Experiment 2 (in the presence of metabolic activation only, post washing). 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 dose-related (linear-trend) increases in the mutant frequency at any of the dose levels, either with or without metabolic activation, in either the first or the second experiment. It was concluded that the test item did not induce any toxicologically significant increases in the mutant frequency at the TK +/- locus in L5178Y cells.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: This GLP study was done per standard OECD and EU test guidelines, but the study report did not include the CAS number or the Certificate of Analysis of the test article.

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 472 (Genetic Toxicology: Escherichia coli, Reverse Mutation Assay)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Deviations:

no

Guideline:

other: Environmental Protection Agency, Code of Federal Regulations, Title 40, Subpart F-Genetic Toxicity, Revision July 1, 1986 "The salmonella typhimurium reverse mutation assay".

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

other: Salmonella typhimurium strains TA 1535, TA 1537, TA 1538, TA 98, and TA 100; Escherichia coli WP2

Metabolic activation:

with and without

Metabolic activation system:

S9 (rat liver)

Test concentrations with justification for top dose:

10.0; 100.0; 333.3; 1000.0; and 5000.0 ug/plate

Vehicle / solvent:

On the day of the experiment, the test article DI-(4-METHYLBENZOYL)-PEROXID (INTEROX-PMBP) was dissolved in Aceton. The solvent was chosen because of its solubility properties and its relative non-toxicity for the bacteria.

Untreated negative controls:

yes

Remarks:

Concurrent untreated and solvent controls were performed

Negative solvent / vehicle controls:

yes

Remarks:

Concurrent untreated and solvent controls were performed

True negative controls:

yes

Positive controls:

yes

Remarks:

Positive Control, without metabolic activation: Strains: TA 1535, TA 100; Positive control substance: sodium azide, NaN3 (from SERVA, D-6900 Heidelberg, F.R.G.; Cat. No.: 30175), dissolved in aqua dest. at 10 ug/plate, on the day of experiment.

Positive control substance:

sodium azide

methylmethanesulfonate

Remarks:

Positive Control, without metabolic activation: Strain: WP2; Positive control: methyl methane sulfonate, MMS, (Merck-Schuchardt, D-8011 Hohenbrunn, F.R.G., Cat.No. 820775), dissolved in aqua dest. at 10 uL/plate, prepared on the day of experiment.

Positive controls:

yes

Remarks:

Positive Control: Strains: TA 1537, TA 1538, TA 98; Substance: 4-nitro-o-phenylene-diamine, 4-NOPD (from SIGMA, D-8024 Deisenhofen, F.R.G; Cat. No. N9504); Dissolved in DMSO at 50 ug/plate on the day of experiment.

Positive control substance:

other: 2-aminoanthracene, 2-AA and 4-nitro-o-phenylene-diamine, 4-NOPD

Remarks:

Positive Control, with metabolic activation: Strains: TA 1535, TA 1537, TA 1538, TA 98, TA 100, WP2; Substance: 2-aminoanthracene, 2-AA (SIGMA, D-8024 Deisenhofen, F.R.G., Cat. No.: A 1381), dissolved in DMSO at 2.5 ug/plate on the day of experiment.

Details on test system and experimental conditions:

Start of Experiment I: August 21, 1991
End of Experiment I: August 30, 1991
Start of Experiment II: September 04, 1991
End of Experiment II: September 16, 1991
Date of Draft: September 19, 1991
Date of Report: October 17, 1991

AIMS OF THE STUDY: The experiments were performed to assess the potential of the test article to induce gene mutations by means of two independent Salmonella typhimurium and Escherichia coli reverse mutation assays. The most widely used assays for detecting gene mutations are those using bacteria. They are relatively simple and rapid to perform, and give reliable data on the ability of an agent to interact with DNA and produce mutations. Since the bacteria most commonly used in these assays do not possess the enzyme systems which, in mammals, are known to convert promutagens into active DNA damaging metabolites, an exogenous metabolic system is added in form of mammalian microsome enzyme activation mixture. In spite of great differences between bacterial and eukaryotic cells with respect to structure and function there is an association between mutagenicity in bacteria and carcinogenicity in mammals described in literature (8,9).

Reverse mutation assays determine the frequency at which an agent abolishes or suppresses the effect of the forward mutation. The genetic target presented to an agent is therefore small, specific and selective. Several bacterial strains or a single strain with multiple markers are necessary to overcome the effects of mutagen specificity. The reversions of bacteria from growth-dependence on a particular amino acid to growth in the absence of that amino acid (reversion from auxothrophy to prototrophy) is the most widely used marker. The Salmonella typhimurium histidine (his) and the E. coli tryptophan (trp) reversion system measures his- --> his+ and trp- -->trp+ reversions, respectively. The S. typhimurium strains are constructed to differentiate between base pair (TA 1535, TA 100) and frameshift (TA 1537, TA 1538, TA 98) mutations.

According to the direct plate incorporation method the bacteria are exposed to the test article with and without metabolic activation and plated on selective medium. After suitable period of incubation, revertant colonies are counted. To establish a dose response effect at least 5 dose levels with adequate spaced intervals are tested. The maximum dose level is 5000.0 ug/plate, unless limited by toxicity or solubility of the test article. To validate the test, reference mutagens are tested parallel to the test article.

THE TEST SYSTEM
Characterisation of the Salmonella typhimurium Strains and E.coli strain: The strains are derived from S. typhimurium strain LT2 and due to a mutation in the histidine locus are histidine dependent. Additionally, due to the "deep rough" (rfa-minus) mutation they possess a faulty lipopolysaccharide envelope which enables substances to penetrate the cell wall more easily. A further mutation causes a reduction in the activity of an excision repair system. The latter alteration includes mutational processes in the nitrate reductase and biotin genes produced in a UV-sensitive area of the gene named "uvrB-minus". In the strains TA 98 and TA 100 the R-factor plasmid pKM 101 carries
the ampicillin resistance marker.

The uvrA derivate of the E. coli strain WP2 is deficient in the DNA repair process (excisable repair damage). Such a repair-deficient strain may be more readily mutated by agents. When summarized the mutations of the TA strains and the E. coli strain used in this study can be described as follows:

Salmonella typhimurium: Sensitive to mutagens inducing:
TA 1537: his C 3076; rfa-; uvrB-; :frame shift mutations
TA 1538: his D 3052; rfa-; uvrB-; : " "
TA 98: his D 3052; rfa-; uvrB-; R-factor: : " "
TA 1535: his G 46; rfa-; uvrB-; :base-pair substitutions
TA 100: his G 46; rfa-; uvrB-; R-factor: : " "

Escherichia coli: Sensitive to mutagens inducing:
WP2: trp; uvrA- :base-pair substitutions

Regular checking of the properties of the strains with regard to membrane permeability and ampicillin resistance as well as normal spontaneous mutation rates is performed in C C R according to Ames et al. (1). In this way it was ensured that the experimental conditions set down by Ames were fulfilled. The bacterial strains were obtained from Dr. Heinz Trager, Knoll AG, D-6700 Ludwigshafen, F.R.G.

Storage: The strain cultures were stored as stock cultures in ampoules with nutrient broth + 5 % DMSO in liquid nitrogen.

Precultures: From the thawed ampoules of the strains 0.5 ml bacterial suspension was transferred to 250 ml Erlenmeyer flasks containing 20 ml nutrient medium. This nutrient medium contains per litre: 8 g Merck Nutrient Broth and 5 g NaCl. The bacterial culture was incubated in a shaking water bath for 10 hours at 370 C.

Selective Agar: 2.0 % Vogel-Bonner-Glucose-Minimal-Agar was used as selective agar. Each petri dish was filled with 20 ml of this nutrient medium. Sterilizations were performed at 1210 C in an autoclave.

Overlay Agar: The overlay agar contains per litre:

For Salmonella strains: For Escherichia coli:
6.0 g Merck Agar Agar 6.0 g Merck Agar Agar
6.0 g NaCl 6.0 g NaCl
10.5 mg L-histidine x HCl x H2O 2.5 mg Tryptophan
12.2 mg Biotin

Sterilizations were performed at 121 degrees C in an autoclave.

MAMMALIAN MICROSOMAL FRACTION S9 MIX

S9 (Preparation by C C R): The S9 liver microsomal fraction was obtained from the liver of 8 - 12 weeks old male Wistar rats, strain WU (SAVO-Ivanovas, med. Versuchstierzuchten GmbH, 0-7964 Kisslegg, F.R.G.; weight approx. 150 - 200 g) which received a single i.p. injection of 500 mg/kg b.w. Aroclor 1254 (Antechnika, 0-7500 Karlsruhe, F.R.G.) in olive oil 5 days previously.

After cervical dislocation the livers of the animals were removed, washed in 150 mM KCl and homogenized. The homogenate, diluted 1+3 in KCl was centrifuged cold at 9,000 g for 10 minutes. A stock of the supernatant containing the microsomes was frozen in ampoules of 2, 3 or 5 ml and stored at -700 C. Small numbers of the ampoules are kept at -200 C for only several weeks before use. The standardization of the protein content was made using the analysis kit of Bio-Rad Laboratories, D-8000 Mlinchen: Bio-Rad protein assay, Catalogue 500 000 6 (7). The protein concentration in the S9 preparation was 36.0 mg/ml (lot 030691).

S9 Mix: Before the experiment an appropriate quantity of S9 supernatant was thawed and mixed with S9 cofactor solution in a ratio 3:7. The composition of the cofactor solution was concentrated to yield the following concentrations in the S9 mix:

8 mM MgCl2, 33 mM KCl, 5 mM glucose-6-phosphate, and 5 mM NADP. all in 100 mM sodium-ortho-phosphate-buffer, pH 7.4. During the experiment, the S9 mix was stored in an ice bath. The S9 mix preparation was performed according to Ames et al. (2).

PRE-EXPERIMENT FOR TOXICITY: To evaluate the toxicity of the test article a pre-study was performed with strains TA 98, TA 100 and WP2. Eight concentrations were tested for toxicity and mutation induction, each with 3 plates. The experimental conditions in this pre-experiment were the same as described below for the experiment. Toxicity of the test article may be evidenced by a reduction in the number of spontaneous revertants, a clearing of the bacterial background lawn, or by degree of survival of treated cultures.

DOSE SELECTION: According to the results of this pre-experiment the concentrations applied in the main experiments were chosen. The concentration range covered three logarithmic decades. The maximum concentration was 5000.0 ug/plate. The concentration range included three logarithmic decades. In this study five adequately spaced concentrations were tested. Two independent experiments were performed. As the results of the pre-experiment are in accordance with the criteria described above, these data are reported as a part of the main experiment I.

EXPERIMENTAL PERFORMANCE: For each strain and dose level, including the controls, a minimum of 3 plates were used. The following materials were mixed in a test tube and poured onto the minimal agar plates:

100 ul Test solution at each dose control, negative control, or solution (positive control),
500 ul S9 mix (for test with metabolic activation) or S9 mix substitution-buffer (for test without metabolic activation),
100 ul Bacteria suspension (cf. test system, pre-culture of the strains),
2000 ul Overlay agar

After solidification the plates were incubated upside down for at least 48 hours at 37°C in the dark.

DATA RECORDING: The colonies were counted using the BIOTRAN III counter (BIOTRONIK, D-6000 Frankfurt, F.R.G.). The counter was connected to an IBM AT compatible PC with printer which printed out the individual values and the means from the plates for each concentration together with standard deviations and enhancement factors as compared to the spontaneous reversion rates (see tables of results). If precipitation of the test article precluded automatic counting the revertant colonies were counted by hand.

Evaluation criteria:

EVALUATION OF RESULTS: The generally accepted conditions for the evaluation of the results are:
- corresponding background growth on both negative control and test plates
- normal range of spontaneous reversion rates .. lmO

Range of spontaneous reversion frequencies (4,6,10)*
1535 1537 1538 98 100 WP2
3 - 37 4 – 31 12 - 37 15 – 60 75 - 200 30-60
* These values refer to the negative control without metabolic activation

Due to international guidelines a statistical evaluation of the results is recommended. However, no evaluated statistical procedure can be recommended for analysis of data from the bacterial assays at this time (6).

A test article is considered as positive if either a dose related increase in the number of revertants or a significant and reproducible increase for at least one test concentration is induced.

A test article producing neither a dose related increase in the number of revertants nor a significant and reproducible positive response at anyone of the test points is considered non-mutagenic in this system.

A significant response is described as follows:

A test article is considered as mutagenic if in the strains TA100 and WP2 the number of reversions will be at least twice as high and in the strains TA 1535, TA 1537, TA 1538 and TA 98 it will be at least three times higher as compared to the spontaneous reversion rate (5).

Also, a dose-dependent increase in the number of revertants is regarded as an indication of possibly existing mutagenic potential of the test article regardless whether the highest dose induced the above described enhancement factors or not.

Statistics:

BIOMETRY: No appropriate statistical method is available (6).

Key result

Species / strain:

other: 1535, 1537, 1538, 98, 100, Wp2

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Additional information on results:

See the "Summary of Results" table under illustration.

The results from Experiments 1 and 2 on individual strains with and without S9 mix are summarised in the "Summary of Results" table under illustration.

Conclusions:

Interpretation of results: negative

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test article did not induce point mutations by base pair changes or frameshifts in the genome of the strains used. Therefore, DI-(4 -METHYLBENZOYL)-PEROXID (INTEROX-PMBP) is considered to be non-mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.

Executive summary:

SUMMARY

This study was performed to investigate the potential of D1- (4-METHYLBENZOYL)-PEROX1D (INTEROX-PMBP) to induce gene mutations according to the plate incorporation test using the Salmonella typhimurium strains TA 1535, TA 1537, TA 1538, TA 98 and TA 100 and in addition the Escherichia coli strain WP2. This GLP study by A. Poth (1991) was done according to OECD 471/472 and EU B.13/24 guidelines as well as the Environmental Protection agency, Code of Federal regulations Title 40, Subpart F-Genetic toxicity, Revision July 1, 1986 " The Salmonella typhimurium reverse mutation assay". A reliability rating of K2 was assigned since the study report did not include the CAS number and the Certificate of Analysis of the test article.

 

The assay was performed, after a preexperiment to determine the toxicity of the test article, in two independent experiments, using identical procedures, both with and without liver microsomal activation. Each concentration, including the controls, was tested in triplicate. The test article was tested at the following concentrations: 10.0; 100.0; 333.3; 1000.0; and 5000.0 ug/plate. The plates incubated with the test article showed normal background growth up to 5000.0 ug/plate with and without metabolic activation in both independent experiments.

 

Toxicity was evidenced by a reduction in the number of revertants at the highest investigated dose in strain TA 1535 (without S9 mix, exp.I), TA 1538 (with S9 mix, expt. I) and TA 98 (with S9 mix, expt. II). A biological irrelevant reduction in the number of revertants was found in strain TA 100 (without S9 mix, expt..I) at 10.0 ug/plate.

 

Up to the highest investigated dose, neither a significant and reproducible increase of the number of revertants was found in any strain as compared to the solvent control nor a concentration-dependent enhancement of the revertant number exists. The presence of liver microsomal activation did not influence these findings. Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies.

 

CONCLUSION: In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test article did not induce point mutations by base pair changes or frameshifts in the genome of the strains used. Therefore, DI-(4 -METHYLBENZOYL)-PEROXID (INTEROX-PMBP) is considered to be non-mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The study was conducted between 27 June 2014 and 26 August 2014.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline 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 locus

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

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

Experiment 1
4-hour without S9: 0.63, 1.25, 2.5, 5, 10, 20 µg/mL
4-hour with S9 (2 %): 20, 40, 60, 80, 120, 160 µg/mL

Experiment 2
24-hour without S9: 0.31, 0.63, 1.25, 2.5, 5, 10 µg/mL
4-hour with S9 (1%): 10, 20, 40, 60, 80, 120 µg/mL

Vehicle / solvent:

Dimethyl sulfoxide (DMSO)

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

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, the test item was accurately weighed and formulated in DMSO prior to serial dilutions being prepared. The test item had a molecular weight of 270.28 therefore the maximum proposed dose level in the solubility test was set at 2703 µg/mL, the maximum recommended 10mM dose level, and the purity of the test item was 75% which was not accounted for at the request of the sponsor. However the maximum achievable dose level was 1351.5 µg/mL due to formulation issues at higher concentrations. 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).

No analysis was carried out to determine the homogeneity, concentration or stability of the test item formulation. The test item was formulated within two hours of it being applied to the test system. It is assumed that the formulation was stable for this duration. This is an exception with regard to GLP and has been reflected in the GLP compliance statement.

Control Preparation
Vehicle and positive controls were used in parallel with the test item. Solvent (DMSO) (CAS No. 67-68-5) treatment groups were used as the vehicle controls. Ethylmethanesulphonate (EMS) (CAS No.62-50-0) 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) (Cas No. 6055-19-2) 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 5.28 to 1351.5 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 2 x 105 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 (0.31 to 40 µg/mL in the absence of metabolic activation and 5 to 160 µg/mL in the presence of metabolic activation), vehicle and positive controls. To each universal was added 2 mL of S9 mix if required, 0.2 mL of the treatment dilutions, (0.2 mL for the positive 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 (1% S9 final concentration) at eight dose levels of the test item (0.31 to 40 µg/mL in the absence of metabolic activation, and 5 to 160 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, 0.2 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.

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 104 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 (%RSG) 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:

Preliminary Cytotoxicity Test
The dose range of the test item used in the preliminary toxicity test was 5.28 to 1351.5 µg/mL in all three of the exposure groups.
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 168.94 µg/mL in both the 4-hour and 24-hour exposure group without metabolic activation (post washing). In the 4-hour exposure group with metabolic activation precipitate can be observed at and above 84.47 µg/mL (post washing). Based on the %RSG values observed, the maximum dose level in the subsequent mutagenicity experiment was limited by test item induced toxicity.

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 no evidence of reductions in viability (%V), therefore indicating that residual toxicity had not occurred. Based on the RTG and %RSG values observed, optimum levels of toxicity were achieved in the presence of metabolic activation only. Optimum levels of toxicity were very nearly achieved in the absence of metabolic activation. The excessive toxicity observed at 40 µg/mL in the absence of metabolic activation, resulted in this dose level not being plated for viability or 5-TFT resistance. The toxicity observed at 20 µg/mL in the in the absence of metabolic activation, and 160 µg/mL in the presence of metabolic activation exceeded the upper acceptable limit of 90%, therefore, these dose levels were excluded from the statistical analysis. 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 levels that exceeded the upper limit of acceptable toxicity), in either the absence or presence of metabolic activation. With no evidence of any toxicologically significant increases in mutant frequency in either the absence or presence of metabolic activation in this Experiment, the test item was considered to have been adequately tested. Precipitate of the test item was not observed at any of the dose levels (post washing).

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 no evidence of marked reductions in viability (%V), therefore indicating that residual toxicity had not occurred. Based on the RTG and / or %RSG values observed, very near to optimum levels of toxicity were considered to have been achieved in both the absence and presence metabolic activation. The excessive toxicity observed at 160 µg/mL in the presence of metabolic activation resulted in this dose level not being plated for viability or 5-TFT resistance. The toxicity observed at 120 µg/mL in the in the presence of metabolic activation, exceeded the upper acceptable limit of 90%, therefore, these dose levels were excluded from the statistical analysis. 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 a 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 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 levels that exceeded the upper limit of acceptable toxicity), in either the absence or presence of metabolic activation. With no evidence of any toxicologically significant increases in mutant frequency in either the absence or presence of metabolic activation in any exposure group, the test item was again, considered to have been adequately tested. Precipitate of the test item was observed at 160 µg/mL in the presence of metabolic activation only (post washing).

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Preliminary Cytotoxicity Test

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
5.28	39	106	66
10.56	25	96	31
21.12	10	88	10
42.23	3	66	0
84.47	1	28	0
168.94	1	2	0
337.88	0	0	0
675.75	0	0	0
1351.5	0	0	0

 

Summary of results

Experiment 1

Treatment (µg/ml)		4-hour-S-9				Treatment (µg/ml)		4-hour+S-9
		%RSG	RTG	MF§				%RSG	RTG	MF§
0		100	1.00	133.98		0		100	1.00	140.04
0.31	Ø	91				5	Ø	90
0.63		82	0.78	160.12		10	Ø	94
1.25		80	0.72	140.17		20		86	0.91	121.22
2.5		73	0.69	148.68		40		68	0.68	130.27
5		57	0.54	135.37		60		48	0.46	138.77
10		39	0.36	114.54		80		31	0.28	150.85
20	X	13	0.08	132.34		120		23	0.19	161.16
40	Ø	3				160	X	10	0.08	120.72
Linear trend				NS		Linear trend				NS
EMS						CP
400		70	0.50	931.58		2		62	0.37	1264.45

Experiment 2

Treatment (µg/ml)		24-hour-S-9				Treatment (µg/ml)		4-hour+S-9
		%RSG	RTG	MF§				%RSG	RTG	MF§
0		100	1.00	141.48		0		100	1.00	125.53
0.31		85	0.96	121.19		5	Ø	84
0.63		79	0.94	103.56		10		91	0.89	119.47
1.25		71	0.75	114.60		20		73	0.72	105.15
2.5		68	0.83	104.07		40		52	0.50	105.25
5		53	0.74	138.65		60		35	0.30	135.24
10		20	0.43	124.19		80		25	0.22	129.32
20	Ø	2				120	X	4	0.02	166.62
40	Ø	0				160	Ø	1
Linear trend				NS		Linear trend				NS
EMS						CP
150		44	0.41	1226.89		2		72	0.40	1014.26

Conclusions:

Interpretation of results: 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…….

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, dimethyl sulfoxide (DMSO), 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 (1% S9) and a 24-hour exposure group in the absence of metabolic activation.

 

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

 

Experiment 1

Group	Concentration of Bis(4-methylbenzoyl)peroxide, 75% in water CAS No. 895-85-2 (µg/mL) plated for mutant frequency
4-hour without S9	0.63, 1.25, 2.5, 5, 10, 20
4-hour with S9 (2%)	20, 40, 60, 80, 120, 160

 

Experiment 2

Group	Concentration of Bis(4-methylbenzoyl)peroxide, 75% in water CAS No. 895-85-2 (µg/mL) plated for mutant frequency
24-hour without S9	0.31, 0.63, 1.25, 2.5, 5, 10
4-hour with S9 (1%)	10, 20, 40, 60, 80, 120

 

Results……..

The maximum dose levels used in the Mutagenicity Test were limited by test item-induced toxicity. A precipitate of the test item was observed at 160 µg/mL in Experiment 2 (in the presence of metabolic activation only, post washing). 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 dose-related (linear-trend) increases in the mutant frequency at any of the dose levels, either with or without metabolic activation, in either the first or the second experiment.

 

Conclusion

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

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:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

In the key in vivo study, the test article suspended in carboxymethylcellulose 1% (vehicle) was administered to NMRI mice orally. Twenty-four, 48 and 72 hours after the single application of the test article, the bone marrow cells were collected for micronuclei analysis. Ten animals (5 males, 5 females) per test group were evaluated for the occurrence of micronuclei. 1000 polychromaticerythrocytes (PCE) per animal were scored for micronuclei. The ratio between polychromatic and normochromatic erythrocytes (NCE) was determined in the same sample and reported as the number of NCE per 1000 PCE. The dose level of the test article was 5000 mg/kg b.w. After treatment with the test article the ratio between PCEs and NCEs was not affected as compared to the corresponding negative controls thus indicating no cytotoxic effects. In comparison to the corresponding negative controls there was no statistically significant enhancement in the frequency of the detected micronuclei at any preparation interval after application of the test article. An appropriate reference mutagen (positive control) was used as positive control which showed a distinct increase of induced micronucleus frequency. Since the test article did not induce micronuclei as determined by the micronucleus test with bone marrow cells of the mouse, DI-(4-METHYLBENZOYL)-PEROXID (INTEROX-PMBP) is considered to be non-mutagenic in this assay.

Link to relevant study records

Reference

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:

August 27, 1991 to September 24, 1991

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

The study report does not mention the CAS number or contain the Certificate of Analysis of the test substance. However, this GLP study was conducted according to the then-existing OECD 474 and EEC B12 test guidelines and has used an approved scientific methodology which satisfies basic scientific requirements of the test.

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

micronucleus assay

Species:

mouse

Strain:

NMRI

Sex:

male/female

Details on test animals or test system and environmental conditions:

THE TEST SYSTEM
Reasons for the Choice of the Experimental Animal Species: The mouse is an animal which has been used for many years as suitable experimental animal in cytogenetic investigations. There are many data available from such investigations which may be helpful in the interpretation of results from the micronucleus test. In addition, the mouse is an experimental animal in many physiological, pharmacological and toxicological studies. Data from such experiments also may be useful for the design and the performance of the micronucleus test (1,2,3,4,5).

Strain: NMRI
Source: BRL Tierfarm Flillinsdorf, CH- 4414 Flillinsdorf/Basel, Switzerland
Number of Animals: 84 (42 males/42 females)
Initial Age at Start of Acclimatization: Minimum 10 weeks
Acclimatization: Minimum 5 days
Initial Body Weight at Start of Treatment: Approximately 30 g

According to the suppliers assurance the animals were in healthy condition. The animals were under quarantine in the animal house of C C R for a minimum of five days after their arrival. During this period the animals did not show any signs of illness or altered behavior. The animals were distributed into the test groups at random and identified by cage number.

Husbandry
The animals were kept conventionally. The experiment conducted under standard laboratory conditions.
Housing: single
Cage Type: Makrolon Type I, with wire mesh top (EHRET GmbH, 0-7830 Emmendingen)
Bedding: Granulated soft wood bedding (ALTROMIN, 0-4937 Lage/Lippe)
Feed: Pelleted standard diet (ALTROMIN 1324, 0-4937 Lage/Lippe)
Water: Tap water, ad libitum (Gemeindewerke, 0-6101 RoEdorf)
Environment: temperature 21 ± 3°C; relative humidity 30-70%; artificial light 6.00 a.m.- 6.00 p.m.

Route of administration:

oral: gavage

Vehicle:

carboxymethylcellulose

Details on exposure:

EXPERIMENTAL PERFORMANCE

Pre-Experiment for Toxicity:
A preliminary study on acute toxicity was performed with the same strain and under identical conditions as in the mutagenicity study.

Dose Selection:
It is generally recommended to use the maximum tolerated dose or the highest dose that can be formulated and administered reproducibly. The volume to be administered should be compatible with physiological space available. The maximum tolerated dose level was determined to be the dose that caused toxic reactions without having major effects on survival within 72 hours.

Study Procedure:
Test Groups: Six males and six females were assigned to each test group. The animals were identified by their cage number as shown below in the table.

Test group
Hours post-treatment

24 h male/female 48h male/female 72 h male/female
Negative control 1-6 / 7-12 37-42 / 43-48 61-66 / 67-72
Test article 13-18 / 19-24 49-54 / 55-60 73-78 / 79-84
Positive control 25-30 / 31-36 -/ - -/ -


Treatment::
Approximately 18 hours before treatment with the test article the animals received no food but water ad libitum. At the beginning of the treatment the animals were weighed and the individual volume to be administered was adjusted to the animal's body weight. The animals received the test article once. Twelve animals, six males and six females, were treated per dose group. Sampling of the bone marrow was done 24, 48 and 72 hours after treatment.

Frequency of treatment:

Single oral exposure

Post exposure period:

24, 48 and 72 hr

Remarks:

Doses / Concentrations:

Basis:
nominal conc.

No. of animals per sex per dose:

Twelve animals, six males and six females, were treated per dose group. Sampling of the bone marrow was done 24, 48 and 72 hours after treatment.Cells from five animals per sex and group were evaluated as described. The remaining animal of each test group was evaluated in case an animal had died in its test group spontaneously or due to gavage error.

Control animals:

yes, concurrent vehicle

Positive control(s):

The Positive Control

Name: CPA; Cyclophosphamide
Supplier: SERVA, 0-6900 Heidelberg
Catalogue no.: 17681
Dissolved in: physiological saline
Dosing: 30 mg/kg b.w.
Route and Frequency
of Administration: Orally, once
Volume Administered: 10 ml/kg b.w.
Solution prepared on day of administration. The stability of CPA at room temperature is good. At 20°C only 1 % of CPA is hydrolyzed per day in aqueous solution.

Tissues and cell types examined:

Bone marrow

Details of tissue and slide preparation:

Preparation of the Animals / cells:
The animals were sacrificed by cervical dislocation. The femora were removed, the epiphyses were cut off and the marrow was flushed out with fetal calf serum, using a 5 ml syringe. The cell suspension was centrifuged at 1,500 rpm for 10 minutes and the supernatant was discarded. A small drop of the resuspended cell pellet was spread on a slide. The smear was air-dried and then stained with May-Grunwald (MERCK, D-6100 Darmstadt)/Giemsa (Gurr, BDH Limited Poole, Great Britain). Cover slips were mounted with EUKITT (KINDLER, D-7800 Freiburg). At least one slide was made from each bone marrow sample.

Analysis of Cells:
Evaluation of the slides was performed using NIKON microscopes with 100x oil immersion objectives. 1000 polychromatic erythrocytes (PCE) were analysed per animal for micronuclei. To describe a cytotoxic effect the ratio between polychromatic and normochromatic erythrocytes was determined in the same sample and expressed in normochromatic erythrocytes per 1000 the PCEs. The analysis was performed with coded slides.

Five animals per sex and group were evaluated as described. The remaining animal of each test group was evaluated in case an animal had died in its test group spontaneously or due to gavage error.

Evaluation criteria:

DATA RECORDING:
The data generated are recorded in the laboratory protocol. The results are presented in tabular form, including experimental groups, negative and positive control. The micronucleated cells per thousand and the ratio of polychromatic to normochromatic erythrocytes are presented for each animal.

EVALUATION OF RESULTS:
A test article is classified as mutagenic if it induces a statistically significant increase in the number of micronucleated polychromatic erythrocytes at for at least one of the test points. A test article producing no statistically significant increase in the number of micronucleated polychromatic erythrocytes at any of the test points is considered rion-mutagenic in this system. This can be confirmed by means of the nonparametric Mann-Whitney test (6). However, both biological and statistical significance should be considered together.

Statistics:

BIOMETRY
Statistical significance at the five per cent level (p < 0.05) was evaluated by means of the non-parametric Mann-Whitney test.

Negative Control versus Test group Significance
5000 mg/kg b.w. .... 24 h .......... n.t. *
5000 mg/kg b .w. .... 48 h .......... n.t. *
5000 mg/kg b.w. .... 72 h .......... -

- = not significant; + = significant; n.t. = not tested
* = mean micronucleus frequencies were not above the mean corresponding negative control value.

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Dose: 500 mg/kg b.w.:
Toxic reactions hours post-treatment male/female
1h 6 h 24h 48h 72h
Reduction of spontaneous activity 1/1 1/1 1/1
Eyelid closure 1/1 0/1
Apathy 0/1 0/1

Dose: 1000 mg/kg b.w.: (only 3 animals (2 males and 1 female used)
Toxic reactions hours post-treatment male/female
1h 6 h 24h 48h 72h
Reduction of spontaneous activity 2/1 2/1 0/1
Eyelid closure 2/1 0/1
Apathy 2/1 0/1

Dose: 2000 mg/kg b.w.:
Toxic reactions hours post-treatment male/female
1h 6 h 24h 48h 72h
Reduction of spontaneous activity 2/1 2/2 1/1
Eyelid closure 2/1 2/1
Apathy 2/1 2/1

Dose: 3000 mg/kg b.w.:
Toxic reactions hours post-treatment male/female
1h 6 h 24h 48h 72h
Reduction of spontaneous activity 2/2
Eyelid closure 1/1
Apathy 1/1

Dose: 4000 mg/kg b.w.:
Toxic reactions hours post-treatment male/female
1h 6 h 24h 48h 72h
Reduction of spontaneous activity 2/2
Eyelid closure 2/1
Apathy 2/1

Dose: 5000 mg/kg b.w.:
Toxic reactions hours post-treatment male/female
1h 6 h 24h 48h 72h
Reduction of spontaneous activity 2/2
Eyelid closure 2/2
Apathy 2/1

Higher dosing was not attainable: a) Appropriate suspensions (homogeneity, viscosity) could be obtained only up to 250 mg/ml; b) Application volumes higher than 20 ml/kg b.w. were not justifiable for the rodents used.

SUMMARY OF RESULTS:

test group Dose, sampling PCEs with range PCE/NCE
mg/kg b.w. time (h) micronuclei

suspending agent 0 24 0.19% 0-6 1000/ 796
test article 5000 24 0.06% 0-2 1000/ 806
cyclo-phosphamide 30 24 1.43% 5 -23 1000/ 737
suspending agent 0 48 0.10% 0-2 1000/ 877
test article 5000 48 0.04% 0-2 1000/ 848
suspending agent 0 72 0.06% 0-1 1000/ 680
test article 5000 72 0.07% 0-2 1000/ 786

Conclusions:

Interpretation of results: negative
CONCLUSIONS
The test article DI-(4-METHYLBENZOYL)-PEROXID (INTEROX-PMBP) was assessed in the micronucleus assay for its potential to induce micronuclei in polychromatic erythrocytes (PCE) in the bone marrow of the mouse.

The test article was suspended in carboxymethylcellulose (CMC, 1%). This suspending agent was used as negative control. The volume administered orally was 20 ml/kg b.w. At 24 h, 48 h and 72 h after a single application of the test article the bone marrow cells were collected for micronuclei analysis. Ten animals (5 males, 5 females) per test group were evaluated for the occurrence of micronuclei. 1000 polychromatic erythrocytes (PCE) per animal were scored for micronuclei. To describe a cytotoxic effect due to the treatment with the test article the ratio between polychromatic and normochromatic erythrocytes (NCE) was determined in the same sample and reported as the number of NCE per 1000 PCE. The following dose level of the test article was investigated: 24 h, 48 h, and 72 h preparation interval: 5000 mg/kg b.w. In pre-experiments this dose level was estimated to be the maximum attainable dose. The animals expressed slight toxic reactions. The mean number of normochromatic erythrocytes was not increased after treatment with the test article as compared to the mean values of NCEs of the corresponding negative controls, indicating that DI-(4-METHYLBENZOYL)-PEROXID (INTEROX-PMBP) had no cytotoxic properties. In comparison to the corresponding negative controls there was no statistically significant enhancement in the frequency of the detected micronuclei at any preparation interval after application of the test article. The mean values of micronuclei observed after treatment with DI-(4-METHYLBENZOYL)-PEROXID (INTEROX-PMBP) were in the same range as compared to the negative control groups. 30 mg/kg b.w. cyclophosphamide administered per os was used as positive control which showed a distinct increase of induced micronucleus frequency.

In conclusion, it can be stated that during the study described and under the experimental conditions reported, the test article, DI-(4-METHYLBENZOYL)-PEROXID (INTEROX-PMBP) , did not induce micronuclei as determined.

Executive summary:

This study was performed to investigate the potential of DI-(4 -METHYLBENZOYL)-PEROXID (INTEROX-PMBP) to induce micronuclei in polychromatic erythrocytes (PCE) in the bone marrow of the mouse. The occurrence of micronuclei in interphase cells provides an indirect but easy and rapid measure of chromosomal damage. The mouse has been used for many years as suitable experimental animal in cytogenetic investigations. This study followed internationally accepted guidelines: (1) First Addendum to the OECD 474 (1983), (2) EEC Directive 84/449, L 251, B 12, and (3) Environmental Protection Agency, CFR, Title 40, (1986) "In vivo mammalian bone marrow cytogenetics tests: Micronucleus assay." A reliability rating of K2 was assigned since the study report did not provide the CAS number and the Certificate of Analysis of the test article.

 

The test article was suspended in carboxymethylcellulose (CMC, 1%). This suspending agent was used as negative control. The volume administered orally was 20 ml/kg b.w. and 24 h, 48 h and 72 h after a single application of the test article the bone marrow cells were collected for micronuclei analysis. Ten animals (5 males, 5 females) per test group were evaluated for the occurrence of micronuclei. 1000 PCE per animal were scored for micronuclei. The ratio between polychromatic and normochromatic erythrocytes (NCE) was determined in the same sample and reported as the number of NCE per 1000 PCE. The dose level of the test article investigated at 24 h, 48 h, and 72 h preparation intervals was 5000 mg/kg b.w. In pre-experiments this dose level was estimated to be the maximum attainable dose. The animals expressed slight toxic reactions. After treatment with the test article the ratio between PCEs and NCEs was not affected as compared to the corresponding negative controls thus indicating no cytotoxic effects. In comparison to the corresponding negative controls there was no statistically significant enhancement in the frequency of the detected micronuclei at any preparation interval after application of the test article. An appropriate reference mutagen (positive control) was used as positive control which showed a distinct increase of induced micronucleus frequency.

 

CONCLUSION

During the study described and under the experimental conditions reported, the test article did not induce micronuclei as determined by the micronucleus test with bone marrow cells of the mouse. Therefore, DI-(4-METHYLBENZOYL)-PEROXID (INTEROX-PMBP) is considered to be non-mutagenic in this assay.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vivo:

In the key in vivo mammalian study, Polkner (1991) investigated the potential of DI-(4 -METHYLBENZOYL)-PEROXID (INTEROX-PMBP) to induce micronuclei in polychromatic erythrocytes (PCE) in the bone marrow of mice. The occurrence of micronuclei in interphase cells provides an indirect but easy and rapid measure of chromosomal damage. Since the test article did not induce micronuclei, DI-(4 -METHYLBENZOYL)-PEROXID (INTEROX-PMBP) is considered to be non-mutagenic in this assay.

Additionally, a bacterial gene mutation study (Ames Test) was was performed by Poth (1991) to investigate the potential of D1- (4-METHYLBENZOYL)-PEROX1D (INTEROX-PMBP) to induce gene mutations using the Salmonella typhimurium strains TA 1535, TA 1537, TA 1538, TA 98 and TA 100 and in addition Escherichia coli strain WP2. Up to the highest investigated dose, neither a significant and reproducible increase of the number of revertants was found in any strain as compared to the solvent control nor a concentration-dependent enhancement of the revertant number exists. The presence of liver microsomal activation did not influence these findings. Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies. It was concluded that during the described mutagenicity test and under the experimental conditions reported, the test article did not induce point mutations by base pair changes or frameshifts in the genome of the strains used. Therefore, DI-(4 -METHYLBENZOYL)-PEROXID (INTEROX-PMBP) is considered to be non-mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.

In a recent in vitro mammalian cell mutagenicity study (Bis(4-methylbenzoyl)peroxide, 75% in water CAS No. 895-85-2: L5178Y TK +/- Mouse Lymphoma Assay, Harlan Laboratories, 2014), the test item showed negative results. The maximum dose levels used in the Mutagenicity Test were limited by test item-induced toxicity. A precipitate of the test item was observed at 160 µg/mL in Experiment 2 (in the presence of metabolic activation only, post washing). 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 dose-related (linear-trend) increases in the mutant frequency at any of the dose levels, either with or without metabolic activation, in either the first or the second experiment. It was concluded that the test item did not induce any toxicologically significant increases in the mutant frequency at the TK +/- locus in L5178Y cells.

Justification for selection of genetic toxicity endpoint
This is a well-done key in vivo study (GLP) on mammalian cell chromosome abberation that detects the clastogenic and aneugenic potential of the test item.

Justification for classification or non-classification

All the three mutations assays (in vitro bacterial gene mutation assay, in vitro mammalian cell mutation assay and the in vivo mammalian chromosome aberration assay) showed negative results with the test article. Therefore, DI-(4 -METHYLBENZOYL)-PEROXID (INTEROX-PMBP) is considered non-mutagenic in mammalian cells. Thus, the data are conclusive but not sufficient for classification.