Bis(4-methylbenzoyl)peroxide

Genetic toxicity: in vitro

Administrative data

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.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1991

Report date:

1991

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 4

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

Test material

Test material information

Constituent 1

Reference substance name:

DI-(4-METHYLBENZOYL)-PEROXID (INTEROX-PMBP)

IUPAC Name:

DI-(4-METHYLBENZOYL)-PEROXID (INTEROX-PMBP)

Constituent 2

Reference substance name:

Bis(4-methylbenzoyl)peroxide

EC Number:

407-950-9

EC Name:

Bis(4-methylbenzoyl)peroxide

Cas Number:

895-85-2

Molecular formula:

C16H14O4

IUPAC Name:

4-methylbenzoyl 4-methylbenzene-1-carboperoxoate

Test material form:

other: solid white

Details on test material:

- Name of test material (as cited in study report): Di-(4-Methylbenzoyl)-peroxid (INTEROX-PMBP)
- Physical state: Solid, white
- Analytical purity: Water damped powder 72% PMBP
- Batch No.: IR 240001
- Stability of test article
Pure: Stable for 6 months
In vehicle: At least 48 hours in water, polyethylene glycol and carboxymethyl cellose.

-Expiration date: November, 1991
-Storage condition of test material: Room temperature

Method

Species / strain

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.

Controls

Controls 1

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.

Controls 2

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).

Results and discussion

Test results

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.

Any other information on results incl. tables

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.

Applicant's summary and conclusion

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.