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Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2012-04-17 to 2012-11-09
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:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
- Name of test material (as cited in study report): PEROXAN BCC
- Substance type: pure active substance
- Physical state: solid
- Analytical purity: 98.5%
- Lot/batch No.: 1092801
- Storage condition of test material: 2 to 8 C, protected from light

Test animals

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 - 193 g, females: 131 - 149 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:
corn oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS: the formulations (test item in corn oil) were prepared daily before administration to the animals.

DIET PREPARATION
- Rate of preparation of diet (frequency): not relevant; the test material was given to the animals by gavage

VEHICLE
- Justification for use and choice of vehicle (if other than water): selected as suggested by the sponsor in accordance with thest material's charcteristics and the testing guideline.
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 once in the 1st and once in the 3rd week. Stability was tested once in the start of treatment period. 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 (LD), 500 (MD), 1000 (HD) 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
Total volume given per dose: 5 mL/kg bw
Gavage administrations of corn oil to control animals

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.

OPHTHALMOSCOPIC EXAMINATION: Yes, 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, overnight
- 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: prothrombin time and 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
- 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 sacrificed one day after the last treatment, and a gross necropsy was performed.
Spontaneous gross pathological findings like discolored red axillary lymph nodes (1/5 in C) and red spots on jejunum (1/5 in MD) in males and fluid distension of uterus (1/5 In HD) and cyst on ovary (1/5 in MD) were observed in females.
In males, statistical analysis of organ weight data revealed significant increase in absolute and relative (to brain weight and body weight) liver weights in MD and HD dose group when compared with controls although statistical significance was not achieved for increase in absolute and relative (to brain weight) liver weight in MD group. There was also statistically significant decrease in absolute heart weight but there was no effect on relative heart weights. Statistically significant increase in relative (to body weight) total kidney weights was observed in MD and HD group when compared to the controls. The absolute and relative (to brain weight) thymus weights were statistically significantly reduced in all treatment groups when compared with controls. There was also statistically significant increase in relative (to body weight) testes weight was observed in HD group when compared with controls.
In females, substantial increase in absolute and relative (to body weight) liver weights was observed in HD group although statistical significance was not achieved for absolute liver weights. There was also statistically significant increase in absolute total adrenal weight observed in HD group when compared with controls.
 
 
HISTOPATHOLOGY:
In the liver, minimal centrilobular hepatocellular hypertrophy was noted in a proportion of males and females treated at 1000 mg/kg/day and were considered to be correlated to the observed higher liver weight in this group. It was interpreted as a minor adaptive change of the liver, probably involving the induction of hepatic microsomal enzymes. In the thyroid gland, a minimal diffuse follicular cell hypertrophy was observed in two males treated at 1000 mg/kg/day and one single male at 500 mg/kg/day. In view of the concurrent effect on the liver this minor change was considered to be most probably related to increased clearance of thyroid hormones by increased activity of hepatic microsomal enzymes and subsequent thyroid gland stimulation. It was therefore considered to be secondary to the liver change seen in this study.
In the kidney of males only, hyaline droplets in corticotubular cells were seen in a dose-related manner in all treated groups. These were considered to representα2-microglobulin. This change was associated with minimal degeneration of corticotubular epithelial cells, multifocal debris-filled tubules in the inner cortex and an increased incidence and severity of cortical basophilic tubules, all of which were considered to be most likely the consequence of corticoepithelial overload withα2-microglobulin. As the renal storage ofα2-microglobulin is commonly recognized to be a male rat-specific event, its test item-related increase in severity and associated secondary renal lesions in this study are regarded to be of no relevance for man.
In the thymus of males treated at 1000 mg/kg/day, there was a minimal tendency towards more prominent atrophy/regression of the thymus, when compared to the control group. This was considered to most likely be an unspecific borderline effect and to be secondary to the prominent renal pathology observed in this male group.
As a conclusion, kidney and secondary thymus effects noted in this study were not considered relevant for humans. Histopathological liver changes and associated thyroid gland changes were minimal in degree and not seen at 100 mg/kg/day. As the liver weight increase remained very limited at 500 mg/kg/day, the dose of 500 mg/kg/day is considered to be the NOAEL (No Observed Adverse Effect Level) for pathology under the conditions of this study.
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 was performed for each gender by comparing values of dosed with control animals using a one-way ANOVA and a post-hoc Dunnett Test. The statistics was performed with GraphPad Prism V.5.01 software or IDBS Workbook 8.1.2, ANOVA v8.8 software (p<0.05 is considered as statistically significant).

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
no effects observed
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
no effects observed
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; no treatment related clinical effects.

BODY WEIGHT AND WEIGHT GAIN: slight changes in the body weight of males, still not statistically significant

FOOD CONSUMPTION: no effects

OPHTHALMOSCOPIC EXAMINATION: no effects

HAEMATOLOGY (see tables in the attached document): Statistically significant changes were seen in the MCV, MCHC, PLT, WBC, basophils of females in the MD group, and in PLT, neutrophils and lymphocytes in the HD group. No dose-dependent pattern was observed, and the values were within the normal ranges for the female rat. Increase in mean HB, lymphocytes and monocytes were seen in the male HD group; still values were within the historical control ranges. Hence, such effects were not related to the treatment with the test item.

CLINICAL CHEMISTRY: In males, statistically significant alterations in TP and TBIL was recorded in MD group; however, not in a dose-dependent manner. Creatinine increases in HD group, were corrobated by histopathological findings in the kidneys. In females, statistically significant changes were seen in ASAT in MD group and glucose in MD and HD group, when compared with controls. There was also a statistically significant increase in cholesterol was observed in MD and HD group. All group mean and majority of individual values were within the historical control data range and due to lack of dose dependency, these statistically significant effects, were not assumed to be of any toxicological significance.

URINALYSIS: In males, the high level of erythrocytes were noted in LD (4/5 animals), MD (2/5) and HD 5/5) ; high level of bilirubin in LD (4/5), in MD (4/5) and in HD (5/5); high level of protein in Control (1/5), LD (3/5), MD (5/5) and HD (3/5); high level of ketone in LD (1/5), MD (4/5) and HD (3/5); high levels of glucose in LD (3/5), in MD (2/5) and HD (5/5); low urine pH in Control (1/5), LD (1/5), MD (1/5) and HD (1/5); high level of leucocytes in LD (5/5), MD (5/5) and HD (5/5): positive nitrite in Control (1/5), LD (1/5) and MD (1/5).

Urine parameter changes in males were corrobated by the rat-specific kidney effects detcted in the histopathological examination.

In females, the high level of erythrocytes were noted in LD (1/5 animals) and MD (1/5) ; high level of bilirubin in HD (1/5); high level of protein in Control (1/5) and HD (1/5); low urine pH in Control (1/5), LD (1/5), MD (3/5) and HD (1/5); high level of leucocytes in MD (1/5); positive nitrite in Control (2/5), LD (2/5), MD (3/5) and HD (1/5).

NEUROBEHAVIOUR: no effects

ORGAN WEIGHTS (see tables in the attached document): In males, significant increases were recorded in absolute and relative liver weights in MD and HD dose group when compared with controls.This effect on liver weight could be considered as treatment related effect which was correlated with the minimal histopathological changes observed in liver. Statistically significant decrease, still minimal, was seen in absolute heart weight. The effect was not considered of toxicological significance since relative weights were not changed. Significant increase in relative (to body weight) total kidney weights was observed in MD and HD group. The absolute and relative (to brain weight) thymus weights were statistically significantly reduced in all treatment groups, but it was considered most likely to be an unspecific borderline effect and, therefore, non adverse and of no relevance for man . Histopathologically, effect on kidney was regarded as male rat specific and therefore, of no relevance for humans. Relative testis weight was increased in the HD group.

In females, increase in absolute and relative (to body weight) liver weights was observed in HD group although statistical significance was not achieved for absolute liver weights. There was also statistically significant increase in absolute total adrenal weight observed in HD group when compared with controls.

GROSS PATHOLOGY: Spontaneous gross pathological findings like discolored red axillary lymph nodes (1/5 in C) and red spots on jejunum (1/5 in MD) in males and fluid distension of uterus (1/5 In HD) and cyst on ovary (1/5) in MD were observed in females.

HISTOPATHOLOGY: Histopathological findings considered to be test item-related were seen in the kidney, liver, thyroid gland and thymus. Out of these, only the liver and thyroid gland changes were considered to be relevant for humans. In the liver minimal centrilobular hepatocellular hypertrophy was seen in both sexes treated with the highest dose level, which was considered to be a metabolic adaptation.In the thyroid gland, a minimal diffuse follicular cell hypertrophy was observed in two males treated at 1000 mg/kg/day and one single male at 500 mg/kg/day. In view of the concurrent effect on the liver this minor change was considered to be most probably related to increased clearance of thyroid hormones by increased activity of hepatic microsomal enzymes and subsequent thyroid gland stimulation. It was therefore considered to be secondary to the liver change seen in this study. Hyaline droplets in corticotubular cells of the male kidneys were considered to represent a2-microglobulin, a male rat-specific protein. The effect is not deemed as relevant for humans. A minimal tendency towards more prominent atrophy/regression of the thymus, was seen in males treated with 1000 mg/kg bw/day. This was also considered an unspecific borderlined and secondary effect.

Effect levels

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Dose descriptor:
NOAEL
Effect level:
500 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 (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Histopathological liver changes and associated thyroid gland changes

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 500 and 1000 mg/kg bw), 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). This is 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:
Based on this study the NOAEL of the test material is considered to be 500 mg/kg body weight/day for the 28-day repeated dose oral toxicity study in male and female rats.
Executive summary:

In a subacute toxicity study Bis(4-tert-butylcyclohexyl)peroxydicarbonate (98.5%) was administered to 5 Wistar Rats/sex/dose in by gavage at dose levels of 0, 100, 500, 1000 mg/kg bw/day. 

No mortality and no treatment-related clinical symptoms were observed. Slight changes in male body weights observed, were not significant and were not considered treatment related. Several haematological and biochemical (blood and urine) parameters appeared altered in the treated groups. However, all group mean and majority of individual values were within the historical control data range and due to lack of dose dependency, these statistically significant effects, were not assumed to be of any toxicological significance. Spontaneous gross pathological findings like discolored red axillary lymph nodes (1/5 in C) and red spots on jejunum (1/5 in MD) in males and fluid distension of uterus (1/5 In HD) and cyst on ovary (1/5 in MD were observed in females.The LOAEL is 1000 mg/kg bw/d, based on histopathological findings in the kidney, liver, thyroid gland and thymus. Out of these, only the liver and thyroid gland changes were considered to be relevant for humans.The NOAEL is 500 mg/kg/d.

This subacute toxicity study in the rat is acceptable and satisfies the guideline requirement for a subacute oral study (OECD 407) in rat.