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Repeated dose toxicity: oral

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Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
9th October 1991 to 7th December 1992
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study following GLP.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1993
Report Date:
1993

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
Principles of method if other than guideline:
The potential subchronic toxicity (Phase A) & neurotoxicity (Phase B) related to the administration of the test material in the Sprague-Dawley Crl: CD®BR rat were evaluated in this combined repeated dose study.
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
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:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: The Charles River Breeding Laboratories, Inc., Portage, Michigan 49081
- Age at study initiation: Young adult, 50 days old.
- Weight at study initiation: Males, 197g - 240g; Females, 145g - 179g
- Housing: Animals were housed individually in suspended, stainless steel wire-mesh cages.
- Diet/water (e.g. ad libitum): Purina® Certified Rodent Chow #5002 and municipal water were provided did libitum.
- Acclimation period: The animals were acclimated to laboratory conditions for 13 days prior to study initiation. The acclimation period included a one week pretest period for each phase. The animals were observed twice daily for mortality and moribundity.

ENVIRONMENTAL CONDITIONS
- Temperature : 68-76°F
- Humidity (%): 30-70%
occasional deviations from set temperature and humidity levels did not apparently effect the outcome of the study
- Air changes (per hr): approximately 10 fresh air changes per hour.
- Photoperiod (hrs dark / hrs light): 12 hours light/ 12 dark

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
peanut oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
The required amount of test article for each group was drawn into a syringe. A specified amount of vehicle was placed in a storage container. The test article was added to the vehicle while mixing with a laboratory mixer until the test material had been incorporated. The preparation was then stirred (magnetic stir bar) throughout the sampling, dispensation and dosing procedures, and periods of non-use. Storage containers were wrapped in foil for additional light protection. Test article preparations were formulated weekly; the prepared suspensions for each dose group were stirred at least 30 minutes prior to sampling.

Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analytical Tests: Homogeneity, 8 and 14 day stability and test article concentration analyses were conducted on test material preparations.

Strata Sampled: The top, middle and bottom of the test material preparations were sampled for homogeneity and stability; the middle of a preparation was sampled for concentration.

Sample Volume: 10 ml

Samples were taken in duplicate. One set of samples was sent to the sponsor for analysis; the second set was retained at WIL Research Laboratories, Inc., at ambient temperature until issuance of the final report.

Samples were collected prior to study initiation (homogeneity and stability analyses) and from each dosing preparation on the first and last day of use (concentration analyses).
Duration of treatment / exposure:
28 days for all dose groups and 42 days for the control and high dose recovery groups
Frequency of treatment:
daily
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 50, 200 and 1000 mg/kg b.wt./day
Basis:
actual ingested
No. of animals per sex per dose:
There were 5 rats/sex/group for the 4-week exposure groups, and initially the same group size for the control and high dose 2-week recovery
groups. In addition there were control and high dose satellite groups of 10 males for neurotoxicity evaluations.
Control animals:
yes, concurrent vehicle
Details on study design:
Dose concentrations were 0, 50, 200 and 1000 mg/kg b.wt./day administered in a dose volume of 5 mL/kg b.wt./day.
Positive control:
No data

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: all animals in all study phases were observed approximately one hour following dosing during the treatment period. Significant findings were noted.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Detailed clinical observations were recorded daily for all animals, prior to dose administration.

BODY WEIGHT: Yes
- Time schedule for examinations: Individual body weights were recorded weekly, beginning one week prior to treatment.

HAEMATOLOGY: Yes
- Time schedule for collection of blood:
Clinical pathologic parameters were evaluated on all surviving rats (5/sex/group) selected for a scheduled necropsy following 28 days of test article administration. The same parameters were evaluated on all recovery animals in the control group (4 females; 5 males) and 1000 mg/kg/day group (5/sex/group) selected for necropsy on study day 42. Animals were fasted overnight before taking blood and urine samples. Blood was collected from the inferior vena cava at the time of necropsy. Urine was collected in metabolism cages; urine volume was determined from an overnight sample.
- Animals fasted: Yes
- Parameters examined:
Total Leukocyte Count (White Cell)
Erythrocyte Count (Red Cells)
Hemoglobin
Hematocrit
Mean Corpuscular Volume (MCV)
Mean Corpuscular Hemoglobin (MCH)
Mean Corpuscular Hemoglobin Concentration (MCHC)
Reticulocyte Count (Reticulocyte)
Platelet Count (Platelet)
RBC Morphology
Platelet Estimate
Differential WBC Count - Percent and Absolute
- Segmented Neutrophil (Segmented)
- Lymphocyte
- Monocyte
- Eosinophil
- Unsegmented Neutrophil (Unsegmented)
- Prothrombin Time (Pro Time)
Activated Partial Thromboplastin Time (APTT)


CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood:
Clinical pathologic parameters were evaluated on all surviving rats (5/sex/group) selected for a scheduled necropsy following 28 days of test article administration. The same parameters were evaluated on all recovery animals in the control group (4 females; 5 males) and 1000 mg/kg/day group (5/sex/group) selected for necropsy on study day 42. Animals were fasted overnight before taking blood and urine samples. Blood was collected from the inferior vena cava at the time of necropsy. Urine was collected in metabolism cages; urine volume was determined from an overnight sample.
- Animals fasted: Yes
- Parameters examined:
Glucose
Urea Nitrogen
Creatinine
Sodium
Potassium
Serum Aspartate Aminotransferasea (Aspartat Transfer)
Chloride
Calcium
Globulin
A/G Ratio
Lactate Dehydrogenase (Lactate 'Genase)
Triglycerides
Uric Acid
B/C Ratio
Serum Alanine Aminotransferaseb (Alanine Transfer)
Serum Alkaline Phosphatase (Alkaline Phos'tse)
Total Bilirubin (Total Bili)
Total Cholesterol (Cholesterol)
Total Protein
Phosphorus
Albumin
Creatine Kinase
Gamma Glutamyltransferase (Glutamyl Transfer)
Plasma Cholinesterase (Cholin Plasma)

URINALYSIS: Yes
- Time schedule for collection of urine:
Clinical pathologic parameters were evaluated on all surviving rats (5/sex/group) selected for a scheduled necropsy following 28 days of test article administration. The same parameters were evaluated on all recovery animals in the control group (4 females; 5 males) and 1000 mg/kg/day group (5/sex/group) selected for necropsy on study day 42. Animals were fasted overnight before taking blood and urine samples. Blood was collected from the inferior vena cava at the time of necropsy. Urine was collected in metabolism cages; urine volume was determined from an overnight sample.
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters examined:
Volume
Color (CLOR)
Appearance (APP)
Specific gravity (SG)
pH
Protein (PRO)
Glucose (GLU)
Ketones (KET)
Bilirubin (BIL)
Occult blood (BLD)
Nitrites (NIT)
Leukocytes (WBC)
Microscopy of sediment (MICRO)

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations:
Observations on male rats in the control and 1000 mg/kg/day groups were made pre-study and then 14, 28 and 42 (recovery animals only) days after the first exposure to the test article. All observations were performed prior to test article administration. Testing was performed by the same technicians without knowledge of the animal group assignment. The FOB was performed in a sound-proofed room equipped with a white noise generator set to operate at 70 ± 10 db. with one exception; home cage observations were performed in the animal room.
- Battery of functions tested:
Hindlimb extensor strength
Grip strength-hind and forelimb
Hindlimb foot splay
Rotarod performance
Approach response
Touch response
Startle response
Tail pinch
Pupil response
Eye blink response
Forelimb extension
Hindlimb extension
Air righting reflex
Olfactory orientation



Sacrifice and pathology:
GROSS PATHOLOGY: Yes.
For the subchronic toxicity phase, 5 animals/sex/group were necropsied on study day 28 and the remaining animals in the control group (4 females; 5 males) and 1000 mg/kg/day group (5/sex/group) were necropsied on study day 42 (recovery).
The necropsy included an examination of the external surface, all orifices, and the cranial, thoracic, abdominal and pelvic cavities including the viscera.
The following organs were weighed for animals at the scheduled necropsy. Absolute weights, and organ to fmal body weight and brain weight were calculated.
Adrenals
Liver
Brain
Pituitary (Phase C only)
Kidneys
Ovaries or Testes

HISTOPATHOLOGY: Yes.
Microscopic examination of hematoxylin-eosin stained paraffin sections were conducted on the following tissues from control and 1000 mg/kg/day:
Adrenal Glands
All gross lesions
Brain
Ovaries
Epididymides
Pituitary Gland
Heart
Prostate
Kidneys
Seminal Vesicles
Liver
Spleen Testes
Other examinations:
A functional observation battery (home cage, handling, open field, sensory, neuromuscular and physiological observations) was conducted
pre-study and on days 14, 28 and 42 on 5 males of the control and high dose satellite groups.
Statistics:
Body weights, body weight changes, food consumption, and absolute and relative organ weights were analyzed by a one-way analysis of variance (ANOVA). If significant differences were indicated by the ANOVA, Dunnett's test was used to compare the control and treated groups.

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Mortality:
mortality observed, treatment-related
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:
effects observed, treatment-related
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:
no effects observed
Histopathological findings: neoplastic:
not examined
Details on results:
CLINICAL SIGNS AND MORTALITY
No mortalities were observed in either sex at dose levels of 50, 200 or 1000 mg/kg/day.
The predominant clinical signs associated with test material administration occurred one hour following treatment at the 1000 mg/kg/day level in both males and females. These findings included salivation, clear material around the mouth and red or yellow staining around the mouth. These clinical signs were initially observed within the first two weeks of dosing and generally persisted until cessation of dose administration. Clear material around the mouth was observed as single incidents for two males and one female in the 200 mg/kg/day group.
Other clinical fmdings were observed during the study, but the frequency in the treated groups was similar to the control group or the findings were limited to a few animals. Therefore, these observations were considered to be incidental to test material administration.

BODY WEIGHT AND WEIGHT GAIN
Weekly group mean body weights in the 1000 mg/kg/day males were comparable to the control group values during the pretest week and for study weeks 0, 1 and 2. During study-weeks 3 and 4, however, group mean body weights at this high dose level were slightly lower (3-5 %) than the control group data. Correspondingly, cumulative group mean body weight changes (from study day 0) were slightly reduced during the intervals 0-3 and 0-4 weeks in the 1000 mg/kg/day males. Although these reductions were not statistically significant, they were considered to be treatment-related. A marginal improvement in cumulative mean body weight gains was noted during weeks 0-5 and 0-6, when the animals were not administered the test article.
Weekly group mean body weights and cumulative group mean body weight changes in the 50 and 200 mg/kg/day males and in the 50, 200 and 1000 mg/kg/day females were comparable to the control group values.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
No adverse effects were apparent on food consumption (evaluated as g/animal/day and g/kg/day) for males or females at dose levels of 50, 200 or 1000 mg/kg/day.

HAEMATOLOGY
Hematology parameters in the treated groups were similar to those values observed for the control group. Hemoglobin and hematocrit week 6 (recovery) values in the 1000 mg/kg/day males were significantly lower (p < 0.05) than the concurrent control group values. These reductions were not considered to be treatment-related because:
1) the week 6 values were comparable to the week 4 values;
2) the values were within the observed ranges of the WIL historical control data for hemoglobin (10.76-22.24 g/dL) and hematocrit (35.66-65.34%);
3) a decrease in these parameters at week 4, not week 6, could have indicated a treatment-related effect.

CLINICAL CHEMISTRY
No test article related changes were apparent in serum chemistry parameters at any dose level tested. Glucose levels were lower in the 1000 mg/kg/day males (21 %) and in the 50 (37 %), 200 (41 %) and 1000 mg/kg/day females (27%) than the concurrent control group values at the week 4 evaluation. The reductions in the treated female groups were statistically significant (p < 0.05 or p < 0.01) but the response was not dose-related. The values were, however, within the observed ranges of the WIL historical control data for glucose in males (1.2 - 270.8 mg/dL) and females (16.2 - 215.8 mg/dL). (All clinical pathology historical control "ranges" consist of the mean value ± 2 standard deviations.) Cholesterol values for males and females at the 1000 mg/kg/day dose level were significantly reduced (p < 0.05) below concurrent control group values at the week 4 evaluation. These values were within the observed ranges of the WIL historical control data for males (22.0 - 88.0 mg/dL) and females (26.6 - 101.4 mg/dL). The toxicological significance of these reductions was uncertain.

URINALYSIS
No test article related differences were apparent in the urinalysis parameters between the control and treated groups.

NEUROBEHAVIOUR
No remarkable differences were apparent between the control group and the treated group when sensorimotor responses were evaluated for weeks -1 (pretest), 2, 4 and 6 (recovery).

ORGAN WEIGHTS
Slight increases in group mean absolute adrenal gland weights at the 1000 mg/kg/day dose level were noted at the week 4 necropsy in males (12%) and females (6%), and at the week 6 (recovery) necropsy in males (14%) and females (19%). When evaluated relative to the final body weight and brain weight, increases in the group mean adrenal gland weight were apparent; the weight relative to the fmal body weight for the 1000 mg/kg/day recovery females was statistically significant (p <0.01). These adrenal weight increases were interpreted to be a potential effect of test material administration. No apparent effects on group mean adrenal gland weights were noted in the males and females at the 50 and 200 mg/kg/day dose levels.
Group mean kidney, liver, testes and ovaries weights were apparently unaffected by test material administration. At week 4, statistically significant elevations in group mean kidney weight in the 1000 mg/kg/day males (p < 0.05) and group mean liver weight in the 1000 mg/kg/day females (p < 0.01) were noted when organ weights were evaluated-relative to the final body weight. However, concurrent increases in the organ weights on an absolute and relative to brain weight basis were not observed.

GROSS PATHOLOGY
No treatment-related internal findings were observed in animals at the scheduled week 4 or week 6 (recovery) necropsies. Macroscopic tissue changes were noted similarly in the control and treated animals or were generally single fmdings.

HISTOPATHOLOGY: NON-NEOPLASTIC
Microscopic examination of the gross lesions observed at the week 4 and week 6 necropsies did not reveal any effects of test material administration. Findings were noted in a limited number of animals in the treated groups or in control and treated groups at a similar incidence.

Effect levels

Dose descriptor:
NOAEL
Effect level:
200 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male
Basis for effect level:
clinical signs
body weight and weight gain
organ weights and organ / body weight ratios

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
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.
Executive summary:

The potential subchronic toxicity, neurotoxicity and reproductive toxicity related to the administration of the test material in the Sprague-Dawley Crl: CD®BR rat were evaluated in this combined repeated dose study conducted in line with OECD guideline 422 with slightly expanded scope. The test article was administered by the oral route (gavage) daily for 28 consecutive days to animals in the subchronic toxicity (5/sex/group) and neurotoxicity phases (5 males/group), followed by a 14 day recovery period in the control and high dose groups.

Dose levels were 50, 200 and 1000 mg/kg/day. A concurrent control group received the vehicle, peanut oil, on a comparable regimen. A dose volume of 5 ml/kg was used for all groups. Parameters common to all study phases included viability, clinical signs, body weights, food consumption and necropsy evaluations. Clinical pathology and organ weight data were collected in the subchronic toxicity phase. Neuropathology and functional observational battery data were collected in the neurotoxicity phase.

The adverse effects observed in this study are summarized below:

* Salivation, clear material around the mouth, red or yellow staining around the mouth and/or red material around the nose were treatment-related clinical signs noted one-hour following dosing in males and females receiving 1000 mg/kg/day.

* Weekly group mean body weights and body weight gains in males were decreased at a dose level of 1000 mg/kg/day. This occurred during weeks 3-4 in the subchronic toxicity phase & weeks 2-4 in the neurotoxicity phase. A possible recovery was noted by the 1000 mg/kg/day males during weeks 5 and 6 in the subchronic toxicity and neurotoxicity phases.

* Increased group mean adrenal gland and pituitary gland weights (absolute and relative to brain weights) were observed in males receiving 1000 mg/kg/day during the reproduction phase. A potential effect on adrenal gland weight was apparent also for females at the 1000 mg/kg/day level, noted during the subchronic toxicity phase. No other organ weights (brain, liver, kidneys, testes or ovaries) were adversely affected.

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.