3,5-dimethyl-1,2-dioxolane-3,5-diol

Repeated dose toxicity: oral

Administrative data

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

Data source

Reference

Reference 1

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Reference 2

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2010

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

3,5-dimethyl-1,2-dioxolane-3,5-diol

Cas Number:

13784-51-5

IUPAC Name:

3,5-dimethyl-1,2-dioxolane-3,5-diol

Test material form:

other: Liquid

Details on test material:

- Identification: 2,4-Pentanedione, peroxide, 30% in solvent mixture
- Description: Liquid
- Batch Number: 1103425149 per REACH CofA

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system 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.

Administration / exposure

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

Doses / concentrations

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

Examinations

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.

Results and discussion

Results of examinations

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.

Effect levels

1

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'

2

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.

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

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 SeverityGrade	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 SeverityGrade	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 SeverityGrade	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

 

 

 

Applicant's summary and conclusion

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.