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Repeated dose toxicity: oral

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Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
4th November 2008 to the 3rd April 2010
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Guideline study conducted to GLP. Read across from analog substance.
Justification for type of information:
Category and read across justification attached in section 13 and in appendix of CSR

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Report date:
2010

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Deviations:
yes
Principles of method if other than guideline:
Deviation:
- Functional observational batteries (FOBS) were not conducted.
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
Phenol, paraalkylation products with C10-15 branched olefins (C12 rich) derived from propene oligomerization, carbonates, calcium salts, overbased, sulfurized including distillates (petroleum), hydrotreated, solvent-refined, solvent-dewaxed, or catalytic dewaxed, light or heavy paraffinic C15-C50
EC Number:
701-251-5
Molecular formula:
Formula for a representative structure is C36H58Ca2O4Sx where x = 1,2. Actual molecular formula is not possible to generate. Substance is a UVCB.
IUPAC Name:
Phenol, paraalkylation products with C10-15 branched olefins (C12 rich) derived from propene oligomerization, carbonates, calcium salts, overbased, sulfurized including distillates (petroleum), hydrotreated, solvent-refined, solvent-dewaxed, or catalytic dewaxed, light or heavy paraffinic C15-C50
Details on test material:
- Name and of test material: Phenol, dodecyl-, sulfurized, carbonates, calcium salts, overbased (alternate name: Phenol, tetrapropenyl-, sulfurized, carbonates, calcium salts, overbased)
- CAS number of test material: 68784-26-9 (alternate CASRN 122384-87-6)
Testing was performed on a commercial sample of this material. Typical composition of this UVCB material as distributed in commerce is the same as identified in Section 1.2 of IUCLID. Residual calcium carbonate (overbasing) of the commercial substance is typically 12 wt%.

Test animals

Species:
rat
Strain:
other: Crl:CD(SD)
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Inc., Raleigh, NC
- Age at study initiation: approximately 6 weeks old
- Weight at study initiation: Mean bodyweights: Males - 188-189g; Females - 144-147g
- Fasting period before study: No, however it should be noted that animals were fasted prior to obtaining blood samples.
- Housing: upon arrival, all animals were housed 3 per cage by sex for approximately 3 days. Thereafter, all animals were housed individually in clean, stainless steel, wire-mesh cages suspended above cage-board.
- Diet: PMI Nutrition International, LLC, Certified Rodent LabDiet® 5002, ad libitum
- Water (e.g. ad libitum): reverse osmosis-treated (on-site) drinking water, delivered by an automatic watering system was provided ad libitum during acclimation and throughout the study
- Acclimation period: 14-days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21.2 C to 21.5 °C
- Humidity (%): 32.5% to 61.7%
- Air changes (per hr): Air handling units were set to provide a minimum of 10 fresh air changes per hour.
- Photoperiod (hrs dark / hrs light): Fluorescent lighting provided illumination for a 12-hour light (0600 hours to 1800 hours)/12-hour dark photoperiod. The 12-hour light/12-hour dark photoperiod was interrupted as necessary to allow for the performance of protocol-specified activities

Administration / exposure

Route of administration:
oral: feed
Vehicle:
corn oil
Details on oral exposure:
DIET PREPARATION
- Rate of preparation of diet (frequency): The control diet admixture was prepared approximately weekly for administration to the control group (Group 1) and was stored at room temperature. The test diet formulations were weight/weight (test substance/diet) admixtures. The test substance formulations were prepared approximately weekly as single formulations for each dosage level and stored at room temperature. The test substance formulations were stirred as necessary throughout preparation.
- Mixing appropriate amounts with (Type of food): PMI Nutrition International, LLC, Certified Rodent LabDiet® 5002 (meal) mixed with an appropriate amount (2%) of corn oil was used for administration to the control group. The vehicle was mixed as necessary during preparation in a Hobart mixer using a designated vehicle bowl and paddle.
Dosing formulations were prepared at the test substance concentrations indicated in the following table:
Group Number Dosage Level (mg/kg/day) a
2 125
3 250
4 500
5 1000

a = The dosing formulations were not adjusted for purity.

Prior to formulating the test diet for all groups, the test substance and corn oil were placed into an incubator at approximately 50°C to aid in mixing. The calculated amount of test substance was weighed into tared glass jars. The appropriate amount (2%) of corn oil was weighed, added to the test substance for each group, and mixed using a magnetic stirrer to obtain a uniform mixture. A predetermined amount of basal diet was added to the test substance mixture and mixed for an appropriate amount of time. The calculated remaining basal diet was added to the pre-mixture to bring the batch size to the final weight and the mixture was mixed in a V-blender for a specified amount of time.
- Storage temperature of food: room temperature

VEHICLE
The vehicle used in preparation of the test diet formulations and for administration to the control group was Certified Rodent LabDiet® 5002 (meal), PMI Nutrition International, LLC and corn oil (lot nos. 117K0127, 028K0044, and 058K0070 [2 receipt dates], exp. dates: 22 July 2009, 11 August 2009, 1 December 2009, and 31 December 2009, respectively, received from Sigma Aldrich, St. Louis, MO).
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Homogeneity and stability of the test substance in batch sizes of 10 kg was analyzed prior to the initiation of dose administration in a previous study, WIL-187079 (Bodle, 2009). Duplicate samples (100 g) from the top, middle, and bottom strata of formulations prepared on 23 September 2008 and 28 October 2008 at target concentrations of 0.4 and 25 g test material/kg of diet were assessed for calcium phenate and tetrapropenyl phenol homogeneity. Dietary admix formulations prepared on 28 October 2008 were analyzed for stability following up to 15 days of room temperature or frozen storage. Prior to the initiation of dose administration for the current study, duplicate samples (100 g) for homogeneity determination in batch sizes of 4 kg were collected from the top, middle and bottom strata of the 0.4 and 25 g/kg diet preparations (6 November 2008). Duplicate samples (100 g) for concentration analysis were collected weekly during the first 4 weeks (study weeks 0 through 3) of the study and monthly thereafter from the middle stratum of each dosing formulation (including the control group) at the time of preparation. During study week 4 (the fifth study preparation), additional samples were collected from all groups (by sex) and analyzed for concentration due to the low analytical concentration results (approximately 75% of target) achieved for the 125 mg/kg/day group females during study week 3. In addition, samples (100 g) were collected at the time of preparation from the middle stratum of each test diet batch (including controls) during study weeks that concentration analysis were not conducted and stored frozen under nitrogen for possible future analysis. All analyses were conducted by the Analytical Chemistry Department, WIL Research Laboratories, LLC using a validated high performance liquid chromatography method using fluorescence detection.
Duration of treatment / exposure:
91-92 days
Frequency of treatment:
ad libitum in diet
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 125, 250, 500 & 1000 mg/kg
Basis:
nominal in diet
No. of animals per sex per dose:
10 animals/sex/group
Control animals:
yes, concurrent vehicle
Positive control:
n/a

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: All animals were observed twice daily, once in the morning and once in the afternoon, for mortality and moribundity.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Clinical examinations were performed once daily. The absence or presence of findings was recorded for all animals. Detailed physical examinations were conducted on all animals approximately weekly, beginning 1 week prior to randomization, at randomization, and prior to the scheduled necropsy. Once daily clinical observations were not performed on days when detailed physical examinations were conducted. A separate computer protocol was used to record any observations noted outside of the above-specified intervals.

BODY WEIGHT: Yes
- Time schedule for examinations: Individual body weights were recorded approximately weekly during pretest and throughout the study. Mean body weights and mean body weight changes were calculated for the corresponding intervals. Final body weights (fasted) were recorded on the day of the scheduled necropsy for all animals.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated: Yes, individual food consumption was recorded approximately weekly during pretest and throughout the study. Food intake was calculated as g/animal/day for the corresponding body weight intervals. When food consumption could not be measured for a given interval (due to spillage, weighing error, obvious erroneous value, etc.), the appropriate interval was footnoted as "NA" (Not Applicable) on the individual tables.
The mean amounts of the test material consumed (mg/kg/day) by each sex per dose group were calculated from the mean food consumed (g/kg/day) and the appropriate target concentration of test substance in the food (mg/kg).

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: study week 13
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes
- How many animals: all
- Parameters checked:
Total leukocyte count (White Cells); Erythrocyte count (Red Cells); Hemoglobin; Hematocrit; Mean corpuscular volume (MCV); Mean corpuscular hemoglobin
(MCH); Mean corpuscular hemoglobin; concentration (MCHC); Platelet count (Platelet); Prothrombin time (ProTime); Activated partial thromboplastin time
(APTT); Reticulocyte count; Percent (Reticulocyte); Absolute (Retic Absolute); Differential leukocyte count - Percent and absolute, -Neutrophil, -Lymphocyte, -Monocyte, -Eosinophil, -Basophil, -Large unstained cell; Platelet estimate; Red cell morphology; (RBC Morphology).

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: study week 13
- Animals fasted: Yes
- How many animals: all
- Parameters checked:
Albumin; Total protein; Globulin [by calculation]; Albumin/globulin ratio (A/G Ratio) [by calculation]; Total bilirubin (Total Bili); Urea nitrogen; Creatinine; Alkaline phosphatase (AlkalinePhos’tse); Alanine aminotransferase (Alanine Transfer); Aspartate aminotransferase (AspartatTransfer); Glucose; Total cholesterol (Cholesterol); Calcium; Chloride; Phosphorus; Potassium; Sodium Triglycerides (Triglyceride).

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
A complete necropsy was conducted for all animals. Animals were euthanized by carbon dioxide inhalation followed by exsanguination. The necropsies included, but were not limited to, examination of the external surface, all orifices, and the cranial, thoracic, abdominal and pelvic cavities, including viscera. The following tissues and organs were collected and placed in 10% neutral-buffered formalin (except as noted):

Adrenals (2); Aorta; Bone with marrow; Femur; Sternum; Bone marrow smear (from femur)[a]; Brain [b]; Cerebrum 2 levels; Cerebellum with medulla/pons; Cervix; Epididymides (2) [c]; Eyes with optic nerve (2) [d]; Gastrointestinal tract; Esophagus; Stomach; Duodenum; Jejunum; Ileum; Peyer’s patches [e]; Cecum; Colon; Rectum; Heart; Kidneys (2); Liver (sections of 2 lobes); Lungs (including bronchi, fixed by inflation with fixative); Lymph nodes; Mandibular (2); Mesenteric; Nasal Cavity [f]; Ovaries with oviducts (2) [g]; Pancreas; Peripheral nerve (sciatic); Pituitary; Prostate; Salivary glands (mandibular [2]); Seminal vesicles with coagulating glands (2); Skeletal muscle (rectus femoris); Skin (with mammary gland) [h]; Spinal cord (cervical, thoracic, lumbar) [i]; Spleen; Testes (2) [c]; Thymus; Thyroid (with parathyroids, if present [2]) [g]; Trachea; Urinary bladder; Uterus; Vagina; Gross lesions (when possible).

[a] - Bone marrow smears were obtained at scheduled necropsy, but not placed in formalin; slides were not examined.
[b] - All levels of the brain were designated as “Brain” in the raw data.
[c] - Fixed in Bouin’s solution.
[d] - Fixed in Davidson’s solution.
[e] - Peyer’s patches were examined microscopically if in the plane of section of the jejunum. If not in the plane of section of the jejunum, then patches were examined if in the place of section of the ileum.
[f] - Sectioning of nasal cavity was in accordance with the method of Young (1981).
[g] - Oviducts and parathyroids were examined microscopically if in the plane of section and in all cases where gross lesions were present.
[h] - For females; a corresponding section was taken from the same anatomic area for males.
[i] - Cervical, thoracic and lumbar spinal cord was designated as “Spinal cord” in the raw data.

HISTOPATHOLOGY: Yes
After fixation, protocol-specified tissues were trimmed according to standard operating procedures and the protocol. Trimmed tissues were processed into paraffin blocks, sectioned at 4 to 8 microns, mounted on glass microscope slides and stained with hematoxylin and eosin.
Microscopic examination was performed on all tissues listed above, from all animals in the control and 1000 mg/kg/day groups at the scheduled necropsy and gross lesions were examined from all animals in the 125, 250 and 500 mg/kg/day groups.
Missing tissues were identified as not found at necropsy, lost at necropsy, lost during processing or other designations as appropriate. Tissues may appear on the report tables as not examined due to the tissue not being in the plane of section, not present at trimming, etc. Microscopic examination was performed by John Boyce, DVM, PhD, DACVP, DACLAM, WIL Research Laboratories, LLC.
Other examinations:
The following organs were weighed from all animals at the scheduled necropsy:
Adrenals; Brain; Epididymides; Kidneys; Liver; Ovaries with oviducts; Prostate; Seminal vesicles with coagulating glands; Testes; Thyroid with parathyroids*; Uterus.

Paired organs were weighed together. Designated organs (*) were weighed after fixation.
Organ-to-final-body-weight and organ-to-brain-weight ratios were calculated.
Statistics:
All statistical tests were performed using appropriate computing devices or programs.
Analyses were conducted using two-tailed tests (except as noted otherwise) for minimum significance levels of 1% and 5%, comparing each test substance-treated group to the control group by sex. Each mean was presented with the standard deviation (S.D.), standard error (S.E.) and the number of animals (N) used to calculate the mean. In addition, percent difference from the control group is presented for body weights, clinical pathology parameters and organ weights. Due to the different rounding conventions inherent in the types of software used, the means and standard deviations on the summary and individual tables may differ by ±1 in the last significant figure.
Body weight, body weight change, food consumption, clinical pathology and organ weight data were subjected to a parametric one-way analysis of variance (ANOVA) (Snedecor and Cochran, 1980) to determine intergroup differences. If the ANOVA revealed statistically significant (p<0.05) intergroup variance, Dunnett's test (Dunnett, 1964) was used to compare the test substance-treated groups to the control group.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
not specified
Details on results:
CLINICAL SIGNS AND MORTALITY
All animals survived to the scheduled necropsy. There were no test substance-related clinical observations. All clinical findings in the test substance-treated groups were noted with similar incidence in the control group, were limited to single animals, were not noted in a dose-related manner and/or were common findings for laboratory rats of this age and strain.

BODY WEIGHT AND WEIGHT GAIN
Test substance-related effects of lower body weights were noted in the 250, 500, and 1000 mg/kg/day test substance-treated groups.
Lower mean body weight gains were noted for the 250, 500, and 1000 mg/kg/day group males and females as early as study week 0 to 1; some of these intervals were statistically significant when compared to the control group. By the end of the study (study week 13), mean body weights for the 250, 500, and 1000 mg/kg/day groups were 3.8%, 5.6%, and 9.1% lower for males, respectively, and 4.4%, 6.7%, and 8.4% lower for females, respectively, than the corresponding control group. However, since the differences between the absolute mean body weights and the control group at the end of the study did not exceed 10%, these body weight effects were considered to be non-adverse.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
Food consumption was unaffected by test substance administration. There were no statistically significant differences when the control and test substance-treated groups were compared.

Theoretical Dietary Level (mg/kg/day) Average Test Substance Consumption (mg/kg/day)
Male Female
0 0 0
125 129 125
250 259 255
500 518 506
1000 1013 1015


HAEMATOLOGY
Hematology parameters were unaffected by test substance administration. While there were several statistically significant differences between the group means, none were considered evidence of a test substance-related change because all mean values for the test substance-treated groups for all parameters were within the WIL Historical Control Summary of Clinical Pathology Database (version 2.8) minimum/maximum ranges for group means.

CLINICAL CHEMISTRY
Serum chemistry parameters were unaffected by test substance administration. While there were several statistically significant differences between the group means, none were considered evidence of a test substance-related change because all mean values for the test substance-treated groups for all parameters were within the WIL Historical Control Summary of Clinical Pathology Database (version 2.8) minimum/maximum ranges for group means.

GROSS PATHOLOGY/ORGAN WEIGHTS
There were no test substance-related alterations in organ weights.
Mean final body weights for the 1000 mg/kg/day group males and females were 9.3% and 8.7% lower, respectively, than the corresponding control group values. The final body weight differences between these groups were not statistically significant; however, the differences were great enough to cause statistically significant differences in male and female liver weights and male kidney weights relative to the final body weights. These significant relative weight differences were not considered direct effects of test substance administration since there were no correlating effects on serum chemistry parameters and/or histopathological findings.
The absolute mean seminal vesicle/coagulating gland weight in the 1000 mg/kg/day group was statistically significantly lower than the control group; however, only 1/10 animals in the 1000 mg/kg/day group had a value below the WIL Historical Control reference range lower limit (version 2.8). The low gland weight value of this single animal (male no. 9287) was most likely attributed to a loss of seminal fluid prior to weighing and therefore, was not considered evidence of a test substance effect.
There were no other statistically significant differences when the control and test substance-treated groups were compared.

HISTOPATHOLOGY: NON-NEOPLASTIC
There were no test substance-related microscopic findings. All findings observed were consistent with normal background lesions in clinically normal rats of the age and strain used on this study and were considered spontaneous and/or incidental in nature and unrelated to test substance administration

HISTORICAL CONTROL DATA (if applicable)
Historical control values for this laboratory were consulted to refine data interpretation.

Effect levels

open allclose all
Dose descriptor:
NOEL
Effect level:
125 mg/kg diet
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: based on lower body weights and body weight gains at dose level ≥ 250 mg/kg/day
Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg diet
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other:
Remarks on result:
not determinable due to absence of adverse toxic effects

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
Based on the results of this study, the no-observed-effect level (NOEL) for oral (dietary) administration of the test material offered ad libitum via the basal diet for 91 or 92 consecutive days to Crl:CD(SD) rats was 125 mg/kg/day based on lower body weights and body weight gains at dose levels ≥ 250 mg/kg/day. Given that clinical observations, macroscopic and microscopic examinations, food consumption, hematology and serum chemistry parameters, and organ weights were unaffected by test substance administration and body weight effects were considered non-adverse, the no-observed-adverse-effect level was considered to be 1000 mg/kg/day.
Executive summary:

The objective of this study was to establish dose levels for a proposed dietary two-generation reproductive toxicity study by evaluating the potential toxic effects of the test substance when administered via the diet to rats for 90 days. The protocol was designed to be in general accordance with the Organisation for Economic Cooperation and Development (OECD) Guidelines for Testing of Chemicals, Health Effects Test Guidelines, Section 408, adopted 2 1 September 1998 with the following exception; Functional observational batteries (FOBS) were not conducted.

The test material was offered ad libitum via the basal diet for a minimum of 90 consecutive days to 4 toxicology groups (Groups 2-5) of Crl:CD(SD) rats. Dosage levels were 125, 250, 500, and 1000 mg/kg/day. A concurrent control group (Group 1) received the basal diet on a comparable regimen. Groups 1-5 each consisted of 10 animals/sex. Following 91 or 92 days of test diet administration, all animals were euthanized and subjected to a complete necropsy.

All animals were observed twice daily for mortality and moribundity. Clinical examinations were performed daily, and detailed physical examinations were performed approximately weekly. Individual body weights and food consumption were recorded approximately weekly. Clinical pathology evaluations (hematology, coagulation and serum chemistry) were performed on all animals at the scheduled necropsy (study week 13). Complete necropsies were conducted on all animals, and selected organs were weighed at the scheduled necropsy. Selected tissues were examined microscopically from all animals in the control and 1000 mg/kg/day groups. In addition, gross lesions were examined from all animals in the 125, 250 and 500 mg/kg/day groups.

All animals survived to the scheduled necropsy. There were no test substance-related clinical observations, macroscopic or microscopic findings noted. Food consumption, hematology and serum chemistry parameters, and organ weights were unaffected by test substance administration.

Test substance-related effects of lower body weights were noted in the 250, 500, and 1000 mg/kg/day test substance-treated groups. Lower mean body weight gains were noted for the 250, 500, and 1000 mg/kg/day group males and females as early as study week 0 to 1. Throughout the dosing period, lower mean body weights were observed in a dose-related manner in all test substance-treated groups when compared to the control group. Mean body weights for the 250, 500, and 1000 mg/kg/day group males and females were lower than the corresponding control group at the end of the study; however, these body weight effects were non-adverse as the absolute body weights at the end of the study were lower by < 10% compared to the control group.

Based on the results of this study, the no-observed-effect level (NOEL) for oral (dietary) administration of the test material offered ad libitum via the basal diet for 91 or 92 consecutive days to Crl:CD(SD) rats was 125 mg/kg/day based on lower body weights and body weight gains at dose levels ≥ 250 mg/kg/day. Given that clinical observations, macroscopic and microscopic examinations, food consumption, hematology and serum chemistry parameters, and organ weights were unaffected by test substance administration and body weight effects were considered non-adverse, the no-observed-adverse-effect level was considered to be 1000 mg/kg/day.