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Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2015-08-17 to
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Specific details on test material used for the study:
- Batch number: 1198395-01
- Physical state: liquid
- Colour: colourless
- Density: 0.995 g/cm³
- Purity: 98%
- Storage Conditions: -15 to -35 °C, protected from light
- Expiry date: 2015-11-30

Test animals

Species:
rat
Strain:
Wistar
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: 6-8 weeks old
- Weight at study initiation: males: 202-221 g; females: 139-159 g
- Housing: 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
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 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:
corn oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
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 the control item and vortexing it. Formulations or control items were stored at 2 to 8 °C or on crushed ice and were administered within 6 hours after preparation. Prior to administration test item formulations were brought to room temperature.

VEHICLE
- Amount of vehicle (if gavage): 5 mL/kg bw

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.; 10 mL of HD, 15 mL of MD and 65 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 abovementioned) 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).
Duration of treatment / exposure:
90 days
Frequency of treatment:
once per day, 7 days/week
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
vehicle control group
Dose / conc.:
100 mg/kg bw/day (nominal)
Remarks:
low dose (LD)
Dose / conc.:
300 mg/kg bw/day (nominal)
Remarks:
middle dose (MD)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Remarks:
high dose (HD)
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
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. The animals were treated with the test item formulation or vehicle on 7 days per week for a period of 90 days. 10 animals per gender and group were subjected to necropsy one day after the last administration.
Positive control:
no

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least once a day, 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
- Time schedule: 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
- Time schedule for examinations: 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: Yes
- Food consumption was measured weekly during the treatment period.

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

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: 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 (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
One day after the last administration (study day 91) all animals 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.

HISTOPATHOLOGY: Yes
The organs (see table 1) were examined histopathologically after preparation of paraffin sections and haematoxylin-eosin staining for the animals of the groups 1 and 4 sacrificed at the end of the treatment period. These examinations were extended to animals of all other dosage groups for kidneys and livers. Any gross lesion macroscopically identified was examined microscopically in all animals.
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 (40 animals). 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).
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 V.6.01 software or Ascentos 1.1.3 software (p< 0.05 was considered as statistically significant).

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Description (incidence and severity):
see below
Mortality:
no mortality observed
Description (incidence):
see below
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
see below
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
see below
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):
see below
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
see below
Urinalysis findings:
no effects observed
Description (incidence and severity):
see below
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
see below
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
see below
Gross pathological findings:
no effects observed
Description (incidence and severity):
see below
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
see below
Histopathological findings: neoplastic:
not examined
Details on results:
- Mortality:
All animals survived the treatment period of this study.
- Clinical Observations:
Throughout the treatment period there were no clinical signs that could be related to bis(2-ethylhexyl)peroxydicarbonate. Weekly detailed clinical observations revealed no conspicuous findings in bis(2-ethylhexyl)peroxydicarbonate-treated rats.
- Functional Observation Battery:
Bis(2-ethylhexyl)peroxydicarbonate had no effect on neurobehavioral parameters determined in a function observation battery at the end of the treatment period
- Body weight development:
Over the complete treatment period body weight gain was statistically significantly lower in all dose groups, when compared to controls. In male statistically significantly lower weight gain was observed in all dose groups during the first week of treatment, in the HD group also during the second week. Moreover, in the HD group statistically significantly lower weight gain was also seen in weeks 8 and 9 and in the MD body weight even declined slightly in week 12. In female animals statistically significantly lower body weight gain was seen in the LD group in weeks 1 and 4 and in the MD group in week 4. In male animals a slight to moderate effect on body weight was seen at 100 and 300 mg/kg/day leading to a 8 and 9 % lower body weight at the end of the treatment period, whereas a marked effect was observed at 1000 mg/kg/day. At the highest dose level the difference to controls surpassed 10 % after 5 weeks, reached statistical significance from week 9 onwards and finally - after 90 days - was 16 % below controls. In female animals body weights were only slightly lower in the dose groups compared to the control group, leading to differences between 1 and 3 %, which were not statistically significant and not considered adverse.
- Food consumption:
In male – but not female animals food consumption was lower in the dose groups when compared to controls. At the end of the treatment period, approx. 7 and 6 % less food was consumed in LD and HD groups when compared to controls. No considerable effect was seen in the MD group. Also no considerable effect on food consumption was seen in female animals of the dose groups.
- Haematology and Blood Coagulation:
At the end of the treatment period a minimally – but statistically lower count of white blood cells was noted in male – but not female animals – at all dose levels, when compared to controls. The differential blood analysis revealed a slightly – but statistically significantly higher rate of neutrophils associated with a minimally – but statistically significantly lower rate of lymphocytes in male animals of these groups compared to controls. The above-mentioned statistical differences are not assumed to be toxicologically relevant as the values were within the normal range of historical data and a dose-dependency was not observed.
A slightly – but statistically significantly lower rate of eosinophils observed in male animals at 100 and 1000 mg/kg bw compared to controls is not assumed to be toxicologically relevant, as the level was within the normal range of historical control data. Minimally more reticulocytes determined in male animals at 1000 mg/kg bw than in controls are not assumed to be toxicologically relevant. No statistically or biologically relevant differences in haematological parameters and coagulation parameters were found in female animals of each of the dose groups, when compared to controls.
- Clinical Biochemistry:
Bis(2-ethylhexyl)peroxydicarbonate had no toxicologically relevant effect on clinical biochemistry parameters determined at the end of the treatment period of this study.
-Urinalysis:
In the urine of male animals treated with bis(2-ethylhexyl)peroxydicarbonate at 1000 mg/kg/day Bilirubin was increased when compared to controls. Bilirubin was detected in 4/9 animals compared to 0/10 in the control group. At 100 mg/kg/day 2/10 and at 300 mg/kg/day 0/10 animals were affected. Even higher bilirubin levels were seen in female animals of the dose group (9/10, 7/10 and 2/10 animals at 100, 300 and 1000 mg/kg/day, respectively). Although this effect seemed to be inversely dose-dependent and also affected 3/10 control animals, an effect of the test item seems unlikely. Moreover, there were no histopathological correlates in liver or kidney of female animals.
- Pathology:
Single macroscopic findings observed at necropsy at the end of the treatment period were: diaphragmal herniation of the liver in MD animal no. 69, fluid-filled ureter of control animal no. 45, a cyst at the ovary of control animal no. 44. These findings are not assumed to be test item-related. A fluid-filled uterus in 5/10 animals of the LD and MD groups and 1/10 animals of the control and HD groups is related to cyclicity and is not assumed to be caused by the test item.
- Organ weights:
As body weight of male animals of the dose groups was significantly lower than in controls at the end of the treatment period, weights of a number of organs were statistically significantly higher when put in relation to the animal’s body weight and compared to controls: i.e. brain (+18 %), spleen (+28 %), testes (+14 %), kidneys (+11 %), pituitary gland (+37 %), prostate (+21 %), epididymides (+35 %), liver (+9 %). This was also true for kidneys (+11 %), prostate gland (+19 %) and pituitary gland (+45 %) of male animals of the MD group. Absolute weights of these organs, however, did not significantly differ between the groups. Only in liver and kidneys did the significant weight changes coincide with histopathological findings. In female animals liver weight was also statistically significantly higher than in controls, when put in relation to the animal’s body weight (+10 %) and correlated with histopathological findings. Absolute thyroid/parathyroid weight of female animals was slightly – but not statistically significantly higher in the MD and HD groups when compared to controls (+16 %). This was not associated with any histopathological finding in HD animals. A higher heart weight in female animals of the LD group did also not coincide with any histopathological finding and is not assumed to be toxicologically relevant.
- Histopathology:
The test item caused a minimal hepatocellular hypertrophy in the liver of a few animals at 1000 mg/kg bw/day without any further associated adverse lesion. Therefore, liver findings were deemed to be of metabolic adaptation only. In the kidneys, there was an increase of hyaline inclusions and α2-microglobulin in males of all test item-treated groups. The hyaline inclusion that were recorded in hematoxylin and eosins stained sections at increased incidences/severities in all test item treated male groups corresponded with the presence of α2-microglobulin. The increase of this protein was statistically significant in all test item groups compared to controls but not between groups 2 and 3 or 4, and between groups 3 and 4. There were no further changes in the kidneys. The presence of α2-microglobulin is a typical male rat issue. In case of no further lesion, it is deemed to be a non-adverse metabolic change only.

Effect levels

Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
In a 90-day subchronic toxicity study (OECD 408) bis(2-ethylhexyl)peroxydicarbonate (98 % 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 can be established at 1000 mg/kg/day.
Executive summary:

In a 90-day subchronic toxicity study (OECD 408) bis(2-ethylhexyl)peroxydicarbonate (98 % purity) was administered orally to 10 Wistar rats/sex/group at nominal dose levels of 0, 100, 300 and 1000 mg/kg bw/day.

Under the conditions of this study, there was no mortality. No clinical signs of toxicity were noted during the treatment period and no alterations in parameters of clinical pathology and no test item-related gross lesions were recorded at the end of the treatment period. An effect of bis(2-ethylhexyl)peroxydicarbonate on body weight development of male animals was noted which is assumed to be related to species specific histopathological findings in the kidney described below. The test item caused a minimal hepatocellular hypertrophy in the liver of a few animals at 1000 mg/kg without any further associated adverse lesion. Therefore, liver findings were deemed to be of metabolic adaptation only. In the kidneys, there was an increase of hyaline inclusions and α2-microglobulin in males of all test item-treated groups. The increase of this protein was statistically significant in all test item groups compared to controls but not between groups 2 and 3 or 4, and between groups 3 and 4. The positivity for α2-microglobulin increased dose-dependently in males from all test item-treated groups and shows that histomorphological changes in the kidneys can be interpreted as species-specific findings that are not relevant to human. There were no further lesions recorded in kidneys. Based on the pathology evaluation, the NOAEL can be established at 1000 mg/kg/day.

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