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Repeated dose toxicity: oral

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Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Remarks:
combined repeated dose and reproduction / developmental screening
Type of information:
experimental study
Adequacy of study:
key study
Study period:
June 20, 2007 - August 10, 2007
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was conducted according to the following guidelines - OECD Guideline No. 422 (1996) and USEPA OPPTS 870.3650 (2000) and complied with the Principles of GLP.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2008
Report date:
2008

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes

Test material

Constituent 1
Details on test material:
- Name of test material (as cited in study report): Methyl Polyhydroxymethyl Stearate (Imperial Monomer)
- Molecular formula (if other than submission substance): UVCB- not applicable
- Molecular weight (if other than submission substance): UVCB- MW of primary components range from 270- 389 g/mole
- Smiles notation (if other than submission substance): UVCB- not applicablet
- InChl (if other than submission substance): not specified in the report
- Structural formula attached as image file (if other than submission substance): UVCB- not applicable
- Substance type: Mixture of functionalized fatty acid methyl esters (FAMEs) which may be derived from a variety of natural seed oils.
- Physical state: Waxy solid at room temperature
- Analytical purity: 95.2 % as summed concentration of five primary components
- Impurities (identity and concentrations): water, approx. 0.4%
- Composition of test material, percentage of components:
Methyl Palmitate: 9.68 %
Methyl Stearate: 17.8 %
Methyl Hydroxymethyl Stearate: 38.6 %
Methyl Dihydroxymethyl Stearate: 26.7 %
Methyl Trihydroxymethyl Stearate: 2.37 %
- Isomers composition: The balance of unassigned composition can be attributed to both minor isomers of the primary components, as well as trace components resulting from physical/chemical processing of the unrefined natural oil feedstock.
- Purity test date: 22 August 2005
- Lot/batch No.: 200500200-25-4
- Characterisation expiration date: 07 September 2007
- Radiochemical purity (if radiolabelling): not applicable
- Specific activity (if radiolabelling): not applicable
- Locations of the label (if radiolabelling): not applicable
- Expiration date of radiochemical substance (if radiolabelling): Stable
- Stability under test conditions: At room temperature in the dark under nitrogen
- Storage condition of test material: not specified in the report

Test animals

Species:
rat
Strain:
Crj: CD(SD)
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories Inc. (Portage, Michigan)
- Age at study initiation: 8 weeks
- Weight at study initiation: not specified in the report
- Fasting period before study: not specified in the report
- Housing: Acclimation - group housed in two-three animals/cage, experimental - singly, except during breeding (one male and one female)
- Diet (e.g. ad libitum): ad libitum, animals were provided LabDiet Certified Rodent Diet #5002 (PMI Nutrition International, St. Louis, Missouri) in meal form.
- Water (e.g. ad libitum): ad libitum, drinking water obtained from the municipal water department
- Acclimation period: at least one week prior to start of the study.


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22°C with a tolerance of ± 1 C (and a maximum permissible excursion of ± 3°C)
- Humidity (%): 40-70%
- Air changes (per hr): 12-15 times/hour
- Photoperiod (hrs dark / hrs light): 12-hour light/dark

IN-LIFE DATES: From: June 20, 2007 To: August 10, 2007

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS: The test material was administered in a corn oil vehicle, such that a dose volume of 4 ml/kg body weight yielded the targeted dose. Dose volumes were adjusted using the most current body weight. Dose solutions were prepared approximately biweekly throughout the study period, and were used within the stability limits.

VEHICLE
- Justification for use and choice of vehicle: Solubility and recommended as a vehicle by various regulatory agencies
- Concentration in vehicle: 25 mg/ml, 125 mg/ml & 250 mg/ml
- Amount of vehicle (if gavage): max volume = 4 ml/kg
- Lot/batch no. (if required): not specifed in the report
- Purity: not specifed in the report
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analysis to determine concentration of the test material of all dosing solutions from the first mix of the main study was initiated prior to the start of dosing. The low- and high-dose solutions from the first mix of the main study were analyzed to confirm homogenous distribution of the test material concurrent with dose confirmation. Analyses were conducted using GC-FID.
Duration of treatment / exposure:
Females were dosed once daily for two weeks prior to breeding, through breeding (two weeks), gestation (three weeks), and through postpartum day 4 or 5. Females were necropsied on postpartum day 5 or 6. Males were dosed two weeks prior to breeding and continuing through breeding (two weeks) until necropsy (test day 34).
Frequency of treatment:
Once a day
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
0.00 mg/ml (Vehicle control)
Basis:
other: Mean Measured Concentration: < LLQ
Remarks:
Doses / Concentrations:
25.0 mg/ml
Basis:
other: Mean Measured Concentration: 24.8
Remarks:
Doses / Concentrations:
125.0 mg/ml
Basis:
other: Mean Measured Concentration: 123.0
Remarks:
Doses / Concentrations:
250 mg/ml
Basis:
other: Mean Measured Concentration: 247.0
No. of animals per sex per dose:
12 males + 12 females
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The high-dose level of 1000 mg/kg/day was selected based upon data obtained from a preliminary range-finding study and represented a limit dose as defined in the Health Effects Test Guideline of the United States Environmental Protection Agency (OPPTS 870.3650) and the OECD Guidelines for the Testing of Chemicals (OECD 422). The lower dose levels (500 and 100 mg/kg/day) were selected to provide dose response data for any toxicity that may have been observed among the high-dose group rats and to establish a no-observed-effect level (NOEL).

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily
- Cage side observations checked in table were included. yes


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Detailed clinical observations (DCO) were conducted on all rats pre-exposure and weekly throughout the study.

BODY WEIGHT: Yes
- Time schedule for examinations: All rats were weighed at least once during the pre-exposure period and on the first day of dosing. Male body weights continued to be recorded weekly throughout the study. Females were weighed weekly during the pre-mating and mating periods. During gestation, females were weighed on Gestational Days (GD) 0, 7, 14, 17, and 20. Females that delivered litters were weighed on LD 1 and 4. Females that failed to mate or deliver a litter were weighed at least weekly for the remainder of the study. Body weight analyses were conducted for the following days: GD 0, 7, 14, and 20. Body weight gains were determined for the following intervals: GD 0-7, 7-14, 14-20, 0-20, and Lactational Days (LD) 1-4.


FOOD CONSUMPTION:
- Food consumption for each animal determined and calculated as g food/day: Yes
Feed consumed was determined weekly during the two week pre-breeding period for males and females by weighing feed crocks at the start and end of a measurement cycle. Feed consumption was not measured for males or females due to co-housing during breeding. Following breeding, feed consumption was not measured for males. For females during gestation, feed consumption was measured on GD 0, 7, 14, and 20. After parturition, feed consumption was measured on LD 1 and 4. Feed consumption was not recorded for females that failed to mate or deliver a litter.


Other:

CLINICAL PATHOLOGY:

HEMATOLOGY:
Blood samples for a complete blood count were mixed with ethylenediaminetetraacetic acid (EDTA). Blood smears were prepared, stained with Wright-Giemsa stain, cover-slipped prior to evaluation. Hematologic parameters were assayed using an Advia 120 Hematology Analyzer (Bayer Corporation, Tarrytown, New York). Following parameters were analysed - Hematocrit (HCT), Hemoglobin (HGB) concentration, Red blood cell (RBC) count, Total white blood cell (WBC) count, Differential WBC count, Platelet (PLAT) count, Reticulocyte (RET) count, RBC indices: Mean Corpuscular Hemoglobin (MCH), Mean Corpuscular Volume (MCV) and Mean Corpuscular Hemoglobin Concentration (MCHC).
Coagulation - Blood samples were collected in sodium citrate tubes, centrifuged and plasma collected and assayed using an ACL9000 (Instrumentation Laboratory, Lexington, Massachusetts).


CLINICAL CHEMISTRY:
Blood samples were collected and serum was separated from cells as soon as possible. Serum parameters were measured using a Hitachi 912 Clinical Chemistry Analyzer (Roche Diagnostics, Indianapolis, Indiana). Following parameters were analysed - Alkaline phosphatase (ALP), Alanine aminotransferase (ALT), Aspartate aminotransferase (AST), Gamma-glutamyl transpeptidase (GGT), Albumin (ALB), Albumin/Globulin Ratio (ALB/GLOB), Cholesterol (CHOL), Creatinine (CREA), Electrolytes (NA, K, PHOS, CL, and CA), Globulin (GLOB), Glucose (GLU), Total bilirubin (TBIL), Total protein (TP), Triglycerides (TRIG) and Urea nitrogen (UN).

URINANALYSIS:
Urine samples were obtained from all males the week prior to the scheduled necropsy. Animals were housed in metabolism cages and the urine collected overnight (approximately 16 hours). Feed and water were available during this procedure. Following parameters were analysed - Color, appearance, specific gravity (refractometer), and urine volume Semiquantitative analyses (Multistix Reagent Strips, Bayer Corporation, Elkhardt, Indiana on the Clinitek 200+) of: pH, Bilirubin, Glucose, Protein, Ketones and Blood Urobilinogen. Also, urine samples were collected from each male by manual compression of the urinary bladder. The urine samples were pooled from each group, and the microsediment were characterized microscopically.

FUNCTIONAL TESTS:
The functional tests (sensory evaluation, rectal temperature, grip performance, and motor activity) were conducted pre-exposure on days -8 and -7 for males and females, respectively, and at the end of the study for males (day 30) and on LD4 for females that produced a litter.
Sacrifice and pathology:
SACRIFICE
- Male animals: All surviving animals (fasted) were submitted for necropsy after at least four weeks of exposure
- Maternal animals: All surviving animals (fasted) were terminated on LD 5 or 6, or at least 24 days after the end of the mating period for females not producing a litter.


GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera. The uteri of all females were stained with an aqueous solution of 10% sodium sulfide stain (Kopf et al., 1964) for approximately two minutes and was examined for the presence and number of implantation sites. After evaluation, uteri were gently rinsed with saline and preserved in neutral phosphate-buffered 10% formalin.


HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues indicated in Table [1] were prepared for microscopic examination. Weights of the adrenals, brain, epididymides, heart, kidneys, liver, spleen, testes, thymus, thyroid with parathyroids (weighed after fixation) were recorded, and organ:body weight ratios calculated.
Statistics:
Descriptive statistics only (means and standard deviations) were reported for globulin, albumin/globulin ratio, RBC indices, and WBC differential counts. Parental body weights and parental body weight gains, litter mean body weights, feed consumption, urine volume, urine specific gravity, coagulation, clinical chemistry data, appropriate hematologic data, and organ weights (absolute and relative) were first evaluated by Bartlett's test (alpha = 0.01) for equality of variances. Based upon the outcome of Bartlett's test, either a parametric or non-parametricanalysis of variance (ANOVA) was performed. If the ANOVA was significant at alpha = 0.05, a Dunnett's test (alpha = 0.05) or the Wilcoxon Rank-Sum (alpha = 0.05) test with Bonferroni's correction was performed. Feed consumption values were excluded from analysis if the feed was spilled or scratched.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not specified
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
no effects observed
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Details on results:
Mortality
All animals survived until termination.

In-Life Observations
No treatment-related clinical signs were observed at any dose level. There were no notable observations made during the cage-side observations.

Detailed Clinical Observations
Examinations performed on all rats revealed no treatmentrelated or statistically significant findings.

Functional Tests
Sensory Evaluation, Rectal Temperature, Grip Performance and Motor Activity
Examinations performed on males and females at baseline and termination revealed no treatment-related or statistically significant findings.

Body Weights/Body Weight Gains
No statistically identified or treatment-related differences in body weights were observed for males at any dose level. Similarly, there were no statistically identified or treatment-related differences in the body weights or body weight gains of females at any dose level tested during the pre-mating, gestation, or lactation periods.

Feed Consumption
There were no statistically
identified or treatment-related differences in the amount of feed consumed by any of the dose groups when compared to their respective controls throughout the study.

Reproductive Indices, Pup Survival, and Sex Ratio
There were no statistically identified or treatment-related effects at any dose level on any of the reproductive parameters or pup survival indices evaluated.

Litter Observations
Observations recorded in the offspring occurred at low frequency and bore no relationship to treatment.

Litter Size and Pup Body Weights
There were no statistically identified or treatment-related effects on litter size or pup body weights at any dose level tested.

Clinical Pathology

Hematology
There were no treatment related hematologic effects in males or females at any dose level.
Coagulation
There were no treatmentrelated changes in prothrombin times of males and females at any dose level.
Clinical Chemistry
There were no treatmentrelated changes in clinical chemistry parameters for males and females at any dose level.
Urinalysis
There were no treatment-related changes in urinalysis parameters for males and females at any dose level.

Organ Weights

Males and females given 1000 mg/kg/day had higher absolute and relative liver weights. The higher liver weights in males corresponded to an increase in the incidence of very slight hypertrophy of centrilobular and midzonal hepatocytes with altered tinctorial properties (increased eosinophilia of hepatocytes). There was no histopathologic correlate for the higher liver weights of females given 1000 mg/kg/day. Based on the histopathologic findings, these effects were considered treatment-related, but non-adverse.

Gross Pathology
There were no treatment related gross pathologic observations for males and females at any dose level.

Histopathology
The only treatment related histopathologic alteration consisted of an increase in the incidence of very slight hypertrophy of centrilobular and midzonal hepatocytes with altered tinctorial properties (increased eosinophilia of hepatocytes) in males given 1000 mg/kg/day. The very slight hepatocellular hypertrophy was interpreted to be a non-adverse effect, possibly reflective of the induction of hepatic enzymes for the metabolism of methyl polyhydroxymethyl stearate. All other histopathologic observations were interpreted to be spontaneous alterations, unassociated with administration of the test material.

Effect levels

open allclose all
Dose descriptor:
NOEL
Remarks:
for general toxicity
Effect level:
500 mg/kg bw/day (nominal)
Sex:
male/female
Basis for effect level:
other: No effects observed
Dose descriptor:
NOAEL
Remarks:
for general toxicity
Effect level:
1 000 mg/kg bw/day (nominal)
Sex:
male/female
Basis for effect level:
other: see 'Remark'
Dose descriptor:
NOEL
Remarks:
for reproductive and neurological effects
Effect level:
1 000 mg/kg bw/day (nominal)
Sex:
female
Basis for effect level:
other: see 'Remark'

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
Based on these results, the no-observed-effect level (NOEL) for general toxicity was 500 mg/kg/day, and the no-observed-adverse-effect level (NOAEL) was 1000 mg/kg/day. The NOEL for reproductive and neurological effects was 1000 mg/kg/day, the highest dose level tested.
Executive summary:

Groups of 12 male and 12 female Crl:CD(SD) rats were administered methyl polyhydroxymethyl stearate in corn oil daily, by gavage at dose levels of 0 (control), 100, 500, or 1000 mg/kg/day. Females were dosed once daily for two weeks prior to breeding, through breeding (two weeks), gestation (three weeks), and through postpartum day 4 or 5. Females were necropsied on postpartum day 5 or 6. Males were dosed two weeks prior to breeding and continuing through breeding (two weeks) until necropsy (test day 34). Effects on reproductive and neurological function as well as general toxicity were evaluated. In addition, postmortem examinations included a gross necropsy of the adults with collection of organ weights and histopathologic examination of tissues. Litter size, pup survival, sex, body weight, and the presence of gross external abnormalities were also assessed.

The effects of treatment on parameters of general toxicity were limited to minor, nonadverse changes in the liver. Specifically, male and female rats administered 1000 mg/kg/day had increases in absolute and relative liver weights. The higher liver weights in males corresponded to an increase in the incidence of very slight hypertrophy of centrilobular and midzonal hepatocytes with altered tinctorial properties (increased eosinophilia of hepatocytes) at this dose level. In contrast, there was no histopathologic correlate for the higher liver weights observed in females given 1000 mg/kg/day. Based on these histopathological findings, the liver effects were interpreted to be non-adverse effects, possibly reflective of the induction of hepatic enzymes for the metabolism of methyl polyhydroxymethyl stearate.

Gavage administration of methyl polyhydroxymethyl stearate, at dose levels up to and including the limit dose of 1000 mg/kg/day produced no indication of neurological or reproductive toxicity at any dose level. There were no effects on prenatal/early neonatal growth and survival of the offspring.

Based on these results, the no-observed-effect level (NOEL) for general toxicity was 500 mg/kg/day, and the no-observed-adverse-effect level (NOAEL) was 1000 mg/kg/day. The NOEL for reproductive and neurological effects was 1000 mg/kg/day, the highest dose level tested.

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