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Description of key information

As the result of a sub-chronic study in rats a NOAEL of 80 mg/kg was obtained for repeated oral toxicity.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2013.10.22 - 2014.6.20
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP/OECD guildeline study
Qualifier:
according to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
GLP compliance:
yes (incl. certificate)
Limit test:
no
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Strain: Hsd.Brl.Han: of Wistar origin
- Source: Toxi-Coop Zrt. 1103 Budapest, Cserkesz u. 90.
- Age at study initiation: Male animals: 38 – 43 days; Female animals: 38 – 43 days
- Weight at study initiation: Male animals: between range of 141 and 164 g; Female animals: between range of 102 and 135 g
- Housing: 2 or 3 animals of the same sex/ cage, Type III polypropylene/polycarbonate-Size: 22 x 32 x 19 cm, certified laboratory wood bedding changed twice a week
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 8d

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 °C
- Humidity (%): 30 - 70 %
- Air changes (per hr): 10-15 air exchanges/hour by central air-condition system
- Photoperiod (hrs dark / hrs light): Artificial light, from 6 a.m. to 6 p.m
Route of administration:
oral: gavage
Vehicle:
other: sunflower oil
Details on oral exposure:
VEHICLE
- Justification for use and choice of vehicle (if other than water): The test item is not soluble in water in concentrations necessitated for this study. Formulations prepared with the vehicle are suitable for oral administration as it is the anticipated route of human exposure to the test item.
- Amount of vehicle (if gavage): 5 ml/kg bw
- Lot/batch no. (if required): Batch 1: 1305-4630; Batch 2: 1304 - 4573
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analytical control of dosing solutions (concentration and homogeneity) was performed in the Analytical Laboratory of Test Facility twice during the study.
Duration of treatment / exposure:
90-91 days of treatment, 4 weeks recovery for satellite group
Frequency of treatment:
daily
Remarks:
Doses / Concentrations:
20, 80, 320 mg/kg bw/day
Basis:
actual ingested
No. of animals per sex per dose:
10 animals/sex/dose plus 5 additional/sex in the control and highest dose group as a satellite group
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: based on data obtained from a previously performed 28-day oral toxicity study with the test item in rats. Doses were selected with the aim of inducing toxic effects but no mortality or suffering at the highest dose and a NOAEL at the lowest dose
- Rationale for animal assignment (if not random): All male and female animals were sorted according to body weight and divided to weight groups aided by a computerized calculation. There were an equal number of animals from each weight group in each of the experimental groups assigned by randomization to ensure that the mean weight of animals from all test groups was as uniformly as practicable
- Rationale for selecting satellite groups: to assess reversibility
- Post-exposure recovery period in satellite groups: 28d
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily
- Cage side observations checked: morbitidy and mortality

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once daily

BODY WEIGHT: Yes
- Time schedule for examinations: once during acclimation, Days 0 (prior to study start), 7, 14 and twice weekly during weeks 3, 4 and once weekly thereafter, and prior to sacrafice

FOOD CONSUMPTION:
- Food consumption was determined with the measurement of non-consumed diet

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: Yes / No / No data

OPHTHALMOSCOPIC EXAMINATION: Yes
- Examinations occurred during the acclimation period, the eyes of all rats being considered for study were examined; examinations were repeated on control and high dose animals prior to test termination

HAEMATOLOGY: Yes
- Time schedule for collection of blood: conducted at termination of the treatment (i.e. one day after the last treatment) and at the end of the recovery period
- Anaesthetic used for blood collection: No data
- Animals fasted: Yes
- Parameters checked: WBC, RBC, HGB, HTC, MCV, MCH, MCHC, PLT, RET, Differential white blood cell count, and APTT, PT for blood coagulation measures

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: conducted at termination of the treatment (i.e. one day after the last treatment) and at the end of the recovery period
- Animals fasted: Yes
- Parameters checked: ALT, AST, GGT, ALP, TBIL, CREA, UREA, BUN, GLUC, CHOL, BAC, Pi, Ca++, Na+, K+, Cl-, ALB, TPROT, A/G

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

OTHER: The estrous cycle of all female animals was examined during the last three weeks of the treatment period or receovery period;
Sacrifice and pathology:
GROSS PATHOLOGY: Yes:
-Adrenals, aorta, bone marrow (femur), brain (representative regions: cerebrum, cerebellum and pons and medulla oblongata), esophagus, eyes (lachrymal gland with Harderian glands), female mammary gland, gonads (testes with epididymides, ovaries, uterus with vagina), gross lesions, heart, kidneys, large intestines (caecum, colon, rectum, including Peyer’s patches), liver, lungs (with main stem bronchi; Inflation with fixative and then immersion;), lymph nodes (submandibular, mesenteric), muscle (quadriceps), pancreas, pituitary, prostate, salivary glands (submandibular), sciatic nerve, seminal vesicle with coagulating gland, skin, small intestines (representative regions: duodenum, ileum, jejunum), spinal cord (at three levels: cervical, mid-thoracic and lumbar), spleen, sternum, stomach, thymus, thyroid + Parathyroid, trachea, urinary bladder

HISTOPATHOLOGY: Yes
-Histological examination was performed on the preserved organs and tissues of the animals from both the control and high dose groups
Other examinations:
-organ weight: Liver, kidneys, testes, epididymides, uterus and fallopian tubes, thymus, spleen, brain, heart, adrenals, ovaries, thyroid/parathyroid
-sperm examinations: Sperm analysis was made from 10 control and 10 high dose males at the necropsy. One-side testes and epididymides were used for sperm examinations - sperm motility, morphological evaluation of ductus deferens, total number of homogenization testes sperm
Statistics:
-Statistical analysis was done with SPSS PC+ software for: Body weight, Food consumption and feed efficiency, Hematology, Blood coagulation, Clinical chemistry, Sperm parameters, Estrous cycle, Organ weight,
-The heterogeneity of variance between groups was checked by Bartlett's homogeneity of variance test. Where no significant heterogeneity is detected, a one-way ANOVA was carried out. If the obtained result was positive, Duncan's Multiple Range test was used to assess the significance of inter-group differences. Where significant heterogeneity was found, the normal distribution of data was examined by Kolmogorov-Smirnov test.
-In case of a none-normal distribution, the non-parametric method of Kruskal-Wallis One-Way analysis of variance was used. If there was a positive result, the inter-group comparisons were performed using the Mann-Whitney U-test.
-For evaluation of data obtained during the recovery period, the homogeneity of variance between groups was checked by F-test. Depending on the result pooled or separate variance estimate of the Two-Sample t-test was performed
-Frequency of toxic response, ophthalmoscopy, pathological and histopathological findings by sex and dose was calculated.
- Significant was indicated at a probability value of p < 0.05 and < 0.01.
-Male and female rats were evaluated separately
Clinical signs:
effects observed, treatment-related
Mortality:
mortality observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
effects observed, treatment-related
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 AND MORTALITY
-no test item related mortality
-behavior and physical conditions considered normal
-Test item related salivation was observed in male and female animals administered with 20, 80 or 320 mg/kg bw/day with variable frequency within a group but in a dose related manner
-Clinical signs were not detected in the male or female animals of control or 320 mg/kg bw/day group during the entire recovery period.

BODY WEIGHT AND WEIGHT GAIN
-The body weight development was reduced in male and female animals of 320 mg/kg bw/day group during the entire treatment period. The changes in body weight were only reversible in male animals.
-The mean body weight gain significantly exceeded that of the control animals in male animals of 320 mg/kg bw/day group, thus the mean body weight was similar to that of the control group during the entire recovery period. The mean body weight gain of female animals of 320 mg/kg bw/day group was slightly higher than in the control group (no statistical significance) however the difference in the mean body weight with respect to the control observed at the termination of the treatment remained present during the entire recovery period.

FOOD EFFICIENCY
-The mean feed efficiency was less in male and female animals of 320 mg/kg bw/day group with respect to the control during the treatment period.
-The mean feed efficiency was slightly higher in male animals of 320 mg/kg bw/day group with respect to the control during the recovery period. The mean feed efficiency was similar in the female animals of the control and high dose group during the recovery period.

CLINICAL CHEMISTRY
-Male animals: higher mean activity of alanine aminotransferase (ALT) and alkaline phosphatase (ALP), and less mean activity of aspartate aminotransferase (AST) at 320 mg/kg bw/day; higher mean activity of gamma-glutamyltransferase (GGT) at 80 and 320 mg/kg bw/day; higher mean concentrations of total bilirubin (TBIL) and inorganic phosphorous (Pi) at 80 and 320 mg/kg bw/day; higher mean concentrations of urea and blood urea nitrogen (BUN) at 20, 80, 320 mg/kg bw/day; less mean concentrations of creatinine (CREA), cholesterol (CHOL) and chloride (Cl-) and higher mean concentrations of bile acids (BAC) and calcium (Ca2+) at 320 mg/kg bw/day.
-Female animals: higher mean activity of ALT (320 mg/kg bw/day) and GGT (80 and 320 mg/kg bw/day) was observed. The mean concentrations of total bilirubin (80 and 320 mg/kg bw/day), urea and BUN (320 mg/kg bw/day), inorganic phosphorous and potassium (K+) exceeded the values of the control group. The mean concentrations of creatinine (80 and 320 mg/kg bw/day) and glucose (20 and 320 mg/kg bw/day) were slightly less than in the female animals of the control group
-At the end of the recovery period, statistically significant differences were indicated between the control and 320 mg/kg bw/day treated groups for the less mean activity of alanine aminotransferase and for the slightly higher mean activity of gamma-glutamyltransferase in male and female animals, less mean concentrations of glucose, total bilirubin and albumin (ALB) and higher mean concentration of bile acids in female animals.

ORGAN WEIGHTS
-A test item related and reversible changes were detected in the liver and in the kidneys weights in male and female animals administered with 320 mg/kg bw/day.
-The mean epididymides weights (absolute and relative to brain weight) were slightly less than in the control group in male animals of 320 mg/kg bw/day group.
-Statistical significances with respect to the control were also noted for some organ weights (absolute or relative to body or brain weight in male animals, such as, for thymus and adrenals at 80 mg/kg bw/day; for heart, spleen, testes, adrenals and thyroids at 320 mg/kg bw/day and in female animals of 320 mg/kg bw/day group for heart weight relative to body weight.
-Recovery group: The body weight, weight of brain, spleen, thyroid and thymus weight relative to body weight were slightly less than in the control group in female animals of 320 mg/kg bw/day group. The changes in the kidneys and liver weights in male and female animals administered 320 mg/kg bw/day together with the elevated serum levels of some biochemical parameters were considered to be indicative of test item influence on the hepatic and renal functions.

OTHER FINDINGS
-A test item influence on the estrous cycle was not detected.
-Sperm examinations did not point out any test item related influence on the sperm cells at 320 mg/kg bw/day.
Dose descriptor:
NOAEL
Effect level:
80 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
food efficiency
clinical biochemistry
organ weights and organ / body weight ratios
other: salivation
Dose descriptor:
LOAEL
Effect level:
320 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: salivation, changes in body weight and body weight gain, in feed efficiency, clinical chemistry parameters and organ weights (liver and kidneys)
Critical effects observed:
not specified
Conclusions:
The LOAEL 320 mg/kg bw/day based on salivation, changes in body weight and body weight gain, in feed efficiency, clinical chemistry parameters and organ weights (liver and kidneys).
Executive summary:

In a subchronic toxicity study, Dicumyl Peroxide was administered to 10 Wistar rats/sex/dose by oral gavage at dose levels of 0, 20, 80, 320 mg/kg bw/day.

Dicumyl Peroxide (CAS number 80-43-3) caused salivation, changes in body weight and body weight gain, in feed efficiency, clinical chemistry parameters (ALT, GGT, total bilirubin, blood urea nitrogen, bile acid or inorganic phosphorous) and organ weights (liver and kidneys) after repeated dose oral administration to male and female Hsd.Brl.Han: Wistar rats. The LOAEL is 320 mg/kg bw/day. The NOAEL is 80 mg/kg bw/day .

This subchronic toxicity study in the rat is acceptable and satisfies the guideline requirement for a subchronic oral study (OPPTS 870.3100; OECD 408) in rats.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
80 mg/kg bw/day
Study duration:
subchronic
Species:
rat

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

Repeated oral toxicity was investigated in two studies according to OECD Guideline 407 (28 d, using a 14 d recovery period for the satellite group) and OECD 408 (90 d).

In the subchronic toxicity study, Dicumyl Peroxide was administered to 10 Wistar rats/sex/dose by oral gavage at dose levels of 0, 20, 80, 320 mg/kg bw/day. The test item caused salivation, changes in body weight and body weight gain, in feed efficiency, clinical chemistry parameters and organ weights (liver and kidneys). The LOAEL is 320 mg/kg bw/day. The NOAEL is 80 mg/kg bw/day. No effect on estrous cycle was detected. Sperm examinations did not point out any test item related influence on the sperm cells at 320 mg/kg bw/day.

In the sub-acute toxicity study, rats were exposed to 60, 200 and 600 mg/kg. No deaths were observed in any of the treatment groups in either sex. Salivation was observed at 200 and 600 mg/kg in both sexes and body weight gain was suppressed at 600 mg/kg. These changes proved reversible after withdrawal. An increase in serum gamma-GTP was observed in both sexes at 600 mg/kg. Increased ALT was observed in males at 600 mg/kg. Absolute and relative liver weights were increased in both sexes at 600 mg/kg and relative liver weights were increased in females at the 200 mg/kg. Absolute and relative thymus weights were decreased at 600 mg/kg in both sexes. Enlarged livers were grossly observed in females at 600 mg/kg. Histopathologically, hypertrophy of hepatocytes was observed at 200 and 600 mg/kg in both sexes. Degeneration of hepatocytes was also observed at 600 mg/kg in both sexes. Mobilization of Kupffer cells was observed in males at 600 mg/kg. These changes proved reversible after withdrawal.

Data for dermal and inhalation route are not available. Testing of repeated dermal toxicity is not regarded necessary, as the substance shows no acute dermal toxicity and does not exhibit irritating or sensitizing properties. Testing of repeated inhalation toxicity is not regarded necessary, as the substance is a solid and granulometry data indicates there are no significant particles less than 100 microns in size (only 0.25% below 500 microns), demonstrating that there are no significant inhalable particles present and that inhalation is therefore not a likely route of exposure.

Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:

Sub-chronic OECD guideline study.

Justification for classification or non-classification

Based on the currently available data classification according to EU regulation 1272/2008 is not warranted.