Registration Dossier

Toxicological information

Repeated dose toxicity: oral

Currently viewing:

Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
in life dates 6 November - 4 December 1996
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Comparable to guideline study with acceptable method restriction for subchronic hazard determination.
Cross-reference
Reason / purpose:
reference to other study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1998
Report Date:
1998

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Deviations:
not specified
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
(draft)
Deviations:
not specified
Qualifier:
according to
Guideline:
other: Test Guidelines for a Neurotoxicity Screening Battery (Pesticide Assessment Guidelines, Subdivision F, Hazard Evaluation: Human and Domestic Animals, Addendum 10; Neurotoxicity: Series 81-8, 82-7, and 83-1 - excluding motor activity.)
Deviations:
not specified
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
122384-85-4/68855-45-8
Phenol, tetrapropenyl-, sulfurized, calcium salts
Testing was performed on a commercial sample of this material. Typical purity of this material as distributed in commerce is 60% alkyl phenol sulfide and 40% highly refined lubricant base oil.
- Physical state: Dark brown liquid
- Analytical purity: 100.0%
- Expiration date of the lot/batch: 01 June 1997

Documentation of the identity, strength, purity, composition and stability and synthesis, fabrication, and/or derivation of the control vehicle were the responsibility of the Supplier.

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
Sprague-Dawley CD® (Sprague-Dawley derived) [Crl: CD® BR]
- Source: Charles River Laboratories, Portage, Michigan, 49081
- Age at study initiation: 45 days old
- Weight at study initiation:
Male: 238 g (209-262)
Female (Subchronic): 172 g (153-187)
- Housing: Animals were individually housed in elevated, stainless steel, wire mesh cages.
- Diet (e.g. ad libitum): Certified Rodent Diet, No. 5002; (Meal) (PMI Feeds, Inc., St. Louis, Missouri) was available without restriction except for those intervals when animals were fasted. Fresh food was presented weekly or more frequently, as necessary, throughout the study.
- Water (e.g. ad libitum): Water was available without restriction via an automated watering system (Elizabethtown Water Company, Westfield, New Jersey).
- Acclimation period: Animals in the range-finding study were acclimated for eight days and animals for the Subchronic Toxicity, Neurotoxicity and Reproduction Study were acclimated for 1.5 days. All animals were observed twice daily during the acclimation period by the technical staff for any unusual physical or behavioral abnormalities that might preclude their use on study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): Temperature in the animal quarters was monitored and recorded twice daily (morning and afternoon). 20 - 28°C. Over the 85 days of the subchronic toxicity, neurotoxicity and reproduction study (acclimation to study termination) temperature was out of range on one occasion (28°C).
- Humidity (%): Relative humidity in the animal quarters was monitored and recorded once daily: 14 to 68%.
Over the 85 days of the subchronic toxicity, neurotoxicity and reproduction study (acclimation to study termination) relative humidity readings were below the desired range on five occasions (14 -22%). These few instances that relative humidity values were not in range were not considered to have affected the integrity of the study.
- Photoperiod (hrs dark / hrs light): The light cycle in the animal quarters was controlled via automatic timer to maintain a 12 hour light/dark cycle.

- Other: There were no known contaminants in the feed or water which were expected to interfere with the results of this study.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
The appropriate amounts of the test material were weighed directly into calibrated beakers for preparation of dosing mixtures for each dose group. The weight of the beaker with the test material was tared to zero and then brought to volume with the vehicle. The weight of the final mixture was then recorded for each group. The mixtures were then stirred first with a spatula followed by a longer period (approximately two and one-half hours) with a stir bar and magnetic stirrer until thoroughly mixed. The mixtures were then aliquoted into labeled glass jars with teflon coated screw-on plastic lids, one jar was dispensed for each group daily for dosing.

Dosing mixtures were prepared fresh weekly during the range-finding and the subchronic toxicity, neurotoxicity and reproduction studies.

VEHICLE
- Lot/batch no. (if required): 86H0059
- Purity: 100%
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Corn oil dosing solutions were prepared weekly, and their test material concentration, homogeneity and stability verified by chemical analysis.
At each weekly preparation of dosing mixtures, two samples were taken from each dose group and control vehicle. One sample per concentration was sent to the Sponsor's Analytical Laboratory for analyses. The other sample was retained at the Testing Facility and stored at room temperature.
Duration of treatment / exposure:
Subacute toxicity and neurotoxicity phases: animals were treated once daily for 29 consecutive days and 28 consecutive days (neurotoxicity phase).
Frequency of treatment:
daily
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 50, 300 and 1000 mg/kg/day administered in a dose volume of 5 mL/kg
Basis:
actual ingested
No. of animals per sex per dose:
6 rats/sex/group
Control animals:
yes, concurrent vehicle
Details on study design:
The study was a 4-week repeated dose oral toxicity study in rats. Six rats/sex/group were employed for all control, test and recovery groups.
Control rats received corn oil vehicle only.

Post exposure observation period: 15 days for the control and high dose recovery groups.

There were 6 rats/sex/group for the 4-week exposure groups and for the control and high dose 2-week recovery groups. In addition there were
control and high dose satellite groups of 12 males for neurotoxicity evaluations.



Positive control:
no data

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily, once in the morning and once in the afternoon performed at least six hours apart.
Animals were observed, in their cages, for mortality, general appearance and signs of severe toxic or pharmacologic effects.
Observations were made for any gross signs of poor health or toxic or pharmacologic effects (e.g., gross abnormalities in general condition, appearance, activity, behavior, respiration, etc.) approximately 1 hour post-dose.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Pretest (range-finding and subchronic toxicity, neurotoxicity and reproduction study); and weekly thereafter throughout the studies. Animals were removed from their cages and given a thorough examination. Unusual behavior and physical signs were recorded.

BODY WEIGHT: Yes
- Time schedule for examinations: Animals were weighed pretest, on the day treatment initiated, weekly throughout the study (including recovery period) and at terminal sacrifice.

FOOD CONSUMPTION AND COMPOUND INTAKE:
Subchronic toxicity and neurotoxicity phases food consumption was recorded weekly, beginning one week prior to treatment (main study), and continuing through the treatment and post-treatment (recovery) periods.

FOOD EFFICIENCY:
Calculated from weekly body weight and food consumption data.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood:
Termination after four weeks of treatment: Day 30 (05 December 1996)
Completion of the two-week recovery period: Day 45 (20 December 1996)
- Anaesthetic used for blood collection: Yes, blood was obtained via venipuncture of the orbital sinus (retrobulbar venous plexus) under light C02/02 anesthesia. Animals were fasted overnight prior to blood collection.
Blood for hematology studies was collected into tubes containing EDTA anticoagulant. Blood for coagulation studies was collected into tubes containing sodium citrate anticoagulant.
- Animals fasted: Yes
- How many animals:
Termination after four weeks of treatment: 6/sex/Groups I-IV
Completion of the two week recovery period: 6/sex/Groups I and IV
- Parameters checked:
Hemoglobin concentration
Hematocrit
Erythrocyte count
Platelet count
Mean corpuscular volume
Mean corpuscular hemoglobin
Mean corpuscular hemoglobin concentration
Total leukocyte counts
Differential leukocyte count
Absolute lymphocytes (calculated value; total WBC x lymphocyte value / 100)
Absolute segmented neutrophils (calculated value; total WBC x segmented neutrophil / 100)
Prothrombin time
Activated partial thromboplastin time
Reticulocyte count
Erythrocyte morphology

CLINICAL CHEMISTRY: Yes, blood was obtained via venipuncture of the orbital sinus (retrobulbar venous plexus) under light C02/02 anesthesia. Animals were fasted overnight prior to each blood collection interval.
Blood for clinical chemistry studies was collected into tubes with no anticoagulant, allowed to clot, and centrifuged to obtain serum.
- Time schedule for collection of blood:
Termination of the four week treatment period: Day 30 (05 December 1996)
Completion of the two week recovery period: Day 45 (20 December 1996)
- Animals fasted: Yes
- How many animals:
Termination: 6/sex/Groups I - IV
Recovery: 6/sex/Groups I and IV
- Parameters checked:
Aspartate aminotransferase (Kinetic - Modified IFCC Technique)
Alanine aminotransferase (Kinetic - Modified IFCC Technique)
Alkaline phosphatase (AMP Buffer - Modified Bessey-Lowry-Brock Technique)
Lactate dehydrogenase (Lactate-pyruvate Technique)
Blood urea nitrogen (Modified Urease Technique)
Creatinine (Jaffe Reaction - Kinetic - Alkaline Picrate)
Blood urea nitrogen/creatinine ratio (calculated value; blood urea nitrogen / creatinine)
Fasting Glucose (Hexokinase Method)
Creatine kinase (Szasz-NAC Activated Method)
Cholesterol - enzymatic (Cholesterol esterase-cholesterol oxidase Method)
Triglycerides (GPO Triglyceride-lipase Method)
Total protein (Biuret Technique)
Albumin (Bromocresol Green Method)
Globulin (calculated value; total protein - albumin)
Albumin/globulin ratio (calculated value; albumin - globulin)
Uric acid (Uricase Method)
Total bilirubin (Modified Jendrassik and Grof Method)
Direct bilirubin (Modified Jendrassik and Grof Method)
Indirect bilirubin (calculated value; total bilirubin - direct bilirubin)
Sodium (Ion Selective Electrode)
Potassium (Ion Selective Electrode)
Chloride (Ion Selective Electrode)
Calcium (Cresolphthalein Complexone Method)
Inorganic phosphorus (Phosphomolybdate-UV Method)
Gamma-glutamyl transferase (Kinetic - Szasz, G.)
Cholinesterase parameters (Modified Ellman Method - Kinetic) serum

URINALYSIS: Yes
- Time schedule for collection of urine:
Termination after a four week treatment period: Day 29 (06 December 1996)
Completion of a two week recovery period: Day 44 (21 December 1996)
- Metabolism cages used for collection of urine: Yes, urine was collected in metabolism cages or in pans placed beneath each animal's cage. Urinalysis was performed on freshly voided samples.
Urine volume was measured over a 16-hour interval.
- Animals fasted: No data
- Parameters checked:
Appearance (clarity/color)
Specific gravity (Clinical Refractometer, Atago Uricon-N)

A Clinitek 200 Urine Chemistry Analyzer, Bayer Corporation, Diagnostics Division, was used for analyzing the following parameters.
Protein
Glucose
Ketones
Occult blood (semi-quantitatively)
pH
Bilirubin
Urobilinogen

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations:
Pretest and at Days 14 and 28: 12 males/group (Groups I and IV)
Day 42 (end of recovery period): 6 males/group (Groups I and IV)
- Battery of functions tested:
Handling Evaluations: reactivity to general stimuli (handling); assessment of signs of autonomic function: lacrimation, salivation, altered fur appearance, or red/crusty deposits around eyes.

Open Field Evaluations: arousal level and gait; count of urination and defecation; convulsions, tremors, abnormal movements or behaviors, excessive or repetitive actions; piloerection and exophthalmos.

Reflex Assessments: response to visual (approach response) and auditory (finger snap) stimuli; response to a tail pinch; pupillary function.
Hindlimb extensor strength: Each animal was held vertically with the observer's fingers against the bottom of each hindpaw. The animal's hindpaws are pressed towards it's body. The score was based on the amount of resistance shown by the animal.

Grip Strength (Meyer et al., 1979): Fore- and hindlimb Grip strength was measured using a Grip Strength Meter (Columbus Instruments International Corporation, Columbus, Ohio). Each animal was given two trials for evaluation of fore- and hindlimb grip strength.

Landing Foot Splay: A small dot of paint was applied to each animal's hindpaws. Each animal was than dropped on to a flat surface from a height of two feet. The distance between the marks left by the hindpaws was measured in centimeters. Landing Foot Splay was evaluated as two trials for each animal.

Air Righting Ability: Animals were held upside down and dropped from a height of two feet into a container of bedding. The landing position of the animal was recorded.

Body Weight: Animals were removed from their cages and weighed using a Mettler Balance, Model PE4000 (Mettler Instrument Corporation, Hightstown, New Jersey).


Sacrifice and pathology:
GROSS PATHOLOGY: Yes
Complete macroscopic postmortem examinations were performed for all subchronic toxicity animals killed at the scheduled sacrifice interval.
Exsanguination following anesthesia with inhaled carbon dioxide.
HISTOPATHOLOGY: Yes (see tables I and II) in "Overall remarks, attachments".
Statistics:
Body weight, food consumption, feed efficiency, hematology, serum chemistry, urinalysis, and organ weight data were analyzed with Bartlett’s test to determine homogeneity of variances. If group variances were equal, data were analyzed by ANOVA with a Dunnett’s test if significance was observed with ANOVA. If group variances were unequal, data were analyzed by the non-parametric Kruskal-Wallis test with a Dunn’s rank sum test if
significance was observed with Kruskal-Wallis. A test for dose-response trend was also conducted using regression analysis for parametric data andJonckeere’s test for monotonic trend for non-parametric data. Statistical analysis was not performed when the standard deviation of the control
group was zero and dose groups were eliminated when their standard deviation was zero. If only one dose group remained, they were analyzed with astandard t-test if variances were equal, or Welch’s t-test if variances were unequal.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
no effects observed
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:
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 mortality was seen in the control or treated groups in the subchronic toxicity or neurotoxicity phases of study. All of these animals survived to scheduled sacrifice.
Likewise, all subchronic and neurotoxicity animals (control and high-dose group) retained for the recovery period survived to scheduled sacrifice.
In the subchronic toxicity and neurotoxicity phases of study and in the recovery animals (control and high-dose group), no effect of treatment with the test meterial at a dose level up to and including 1000 mg/kg/day was evident from the detailed physical examinations. Clinical findings seen in the treated animals occurred at low incidence or with similar frequency as in the control group and were typical of those seen commonly in this laboratory with this strain and age of rat.

BODY WEIGHT AND WEIGHT GAIN
No adverse effect of treatment with the test material at a dose level up to and including 1000 mg/kg/day was evident from body weight or body weight gain data during the four week treatment period. Mean weekly body weights and mean body weight gain data (week-to-week and cumulative from Week 0, the start of treatment) for the treated groups (males and females) were comparable to control data.
Mean body weight and body weight gain data during the recovery period (subchronic and neurotoxicity animals) were also comparable between the control and high-dose group.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
Mean weekly food consumption values for the treated groups over the four week treatment period were slightly higher than control values and some of these differences were statistically significant (300 mg/kg/day males at Week 1 and 1000 mg/kg/day males and females for Weeks 1-4). This type of response (i.e., increase in food consumption), however, for the treated groups was not considered indicative of an adverse effect of treatment.

FOOD EFFICIENCY
No effect of treatment with the test material was seen from feed efficiency data. There were a few instances when weekly feed efficiency values for one or more of the treated groups were statistically significantly lower than control data but in the absence of a consistent trend to these changes, they were not considered toxicologically significant.

HAEMATOLOGY
No effect of treatment with the test material at a dose level up to and inclusive of 1000 mg/kg/day was evident from terminal hematology data in the subchronic toxicity study. Mean hematology data, total and differential leukocyte counts and erythrocyte morphology data for the treated males were comparable to control data. In the females, a few statistically significant differences were seen in hematology data between the control and one or more of the treated groups (e.g., lower mean corpuscular volume in the 300 mg/kg/day group, lower mean corpuscular hemoglobin values in the 300 and 1000 mg/kg/day groups), but in the absence of a dose-relationship to these changes they were not considered treatment-related. Total and differential leukocyte counts and erythrocyte morphology data for the treated females were considered similar to control data.
During the recovery period, the mean absolute neutrophil level for the 1000 mg/kg/day females was increased in comparison to control data. This difference was statistically significant but was not considered toxicologically significant.

CLINICAL CHEMISTRY
No adverse effect of treatment with the test material at a dose level up to and inclusive of 1000 mg/kg/day was evident from terminal clinical chemistry data in the subchronic toxicity study. In the males, mean cholesterol levels for the 300 and 1000 mg/kg/day groups were statistically significantly lower than control data. The toxicological significance of this type of response, if any, is unclear. Cholesterol levels in the treated females were comparable to control data. In the recovery phase, cholesterol levels for the high-dose males were comparable to control data and were slightly higher than those seen in the high-dose subchronic toxicity males (55 and 45 mg/dL, respectively). A statistically significant reduction in serum cholinesterase level was seen in the 50 mg/kg/day males in comparison to control data in the subchronic toxicity study; however, in the absence of a similar response at the higher dose levels, this was not considered indicative of a treatment-related effect.

URINALYSIS
No effect of treatment with the test material at a dose level up to and inclusive of 1000 mg/kg/day was evident from urinalysis data in the subchronic toxicity study. These data were similar between the control and treated groups. Urinalysis data for animals retained for the recovery period were also comparable between the control and high-dose group.

NEUROBEHAVIOUR
No effect of treatment with the test material at a dose level of 1000 mg/kg/day was seen in the functional observational battery (FOB) data. At pretest, a statistically significant reduction in forelimb and hindlimb grip strength and statistically significant increase in landing foot splay during one of the two trials were seen in the animals designated for treatment in comparison to animals designated to the control group. The reason for these differences between groups at this point in the study is unclear. During the study (Days 14 and 28), fore-and hindlimb grip strength and foot splay data for the treated group were comparable to control data. All other FOB data for the treated group were considered comparable to control data at both the 14 and 28-day evaluation intervals. FOB data were also comparable between the control and high-dose males at completion of the recovery period.

ORGAN WEIGHTS
No effect of treatment with the test material at dose levels of 50 and 300 mg/kg/day was evident from organ weight data in the subchronic toxicity study. Mean organ weight data for these groups both absolute and relative to either the terminal body weight or brain weight were comparable to control data.
In the 1000 mg/kg/day group, the only effect of treatment was an increase in adrenal weights in the females.
In the recovery phase, adrenal weights, both absolute and relative to either terminal body weight or brain weight for the 1000 mg/kg/day males were statistically significantly increased in comparison to control data.

GROSS PATHOLOGY
Macroscopic findings in the subchronic rats of the study following four weeks of treatment and in the recovery phase occurred sporadically. These incidental findings have been seen in untreated control rats of similar strain and age used in other studies conducted in this laboratory.

HISTOPATHOLOGY: NON-NEOPLASTIC
Microscopic examination of gross lesions in the rats from the subchronic study did not indicate any treatment-related microscopic findings.

Effect levels

Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: LOAEL (male/female): 1000 mg/kg bw/day based on increase in mean adrenal weight in females with increased severity of fine vacuolar changes in cells of zona fasciculate in adrenal cortex.

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

NOAEL = 300 mg/kg b.wt./day for males and females

LOAEL = 1000 mg/kg b.wt./day for males and females based on increased adrenal weights and increased food consumption without commensurate body weight gain

Applicant's summary and conclusion

Conclusions:
NOAEL = 300 mg/kg b.wt./day for males and females based on increased adrenal weights and increased food consumption without commensurate
body weight gain.
Executive summary:

This screening study, conducted in line with OECD Guideline 407 was designed to determine the potential subchronic toxicity, including neurotoxicity, of the test material administered to rats orally via gastric intubation once daily for at least four weeks (subchronic and neurotoxicity phases) and to evaluate the potential of the test material to adversely affect male and female reproductive performance (animals treated for four weeks prior to mating with treatment continuing throughout the ensuing mating and gestation period through to lactation Day 4). Dose levels for the subchronic toxicity and reproduction phases were 0 (vehicle [corn-oil] control), 50, 300 and 1000 mg/kg/day; group sizes in the subchronic toxicity study were 6 animals/sex/group and in the reproduction study 12 animals/sex/group.

In this screening study the no-observed effect level (NOEL) for subchronic toxicity was 300 mg/kg/day and for reproductive toxicity and neurotoxicity was 1000 mg/kg/day.