Registration Dossier

Administrative data

Endpoint:
sub-chronic toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
November 2012 - March 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: This is a well documented study conducted according to modern standards of protocol, quality assurance, and good laboratory practices. The test material was >99 % pure.
Cross-reference
Reason / purpose:
reference to same study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report Date:
2013

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)
Deviations:
yes
Remarks:
A micronucleus assay was included
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
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

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
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. 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 in the range-finding study were 295 and 179 grams for male and female animals, respectively. Mean body weights at the start of treatment in the main study 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 three (range-finding study) or five (main study) 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. The duration of the acclimatization period to the conditions in the experimental room prior the first exposure was 16 days (range-finding study) or 10 (males) / 11 (females) days (main 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 the following exceptions. During the main study, the temperature was just below 20°C (minimum 19.8°C) for about half an hour on 22 January 2013.
The relative humidity exceeded 65% during occasional brief periods following cleaning activities. In addition, during the main study, the relative humidity was lower than 45% on three occasions: on 8 December 2012 for about half an hour due to technical malfunction (minimum 25.9%); on 10 January 2013 for about an hour due to maintenance (minimum 34.3%); and on 13 February 2013 for about five hours due to technical malfunction (minimum 20.2%).

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). Sentinel animals were housed similarly (individually in the range-finding study, in groups of four of the same sex in the main study). During exposure, the animals were kept individually in the exposure units. 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, in the main study, 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
Type of inhalation exposure:
nose/head only
Vehicle:
clean air
Remarks on MMAD:
MMAD / GSD: Not applicable, vapour test atmosphere
Details on inhalation 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. In both the rang-finding study and the main study the animals were placed at the top level. Empty ports were used for measurement of temperature and relative humidity (high concentration groups only). 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 of the main study 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: 22.0˚C on the first six days of the rangefinding study; occasionally 22.7 or 22.8˚C in the main study) 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).
In the range-finding study, the test atmospheres for group 2 (low-concentration, target 0.1 g/m3) and 3 (mid-concentration, target 1 g/m3) were obtained by diluting the high-concentration (group 4, target 10 g/m3). For this purpose, a mass flow controlled stream of the high-concentration test atmosphere was supplemented with a mass flow controlled stream of humidified compressed air via an eductor (Fox Eductor from Fox Valve Development Corp., Dover, NJ, USA). The generated test atmospheres were directed to the bottom inlets of the exposure units.
In the main study, 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 (range-finding and main study) 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 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 generation efficiency was calculated from the actual and the nominal concentration (efficiency = actual concentration as percentage of nominal concentration). In the range-finding study, the nominal concentration was calculated only for the high-concentration because the low- and mid-concentration were obtained by diluting the high-concentration.

The chamber airflow of the test atmospheres was recorded about hourly using a Rotameter or a mass flow controller.

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) or were (additionally) measured about hourly by means of a RH/T device (TESTO 635-1, TESTO GmbH & Co, Lenzkirch, Schwarzwald, Germany)
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
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.
Duration of treatment / exposure:
RF study: the animals were exposed on 5 days per week, 6 hours per day, over a 2-week study period (total of 10 exposure days), for the last time on the day before scheduled sacrifice.
Main study: the animals of groups 1-4 (control, low, mid and high concentration) were exposed on 5 days per week, 6 hours per day, over a 13-week study period (65 exposure days in total), for the last time on the day before scheduled sacrifice. The animals of the positive control group received a single intraperitoneal injection with Mitomycin C on the day prior to scheduled sacrifice.
Frequency of treatment:
5 days/week, 6 h/day
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
0, 100, 300 and 1000 mg/m3
Basis:
other: target concentrations
Remarks:
Doses / Concentrations:
0, 101, 299, and 993 mg/m3
Basis:
analytical conc.
No. of animals per sex per dose:
RF study: 5
Main study: 10
Control animals:
yes, concurrent vehicle
Details on study design:
Concentrations for the main study were based on the results of the RF study.

Shortly before initiation of exposure (range finding study: day -1; main 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 (range-finding study: one/sex; main study: four/sex). These animals were discarded at the end of the inlife phase of the study.
Positive control:
For the in vivo MN test (see also section 7.6.2) the animals of the positive control group received a single intraperitoneal injection with Mitomycin C on the day prior to scheduled sacrifice.

Examinations

Observations and examinations performed and frequency:
Clinical signs
On exposure days, each animal was observed daily in the morning, prior to exposure, by cage-side observations and, if necessary, handled to detect signs of toxicity. All animals were thoroughly checked again after exposure. During exposure in the main study, a group-wise observation was made about halfway the 6-hour exposure period. During exposure in the range-finding study, observations were made more frequently on account of abnormalities noted in the groups exposed to the test material (tail trembling and restlessness). During weekends and on public holidays only one check per day was carried out. All abnormalities, signs of ill health, and reactions to treatment were recorded.

Ophthalmoscopic examination
In the main study, ophthalmoscopic observations were made prior to the start of the exposure in all rats (on day -6) and in the last week of the exposure period in all rats of the control group and the high-concentration group (on day 92). Eye examination was carried out using an ophthalmoscope after induction of mydriasis by a solution of atropine sulphate.

Body weight
Range-finding study
The body weight of each animal was recorded once before the start of exposure (these pre-test weights served as a basis for animal allocation, once prior to exposure on the first exposure day (day 0) and twice weekly thereafter, for the last time on the day of scheduled sacrifice.
Main study
The body weight of each animal was recorded once before the start of exposure. These pre-test weights served as a basis for animal allocation. Animals were weighed prior to exposure on the first exposure day (day 0), twice weekly for the first four weeks of exposure (Mondays and Fridays), then once weekly from the fifth week onward. At the end of the in-life phase, animals were weighed the day before overnight fasting prior to scheduled sacrifice. They were weighed again on the day of sacrifice; this was the terminal body weight used for calculation of organ weights relative to body weight.

Food consumption
Food consumption was measured per cage by weighing the feeders. In the rangefinding study, food consumption was measured over two 7-day periods, starting on day 0. In the main study, consumption was measured from day 0, over successive 7-day periods. The results were expressed in g per animal per day.

Haematology
In the main study, haematology was conducted at the end of the treatment period on all animals. Blood samples were taken from the abdominal aorta of overnight fasted rats (water was freely available) whilst under pentobarbital anaesthesia at sacrifice. Potassium-EDTA was used as anticoagulant. In each sample the following determinations were carried out: red blood cells (RBC), haemoglobin (Hb), packed cell volume (PCV), reticulocytes, total white blood cells (WBC), differential white blood cells, prothrombin time (PT), thrombocytes.
The following parameters were calculated: mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), mean corpuscular haemoglobin concentration (MCHC).

Clinical chemistry
In the main study, clinical chemistry was conducted at the end of the treatment period on all animals, after overnight fasting, at the same time blood samples for haematology were collected. Blood was collected from the abdominal aorta in heparinized plastic tubes. Plasma was prepared by centrifugation. The following measurements were made in the plasma: alkaline phosphatase activity (ALP), cholesterol (total), aspartate aminotransferase activity (ASAT), phospholipids, alanine aminotransferase activity (ALAT), triglycerides, gamma glutamyl transferase activity (GGT), creatinine, bilirubin (total), urea, total protein, inorganic phosphate (PO4), albumin, calcium (Ca), ratio albumin to globulin (calculated), chloride (Cl), glucose, potassium (K), sodium (Na).
Sacrifice and pathology:
Pathology
Range-finding study
Macroscopic examination
At the end of the exposure period, the animals were sacrificed in such a sequence that the average time of killing was approximately the same for each group. The animals were sacrificed by exsanguination from the abdominal aorta under pentobarbital anaesthesia and then examined grossly for pathological changes.

Organ weights
At scheduled sacrifice, the following organs of all animals were weighed (paired organs together) as soon as possible after dissection to avoid drying and the relative organ weights (g/kg body weight) were calculated from the absolute organ weights and the terminal body weight: adrenals, heart, kidneys, liver, lung with trachea and larynx, spleen, testes (after weighing, the lung was infused with the fixative).

Tissue preservation
The organs listed above and the complete respiratory tract of all animals were preserved with a 10% solution of Formalin in a neutral aqueous phosphate buffer (final formaldehyde concentration 4 per cent).

Histopathological examination
The main lung lobe (one level), the larynx (three levels, including the base of the epiglottis) and the nasal tissues (levels 3 and 5, as described by Woutersen et al., 1994) of all male animals were examined microscopically. Tissue for microscopy was embedded in paraffin wax, sectioned at 5 μm and stained with haematoxylin and eosin.

Main study
Gross necropsy
On study day 93 (13 and 14 March 2013 for males and females, respectively), animals were fasted overnight but allowed free access to water. The next day (study day 94; 14 and 15 March 2013 for males and females, respectively), the animals were killed in such a sequence that the average time of killing was approximately the same for each group. The animals were anaesthetized by intraperitoneal injection of sodium pentobarbital then killed by exsanguination from the abdominal aorta and examined grossly for pathological changes.

Organ weights
At scheduled necropsy, the following organs were weighed (paired organs together) as soon as possible after dissection to avoid drying and the relative organ weights (g/kg body weight) were calculated from the absolute organ weights and the terminal body weight: adrenals, lung with trachea and larynx, brain, ovaries, epididymides, spleen, heart, testes, kidneys, thymus, liver, thyroid, uterus (after weighing, the lung was infused with the fixative).

Tissue preservation
Samples of the tissues and organs listed below of all animals were preserved in a neutral aqueous phosphate-buffered 4% solution of formaldehyde (10% solution of formalin). The carcass containing any remaining tissues was retained in formalin until completion of the histopathological examination and then discarded. The following were preserved, all gross lesions, ovaries, adrenals, pancreas, aorta, parathyroids, axillary lymph nodes, parotid salivary glands, brain, pharynx, caecum, pituitary, colon, prostate, duodenum, rectum, epididymides, seminal vesicles / coagulating glands, exorbital lachrymal glands, skeletal muscle (thigh), eyes (with optic nerve), skin (flank), femur with joint, spinal cord, NALT (nose associatedlymphoid tissue, spleen, sternum with bone marrow, heart, stomach, ileum, sublingual salivary glands, jejunum, submaxillary salivary glands, kidneys, testes, larynx, thymus, liver, thyroid, lung, tongue, mammary gland (female), trachea, mandibular (cervical) lymph nodes, tracheobronchial (mediastinal) lymph nodes, nasopharyngeal tissues (with teeth), ureter, nerve-peripheral (sciatic), urethra, oesophagus, urinary bladder, olfactory bulb, uterus (with cervix).
- For the micronucleus test, bone marrow cells of one of the femurs (left femur) were collected from the first five male rats of each group
- Brain: Three levels were examined microscopically (brain stem, cerebrum, cerebellum).
- Larynx: Three levels (one including the base of the epiglottis) were examined microscopically.
- Lung: Each lung lobe was examined microscopically at one level, including main bronchi.
- Nasopharyngeal tissues: Six levels (Woutersen et al., 1994) were examined microscopically (one including the nasopharyngeal duct and the draining lymphatic tissue [NALT]).
- Spinal cord: Retained in vertebral column, at least three levels were examined microscopically (cervical, mid-thoracic and lumbar).
- Stomach: Non-glandular (‘forestomach’) and glandular (fundus, pylorus) parts were examined microscopically.
- Trachea: Three transverse sections and three longitudinal sections (at least one through the carina of the bifurcation of the bronchi) were examined microscopically.

Histopathological examination
The tissues for microscopic examination were embedded in paraffin wax, sectioned at 5 μm and stained with haematoxylin and eosin. Histopathological examination (by light microscopy) was performed on all tissues and organs listed above of all animals of the control group and the high-concentration group. In addition, all gross lesions were examined in the low- and mid-concentration groups.
Other examinations:
Micronucleus test (see also section 7.6.2)
In the main study, five male animals per group (negative controls, males treated with test material and positive controls) were used for the micronucleus test (from groups 1-4 the animals with the lowest animal identification numbers were used, i.e. those of the first cage of each group).
Statistics:
Body weight data collected after initiation of treatment:
‘AnCova & Dunnett’s Test’ (abbreviation ANCDUN) with ‘Automatic’ data transformation method (abbreviation AUTO). Day 0 body weight data were used as
covariate in the analysis of the post-treatment data unless removed during data preprocessing. The ANCDUN (AUTO) is an automatic decision tree consisting of: (1) Data preprocessing tests, (2) A group test assessing whether or not group means were all equal (one-way analysis of covariance [Ancova], or one-way analysis of variance [Anova] if the covariate is removed), (3) Post-hoc analysis. If the group test showed significant (p<0.05) nonhomogeneity of group means, pairwise comparisons with the control group were conducted by Dunnett’s multiple comparison test.

Pre-treatment body weight, haematology, clinical chemistry and organ weight data: ‘Generalised Anova/Ancova Test’ (abbreviation GEN AN) with ‘Automatic’ data transformation method (abbreviation AUTO). This test is an automatic decision tree consisting of: (1) Data pre-processing tests, (2) A group test assessing whether or not group means were all equal (parametric for untransformed or log-transformed data: one-way analysis of variance [Anova]; non-parametric for rank transformed data: Kruskal-Wallis test), (3) Post-hoc analysis. If the group test showed significant (p<0.05) nonhomogeneity of group means, pairwise comparisons with the control group were conducted by Dunnett’s multiple comparison test (parametric after Anova, non-parametric after Kruskal-Wallis

Food consumption data main study: Dunnett’s multiple comparison test.
Food consumption data range-finding study: no comparative statistics (one cage per sex per group).

Incidences of histopathological changes: Fisher’s exact probability test.

Tests were performed as two-sided tests with results taken as significant where the probability of the results is <0.05 or <0.01.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
no effects observed
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Haematological findings:
no effects observed
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Details on results:
Clinical signs
All animals survived until scheduled sacrifice. There were no treatment-related clinical signs. The few signs observed were incidental findings unrelated to treatment. No abnormalities were seen at the group-wise observations made about halfway each 6-hour exposure period.

Ophthalmoscopic examination
Ophthalmoscopic examination did not reveal any treatment-related abnormalities.

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

Food consumption
Food consumption was not affected by the exposure to the test material.

Haematology
Red blood cell and coagulation values and total and differential white blood cell values showed no treatment-related changes.

Clinical chemistry
Compared to controls, animals exposed to the test material showed the following statistically significant differences:
- Higher cholesterol in high-concentration group males
- Lower creatinine in high-concentration group females

Organ weights
The organ weight results showed the following statistically significant differences between animals exposed to the test material and controls:
- Higher relative liver weight at the high-concentration in males (relative difference from control 9%). Mean relative liver weight in females of the high-
concentration group was also higher than in controls (9%) but the difference was not statistically significant.
- Higher relative kidney weight at the high-concentration in males (relative difference from control 11%).
- Higher thyroid weights in males (higher absolute weight at the midconcentration, higher relative weight at the low- and high-concentration). In the
absence of a concentration-related response, these differences in thyroid weights were judged to be chance findings.
- Lower absolute lung weight at the mid-concentration in females. This difference was considered to be a chance finding because there was no concentration-related response.

Pathology
Macroscopic examination
There were no macroscopic findings attributable to the exposure to the test material. The few gross changes observed represented background pathology in rats of this strain and age and occurred only incidentally.
Microscopic examination
Microscopic examination did not reveal treatment-related histopathological changes. The histopathological changes observed in the high-concentration group were considered unremarkable because they represented background findings and occurred in only one or two animals or at about the same incidence in the highconcentration group and the control group.

Effect levels

Dose descriptor:
NOAEC
Effect level:
993 mg/m³ air
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: At this level only a few modest changes (increases in liver and kidney weight and altered plasma levels of cholesterol and creatinine); these changes were considered not to constitute adverse effects

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

Table – Clinical chemistry at the end of the exposure period (mean value±SD):

 

Males

Cholesterol (mmol/L)

Creatinine (μmol/L)

0 mg/m3

1.544±0.205

33.6±3.4

100 mg/m3

1.847±0.340

34.5±4.0

300 mg/m3

1.629±0.287

31.4±5.3

1000 mg/m3

1.926±0.312*

29.8±3.8

N=10; * p<0.05

 

Females

Cholesterol (mmol/L)

Creatinine (μmol/L)

0 mg/m3

1.248±0.397

37.7±5.3

100 mg/m3

1.096±0.216

35.6±3.5

300 mg/m3

1.395±0.280

35.8±4.0

1000 mg/m3

1.483±0.424

32.3±3.5*

N=10; * p<0.05

 

Table – Absolute and relative organ weights (mean value±SD):

 

Males

Liver (g)

Liver (g/kg)

Kidneys (g)

Kidneys (g/kg)

0 mg/m3

7.756±0.541

21.50±1.30

1.920±0.070

5.326±0.228

100 mg/m3

7.791±0.618

21.75±1.59

1.992±0.138

5.572±0.501

300 mg/m3

8.240±0.762

21.93±1.14

2.102±0.183

5.592±0.158

1000 mg/m3

8.449±1.052

23.51±1.88*

2.118±0.258

5.891±0.383**

N=10; * p<0.05; ** p<0.01

 

 

Females

Liver (g)

Liver (g/kg)

Kidneys (g)

Kidneys (g/kg)

0 mg/m3

5.064±0.492

24.19±2.15

1.336±0.105

6.378±0.411

100 mg/m3

5.189±0.363

24.97±1.52

1.335±0.069

6.429±0.358

300 mg/m3

5.037±0.470

24.60±2.54

1.284±0.049

6.268±0.276

1000 mg/m3

5.440±0.551

26.45±1.88

1.355±0.066

6.600±0.256

N=10

 

Applicant's summary and conclusion

Conclusions:
Exposure to di-tert-butyl peroxide CAS# 110-05-4 resulted in a few modest changes at the highest concentration tested (increases in liver and kidney weight and altered plasma levels of cholesterol and creatinine). No treatment-related changes were observed at the lower concentrations. Since the changes at the high-concentration were considered not to constitute adverse effects, this exposure level (993 mg/m3 actual concentration) was a No-Observed-Adverse-Effect Concentration (NOAEC).
Executive summary:

The toxicity of di-tert-butyl peroxide CAS# 110-05-4 upon repeated exposure by inhalation was studied in a sub-chronic (90-day) study with Wistar Hannover rats. The study included a micronucleus test. The target concentrations for this study (100, 300 and 1000 mg/m3 as low-, mid- and high-concentration, respectively) were selected on the basis of a 14-day range-finding study in which groups of five male and five female Wistar Hannover rats were exposed to target concentrations of 100, 1000, and 10,000 mg/m3 for 6 hours/day, 5 days/week. In this 14-day study treatment-related findings at 10,000 mg/m3 consisted of signs of discomfort during exposure in rats of both sexes (trembling of the tail, restlessness), slightly retarded growth in males (mainly on exposure days), and increased relative weights of the liver in both sexes and of the adrenals and kidneys in males. Trembling of the tail and restlessness during exposure were also observed at the lower exposure levels but to a lesser extent than at the high exposure level. The only other treatment-related change at the lower exposure levels was a slight increase in relative kidney weight in males of the mid-concentration group.

The sub-chronic (main) study included four groups of 10 rats/sex. The animals were exposed nose-only, 6 hours/day, 5 days/week for 13 consecutive weeks (resulting in 65 exposure days in total) to the above target concentrations or to clean air for the control group. Endpoints to assess toxicity included clinical and ophthalmoscopic observations, growth, food consumption, haematology, clinical chemistry and organ weights. In addition, the animals were examined grossly at necropsy, and a large number of organs and tissues were examined microscopically. Five male rats of each group were used to examine possible damage to the chromosomes and/or mitotic apparatus in bone marrow cells collected at scheduled sacrifice.

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.

All animals survived until scheduled sacrifice. Clinical and ophthalmoscopic observations, growth and food consumption results, haematology values, most clinical chemistry values, most organ weights, and necropsy and histopathology findings showed no treatment-related changes.

Clinical chemistry values showed slight but statistically significant changes in the plasma levels of cholesterol (increased) and creatinine (decreased) at the high concentration in male and female rats, respectively. These findings were considered to be of no toxicological significance.

The relative weights of the liver and kidneys were slightly (about 10%) but statistically significantly increased in male rats of the high-concentration group. In female rats of this group relative liver weight was increased to about the same extent but the difference from controls was not statistically significant. Though these organ weight changes were related to treatment, they were considered not to represent adverse effects of the test material because of the modest magnitude of the increases and the absence of corroborative histopathological alterations or clinical chemical indicators of organ damage.

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 resulted in a few modest changes at the highest concentration tested (increases in liver and kidney weight and altered plasma levels of cholesterol and creatinine). No treatment-related changes were observed at the lower concentrations. Since the changes at the high-concentration were considered not to constitute adverse effects, this exposure level (993 mg/m3 actual concentration) was a No-Observed- Adverse-Effect Concentration (NOAEC).

The micronucleus test incorporated in this study revealed no chromosomal damage and/or damage to the mitotic apparatus of bone marrow erythrocytes.