Bis(4-methylbenzoyl)peroxide

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

28 days oral of dosing

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: This GLP study was done according to test guidelines (OECD 407 and EU Method B.1), but has minor deficiencies: Certificate of Analysis of the test substance and the CAS number of the test article were not provided.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1992

Report date:

1992

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Di-(4-Methylbenzoyl)-peroxid

IUPAC Name:

Di-(4-Methylbenzoyl)-peroxid

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: White solid

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
- Expiration date of the batch: Stable, November, 1991
- Stability under test conditions: Stable for at least 48 hours
- Storage condition of test material: At room temperature in the dark

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

Test system: Rat HanIbm: WIST (SPF)
Rationale: Recognized by the international guidelines as the recommended test system (e.g. OECD, EEC)
Source: BRl, Biological Research laboratories Ltd. Wolferstrasse 4 CH-4414 Fuellinsdorf
Total number of animals per group: 5 males 5 females
Total number of animals: 20 males 20 females
Age at acclimatization: 6 weeks
Age at treatment start: 7 weeks
Body weight and range (at acclimatization): Males: 163 -174 g; Females 145 -158 g
Identification: Individual cage number and corresponding ear tattoo.
Randomization: Computer-generated random algorithm.
Acclimatization: Seven days under laboratory conditions, after veterinary examination. Only animals without any visual signs of illness were used for the study.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

polyethylene glycol

Details on oral exposure:

Method: Oral by gavage. The control animals received the vehicle (PEG 400) without the test article.
Rationale: Accidental oral ingestion is a possible human exposure.
Frequency: Once daily, 7 days per week.
Dose levels:
Group 1 (control): 0 mg/kg and treatment day
Group 2: 50 mg/kg and treatment day
Group 3: 200 mg/kg and treatment day
Group 4: 1000 mg/kg and treatment day

Dose volume: 10 ml/kg body weight and treatment drY.
Duration of acclimatization: 7 days
Duration of treatment: 28 days

Allocation:
The identification number of rats assigned to toxicity testing per group as well as the dose levels daily administered were as follows:
GROUP 1* (0 mg/kg) GROUP 2 (50 mg/kg) GROUP 3 (200 mg/kg) GROUP 4 (1000 mg/kg)
Males:. A 1 -5, 6 -10, 11 -15, 16 -20
Females: A 21 -25, 26 -30, 31 -35, 36 -40
* Control animals were gavaged with the vehicle only (Polyethylene glycol, PEG 400)
A = Toxicity testing (termination after 28 treatment days)

Animal Husbandry (Room No.: 106)
Conditions: Standard Laboratory Conditions. Air-conditioned with 10-15 air changes per hour, and continuously monitored environment with a temperature of 22 +1- 3 degrees centigrade, a relative humidity between 40-70 %, 12 hours artificial fluorescent light (approximately 100 Lux), 12 hours dark, music during the light period.

Accommodation: Individually in Makrolon type-3 cages (size: 22x37.5x15 cm) with standard softwood bedding ('Lignocel', Schill AG, CH-4132 Muttenz)

Water: Community tap-water from Fullinsdorf was available ad 1i bitum. Results of bacteriological, chemical and contaminant analyses are included in the original study report.

Diet: Pelleted standard Kliba no. 343, Batch 86/91 rat maintenance dietl ('Kliba', Klingentalmuehle AG, CH-4303 Kaiseraugst) ad libitum. Results of contaminant analyses are included in the original study report.

PREPARATION OF DOSING SOLUTIONS:

Test article preparation: The test article was weighed into a glass beaker on a tared Mettler PM 480 balance and the vehicle, polyethylene glycol (PEG 400), was added. The mixture (w/v) was prepared using a homogenizer. Homogeneity of the vehicle was maintained during treatment using a magnetic stirrer.

Frequency of preparation: Daily prior to each application

VEHICLE - PEG 400. No other information is provided.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Chemical analysis of dose preparation: Concentration, homogeneity and stability of the test article/vehicle mixtures were determined during acclimatization. Further samples for analysis were taken during week 3 ofithe test and subsequently analyzed. See the dates below. The analyses were performed in the Analytical Laboratories of Umweltchemie AG, according to a method which was supplied by the sponsor.

Accl!matization/Pretest: September 23, 1991
At 3 weeks of the test: October 14, 1991

Duration of treatment / exposure:

28 days

Frequency of treatment:

Frequency: Once daily, 7 days per week.

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Details on study design:

Delivery of Animals: September 19, 1991
Acclimatization: September 19 to 25, 1991
Administration: September 26 to October 23, 1991
Termination: October 24, 1991
Report (Final): March 30, 1992

Dose selection rationale: Based upon data from acute and subacute studies, especially an acute toxicity study (RCC 309431) and a 5-day range-finding study (RCC:310230) in which Di-(4-Methylbenzoyl)-peroxid was administered orally by gavage to rats. There were no satellite or recovery groups.

Positive control:

No

Examinations

Observations and examinations performed and frequency:

DETAILED CLINICAL OBSERVATIONS: Observations for viability and mortality were recorded once daily. Signs of toxicity were assessed once daily. Descriptions of all observations were recorded and the subsequent progress was monitored.

BODY WEIGHT: The body weight of each animal was recorded on the same days as the food consumption using the same recording system. Additionally, terminal body weights were recorded at necropsy.

FOOD CONSUMPTION: The food consumption was recorded once during the acclimatization period and weekly thereafter using an on-line electronic recording system consisting of a Mettler PM 4800 balance connected to the RCC computer.

OPHTHALMOSCOPIC EXAMINATION was done at Week 4, October 22, 1991, on all surviving animals. A description of any abnormality was recorded. Ten to ninety minutes after the application of a mydriatic solution (Dispersa AG, CH-8442 Hettlingen), the cornea, lens, anterior chamber, vitreous body and ocular fundus of both eyes were examined under dimmed light using a Heine BETA 200 Ophthalmoscope (Eisenhut Vet. AG, CH-4123, Allschwil).

CLINICAL LABORATORY INVESTIGATIONS: Blood and urine sampling were done at Week 4 (Oct. 24, 1991).
Blood samples for hematology and clinical biochemistry were collected from all animals under light ether anesthesia. The animals were fasted for approximately 18 hours before blood sampling but, allowed access to water ad libitum. Blood samples were collected between 07.00 and 08.30 a.m. to reduce biological variation caused by circadian rhythms. Blood samples were drawn from the retroorbital plexus using a micro-hematocrit glass capillary tube. The assays of blood and urine parameters were performed under internal laboratory quality control conditions to assure reliable test results. The summary and individual tables were generated by a computer the program of which limits the width of each column to 10 characters. Therefore, the names of some parameters have been abbreviated. Any abbreviation, has been defined in this section under "Parameter" in upper-case letters enclosed by parentheses. Clinical laboratory data were expressed in general accordance with the International System of Units (SI), which in structure comprises base units, derived units and supplementary units. It also includes a series of prefixes by means of which decimal multiples and submultiples of these units can be formed. In some cases non-SI Units or conventional units may be used. The anticoagulants used during blood collection were: EDTA-K2 (hematology); Lithium heparin 30 U.S.P. Units (methemoglobin); and Sodium citrate, 3.8% (coagulation; 1 part anticoagulant to 9 parts blood)

HAEMATOLOGY PARAMETERS: Erythrocyte count (RBC), Hemoglobin (HB), Hematocrit (HCT), Mean corpuscular Volume (MCV), Mean corpuscular hemoglobin (MCH), Mean corpuscular hemoglobin concentration (MCHC), Platelet count (PLATELETS), Reticulocyte count (RETIC.), Nucleated erythrocytes normoblasts (NEN), Heinz bodies (HEINZ-BOD.), Methemoglobin (MET-HB), Total leukocyte count (WBC), Differential leukocyte Count (Diff. WBC Count), Red cell morphology, and the two coagulation parameters, Thromboplastin time (=Prothrombin time) (PT) and Activated partial thromboplastin time (APTT).

CLINICAL CHEMISTRY PARAMETERS: Glucose, Urea, Creatinine, Uric acid, Bilirubin, total (BILl. T.), Cholesterol, total (CHOLEST. T.), Triglycerides (TRIGL.), Phospholipids (PHOS.LIPID), Aspartate aminotransferase (ASAT/GOT), Alanine aminotransferase (ALAT/GPT), Lactate dehydrogenase (LDH), Creatine kinase (CK), Alkaline phosphatase (ALP), Gamma-glutamyltransferase (G-GT), Calcium, Phosphorus, Sodium, Potassium, Chloride, Total protein, Albumin, Globulin, and Albumin/Globulin ratio (A/G RATIO).

URINALYSIS PARAMETERS: Volume (18-hour),Specific gravity (SPEC. GRAV.), Color, pH, Protein, Glucose, Ketone, bilirubin, Blood Urobilinogen (UROBILI.), and Urine Sediment (SED. MICRO.).

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes

NECROPSY was done after 4 weeks (October 24, 1991).
All animals were weighed and necropsied and descriptions of all macroscopic abnormalities were recorded. Prior to necropsy, the animals were fasted for approximately 18 hours, but water was provided. Necropsies were performed by experienced prosectors. All animals surviving to the end of the observation period and all moribund animals were anesthetized by intraperiton1eal injection of sodium pentobarbitone and killed by exsanguination. Samples of the following tissues and organs were collected from all animals at necropsy and fixed in phosphate buffered neutral 4% formaldehyde solution: Adrenals; Aorta; Bone (sternum, femur); Bone marrow (sternum, femur); Brain; Cecum; Colon; Duodenum; Epididymides; Esophagus; Eyes with optic nerve and Harderian gland; Mammary gland area; Femur including joint; Heart; Ileum; Jejunum; Kidneys; Larynx; Lacrimal gland, exorbital; Liver; Lung infused with formalin; Lymph nodes, mandibular, mesenteric; Nasal cavity; Ovaries; Pancreas; Pituitary gland; Prostate gland; Rectum; Salivary gland (mandibular, sublingual); Seminal vesicles; Sciatic nerve; Skeletal muscle; Skin; Spinal cord, (cervical, midthoracic, lumbar); Spleen; Stomach; Testes; Thymus; Thyroid including Parathyroid gland; Tongue; Trachea; Urinary bladder infused with formalin; Uterus; Vagina; and Gross lesions.
 
HISTOTECHNIQUE: All organ and tissue samples, as defined under Histopathology wene processed, embedded, cut at a thickness of 2-4 micrometers, and stained with hematoxylin and eosin.
 
HISTOPATHOLOGY: Slides of Adrenals, Heart, Kidneys, Liver, Spleen and Stomach collected at terminal sacrifice from the animals of the control and high-dose. groups were examined by a pathologist. The same applied to all Gross lesions and to all animals which died spontaneously. All abnormalities were described and included in the report. Due to a suspected toxicity in the liver and the adrenal glands, samples of these organs of all animals of the intermediate and low dose group were processed and microscopically evaluated. 

Statistics:

The following statistical methods were used to analyze the body weights, food consumption, organ weights, all ratios and clinical laboratory data:

Univariate one-way analysis of variance was used to assess the significance of intergroup differences. If the variables could be assumed to follow a normal distribution, the Dunnett test (many to one t-test) based on a pooled variance estimate was applied for the comparison between the treated groups and the control groups for each sex. The Steel-test (many-one rank test) was applied when the data could not be assumed to follow a normal distribution. The Fisher's exact test for 2x2 tables was applied to the overall spontaneous mortality data. Group means were calculated for continuous data and medians were calculated for discrete data (scores) in the summary tables. Individual values, means, standard deviations and statistics were round-off before printing. For example, test statistics were calculated on the basis of exact values for means and pooled variances and then rounded-off to two decimal places. Therefore, two groups may display the same printed means for a given parameter, yet display different test statistics values.

References:
C.W. Dunnett: A Multiple Comparison Procedure for Comparing Several Treatments with a Control, J. Amer. Statist. Assoc. 50, 1096-l121 (1955).
R.G. Miller: Simultaneous Statistical Inference, Springer Verlag, New York (1981).
R.A. Fisher: Statistical Methods for Research Workers, Oliver and Boyd, Edinburgh (1950).

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Description (incidence and severity):

Mortality: Female no. 25 (group control) died spontaneously on treatment day 4. The animal was treated with the test article only twice prior to its spontaeous death. No other premature death occurred. Clinical Signs: One out of five males of group 4 (1000 mg/kg) showed rales on treatment day 3 and one out of five females of control group 1 (0 mg/kg) showed sedation, dyspnea and ruffled fur on treatment day 3. In addition to this findings all treated animals of groups 1, 2, 3 and 4 (0-, 50-, 200- and 1000 mg/kg) showed slight diarrhea during most of the study period starting with day 6. The latter was considered to be related to the use of polyethylene glycol (PEG 400) as the vehicle.

Mortality:

no mortality observed

Description (incidence):

Mortality: Female no. 25 (group control) died spontaneously on treatment day 4. The animal was treated with the test article only twice prior to its spontaeous death. No other premature death occurred. Clinical Signs: One out of five males of group 4 (1000 mg/kg) showed rales on treatment day 3 and one out of five females of control group 1 (0 mg/kg) showed sedation, dyspnea and ruffled fur on treatment day 3. In addition to this findings all treated animals of groups 1, 2, 3 and 4 (0-, 50-, 200- and 1000 mg/kg) showed slight diarrhea during most of the study period starting with day 6. The latter was considered to be related to the use of polyethylene glycol (PEG 400) as the vehicle.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

There were no treatment-related effects on body weight or body weight gain which were considered to be toxicologically significant.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

There were no effects on absolute or relative food consumption, which were considered to be toxicologically significant.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

no effects observed

Description (incidence and severity):

No treatment related abnormalities.

Haematological findings:

no effects observed

Description (incidence and severity):

The assessment of hematological data indicated no changes of toxicological significance at termination of the treatment. All differences in these results were considered to be incidental and of norrmal biological variation for rats of this strain and age.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

The assessment of clinical biochemistry data indicated no changes of toxicological significance at termination of the treatment. All differences in results were considered to be incidental and of norrmal biological variation for rats of this strain / age.

Urinalysis findings:

no effects observed

Description (incidence and severity):

Assessment of urinalysis data indicated no changes of toxicological significance at the end of treatment. All differences in the results of urinalysis parameters were considered incidental and of norrmal biological variation for rats of this strain/age.

Behaviour (functional findings):

not examined

Description (incidence and severity):

Described under "Details on results"

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Described under "Details on results", it was concluded that the changes were not toxicologically relevant.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Described under "Details on results", it was concluded that the changes were not toxicologically relevant.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Described under "Details on results", it was concluded that the changes were not toxicologically relevant.

Histopathological findings: neoplastic:

no effects observed

Details on results:

Details of observations on organ weights, gross pathology, and histopathology:

ORGAN WEIGHTS, ORGAN TO BODY WEIGHTS and ORGAN TO BRAIN WEIGHT RATIOS: The relative adrenal weights (ratios to body and brain weights) of males of group 3 were significantly lower when compared to those of the controls. Females of group 2 (50 mg/kg) showed absolute heart weights and heart to brain weight ratios as well as kidney weights, kidney to brain weight ratios and ovaries to brain weight ratios significantly lower than those of controls. In female rats of group 3 (200 mg/kg) the terminal body weights and the absolute heart weights and heart to brain weight ratios were significantly lower whereas the absolute thyroid weights and their ratios were significantly higher than those of the controls. High-dose female rats of group 4 (1000 mg/kg) showed statistically significantly higher absolute and relative thyroid weights when compared to those of controls.

GROSS PATHOLOGY: Macroscopic findings were largely unremarkable and amongst those commonly recorded in this age and strain of rat. Exceptions to these was the following:

Liver: left lateral lobe: diaphragmatic hernia, d=8 mm (no. 5/ Group 1). During the necropsy the cecum of three males and four females of the control group (0 mg/kg), all animals of group 2 (50 mg/kg), four males and all five females of group 3 (200 mg/mg) and all animals of group 4 (1000 mg/kg} showed a liquid content and/or dilation.

HISTOPATHOLOGY:
Cecum: The cecum of all examined an;mals showed an edema of the submucosa of a minimal to a marked degree. Focal necrosis of cecal mucosa was detected in three animals (female no. 24 / Group 1: grade 2; male no. 10 / Group 2: hemorrhagic necrosis, grade 2; and male no. 12 / Group 3: grade 2).

Adrenal Glands: The incidence and the degree of single cell degeneration, characterized by pycnotic cells seemed to be increased in group 4. However, in comparison with all other animals of the treatment groups this finding was not confirmable.

Liver; There was no detectable accidental or traumatic reason for the dtaphragmatic hernia. The incidence and degree of single cell degeneration seemed to be increased in all treatment groups. However, in comparison with the animals of all other treatment groups, this finding was not confirmable.

Other findings recorded were within the normal range of background lesions which may be observed in this age and strain of rats.

Effect levels

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw (total dose)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No toxicologically significant overall effects (on clinical signs; mortality; body weight; food consumption; food efficiency; ophthalmoscopic examination; haematology; clinical chemistry; urinalysis; gross pathology; organ weights; histopathology).

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

Historical reference values for untreated Wistar Han rats were provided in the report.

Applicant's summary and conclusion

Conclusions:

Based upon the results obtained in this study, the "no-adverse-effect-level" of Di-(4 -Methylbenzoyl)-peroxid (INTEROX-PMBP) is 1000mg/kg body weight for male and female WISTAR rats when administered orally by gavage for a period of 28 days.

Executive summary:

In this subacute 28-day oral toxicity study, Di-(4-Methylbenzoyl)-peroxid (INTEROX-PMBP) was administered daily by oral gavage to SPF-bred Wistar rats. The test article, prepared in PEG 400, was administered to 7-week old rats [(HanIbm: WIST (SPF)] of both sexes (five each) for 28 days at doses of 0 (vehicle only), 50, 200 and 1000 mg/kg body weight. While this GLP study by N. Hoff (1992) was done according to OECD 407 and EU B.7 Test Guidelines, a reliability rating of K2 was assigned due to minor deficiencies in the study report (non-inclusion of the CAS number and the Certificate of Analysis of the test substance).

A control group female (0 mg/kg) animal (No. 25) died spontaneously on treatment day 4. No other premature death occurred. Rales were observed on treatment day 3 in one Group 4 male 1000 mg/kg). Additionally, all treated animals of all groups (1 to 4) showed slight diarrhea for most of the study period. The latter was considered to be related to the use of polyethylene glycol (PEG 400) as the vehicle. There were no toxicologically or statistically significant effects on absolute or relative food consumption in the test article treated animals observed when the results were compared to those of the controls. There were no toxicologically or statistically significant effects on the body weights observed in the test article treated animals which were considered to be treatment related. The observed data reflect the normal pattern for this strain and age of rats used. No treatment related findings on ophthalmoscopy were observed. The assessment of hematological, clinical biochemical and urinalysis data indicated no changes of toxicological significance at termination of the treatment. No treatment-related effects on absolute or relative organ weights were observed in the animals of the test article treated groups 2 to 4, when the results were compared to those of the controls. The statistically significant differences observed were considered to be incidental and of no toxicological relevance. Thus, there was no evidence of any treatment-related macroscopic or microscopic findings.

Based upon the results obtained in this study, the "no-adverse-effect-level" of Di-(4 -Methylbenzoyl)-peroxid (INTEROX-PMBP) is1000mg/kg body weight for male and female rats when administered orally by gavage for a period of 28 days.