Registration Dossier

Administrative data

Description of key information

Two repeated dose toxicity studies are available for the target substance. In a sub-acute 28-day repeated dose toxicity study (OECD 407), the NOAEL was considered to be 500 mg/kg bw/day based on adverse histopathological findings. In a sub-chronic 90-day (OECD 408) repeated dose toxicity study, the NOAEL was considered to be 1000 mg/kg bw/day for females. Due to species-specific findings, which are considered as not relevant for humans, a NOAEL could not be determined for male rats.

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:
2012-04-17 to 2012-11-09
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 - 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
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
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
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).
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.
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
Critical effects observed:
not specified

***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.

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.

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2016-09-30 to 2017-08-21
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)
Version / remarks:
adopted 21 September 1998
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no
Specific details on test material used for the study:
- Batch no.: 1224545-01
- Physical state: Solid
- Colour: White
- Molecular weight: 398.54 g/mol
- Purity: 95.6 %
- Storage conditions: 2-8 °C, protected from light
- Expiry date: Not applicable
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: approx. 7-8 weeks
- Weight at study initiation: males: 155–195 g (mean: 179.33 g ± 20% = 143.46–215.19 g); females: 124–147 g (mean: 138.5 g ± 20% = 110.84–166.26 g)
- Fasting period before study: Not specified
- Housing: Full barrier in an air-conditioned room ; animals were kept in groups of 5 animals/sex/group/cage in type IV polysulphone cages on Altromin saw fibre bedding
- Diet (e.g. ad libitum): Ad libitum, Altromin 1324
- 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
- Humidity (%): 55 ± 10
- Air changes (per hr): 10/h
- Photoperiod (hrs dark/hrs light): 12/12
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
The test item was weighed into a tared plastic vial on a precision balance. The test item formulations were prepared at room temperature by adding the vehicle (corn oil) to give the appropriate final concentration and vortexing it. The test item formulations were prepared freshly on each administration day before the administration procedure. After preparation, the test item formulations and control item were stored at 2 to 8 °C or on crushed water ice and were administered within 6 hours after preparation. Shortly before administration, formulations and control item were brought to room temperature. Formulates were kept under magnetic stirring during the daily administration.

VEHICLE
- Justification for use and choice of vehicle (if other than water): The vehicle was selected as suggested by the sponsor based on the test item’s characteristics, testing guideline and the previously performed 28-day study.
- Concentration in vehicle: 20 mg/mL (LD), 60 mg/mL (MD), 200 mg/mL (HD)
- Amount of vehicle (if gavage): Application volume: 5 mL/kg bw
- Lot/batch no. (if required): MKBQ9948V, MKBV2080V, MKBW9504V
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
During the study samples were collected for the investigation of homogeneity and substance concentration. Samples were taken from the top, middle and bottom of prepared formulations from all dose groups and from the middle of the control group in study week 1, 5, 9 and the last week of the treatment period (in total 40 samples). All samples of dosing formulations were analysed at Eurofins Munich in accordance with GLP under the reference number 165860. After transfer to the analytical department samples were stored at 2-8 °C and were measured the same day within 6 hours after arrival. Exact procedures are mentioned in the analytical phase plan which was amended to the study plan.
Duration of treatment / exposure:
90 days
Frequency of treatment:
once per day, 7 days/week
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
C ( vehicle control)
Dose / conc.:
100 mg/kg bw/day (nominal)
Remarks:
LD (low dose)
Dose / conc.:
300 mg/kg bw/day (nominal)
Remarks:
MD (mid dose)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Remarks:
HD (high dose)
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 animal 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). According to the results of a previous 28-day study the doses were chosen and 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
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: General clinical observations were made at least once a day, preferably at the same time each day. 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
- 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 for each animal was measured at least weekly during the treatment period.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Ophthalmological examinations using an ophthalmoscope were 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 period prior to or as part of the sacrifice of the animals
- 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), mean platelet volume (MPV), white blood cells (WBC), neutrophils (Neu), lymphocytes (Lym), monocytes (Mono), eosinophils (Eos), basophils (Baso), large unstained cells (Luc)
- Coagulation parameters checked: prothrombin time (PT) and 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 period prior to or as part of the sacrifice of the animals
- Animals fasted: Yes, overnight
- 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: A urinalysis was performed with samples collected from all animals prior to or as part of the sacrifice of the animals.
- Animals fasted: Yes, overnight
- Parameters examined: specific gravity, nitrite, pH-value (pH), protein, glucose, ketone bodies (Ket), urobilinogen (UBG), bilirubin (BIL), erythroctes (Ery), leukocytes (Leu), colour/appearance of the urine

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 behavioral observations were made outside the home cage using a functional observational battery of tests
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
One day after the last administration (study day 91) all surviving animals of the treatment period were sacrificed using anesthesia (ketamine/xylazine) and were subjected to a detailed gross necropsy which included 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 in box "Any other information on materials and methods incl. tables") 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. For testis, a detailed qualitative examination was made, taking into account the tubular stages of the spermatogenic cycle at evaluation of additional hematoxylin-PAS (Periodic Acid Schiff) stained slides. Any gross lesion macroscopically identified was examined microscopically in all animals.

ORGAN WEIGHTS: Yes
The wet weight of the organs (see table 2 in box "Any other information on materials and methods incl. tables") of all sacrificed animals was recorded as soon as possible. Paired organs were weighed together. Organ weights of animals found dead or
euthanised for animal welfare reasons were not recorded.
Other examinations:
EXAMINATION OF FERTILITY PARAMETERS:
The oestrous cycle of all female animals was examined daily over a period of 21 days before the day of scheduled necropsy. At necropsy (one day after the last administration), left epididymis, left testis and left vas deferens were separated and used for evaluation of sperm parameters. Epididymal sperm motility and testicular sperm count was evaluated in all male animals using Hamilton Thorne Sperm Analyser (TOX IVOS Version 13.0). Furthermore, sperm morphology slides were prepared from all animals subjected to necropsy one day after the last administration. Sperm from the left vas deferens was transferred to 0.1 % bovine serum albumin solution. For staining, two drops of 1 % aqueous Eosin-Y solution were mixed with six drops of the sperm suspension. The stained sperm suspension was used to prepare smears on slides. After complete drying, the slides were dipped into 0.1 % acetic acid for approximately 30 seconds to intensify the coloring. Sperm morphology examinations were performed in male animals of the control and the high dose group.

Analysis of α2μ-globulin in male kidneys:
Immunohistochemistry of α2μ-globulin was performed in order to investigate species specifity of kidney findings in male animals and to rule out human relevance. Immunohistochemistry of α2μ-globulin and/or a specific staining was performed on kidney of all male animals sacrificed at the end of the treatment period of the study (40 animals). Immunohistochemistry was performed with rat α2μ-globulin antibody (monoclonal mouse IgG1 clone #129736, R&D Systems). The positive area on the total area was measured in μm² per particles 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). Furthermore, all data were statistically by the t-test using the GraphPad system.
Statistics:
A statistical assessment of 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 either a parametric one-way ANOVA and a post-hoc Dunnett's test or a non-parametric Kruskal-Wallis test and a post-hoc Dunn's test, based on the results of homogeneity and normality tests. A statistical assessment of the results of sperm analysis and oestrous cyclicity were performed by comparing values of dosed with control animals using a parametric one-way ANOVA and a post-hoc Dunnett's test. These statistics were performed with Ascentos 1.1.3 software or GraphPad Prism V.6.01 software (p < 0.05 is considered as statistically significant).
Clinical signs:
no effects observed
Description (incidence and severity):
Salivation was noted dose dependently in 2/10 males and 2/10 females of the LD group, in 5/10 males and 5/10 females of the MD group and in 7/10 males and 6/10 females of the HD group on several days of treatment. Furthermore, moving the bedding was occasionally observed in 1/10 males and 1/10 females of the LD group and in 10/10 males and 10/10 females of the HD group. Moving the bedding and salivation were seen transiently in timely relation to dose administration and were considered as slight clinical signs elicited by local effects of the test item formulation and/or attributed to discomfort of the animals due to the oral administration, but not systemic toxicity.
One day before unscheduled euthanasia, one female of the MD group was noted with slight piloerection. On the next day, the animal showed severely reduced health condition with marked piloerection, reduced spontaneous activity, hunched posture, wasp waist, abnormal breathing, red nasal discharge and dry nose and eyes.
One female of the HD group which was euthanised for animal welfare reasons on treatment day 15 showed first signs of reduced health condition on treatment day 7 with moderate piloerection, slightly reduced spontaneous activity and hunched posture. It recovered afterwards until day 12 from which day onwards it was seen with piloerection, wasp waist and abnormal breathing. It was euthanised in a moribund condition with additionally observed hunched posture and avoidance of food intake.
Abnormal breathing was also noted on one single day of treatment in one male of the LD group, on three consecutive days in one male of the MD group and on 7 consecutive days in one female of the MD group. As this was seen only transiently in single animals without clear dose dependency and without further reduced health condition, clinical signs of abnormal breathing in these animals were assumed to be related to the procedure of oral gavaging followed by possible regurgitation with local effects due to incidental aspiration, but not systemic toxicity. One female of the HD group was transiently seen with a reduced health condition with piloerection, hunched posture, slightly reduced spontaneous activity and wasp waist on one day (treatment day 49) and subsequently further slightly reduced spontaneous activity for 2 more days (treatment days 50-51). The animal fully recovered afterwards and there were no remarkable microscopic findings. Local alopecia and crust were noted in single animals irrespective of the group and were considered incidental in nature.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
In the course of the study, two females were euthanised in a moribund condition for animal welfare reasons. One female of the MD group was euthanised on treatment day 32. This animal was seen with sudden respiratory problems and showed an enlarged heart at necropsy. One female of the HD group was euthanized on treatment day 15. This female was noted with worsening health condition and abnormal breathing which at necropsy was related to a fluid filled thorax with pleural adhesions. Microscopic findings were indicative for misgavage. All remaining animals survived the treatment period.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
The test item had no adverse effect on body weight development in this study. In males, slightly to moderately and statistically significantly lower body weight gain was seen in single weeks of treatment when compared to the control group. When related to the whole treatment period (day 1-90), mean body weight gain was 17 %, 18 % and 20 % below controls in the male LD, MD and HD group, respectively. However, this did not achieve statistical significance and had no statistically significant effect on mean body weight. A marginal tendency towards lower mean body weights was noted in all dose groups in the course of the study period. On the last day of the treatment period, mean body weight was 7%, 8% and 10% below controls in the LD, the MD and the HD group, respectively, but without achieving statistical significance. No such effect was noted in females. Due to the missing statistical significance for mean body weight and mslightness of the differences for body weight development between the male dose groups and controls, effects on male body weight were considered as slight effects of the treatment with the test item but not as adverse. Body weight development of the female dose groups was comparable to controls.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
There was no effect of toxicological relevance on food consumption in any of the dose groups. When related to the whole study period, mean daily food consumption was comparable between all male dose groups and controls. Marginally higher mean daily food consumption in the female HD group when compared to the control group was not considered as biologically relevant as values were within the normal range of variation and as there was no significant impact on body weight.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Description (incidence and severity):
There were no ophthalmoscopic findings in any of the animals of this study.
Haematological findings:
no effects observed
Description (incidence and severity):
At the end of the treatment period, marginally but statistically significantly lower mean platelet volume (MPV) was noted in males and females of the HD group (14% and 8% below controls, respectively) when compared to the respective control group. However, platelet count was within the normal range of variation and no microscopic correlate was observed in the bone marrow. Thus, these isolated marginal differences to controls were not considered as toxicologically relevant.
Slightly and statistically significantly higher percentage of reticulocytes was seen in the male LD, MD and HD group when compared to the control group (23, 21, and 29 % above controls). However, as there was no clear dose dependent pattern, as values were within the normal range of variation and as no such effect was seen in females, slight and not consistent differences to controls were not considered as toxicologically relevant but incidental.
Slightly higher number of white blood cell count (WBC) was noted in the female MD group and HD group (48 and 50 % above controls, respectively) when compared to controls with achieving statistical significance in the HD group. In males, a marginal tendency towards higher WBC was seen in all dose groups (up to 13 % above controls) without statistical significance. As mean values were within the normal range of variation and individual values were seen with high inter-individual variability without consistency throughout all groups and without correlating findings in other parameters, differences to controls were considered as incidental and not related to the treatment with the test item.
The isolated but statistically significant finding of higher percentage of monocytes in the female MD and HD group (47 and 55 % above controls, respectively) was considered as incidental with values in the normal range of variation. In males, slightly but not statistically significantly higher percentage of monocytes (35 % above controls) was also within the normal range of variation.
Further slight and not statistically significant changes were seen without consistency and were considered as incidental such as lower percentage of eosinophils in all male dose groups and higher percentage of eosinophils in all female dose groups without dose dependency when compared to the respective controls. Values were within the normal range of variation.
Besides, there were no further statistically significant differences for parameters of haematology. Slight differences between the groups and deviating values in individual animals were considered as incidental in nature.
Markedly deviating values for several red blood cell parameters as well as platelets in a single gfemale of the HD group were considered as outlier.
A statistically significant effect was observed for blood coagulation in males. Prothrombin time (PT) was noted to be marginally but statistically significantly higher in the male LD, MD and HD group when compared to the control group (8, 12, and 15 % above controls, respectively). However, as values were well within the normal range of variation, this marginal difference was not considered as biologically relevant. No effect was seen for activated partial thromboplastin time (aPTT). Blood coagulation in the female dose groups was comparable to controls.
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
At the end of the treatment period, few statistically significant changes for individual parameters were noted without consistency. In males, the mean value for total bile acid (TBA) was moderately and statistically significantly below controls in the HD group (-60 %). In females, no statistically significant difference to controls for TBA was achieved and no dose dependent pattern was followed (female LD group 41 % below controls, female MD group 2 % above controls, female HD group 17 % below controls). No remarkable microscopic findings were noted in the liver except for adaptive metabolic changes. Furthermore, values were in the normal range of variation. Marginally but statistically significantly lower mean value for sodium (Na) was observed in the male HD group when compared to controls (2 % below controls). As values were within the normal range of variation, this marginal difference to controls was not considered as biologically relevant. In females, no statistically significant difference was observed and values showed increased inter-individual variability without consistency.
Though statistically significant, marginally lower mean value for ASAT in the male HD group (22 % below controls) was considered as incidental with females not affected and values in the normal range of variation.
The isolated finding of marginally higher cholesterol in the female MD group (29 % above controls) was considered as incidental. Besides, there were no further statistically significant differences for parameters of clinical biochemistry. Slight differences between the groups and deviating values in individual animals were considered as incidental in nature.
Urinalysis findings:
no effects observed
Description (incidence and severity):
At the end of the treatment period, most parameters of urinalysis of the dose groups were not considerably different to the control group and were within the normal range of variation. However, a slight tendency towards slightly lower urinary pH-value was noted in the HD group when compared to controls. Mean pH-value was 6.6, 6.9 and 5.6 in the female LD, MD and HD group, respectively, when compared to 7.3 in the female control group. In males, mean pH-value was 7.0, 6.4 and 6.4 in the male LD, MD and HD group, respectively, when compared to 7.2 in the male control group. However, individual values did not follow a strict dose dependent pattern and showed no clear consistency. A slightly lower value of a pH of 5 was noted in one female of the LD group, 4 females of the HD group and one male of the MD group. Further slightly deviating values for individual animals throughout all groups were considered as incidental, isolated findings without relation to the treatment with the test item.
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
In the functional observation battery, slight differences between the groups before initiation of treatment were considered as incidental in nature such as marginally but statistically significantly higher number of supported rearings in the male HD group and statistically significant difference for number of animals sleeping/moving in the cage in the female HD group when compared to the respective controls. At the end of the treatment period, there were no effects of toxicological relevance on tested parameters of functional observation battery including gait, strength and reflexes. The isolated finding of statistically significantly higher number of males sleeping during cage-side observation in all dose groups when compared to the respective controls at the end of the treatment period was not observed during weekly detailed clinical observations and was considered as incidental.
There were further marginal but statistically significant differences of the male MD and male HD group in terms of marginally higher spontaneous activity, higher number of supported rearings and lower number of defecations when compared to the control group. As all these findings were within the normal range of variation and were seen without consistency and without impact on other parameters of this study, they were considered as incidental and not biologically significant.
Marginally but statistically significantly lower body temperature of all male dose groups when compared to the control group at the end of the treatment period was seen without clear dose dependency and was not noted in females. As values were within the normal range of biological variation, statistically significant difference to controls was considered as incidental. There were no statistically or biologically significant effects in females at the end of the treatment period.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
At the end of the treatment period, mean liver weight was noted to be slightly and statistically significantly higher in the female HD group when compared to the control group (absolute weight 22 % above controls, relative weight (compared to body weight) 19 % above controls). No such effect was seen in males.
Mean weight of kidneys was observed to be slightly higher in all male dose groups when compared to controls but without following a dose dependent pattern. Absolute weight was 15, 19, and 13 % higher compared to controls in the LD, the MD and the HD group, respectively, with achieving statistical significance in the MD group. Relative weight (to body weight) was statistically significantly 28, 33, and 27 % higher, respectively. No such effect was seen in females. Marginally but statistically significantly higher relative (compared to body weight) but not absolute weight of testes was noted in the male MD and the HD group. However, differences to controls were marginal (both 14 % above controls) and did not follow a clear dose dependent pattern. Furthermore, no microscopic correlate was seen at histopathologic examination and no relevant differences for male fertility parameters were noted between the dose groups and controls. Absolute weight of testes was in the normal range of variation. Thus, marginal differences for weight of testes were not considered as toxicologically relevant. Markedly deviating organ weight values in one single animal 13 of the LD group for reproductive organs and liver were seen in correlation to macroscopic gross lesions and were considered as outlier. Mean absolute weight of thyroid/parathyroid glands was 21 % lower in the male HD group compared to the control group. However, there was no statistically significant difference, values were in the normal range of variation and no such effect was seen in females.
Mean absolute weight of pituitary gland was 20 % below controls in the male HD group. Based on high inter-individual variability no statistical significance was achieved. Values were in the normal range of variation and no such effect was seen in females.
Marginally but statistically significantly higher relative (compared to body weight) weight of heart was seen in the male HD group (14 % above controls). Differences to controls in the dose groups did not follow a dose dependent pattern and mean absolute values were comparable between the dose groups and controls with values in the normal range of variation. There was no statistically significant effect in females.
Uterus weight was seen with high inter-individual variability but with values within the normal range of variation so that slightly but not statistically significantly higher mean absolute uterus weight in the female LD and HD group was considered as incidental (27 and 21 % above controls, respectively).
Without correlating microscopic lesions, marginal to slight weight differences to controls in testes, thyroid/parathyroid glands, pituitary gland and heart were not assumed to be biologically relevant. There were no further statistically or biologically significant differences between the dose groups and the control group for the remaining organ weight data.
Gross pathological findings:
no effects observed
Description (incidence and severity):
One female of the MD group which was euthanised in a moribund condition with sudden respiratory problems showed an enlarged heart at necropsy. One female of the HD group which was also euthanised in a moribund condition including the clinical sign of abnormal breathing showed a fluid filled thorax with pleural adhesions at necropsy. Few gross pathological changes were recorded at necropsy in the surviving animals at the end of the treatment period without following a dose-dependent pattern. One male of the LD group was seen with several gross lesions (abnormally coloured (spotted) liver, bladder stone and discoloured (red) bladder as well as small sized testes, epididymides, prostate gland and seminal vesicles with coagulating glands). By microscopic examination findings correlated with urothelial hyperplasia, tubular degeneration in testis, hypospermia and atrophy of accessory sex organs. One further male of the MD group was noted with discoloured (pink) testes with a small sized right testis and right epididymis. Further single gross pathological changes were recorded in one male of the LD group (discoloured (pale) kidneys) and one further male of the MD group (enlarged thyroid gland). One female of the MD group was seen with pleurodiaphragmatic adhesions, adhesion of thymus with diaphragma and local discolouration of the colon (diaphragmatic flexure). Absent left side of testis and epididymis was observed in one mail of the LD group which was considered as biological variation, not related to the treatment with the test item. Dilated uterus was seen in single females at terminal sacrifice irrespective of the dose group.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Under the conditions of this study, the test item caused a minimal hepatocellular hypertrophy in the liver of a few HD animals and an increase of α2-microglobulin in male kidneys of all test item-treated groups. The hyaline inclusions 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. The findings were associated with tubular basophilia and tubular cell necrosis. There were no accompanying lesions noted in the livers of animals with hepatocellular hypertrophy. The latter was considered therefore an adaptive metabolic effect. A minor increase of thymic atrophy is considered to be indicative for stress. After histopathological evaluation, gross lesions at necropsy were unremarkable and could not be attributed to the treatment with the test item. The distributions among the groups were considered to be incidental, reflecting the usual individual variability.
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed
Description (incidence and severity):
FERTILITY PARAMETERS:
The test item had no effect on epididymal sperm motility or testicular sperm count analyzed at the end of the treatment period of this study. The statistical analysis showed no significant changes between the control group and any of the dose groups neither in the percentage of motile, static or rapidly moving epididymal sperms nor in testicular number of sperms/g testis. Sperm staging and evaluation of sperm morphology did not reveal any indicator for toxicity induced by the test item. Bis(4-tert-butylcyclohexyl) peroxydicarbonate had no statistically or biologically significant effect on the oestrous cycle evaluated over a period of 21 days before the day of scheduled necropsy. There were no considerable, consistent differences in the length or sequence of cycle stages between the dose groups and the control group. Deviations from the physiological 4 or 5 day cycle in the rat were observed rarely, mainly as irregularly short cycles irrespective of the dose group. A 3 day cycle was observed in one female of the control group, one of the LD group and one of the MD group. Another female of the control group also showed an irregular sequence with two consecutive 2 day cycles. In the HD group, one female was observed with a period of persistent diestrous for almost two weeks. Cyclicity normalized again at the end of the treatment period with a normal 4 day cycle. As the observed period of transiently prolonged diestrous was a single case in a single female, it was considered as incidental and not toxicologically relevant. There were no microscopic abnormalities in the reproductive organs of this female.
Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: no treatment-related findings observed in females
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
Relevant for humans:
no

Dose formulation analysis

Concentration and homogeneity analysis of formulation samples was performed in study weeks 1, 5, 9 and last week of the study for all dose groups. The mean recoveries observed for the LD group were between 101.5 % and 119.9 % of the nominal value, between 94.6 % and 101.1 % for the MD group and between 102.9 % and 112.9 % of the nominal value for HD group. The mean recoveries observed in the LD, MD and HD groups were 108.4, 98.1, and 108.3 % of the nominal concentration, respectively. The coefficients of variation of the different sampling locations (top, middle, bottom) was between 2.9 and 12.0 % in the LD group, between 1.5 and 3.1 % in the MD group, and between 1.2 and 3.9 % in the HD group.

Conclusions:
In a 90-day subchronic toxicity study (OECD 408), bis(4-tert-butylcyclohexyl) peroxydicarbonate (98.5 % 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 bw/day. Based on the pathology evaluation a NOAEL could not be established for males.
Executive summary:

In a 90-day sub-chronic toxicity study (OECD 408), bis(4-tert-butylcyclohexyl) peroxydicarbonate (98.5 % purity) was administered orally to 10 Wistar rats/sex/group at nominal dose levels of 0, 100, 300 and 1000 mg/kg bw/day. No clinical signs of systemic toxicity were observed in this study. The test item had no effect on body weight development. At a dose level of 1000 mg/kg body weight/day a tendency towards slightly lower body weight gain was observed in male but not female animals. As there was just a marginal impact on mean body weight of males (≤ 10 % difference to controls) without achieving statistical significance, effects were considered as slight effects of the treatment with the test item but not as adverse. The test item caused a minimal hepatocellular hypertrophy in the liver of a few HD animals without any further associated adverse lesions. Therefore, liver findings were deemed to represent metabolic adaptation only. A minor increase of thymic atrophy was considered to be indicative for stress. In the kidneys, there was a dose-dependent increase of hyaline inclusions and α2-microglobulin in males of all test item-treated groups. The findings were associated with tubular cell necrosis and tubular basophilia in all test item-treated groups. Based on the pathology evaluation, the NOAEL for male rats could not be established. Nevertheless, a LOAEL of 100 mg/kg bw/day for male rats is assumed based on the species-specific findings. The presence of α2-microglobulin and subsequent secondary findings like cell necrosis and tubular basophilia are species-specific findings that are not relevant for humans. The NOAEL for female rats was considered to be 1000 mg/kg bw/day.

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:
NOAEL
500 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
GLP guideline study

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

Two repeated dose toxicity studies are available for bis(4-tert-butylcyclohexyl) peroxydicarbonate.

In a sub-acute toxicity study the test substance was administered to 5 Wistar rats/sex/dose by gavage at nominal 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 to be altered in the treated groups. However, all group means and the 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 discoloured 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/day, 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 bw/day.

In a 90-day subchronic toxicity study (OECD 408), bis(4-tert-butylcyclohexyl) peroxydicarbonate was administered orally to 10 Wistar rats/sex/group at nominal dose levels of 0, 100, 300 and 1000 mg/kg bw/day. No adverse clinical signs of systemic toxicity were observed in this study. The test item had no effect on body weight development. At a dose level of 1000 mg/kg body weight/day a tendency towards slightly lower body weight gain was observed in male but not female animals. As there was just a marginal impact on mean body weight of males (≤ 10 % difference to controls) without achieving statistical significance, effects were considered as slight effects of the treatment with the test item but not as adverse. The test item caused a minimal hepatocellular hypertrophy in the liver of a few HD animals without any further associated adverse lesion. Therefore, liver findings were deemed to represent metabolic adaptation only. A minor increase of thymic atrophy was considered to be indicative for stress. In the kidneys, there was a dose-dependent increase of hyaline inclusions and α2-microglobulin in males of all test item-treated groups. The findings were associated with tubular cell necrosis and tubular basophilia in all test item-treated groups. Based on the pathology evaluation, the NOAEL for male rats could not be established. Nevertheless, a LOAEL of 100 mg/kg bw/day for male rats is assumed based on the species-specific findings. The presence of α2-microglobulin and subsequent secondary findings like cell necrosis and tubular basophilia are species-specific findings that are not relevant for humans. Thus, this adverse effect observed in male rats will not be further considered for hazard and risk assessment purposes. The NOAEL for female rats was considered to be 1000 mg/kg bw/day.

Justification for classification or non-classification

Based on the available data from two repeated dose toxicity studies (OECD 407, OECD 408), bis(4-tert-butylcyclohexyl) peroxydicarbonate does not warrant classification for specific target organ toxicity.