Fatty acids, tall-oil, reaction products with ethanolamine, ethoxylated

Repeated dose toxicity: dermal

Administrative data

Endpoint:

sub-chronic toxicity: dermal

Type of information:

migrated information: read-across based on grouping of substances (category approach)

Adequacy of study:

key study

Study period:

From 02 June 1992 to 24 September 1992

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study well documented, meets generally accepted scientific principles, acceptable for assessment; compliant with GLP.

Justification for data waiving:

other:

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

reference to same study

Cross-reference 2

Reason / purpose for cross-reference:

reference to other study

Data source

Reference

Reference Type:

secondary source

Title:

Unnamed

Year:

1999

Materials and methods

Test guideline

Qualifier:

no guideline followed

Principles of method if other than guideline:

A 13-week toxicity study in F344/N rats was conducted by NTP to evaluate the cumulative toxic effects of repeated dermal exposure to the test substance.

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

N,N-bis(2-hydroxyethyl)oleamide

EC Number:

202-281-7

EC Name:

N,N-bis(2-hydroxyethyl)oleamide

Cas Number:

93-83-4

IUPAC Name:

N,N-bis(2-hydroxyethyl)octadec-9-enamide

Constituent 2

Reference substance name:

Oleic acid diethanolamine condensate

IUPAC Name:

Oleic acid diethanolamine condensate

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 or test system and environmental conditions:

- Source: Taconic Farms (Germantown, NY)
- Age at study initiation: 8 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: Males: 23 d and Females: 24 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: 21.1-22.8°C
- Relative humidity: 37-65 %
- Air changes: 10/h
- Photoperiod: 12 h dark/12 h light

IN-LIFE DATES: From: 1992-06-02 To: 1992-09-24 (males); 1992-06-02 To: 1992-09-25 (females)

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 (i.e., by stirring or sonicating) the test substance with 95% ethanol to give the required concentrations. The test substance formulations were applied on shaved interscapular skin of the test animals.
- Storage of dose formulations: The dose formulations were stored at room temperature, protected from light, in amber glass bottles for up to 28 days.
- Stability studies of a 10 mg/mL formulation prepared from lot CH1F980 (not used) were performed by the study laboratory using high-performance liquid chromatography. Stability of the dose formulation 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.

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 study were analyzed using HPLC. All the samples from the formulations analyzed during the 13 week study 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:

13 wks

Frequency of treatment:

5 exposures/wk

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 25, 50, 100, 200, or 400 mg/kg bw/day (0, 30, 61, 121, 243, or 485 mg/mL in ethanol)
Basis:
nominal per unit body weight

No. of animals per sex per dose:

10/sex/dose

Control animals:

yes, concurrent vehicle

Details on study design:

Method of animal grouping: Distributed randomly into groups of approximately equal initial mean body weights.

Positive control:

Not used in the study

Examinations

Observations and examinations performed and frequency:

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Clinical findings were recorded weekly
BODY WEIGHT: Yes
- Time schedule for examinations: Weighed initially, weekly, and again at the end of the study
HAEMATOLOGY: Yes
- Time schedule for collection of blood: On Days 5 and 19 and all core study rats surviving to the end of the study
- Method: Blood was collected from the retro-orbital sinus of special study rats from each dose group
- Anaesthetic used for blood collection: Yes, carbon dioxide/oxygen mixture
- How many animals: All animals
- Parameters examined.: Hematocrit; hematocrit; hemoglobin; erythrocyte, nucleated erythrocyte, reticulocyte, and platelet counts; mean cell volume; mean cell hemoglobin; mean cell hemoglobin concentration; leukocyte counts and differentials

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: On Days 5 and 19 (blood was collected from the retro-orbital sinus of special study rats from each dose group) and all core study rats surviving to the end of the study.
- How many animals: All animals
- Parameters examined: Urea nitrogen, creatinine, alanine aminotransferase, alkaline phosphatase, sorbitol dehydrogenase, total protein, albumin, and bile salts

OTHER: Organs weight: Organ weights were determined for heart, right kidney, liver, lung, right testis, and thymus

Sacrifice and pathology:

SACRIFICE: At the end of the 13-week study animals were sacrificed by carbon dioxide asphyxiation.

GROSS PATHOLOGY: Yes

HISTOPATHOLOGY: Yes.

- Complete histopathology was performed on 0 and 400 mg/kg bw/day study rats. In addition to gross lesions and tissue masses, the tissues examined were: adrenal gland, bone and marrow, brain, clitoral gland, esophagus, heart, large intestine (cecum, colon, rectum), small intestine (duodenum, jejunum, ileum), kidney, liver, lung, lymph nodes (mandibular and mesenteric), mammary gland, nose, ovary, pancreas, parathyroid gland, pituitary gland, preputial gland, prostate gland, salivary gland, spleen, stomach (forestomach and glandular), testis (and epididymis and seminal vesicle), thymus, thyroid gland, trachea, urinary bladder, and uterus. In addition, the skin was examined in all rats.
- 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.

Other examinations:

Sperm Motility and Vaginal Cytology:
- Samples were collected for sperm motility or vaginal cytology from all rats in the 0, 100, 200, and 400 mg/kg bw/day groups.
- The sperm motility parameters evaluated was: Sperm concentration, sperm motility, sperm count, spermatid heads per testis, and spermatid heads per gram of testis. The left cauda, epididymis, and testis were weighed.
- Vaginal samples were collected for 12 consecutive days prior to the end of the studies for vaginal cytology evaluations.
- The parameters observed were: Estrous cycle length and relative frequency of estrous stage.

Statistics:

Statistical analyses for possible dose-related effects on survival used Cox’s (1972) method for testing two groups for equality and Tarone’s (1975) life table test to identify dose-related trends.

- 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). Hematology, clinical chemistry, spermatid, and epididymal spermatozoal data were analysed using the nonparametric multiple comparison methods of Shirley (1977) and Dunn (1964). Prior to statistical analysis, extreme values identified by the outlier test of Dixon and Massey. Average severity values were analyzed for significance with the Mann-Whitney U test (Hollander and Wolfe, 1973). Treatment effects were investigated by applying a multivariate analysis of variance (Morrison, 1976) to the transformed data to test for simultaneous equality of measurements across dose levels.

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Description (incidence and severity):

(all animals survived to the end of the study)

Dermal irritation:

effects observed, treatment-related

Description (incidence and severity):

(irritation of the skin at the site of application in males administered 100 mg/kg bw/day or greater and in females administered 50 mg/kg bw/day or greater)

Mortality:

no mortality observed

Description (incidence):

(all animals survived to the end of the study)

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

(reduced final mean body weights and body weight gains of males and/or females at 200 or 400 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:

effects observed, treatment-related

Description (incidence and severity):

(increased segmented neutrophil counts in the 400 mg/kg bw/day male group on Day 5 and 19, in the 200 mg/kg bw/day female group on Day 19 and at wk 13 and in the 400 mg/kg bw/day female group on Day 5 and 19 and at wk 13).

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

(significant increase in alkaline phosphatase activity on Day 19 in males at 200 mg/kg bw/day, at wk 13 in females at 200 mg/kg bw/day and at wk 13 in males and females at 400 mg/kg bw/day)

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

(Increased kidney weights in females at 200 and 400 mg/kg bw/day; decreased heart, liver, lung and thymus weights in males and females in the 200 and 400 mg/kg bw/day groups were associated with the lower mean body weights of these groups)

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

(dose dependent increase in the incidences of non-neoplastic lesions of the skin (including hyperplasia of the epidermis, hypertrophy of sebaceous gland, parakeratosis, chronic active inflammation of the dermis and suppurative epidermal inflammation); In

Histopathological findings: neoplastic:

no effects observed

Details on results:

None

Effect levels

1

Dose descriptor:

NOAEL

Remarks:

(local effects)

Effect level:

25 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: see 'Remark'

2

Dose descriptor:

NOAEL

Remarks:

(systemic effects)

Effect level:

100 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: overall effects (i.e., decreased body weight and body weight gain, increased segmented neutrophil counts, increased alkaline phosphatase concentrations, increased kidney weights in males and/or females at ≥200 mg/kg bw/day)

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

- Non-neoplastic lesions: Lesions of the skin were also present at the site of application in groups administered 100 mg/kg bw/day; however, the incidences were somewhat less than those observed in the 200 and 400 mg/kg bw/day groups. In addition, the severities of the lesions were increased only slightly in the 200 and 400 mg/kg bw/day groups compared to the severities in the 100 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. In groups treated with 50 mg/kg bw/day, the incidences of skin lesions diminished considerably and lesion severities were minimal.

- Other observed parameters (sperm and vaginal): There were no significant differences in sperm motility and vaginal cytology parameters between dosed and vehicle control groups.

Applicant's summary and conclusion

Conclusions:

Under the conditions of the 13-week study in rats, the NOAEL for systemic and local effects can be considered to be at 100 and 25 mg/kg bw/day respectively.

Executive summary:

A study was conducted by NTP to evaluate the cumulative toxic effects following repeated dermal exposure to oleic acid diethanolamine condensate (ODEA) in F344/N rats.

Groups of 10 male and 10 female rats were dermally exposed to 0, 25, 50, 100, 200, or 400 mg test substance/kg bw/day in ethanol at a frequency of 5 d/wk for a period of 13 weeks. Survival, clinical findings, body weight, hematology, clinical chemistry and histopathology were evaluated at specific time intervals.

All animals survived until study termination. The final mean body weights and body weight gains of 200 and 400 mg/kg bw/day males and the mean body weight gain of 400 mg/kg bw/day females were significantly less than those of the vehicle controls. The only chemical-related clinical finding was irritation of the skin at the site of application in most males administered 100 mg/kg bw/day or greater and in all females administered 50 mg/kg bw/day or greater. Segmented neutrophil counts and alkaline phosphatase concentrations were increased significantly on specific days in the 200 and/or 400 mg/kg bw/day male and female groups. Kidney weights of 200 and 400 mg/kg females were significantly greater than those of the vehicle controls. There was a dose dependent increase in the incidence of non-neoplastic lesions of the skin at the site of application, including epidermal hyperplasia, parakeratosis, chronic active dermal inflammation, suppurative epidermal inflammation, and sebaceous gland hypertrophy in dosed rats. However, these effects were minimal in the 50 mg/kg bw/day dose group.

Hence, under the conditions of the 13-week study in rats, the NOAEL for systemic and local effects can be considered to be at 100 and 25 mg/kg bw/day respectively.