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Administrative data

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.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1991
Report date:
1991

Materials and methods

Test guidelineopen allclose all
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

Test material

Constituent 1
Reference substance name:
Di-(4-Methylbenzoyl)-peroxid (INTEROX-PMBP)
IUPAC Name:
Di-(4-Methylbenzoyl)-peroxid (INTEROX-PMBP)
Constituent 2
Chemical structure
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:
bis(4-methylbenzoyl)peroxide
Test material form:
other: solid
Details on test material:
Name of test material (as cited in study report): Di-(4-Methylbenzoyl)-peroxid (INTEROX-PMBP)
- Aggregate state at room temperature: Solid
-Color: white
- Purity: Water damped powder 72% PMBP
- Batch No.: IR 240001
- Analysis: cf. analytical certificate in Sponsor's file.
- Stability: Pure: Stable for 6 months; In vehicle: at least 48 hrs in water, polyethylene glycol and carboxymethylcellulose (CMC).
- Expiration date: November, 1991
- Storage: Room temperature

On the day of the experiment, the test article was suspended in CMC (1%). The vehicle was chosen due to its nontoxicity for the animals. All animals received a single standard volume of 20 ml/kg body weight orally.

Test animals

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.

Administration / exposure

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
Doses / concentrations
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.

Examinations

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.

Results and discussion

Test results
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


Applicant's summary and conclusion

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.

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