Bis(3,5,5-trimethylhexanoyl) peroxide

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2012-04-11 to 2012-11-13

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Bis(3,5,5-trimethylhexanoyl) peroxide

EC Number:

223-356-0

EC Name:

Bis(3,5,5-trimethylhexanoyl) peroxide

Cas Number:

3851-87-4

Molecular formula:

C18H34O4

IUPAC Name:

3,5,5-trimethylhexanoyl 3,5,5-trimethylhexaneperoxoate

Constituent 2

Reference substance name:

Bis(3,5,5-trimethylhexanoyl)peroxide

IUPAC Name:

Bis(3,5,5-trimethylhexanoyl)peroxide

Test material form:

gas under pressure: refrigerated liquefied gas

Details on test material:

- Name of test material (as cited in study report): PEROXAN NPO
- Substance type: formulation
- Physical state: liquid
- Analytical purity: 75% of the active substance (3851-87-4) and 20-25% solvent (isododecan)
- Lot/batch No.:
- Expiration date of the lot/batch:
- Storage condition of test material: -8 to 0 C, protected from light

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system 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

Administration / exposure

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

Doses / concentrations

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

Examinations

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).

Results and discussion

Results of examinations

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.

Effect levels

1

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'

2

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

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

**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.

Applicant's summary and conclusion

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.