Registration Dossier

Administrative data

Description of key information

The substance was administered daily by oral gavage to Wistar rats of both sexes at at 100, 300 and 1000 mg/kg body weight/day for 28 days (5 animals / group), and a control group (vehicle, double distilled water). Test item-related non-adverse findings noted at 1000 mg/kg/day were restricted to slightly diminished fore limb grip strength in males only, reduced locomotor activity, reduced food consumption, and body weight development in males and females. Non-adverse findings noted in hematology parameters at 1000 mg/kg/day included compensatory reticulocytosis, whereas females also showed mild anemia. Mild reticulocytosis was also noted in females at 300 mg/kg/day. These findings are generally recognized as reversible after exposure is discontinued.

Males and females at 1000 mg/kg/day showed mild aberrations in lipid metabolism that were attributed to metabolic adaptation (therefore non-adverse). Increased organ weights included relative liver weights (considered to be an adaptive change and not adverse) and relative kidney weights in males and females and relative spleen weights in males only at 1000 mg/kg/day. Test item-related non-adverse and typically adaptive microscopic findings included hypertrophy of mucosal epithelium of duodenum, centrilobular hepatocellular hypertrophy and increased incidence and severity of extramedullary erythrocytic hemopoiesis (i.e. erythropoiesis) in both sexes treated with 1000 mg/kg/day, increased erythropoiesis in femur bone marrow in females treated with 1000 mg/kg/day, and enhanced hyaline droplets in renal proximal tubules as well as increased incidence and severity of tubular basophilia in the kidney in males treated with 300 and 1000 mg/kg/day. A (NOEL) of 100 mg/kg body weight/day and NOAEL of 300 mg/kg body weight/day were reported in the above study. However, based on human relevance and typical adaptive nature of some of the effects, a NOAEL of 1000 mg/kg bw/d was established as the correct NOAEL.

 

 

The substance was administered orally, by gavage, to Han Wistar rats for 13 weeks. Three groups, each comprising ten males and ten females, received the test item at doses of 80, 250 or 750 mg/kg/day and a similarly constituted control group received the vehicle (purified water) at the same dose volume.

The appearance and behavior of the animals were unaffected by treatment, there were no treatment-related findings at the sensory activity, grip strength and motor activity assessment in Week 12 and no deaths occurred during the treatment period. Overall body weight gain was reduced by approximately 15% in males receiving 750 mg/kg/day but there was no effect on their food consumption. Females were unaffected.

There were no treatment-related ophthalmoscopic findings.

The hematological examination in Week 13 indicated high reticulocyte count in animals receiving 750 mg/kg/day which associated with increased red cell distribution width in males and an increased mean cell hemoglobin in females but all other erythrocyte indices were unaffected. Males and females receiving 750 mg/kg/day had high neutrophil, lymphocyte, monocyte and large unstained cell counts, with males also showing increased eosinophil count and females showing increased basophil count, and there was also an increase of lymphocyte and basophil count in females receiving 250 mg/kg/day. As a consequence, total leucocyte counts were higher than controls in females receiving 250 mg/kg/day and in males and females receiving 750 mg/kg/day.

Treatment-related changes in the blood plasma in Week 13 comprised: high bile acid concentration in animals receiving 750 mg/kg/day; high urea/blood urea nitrogen concentrations in males receiving 250 mg/kg/day and in animals receiving 750 mg/kg/day; low creatinine concentration in females receiving 250 mg/kg/day and in males and females receiving 750 mg/kg/day; high glucose concentrations in animals receiving 750 mg/kg/day; increased high and low density lipoprotein concentrations with an associated increase of total cholesterol concentration in males and females receiving 750 mg/kg/day and which associated with high triglyceride concentrations in females; low calcium concentrations in animals receiving 750 mg/kg/day; high phosphorus concentrations in males receiving 750 mg/kg/day; low sodium and chloride concentrations in females receiving 750 mg/kg/day.

Urinary pH was slightly low in Week 13 in males and females receiving 750 mg/kg/day.

 

Acetylacetone Peroxide had no effect on estrus cycle or on spermmotility, morphology and sperm count in the testis and cauda epididymis.

 

Serum triiodothyronine (T3), thyroxine (T4) and thyroid stimulation hormone (TSH) concentrations were unaffected by treatment.

 

After 13 weeks of treatment kidney weights were high in females given 250 mg/kg/day and in males and females given 750 mg/kg/day, liver weights were high in males and females given 750 mg/kg/day and, in addition, males given 750 mg/kg/day had high spleen and low combined prostate/seminal vesicle/coagulating gland weight.

 

Pale kidneys were reported at the macroscopic examination in two males given 250 mg/kg/day and in eight males given 750 mg/kg/day.

 

Treatment-related histopathological findings were confined to the kidneys of males where tubular hyaline droplet accumulation was observed in four males given 80 mg/kg/day and all males given 250 or 750 mg/kg/day which, in some of the high dose males, was accompanied by an adverse finding of tubular granular cast or tubular basophilia.

 

It is concluded that oral administration of Acetylacetone Peroxide to Han Wistar rats for 13 weeks at doses up to 750 mg/kg/day was welltolerated. At the highest dose, there was low overall body weight gain (15%) and hyaline droplet accumulation with nephropathy (granular cast and basophilic tubules) in males that were considered adverse. Consequently, the noobserved-adverse-effect level (NOAEL) in this study was considered to be 250 mg/kg/day.

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:
21-Feb-2012 to 19-Nov-2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted according to GLP in compliance with internationally accepted test guidelines
Qualifier:
according to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))
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

- Animals: Rat, RccHanTM: WIST(SPF)
- Rationale: Recognized by international guidelines as a recommended test system.
- Breeder: Harlan Laboratories B.V., Kreuzelweg 53, 5961 NM Horst / Netherlands
- Total Number of Animals ordered: 21 males and 21 females. Groups 1 to 4, 5 males and 5 females in each. Group 10, 1 male and 1 female reserve animals
- Age (at Delivery): Ca. 7 weeks
- Body Weight Range (on the first day of acclimatization): 197 to 228 g (mean 213 g) in males and 134 to 150 g (mean 141 g) in females
- Identification: During acclimatization cage card and tail mark (later ear tattoo), during treatment cage card and individual ear tattoo
- Randomization: Randomly allocated to groups by body weight.
- Acclimatization: Under test conditions after health examination. Only animals without any visible signs of illness were used for the study.

ENVIRONMENTAL CONDITIONS

- Conditions: Standard laboratory conditions. Air-conditioned with 10 - 15 air changes per hour, continuously monitored environmental conditions (temp. range: 22 ± 3 °C; relative humidity range: 30 - 70%). Values outside of these ranges occasionally occurred, usually following room cleaning, and are considered not to have any influence on the study. Therefore, these data are not reported but are retained at Harlan Laboratories Ltd. The light cycle was set to 12-hour fluorescent light / 12-hour dark cycle with at least eight hours music during the light period.
- Accommodation: In groups of five in Makrolon type-4 cages with wire mesh tops and standard softwood bedding (J. Rettenmaier & Söhne GmbH & Co. KG, 73494 Rosenberg / Germany, imported by Provimi Kliba AG, 4303 Kaiseraugst / Switzerland) including paper enrichment (Enviro-dri from Lillico, Biotechnology, Surrey / UK).
- Diet: Pelleted standard Harlan Teklad 2914C (batch no. 73/11 rat / mouse maintenance diet (Provimi Kliba AG, 4303 Kaiseraugst / Switzerland) was available ad libitum. The feed batch was analyzed for contaminants.
- Water: Community tap-water from Itingen was available ad libitum in water bottles. A bacteriological assay, chemical and contaminant analyses of respective samples were performed.
Route of administration:
oral: gavage
Vehicle:
other: Double distilled water
Details on oral exposure:
DOSE FORMULATIONS

The test item was used as supplied by the Sponsor. The purity was not corrected.

The dose formulations were prepared weekly. Based upon the results of dose formulation analyses performed during a non-GLP dose range finding study (Harlan Laboratories study D43060), the stability of the test item formulations was considered to be sufficient to justify weekly preparation.

The test item, 2,4-Pentanedione, peroxide (CAS#37187-22-7), 30% in solvent mixture, was weighed into a glass beaker on a tared Mettler balance and the vehicle added. Homogeneity of the test item in the vehicle was maintained during the daily administration period using a magnetic stirrer.

STORAGE OF DOSE FORMULATIONS

The dose formulations were stored in glass beakers at room temperature (20 ± 5 °C).

TREATMENT

- Method: Oral, by gavage
- Rationale for Method: Administration by gavage is a common and accepted route of exposure for studies of this type.
- Frequency of Administration: Daily.
- Daily Target Dose Level: 0 mg/kg/day (Group 1), 100 mg/kg/day (Group 2), 300 mg/kg/day (Group 3) and 1000 mg/kg/day (Group 4)
- Rationale for Dose Level Selection: The dose levels were selected based on a previous non-GLP dose range finding toxicity study in Wistar rats, Harlan Laboratories study D43060.
- Dose Volume: 10 mL/kg body weight
- Dose Concentrations: 0 mg/mL (Group 1), 10 mg/mL (Group 2), 30 mg/mL (Group 3) and 100 mg/mL (Group 4)
- Duration of Pre-Randomization Phase: 1 day
- Duration of Acclimatization Phase: At least 5 days
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
METHOD

The analysis was performed by Harlan Laboratories Ltd. using a HPLC method provided by the Sponsor.

After experimental start and during week 3, duplicate samples of the control group as well as three samples (top, middle and bottom) of about 2 g of each concentration were taken prior to dosing for analysis of homogeneity and concentration. Duplicate samples of about 2 g of each concentration were taken to confirm stability (4 hour and 8 days).

The samples were delivered to the analytical department (Harlan Laboratories Ltd., Analytics, Zelgliweg 1, 4452 Itingen / Switzerland) and stored there at 20 ± 5 °C until analysis.

Freshly prepared test item formulations were used as analytical standard.

RESULTS

The linearity of the analytical systems used for sample analyses was demonstrated with a good relationship between peak areas measured and working standard concentrations. All calibration points used met the acceptance limit of ±20% variation from the calibration curve derived by linear regression analysis. The regression coefficients (R2) calculated were found to exceed 0.99.

The 2,4-Pentanedione, peroxide (CAS#37187-22-7), 30% in solvent mixture peak was identified in sample chromatograms by comparison to that of working standards. In blank sample chromatograms, no peak appeared at the retention time of 2,4-Pentanedione, peroxide (CAS#37187-22-7), 30% in solvent mixture, confirming that the test item was not present in the vehicle control samples.

The application formulations investigated during the study were found to comprise 2,4-Pentanedione, peroxide (CAS#37187-22-7), 30% in solvent mixture in the range of 92.9% to 99.7%, meeting the required content limit of ±20% of the nominal content.

Because single results found did not deviate more than 2.7% (acceptance criterion: <15%) from the corresponding mean, 2,4-Pentanedione, peroxide (CAS#37187-22-7), 30% in solvent mixture was considered to be homogeneously distributed in the vehicle.

In addition, the test item was found to be stable in application formulations when kept up to eight days at room temperature. Recoveries met the variation limit of 10% from the time-zero (homogeneity) mean.

In conclusion, the results indicate the accurate use of the test item 2,4-Pentanedione, peroxide (CAS#37187-22-7), 30% in solvent mixture and double distilled water as vehicle during this study. The dose formulations were found to be homogeneously prepared and sufficiently stable under storage conditions.
Duration of treatment / exposure:
28 days
Frequency of treatment:
Once daily
Remarks:
Doses / Concentrations:
0, 100, 300 and 1000 mg/kg bw/day
Basis:
other: purity not corrected
No. of animals per sex per dose:
5 males and 5 females
Control animals:
yes, concurrent vehicle
Details on study design:
The purpose of this oral toxicity study was to assess the cumulative toxicity of 2,4-Pentanedione, peroxide (CAS#37187-22-7), 30% in solvent mixture when administered daily to rats by gavage for a period of 28 days.

This study should provide a rational basis for toxicological risk assessment in man.

In this subacute toxicity study, 2,4-Pentanedione, peroxide (CAS#37187-22-7), 30% in solvent mixture was administered daily by oral gavage to SPF-bred Wistar rats of both sexes at dose levels of 100, 300 and 1000 mg/kg body weight/day for a period of 28 days. A control group was treated similarly with the vehicle, double distilled water, only.

The groups comprised 5 animals per sex which were sacrificed after 28 days of treatment.

Clinical signs, outside cage observation, food consumption and body weights were recorded periodically during the pretest and treatment periods. Functional observational battery, locomotor activity and grip strength were performed during week 4.
At the end of the dosing period, blood samples were withdrawn for hematology and plasma chemistry analyses. Histological examinations were performed on organs and tissues from all control and high dose animals, and all gross lesions from all animals.

Because of possible test-item-related-findings that were noted in animals treated with 1000 mg/kg/day, the stomach, duodenum, liver and spleen from both sexes, kidneys from males and femur bone marrow from females treated with 100 or 300 mg/kg/day were also examined to establish a no-effect level.
Positive control:
Not required
Observations and examinations performed and frequency:
VIABILITY/MORTALITY

Observations for viability / mortality were recorded twice daily.

DAILY OBSERVATIONS

The animals were observed for clinical signs once before commencement of administration as well as daily on days 1 - 28 (twice daily during days 1 - 3) during the treatment period.


WEEKLY BEHAVIORAL OBSERVATIONS

The animals were observed in their home cages, outside their home cages in a standard arena and in the hand. These observations were performed once before commencement of administration and once weekly (weeks 1 to 3) thereafter. More details on the investigations can be found in the section “Any other information on materials and methods incl. tables” below.


FUNCTIONAL OBSERVATIONAL BATTERY (SCREEN)

During week 4, relevant parameters from a modified Irwin screen test were evaluated in all animals.

- Grip Strength: Forelimb and hindlimb grip strength measurements were performed using a push-pull strain gauge (Mecmesin, AFG 25N). The animals were placed with the forepaws inside a triangular grasping ring and with the hind paws outside a triangular grasping ring. Using one hand, the animals were held towards the base of the tail and steadily pulled away or towards the ring until the grip was broken. Each measurement was repeated three times, the means were calculated and recorded.

- Locomotor Activity: Locomotor (decreased or increased) activity was measured quantitatively with AMS Föhr Medical Instruments GmbH (FMI) and DeMeTec GmbH Activity Monitor System. Animals were monitored during the fourth treatment week for a 60-minute period and the total activity of this time period was recorded. Low beams count was reported in 10-minute intervals as well as the total activity of the measuring period.

FOOD CONSUMPTION

The food consumption was recorded once during the acclimatization period and weekly thereafter, using an on-line electronic recording system consisting of a Mettler balance connected to the Harlan Laboratories computer.

BODY WEIGHTS

Body weights were recorded once during pre-randomization (to allow allocation by weight), weekly during the acclimatization and treatment periods and on the day prior to necropsy and immediately before necropsy, using an on-line electronic recording system consisting of a Mettler balance connected to the Harlan Laboratories computer.

CLINICAL LABORATORY INVESTIGATIONS

Blood Sampling at 4 Weeks of Treatment: 27-Mar-2012

Blood samples were drawn from the retro-orbital plexus from all animals under light isoflurane anesthesia. The animals were fasted in metabolism cages for approximately 18 hours before blood sampling but allowed access to water ad libitum. The samples were collected early in the working day to reduce biological variation caused by circadian rhythms. Blood samples were drawn from the retro-orbital plexus using a micro-hematocrit glass capillary tube.

Clinical laboratory data are expressed, with a few exceptions, in general accordance with the International System of Units (SI).


HEMATOLOGY

The following hematology parameters were determined:

Complete Blood Cell Count
- Erythrocyte count
- Hemoglobin
- Hematocrit
- Mean corpuscular volume
- Red cell volume distribution width
- Mean corpuscular hemoglobin
- Mean corpuscular hemoglobin concentration
- Hemoglobin concentration distribution width
- Reticulocyte count
- Reticulocyte maturity index (low, medium, high fluorescence)
- Leukocyte count, total
- Differential leukocyte count:
- Neutrophils
- Eosinophils
- Basophils
- Lymphocytes
- Monocytes
- Large unstained cells
- Platelet count

Hemoglobin Derivatives
- Methemoglobin
- Heinz bodies (slides were prepared but not evaluated)

Coagulation
- Prothrombin time (= thromboplastin time)
- Activated partial thromboplastin time

CLINICAL BIOCHEMISTRY

The following clinical biochemistry parameters were determined:

- Glucose
- Urea
- Creatinine
- Bilirubin, total
- Cholesterol, total
- Triglycerides
- Phospholipids
- Aspartate aminotransferase
- Alanine aminotransferase
- Lactate dehydrogenase
- Alkaline phosphatase
- Bile acids
- Gamma-glutamyl-transferase
- Creatine kinase
- Sodium
- Potassium
- Chloride
- Calcium
- Phosphorus
- Protein, total
- Albumin
- Globulin
- Albumin/Globulin ratio
Sacrifice and pathology:
NECROPSY

Sacrifice after 4 weeks of treatment: 27-Mar-2012

All animals were weighed and necropsied. Descriptions of all macroscopical abnormalities were recorded. All animals were anesthetized by intraperitoneal injection of pentobarbitone and killed by exsanguination.

A blood sample was taken from each animal by heart puncture (ca. 2 mL) into appropriate tubes (Vacutainer glass tubes containing SST-Gel and Clot Activator or the equivalent) for serum preparation and cooled. Following centrifugation, the serum was divided in aliquots of at least 350 µL and transferred to plastic (polypropylene) tubes. These samples were stored at approximately -80 °C and protected from light. The samples were not evaluated since there were no changes in thyroid-related parameters.

Samples of the following tissues and organs were collected from all animals at necropsy and fixed in neutral phosphate buffered 4% formaldehyde solution unless indicated otherwise.

- Adrenal glands
- Aorta
- Bone (sternum, femur including joint)
- Bone marrow (femur)
- Brain - including section of medulla/pons, cerebral and cerebellar cortex
- Cecum
- Colon
- Duodenum
- Epididymides (fixed in Bouin's solution)
- Esophagus
- Eyes w/optic nerve (fixed in Davidson's solution)
- Harderian gland (fixed in Davidson's solution)
- Heart including auricles
- Ileum, with Peyer's patches
- Jejunum with Peyer's patches
- Kidneys
- Larynx
- Lacrimal gland, exorbital
- Liver
- Lungs, filled w/formalin at necropsy
- Lymph nodes – mesenteric and mandibular
- Mammary gland area
- Nasal cavity
- Ovaries
- Pancreas
- Pharynx
- Pituitary gland
- Seminal vesicles/Prostate gland incl. coagulating glands
- Rectum
- Salivary glands - mandibular, sublingual
- Sciatic nerve
- Skeletal muscle
- Skin
- Spinal cord - cervical, midthoracic, lumbar
- Spleen
- Stomach
- Testes (fixed in Bouin's solution)
- Thymus
- Thyroid (incl. parathyroid gland, if possible)
- Tongue
- Trachea
- Urinary bladder, filled w/formalin at necropsy
- Uterus, with cervix, vagina and oviducts
- All gross lesions

ORGAN WEIGHTS

The organs listed below were weighed before fixation and recorded on the scheduled dates of necropsy. Relative organ weights were calculated on the basis of the body weight and brain weight.

- Adrenal glands
- Brain - including section of medulla/pons, cerebral and cerebellar cortex
- Epididymides (fixed in Bouin's solution)
- Heart including auricles
- Kidneys
- Liver
- Ovaries
- Pituitary gland
- Seminal vesicles/Prostate gland incl. coagulating glands
- Spleen
- Stomach
- Testes (fixed in Bouin's solution)
- Thymus
- Thyroid (incl. parathyroid gland, if possible)
- Uterus, with cervix, vagina and oviducts

The terminal body weight was recorded immediately prior to necropsy and the organ to terminal body weight ratios as well as organ to brain weight ratios were determined.

HISTOTECHNIQUE

All organ and tissue samples, as defined under Histopathology below were processed, embedded and cut at an approximate thickness of 2 to 4 micrometers and stained with hematoxylin and eosin.

HISTOPATHOLOGY

Slides of all organs and tissues listed below collected at scheduled sacrifices from all animals of the control and high-dose groups and all gross lesions from all animals were examined by the study pathologist.

- Adrenal glands
- Bone marrow (femur)
- Brain - including section of medulla/pons, cerebral and cerebellar cortex
- Cecum
- Colon
- Duodenum
- Epididymides (fixed in Bouin's solution)
- Eyes w/optic nerve (fixed in Davidson's solution)
- Heart including auricles
- Ileum, with Peyer's patches
- Jejunum with Peyer's patches
- Kidneys
- Liver
- Lungs, filled w/formalin at necropsy
- Lymph nodes – mesenteric and mandibular
- Ovaries
- Pituitary gland
- Seminal vesicles/Prostate gland incl. coagulating glands
- Rectum
- Sciatic nerve
- Skeletal muscle
- Spinal cord - cervical, midthoracic, lumbar
- Spleen
- Stomach
- Testes (fixed in Bouin's solution)
- Thymus
- Thyroid (incl. parathyroid gland, if possible)
- Trachea
- Urinary bladder, filled w/formalin at necropsy
- Uterus, with cervix, vagina and oviducts
- All gross lesions

Attempts were made to correlate gross observations with microscopic findings. Microscopically, the treatment-related findings recorded in stomach, duodenum, liver and spleen of both sexes of animals, kidneys of male animals and femur bone marrow of female animals required evaluation of these organs in the low and middle dose groups.

A peer review was performed. The findings of the study pathologist and the peer-reviewing pathologist compared favorably.
Statistics:
The following statistical methods were used to analyze body weight, grip strength, locomotor activity, clinical laboratory data, organ weights and ratios as well as macroscopic findings:

- The Dunnett-test (many to one t-test) based on a pooled variance estimate was applied if the variables could be assumed to follow a normal distribution for the comparison of the treated groups and the control groups for each sex.

- The Steel-test (many-one rank test) was applied instead of the Dunnett-test when the data could not be assumed to follow a normal distribution.

- Fisher's exact-test.
Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Mean body weights of male rats at 1000 mg/kg/day lower than controls throughout treatment. Females at 1000 mg/kg/day had lower mean body weights on the first day of the treatment period, but generally similar to controls thereafter.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Mean daily food consumption of males and females at 1000 mg/kg/day was lower throughout the study when compared with the other treated groups and the respective controls.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
At 1000 mg/kg bw/day elevated reticulocytes in males and in females indicators of mild anemia. At 300 mg/kg bw/day in females increased mean relative reticulocyte count.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Both genders at 1000 mg/kg/day, elevated mean cholesterol and phospholipid levels and elevated triglyceride levels in females. Elevated mean albumin level and albumin/globulin ratio males. All exceeding the upper limit of the historical control data.
Urinalysis findings:
not examined
Behaviour (functional findings):
effects observed, treatment-related
Description (incidence and severity):
slightly diminished fore limb grip strength in males only, reduced locomotor activity in males and females.
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
At 1000 mg/kg bw/day: in both genders higher mean liver/bw ratio deemed adaptive test item related change. Mean kidney/bw ratios of both genders elevated and in males elevated mean spleen/bw ratio considered to be test item-related.
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
hypertrophy of mucosal epithelium of duodenum, centrilobular hepatocellular hypertrophy and increased incidence and severity of extramedullary erythrocytic hemopoiesis (i.e. erythropoiesis) in both sexes treated with 1000 mg/kg/day.
Histopathological findings: neoplastic:
not examined
Details on results:
1. IN-LIFE DATA

VIABILITY/MORTALITY

There were no test item-related deaths. One female rat of the control group died following blood sampling on the day of necropsy.

DAILY OBSERVATIONS

At 300 mg/kg/day, salivation was noted in one male during week 3 and in one male and one female during week 4.

At 1000 mg/kg/day, salivation was seen in two males and three females during week 3 and in two males and one female during week 4

In view of the limited incidence of these findings, they were considered to be toxicologically irrelevant.

WEEKLY BEHAVIORAL OBSERVATIONS

There were no findings noted during the weekly behavioral observations performed at weeks 1, 2 or 3 of treatment.

FUNCTIONAL OBSERVATIONAL BATTERY

There were no findings evident during the functional observational battery performed at week 4.

- Grip Strength: The mean forelimb grip strength value of the males treated with 1000 mg/kg/day was significantly lower (p<0.05) than that of the control males. In the absence of a similar change in the mean hind limb grip strength value, this difference was considered to be due to the slightly lower mean body weight of the males at 1000 mg/kg/day. The mean hind limb grip strength was unaffected.
The mean fore- and hind limb grip strength values of the males and females at 100 mg/kg/day and 300 mg/kg/day, as well as the females at 1000 mg/kg/day were unaffected by the test item administration.

- Locomotor Activity: The mean locomotor activity of the males and females treated with 1000 mg/kg/day was slightly, but consistently, lower than that of the respective controls. Although the differences did not attain statistical significance, the locomotor activity of both sexes at 1000 mg/kg/day were lower during all measurement intervals (with the exception of the first measurement interval in males). The overall locomotor activity (0-60 minutes) was clearly reduced in both sexes treated with 1000 mg/kg/day.
Rats treated at 100 mg/kg/day and 300 mg/kg/day were considered to be unaffected by the test item.

FOOD CONSUMPTION

The mean daily food consumption of the males and females treated with 1000 mg/kg/day was lower throughout the study when compared with the other treated groups and the respective controls, and was considered to be a test item-related effect. The remaining groups were unaffected.

The slight increase in the mean daily food consumption of the control males was considered to be the result of food wastage.

BODY WEIGHTS

The mean body weights of the male rats treated with 1000 mg/kg/day remained lower than the control males throughout the treatment period. This was also reflected in the mean body weight gain, which was reduced with statistical significance on treatment days 8 (p<0.01), 22 (p<0.05) and 28 (p<0.05), when compared with the controls. Females treated with 1000 mg/kg/day had significantly lower mean body weights on the first day of the treatment period (p<0.01), but were generally similar thereafter.

The mean body weights of the remaining males and females were similar to those of the controls.

2. CLINICAL LABORATORY INVESTIGATIONS

HEMATOLOGY

Males treated with 1000 mg/kg/day showed slightly elevated mean relative and absolute reticulocyte counts (p<0.05 and p<0.01, respectively) and a compensatory ‘left-shift’ towards high fluorescence reticulocytes (p<0.05) when compared with the controls. Females treated with 1000 mg/kg/day showed findings that are generally indicative of mild anemia with compensatory reticulocytosis: significantly reduced red cell count (p<0.05) and hematocrit (p<0.05) with significantly increased mean relative and absolute reticulocyte counts (p<0.05 and p<0.01, respectively), and slight ‘left-shift’ towards high fluorescence reticulocytes. Although the latter finding did not attain statistical significance, it was considered to be related to the other changes in the hematology parameters and therefore test item related.

Test item-unrelated statistical differences included decreased monocyte counts (p<0.05) and a foreshortened partial thromboplastin time (p<0.05) in males treated with 1000 mg/kg/day, as well as a significantly elevated ‘large unstained cell’ counts (p<0.01) in females treated with 1000 mg/kg/day. The latter difference exceeded the range of the historical control data.

At 300 mg/kg/day, the mean relative reticulocyte count of the females was significantly increased (p<0.05) when compared with the controls. The difference was dose-related and a slight if statistically insignificant difference in the absolute reticulocyte counts was noted; this was considered to be a mild test item-related effect. Males were unaffected.

At 100 mg/kg/day, there were no test item-related effects.

CLINICAL BIOCHEMISTRY

At 1000 mg/kg/day, a decreased mean creatinine level in males was considered likely to be the result of increased clearance. Bile acids were increased in males and females but statistically significant only in females (p<0.05), were considered to be of indefinable toxicological relevance since this parameter is subject to large range of normal variation.

The mean cholesterol and phospholipid levels for males and females treated with 1000 mg/kg/day were elevated (both p<0.01) in females only when compared with the respective controls. These exceeded the upper limit of the historical control data, as did the elevated triglyceride levels in females (p<0.01).

The mean albumin level and albumin/globulin ratio of the males were elevated (p<0.01 and p<0.05, respectively) and out of range of the historical control data values. Females were unaffected.

The decreased mean calcium level noted in the females treated with 300 mg/kg/day (p<0.05) and 1000 mg/kg/day (p<0.01) remained within the ranges of the historical control data and were considered to be of no toxicological relevance.

All remaining values were considered to be without any toxicological relevance.

3. PATHOLOGY

ORGAN WEIGHTS

In males treated with 1000 mg/kg/day, the mean absolute brain weights were significantly reduced (p<0.01) when compared with the controls. The mean brain-to-body weight ratio was similar to that of the control males, confirming that this was most likely related to the lower mean body weights. All other mean absolute organ weights compared favorably with those of the controls.

The marginally, but significantly elevated heart-to-body weight ratio was considered to an artifact of the lower mean body weight. Females were unaffected.

The mean liver-to-body weight ratio of the males and females treated with 1000 mg/kg/day were significantly higher than those of the respective controls, and was considered to be a test item-related effect that was an adaptive change rather than one of systemic and toxicological relevance.

The mean kidney-to-body weight ratios of males and females at 1000 mg/kg/day were significantly elevated (p<0.01 in males and p<0.05 in females) when compared to the controls and considered to be a test item-related effect.

The increased mean spleen-to-body weight ratio in the males was also considered to be test item-related and correlated with the extramedullary hemopoiesis that was noted in this organ.

The mean organ-to-brain weight ratios of the rats treated with 1000 mg/kg/day were similar to those of the respective controls. The significantly elevated heart-to-brain weight ratio (p<0.01) and significantly elevated spleen-to-brain weight ratio (p<0.05) noted in the males treated with 100 mg/kg/day were considered to be an incidental finding in the absence of any corroborating microscopical changes.

MACROSCOPIC FINDINGS

There were no gross lesions that could be attributed to treatment with the test item.

All gross lesions recorded were within the range of normal background alterations which may be recorded in animals of this strain and age.

MICROSCOPIC FINDINGS

Microscopically, test item-related findings were recorded in stomach, duodenum, liver and spleen of both sexes of animals, kidneys of male animals and femur bone marrow of female animals.

In a female animal (No. 25) which died just before necropsy, there were no particular changes that should be considered.

- Stomach and Duodenum: Diffuse squamous hyperplasia of the forestomach as well as hyperkeratosis was recorded at a minimal severity in both sexes treated with 1000 mg/kg/day. Necrosis of glandular stomach mucosa near the limiting ridge was also observed in one male and one female animals treated with 1000 mg/kg/day. In addition, hypertrophy of duodenal mucosal epithelium was recorded at a minimal severity in both sexes treated with 1000 mg/kg/day. See also table 1 in section “Any other information or results incl. tables” below.

- Liver: Centrilobular hypertrophy of hepatocytes was recorded in both sexes treated with 1000 mg/kg/day. There were no further indicators of liver injury. See also table 2 in section “Any other information or results incl. tables” below.

- Kidneys: Increased incidence and severity of hyaline droplets were recorded together with increased incidence and severity of tubular basophilia in male animals treated with 300 or 1000 mg/kg/day. See also table 3 in section “Any other information or results incl. tables” below.

- Spleen and Femur Bone Marrow: Increased severity of erythrocytic extramedullary hemopoiesis (erythroid hyperplasia) was recorded in the spleen of both sexes treated with 1000 mg/kg/day, and increased erythropoiesis in the femur bone marrow was recorded in female animals treated with 1000 mg/kg/day. See also table 4 in section “Any other information or results incl. tables” below.

- Vaginal Estrus Stages: No specific direction of estrus cycle was observed in animals treated with 1000 mg/kg/day, and therefore it was judged that there were no treatment-related effects on the estrus cycles. See also table 5 in section “Any other information or results incl. tables” below.

- Other Findings: The remainder of findings recorded was within the range of normal background lesions which may be recorded in animals of this strain and age.
Dose descriptor:
NOEL
Effect level:
100 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: see 'Remark'
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Test item-related adverse microscopical findings included squamous hyperplasia and hyperkeratosis of forestomach, as well as mucosal necrosis of glandular stomach in males and females treated with 1000 mg/kg/day.
Critical effects observed:
not specified

TABLES OF MICROSCOPIC FINDINGS

 

Table 1: Incidence and Mean Severity Grade of Main Findings in Stomach and Duodenum

 

Finding

Incidence /

Mean SeverityGrade

Group 1

Group 2

Group 3

Group 4

Stomach

5M

5F

5M

5F

5M

5F

5M

5F

Squamous hyperplasia, forestomach, diffuse
Incidence/Mean Severity

0

0

0

0

0

0

4/1.0

3/1.0

Hyperkeratosis, forestomach, diffuse
Incidence/Mean Severity

0

0

0

0

0

0

4/1.0

3/1.0

Necrosis, glandular stomach mucosa, near the limiting ridge
Incidence/Mean Severity

0

0

0

0

0

0

1/1.0

1/1.0

Duodenum

5M

5F

5M

5F

5M

5F

5M

5F

Hypertrophy, mucosal epithelium
Incidence/Mean Severity

0

0

0

0

0

0

4/1.0

2/1.0

 

Table 2: Incidence and Mean Severity Grade of Main Findings in Liver

 

Finding
Incidence /
Mean Severity
Grade

Group 1

Group 2

Group 3

Group 4

 

5M

5F

5M

5F

5M

5F

5M

5F

Hypertrophy, hepatocytes, centrilobular 
Incidence/Mean Severity

0

0

0

0

0

0

5/1.6

5/1.0

 

Table 3: Incidence and Mean Severity of Main Findings in Kidneys

 

Finding
Incidence /
Mean Severity
Grade

Group 1

Group 2

Group 3

Group 4

 

5M

5F

5M

5F

5M

5F

5M

5F

Hyaline droplets, proximal tubules
Incidence/Mean Severity

4/1.3

0

3/1.0

0

5/1.8

0

5/3.0

0

 

5M

5F

5M

5F

5M

5F

5M

5F

Tubular basophilia
Incidence/Mean Severity

1/1.0

0

2/1.0

2/1.5

4/1.3

2/1.0

5/1.8

1/1.0

 

Table 4: Incidence and Mean Severity of Main Findings in Spleen and Femur Bone Marrow

 

Finding
Incidence /
Mean Severity
Grade

Group 1

Group 2

Group 3

Group 4

Spleen

5M

5F

5M

5F

5M

5F

5M

5F

Extramedullary hemopoiesis , erythrocytic
Incidence/Mean Severity

5/1.2

5/1.0

5/1.2

5/1.4

5/1.4

5/1.4

5/1.8

5/2.4

Femur bone marrow

5M

5F

5M

5F

5M

5F

5M

5F

Increased erythropoiesis
Incidence/Mean Severity

0

0

0

0

0

0

0

3/1.0

 

Table 5: Incidence of Estrus Stages in Vagina

 

Finding(Estrus stage)
Incidence

Group 1

Group 2

Group 3

Group 4

 

5F

 

 

5F

Proestrus

1

2

Estrus

0

1

Metestrus

1

1

Diestrus

3

1

 

 

 

Conclusions:
Oral administration of the substance to Wistar rats at doses of 100, 300 and 1000 mg/kg/day, for 28 days resulted in no test item-related deaths, no test item-related clinical signs during daily observations, weekly observations or functional observational battery, and no macroscopical findings of toxicological relevance.

Test item-related non-adverse findings noted at 1000 mg/kg/day during the in-life phase were restricted to slightly diminished fore limb grip strength in males only, reduced locomotor activity in males and females, reduced food consumption in males and females and reduced body weight development in males and females.

Non-adverse findings noted in hematology parameters of males and females at 1000 mg/kg/day included compensatory reticulocytosis, whereas females also showed mild anemia. Mild reticulocytosis was also noted in females at 300 mg/kg/day. These findings are generally recognized as reversible after exposure is discontinued. The clinical biochemistry parameters of males and females at 1000 mg/kg/day showed mild aberrations in lipid metabolism that were attributed to metabolic adaptation and therefore considered to be non-adverse.

Increased organ weights included relative liver weights (considered to be an adaptive change and not adverse) in males and females, relative kidney weights in males and females and relative spleen weights in males only at doses of 1000 mg/kg/day.

Test item-related non-adverse and typically adaptive microscopical findings included hypertrophy of mucosal epithelium of duodenum, centrilobular hepatocellular hypertrophy and increased incidence and severity of extramedullary erythrocytic hemopoiesis (i.e. erythropoiesis) in both sexes treated with 1000 mg/kg/day, increased erythropoiesis in femur bone marrow in females treated with 1000 mg/kg/day, and enhanced hyaline droplets in renal proximal tubules as well as increased incidence and severity of tubular basophilia in the kidney in males treated with 300 and 1000 mg/kg/day. These findings were used to establish the no-observed-effect-level (NOEL) of 100 mg/kg body weight/day of 2,4-Pentanedione, peroxide (CAS#37187-22-7), 30% in solvent mixture.

Test item-related microscopical findings included squamous hyperplasia and hyperkeratosis of forestomach, as well as mucosal necrosis of glandular stomach in males and females treated with 1000 mg/kg/day. These findings were used establish the no-observed-adverse-effect-level (NOAEL) of 300 mg/kg body weight/day of 2,4-Pentanedione, peroxide (CAS#37187-22-7), 30% in solvent mixture. However, based on human relevance and adaptive nature of some of the effects, a NOAEL of 1000 mg/kg/d was established as the correct NOAEL
Executive summary:

GENERAL

 

In this subacute toxicity study, of the substance, was administered daily by oral gavage to SPF-bred Wistar rats of both sexes at dose levels of 100, 300 and 1000 mg/kg body weight/day for a period of 28 days. A control group was treated similarly with the vehicle, double distilled water, only. The groups comprised 5 animals per sex which were sacrificed after 28 days of treatment.

 

Clinical signs, outside cage observation, food consumption and body weights were recorded periodically during the pretest and treatment periods. Functional observational battery, locomotor activity and grip strength were performed during week 4. At the end of the dosing period, blood samples were withdrawn for hematology and plasma chemistry analyses. Histological examinations were performed on organs and tissues from all control and high dose animals, and all gross lesions from all animals. Because of possible test-item-related-findings that were noted in animals treated with 1000 mg/kg/day, the stomach, duodenum, liver and spleen from both sexes, kidneys from males and femur bone marrow from females treated with 100 or 300 mg/kg/day were also examined to establish a no-effect level.

 

RESULTS:

MORTALITY/VIABILITY: There were no test item-related deaths.

 

CLINICAL SIGNS (DAILY AND WEEKLY): There were no findings seen during the daily observations that were considered to be related to systemic toxicity and there were no findings noted during the weekly behavioral observations (weeks 1, 2 or 3).

 

FUNCTIONAL OBSERVATIONAL BATTERY: There were no findings evident during the functional observational battery performed at week 4.

 

GRIP STRENGTH: The mean forelimb grip strength value of the males treated with 1000 mg/kg/day was lower than that of the control males, and was considered to be secondary to the slightly lower mean body weight of the males at 1000 mg/kg/day. The mean hind limb grip strength of these males was unaffected. The mean fore- and hind limb grip strength values of the males and females at 100 mg/kg/day and 300 mg/kg/day, as well as the females at 1000 mg/kg/day were unaffected.

 

LOCOMOTOR ACTIVITY: The mean locomotor activity values of the males and females treated with 1000 mg/kg/day were lower during all measurement intervals (with the exception of the first measurement interval in males). The overall locomotor activity (0-60 minutes) was clearly reduced in both sexes treated with 1000 mg/kg/day, and considered to be test item-related. Rats treated at 100 mg/kg/day and 300 mg/kg/day were unaffected.

 

FOOD CONSUMPTION: The mean daily food consumption of the males and females treated with 1000 mg/kg/day was lower throughout the study when compared with the other treated groups and the respective controls, and was considered to be a test item-related effect. The remaining groups were unaffected.

 

BODY WEIGHTS: The mean body weights of the male rats treated with 1000 mg/kg/day remained lower than the control males throughout the treatment period. This was also reflected in the reduced mean body weight gain seen on treatment days 8, 22 and 28. Females treated with 1000 mg/kg/day had lower mean body weights on the first day of the treatment period, but were generally similar thereafter. The mean body weights of the remaining males and females were similar to those of the controls.

 

CLINICAL LABORATORY INVESTIGATIONS:

- Hematology: males treated with 1000 mg/kg/day showed slightly elevated mean relative and absolute reticulocyte counts and a compensatory ‘left-shift’ towards high fluorescence reticulocytes when compared with the controls. Females treated with 1000 mg/kg/day showed findings that are generally indicative of mild anemia with compensatory reticulocytosis: reduced red cell count and hemoglobin with increased mean relative and absolute reticulocyte counts and slight ‘left-shift’ towards high fluorescence reticulocytes. These findings were considered to be related to the test item administration. All other parameters were considered to be unaffected.

At 300 mg/kg/day, the mean relative reticulocyte count of the females was increased when compared with the controls. The difference was dose-related and a slight difference in the absolute reticulocyte counts was noted; this was considered to be a mild test item-related effect that commonly reverts to normal levels after exposure is discontinued. Males, in comparison, were unaffected. All other parameters were considered to be unaffected.

At 100 mg/kg/day, there were no test item-related effects.

 

- Clinical Biochemistry: differences to the control values were noted in males and females treated at 1000 mg/kg/day. The mean cholesterol and phospholipid levels were elevated and exceeded the upper limit of the historical control data, as did the elevated triglyceride levels in females. The mean albumin level and albumin/globulin ratio of the males were elevated and out of range of the historical control data values. Females were unaffected.

All remaining values were considered to be without any toxicological relevance.

 

ORGAN WEIGHTS AND RATIOS: The mean liver-to-body weight ratio of the males and females treated with 1000 mg/kg/day were higher than those of the respective controls, and was considered to be a test item-related effect that was an adaptive change rather than one of systemic and toxicological relevance. The mean kidney-to-body weight ratios of males and females at 1000 mg/kg/day were elevated when compared to the controls and considered to be a test item-related effect. The elevated mean spleen-to-body weight ratio (p<0.01) in the males at 1000 mg/kg/day was also considered to be test item related and correlated with the extramedullary hemopoiesis that was noted in this organ.

 

The remaining organ weights were considered to be unaffected by the treatment with the test item.

 

MACROSCOPIC / MICROSCOPIC FINDINGS:

There were no gross lesions that could be attributed to treatment with the test item.

Test item-related microscopical findings included squamous hyperplasia and hyperkeratosis of forestomach, mucosal necrosis of glandular stomach, hypertrophy of mucosal epithelium of duodenum, centrilobular hepatocellular hypertrophy and increased incidence and severity of extramedullary erythrocytic hemopoiesis (i.e. erythropoiesis) in both sexes treated with 1000 mg/kg/day, increased erythropoiesis in femur bone marrow in females treated with 1000 mg/kg/day, and enhanced hyaline droplets in renal proximal tubules as well as increased incidence and severity of tubular basophilia in the kidney in males treated with 300 and 1000 mg/kg/day. Increased incidence and severity of extramedullary erythropoiesis in the spleen and increased erythropoiesis in the femur bone marrow correlated with increased reticulocyte count in the hematology.

 

The lesions observed in the forestomach, glandular stomach and duodenum were considered to be a local reaction to a repeated administration of the test item by gavage.

 

In the liver, centrilobular hepatocellular hypertrophy was recorded, but there were no further indicators of liver injury, hence this was considered to be of metabolic nature and of adaptive character.

Increased incidence and severity of hyaline droplets in the renal proximal tubules were considered to be induced by overload of synthetized protein that is specific in male rat (such as alpha-2 microglobulin) derived from hyperfunction of the liver. Hepatocellular hypertrophy was not recognized histologically in males treated with 300 mg/kg/day, but increased organ/body weight ratio (but not absolute liver weight) was observed in these males, as well as both sexes treated with 1000 mg/kg/day, which suggests that the hyperfunction had occurred in the liver of males treated with 300 mg/kg/day as well as both sexes treated with 1000 mg/kg/day. Increased incidence and severity of renal tubular basophilia were considered to be caused by the enhanced hyaline droplet deposition.

 

CONCLUSION

 

Test item-related non-adverse findings noted at 1000 mg/kg/day during the in-life phase were restricted to slightly diminished fore limb grip strength in males only, reduced locomotor activity in males and females, reduced food consumption in males and females and reduced body weight development in males and females.

 

Non-adverse findings noted in hematology parameters of males and females at 1000 mg/kg/day included compensatory reticulocytosis, whereas females also showed mild anemia. Mild reticulocytosis was also noted in females at 300 mg/kg/day. These findings are generally recognized as reversible after exposure is discontinued. The clinical biochemistry parameters of males and females at 1000 mg/kg/day showed mild aberrations in lipid metabolism that were attributed to metabolic adaptation and therefore considered to be non-adverse.

 

Increased organ weights included relative liver weights (considered to be an adaptive change and not adverse) in males and females, relative kidney weights in males and females and relative spleen weights in males only at doses of 1000 mg/kg/day.

 

Test item-related non-adverse and typically adaptive microscopical findings included hypertrophy of mucosal epithelium of duodenum, centrilobular hepatocellular hypertrophy and increased incidence and severity of extramedullary erythrocytic hemopoiesis (i.e. erythropoiesis) in both sexes treated with 1000 mg/kg/day, increased erythropoiesis in femur bone marrow in females treated with 1000 mg/kg/day, and enhanced hyaline droplets in renal proximal tubules as well as increased incidence and severity of tubular basophilia in the kidney in males treated with 300 and 1000 mg/kg/day. These findings were used to establish the no-observed-effect-level (NOEL) of 100 mg/kg body weight/day of 2,4-Pentanedione, peroxide (CAS#37187-22-7), 30% in solvent mixture.

 

Test item-related microscopical findings included squamous hyperplasia and hyperkeratosis of forestomach, as well as mucosal necrosis of glandular stomach in males and females treated with 1000 mg/kg/day. These findings were used in the report to suggest a no-observed-adverse-effect-level (NOAEL) of 300 mg/kg body weight/day of 2,4 -Pentanedione, peroxide (CAS#37187 -22 -7), 30% in solvent mixture. However, based on human relevance and typical adaptive nature of some of the effects, a NOAEL of 1000 mg/kg/d was established as the correct NOAEL.

 

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Experimental start date (animal arrival) 30 January 2019 Treatment commenced 13 February 2019 Necropsy 15 to 16 May 2019 Experimental completion date (Pathology) 17 July 2019
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)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no
Specific details on test material used for the study:
Test item Acetylacetone Peroxide
Test item identity (including alternative names) Acetylacetone Peroxide
2,4-Pentanedione, peroxide (upper limit: 42% w/w; typical
concentration: 30% w/w)
Trigonox 44B
CAS number 13784-51-5
Intended use Substance used in industry.
Appearance Clear, colorless liquid.
Storage conditions Ambient temperature 15-25°C in the dark.
Supplier Sponsor.
Batch number 1809428243
Expiry date 13 June 2021
Supplier’s responsibilities Characterization of the test item and the documentation of the methods of synthesis, fabrication or derivation and stability.
Archive sample A 0.5 g representative sample was taken from the batch of test item used. This sample was placed in a well closed glass container and stored in the archives under the same conditions as the bulk material.
Species:
rat
Strain:
Wistar
Details on species / strain selection:
The rat was chosen as the test species because it is accepted by regulatory agencies. The Han Wistar (RccHan™;WIST) strain was used because of the historical control data available at this laboratory.
Sex:
male/female
Details on test animals and environmental conditions:
Strain/Species: RccHan™:WIST rat.
Supplier: Envigo RMS Limited.
Number of animals: 45 males and 45 females.

Spare animals were removed from the study room after treatment commenced.

Duration of acclimatization 14 days before commencement of treatment.

Age of the animals at start of treatment 43 to 49 days.

Weight range of the animals at the start of treatment
Males: 130 to 194 g
Females: 114 to 155 g

Allocation and Identification
Allocation Randomly allocated on arrival.

Using the sequence of cages in the battery, one animal at a time was placed in each cage with the procedure being repeated until each cage held the appropriate number of animals. Each sex was allocated separately.

Identification of animals Each animal was assigned a number and identified uniquely within the study by a microchip inserted shortly after arrival.

Identification of cages Each cage label was color-coded according to group and was numbered uniquely with cage and study number, as well as the identity of the occupants.

Animal Replacement
On Day 1 (before dosing) variations in body weight of the animals were checked to ensure that they did not exceed 20% of the mean for the appropriate sex. No replacements were required.

Animal Care and Husbandry
Environmental Control
Animal facility Limited access - to minimize entry of external biological and chemical agents and to minimize the transference of such agents between rooms.
Air supply Filtered fresh air which was passed to atmosphere and not recirculated.
Temperature and relative humidity Monitored and maintained within the range of 20-24ºC and 40-70%.

Although conditions were occasionally outside the indicated ranges, these deviations were minor and/or of short duration and were not considered to have influenced the health of the animals and/or the outcome of the study.

Lighting Artificial lighting, 12 hours light : 12 hours dark.
Electricity supply Public supply with automatic stand-by generators.

Animal Accommodation
Cages Polycarbonate body with a stainless steel mesh lid, changed at appropriate intervals.
Cage distribution Males and females were blocked by sex and the cages constituting each group were dispersed in batteries so that possible environmental influences arising from their spatial distribution were equilibrated, as far as was practicable. The positions of the cage batteries in the room were changed weekly, following a rotation plan, to further minimize possible effects of spatial variations.
Number of animals per cage Three or four of the same sex.
Bedding Wood based bedding which was changed at appropriate intervals each week.

Environmental Enrichment
Aspen gnawing material Provided to each cage throughout the study and replaced when necessary.
Plastic shelter Provided to each cage throughout the study and replaced when necessary.

Diet Supply
Diet Teklad 2014C, pelleted diet.
Availability Non-restricted (removed overnight before blood sampling for hematology or blood chemistry and during the period of urine collection).

Water Supply
Supply Potable water from the public supply via polycarbonate bottles with sipper tubes. Bottles were changed at appropriate intervals.
Availability Non-restricted (except during the period of urine collection).

Supplier Certificates of Analysis
Certificates of analysis for the diet were scrutinized and approved before any batch of diet was released for use. Certificates of analysis are routinely provided by the water supplier.

Certificates of analysis were also received from the suppliers of the softwood based bark-free fiber bedding and Aspen gnawing material.

No specific contaminants were known that may have interfered with or prejudiced the outcome of the study and therefore no special assays were performed.
Route of administration:
oral: gavage
Details on route of administration:
The oral route of administration was chosen as it is a possible route of human exposure during manufacture, handling or use of the test item. The test item was administered by gavage.
Vehicle:
water
Remarks:
Purified
Details on oral exposure:
Method of preparation
Acetylacetone Peroxide was prepared for administration by dilution of individual weighings of the test item. The required amount of test item was weighed out into a suitable container and approximately 50% of the final volume of vehicle was added and mixed with a magnetic stirrer until the formulation appeared visibly homogenous. The solution was then made up to the required volume with vehicle and stirred with a magnetic stirrer until homogenous.

Frequency of preparation Weekly.

Storage of formulation Refrigerated (2-8°C) for up to 15 days.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Stability and homogeneity Before commencement of treatment, the suitability of the proposed mixing procedures was determined and specimen formulations were analyzed to assess the stability and homogeneity of the test item in the liquid matrix.

Achieved concentration Samples of each formulation prepared for administration in Weeks 1, 2 and 12 of treatment were analyzed for achieved concentration of the test item.

Analytical Procedure
The samples were analyzed in accordance with the validated Covance Analytical Procedure(DFA/M002/19). The analytical method involved extraction and dilution in acetonitrile/water 5/95 v/vfollowed by reverse phase high performance liquid chromatographic analysis with ultra violet
detection at 195 nm. Sample concentrations were determined with reference to external standards prepared in the concentration range 40 μg/mL to 400 μg/mL.

The formulations for Week 1, Week 2 and Week 12 were sampled, 2 × 3 mL (accuratelyweighed), from the middle of the formulation by Pharmacy personnel.Duplicate aliquots from the first sample were analyzed in accordance with the analytica procedure. The remaining samples were retained for contingency. Contingency samples wereanalyzed for Week 1 Group 2 and Week 12 Group 4. Duplicate aliquots from the second
sample were analyzed in accordance with the analytical procedure. Samples were disposed of once satisfactory results were achieved.

RESULTS AND CONCLUSION
The mean concentrations were within ±10% of the nominal concentration, confirming the accuracy of formulation with the exception of Week 1 Group 2 and Week 12 Group 4. The difference from mean remained within 5%, confirming precise analysis. The coefficient of variation remained within 1%, confirming precise analysis. Week 1 Group 2 was +21.3% of the nominal concentration. Contingency samples were analyzed which confirmed the result. Week 12 Group 4 was -22.5% of the nominal concentration. Re-dilutions were performed alongside analysis of contingency samples. The re-dilutions and contingency analysis were all within the acceptable range. Suggesting there was an analytical error with the original dilutions performed. This was performed outside of the known stability period and therefore will be reported for information only.






.


Duration of treatment / exposure:
13 weeks
Frequency of treatment:
Daily
Dose / conc.:
0 mg/kg bw/day (nominal)
Dose / conc.:
80 mg/kg bw/day (nominal)
Dose / conc.:
250 mg/kg bw/day (nominal)
Dose / conc.:
750 mg/kg bw/day (nominal)
No. of animals per sex per dose:
Ten males and ten females per dose group
Control animals:
yes, concurrent no treatment
Details on study design:
Rationale for Dose Level Selection
The doses used in this study (0, 80, 250 and 750 mg/kg/day) were selected in conjunction with the Sponsor.

Administration
Route Oral, by gavage, using a suitably graduated syringe and a rubber catheter inserted via the mouth.
Treated at Constant doses in mg/kg.
Dose volume 10 mL/kg body weight.
Individual dose volume Calculated from the most recently recorded scheduled body weight.
Control (Group 1) Vehicle at the same dose volume as the treated groups.
Frequency Once daily at approximately the same time each day.
Formulation Formulations were stirred using a magnetic stirrer before and throughout the dosing procedure.

A daily record of the usage of formulation was maintained based on weights. This balance was compared with the expected usage as a check of correct administration. No significant discrepancy was found.

Dose levels were selected for this study, based on the results from a four-week toxicity study (Envigo (Switzerland) Study Number: D43071) in which treatment at 0, 100, 300 or 1000 mg/kg/day was well-tolerated by RccHanTM: WIST(SPF) rats. In that study there were no deaths. When compared with control, body weight gain and food intake was low at 1000 mg/kg/day in males and females, though for females the effect on body weight occurred only after the first dose. Reticulocyte count was high in males at 1000 mg/kg/day and in females at 300 or 1000 mg/kg/day and this finding correlated with extramedullary hemopoiesis in the spleen and increased erythropoiesis in the femoral bone marrow in females at 1000 mg/kg/day. At 1000 mg/kg/day plasma cholesterol and phospholipid concentrations were high in males and females, triglyceride concentrations were high in females and albumin concentrations and albumin:globulin ratios were high in males. There were no macroscopic findings, but liver and kidney weights were high in males and females and spleen weights were high in males given 1000 mg/kg/day. Microscopic examination revealed the presence of diffuse squamous hyperplasia of the forestomach and minimal hyperkeratosis at 1000 mg/kg/day, with necrosis of glandular stomach mucosa near the limiting ridge also being observed in one male and one female at this dose, and minimal hypertrophy of duodenal mucosal epithelium at 1000 mg/kg/day. The findings in the stomach were considered adverse. In addition, there was centrilobular hepatocellular hypertrophy and an increased incidence and severity of erythropoiesis at 1000 mg/kg/day. The conclusion from this study was that as there were no adverse findings in the low and intermediate dose animals, the no-observed-adverse-effect level (NOAEL) was 300 mg/kg/day.

In view of the presence of adverse findings in the stomach that was attributed to the administration of an irritant test formulation, the highest dose in this 13 week study was slightly lower than 1000 mg/kg/day. Consequently, doses of 0, 80, 250 and 750 mg/kg/day were selected for this study.
Positive control:
None
Observations and examinations performed and frequency:
Serial Observations
Clinical and Behavioral Observations
Animals were inspected visually at least twice daily for evidence of ill-health or reaction to treatment. Cages were inspected daily for evidence of animal ill-health amongst the occupants. Any deviation from normal was recorded at the time in respect of nature and severity, date and time of onset, duration and progress of the observed condition, as appropriate.

During the acclimatization period, observations of the animals and their cages were recorded at least once per day.

Signs Associated with Dosing
Daily during the first week of treatment, twice weekly during Weeks 2 to 4 (middle and end of each week) and weekly thereafter, detailed observations were recorded at the following times in relation to dose administration:

Immediately before dosing (Pre-dose).
At the end of dosing each group.
One to two hours after completion of dosing all groups.
As late as possible in the working day.

Detailed Physical Examination and Arena Observations
Before treatment commenced and during each week of treatment, detailed physical examination and arena observations were performed on each animal. On each occasion, the examinations were performed at approximately the same time of day (before dosing during the treatment period), by an observer who was unaware of the experimental group identities.

After removal from the home cage, animals were assessed for physical condition and behavior during handling and after being placed in a standard arena. Any deviation from normal was recorded with respect to the nature and, where appropriate, degree of severity. Particular attention was paid to possible signs of neurotoxicity, such as convulsions, tremor and abnormalities of gait or behavior.

Findings were either reported as "present" or assigned a severity grade - slight, moderate or marked.

Sensory Reactivity Observations and Grip Strength
Sensory reactivity and grip strength assessments were performed (before dosing) on all animals during Week 12 of treatment. Animals were tested by an observer who was unaware of the treatment group to which each animal belonged. Before the start of observations, cage labels showing the treatment group were replaced by labels stating only the study, animal and cage numbers. Animals were not necessarily all tested on the same day, but the numbers of animals and the times of testing were balanced across the groups on each day of testing.

The following measurements, reflexes and responses were recorded:
Approach response
A blunt probe was brought towards the animal’s head until it was close to the animal’s nose (but not touching the whiskers). The animal’s reaction was recorded as:
1 No reaction or ignores/walks past probe
2 Normal awareness and reaction e.g. approaches and/or sniffs probe
3 Active avoidance, abnormally fearful or aggressive reaction

Pinna reflex
The inside of one ear was touched lightly with a nylon filament and the reaction recorded as:
1 No response
2 Normal response e.g. ear twitches/flattens or animal shakes its head
3 Abnormally fearful or aggressive response

Auditory startle reflex
The animal’s response to a sudden sharp noise was assessed and scored as:
1 No response
2 Weak response e.g. ear twitch only
3 Normal response e.g. obvious flinch or startle
4 Exaggerated response e.g. all feet off floor

Tail pinch response
The animal’s tail was pinched sharply with forceps approximately one third from the tip and the response graded as:
1 No response
2 Weak response e.g. turns around slowly or weak vocalization without moving away
3 Normal response e.g. jumps forward or turns around sharply, usually with vocalization
4 Exaggerated response e.g. excessive vocalization, body movement or aggression

Grip strength
Forelimb and hindlimb grip strength was measured using Mecmesin Basic Force Gauges. Three trials were performed.

At any point during the observations, additional comments were made as free text where considered appropriate.

Motor Activity
During Week 12 of treatment (before dosing), the motor activity of each animal was measured using a Rodent Activity Monitoring System (Version 2.0.6), with hardware supplied by Pearson Technical Services and software developed and maintained by Covance.

Animals were tested individually in clear polycarbonate cages and motor activity was measured by counting infra-red beam breaks over ten 6-minute intervals (one hour total). Ten beams were set at two height levels (five low and five high) to detect cage floor and rearing activity respectively. Animals were not necessarily all tested on the same day, but the numbers of animals and the times of testing were balanced across the groups on each day of testing.

Body Weight
The weight of each animal was recorded one week before treatment commenced, on the day that treatment commenced (Week 0), weekly throughout the study and before necropsy.

Food Consumption
The weight of food supplied to each cage, that remaining and an estimate of any spilled was recorded for the week before treatment started and for each week throughout the study.

Water Consumption
Fluid intake was assessed by daily visual observation. No effect was observed and, consequently, quantitative measurements were not performed.

Ophthalmic Examination
The eyes of the animals were examined by means of a binocular indirect ophthalmoscope as follows:

Occasion Animals
Pretreatment All animals
Week 12 All animals of Groups 1 and 4

Prior to each examination, the pupils of each animal were dilated using tropicamide ophthalmic solution (Mydriacyl). The adnexae, conjunctiva, cornea, sclera, anterior chamber, iris (pupil dilated), lens, vitreous and fundus were examined.

Hematology, Peripheral Blood
Blood samples were collected after overnight withdrawal of food and prior to dosing. Sampling was performed on the morning after overnight collection of urine; therefore the animals were also deprived of water overnight but had access to water for a minimum period of one hour prior to the commencement of blood sampling procedures. Samples were collected at the following occasion:

Occasion Animals
Week 13 All animals

Animals were held under light general anesthesia induced by isoflurane. Blood samples (nominally 0.5 mL) were withdrawn from the sublingual vein, collected into tubes containing EDTA anticoagulant and examined for the following characteristics using a Bayer Advia 120 analyzer:

Hematocrit (Hct)*
Hemoglobin concentration (Hb)
Erythrocyte count (RBC)
Absolute reticulocyte count (Retic)
Mean cell hemoglobin (MCH)*
Mean cell hemoglobin concentration (MCHC)*
Mean cell volume (MCV)
Red cell distribution width (RDW)
Total leucocyte count (WBC)
Differential leucocyte count:
Neutrophils (N)
Lymphocytes (L)
Eosinophils (E)
Basophils (B)
Monocytes (M)
Large unstained cells (LUC)
Platelet count (Plt)
* Derived values calculated in ClinAxys

Blood film (prepared for all samples) - Romanowsky stain, examined for abnormalities by light microscopy, in the case of flags from the Advia 120 analyzer. Confirmation or a written description from the blood film was made where appropriate.

Additional blood samples (nominally 0.5 mL) were taken into tubes containing citrate anticoagulant and examined using a Stago STA Compact Max analyzer and appropriate reagent in respect of:
Prothrombin time (PT) - using IL PT Fibrinogen reagent.
Activated partial thromboplastin time (APTT) - using IL APTT reagent.

Blood Chemistry
Blood samples were collected after overnight withdrawal of food and prior to dosing. Sampling was performed on the morning after overnight collection of urine; therefore the animals were also deprived of water overnight but had access to water for a minimum period of one hour prior to the commencement of blood sampling procedures. Samples were collected at the following occasion:

Occasion Animals
Week 13 All animals

Animals were held under light general anesthesia induced by isoflurane. Blood samples (nominally 0.7 mL) were withdrawn from the sublingual vein and collected into tubes containing lithium heparin as anticoagulant. After separation, the plasma was examined using a Roche P Modular Analyzer in respect of:

Alkaline phosphatase (ALP)
Alanine aminotransferase (ALT)
Aspartate aminotransferase (AST)
Total bilirubin (Bili)
Bile acids (Bi Ac)
Urea*
Creatinine (Creat)
Glucose (Gluc)
Total cholesterol (Chol)
Cholesterol HDL (HDL)
Cholesterol LDL (LDL)
Triglycerides (Trig)
Sodium (Na)
Potassium (K)
Chloride (Cl)
Calcium (Ca)
Inorganic phosphorus (Phos)
Total protein (Total Prot)
Albumin (Alb)
Albumin/globulin ratio (A/G Ratio) was calculated from total protein concentration and analyzed albumin concentration.
*Numerically equivalent to blood urea nitrogen (BUN)

Urinalysis
Animals were placed in an individual metabolism cage, without food or water. Urine samples were collected overnight at the following occasion:

Occasion Animals
Week 13 All animals

The individual samples were examined for the following characteristics:

Using manual methods:
Clarity and Color (App) - by visual assessment
Volume (Vol) - using a measuring cylinder
pH - using a pH meter
Specific gravity (SG) - by direct refractometry using a SG meter
Using Multistix reagent strips interpreted using the Clinitek®500 instrument:
Ketones (Keto)
Bile pigments (Bili)
Blood pigments (UBld)
Using a Roche P Modular Analyzer:
Protein (T-Prot and Prot)

A microscopic examination of the urine sediment was performed. An aliquot of the urine sample was centrifuged, stained with Kova stain and the resulting deposit spread on a microscope slide. The number of elements seen in nine high or low power fields (HPF or LPF) was recorded in the raw data and entered onto the database and the number seen /HPF or /LPF was derived from these data as described below.

Epithelial cells (Epi)
Leucocytes (WBC)
Erythrocytes (RBC)
Casts
Other abnormal components (A)

The slide was also examined for abnormalities in spermatozoa and crystals.

Estrous Cycles - Vaginal Smears
Wet smears Smears were taken for at least three weeks (from Week 11 and included the day of scheduled termination, using pipette lavage. Smears were subsequently examined to investigate estrous cyclicity.

Thyroid Hormone Analysis
Occasion Animals
At termination All animals

Sequence of blood sampling on each occasion To minimize any potential confounding effect of the time of day of blood sampling, the time of blood sampling was controlled to allow satisfactory inter-group comparison.
Conditions No overnight deprivation of food.
Blood sample site Sublingual vein.
Anesthetic Isoflurane. The animals were not allowed to recover from the anesthesia.
Anticoagulant None.
Blood tube Grenier Minicollect tubes with clotting activator.
Blood volume 1.0 mL
Temporary storage conditions Samples were kept at ambient temperature (15 to 25°C) for a minimum of 30 minutes prior to centrifugation.
Centrifugation conditions At 2000 g for 10 minutes at 4°C.
Aliquot volumes Aliquot 1: T3 and T4: 0.2 mL of serum
Aliquot 2: TSH: all remaining serum
Final storage conditions Deep frozen (approximately -60°C to -90°C).
Fate of samples Dispatched to the Department of Biomarkers, Bioanalysis and Clinical Sciences, Covance.
T3, T4 and TSH Analysis Performed by the Department of Department of Biomarkers, Bioanalysis and Clinical Sciences, Covance.
The method of analysis and results are presented in Annex 3, Annex 4.

Additional Hormone Sampling
Occasion Animals
At termination All animals

Sequence of blood sampling on each occasion To minimize any potential confounding effect of the time of day of blood sampling, the time of blood sampling was controlled to allow satisfactory inter-group comparisons.
Conditions No overnight deprivation of food.
Blood sample site Sublingual vein.
Anesthetic Isoflurane. The animals were not allowed to recover from the anesthesia.
Anticoagulant None.
Blood tube Grenier Minicollect tubes with clotting activator.
Blood volume 1.0 mL
Temporary storage conditions Samples were kept at ambient temperature (15 to 25°C) for a minimum of 30 minutes prior to centrifugation.
Centrifugation conditions At 2000 g for 10 minutes at 4°C.
Final storage conditions Serum samples were stored deep frozen (approximately -60°C to -90°C) pending any future requirement for analysis.
Retention The samples were transferred to archives and will be retained for the same period as the study raw data.
Sacrifice and pathology:
Terminal Investigations

Method of Kill
Carbon dioxide asphyxiation with subsequent exsanguination.

Necropsy
All animals were subject to a detailed necropsy. After a review of the history of each animal, a full macroscopic examination of the tissues was performed. All external features and orifices were examined visually. Any abnormality in the appearance or size of any organ and tissue (external and cut surface) was recorded and the required tissue samples preserved in appropriate fixative.

The retained tissues were checked before disposal of the carcass.

Schedule Animals were killed following 13 weeks of treatment.
Sequence To allow satisfactory inter-group comparison.

The organs weighed, tissue samples fixed and sections examined microscopically are detailed as follows:
Tissue and regions examined Necropsy Histology Pathology
Weigh Fix Light microscopy
Abnormalities * * *
Adrenals * * * *
Aorta * * *
Brain (cerebellum, cerebrum, midbrain) * * * *
Bone marrow smear * a)
Cecum * * *
Coagulating glands * b) * * *
Colon * * *
Duodenum * * *
Epididymides * L+R c) * R * R * R
Esophagus * * *
Eyes *
Femur (femorotibial joint) * d) † †
Head * e)
Heart (including auricular and ventricular regions) * * * *
Ileum * * *
Jejunum * * *
Kidneys * * * *
Liver (section from two lobes) * * * *
Lungs (section from two major lobes including bronchi) * * *
Lymph nodes - mesenteric * * *
- left axillary * * *
Tissue and regions examined Necropsy Histology Pathology
Weigh Fix Light microscopy
Ovaries * * * *
Pancreas * * *
Pituitary * * *
Prostate * b) * * *
Salivary glands - submandibular * † †
- sublingual * † †
- parotid * † †
Sciatic nerves * † †
Seminal vesicles * b) * * *
Skin with mammary glands (inguinal area) * * *
Spinal cord (transverse and longitudinal sections at the cervical, thoracic and lumbar levels) * * *
Spleen * * * *
Sternum (and bone marrow) * * *
Stomach * * *
Testes * L+R c) * R * R * R
Thymus * * * *
Thyroid with parathyroids * f) * * *
Trachea * * *
Urinary bladder * * *
Uterus with cervix * * * *
Vagina * * *
a) See Section 3.7.2.
b) Prostate, seminal vesicles and coagulating gland weighted together.
c) Left testis and left epididymis processed as indicated in Section 3.7.2.
d) Both hind limbs retained, one sectioned where appropriate.
e) Including nasal cavity, paranasal sinuses and nasopharynx.
f) Weighed after partial fixation.
* Organs weighed, samples fixed or sections examined microscopically.
† Only one examined.
L&R Left and right. Animal ID retained.

Bone Marrow
Bone marrow smears were prepared immediately following death, on completion of the scheduled treatment period.
Fixation Smears were air dried and subsequently fixed in methanol.
Analysis No examinations were performed, however, the smears were retained for possible future examination.
Retention The smears were transferred to archives and will be retained for the same period as the study raw data.

Sperm Analysis
Immediately after scheduled sacrifice of each male and collection of bone marrow, the left vas deferens, epididymis and testis were removed and the epididymis and testis were weighed.

The following tests were performed:
Sperm motility - Groups 1-4 A sample of sperm was expressed from the left vas deferens into prewarmed (target 37°C) medium M199, which contained 0.5% w/v bovine serum albumin (BSA Fraction V). A sample for assessment was taken into a 100 µm depth cannula by capillary action and at least 200 sperm per animal analysed using the Hamilton Thorne IVOS II Computer Assisted Sperm Analyser (CASA).
Sperm morphology - Groups 1 and 4 A 200 L aliquot of the sperm/medium mixture (described above) was diluted with 800 L of 10% neutral buffered formalin. After staining with nigrosine and eosin an air dried smear was prepared. Slides were examined by light microscopy for the assessment of sperm morphology. At least 200 sperm were assessed for each male.
- Groups 2 and 3 Fixed samples retained for possible future assessment.
Sperm count - all groups The left cauda epididymis of each male was weighed and then the tunica was removed. Samples were then frozen for possible future assessment.
Homogenization-resistant spermatids count - all groups After removal of the tunica, the left testis of each male samples were then frozen for possible future assessment.
Retention The samples were transferred to archives and will be retained for the same period as the study raw data.

Organ Weights
For bilateral organs, left and right organs were weighed together, unless specified above. Requisite organs were weighed for animals killed at the end of the treatment period.

Fixation
Tissues were routinely preserved in 10% Neutral Buffered Formalin with the exception of those detailed below:
Testes In modified Davidson’s fluid.
Eyes In Davidson’s fluid.

Histology
Processing Tissue samples were dehydrated, embedded in paraffin wax and sectioned at a nominal four to five micron thickness. For bilateral organs, sections of both organs were prepared. A single section was prepared from each of the remaining tissues required.

Full List Animals of Groups 1 and 4 killed at the end of the treatment period.
Male kidney Animals of Groups 2 and 3 killed at the end of the treatment period.
Abnormalities only Animals of Groups 2 and 3 killed at the end of the treatment period.
Routine staining Sections were stained with hematoxylin and eosin.

Light Microscopy
Tissues preserved for examination were examined as follows:
Category Animals Tissues
Scheduled kill All animals of Groups 1 and 4.
All animals of Groups 2 and 3. Male kidney

Abnormalities.
Findings were either reported as "present" or assigned a severity grade. In the latter case one of the following five grades was used - minimal, slight, moderate, marked or severe. A reviewing pathologist undertook a peer review of the microscopic findings.

Stage-dependent Evaluation of Spermatogenesis
Stage dependent evaluation of spermatogenesis was conducted on sections of testes from all animals prepared and stained using the PAS method. A qualitative examination of spermatogenic stages was made for normal progression of the stages of the spermatogenic cycle, cell associations, and proportions expected to be present during normal spermatogenesis.
Statistics:
Data Evaluation
This report contains serial observations pertaining to all weeks of study completed, together with signs data collected during the necropsy period. In the case of detailed physical examination and arena observations, body weight and food consumption data, only information from the final week of the acclimatization period is presented.

Summary statistics (e.g. means and standard deviations) presented in this report were calculated from computer-stored individual raw data. Group mean values and standard deviations were frequently calculated using a greater number of decimal places than presented in the appendices. It is, therefore, not always possible to derive exact group values from the data presented in the appendices.
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
The appearance and behavior of the animals were unaffected by treatment and there were no deaths during the treatment period.

There were isolated incidences of chin-rubbing, paddling of forepaws and salivation at 750 mg/kg/day and of chin rubbing at 250 mg/kg/day, occurring immediately after dose administration. These are commonly observed signs in studies where test formulations have been administered by gavage and are considered of no toxicological significance.
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Overall body weight gain was low, compared to controls, in males receiving 750 mg/kg/day with the extent of the reduction being approximately 15% that was considered adverse.

The body weight gains of males and females receiving 80 or 250 mg/kg/day and of females receiving 750 mg/kg/day were unaffected by treatment. The overall weight gains of males receiving 80 mg/kg/day were higher than controls but this was attributed mainly to weight gains in three animals (Nos. 6, 8 and 10) that were higher than those reported for the control males

PLEASE REFER TO ATTACHED TABLE OF RESULTS
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
Food consumption was considered to have been unaffected by treatment.

Overall food consumption by males receiving 750 mg/kg/day was marginally, but statistically significantly lower than that of the controls during the treatment period, with the extent of the reduction being approximately 8%, but this reflected a trend that was observed during the pre-treatment period and was therefore not clearly attributable to treatment. Food intake was also slightly higher than controls in males receiving 80 mg/kg/day but as there was no similar trend at the higher doses this finding was also not attributed to treatment.
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 that were attributable to treatment.
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
Hematology, Peripheral Blood
The hematological examination in Week 13 revealed, when compared to controls, high reticulocyte count in males and females receiving 750 mg/kg/day which resulted in an increased red cell distribution width in males and an increased mean cell hemoglobin in females. There was, however, no effect upon erythrocyte count, hemoglobin concentration and hematocrit or any other erythrocyte indices and, consequently, these minor differences from controls were considered non adverse.

Males and females receiving 750 mg/kg/day had high neutrophil, lymphocyte, monocyte and large unstained cell counts, when compared to controls, with males also showing an increase of eosinophil count and females showing in increase of basophil count. There was also an increase of lymphocyte and basophil count in females receiving 250 mg/kg/day. As a consequence, total leucocyte counts were higher than controls in females receiving 250 mg/kg/day and in males and females receiving 750 mg/kg/day.

All other differences from control were minor, lacked dose-relationship or were confined to one sex and were therefore attributed to normal biological variation. Such differences included the longer activated partial thromboplastin times in females receiving 250 or 750 mg/kg/day as there was no dose-response, no similar finding in males (the activated partial thromboplastin times of males tended to be lower than controls), and prothrombin times were all similar to controls

PLEASE REFER TO ATTACHED TABLE OF RESULTS
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Blood Chemistry
The biochemical examination of the blood plasma in Week 13 revealed, when compared to Control, high bile acid concentration in males and females receiving 750 mg/kg/day. In females there was also an increase of total bilirubin concentration but the extent of the difference from controls was minimal and none of the individual values was abnormal.

Urea/blood urea nitrogen concentrations were high, compared to controls, in males and females receiving 750 mg/kg/day, with urea/blood urea nitrogen concentrations also being high in males receiving 250 mg/kg/day. There was also a reduction of creatinine concentration in females receiving 250 mg/kg/day and in males and females receiving 750 mg/kg/day.

Males and females receiving 750 mg/kg/day had higher plasma glucose concentrations than the controls.

There was an increase, compared to controls, of plasma high and low density lipoprotein concentrations in males and females receiving 750 mg/kg/day that resulted in high total plasma cholesterol concentration in these animals. This finding associated with high triglyceride concentrations in females.

There were minor variations in the plasma concentrations of some electrolytes. Plasma calcium concentrations were lower than controls in animals receiving 750 mg/kg/day and in males this associated with high phosphorus concentrations. Females receiving 750 mg/kg/day also had reduced sodium and chloride concentrations. There was also a statistically significant reduction of plasma chloride concentration in males receiving 750 mg/kg/day but this was not attributed to treatment as the difference from controls was minimal and there was no associated alteration of sodium concentration.

All other differences from control were minor or were confined to one sex and were therefore attributed to normal biological variation. Such differences included a minimal increase of albumin concentration in males receiving 750 mg/kg/day as there was no effect on total protein concentration or albumin to globulin ratio and there was no similar finding in females, and a decrease of plasma alkaline phosphatase activity in males receiving 750 mg/kg/day since this is not a toxicologically significant finding.

PLEASE REFER TO ATTACHED TABLE OF RESULTS
Urinalysis findings:
effects observed, treatment-related
Description (incidence and severity):
The analysis of urine samples at the end of the treatment period indicated, when compared to controls, slightly low pH in males and females receiving 750 mg/kg/day, though this was statistically significant only in males.

All other differences from control were minor or were confined to one sex and were therefore attributed to normal biological variation. Such differences included the variation of protein output in males where there was no dose response (despite the small increase being statistically significant at the highest dose) and there was no similar trends in females. They also included the small increase of specific gravity in males receiving 750 mg/kg/day as this could not be accounted for by any reduction of urinary volume and there was no similar finding in females.

PLEASE REFER TO ATTACHED TABLE OF RESULTS
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
Sensory Reactivity Observations and Grip Strength
Sensory reactivity and grip strength were unaffected by treatment.

The hindlimb grip strength of males receiving 750 mg/kg/day was statistically significantly lower than the controls but the mean value (0.46 kg) was well within the historical control range (0.35 to 0.66 kg in 29 studies conducted between 2015 and 2019) and there was no similar finding reported for the forelimb grip strength of these animals. In addition, this trend was not evident in females. Consequently, the slightly low hindlimb grip strength in the males was attributed to normal biological variation.

Motor Activity
Motor activity in Week 12 was considered to have been unaffected by treatment.

When compared to controls the low and high beam rearing activity were reduced at all doses in females but this was attributed to higher than expected values for the control females that exceeded the range of the historical control data for high beam (205.8 to 404.8 in 29 studies conducted between 2015 and 2019) and was close to the maximum value for the low beam (504.4 to 934.9 in 29 studies conducted between 2015 and 2019) whilst the results for females receiving Acetylacetone Peroxide tended in the majority of cases to be within these historical ranges.
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
The analysis of organ weights after 13 weeks of treatment indicated, when compared to controls, high bodyweight adjusted kidney weights in females given 250 mg/kg/day and in males and females given 750 mg/kg/day, and high body weight-adjusted liver weight in males and females given 750 mg/kg/day. In addition, males given 750 mg/kg/day had high body weight adjusted spleen weight and low prostate/seminal vesicle/coagulating gland combined weight.

All other differences from control were minor or (where applicable) were confined to one sex and were therefore attributed to normal biological variation. Such differences included the small but statistically significant increase of testis weight at 750 mg/kg/day since there was no dose-response (the group mean value for the low dose males was similar to that of the high dose males, but for the intermediate dose males the group mean value was similar to controls).

PLEASE REFER TO ATTACHED TABLE OF RESULTS
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Macropathology
The macroscopic examination performed after 13 weeks of treatment revealed the following changes in the kidneys.

Kidneys
Pale kidneys were seen in two males receiving 250 mg/kg/day and in eight males receiving 750 mg/kg/day.

The incidence and distribution of all other findings were considered to be unrelated to treatment.

PLEASE REFER TO ATTACHED TABLE OF RESULTS
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Changes related to treatment with Acetylacetone Peroxide were confined to the kidneys.

Hyaline droplet accumulation was observed in all males given 750 mg/kg/day. In a few animals this finding was accompanied by tubular granular cast or tubular basophilia. Tubular hyaline droplet accumulation was also seen in all males given 250 mg/kg/day and four males given 80 mg/kg/day. This finding was not accompanied by tubular granular cast or tubular basophilia.

Incidental Findings
The incidence and distribution of all other findings were considered to be unrelated to treatment.

PLEASE REFER TO ATTACHED TABLE OF RESULTS
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed
Description (incidence and severity):
Thyroid Hormone Analysis
The serum triiodothyronine (T3), thyroxine (T4) and thyroid stimulation hormone (TSH) concentrations at the end of the treatment period were unaffected by treatment.
Key result
Dose descriptor:
NOAEL
Effect level:
250 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
gross pathology
histopathology: non-neoplastic
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
250 mg/kg bw/day (nominal)
System:
urinary
Organ:
kidney
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
not specified

 Estrous cycles

Estrous cycles were unaffected by treatment, with all females having regular 4 or 5 day cycles.

Sperm analysis

The motility, morphology and the sperm counts in the testis and cauda epididymis were not affected by treatment.

There was an increase at 750 mg/kg/day of abnormal head, mid-piece and tail morphology, when compared with control, but as the overall number of abnormal sperm was within the Historical Control Data (HCD) range this was not clearly attributable to treatment.

Conclusions:
It is concluded that oral administration of Acetylacetone Peroxide to Han Wistar rats for 13 weeks at doses up to 750 mg/kg/day was well tolerated. At the highest dose, there was low overall body weight gain and hyaline droplet accumulation with nephropathy (granular cast and basophilic tubules) in males that were considered adverse. Consequently, the no observed-adverse-effect level (NOAEL) in this study was considered to be 250 mg/kg/day.
Executive summary:

Summary

The purpose of this study was to assess the systemic toxic potential of Acetylacetone Peroxide, a substance used in industry, when administered orally, by gavage, to Han Wistar rats for 13 weeks. Three groups, each comprising ten males and ten females, received the test item at doses of 80, 250 or 750 mg/kg/day and a similarly constituted control group received the vehicle (purified water) at the same dose volume.

During the study, detailed physical examination and arena observations, sensory reactivity, grip strength, motor activity, body weight, food consumption, visual water consumption, ophthalmic examination, hematology (peripheral blood), blood chemistry, urinalysis, estrus cycle, seminology, organ weight, macropathology and histopathology investigations were undertaken.

Results

The appearance and behavior of the animals were unaffected by treatment, there were no treatment-related findings at the sensory activity, grip strength and motor activity assessment in Week 12 and no deaths occurred during the treatment period.

Overall body weight gain was reduced by approximately 15% in males receiving 750 mg/kg/day but there was no effect on their food consumption. Females were unaffected.

There were no treatment-related ophthalmoscopic findings.

The hematological examination in Week 13 indicated high reticulocyte count in animals receiving 750 mg/kg/day which associated with increased red cell distribution width in males and an increased mean cell hemoglobin in females but all other erythrocyte indices were unaffected. Males and females receiving 750 mg/kg/day had high neutrophil, lymphocyte, monocyte and large unstained cell counts, with males also showing increased eosinophil count and females showing increased basophil count, and there was also an increase of lymphocyte and basophil count in females receiving 250 mg/kg/day. As a consequence, total leucocyte counts were higher than controls in females receiving 250 mg/kg/day and in males and females receiving 750 mg/kg/day.

Treatment-related changes in the blood plasma in Week 13 comprised: high bile acid concentration in animals receiving 750 mg/kg/day; high urea/blood urea nitrogen concentrations in males receiving 250 mg/kg/day and in animals receiving 750 mg/kg/day; low creatinine concentration in females receiving 250 mg/kg/day and in males and females receiving 750 mg/kg/day; high glucose concentrations in animals receiving 750 mg/kg/day; increased high and low density lipoprotein concentrations with an associated increase of total cholesterol concentration in males and females receiving 750 mg/kg/day and which associated with high triglyceride concentrations in females; low calcium concentrations in animals receiving 750 mg/kg/day; high phosphorus concentrations in males receiving 750 mg/kg/day; low sodium and chloride concentrations in females receiving 750 mg/kg/day.

Urinary pH was slightly low in Week 13 in males and females receiving 750 mg/kg/day.

Acetylacetone Peroxide had no effect on estrus cycle or on spermmotility, morphology and sperm count in the testis and cauda epididymis.

Serum triiodothyronine (T3), thyroxine (T4) and thyroid stimulation hormone (TSH) concentrations were unaffected by treatment.

After 13 weeks of treatment kidney weights were high in females given 250 mg/kg/day and in males and females given 750 mg/kg/day, liver weights were high in males and females given 750 mg/kg/day and, in addition, males given 750 mg/kg/day had high spleen and low combined prostate/seminal vesicle/coagulating gland weight.

Pale kidneys were reported at the macroscopic examination in two males given 250 mg/kg/day and in eight males given 750 mg/kg/day.

Treatment-related histopathological findings were confined to the kidneys of males where tubular hyaline droplet accumulation was observed in four males given 80 mg/kg/day and all males given 250 or 750 mg/kg/day which, in some of the high dose males, was accompanied by an adverse finding of tubular granular cast or tubular basophilia.

Conclusion

It is concluded that oral administration of Acetylacetone Peroxide to Han Wistar rats for 13 weeks at doses up to 750 mg/kg/day was well‑tolerated. At the highest dose, there was low overall body weight gain and hyaline droplet accumulation with nephropathy (granular cast and basophilic tubules) in males that were considered adverse. Consequently, the no‑observed-adverse-effect level (NOAEL) in this study was considered to be 250‑mg/kg/day.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
250 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
K1 OECD 408 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

Mode of Action Analysis / Human Relevance Framework

In the 28 -day study, although the study report shows 300 mg/kg/d as the NOAEL, the following findings were re-considered and regarded as irrelevant to humans or typical adaptive changes or non-adverse in nature. The lesions in the forestomach, glandular stomach and duodenum were considered to be a local reaction of the epithelial lining to the repeated gavage administration of the test item. This reasoning, along with the known significant differences in the functional anatomy of rodent and human digestive system suggest that the above rat study findings may be considered not relevant to human exposure conditions (“Mode-of-action framework for evaluating the relevance of rodent forestomach tumors in cancer risk assessment” by Proctor, DM., Gatto, NM., Hong, SJ., and Allamneni, KP., Toxicological Sci., 98(2): 313-326, 2007). The hematology parameter changes are generally recognized as reversible after exposure is discontinued. The clinical biochemistry parameters of males and females at 1000 mg/kg/day showed mild aberrations in lipid metabolism that was attributed to metabolic adaptation. The centrilobular hepatocellular hypertrophy was considered to be of metabolic nature and of adaptive character. Increased renal tubular basophilia were deemed to be caused by the enhanced hyaline droplet deposition, which in turn was considered to be due to the overload of synthetic protein that is specific to male rats resulting from liver hyperfunction. Alpha2μglobulin, a normal protein in male rats which undergoes reabsorption in the proximal cortical tubules (Alden, CL and Frith CH. Urinary System, Ch. 15, pp 315-387. In: Handbook of Toxicologic Pathology Eds. Haschek WM and Rousseaux CG. Academic Press, San Diego. 1991) is a typical example. A range of chemicals are known to increase hyaline droplet formation beyond the physiological capacity of the tubular epithelium which may then result in tubular epithelial cell damage (hyaline droplet nephropathy) which was evident in this study. Based on these considerations, the NOAEL for DNEL calculations was set a 1000 mg/kg/day.

Additional information

Justification for classification or non-classification

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