Registration Dossier

Toxicological information

Genetic toxicity: in vivo

Currently viewing:

Administrative data

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
November 2012 - March 2013
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Well documented study conducted according to modern standards of internationally accepted protocol and Good Laboratory Practice. Test Material analytically confirmed for purity
Reason / purpose:
reference to same study

Data source

Reference Type:
study report
Report Date:

Materials and methods

Test guideline
according to
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Principles of method if other than guideline:
Animals were exposed for 90 days via inhalation.
GLP compliance:
yes (incl. certificate)
Type of assay:
micronucleus assay

Test material

Test material form:
other: vapour
Details on test material:
Name: Di-tert-butyl peroxide CAS# 110-05-4
Chemical names: di-tert-butyl peroxide; peroxide, bis(1,1-dimethylethyl)
Trade name: Trigonox B
CAS reg number: 110-05-4
Appearance: clear, colourless liquid
Molecular formula: C8H18O2
Molecular weight: 146.2 g/mol
Specific gravity: 0.8 g/cm3 at 25°C
Vapour pressure: 3500 Pa at 25°C (equals >200 g/m3 at 25°C)
Viscosity: 0.9 mPa.s
Storage conditions: <-18°C, protected from light
The material will decompose if heated; selfaccelerating decomposition temperature is 80°C; product may be safely stored at controlled ambient temperature; storage at <-18°C is not required for safety but significantly extends shelf life
Hygroscopy: Absent
Purity: >99%
Total quantity: 10 kg
Quantity per unit package: 2 kg
Expiry date: 1 September 2022
Batch number: 1208250897
Supplier: Sponsor

Test animals

Details on test animals and environmental conditions:
The study was conducted with albino rats. Young adult, male and female Wistar Hannover outbred rats (RccHan®:WIST) were obtained from a colony maintained under specific pathogenfree (SPF) conditions at Harlan Laboratories, The Netherlands (for the in vivo MN test, only male rats were used). On the day of randomization (shortly before the first exposure day), the age of the rats was about 7-8 weeks, and the initial body weight variation did not exceed ± 20% of the mean weight for each sex. Mean body weights at the start of treatment were 264 and 176 grams for male and female animals, respectively.

Upon arrival, the rats were taken to a quarantine room and checked for overt signs of ill health and anomalies. During the quarantine period, serological investigation of the microbiological status was conducted in blood samples taken from five randomly selected animals. Two days after arrival, the
results of serological tests were passed on by telephone and indicated an acceptable microbiological status. Subsequently, the animals were released for experimental use and moved to their definitive room. The duration of the acclimatization period to the conditions in the experimental room
prior the first exposure was 10 days (males) or 11 days (females). Shortly before initiation of exposure (study days -4 and -5 for males and females, respectively), the animals were allocated to the various groups by computer randomization proportionally to body weight (males and females separately). The surplus animals were kept in reserve to serve as sentinels (four/sex). These animals were discarded at the end of the in-life phase of the study.

From their arrival, the rats were housed under conventional conditions in one room separated by sex. No other test system was housed in the same room during the study. Lighting was artificial (fluorescent tubes) with a sequence of 12 hours light and 12 hours dark. The room was ventilated with about 10 air changes per hour. The temperature and relative humidity in the room were 22 ± 2°C and 45-65%, respectively, with a few exceptions.

The animals were housed in groups of five, separated by sex, in Makrolon® cages (type IV) with a bedding of wood shavings (Lignocel, Rettenmaier & Söhne GmbH & Co, Rosenberg, Germany) and strips of paper (Enviro-dri, Shepherd Specialty Papers, Michigan, USA) and a wooden block (ABEDD, Vienna, Austria) as environmental enrichment (Lillico, Betchworth, England). During exposure, the animals were kept individually in the exposure unit. Immediately after each exposure, the animals were returned to their home cages. After treatment with the mutagen Mitomycin C, the five animals of the positive control group were kept in smaller Makrolon® cages with filter tops (one or two animals per cage; bedding: wood shavings; enrichment: strips of paper) until sacrifice the next day.

Feed and drinking water were provided ad libitum from the arrival of the animals until the end of the study, except during inhalation exposure and during the fasting period before scheduled sacrifice. The animals received a proprietary cereal-based rodent diet (Rat & Mouse No. 3 Breeding Diet, RM3) from a commercial supplier (SDS Special Diets Services, Whitham, England). Each batch of RM3 diet is analysed by the supplier for nutrients and contaminants. The feed was provided as a powder in stainless steel cans, covered by a perforated stainless steel plate to prevent spillage. The feed in the feeders was replaced with fresh portions once weekly and filled as needed. Each cage was supplied with domestic mains tap-water suitable for human consumption (quality guidelines according to Dutch legislation based on EC Council Directive 98/83/EC). The water was given in polypropylene bottles, which were cleaned weekly and filled as needed. Results of the routine physical, chemical and microbial examination of the drinking water as conducted by the supplier are made available to the test facility. In addition, the supplier periodically (twice per year) analyses water samples taken on the premises of the test facility for a limited number of variables.

Administration / exposure

Route of administration:
inhalation: vapour
Details on exposure:
The animals were exposed to the test atmosphere in a nose-only inhalation chamber (Institute’s design) consisting of a cylindrical PVC column with a volume of about 75 litres, surrounded by a transparent hood. The test atmosphere was introduced at the bottom of the central column and was exhausted at the top. Each column included three rodent tube levels of 20 ports each. The animals were placed at the top level. Empty ports were used for measurement of temperature and relative humidity. The animals were secured in plastic animal holders (Battelle), positioned radially through the outer hood around the central column. Only the nose of the rats protruded into the interior of the column. The remaining ports were closed. Male and female rats were placed in alternating order. Animals were rotated weekly with respect to the position in the column. From 25 February 2013, larger sized animal holders were used for the male rats because these animals had reached a size for which the standard holders were too small.
The animal's body does not exactly fit in the animal holder which always results in some leakage from the high to the low pressure side. By securing
a positive pressure in the central column and a slightly negative pressure in the outer hood, which encloses the entire animal holder, air leaks from nose to thorax rather than from thorax to nose. This way, dilution of test atmosphere at the animals’ noses was avoided.
The units were illuminated externally by normal laboratory fluorescent tube lighting. The total airflow through the unit was at least 1 litre/min per animal. The air entering the unit was maintained between 22 ± 3˚C and the relative humidity between 30% and 70%.

The inhalation equipment was designed to expose rats to a continuous supply of fresh test atmosphere. To generate the test atmospheres, a liquid flow of test material, controlled by a peristaltic pump (Gilson France SA, Villiers le Bel, France), was evaporated in a glass evaporator. The temperature of the evaporator was controlled at 22.5˚C (exceptions: 22.7 or 22.8˚C on a few occasions) using a temperature controlled flow of circulating water. The vapour was transported in a stream of humidified compressed air, the flow of which was controlled by a mass flow controller (Bronkhorst, Hi Tec, Ruurlo, The Netherlands). All three test atmospheres (target concentrations 0.1, 0.3 and 1 g/m3) were obtained by diluting a pre-mixture containing about 3 g/m3 of the test material in humidified compressed air. First, mass flow controlled streams of the pre-mixture were supplemented with a mass flow controlled stream of humidified compressed air via an eductor to obtain the low- and mid-concentration. Next, the remaining stream of the pre-mixture was diluted with a mass flow controlled stream of humidified compressed air to obtain the high-concentration. The generated test atmospheres were directed to the bottom inlets of the exposure units. The exposure unit for the control animals was supplied with a measured stream of humidified compressed air only. The animals were placed in the exposure unit after stabilization of the test atmosphere.

The actual concentration of the test material in the test atmospheres was measured by total carbon analysis (Sick Maihak EuroFID total hydrocarbon analyser; Sick Instruments Benelux, Hedel, the Netherlands). The response of the analyser was recorded on a PC every minute using a CAN transmitter (G. Lufft Mess- und Regeltechnik GmbH, 70719 Felbach, Germany). The responses were converted to concentrations by means of calibration graphs. For each exposure day, the mean concentration was calculated from the values determined every minute. Representative test atmosphere samples were taken continuously from the exposure unit at the animals’ breathing zone and were passed to the total carbon analyser (TCA) through a sample line.
Prior to the first exposure, the output of the flame ionization detector of the TCA was calibrated using PET sample bags with known volumes of clean dry air and known amounts (by weighing) of test material. For each target concentration three calibration concentrations were prepared, at least in duplicate, and analysed (approximately 80, 100 and 120% of the target concentration). The calibrations were checked weekly by measuring the concentration in a sample bag with a theoretical concentration close to the target concentration. If the measured concentration deviated more than 5% from the theoretical concentration and this was confirmed with a second sample bag, the TCA was recalibrated.

The nominal concentration was determined, for each exposure day, by dividing the total amount of test material used (by weight) by the total volume of air passed through the exposure unit. The nominal concentration was calculated for the low-, mid- and high-concentration as well as for the pre-mixture which was diluted to obtain the test atmospheres. The generation efficiency was calculated from the actual and the nominal concentration (efficiency = actual concentration as percentage of nominal concentration).

The chamber airflow of the test atmospheres was recorded about hourly using a Rotameter (group 1) or a mass flow controller (groups 2-4). The temperature and the relative humidity of the test atmospheres were measured continuously and recorded every minute using a CAN transmitter with temperature and relative humidity probes (G.Lufft Mess- und Regeltechnik GmbH, 70719 Fellbach, Germany). For group 4 of the sub-chronic study the temperature and relative humidity were additionally measured about hourly by means of a RH/T device (TESTO 635-1, TESTO GmbH & Co, Lenzkirch, Schwarzwald, Germany).

The overall mean actual concentrations (+/- standard deviation) of the test material in the test atmospheres as measured by total carbon analysis were 101 (± 3), 299 (± 3) and 993 (± 10) mg/m3 for the low-, mid- and high-concentration, respectively. These actual concentrations were very close to the target concentrations (100, 300 and 1000 mg/m3).

The overall mean nominal concentrations, calculated from the daily consumption of test material, the airflow and the duration of test atmosphere generation, were 96, 290 and 1036 mg/m3 for the low-, mid- and high-concentration, respectively. The corresponding generation efficiencies were close to the expected 100%, namely 105, 103 and 96%, respectively.

The overall mean (± standard deviation) chamber airflows were 27.8 (± 0.0), 26.2 (± 0.2), 27.6 (± 0.3) and 24.3 (± 0.2) L/min for exposure chambers 1 (control), 2 (low), 3 (mid) and 4 (high), respectively.

The air temperature in the exposure chambers during exposure was within the target range of 19 – 25°C. The overall mean temperature was about 23°C for each chamber. The relative humidity during exposure was within the target range of 30-70%. The overall mean relative humidity was 46, 40, 39 and 44% in exposure chambers 1 (control), 2 (low), 3 (mid) and 4 (high), respectively.
Duration of treatment / exposure:
90 days (65 exposure days)
Frequency of treatment:
6 h/day, 5 days/week
Post exposure period:
Not applicable
Doses / concentrationsopen allclose all
Doses / Concentrations:
0, 100, 300 and 1000 mg/m3
other: target concentrations
Doses / Concentrations:
0, 101, 299, and 993 mg/m3
analytical conc.
No. of animals per sex per dose:
5 males/concentration
5 males positive control
Control animals:
yes, concurrent vehicle
Positive control(s):
Mitomycin C was administered by a single intraperitoneal injection at a dose level of 1.5 mg/kg body weight as a solution in physiological saline (dose volume 10 ml/kg body weight; concentration 0.15 mg/ml).


Tissues and cell types examined:
At sacrifice, bone marrow cells of one of the femurs (left femur) were collected from five male animals per group.
Details of tissue and slide preparation:
The bone marrow cells were immediately collected into foetal calf serum and processed into glass drawn smears according to the method described by Schmid (1976). Two bone marrow smears per animal were prepared, air-dried and fixed in methanol. One smear per animal was stained with a May-Grünwald-Giemsa solution. The other fixed unstained smear was kept in reserve and discarded after completion of analysis.

The bone marrow smears of the males of groups 1-5 were examined microscopically. The slides were randomly coded by a person not involved in the
scoring of slides. The slides (one slide per animal) were read by moving from the beginning of the smear (label end) to the leading edge in horizontal lines, taking care that areas selected for evaluation were evenly distributed over the whole smear.
The following criteria were used for the scoring of cells:
˗ A polychromatic erythrocyte (PE) is an immature erythrocyte that still contains ribosomes and can be distinguished from mature, normochromatic erythrocytes by a faint blue stain.
˗ A normochromatic erythrocyte (NE) is a mature erythrocyte that lacks ribosomes and can be distinguished from immature, polychromatic erythrocytes by a yellow stain.
˗ A micronucleus is a small, normally round, nucleus with a diameter of circa 1/20 to 1/5 of an erythrocyte, distinguished from the cytoplasm by a dark blue stain. The numbers of polychromatic and normochromatic erythrocytes (PE and NE, respectively) were recorded in a total of 200 erythrocytes (E) per animal. If micronuclei were observed, these were recorded as micronucleated polychromatic erythrocytes (MPE) or micronucleated normochromatic erythrocytes (MNE). Once a total number of 200 E (PE + NE) had been scored, an additional number of PE was scored for the presence of micronuclei until a total number of 2000 PE had been scored. The incidence of MPE was recorded in a total of 2000 PE per animal and the number of MNE was recorded in the number of NE.
Evaluation criteria:
The test was considered valid if the positive controls showed a statistically significant increase in the mean number of MPE/2000 PE and the negative controls were within the historical range.
A test material was considered to cause chromosomal damage and/or damage to the mitotic apparatus if it showed a dose-related positive response or a clear increase of micronucleated cells in a single dose group.
A test material was considered to be negative in the micronucleus test if it did not produce a positive response at any of the dose levels analysed.
The test material or its metabolites were considered to be cytotoxic to the bone marrow via the general circulation, if the test material statistically significantly reduced the mean number of PE.
Both statistical significance and biological relevance were considered together in the evaluation.
Statistical tests were performed using GraphPad Prism®, Version 5.03, Copyright © 1992-2010 GraphPad Software, Inc., CA, USA.. In all tests a significance level of 5% was used (α = 0.05). Data on PE and MPE (PE/200 E and MPE/2000 PE) were analysed by one-way analysis of variance [Anova]. Prior to Anova, it was checked if the Anova assumptions were met (i.e. variances equal). In case assumptions were not met non-parametric testing was performed using the Mann-Whitney test (positive control compared with negative control) or Kruskal-Wallis analysis of variance (test substance groups compared with negative control). Two Anova models were applied for both PE/200 E and MPE/2000 PE. In the first Anova model it was tested if the positive control differed from the negative control (t-test). In the second Anova model (including Dunnett’s test as post-hoc test) it was tested if the test material (different doses) differed from the negative control.

Results and discussion

Test results
no effects
Vehicle controls validity:
Positive controls validity:
Additional information on results:
MPE results (see Table 5 below)
The mean number of MPE/2000 PE in the negative control (group 1) was within the historical range. The mean number of MPE/2000 PE in the positive control group treated with mitomycin C (group 5) was within the historical positive control range and statistically significantly increased (p value: 0.0097) compared to the concurrent negative control (group 1). This indicates that the positive control substance mitomycin C reached the bone marrow and induced damage to the chromosomes and/or to the spindle apparatus of the bone marrow cells under the conditions of this study. These results, together with the normal MPE/PE ratio in the negative control group, demonstrate the validity of the test system.
The mean numbers of MPE/2000 PE in the groups exposed to the test material (groups 2-4) did not differ statistically significantly from the mean MPE/2000 PE in the negative control group (group 1). This indicates that treatment with the test material under the conditions of this study did not result in damage to the chromosomes and/or to the spindle apparatus of the bone marrow cells.

PE results (see Table 5 below)
Compared with the negative control group (group 1), positive controls treated with mitomycin C showed a statistically significant decrease (p value: 0.0001) in the number of PE/200 E, indicating that mitomycin C was cytotoxic to the bone marrow. The mean numbers of PE/200 E in the groups exposed to the test material did not differ statistically significantly from the mean PE/200 E in the negative control (group 1). This indicates that treatment with the test material under the conditions of this study did not result in cytotoxicity to the bone marrow.

Any other information on results incl. tables

Clinical signs and mortality

All animals survived until scheduled sacrifice. No treatment-related clinical signs were observed in animals exposed to the test material or treated with the positive control substance. The few signs observed were incidental findings unrelated to treatment.

Body weight

Mean body weights of animals exposed to the test material showed no biologically or statistically significant differences from controls.

Organ weights

The organ weight results for male animals showed the following statistically significant differences between animals exposed to the test material and controls:

- Higher relative liver weight at the high-concentration (relative difference from control 9%).

- Higher relative kidney weight at the high-concentration (relative difference from control 11%).

Applicant's summary and conclusion

Interpretation of results: negative
The results of this micronucleus test incorporated in a sub-chronic (13-week) toxicity study did not provide any indication of chromosomal damage or damage to the mitotic spindle apparatus of the bone marrow target cells of male rats exposed via inhalation to di-tert-butyl peroxide CAS# 110-05-4 for 6 hours/day, 5 days/week (total of 65 exposure days) at concentrations of 101 (± 3), 299 (± 3) or 993 (± 10) mg/m3 (mean actual concentrations ± standard deviation, determined by total carbon analysis). As treatment-related systemic effects were observed in male rats of the high-concentration group (increased weights of the liver and kidneys), there is no reason to assume that the negative bone marrow response was due to lack of systemic exposure.
Executive summary:

The purpose of this mammalian in vivo micronucleus test was to examine the potential of di-tert-butyl peroxide CAS# 110-05-4 to cause damage to the chromosomes and/or the mitotic apparatus of erythroblasts (micronuclei). This micronucleus test was part of a sub-chronic (13-week) inhalation toxicity study in which Wistar Hannover rats were exposed nose-only to target concentrations of 0 (control, clean air), 100, 300 and 1000 mg/m3 of the test material 6 hours/day, 5 days/week for 13 consecutive weeks (resulting in 65 exposure days in total).

The micronucleus test was conducted in accordance with the OECD Guideline for the Testing of Chemicals 474. Mammalian Erythrocyte Micronucleus Test, adopted 21st July 1997. At scheduled necropsy at the end of the 13-week study period, bone marrow cells of one of the femurs of five male rats per group (negative control, low, mid and high concentration) were collected, processed into smears and examined microscopically. The study included a positive control group of five male rats treated with the mutagen Mitomycin C (single intraperitoneal injection; 1.5 mg/kg body

weight) and sacrificed 24 hours after administration of the mutagen.

The target concentrations were accurately achieved as demonstrated by the results of total carbon analysis of the test atmospheres. The overall mean actual concentrations (± standard deviation of the daily mean concentration) were 101 (± 3), 299 (± 3) and 993 (± 10) mg/m3 for the low-, mid- and highconcentration level respectively.

Di-tert-butyl peroxide CAS# 110-05-4 did not adversely affect the general health, appearance or body weight development of the animals. Microscopic examination of bone marrow smears of male rats revealed no signs of toxicity to the bone marrow and no evidence of chromosomal damage and/or

damage to the mitotic apparatus of bone marrow erythrocytes. There was no reason to assume that the negative bone marrow response was due to lack of systemic exposure because treatment-related systemic effects (including increases in liver and kidney weight) occurred in male rats of the high-concentration group. Positive controls (five male rats treated with the mutagen Mitomycin C) showed the expected bone marrow response (cytotoxicity and increased number of micronucleated polychromatic erythrocytes).

Under the conditions of this study exposure to di-tert-butyl peroxide CAS# 110-05-4 did not induce chromosomal damage or damage to the mitotic apparatus of bone marrow erythrocytes of male rats.