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Toxicological information

Repeated dose toxicity: dermal

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Administrative data

Endpoint:
chronic toxicity: dermal
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 06 May 1993 to 16 May 1995
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
test procedure in accordance with generally accepted scientific standards and described in sufficient detail
Cross-reference
Reason / purpose:
reference to other study

Data source

Reference
Reference Type:
publication
Title:
Unnamed
Year:
1999

Materials and methods

Principles of method if other than guideline:
A 2-year study was conducted in F344/N rats to evaluate the the chronic dermal toxicity of the test substance.

GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Details on test material:
- Name of test material (as cited in study report): Oleic acid diethanolamine condensate
- Source of the test material: Obtained from Henkel Corporation, Emery Group (Cincinnati, OH)
- Molecular weight: 387.68
- Physical state: Clear liquid
- Analytical purity: Purity of 47.5% was obtained when analyzed by HPLC
- Impurities (identity and concentrations): Analyzed by HPLC/mass spectrometry. Impurities identified were fatty acid alkanolamides (approximately 30%), other fatty acids or unidentified organic impurities, one polar nitrosamines (i.e., nitrosodiethanolamine was detected at a concentration of 68 ppb), free diethanolamine at 0.19%; no non-polar nitrosamine were found.
- Lot/batch No.: 1H01722285 (for purity determination by HPLC method) and DA-021 (for stability studies by gas chromatography)
- Stability under test conditions: Stable when stored in glass vials for 2 wk at 25 °C but unstable at 60 °C.
- Storage condition of test material: At room temperature, protected from ultraviolet light, in amber glass bottles with Teflon®-lined lids
- Other: Solubility: soluble in alcohols, glycols, ketones, chlorinated solvents, and other aliphatic hydrocarbon solvents.

Test animals

Species:
rat
Strain:
Fischer 344
Sex:
male/female
Details on test animals and environmental conditions:
- Source: Taconic Laboratory Animals and Services (Germantown, NY)
- Age at study initiation: 7 wk
- Housing: Housed individually in Polycarbonate cages
-Method of distribution: Animals were distributed randomly into groups of approximately equal initial mean body weights.
- Bedding: Sani-Chip® heat-treated hardwood chips (P.J. Murphy Forest Products Corp., Montville, NJ), changed weekly
- Diet: NIH-07 open formula pelleted diet (Zeigler Brothers, Inc., Gardners, PA), available ad libitum
- Water: Tap water (Columbus municipal supply) via automatic watering system (Edstrom Industries, Inc., Waterford, WI), available ad libitum
- Acclimation period:Time held before studies: Males: 13 d and Females: 14 d
- Cages: Polycarbonate (Lab Products, Inc., Maywood, NJ), changed weekly and rotated every 2 wks
- Animal number per cage: 1
- Cage Filters: Spun-bonded polyester Du Pont 2024 (Snow Filtration, Co., Cincinnati, OH), changed every 2 wks
- Racks: Stainless steel drawer-type (Lab Products, Inc., Maywood, NJ), changed and rotated every 2 wks

ENVIRONMENTAL CONDITIONS
- Temperature: 21.1-23.3°C
- Relative humidity: 31-73 %
- Air changes: 10/h
- Photoperiod: 12 h dark/12 h light

IN-LIFE DATES: From: 1993-05-06 To: 1995-05-16

Administration / exposure

Type of coverage:
open
Vehicle:
ethanol
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: The dose formulations were prepared every 3 weeks by mixing the test substance by stirring or sonicating with 95% ethanol to give the required concentrations. The test substance formulations were applied on shaved skin of the test animals.
-The dose formulations were stored at room temperature, protected from light, in amber glass bottles for up to 28 days.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Periodic analyses of the dose formulations of the test substance from the beginning, middle, and end of the studies were analyzed at the study laboratory using HPLC. All the samples from the formulations were analysed every 9 weeks during the 2-year study and were within 10% of the target concentration.

-Stability of dose formulations: Stability was confirmed for at least 28 days when stored in sealed containers, protected from ultraviolet light, at up to room temperature or for 3 hours when stored open to air and light.
Duration of treatment / exposure:
2-yr
Frequency of treatment:
5 exposures/wk

Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Dose / conc.:
50 mg/kg bw/day (actual dose received)
Dose / conc.:
100 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
50/sex/dose
Control animals:
yes, concurrent vehicle
Details on study design:
Dose selection rationale: The dose selection was based primarily on the incidences and severities of skin lesions observed at the site of application during the 13-week dermal studies. The doses of 200 and 400 mg/kg bw/day exhibited reduced mean body weight and body weight gain along with high incidences of skin lesions at the site of application; thus were considered inappropriate for a 2-year study. Further, lesions of the skin were also present at the site of application in groups administered 100 mg/kg bw/day; however, the incidence were less than those observed in the 200 and 400 mg/kg bw/day groups. Moreover, it was considered unlikely that these lesions would progress and become life threatening over the period of a 2-year study. Therefore, 100 mg/kg bw/day was selected as the high dose for rats in the 2-year study. In groups treated with 50 mg/kg bw/day, the incidences of skin lesions diminished considerably and lesion severities were minimal. Therefore, 50 mg/kg bw/day was selected as the low dose.

Examinations

Observations and examinations performed and frequency:
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Clinical findings were recorded monthly and at the end of the studies.
BODY WEIGHT: Yes
- Time schedule for examinations: Weighed initially, weekly for 13 weeks, approximately monthly thereafter and again at the end of the studies
HAEMATOLOGY: No
CLINICAL CHEMISTRY: No


Sacrifice and pathology:
SACRIFICE: At the end of the 2-year study animals were sacrificed by carbon dioxide asphyxiation.

GROSS PATHOLOGY: Yes

HISTOPATHOLOGY: Yes

- Complete histopathology was performed on all the rats at the end of the study. In addition to gross lesions and tissue masses, the tissues examined were: adrenal gland, bone with marrow, brain, clitoral gland, esophagus, gallbladder (mice), heart with aorta, large intestine (cecum, colon and rectum), small intestine (duodenum, jejunum and ileum), kidney, liver, lung, lymph nodes (mandibular and mesenteric), mammary gland, nose, ovary, pancreas, parathyroid gland, pituitary gland, preputial gland, prostate gland, salivary gland, skin (site of application), spleen, stomach (forestomach and glandular), testis with epididymis and seminal vesicle, thymus, thyroid gland, trachea, urinary bladder, and uterus.

- All major tissues were fixed and preserved in 10% neutral buffered formalin processed and trimmed, embedded in paraffin, sectioned to a thickness of 5 to 6 μm, and stained with hematoxylin and eosin for microscopic examination.
Statistics:
Survival Analyses: The probability of survival was estimated by the product-limit procedure of Kaplan and Meier (1958). Possible dose-related effects on survival were analysed by Cox’s (1972) method for testing two groups for equality and Tarone’s (1975) life table test to identify dose-related trends. All reported P values for the survival analyses were two sided.

- Analysis of neoplasm and non-neoplastic lesion incidences: The Poly-k test (Bailer and Portier, 1988; Portier and Bailer, 1989; Piegorsch and Bailer, 1997) was used to assess neoplasm and nonneoplastic lesion prevalence. Tests of significance included pair wise comparisons of each dosed group with controls and a test for an overall dose-related trend. Continuity-corrected tests were used in the analysis of lesion incidence, and reported P values are one sided. Values of P greater than 0.5 are presented as 1-P with the letter N added to indicate a lower incidence or negative trend in neoplasm occurrence relative to the control group (e.g., P=0.99 is presented as P=0.01N).

Analysis of Continuous Variables: Organ and body weight data were analysed using the parametric multiple comparison procedures of Dunnett (1955) and Williams (1971, 1972).

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Dermal irritation:
effects observed, treatment-related
Description (incidence and severity):
Mild to moderate irritation of the skin at the site of application in dosed males and females; vehicle control, 0/50; 50 mg/kg bw/day, 17/50; 100 mg/kg bw/day, 32/50; females: 3/50, 46/50, 50/50.
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Slightly educed mean body weights of males and females at 100 mg/kg bw/day
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
See below under the 'details on results' section.
Histopathological findings: neoplastic:
no effects observed
Description (incidence and severity):
See below under the 'details on results' section.
Other effects:
no effects observed
Details on results:
Histopathology:
- Non-neoplastic lesions:
- Skin lesions: Minimal to moderate non-neoplastic lesions of the skin at the site of application were observed. The major alterations included (thickening of the epidermis, sebaceous gland and epidermal hyperplasia, hyperkeratosis, parakeratosis, chronic active dermal inflammation and ulcer) which were significantly increased in dosed males and females relative to the vehicle control.
- Forestomach lesions: No significant treatment related effects were observed. The increased incidence of hyperkeratosis and ulceration in 50 mg/kg bw/day males were not considered to be treatment related as these effects were not observed in females.
- Testis: No significant treatment related effects were observed.
- Thyroid gland lesions: No significant treatment related effects were observed.

- Neoplastic lesions:
- Skin lesions: No significant treatment related effects were observed. Few observed skin neoplasms (one subcutaneous fibroma in one vehicle control male and one subcutaneous fibrosarcoma in each of the 50 and 100 mg/kg bw/day male groups) were not considered to be significant as the incidences did not follow a pattern indicative of an association with the test substance.
- Testis: No significant treatment related effects were observed. Increased interstitial cell adenoma in males at 100 mg/kg bw/day was not considered significant as similar incidences were reported in vehicle controls in historical NTP dermal studies.
- Thyroid gland: No significant treatment related effects were observed. Marginal increase in the incidence of follicular cell adenoma or carcinoma observed in males at 50 mg/kg bw/day was not considered dose related and no follicular cell hyperplasias were observed .

Effect levels

open allclose all
Key result
Dose descriptor:
NOAEL
Remarks:
(systemic effects)
Effect level:
50 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: body weight changes at the LOAEL
Key result
Dose descriptor:
LOAEL
Remarks:
(local effects)
Effect level:
50 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: irritation and non-neoplastic lesion of the skin at both the tested doses

Target system / organ toxicity

Key result
Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
Under the condistions of the study, the NOAEL for systemic effects can be considered to be 50 mg/kg bw/d and the LOAEL fo r local effects can be considered to be 50 mg/kg bw/d.

Executive summary:

A study was conducted to evaluate the effects of chronic exposure to the test substance in F344/N rats. Groups of 50 male and 50 female rats were dermally exposed to 0, 50 or 100 mg/kg bw/day in ethanol at a frequency of 5 d/wk for a period of 104 weeks. Survival, clinical findings, body weight and histopathology of different organs were assessed at specific time intervals. Survival of the dosed male and female rats was similar to that of the vehicle control groups. The mean body weights of males and females (Week 24 onwards) were reduced than those of the vehicle control group at 100 mg/kg bw/day. A dose dependent increase in irritation (mild to moderate) and non-neoplastic lesions (minimal to moderate) of the skin were observed at the site of application in all animals. The non-neoplastic lesions included epidermal hyperplasia, sebaceous gland hyperplasia, hyperkeratosis, parakeratosis, chronic active dermal inflammation and ulcer. No significant neoplastic lesions or evidence of carcinogenic activity was observed at any tested dose levels in skin, testis and thyroid gland. Under the conditions of the study, the NOAEL for systemic effects can be considered to be 50 mg/kg bw/d and the LOAEL for local effects can be considered at the lowest dose of 50 mg/kg bw/d (Irwin, 1999).