N,N-bis(2-hydroxyethyl)dodecanamide

Repeated dose toxicity: dermal

Administrative data

Endpoint:

sub-chronic toxicity: dermal

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From December 29, 1991 to April 16, 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:

1999

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 411 (Subchronic Dermal Toxicity: 90-Day Study)

Deviations:

not specified

Principles of method if other than guideline:

A 14-week toxicity study in mice was conducted 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

impurity 1

Reference substance name:

Unknown impurities

IUPAC Name:

Unknown impurities

Constituent 1

Reference substance name:

N,N-bis(2-hydroxyethyl)dodecanamide

EC Number:

204-393-1

EC Name:

N,N-bis(2-hydroxyethyl)dodecanamide

Cas Number:

120-40-1

Molecular formula:

C16H33NO3

IUPAC Name:

N,N-bis(2-hydroxyethyl)dodecanamide

impurity 2

Reference substance name:

2,2'-iminodiethanol

EC Number:

203-868-0

EC Name:

2,2'-iminodiethanol

Cas Number:

111-42-2

Molecular formula:

C4H11NO2

IUPAC Name:

2,2’-iminodiethanol

Test material form:

other: Viscous light yellow liquid or a white to light yellow, waxy solid

Test animals

Species:

mouse

Strain:

B6C3F1

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS:
- Source: Taconic Farms (Germantown, NY)
- Age at study initiation: Approximately 7 wk
- Housing: Housed individually in Polycarbonate cages
- Bedding: Sani-Chip® heat-treated hardwood chips (PJ Murphy Forest Products Corp., Montville, NJ), changed weekly
- Diet: NIH-07 open formula pelleted diet (Zeigler Brothers, Inc., Gardners, PA), available ad libitum (and changed weekly)
- Water: Tap water (Columbus municipal supply) via automatic watering system (Edstrom Industries, Inc., Waterford, WI), available ad libitum (cleaned every 2 wks)
- Acclimatization period: 14 d
- Cages: Polycarbonate (Lab Products, Inc., Maywood, NJ), changed weekly and rotated every 2 weeks
- Animal number per cage: 1 Bedding: Sani-Chip® heat-treated hardwood chips (PJ Murphy Forest Products Corp., Montville, NJ), changed weekly
- Cage Filters: Spun-bonded polyester Du Pont 2024 (Snow Filtration, Co., Cincinnati, OH), changed every 2 weeks
- Racks: Stainless steel drawer-type (Lab Products, Inc., Maywood, NJ), changed and rotated every 2 weeks

ENVIRONMENTAL CONDITIONS
- Temperature : 21.1-23.9°C
- Relative humidity : 42-57%
- Air changes : 10/hr
- Photoperiod : 12 h dark/12 h light

IN-LIFE DATES: From: 1992-01-13 To: 1992-04-14

Administration / exposure

Type of coverage:

open

Vehicle:

ethanol

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: The dose formulations were prepared every 3 weeks by liquefying and stirring the test substance at approximately 70°C. A weighed amount of the test substance was mixed with approximately half the required 95% ethanol, and the mixture was sonicated until it appeared to be in solution. The solution was allowed to cool and was then diluted to volume with 95% ethanol to give the required concentrations.The test substance formulations were applied on shaved skin of the test animals.
-Stability studies of the 10 mg/mL dose formulation were performed by the study laboratory using HPLC. When stored in sealed glass containers and protected from light, the dose formulations were stable for at least 28 days between -20°C and room temperature.

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 within 10% of the target concentration.
-Stability studies of the 10 mg/mL dose formulation were performed by the study laboratory using HPLC. When stored in sealed glass containers and protected from light, the dose formulations were stable for at least 28 days between -20°C and room temperature.

Duration of treatment / exposure:

14 wks

Frequency of treatment:

5 exposures/wk

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

25 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

50 mg/kg bw/day (nominal)

Doses / concentrations 4

Dose / conc.:

100 mg/kg bw/day (nominal)

Doses / concentrations 5

Dose / conc.:

200 mg/kg bw/day (nominal)

Doses / concentrations 6

Dose / conc.:

400 mg/kg bw/day (nominal)

No. of animals per sex per dose:

10 per sex per 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: Daily

BODY WEIGHT: Yes
- Time schedule for examinations: Weighed initially, weekly, and at the end of the studies

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 14 wk study, blood was collected from the retroorbital sinus of all core study mice for hematology and clinical chemistry analyses. Thereafter the test animals were anesthetised with a carbon dioxide/oxygen mixture.

GROSS PATHOLOGY: Yes

HISTOPATHOLOGY: Yes. Complete histopathology was performed on 0 and 400 mg/kg bw core study mice. In addition to gross lesions and tissue masses, the following tissues were examined: 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.

Other examinations:

Sperm motility and vaginal cytology: At the end of the studies, samples were collected for sperm motility or vaginal cytology from all mice in the 0, 100, 200, and 400 mg/kg bw groups for evaluations.
- The following sperm motility parameters was evaluated: spermatid heads per gram of testis, spermatid heads per testis, spermatid count, motility, and concentration in cauda epididymis. 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 length of the estrous cycle and the length of time spent in each stage of the cycle were evaluated.

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 analyzed 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):

Irritation of the skin at the site of application was noted in all males and females administered 400 or 800 mg/kg bw/day.

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

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

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Increased absolute and/or relative kidney weights of in males at ≥100 mg/kg bw/day and in females at 800 mg/kg bw/day; increased liver weights in females at ≥200 mg/kg bw/day and reduced thymus weights in males at 400 and 800 mg/kg bw/day.

Gross pathological findings:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Including epidermal and sebaceous gland hyperplasia, chronic inflammation, parakeratosis and ulcer in males and females at ≥200 mg/kg bw/day.

Histopathological findings: neoplastic:

no effects observed

Details on results:

There were no significant differences in reproductive tissue evaluations or in estrous cycle characterization between dosed and vehicle control groups.

Effect levels

1

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:

organ weights and organ / body weight ratios

2

Key result

Dose descriptor:

NOAEL

Remarks:

(local effects)

Effect level:

ca. 100 mg/kg bw/day (nominal)

Sex:

male/female

Basis for effect level:

other: see 'Remark'

Target system / organ toxicity

Critical effects observed:

no

Any other information on results incl. tables

For detailed tables kindly refer to the attached background materials section of the IUCLID.

Applicant's summary and conclusion

Conclusions:

Under the study conditions, the 14-weeks NOAELs for systemic and local effects in mice were 50 and 100 mg/kg bw/day, respectively.

Executive summary:

A study was conducted to determine the repeated dose dermal toxicity of the test substance, C12 DEA, according to design based on OECD Guidance 411, in compliance with GLP. 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 for 5 d/week during a period of 14 weeks. Mortality, clinical findings, body weight and histopathology were evaluated at specific time intervals. All animals survived until the end of the study and final mean body weights and body weight gains of dosed mice were generally similar to those of the vehicle control groups. Skin irritation at the site of application was noted in all males and females administered 400 or 800 mg/kg bw/day. The kidney weights of males receiving 100, 400, or 800 mg/kg bw/day and females receiving 800 mg/kg bw/day were significantly greater than those of the vehicle controls. Liver weights of females at 200 mg/kg bw/day or greater were significantly higher than those of vehicle controls. Increased incidences of non-neoplastic lesions of the skin at the site of application, including epidermal and sebaceous gland hyperplasia, chronic inflammation parakeratosis and ulcer, were observed in animals receiving 200 mg/kg bw/day or greater. Under the study conditions, the 14-week NOAELs for systemic and local effects in mice were 50 and 100 mg/kg bw/day, respectively (NTP, 1999).