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Description of key information

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
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:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study conducted to GLP.
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
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
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
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
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.
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.
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
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg diet
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse effects observed
Critical effects observed:
not specified
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.

 

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
Klimisch code 1 (reliable without restriction)

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - systemic effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: dermal
Type of information:
experimental study
Adequacy of study:
key study
Study period:
3rd April to 3rd May 1985
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study following GLP.
Qualifier:
according to guideline
Guideline:
OECD Guideline 410 (Repeated Dose Dermal Toxicity: 21/28-Day Study)
Deviations:
not specified
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: The Charles River Breeding Laboratories, Inc., 251 Ballardvale Street, Wilmington, Massachusetts 01887
- Age at study initiation: Males: 74 Days Females: 82 Days
- Weight at study initiation: Males: 302-421 grams Females: 210-263 grams
- Housing: All animals were individually housed in wire-bottom ventilated cages with automatic watering systems. Cage exhaust static pressure ranged from 0.39-0.60 negative inches of water.
- Diet/water (e.g. ad libitum): The animals had free access to Purina Certified Rodent Chow #5002 and water. Food was in pellet form until Day -1 and in powdered form thereafter.
- Acclimation period: quarantined for two weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.8-23.9°C
- Humidity (%): 36.7-38.2%
- Photoperiod (hrs dark / hrs light): 12-hour light/dark cycle

Type of coverage:
semiocclusive
Vehicle:
other: mineral oil
Details on exposure:
TEST SITE
- Area of exposure: Dorsal trunk
- Type of wrap if used: An 8.5 x 3 inch gauze sponge was placed over the site and secured with one-inch Zonas porous tape.

REMOVAL OF TEST SUBSTANCE
- Washing (if done): Six hours after the application, the collars and dressings were removed and the skin site was wiped with a gauze sponge moistened with mineral oil.
- Time after start of exposure: 6 hours

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): A dose of 1 ml/kg of the appropriate suspension was applied by means of a Pipetman 1000. The dose was gently spread with a metal spatula to cover the site.

USE OF RESTRAINERS FOR PREVENTING INGESTION: yes; plastic collars
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Five, five-gram samples of each dosing suspension were taken each week on the day following preparation. Another five samples of each dose level were taken on the last day each batch of mixture was used. For the first and last week of dosing, three of the five samples taken the day after preparation were refrigerated and transmitted to Chevron Research Company for analysis.
Duration of treatment / exposure:
The animals were dosed Mondays through Fridays for 28 days beginning on a Wednesday and ending on a Tuesday.
Frequency of treatment:
6 hours/day, 5 days/week
Remarks:
Doses / Concentrations:
0, 2, 10 and 25 % w/w in mineral oil administered in a dose volume of 1 mL/kg b.wt./day
Basis:
analytical per unit body weight
No. of animals per sex per dose:
12 rats/sex/group.
Control animals:
yes, concurrent vehicle
Details on study design:
No recovery group was employed.

All guideline recommendations were exceeded, except for the following: Surface area of dermal application site was not provided.

The actual doses received were approximately 0, 20, 100 and 250 mg/kg b.wt./day.
Positive control:
No data
Observations and examinations performed and frequency:
Body Weights: All animals were weighed prior to dosing on Day 0 and every Tuesday and Friday thereafter for the remainder of the dosing period. At the end of the study, fasting terminal body weights were measured on the day each animal was killed.

Food Consumption: Food consumption was measured at weekly intervals from Day -1 through Day 27. Clear glass food hoppers were filled with fresh powdered food and weighed each Tuesday; they were reweighed the following Tuesday and replaced with clean, filled hoppers.

Signs of Toxicity: Each animal was observed once daily for signs of toxicity following unwrapping. A viability check was made daily prior to dosing. On weekends, observations were performed in the morning and viability checks in the afternoon. A detailed examination for changes in skin and fur, eyes and mucous membranes, respiratory system, circulatory system, autonomic and central nervous system, somatomotor activity, and behavior patterns was conducted weekly on Thursdays and on the last day of dosing. The pupil response of each animal was examined the first and last days of dosing and weekly during the study.

Skin Irritation: Immediately after the initial six-hour exposure, each Friday thereafter, and following the final exposure, the animals' skins were scored for skin irritation, using the modification of the Draize et al.

Sacrifice: The animals were fasted overnight and killed by exsanguination under pentobarbital anesthesia (Sleepaway, Fort Dodge Laboratories, Inc., Fort Dodge, Iowa). Five rats/sex/group/day were killed on Days 28-30.

Blood Samples: Prior to sacrifice at the end of the study, blood was obtained from 10 rats of each sex from each treatment group.

Samples were taken from the descending aorta of fasted rats. Approximately three ml of whole blood were centrifuged at 2000 x g for 10-15 minutes. The serum was removed, refrigerated, and submitted for a serum chemistry profile. Approximately one ml of whole blood with EDTA was refrigerated and submitted for hematology. A blood smear was prepared for the differential white blood cell count.

California Veterinary Diagnostics, Inc., West Sacramento, California, performed all hematology and serum chemistry analyses. The white blood cell count, red blood cell count, hemoglobin, hematocrit, mean cell volume, mean cell hemoglobin, and mean corpuscular hemoglobin concentration were determined on a Coulter Counter Model SR. The platelet and differential white blood cell counts were performed manually. A Boehringer Mannheim Diagnostics Group 8600R was used to determine the sodium, potassium, chloride, calcium, phosphorus, uric acid, blood urea nitrogen, creatinine, total protein, albumin, globulin, glucose (fasting), total bilirubin, direct bilirubin, alkaline phosphatase, lactate dehydrogenase, aspartate aminotransferase, alanine amino-transferase, creatine phosphokinase activity, cholesterol, and triglycerides levels.

Organ Weights: The following organs were weighed and examined for gross pathological changes: liver, kidneys (left and right), adrenals (left and right), testes (left and right), ovaries (left and right), and brain.


Sacrifice and pathology:
GROSS PATHOLOGY: Yes
A full gross necropsy (including examination of the external surface of the body, all orifices, and the cranial, thoracic, and abdominal cavities and their contents) was performed on all animals found dead during the course of the study and on all animals at the final sacrifice.
The following tissues were also examined for gross pathological changes: thyroid/parathyroid, thymus, lungs, heart, salivary glands, spleen, seminal vesicles, pancreas, uterus, skin (untreated and treated), stomach, small intestine (duodenum, jejunum, ileum), large intestine (cecum, colon), urinary bladder, and gross lesions.
Tissue Preservation: The following organs were preserved in neutral buffered formalin: lungs, liver, spleen, brain, kidneys, testes, ovaries, skin (untreated and treated), and gross lesions.

HISTOPATHOLOGY: Yes
All preserved tissues from the control and high-dose groups were submitted for histopathological examination. Slides were prepared at Histopathology Reference Laboratory, Oakland, California.
Statistics:
Body weight, food consumption, hematology, serum chemistry, and organ weight data were analyzed by ANOVA with a Dunnett’s post-hoc test if
significance was observed with ANOVA, except for hematology and serum chemistry data where the post-hoc test used was the Least Significant
Difference test.
Clinical signs:
no effects observed
Dermal irritation:
effects observed, treatment-related
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:
no effects observed
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
not specified
Behaviour (functional findings):
not specified
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:
Mortality and Signs of Toxicity:
No deaths occurred and no signs of toxicity were observed during the study. Chromodacryorrhea and chromorhinorrhea were seen in almost all animals, resulting from stress induced by the plastic collars. These signs appeared intermittently in some animals and throughout the study in others, but disappeared on weekends when the collars were not used. One-sixth to one-half of the males in each group developed alopecia, sores, or scabs on the skin over the throat after the first week of dosing. These skin effects were apparently due to and aggravated by collaring; animals having sores or scabs in this region were therefore not collared following observation of the lesion.
Several sporadic observations were made: one low-dose female, one high-dose female, and one high-dose male showed colorless ocular discharge; one mid-dose male had bloody penile discharge; and one low-dose and two high-dose females had hunched posture. All animals recovered overnight. All animals had normal pupil responses during the study.

Skin Irritation:
Irritation was observed in all dose groups. The degree and incidence of irritation were highest after the first and second weeks of dosing. Males treated with the test material showed slightly higher incidence and severity of irritation than control males; treated and control females showed approximately equal irritation. During the course of the study, 5 of 12 male and 12 of 12 female control animals showed slight to well-defined erythema with no to slight edema. In the low-dose group, 10 of 12 males and 11 of 12 females had slight to well-defined erythema and no edema. Ten of twelve mid-dose males and 10 of 12 mid-dose females had slight to well defined erythema with no to slight edema. All high-dose animals
showed slight to well-defined erythema and no to slight edema.

Single or multiple small scabs, apparently due to scratching or biting, were observed in all dose groups by Day 9. Scabbing was seen most frequently in control animals, suggesting that mineral oil, the vehicle, was irritating to some animals. Abrasions on the application site were seen on Day 2 in all groups; these abrasions corresponded, for the most part, with scabs or flakiness by Day 9. Dryness and flakiness were observed frequently during the study in all groups.

Body Weights:
There were no significant differences in mean body weight or body weight gain during the study.

Food Consumption:
No significant differences in mean food consumption were observed at any time during the study.

Hematology:
Platelet counts in the low- and mid-dose females and the eosinophil count in the low-dose females were significantly greater than controls. These values were, however, within the range of historical control values in similar studies conducted at CEHC, Inc. Comparison to historical control values and the lack of any dose-related trend indicates that these findings were probably not related to treatment with the test material.

Clinical Chemistry:
The mean SGPT level in the high-dose males was significantly greater than that of the controls. For low- and high-dose females, the mean alkaline phosphatase levels were significantly less than the controls. Values for both SGPT and alkaline phosphatase were within range of historical control values. This comparison, in addition to the lack of a dose-related trend in the alkaline phosphatase levels, indicate that the differences were probably not treatment-related.

Organ Weights and Organ/Body Weight Ratios:
There were no significant differences in organ weights or organ/body weight ratios during the study.

Pathology:
Gross skin observations at necropsy consisted of dryness and flakiness at all dose levels, with sporadic observations of scabbing, necrosis, alopecia, and discoloration. At least one male at each dose level had a dilated renal pelvis. One mid-dose male had granular material in the dilated pelvis; one control male had an atropied kidney, an enlarged kidney, and a nodular renal mass, and another had a lesion on the ventral surface of the neck. One high-dose male had a blanched liver; black pulmonary foci were seen in a low-dose female; a thickened urinary bladder wall and an ovarian hydrocoel were also seen in low-dose females.

There were no dose-related trends. The skin findings were probably due to the technical procedure; other lesions appeared to be due to spontaneous disease.

Histopathology:
Histopathological examination of control and high-dose animals showed trace to mild acanthosis at the treatment site in 11 of 12 male and 7 of 12 female control animals and in all high-dose animals. Other skin findings were dermatitis and surface exudate. Trace renal lymphocytic infiltration was seen-in one control male, one high-dose male, and one high-dose female; trace to mild hepatic lymphocytic infiltration was seen in three control males, two control females, and two high-dose males. One control male had one enlarged and one atrophied kidney; two high-dose males had hydrone¬phrosis, one had renal pyelitis, and one showed renal regeneration.

There was a slight increase in incidence and severity of acanthosis in the high-dose males and females when compared to the controls. There were no other compound-related lesions; the lesions observed were those of spontaneous disease.
Dose descriptor:
NOAEL
Effect level:
ca. 250 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: 250 mg/kg/bw/day was highest dose tested. No LOAEL was determined.
Critical effects observed:
not specified
Conclusions:
Repeated dermal applications of the test material caused no observable toxicity, with the exception of a slightly increased incidence and severity of skin irritation when compared to controls.
NOAEL is >250 mg/kg b.wt./day for males and female rats, highest dose tested.
Executive summary:

In a GLP compliant study conducted to OECD guideline 410, groups of 12 male and 12 female rats were treated dermally six hours per day, five days per week for four weeks with 1.0 ml/kg of the test material weight/weight (w/w) in mineral oil at the following concentrations: 2% (low-dose), 10% (mid-dose), and 25% (high-dose). Twelve rats of each sex were treated with mineral oil and served as controls.

Males treated with the test material showed a slightly higher incidence and severity of skin irritation (no to well-defined erythema with no to slight edema) than control males (no to well-defined erythema with no edema). Treated and control females showed approximately equal irritation. Both sexes had the highest incidence and severity after the first and second weeks of dosing. There were no other clinical signs of toxicity.

No significant differences in body weight, weekly body weight gain, weekly food consumption, organ weights, or organ/body weight ratios were seen between treated and control animals.

Mean SGPT levels in the high-dose males were significantly different from control values; females showed significant differences in alkaline phosphatase levels, platelet counts, and eosinophil counts. These findings were not considered treatment-related due to the lack of dose-related trends and/or based on comparison of values with historical control values.

At necropsy, animals from all groups showed dryness and flakiness at the treated site. Scabbing, necrosis, alopecia, and discoloration were seen in some animals. Histopathological examination of skin sections showed trace to mild acanthosis at the treated site in high-dose and control animals; the severity and incidence of acanthosis were slightly greater for the high-dose animals as compared to the controls. There were no other gross or microscopic treatment-related findings.

In conclusion, repeated dermal applications of the test material caused no observable toxicity in rats, with the exception of slight skin irritation during the first two weeks of the study.

The NOAEL was determined to be >250 mg/kg b.wt./day for males and female rats, the highest dose tested.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
250 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
Klimisch code 1 (reliable without restriction)

Additional information

Oral:

In the key study for repeat dose toxicity, oral exposure (Haas, 2010), the study was conducted to the OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents) and GLP. A reliability rating of 1 was assigned according to the criteria of Klimisch, 1997. This study was used for classification purposes as it is considered the most robust and reliable study.

A 28 day (Lamb, 1993) study is also available as supporting information, the study was conducted to the OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test) and GLP. A reliability rating of 1 was assigned according to the criteria of Klimisch, 1997. This study is not considered the key study as a more robust 90 day study is available.

Results: Based upon the results of this study, the apparent NOAEL (no observable adverse effect level) for subchronic toxicity & neurotoxicity was considered to be 200 mg/kg/day. This was based on increased adrenal and pituitary gland weights, which are effects that are attributed to the residual TPP that is present in phenates. This is consistent with no effects observed in the pituitary or adrenal glands in the 1 -generation reproductive toxicity study with a phenate depleted of TPP. Therefore, the 90 day study with a NOAEL of 1000 is the most appropriate key study to select for repeat dose toxicity.

Dermal:

In the key study for repeat dose toxicity, dermal exposure (Korenaga et al, 1986, report number: SOCAL 2319), the study was conducted to the OECD Guideline 410 (Repeated Dose Dermal Toxicity: 21/28-Day Study) and GLP. A reliability rating of 1 was assigned according to the criteria of Klimisch, 1997.

Supporting studies available:

- The Biodynamics study, 1981 was not considered the key study as it was conducted less recently than the above key study, there was no GLP data and no guideline was specified although it seems similar to OECD 410 with deviations. Deviations are as follows: Only 2 dose groups were used, dressing was only a paper towel, not gauze as specified in the OECD guideline and the test site was wiped with a paper towel, no solvent used to remove test material. A reliability rating of 2 was assigned according to the criteria of Klimisch, 1997.

Treatment-related effects were evident in the low- and high-dose males treated with the test material as indicated by marked, statistically significant, dose-related decreases in the absolute and relative (organ/body weight ratios) weights of the testes (absolute weight decreases of approximately 70% and 80%) and epididymides (absolute weight decreases of approximately 40% and 50%) at the end of the treatment period.

The results of the microscopic examinations indicated that the topical administration of 25% and 100% (w/v) of the test material, for the period used in this study, causes topical irritating skin lesions in both male and female rabbits. Male rabbits, receiving 25% and 100% (w/v) of the test material, had an increased incidence of aspermatogenesis, reduced numbers of spermatids and a diffuse tubular hypoplasia when compared to control males. These changes had not completely resolved themselves in the four week recovery rabbits receiving 25% and 100% (w/v) of the test material.

- The Biodynamics study, 1983 was not considered the key study as it was conducted less recently than the above key study and no guideline was specified although it seems similar to OECD 410 with deviations. Deviations are as follows: Only 2 dose groups were used there was no data on dressing or removal of test material at the test site and only males were used in this study, OECD specifies 5 males/females per dose. A reliability rating of 2 was assigned according to the criteria of Klimisch, 1997.

Increased incidence of desquamation, red raised pustules and red raised areas of eschar noted in the high dose group animals when compared to the control group animals. At necropsy, 2 animals in each of the treated groups were noted to have scabs, sores or necrotic areas on the dorsal cervical skin.

The Group 2 animals showed an increase in mean epididymal weights while the group 3 animals showed a decrease in mean epididymal weights when compared to control group animals. These findings are statistically significant.

Total bilirubin and total protein levels were decreased in group 2 animals when compared to the control group values. Total bilirubin and glucose levels and the albumin/globulin ratio were decreased in Group 3 animals when compared to the control group animals. These findings are statistically significant.

Inhalation:

In accordance with column 2 of REACH Annex XI, section 8.6, it is considered justifiable to omit the acute toxicity: inhalation study. The substance only exists in liquid form and it will not be aerosolized in its normal use pattern and does not exist as small particles or droplets. As such testing via the inhalation route is not considered appropriate for this substance.

Justification for classification or non-classification

Based on no adverse effects observed in a well-conducted 90 day study, the classification criteria for target organ toxicity are not met. This is consistent with no adverse effects in the key 28 -day dermal toxicity study.