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Toxicological information

Repeated dose toxicity: dermal

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Administrative data

Endpoint:
sub-chronic toxicity: dermal
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 02 June 1992 to 22 September 1992
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 for cross-reference:
reference to other study

Data source

Reference
Reference Type:
publication
Title:
Unnamed
Year:
1992

Materials and methods

Principles of method if other than guideline:
A 13-week toxicity study in B6C3F1 mice was conducted to evaluate the effects of repeated dermal exposure to the test substance.
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
(9Z)-N,N-bis(2-hydroxyethyl)octadec-9-enamide
EC Number:
700-972-2
Cas Number:
93-83-4
Molecular formula:
C22H43NO3
IUPAC Name:
(9Z)-N,N-bis(2-hydroxyethyl)octadec-9-enamide
Test material form:
liquid
Details on test material:
No details reported

Test animals

Species:
mouse
Strain:
B6C3F1
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:Time held before studies: Males: 21 d and Females: 22 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: 20.6-25.6°C
- Relative humidity: 39-65 %
- Air changes: 10/h
- Photoperiod: 12 h dark/12 h light

IN-LIFE DATES: From: 1992-06-02 To: 1992-09-22

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.
- 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 / 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)
Dose / conc.:
200 mg/kg bw/day (actual dose received)
Dose / conc.:
400 mg/kg bw/day (actual dose received)
Dose / conc.:
800 mg/kg bw/day (actual dose received)
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.

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: No

CLINICAL CHEMISTRY: No

OTHER: Organs weighed were 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 800 mg/kg bw/day mice. 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 mice.
- 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 withhematoxylin and eosin for microscopic examination.
Other examinations:
Sperm Motility and Vaginal Cytology:
- Samples were collected for sperm motility or vaginal cytology from all mice in the 0, 200, 400, and 800 mg/kg bw/day dose 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 evaluated were: the length of the estrous cycle and the length of time spent in each stage of the cycle.
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 analyzed 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
Dermal irritation:
effects observed, treatment-related
Description (incidence and severity):
Occurred at the site of application in all dosed males and in most of the females administered ≥100 mg/kg bw/day. Irritation progressed to ulcer in one male in the 800 mg/kg bw/day dose group.
Mortality:
no mortality observed
Description (incidence):
All animals except one male mice at 800 mg/kg bw/day survived until 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 at 800 and females at ≥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:
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:
effects observed, treatment-related
Description (incidence and severity):
See below under the 'details on results' section
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
Other effects:
not examined
Details on results:
Organ weights:
- Increased absolute and relative heart weights of males at 400 and 800 mg/kg bw/day and of females at ≥200 mg/kg bw/day. Further, the absolute heart weights were increased for females in ≥50 mg/kg bw/day dose groups.
- Increased liver weights in all dosed groups
- Increased kidney weights of males at ≥50 mg/kg bw/day.
- Decreased absolute thymus weight of males at ≥200 mg/kg bw/day and of females at ≥400 mg/kg bw/day. Decreased relative thymus weight was observed with females only the 800 mg/kg bw/day dosed groups.

Histopathology:
Non-neoplastic lesions:
- Skin: Dose dependent increase in the incidences of lesions (including epidermal hyperplasia, parakeratosis, suppurative epidermal inflammation, chronic active dermal inflammation, sebaceous gland hypertrophy, and ulcer in males and females) were observed.
- Bone marrow myeloid hyperplasia in males and females at 800 mg/kg bw/day and increased hematopoietic cell proliferation of spleen in males at 800 mg/kg bw/day and in females at 400 and 800 mg/kg bw/day were observed.

Effect levels

open allclose all
Key result
Dose descriptor:
LOAEL
Remarks:
(systemic effects)
Effect level:
50 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: Altered organ weights (with increased liver weights in all dosed groups) and reduced body weights and body weight gains at 800 mg/kg bw/day in males and ≥400 mg/kg bw/day in females
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 of the skin at the site of application in all dosed males and in most of the females administered ≥100 mg/kg bw/day.

Target system / organ toxicity

Key result
Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
Under the conditions of the study, the LOAEL for both systemic and local effects was considered to be 50 mg/kg bw/day.
Executive summary:

A study was conducted to evaluate the effects of repeated dermal exposure to the test substance in B6C3F1 mice. Groups of 10 male and 10 female mice were dermally exposed to 0, 50, 100, 200, 400 or 800 mg/kg bw/day in ethanol at a frequency of 5 d/wk for a period of 13 weeks. Mortality, clinical findings, body weight and histopathology were evaluated at specific time intervals. All male and female mice except one 800 mg/kg bw/day male survived until the end of the study. Final mean body weights and body weight gains of 800 mg/kg bw/day males and females and 400 mg/kg bw/day females were significantly lower than those of the vehicle controls. Clinical findings in dosed mice included irritation of the skin at the site of application. Irritation occurred in all surviving dosed males and in most females administered ≥100 mg/kg bw/day. The heart weights of 400 and 800 mg/kg bw/day males and females and 200 mg/kg bw/day females and the kidney weights of 50, 100, and 400 mg/kg bw/day males were significantly greater than those of the vehicle controls. Relative to the vehicle controls, the liver weights were increased in all dosed groups. A dose-dependent increase in the incidences of non-neoplastic skin lesions included epidermal hyperplasia, parakeratosis, suppurative epidermal inflammation, chronic active dermal inflammation, sebaceous gland hypertrophy and ulcer. Under the conditions of the study, the LOAEL for both systemic and local effects was considered to be 50 mg/kg bw/day (Irwin, 1999).