Registration Dossier

Administrative data

Description of key information

Two repeated dose toxicity studies are available for the target substance. In a subactue 28-day study in Wistar rats (OECD 407), the NOAEL was determined to be 300 mg/kg bw/day. In a subchronic 90 -day repeated dose study (OECD 408) in Wistar rats, a NOAEL of 1000 mg/kg bw/day was established for females, while a LOAEL of 100 mg/kg bw/day was determined for males due to tubular cell necrosis.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Referenceopen allclose all

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2012-04-11 to 2012-11-13
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study
Qualifier:
according to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River, 97633 Sulzfeld, Germany
- Age at study initiation: 7-8 weeks
- Weight at study initiation: males: 172 - 200 g, females: 129 - 147 g
- Housing: individually in IVC cages, type III H, polysulphone cages on Altromin saw fibre bedding
- Diet: ad libitum, Altromin 1324
- Water: ad libitum, tap water
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 55 ± 10
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: gavage
Vehicle:
paraffin oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS: the test item formulations were prepared daily, just before the administration to the animals. The test item was weighed into a tarred plastic vial on a precision balance.

DIET PREPARATION
- Rate of preparation of diet (frequency): not relevant; test item given by gavage and through the feed
- Mixing appropriate amounts with (Type of food):
- Storage temperature of food:

VEHICLE
- Justification for use and choice of vehicle (if other than water): based on the test item's characterisitcs
- Lot/batch no. (if required): 5098101
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
For the determination of concentrations, samples were analysed from all dose groups every week. Stability was tested in the start of treatment period (0hr) and 6 hr thereof. Homogeneity was also checked during the 1st week.
Duration of treatment / exposure:
28 days
Frequency of treatment:
daily
Remarks:
Doses / Concentrations:
0, 100, 300, 1000 mg/kg bw/day
Basis:
actual ingested
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: based on a dose range finding study
Application volume of 3 mL/kg bw
Dose groups referred to as: 100 (LD), 300 (MD), and 1000 (HD) mg/kg bw/day
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least once a day throught the whole treatment period

DETAILED CLINICAL OBSERVATIONS: Yes (spontaneous activity, lethargy, recumbent position, convulsions, tremors, apnoea, asphyxia, vocalisation, diarrhoea, changes in skin and fur, eyes and mucous membranes- salivation, discharge-, piloerection and pupil size)
- Time schedule: once before the first administration and at least once per week during treatment

BODY WEIGHT: Yes
- Time schedule for examinations: once before treatment, 1st day of treatment, and weekly during the treatment period

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal was measured weekly during the treatment period.

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No data

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No data

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule: before the first administration and in the last week of treatment

HAEMATOLOGY: Yes
- Time schedule for collection of blood: one day after the last administration
- Anaesthetic used for blood collection: No data
- Animals fasted: Yes
- How many animals: all
- Parameters examined: haematocrit value (Hct), haemoglobin content (Hb), red blood cell count (RBC), mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), mean corpuscular haemoglobin concentration (MCHC), reticulocytes (Re), platelet count (PLT), white blood cells (WBC), neutrophils (Neu), lymphocytes (Lym), monocytes (Mono), eosinophils (Eos), basophils (Baso)
Coagulation parameters checked: prothrobine time, activated partial thromboplastin time

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: one day after the last administration
- Animals fasted: Yes
- How many animals: all
- Parameters examined: alanine aminotransferase (ALAT), aspartate-aminotransferase (ASAT), alkaline phosphatase (AP), creatinine (Crea), total protein (TP), albumin (Alb), urea, total bilirubin (TBIL), total bile acids (TBA), total cholesterol (Chol), glucose (Gluc), sodium (Na), potassium (K)

URINALYSIS: Yes
- Time schedule for collection of urine: prior to or as part of the sacrifice
- Metabolism cages used for collection of urine: No data
- Animals fasted: Yes
- Parameters examined: specific gravity, nitrite, pH-value (pH), protein, glucose, ketone bodies (Ket), urobilinogen (UBG), bilirubin (BIL), erythroctes (Ery), leukocytes (Leu)

NEUROBEHAVIOURAL EXAMINATION: Yes (functional observational battery)
- Time schedule for examinations: before exposure and in the 4th treatment week
- Dose groups that were examined: all
- Battery of functions tested: sensory activity, grip strength, motor activity
Sacrifice and pathology:
GROSS PATHOLOGY: All animals (including control) were sacrifised one day after the last treatment, and a gross necropsy was performed.

Few specific gross pathological changes were recorded for the male and female animals during necropsy. Male: LD- yellow spots on jejunum/ileum (1/5 animals); enlarged thyroid/parathyroid glands (1/5 animals). MD- pale liver (3/5 animals); dark spots on spleen (1/5 animals); enlarged and pale kidneys (1/5 animals). HD- pale liver (5/5 animals); small testis (left); discoloured jejunum (ileum (1/5 animals). Female: control- cyst on oviduct (2mm) (1/5 animals). LD- fluid distended uterus with oviduct and cervix (1/5 animals), slight fluid distended uterus with oviduct and cervix (1/5 animals), small cyst on pituitary. MD- pale liver (1/5 animals). HD- pale liver (2/5 animals).
The macroscopic liver change in both male and female animals correlated with histopathological changes and findings were related to treatment.
Organ weight changes were noted for liver, kidney, thymus and prostrate (including seminal vesicle and coagulating glands) weight in males and for liver and thymus weight in females in MD or HD groups, which in the wake of microscopic changes were considered to be related to treatment. In the absence of significant changes in clinical biochemistry parameters in MD group animals, the histological changes in liver of MD group was assumed to be due to an adaptive response of liver. Hence, the increased liver weight in MD group was related to treatment with no adversity.

HISTOPATHOLOGY: A full histopathological evaluation of the tissues was performed on high dose and control animals. Organs showing gross alterations and liver and kidneys of the medium and low dose groups were also examined histopathologically.

Histopathological evaluation revealed test item-related findings in the liver, kidney, seminal vesicle, coagulating gland and thymus. In the liver, minimal or mild diffuse hepatocellular hypertrophy was noted in a dose-related manner in both sexes of MD and HD groups. Diffuse hepatocellular microvacuolation was observed in the same groups and in one female of LD group, but was not dose related. However, in the absence of significant changes in clinical biochemistry parameters in MD group, the changes in MD group was not considered adverse. In addition, in males of HD group, a minimal degree of hepatocellular single cell death (mainly in the centrilobular zones of hypertrophy) was seen.
In the kidney of males only, hyaline droplets in corticotubular cells were seen in a dose related manner in LD, MD and HD groups. These were considered to represent α2-microglobulin. They were associated with multifocal debris-filled tubules in the inner cortex and with diffuse dilation of medullary tubules, considered to be most likely the consequence of excretion of degenerated and exfoliated corticoepithelial cells. Furthermore, in MD and HD group males, minimal multifocal corticotubular degeneration was also observed. As the renal storage of α2-microglobulin is commonly recognized to be a male rat-specific event, its test item-related increase in severity and associated secondary renal changes in this study are regarded to be of no relevance for man.
In HD group, in the seminal vesicle and coagulating gland, the incidence of epithelial single cell death was increased, and in the thymus, in both sexes and confirming minor weight decreases, a minimal tendency towards more prominent atrophy/regression was noted.
Statistics:
A statistical assessment of the results of the body weight, food consumption, parameters of haematology, blood coagulation and clinical biochemistry and absolute and relative organ weights were performed for each gender by comparing values of dosed with control animals of the main groups using a one-way ANOVA and a post-hoc Dunnett Test. These statistics were performed with GraphPad Prism 5.01 software (p<0.05 was considered as statistically significant).
Clinical signs:
no effects observed
Description (incidence and severity):
no mortality and no treatment related clinical effects
Mortality:
no mortality observed
Description (incidence):
no mortality and no treatment related clinical effects
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
decrease on body weight gain in males
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
decreased of food consumption (males)
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Haematological findings:
no effects observed
Description (incidence and severity):
changes in MCV were not considered treatment related
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
changes in several parameters (see below)
Urinalysis findings:
effects observed, treatment-related
Description (incidence and severity):
changes in several parameters (see below)
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
no effects observed
Details on results:
CLINICAL SIGNS AND MORTALITY: no mortality was observed throught the treatment period. Alopecia and mild salivation seen in some animals was not considered related to the administration of the test item.

BODY WEIGHT AND WEIGHT GAIN: there was a statistically significant decrease in body weight gain in male animals treated at 1000 mg.kg bw; this was not observed in females.

FOOD CONSUMPTION: a slight decrease in food intake was seen in males and females treated at 300 and 1000 mg/kg bw, (MD and HD, respectively).

OPHTHALMOSCOPIC EXAMINATION: no effects

HAEMATOLOGY: all parameters were found to be within the normal range for both sexes, except for mean MCV, which appeared elevated in male LD group. No dose-dependent pattern was observed. The finding was not considered a substance specific toxic effect.
Mean prothrombin time was increased in the male HD group.

CLINICAL CHEMISTRY: the parameteres mean ASAT, CREA and UREA were significantly elevated, while mean TP, CHOL and GLU were significantly decreased, in the male HD group. Statistically significant decrease in mean TBA in male LD, MD and HD groups when compared to the corresponding controls was also observed. The individual and mean TBA values were within the historical control range and in addition the values showed high heterogeneity. Hence, these changes were not considered adverse.

In females, there was a slight increase in mean AP value in MD and HD groups; substantial decrease in mean TBA value in LD group. The individual animal values of TBA and AP were within the normal range. The change in TBA value was not attributed to toxicity.

URINALYSIS: in males, the high level of erythrocytes was noted in LD (1/5 animals) and HD (4/5 animals); high level of protein in Control (1/5 animals), MD (4/5 animals) and HD (4/5 animals); high level of ketone in HD (1/5 animals); low urine pH in Control (3/5 animals), LD (3/5 animals), MD (5/5 animals), HD (5/5 animals); the slightly increased specific gravity in MD and HD group animals (see table 43 in the attached document).

The alterations were related to the histopathological findings on the kidneys and liver.

In females, no treatment related changes were detected.

NEUROBEHAVIOUR: no effects

ORGAN WEIGHTS: statistically significant changes were seen in several organ weights (absolute and relative) of males and females (for details see tables 23, 24, 25, 26, 27, 28 in the attachment). Treatment related changes were considered for liver, kidney, thymus and prostrate (including seminal vesicle and coagulating glands) weight in males and for liver and thymus weight in females in MD or HD groups.

GROSS PATHOLOGY: a gross pathological change related to the administration of the test item was pale liver seen in male and females MD and HD groups.

HISTOPATHOLOGY: histopathological changes were detected in the liver, kidney, seminal vesicle, coagulating gland and thymus. A dose-related diffuse hepatocellular hypertrophy was seen in males and females of MD and HD groups. Diffuse hepatocellular microvacuolation was observed in the same groups and in one female of LD group, but was not dose-related. In males of HD group, a minimal degree of hepatocellular single cell death (mainly in the centrilobular zones of hypertrophy) was seen. Hyaline droplets in the renal corticotubular cells of males (alpha 2u-globulin accumulation), was considered to be a rat-specific effect, with no relevance for humans. Single cell death of the epithelium in the seminal vesicle and coagulating gland was seen in the male HD group. The thymus showed a minimal tendency towards a more prominent atrophy/ regression in the HD group of both sexes.
Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: see 'Remark'
Dose descriptor:
LOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: histopathology: microscopic changes in the liver, kidney, seminal vesicle, coagulating gland and thymus
Critical effects observed:
not specified

**The hyaline droplets observed in the corticotubular renal cells of the kidneys in male treated animals (at all dose levels), represented the accumulation of the male rat-specific protein, alpha 2µ-globulin, in the renal tubules.Chemicals that can bind to alpha 2µ-globulin interfere with intra-renal degradation of the protein by lysosomal hydrolases, causing lysosomal accumulation of alpha 2µ-globulin, which is visible as hyaline droplets (hyaline droplet nephropathy or hyaline droplet accumulation).Thisis a male rat specific effect, and it does not have any relevance to humans, since the protein is synthesized predominantly in the liver of the male rat.

Conclusions:
Under the conditions of this study, the dose of 300 mg/kg/day was considered to be the NOAEL.
Executive summary:

In this repeat-dose oral toxicity study, bis(3,5,5-trimethylhexanoyl)peroxide) was administered by gavage to male and female Wistar rats (5 animals/dose/sex) at dose levels of 0, 100, 300, 1000 mg/kg bw/day, for 7 days/week during a 28-day period. No mortality was observed. Clinical symptoms, such as mild salivation (male MD group) and alopecia (female HD group) were assumed to be incidental. In males, slight change in body weight gain was recorded in HD group during treatment period, which corroborated with food intake during the corresponding days of body weight. In females, no such changes were noted. No changes in the haematological parameters were associated with the administration of the test material. In males of the HD group, statistically significant alterations were recorded in several urine parameters. Statistically significant decrease in mean TBA was seen in all dose groups. Such changes were within the normal ranges for historical controls. In females, there was slight increase in mean AP value in MD and HD groups. Such changes were attributed to the histopathological changes seen in the liver and kidney. Histopathological findings considered to be test item-related were seen in the liver, kidney, seminal vesicle, coagulating gland and thymus. The kidney changes were considered to be of no toxicological relevance for humans. The NOAEL of the study was set at 300 mg/kg bw/d based on microscopic changes in the liver, kidney, seminal vesicle, coagulating gland and thymus. This subacute toxicity study in the rat is acceptable and satisfies the guideline requirement for a subacute oral study (OECD 407) in rat.

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2015-08-07 to 2017-12-04
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
GLP compliance:
yes (incl. certificate)
Limit test:
no
Specific details on test material used for the study:
- Name: Bis(3,5,5-trimethylhexanoyl)peroxide
- CAS No.: 3851-87-4
- Chemical Name: Bis(3,5,5-trimethylhexanoyl)peroxide
- Batch No.: 1198394-01
- Physical State: liquid
- Colour: colourless
- Density: 0.87 g/cm³
- Purity: 75%
- Solvent: 20-25% Isododecan, CAS No. 93685-81-5
- Storage Conditions: -8 to 0 °C, protected from light
- Expiry Date: 30 January 2016
- Safety Precautions: For safety reason special handling is required. The test item was kept at temperatures of -8 to 0 °C continuously. Self-accelerating decomposition temperature: 20 °C. Prior to handling personnel was trained regarding emergency plan.
Species:
rat
Strain:
Wistar
Details on species / strain selection:
healthy Wistar rats, Crl: WI(Han) (Full Barrier)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River, 97633 Sulzfeld, Germany
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: approx. 7-8 weeks old
- Weight at study initiation: males: 148 – 169 g
(mean: 159.83 g, ± 20 % = 127.86 – 191.79 g)
females: 127 – 145 g
(mean: 138.40 g, ± 20 % = 110.72 – 166.08 g)
- Housing: Full barrier in an air-conditioned room; the animals were kept in groups of 5 animals / sex / group / cage in IVC cages (type IV, polysulphone cages) on Altromin saw fibre bedding (lot no. 02102150411)
- Diet (e.g. ad libitum): ad libitum, Altromin 1324 maintenance diet for rats and mice (lot no. 050815/1239)
- Water (e.g. ad libitum): ad libitum; tap water, sulphur acidified to a pH of approximately 2.8 (drinking water, municipal residue control, microbiological controls at regular intervals)
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 °C
- Humidity (%): 55 ± 10 %
- Air changes (per hr): 10 x / hour
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: gavage
Details on route of administration:
The test item formulation or vehicle was administered at a single dose to the animals by oral gavage. The application volume for these groups was 3 mL/kg body weight. In control group C1 corn oil was administered at a single dose to the animals by oral gavage using an application volume of 5 mL/kg bw. For each animal the individual dosing volume was calculated on the basis of the body weight most recently measured.
Vehicle:
paraffin oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
The test item formulations were prepared freshly on each administration day before the administration procedure. The time of preparation and time of dosing was recorded for all dosing formulations. For preparation, the test item was weighed into a tarred plastic vial on a precision balance. The test item formulations were prepared at room temperature by adding the required volume of vehicle and vortexing it. Corn oil was additionally used as control item. Formulations or control items were stored at 2 to 8 °C or crushed ice and were administered within 6 hours after preparation (see stability data in study 114850). Prior to administration test item formulations were brought to room temperature.

VEHICLE (Group C2)
- Justification for use and choice of vehicle (if other than water): The vehicle and control item were selected as suggested by the sponsor based on the test item’s characteristics and testing guideline.
- Name: paraffinum perliquidum
- Manufacturer: AppliChem Panreac
- Batch No.: 000055878
- Expiry Date: 17/09/2015

- Amount of vehicle (if gavage): 3 mL/kg body weight

Additional control group C1: corn oil
- Manufacturer: Sigma
- Batch No.: MKBP7039V
- Expiry Date: 18 December 2015

- Amount of vehicle (if gavage): 5 mL/kg body weight
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
For determination of the concentration of test item in dosing formulations, samples of approx. 8 mL of HD, 12 mL of MD and 35 mL of LD were retained from all groups once in week 1, 5, 9 and 13 of the treatment period (in total 16 samples). Sample analysis was performed on the same day. In the 1st, 5th and 13th week of treatment, samples for the testing of homogeneity were taken (volume as above mentioned) from the top, middle and bottom of the freshly prepared high and low dose formulations and analysed on the same day (in total 18 samples). Each sample was retained only once (sample A). All samples of dosing formulations were analysed at Eurofins Munich in accordance with GLP under the reference number 151793.
Duration of treatment / exposure:
90 days
Frequency of treatment:
once per day, 7 days/week
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
Corn oil (control C1)
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
Parrafin oil (control/vehicle C2)
Dose / conc.:
100 mg/kg bw/day (nominal)
Remarks:
Test substance (LD)
Dose / conc.:
300 mg/kg bw/day (nominal)
Remarks:
Test substance (MD)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Remarks:
Test substance (HD)
No. of animals per sex per dose:
10 animals/sex/dose group
Control animals:
yes
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
- Rationale for animal assignment (if not random): Prior to the start of the treatment period a detailed clinical observation outside the home cage was made. No animals showed pathological signs before the first administration. Before the first administration all animals used for the study were weighed and assigned to the experimental groups with achieving a most homogenous variation in body weight throughout the groups of males and females, respectively (randomisation was performed with IDBS Workbook 9.4.0 software).

- Control groups: Two control groups were used in this study. C2, the vehicle control group was administered daily with paraffinum perliquidum. To control for possible vehicle-related effects a second control group was used. C1 was a control group treated daily with corn oil. This control group was shared from study no. 154433.
Positive control:
not applicable
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
All animals were observed for clinical signs during the entire treatment period of 90 days. General clinical observations were made at least once a day, preferably at the same time each day and considering the peak period of anticipated effects after dosing. The health condition of the animals was recorded. Twice daily all animals were observed for morbidity and mortality except on weekends and public holidays when observations were made once daily.

DETAILED CLINICAL OBSERVATIONS: Yes
Detailed cage side observations considering spontaneous activity, lethargy, recumbent position, convulsions, tremors, apnoea, asphyxia, vocalisation, diarrhoea, changes in skin and fur, eyes and mucous membranes (salivation, discharge), piloerection and pupil size were made outside the home cage in a standard arena once before the first administration and at least once a week thereafter.

BODY WEIGHT: Yes
The body weight was recorded once before the assignment to the experimental groups, on the first day of administration and weekly during the treatment period.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
Food consumption was measured weekly during the treatment period.

OPHTHALMOSCOPIC EXAMINATION: Yes
Ophthalmological examination, using an ophthalmoscope was made on all animals before the first administration and in the last week of the treatment period

HAEMATOLOGY: Yes
Time schedule for collection of blood: Haematological parameters were examined at the end of the treatment prior to or as part of the sacrifice of the animals
- Animals fasted: Yes
- How many animals: all animals
- Parameters checked were: haematocrit value (Hct), haemoglobin content (Hb), red blood cell count (RBC), mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), mean corpuscular haemoglobin concentration (MCHC), reticulocytes (Re), platelet count (PLT), white blood cells (WBC), neutrophils (Neu), lymphocytes (Lym), monocytes (Mono), eosinophils (Eos), basophils (Baso), large unstained cells (Luc).
- Coagulation parameters were examined at the end of the treatment prior to or as part of the sacrifice of the animals: prothrombin time (PT), activated partial thromboplastin time (aPTT)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Parameters of clinical biochemistry were examined at the end of the treatment prior to or as part of the sacrifice of the animals. After overnight fasting, blood from the abdominal aorta of the animals was collected in serum separator tubes. Additionally, at necropsy serum samples of all animals were retained at the end of the treatment and recovery period and stored at -20 °C for an evaluation of test item-related effects on the pituitary-thyroid axis and thyroid hormones.
- Animals fasted: Yes
- How many animals: all animals
- Parameters checked were: alanine aminotransferase (ALAT), aspartate-aminotransferase (ASAT), alkaline phosphatase (AP), creatinine (Crea), total protein (TP), albumin (Alb), urea, total bilirubin (TBIL), total bile acids (TBA), total cholesterol (Chol), glucose (Gluc), sodium (Na), potassium (K).

URINALYSIS: Yes
- Time schedule for collection of urine: A urinalysis was performed with samples collected from all animals prior to or as part of the sacrifice of the animals. Additionally, urine colour/ appearance was recorded.
- Animals fasted: Yes
- Parameters checked were: specific gravity, nitrite, pH-value (pH), protein, glucose, ketone bodies (Ket), urobilinogen (UBG), bilirubin (BIL), erythroctes (Ery), leukocytes (Leu).

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Once before the first exposure and once in the last week of exposure
- Dose groups that were examined: all animals
- Battery of functions tested: multiple detailed behavioural observations were made outside the home cage using a functional observational battery of tests.

IMMUNOLOGY: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes (see table 1, box "Any other information on material and methods")
One day after the last administration (study day 91) all surviving animals of the treatment period were sacrificed using anesthesia (ketamine, medistar Arzneimittel, lot no: 00815, expiry date: Nov. 2017 and xylazin, Rebopharm, lot no. 400260/1, expiry date: Jan. 2017) and were subjected to a detailed gross necropsy which includes careful examination of the external surface of the body, all orifices and the cranial, thoracic and abdominal cavities and their contents.
The wet weight of the following organs (Table 1) was taken of all sacrificed animals as soon as possible. Paired organs were weighed together. Organ weights of animals found dead or euthanised for animal welfare reasons were not recorded.
The following tissues (Table 2) from all animals were preserved in 4 % neutral-buffered formaldehyde except eyes, testes and epididymides which were fixed in Modified Davidson’s fixative for approximately 24 hours before they were transferred to 70 % ethanol.
All animals found dead and/or intercurrently euthanised for animal welfare reasons were also subjected to a gross necropsy and the organs preserved for a histopathological examination.

HISTOPATHOLOGY: Yes (see table 2, box "Any other information on material and methods")
The afore-listed organs (Table 2) were examined histopathologically after preparation of paraffin sections and haematoxylin-eosin staining for the animals of the groups 2 and 5 sacrificed at the end of the treatment period and any animal found dead or euthanised before the planned day of sacrifice. Data on histopathological evaluation of group 1 were collected within Eurofins / BSL Munich Study No. 154433.
These examinations were extended for kidneys to animals of all other dosage groups and for livers, thymus, and adrenal glands to animals of the low dose group.
Liver, kidney, thymus and adrenal glands was examined in all animals of groups 3 and 4. Additionally, Oil Red O staining was performed on liver and kidney of animals no. 81-120 and 121-160. Additionally, immunohistochemistry of catalase and LAMP 2 was performed on liver and kidney of animals no. 81-85 and 111-115. Histological processing of tissues to microscope slides was performed at the GLP certified contract laboratory AnaPath GmbH, AnaPath Services, Hammerstrasse 49, 4410 Liestal, Switzerland (test site for tissue processing). Histopathological evaluation was performed at the GLP-certified contract laboratory AnaPath GmbH, Buchsweg 56, 4625 Oberbuchsiten, Switzerland (test site for histopathology). The study phases from test site 1 and 2 was performed in compliance with the Swiss Ordinance relating to Good Laboratory Practice adopted 18 May 2005 [SR 813.112.1] (Status as of 01 December 2012) [10]. Blocking, embedding, cutting, H&E staining and scientific slide evaluation were performed according to the corresponding SOP’s of the test sites.
The principal investigator for histopathological tissue processing sent all raw data (including blocks, slides, paper raw data, statement of compliance and quality assurance statement) to the study director. The principal investigator for histopathological evaluation provided the histopathology results to the study director by e-mail and sent a pathology phase report to the study director upon the completion of the study.
Other examinations:
Analysis of α2μ-Globulin in Male Kidneys:
Immunohistochemistry of α2μ-globulin and/or a specific staining were performed on kidney of all male animals sacrificed at the end of the treatment period of the study (50 animals). The processing of tissues to microscope slides was performed at the GLP certified contract laboratory AnaPath GmbH, AnaPath Services, Hammerstrasse 49, 4410 Liestal, Switzerland (test site for tissue processing). Immunohistochemistry was performed with Rat α2μ-Globulin Antibody (Monoclonal Mouse IgG1 Clone #129736, R&D Systems). The image analysis was performed by the CELL Analysis System. Pictures of kidney slides were taken by an Olympus UC30 camera at a magnification of x20. The positive area on the total area was measured in mm2 and calculated as % on the total area. The relative values of positive structures (α2μ-globulin) were used for descriptive statistics (mean, standard deviation, minimum, maximum).


Analysis of Oil Red O, Lamp-2 and catalase:
Oil Red O staining was performed on liver of all animals sacrificed at the end of the treatment period of the study (50 animals). Lamp-2 and catalase immunohistochemistry was performed on kidney and liver of first 5 male animals of the control and high dose group sacrificed at the end of the treatment period of the study (10 animals). The processing of tissues to microscope slides was performed at the GLP-certified contract laboratory AnaPath GmbH, AnaPath Services, Hammerstrasse 49, 4410 Liestal, Switzerland (test site for tissue processing). Sections stained with antibodies against LAMP-2, Catalase, and Oil Red O were evaluated at a semi quantitative manner as hematoxylin and eosin stained sections.
Statistics:
The findings of this study were evaluated in terms of the observed effects. Parameters like body weight gain and food consumption were calculated for each animal as the difference in weight measured from one week to the next. Mean body weights are also presented as figures.
The relative organ weights were calculated in relation to the brain weight and in relation to the body weight (measured at necropsy) and are presented as percentage. All results are reported in a tabular form (summarised in mean or summary tables and/or listed in individual data tables).
Analytical results and histopathological findings are presented in separate phase-reports. Toxicology and pathology data were captured either on paper according to appropriate SOPs or using the validated computerised system Ascentos® System (version 1.1.3, Pathology Data Systems Ltd.). A statistical assessment of the results of the body weight, food consumption, parameters of haematology, blood coagulation and clinical biochemistry and absolute and relative organ weights were performed for each gender by comparing values of dosed with control animals of the main groups using a one-way ANOVA and a post-hoc Dunnett Test. Moreover, the results of the same parameters were statistically compared between C1 and C2 control groups. These statistics were performed with GraphPad Prism V.6.01 software or Ascentos 1.1.3 software (p<0.05 was considered as statistically significant).
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
For details see "Any other informations on results"
Mortality:
mortality observed, treatment-related
Description (incidence):
Test item-related mortality occurred at 1000 mg/kg bw/day in 1/10 male animals, with a slight chronic forestomach inflammation associated with moderate forestomach epithelial hyperplasia that coincided with marked clinical symptoms prior to death. Mortality of 2 further animals is considered not test item related. For details see "Any other informations on results"
Body weight and weight changes:
no effects observed
Description (incidence and severity):
For details see "Any other informations on results"
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
No toxicologically relevant effect of Bis(3,5,5-trimethylhexanoyl)peroxide on food consumption was observed. For details see "Any other informations on results"
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Description (incidence and severity):
For details see "Any other informations on results"
Haematological findings:
no effects observed
Description (incidence and severity):
For details see "Any other informations on results"
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
For details see "Any other informations on results"
Urinalysis findings:
no effects observed
Description (incidence and severity):
For details see "Any other informations on results"
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
For details see "Any other informations on results"
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Dose-dependently, but not statistically significantly liver weight was increased in females from 100 mg/kg bw/day and in male animals from 300 mg/kg bw/day upwards. A slight but not statistically significantly higher kidney weight increase was seen in male – but not female animals at 1000 mg/kg bw/day. For details see "Any other informations on results"
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
At scheduled necropsy pale and marbled livers and kidneys were observed in males and females of all dose groups. The incidence was dose-dependent for livers with a higher sensitivity observed in female animals. In kidneys this finding was seen independently of dose. Besides, there were no macroscopic findings that could be related to daily oral dosing of Bis(3,5,5-trimethylhexanoyl)peroxide. For details see "Any other informations on results"
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
no effects observed
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
female
Remarks on result:
not determinable due to absence of adverse toxic effects
Dose descriptor:
LOAEL
Effect level:
100 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male
Basis for effect level:
histopathology: non-neoplastic
Critical effects observed:
yes
Lowest effective dose / conc.:
100 mg/kg bw/day (nominal)
System:
urinary
Organ:
kidney
Treatment related:
yes
Dose response relationship:
no

- Mortality

During the treatment period mortality occurred in 1/10 female animals at a daily dose of 300 mg/kg bw/day and in 1/10 male and 1/10 female animals at 1000 mg/kg bw/day. Male animal no. 118 of the high dose group showed severe piloerection and slightly reduced spontaneous activity. On the same day (day 5) the animal was euthanized for animal welfare reasons. The cause of morbidity was likely related to a slight chronic forestomach inflammation associated with moderate forestomach epithelial hyperplasia. Female animal no. 156 of the high dose group was found dead on study day 10. On study day 9 severe piloerection, abnormal breathing, moderately reduced spontaneous activity and hunched posture was seen on this animal. At necropsy, a slight chronic inflammation in the esophagus was seen. It may be considered that this animal died in a consequence of misgavaging.

Female no. 148 of the medium dose group was found dead on study day 67. No critical clinical signs were seen in this animal on the days prior to death. It is assumed that misgavaging is the cause of death.

- Clinical Observations

Besides, clinical symptoms seen in male animal no. 118 (see mortality), there were no test item related clinical signs of toxicity observed in the treatment period of this study. Occasionally, alopecia and/or crust was observed transiently on the cheek or neck or forelimbs of animals at 300 or 1000 mg/kg bw/day. These are common background findings noted in socially housed rats and are not considered to be related to treatment with the test item. Swelling on the left cheek or a crust at the left flank was observed in single control animals.

Animals no. 151 to 155 and 157 to 160 of the high dose group were observed moving the bedding of on days 50 or 51 of the treatment period. This temporally isolated finding is not assumed to be related to test item. Slight piloerection was observed in female control animal no. 125 on study day 53, female animal no. 147 of the high dose group on study day 90 and female animal no. 155 of the high dose group on day 5. These signs of impaired health condition are assumed to be related to small lesions caused during oral gavaging. All above-mentioned animals had recovered on the next day.

- Functional Observation Battery

Bis(3,5,5-trimethylhexanoyl)peroxide had no effect on neurobehavioural parameters determined in a function observation battery at the end of the treatment period. No biologically relevant differences were observed in neurobehavioural parameters between paraffinum perliquidum and corn oil controls.

- Body Weight Development

Bis(3,5,5-trimethylhexanoyl)peroxide had no significant effect on body weight development in this study. Throughout the treatment period body weights of male animals were slightly lower – but not statistically different at the high dose level when compared to the paraffinum perliquidum control group. At the end of the treatment period, the difference reached 10 %. Over the total treatment period body weight gain was statistically significantly lower in male animals at the high dose level compared to the paraffinum perliquidum controls. Such an effect was not found in female animals.

Throughout the treatment period, body weight of paraffinum perliquidum controls were not considerable different from body weights of the corn oil controls. A statistically significantly higher body weight gain in female paraffinum perliquidum controls compared to the corn oil controls is not assumed to be of toxicological relevance.

- Food Consumption

Bis(3,5,5-trimethylhexanoyl)peroxide had no significant effect on food consumption in this study. From treatment week 2 onwards food consumption of female animals was slightly higher – but not statistically different at the high dose level when compared to the paraffinum perliquidum control group (between approx. 10% and 15% above paraffinum perliquidum controls). This is not considered to be adverse as values were within historical range.

Throughout the treatment period food consumption of male animals of the paraffinum perliquidum control group was slightly higher than in corn oil controls (in average approx. 23 % above corn oil controls in males and in average approx. 17 % above corn oil controls in females). This was not correlated with an adequately higher body weight gain and reflects a compensation for the higher volume of corn oil (5 mL/kg bw) compared to paraffinum perliquidum (3 mL/kg bw) administered. This is not considered adverse.

- Haematology and Blood Coagulation

No toxicologically relevant test item related effects of Bis(3,5,5-trimethylhexanoyl)-peroxide on haematological parameter were observed at the end of the treatment period.

A slight but statistically significantly lower mean corpuscular volume in male animals at 300 mg/kg bw/day and mean cell haemoglobin in male animals at 300 and 1000 mg/kg bw/day are not assumed to be toxicologically relevant, as the values ranged within the historical background data.

In haematological parameters of female animals, mean corpuscular haemoglobin concentration and monocytes were slightly - but statistically significantly higher at 1000 mg/kg bw/day when compared to paraffinum perliquidum controls and platelet count was slightly – but statistically significantly lower at 100 mg/kg bw/day. These differences were not assumed to be toxicologically relevant as the values were within the range of historical control data or only very slightly above (monocytes).

No biologically relevant differences were observed in haematological or coagulation parameters between paraffinum perliquidum and corn oil control groups.

- Clinical Biochemistry

No toxicologically relevant test item related effects of Bis(3,5,5-trimethylhexanoyl)-peroxide on parameters of clinical biochemistry were observed at the end of the treatment period.

There were no statistically or biologically significant differences in any of the clinical biochemistry parameters of male animals between the dose groups and the paraffinum perliquidum control group of this study.

A slightly and statistically significantly higher serum urea level was observed in female – but not male – animals at 300 and 1000 mg/kg bw/day. Although a relation to the test item cannot be excluded, this did not correlate with histopathological findings and is not assumed to be toxicologically relevant.

Serum Chol levels of female animals were slightly and statistically significantly higher at 1000 mg/kg bw/day but slightly and statistically significantly lower at 300 mg/kg bw/day. Crea levels were minimally – but significantly higher and TP and Gluc levels were slightly and statistically significantly lower at 1000 mg/kg bw/day, than the respective controls. As these values ranged within the historical control data, they are not assumed to be toxicologically relevant.

No biologically relevant differences were observed in parameters of clinical biochemistry between paraffinum perliquidum and corn oil control groups. Slightly but statistically significantly higher serum levels of ASAT (both genders), ALAT (males), AP (males), Crea (males), TP (both genders), Alb (males), Urea (both genders), TBil (males), TBA (males), Chol (males) and Na (females) are not assumed to be biologically relevant as all values were within the normal range of historical control data.

- Urinalysis

Bis(3,5,5-trimethylhexanoyl)peroxide had no effect on urinary parameters analysed at the end of the treatment period of this study.

High count of erythrocytes was seen in single animals of all dose groups – but also of the control group. Besides, there were no conspicuous values in any of the parameters analysed.

No biologically relevant differences were observed in urinary parameters between paraffinum perliquidum and corn oil control groups.

- Pathology

Animal no. 148 was found dead on day 67 and showed autolysis of brain, spinal cord and sciatic nerve and its heart, liver and kidneys were cannibalized. Autolysis was also seen in organs (i.e. brain, spinal cord, sciatic nerves) of animal no. 156 that was found dead on study day 10.

Pale and marbled livers and kidneys were observed in males and females of all dose groups, as listed in Table 3. The incidence was dose-dependent for livers with a higher sensitivity observed in female animals. In kidneys this finding was seen independently of dose.

Table 3: Main Macroscopic Findings at Scheduled Necropsy

Groups C1 C2 LD
(100 mg/kg bw/day)
MD
(300 mg/kg bw/day)
HD
(1000 mg/kg bw/day)
Pale / marbled Liver Male 0/10 0/10 0/10 7/10 9/10
Female 0/10 0/10 7/10 9/10 8/10
Pale / marbled Kidneys Male 0/10 0/10 7/10 9/10 8/10
Female 0/10 0/10 3/10 5/10 2/10

Besides, there were no macroscopic findings that could be related to daily oral dosing of Bis(3,5,5-trimethylhexanoyl)peroxide.

- Organ Weight

Daily oral administration of Bis(3,5,5-trimethylhexanoyl)peroxide had an influence on liver weight of male and female animals at the end of the 90-day treatment period. Dose-dependently, but not statistically significantly liver weight was increased in females from 100 mg/kg bw/day and in male animals from 300 mg/kg bw/day.

A slight but not statistically significantly higher kidney weight increase was seen in male but not female animals at the high dose level, when related to body weight (18 % higher than controls). A relation to histopathological findings cannot be excluded.

Slightly lower thymus weight was also seen in male animals of the high dose group (absolute thymus weight 21 % below controls). This might reflect a stress-related response. Stress might also be the reason for increased weight of adrenal glands and of decreased weight of spleen in female animals at all dose levels.

There were no biologically relevant differences in organ weight between paraffinum perliquidum and corn oil controls of this study. Slightly – but statistically significantly higher kidney weights in relation to body weight in male paraffinum perliquidum control animals (7 % above controls) are not considered to be biologically relevant.

- Histopathology

Kidneys:

In males and females of all test item-treated groups, there was a minor severity of tubular swelling/vacuolation. In males, almost all animals were affected. In females, there was an increased incidence with increasing dose.

In males, there was a strongly increased incidence and severity of hyaline inclusions in tubular cells in all test item-treated groups. In addition, there was a strongly increased incidence of minimal tubular basophilia at 100 and 300 mg/kg/day that decreased to control levels at 1000 mg/kg/day. This finding was associated with tubular cell necrosis and the formation of granular casts in males from all test item-treated groups.

Under immunohistochemistry, there was a decreased immunoreactivity of hyaline inclusions against α2-microglobulin at 1000 mg/kg/day. Lamp-2 reactivity increased at this dose, but there was no significant difference in catalase-reactivity between controls and animals treated at 1000 mg/kg/day.

Liver:

There was a minimal hepatocellular hypertrophy (mainly centrilobular) in the liver of test item-treated animals, whereby incidence and/or severity increased with dose. This finding was associated with an increased (severity/incidence) vacuolation. By OilRed O, the vacuolation appeared to be due to increasing intracytoplasmic lipid deposition in hepatocytes of all liver lobular regions. There were no changes in Lamp-2 or Catalase reactivity between controls and test item-treated animals.

Lung:

There was a number of inflammatory and reactive changes recorded in the lungs of control and high dose animals. They consisted of interstitial inflammation in several control animals, associated with alveolar/bronchiolar hyperplasia. In contrast, in a few high dose animals, there was a minor focal fibrosis. In both groups, reactive alveolar macrophages were noted in several animals.

Thymus:

An increased incidence and/or severity of atrophy was noted in a not dose-dependent manner in all test item-treated groups.

Adrenal Cortices:

Diffuse hypertrophy of the zona fascilulata was noted in low and high dose females, but also in high dose males.

- Dose Formulation Analysis

Concentration analysis of formulation samples was determined in study weeks 1, 5, 9 and 13 for all dose groups. The mean recoveries observed in LD, MD and HD groups were 109.5 %, 104.6 % and 94.4 % of the nominal concentration, respectively. The acceptance criteria were met.

Homogeneity of formulation samples was determined in study weeks 1 5 and 13 for LD and HD dose groups. The mean recovery observed was between 98.5 %, and 113.0 % of nominal value for LD dose group and between 89.9 % and 98.4 % for HD dose group.

The coefficients of variation of the different sampling locations (top, middle, bottom) were between 1.7 % and 2.2 % in LD dose group and between 0.5 % and 1.4 % in HD dose group. The acceptance criteria were met.

Conclusions:
In a 90-day subchronic toxicity study (OECD 408) bis(3,5,5-trimethylhexanoyl)peroxide (75 % purity) was administered orally to 10 Wistar rats/sex/group at nominal dose levels of 0, 100, 300 and 1000 mg/kg bw/day. Based on the pathology evaluation, the NOAEL, for females only, can be established at 1000 mg/kg/day. Based on the pathology evaluation and the kidney findings of tubular cell necrosis that was still present in three animals at the low dose group, a NOAEL could not be established for males. The LOAEL for male rats was determined to be 100 mg/kg bw/day.
Executive summary:

In a 90-day subchronic toxicity study (OECD 408) bis(3,5,5-trimethylhexanoyl)peroxide (75 % purity) was administered orally to 10 Wistar rats/sex/group at nominal dose levels of 0, 100, 300 and 1000 mg/kg bw/day. There were three decedents, one male and one female animal at 1000 mg/kg bw/day and one female animal at 300 mg/kg bw/day.

For the male that died, it may be considered that a forestomach inflammation was contributing to morbidity. In one female of the high dose group that died, there was a slight chronic inflammation in the esophagus that may be considered an indicator for misgavage. Misgavaging is also assumed to be the reason for death of the female animal of the medium dose group. Pale kidneys in all test item-treated groups (both sexes) were deemed to be test item-related. This lesion was in males and females of all test item-treated groups associated by a minor severity of tubular swelling/vacuolation. In males, there was a strongly increased incidence and severity of hyaline inclusions in tubular that was not due to increasing α2-microglobulin. Lamp-2 reactivity increased at this dose, but there was no significant difference in catalase-reactivity between controls and animals treated at 1000 mg/kg bw. Therefore, the hyaline inclusions are deemed to be due to increasing lysosome activity storing a substance that may be considered the test item or a metabolite thereof. These findings were associated with with tubular cell necrosis and the formation of granular casts in males from all test item-treated groups. An increasing incidence of minimal tubular basophilia at 100 and 300 mg/kg bw is deemed to be indicator of regeneration. In animals at 1000 mg/kg bw, the tubular basophilia was recorded at control levels that most likely indicates limited regenerative ability.

Mottled and pale livers in females at 100 mg/kg bw and in both sexes at all higher doses was associated with minimal hepatocellular hypertrophy (mainly centrilobular) in test item-treated animals, whereby incidence and/or severity increased with dose. This finding was associated with an increased (severity/incidence) vacuolation. By OilRed O staining, the vacuolation appeared to be due to increasing intracytoplasmic lipid deposition in hepatocytes of all liver lobular regions. There were no changes in Lamp-2 or Catalase reactivity between controls and test item-treated animals. Therefore, a storage in activated lysosomes may be excluded as cause of the vacuolation.

Both, the inflammatory and degenerative findings in the liver and, especially in male kidneys are deemed to be related to the chemical abilities of the test item, i.e. lipid peroxidation is considered to play a role in pathogenesis.

Inflammatory and reactive changes in the lungs consisting of interstitial inflammation in control animals, associated with alveolar/bronchiolar hyperplasia, and minor focal fibrosis in high dose animals, as well as alveolar macrophages accumulation are deemed to be indicators for accidental aspiration, and related to the vehicle that may be enhanced by the test item in the high dose group. Control group 1 was treated with corn oil at an application volume was 5 mL/kg bw. that did not cause comparable incidences of inflammatory lesions. These data are supportive for an effect in the lungs of the present study by accidental aspiration, i.e., these lesions are not primarily test item-related.

An increasing incidence and/or severity of thymic atrophy and diffuse hypertrophy of the adrenal cortex zona fasciculata are deemed to be stress-related findings. Based on the pathology evaluation and the kidney findings of tubular cell necrosis that was still present in three animals at the low dose group, a NOAEL could not be established for males. In females, the NOAEL may be established at 1000 mg/kg bw/day due to the absence of necrosis. Therefore the observed liver and kindney effects can be interpreted as adaptive and reversible in females.

The fact that histopathologically hyaline inclusions in tubular cells was only evident in male and not female animals indicates the possibility of an identical mechanism of entry into the cell for test item (or a metabolite thereof) and α2-microglobulin. Thus, further studies are recommended to test if hyaline inclusions in male animals and subsequent secondary findings like granular casts and cell necrosis are species-specific findings and not relevant to human. But nonetheless, a LOAEL of 100 mg/kg bw/day for male rats is assumed based on the study results until species-specifity of the hyaline inclusions in tubular cells is confirmed.

This subchronic toxicity study in the rat is acceptable and satisfies the guideline requirement for a subchronic oral study OECD 408 in rats. 

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
LOAEL
100 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
GLP guideline study
Organ:
kidney

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

In a 28-day repeated-dose oral toxicity study, bis(3,5,5-trimethylhexanoyl)peroxide was administered by gavage to male and female Wistar rats (5 animals/dose/sex) at dose levels of 0, 100, 300, 1000 mg/kg bw/day, for 7 days/week during a 28-day period. No mortality was observed. Clinical symptoms, such as mild salivation (male MD group) and alopecia (female HD group) were assumed to be incidental. In males, slight change in body weight gain was recorded in HD group during treatment period, which corroborated with food intake during the corresponding days of body weight. In females, no such changes were noted. No changes in the haematological parameters were associated with the administration of the test material. In males of the HD group, statistically significant alterations were recorded in several urine parameters. Statistically significant decrease in mean TBA was seen in all dose groups. Such changes were within the normal ranges for historical controls. In females, there was slight increase in mean AP value in MD and HD groups. Such changes were attributed to the histopathological changes seen in the liver and kidney. Histopathological findings considered to be test item-related were seen in the liver, kidney, seminal vesicle, coagulating gland and thymus. The kidney changes were considered to be of no toxicological relevance for humans. The NOAEL of the study was set at 300 mg/kg bw/day based on microscopic changes in the liver, kidney, seminal vesicle, coagulating gland and thymus. This subacute toxicity study in the rat is acceptable and satisfies the guideline requirement for a subacute oral study (OECD 407) in rat.

In a 90-day subchronic toxicity study (OECD 408), bis(3,5,5 -trimethylhexanoyl)peroxide (75 % purity) was administered orally to 10 Wistar rats/sex/group at nominal dose levels of 0, 100, 300 and 1000 mg/kg bw/day.

There were three decedents, one male and one female animal at 1000 mg/kg bw/day and one female animal at 300 mg/kg bw/day.

For the male that died, it may be considered that a forestomach inflammation was contributing to morbidity. In one female of the high dose group that died, there was a slight chronic inflammation in the esophagus that may be considered an indicator for misgavage. Misgavaging is also assumed to be the reason for death of the female animal of the medium dose group.

Pale kidneys in all test item-treated groups (both sexes) were deemed to be test item-related. This lesion was in males and females of all test item-treated groups associated by a minor severity of tubular swelling/vacuolation. In males, there was a strongly increased incidence and severity of hyaline inclusions in tubular that was not due to increasing α2-microglobulin. Lamp-2 reactivity increased at this dose, but there was no significant difference in catalase-reactivity between controls and animals treated at 1000 mg/kg. Therefore, the hyaline inclusions are deemed to be due to increasing lysosome activity storing a substance that may be considered the test item or a metabolite thereof.

These findings were associated with with tubular cell necrosis and the formation of granular casts in males from all test item-treated groups. An increasing incidence of minimal tubular basophilia at 100 and 300 mg/kg is deemed to be indicator of regeneration. In animals at 1000 mg/kg, the tubular basophilia was recorded at control levels that most likely indicates limited regenerative ability.

Mottled and pale livers in females at 100 mg/kg and in both sexes at all higher doses was associated with minimal hepatocellular hypertrophy (mainly centrilobular) in test item-treated animals, whereby incidence and/or severity increased with dose. This finding was associated with an increased (severity/incidence) vacuolation. By OilRed O staining, the vacuolation appeared to be due to increasing intracytoplasmic lipid deposition in hepatocytes of all liver lobular regions. There were no changes in Lamp-2 or Catalase reactivity between controls and test item-treated animals. Therefore, a storage in activated lysosomes may be excluded as cause of the vacuolation.

Both, the inflammatory and degenerative findings in the liver and, especially in male kidneys are deemed to be related to the chemical abilities of the test item, i.e. lipid peroxidation is considered to play a role in pathogenesis.

Inflammatory and reactive changes in the lungs consisting of interstitial inflammation in control animals, associated with alveolar/bronchiolar hyperplasia, and minor focal fibrosis in high dose animals, as well as alveolar macrophages accumulation are deemed to be indicators for accidental aspiration, and related to the vehicle that may be enhanced by the test item in the high dose group. Control group 1 was treated with corn oil at an application volume was 5 mL/kg bw. that did not cause comparable incidences of inflammatory lesions. These data are supportive for an effect in the lungs of the present study by accidental aspiration, i.e., these lesions are not primarily test item-related.

An increasing incidence and/or severity of thymic atrophy and diffuse hypertrophy of the adrenal cortex zona fasciculata are deemed to be stress-related findings.

Based on the pathology evaluation and the kidney findings of tubular cell necrosis that was still present in three animals at the low dose group, a NOAEL could not be established for males. In females, the NOAEL may be established at 1000 mg/kg bw/day due to the absence of necrosis. Therefore the observed liver and kindney effects can be interpreted as adaptive and reversible in females.

The fact that histopathologically hyaline inclusions in tubular cells was only evident in male and not female animals indicates the possibility of an identical mechanism of entry into the cell for test item (or a metabolite thereof) and α2 -microglobulin. Thus, further studies are recommended to test if hyaline inclusions in male animals and subsequent secondary findings like granular casts and cell necrosis are species-specific findings and not relevant to human. But nonetheless, a LOAEL of 100 mg/kg bw/day for male rats is assumed based on the study results until species-specifity of the hyaline inclusions in tubular cells is confirmed.


Justification for classification or non-classification

Based on the results observed in a 90-day repeated dose toxicity study, no classification is warranted.