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Repeated dose toxicity: dermal

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Administrative data

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:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Guideline study following GLP. Read across.
Justification for type of information:
Category and read across justification attached in section 13 and in appendix of CSR

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1986
Report date:
1986

Materials and methods

Test guideline
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

Test material

Constituent 1
Chemical structure
Reference substance name:
Phenol, paraalkylation products with C10-15 branched olefins (C12 rich) derived from propene oligomerization, carbonates, calcium salts, overbased, sulfurized including distillates (petroleum), hydrotreated, solvent-refined, solvent-dewaxed, or catalytic dewaxed, light or heavy paraffinic C15-C50
EC Number:
701-251-5
Molecular formula:
Formula for a representative structure is C36H58Ca2O4Sx where x = 1,2. Actual molecular formula is not possible to generate. Substance is a UVCB.
IUPAC Name:
Phenol, paraalkylation products with C10-15 branched olefins (C12 rich) derived from propene oligomerization, carbonates, calcium salts, overbased, sulfurized including distillates (petroleum), hydrotreated, solvent-refined, solvent-dewaxed, or catalytic dewaxed, light or heavy paraffinic C15-C50
Details on test material:
- Name and of test material (as cited in study report): Phenol, tetrapropenyl-, sulfurized, carbonates, calcium salts, overbased
- CAS number of test material (as cited in study report): 122384-87-6
Testing was performed on a commercial sample of this material. Typical purity of this material as distributed in commerce is 50% alkyl phenol sulfide and 50% highly refined lubricant base oil.

Test animals

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

Administration / exposure

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
Doses / concentrations
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

Examinations

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.

Results and discussion

Results of examinations

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.

Effect levels

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.

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

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 approximately >250 mg/kg b.wt./day 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 approximately >250 mg/kg b.wt./day males and female rats, the highest dose tested.