Amides, vegetable-oil, N,N-bis(hydroxyethyl)

Repeated dose toxicity: dermal

Administrative data

Endpoint:

sub-chronic toxicity: dermal

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Read across study hence maximum reliability rating of 2 assigned according to ECHA guidance, although study is well documented, meets generally accepted scientific principles, acceptable for assessment

Justification for type of information:

Read across approach adequate to support 7.6.1 according to "Guidance on information requirements and chemical safety assessment Chapter R.6: QSARs and grouping of chemicals" (ECHA 2008) and Read-Across Assessment
Framework (RAAF) (ECHA 2017)

Cross-reference

Reason / purpose for cross-reference:

read-across source

Reference

Endpoint:

chronic toxicity: dermal

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Read across study hence maximum reliability rating of 2 assigned according to ECHA guidance, although study is well documented, meets generally accepted scientific principles, acceptable for assessment

Justification for type of information:

Read across approach adequate to support 7.6.1 according to "Guidance on information requirements and chemical safety assessment Chapter R.6: QSARs and grouping of chemicals" (ECHA 2008) and Read-Across Assessment
Framework (RAAF) (ECHA 2017)

Reason / purpose for cross-reference:

read-across source

Principles of method if other than guideline:

A two-year dermal study was conducted in rat to evaluate the carcinogenic potential of the test substance.

GLP compliance:

yes

Limit test:

no

Specific details on test material used for the study:

- Name of test material (as cited in study report): Coconut oil acid diethanolamine condensate
- Physical state: Viscous yellow liquid
- Composition of test material, percentage of components: Composed primarily of diethanolamides of coconut oil acids, with unreacted diethanolamine, alkanolamides of unsaturated acids, and amine salts of the acids. The polar nitrosamine, N-nitrosodiethanolamine, was detected at a concentration of 219 ppb
- Lot/batch No.: 1G01742286
- Stability: Unstable when stored in glass tubes for 2 wk at 60°C
- Storage condition of test material: At room temperature, protected from light, in amber glass bottles sealed with Teflon- lined caps

Species:

rat

Strain:

Fischer 344

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Taconic Laboratory Animals and Services, (Germantown, NY)
- Age at study initiation: 6 wk
- Housing: Housed individually in Polycarbonate cages (Lab Products, Inc., Maywood, NJ)
- Bedding: Sani-Chip heat-treated hardwood chips (P.J. Murphy Forest Products Corp., Montville, NJ)
- Diet : NIH-07 open formula pelleted diet (Zeigler Brothers, Inc., Gardners, PA), ad libitum
- Water : Tap water (Columbus municipal supply), ad libitum
- Acclimation period: 11 to 12 d

ENVIRONMENTAL CONDITIONS
- Temperature : 20.0 -23.9 °C
- Humidity : 33-70 %
- Air changes : 10/hr
- Photoperiod : 12 h dark/12 h light

IN-LIFE DATES: From: Feb. 1, 1993 To: Jan. 31, 1995

Type of coverage:

open

Vehicle:

ethanol

Details on exposure:

The test substance formulation was applied to the shaved skin of test animals. No further details provided.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Dose formulations were analysed approximately every 2 months using HPLC. In addition to dose formulation analysis prior to dosing, samples collected after dosing (animal room samples) were analysed periodically.

Duration of treatment / exposure:

104 w

Frequency of treatment:

Five exposures per week

Remarks:

Doses / Concentrations:
0, 50 or 100 mg/kg bw/d (0, 85, or 170 mg/mL in ethanol)
Basis:
nominal per unit body weight

No. of animals per sex per dose:

50/sex/dose

Control animals:

yes, concurrent no treatment

Details on study design:

Dose selection rationale: Doses of 200 or 400 mg/kg bw/d in the 14 wk study were associated with reduced mean body weights, mild anemia, and significantly increased incidences and severities of lesions of the skin at the site of application. Therefore, these doses were considered inappropriate for a 2-year study. At 100 mg/kg bw/d, the incidences of skin lesions, especially ulceration, were less than at 200 mg/kg bw/d, and in general, the severities were minimal to mild. Therefore, 100 mg/kg bw/d was selected as the high dose for this 2-yr study.

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Observed twice daily
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Clinical findings were recorded initially, at 4 wk intervals during the study, and at the end of the study


DERMAL IRRITATION (if dermal study): Yes


BODY WEIGHT: Yes
- Time schedule for examinations: Animals were weighed initially, weekly during Week 1 through 13, at 4 wk intervals thereafter, and at the end of the studies

Sacrifice and pathology:

SACRIFICE: Carbon dioxide asphyxiation
GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes, Complete histopathology was performed on all rats. 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, skin, spleen, stomach (forestomach and glandular), testis (and
epididymis and seminal vesicle), thymus, thyroid gland, trachea, urinary bladder, and uterus

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 Nonneoplastic 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 pairwise 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.
Analysis of Continuous Variables: Organ and body weight data, which historically have approximately normal distributions, were analyzed with the parametric multiple comparison procedures of Dunnett (1955) and Williams (1971, 1972). Jonckheere's test (Jonckheere, 1954) was used to assess the significance of the dose-related trends. Average severity values were analyzed for significance with the Mann-Whitney U test. 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

Clinical signs:

effects observed, treatment-related

Dermal irritation:

effects observed, treatment-related

Mortality:

mortality observed, treatment-related

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:

no effects observed

Gross pathological findings:

effects observed, treatment-related

Neuropathological findings:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Details on results:

CLINICAL SIGNS AND MORTALITY: Survival rates of dosed male and female rats were similar to those of the vehicle controls. The only clinical finding attributed to dosing was irritation of the skin at the site of application in 100 mg/kg bw/d females.


BODY WEIGHT AND WEIGHT GAIN: The mean body weights of dosed male and female rats were similar to those of the vehicle controls throughout the study.


HISTOPATHOLOGY:
Skin: No neoplasms of the skin were attributed to treatment with test material. Incidences of squamous cell papilloma, keratoacanthoma, trichoepithelioma, basal cell adenoma, or carcinoma (combined) were significantly decreased in 100 mg/kg bw/d male rats. Incidences of epidermal hyperplasia, sebaceous gland hyperplasia, parakeratosis, and hyperkeratosis in all dosed groups were significantly greater than those in the vehicle control groups. The severities of these lesions generally increased with increasing dose and ranged from minimal to mild. Females in the 100 mg/kg bw/d group had a significantly greater incidence of ulceration at the site of application than did the vehicle controls.

Kidney: Incidences of renal tubule hyperplasia in dosed females were significantly greater than those of the vehicle controls, and the incidence of renal tubule adenoma in 50 mg/kg bw/d males was marginally increased. Incidences of chronic nephropathy were similar between vehicle control and dosed groups of male and female rats; however, the severity of nephropathy increased with increasing dose in female rats. The incidences of renal tubule adenoma in all groups of males and of renal tubule carcinoma in 50 mg/kg bw/d females exceeded the historical control ranges. An extended evaluation of the kidney revealed additional renal tubule adenomas in vehicle control and dosed males, and renal tubule adenomas and/or carcinomas in dosed females. When the single and step sections were combined, the incidences of renal tubule hyperplasia in dosed females and of renal tubule adenoma or carcinoma (combined) in 50 mg/kg bw/d females were significantly greater than those of the controls. In female rats, the combined single and step section evaluations of the kidney revealed a significant dose- related increase in the incidence of renal tubule hyperplasia and two adenomas and two carcinomas in the 50 mg/kg bw/d group but only one neoplasm (an adenoma), in the 100 mg/kg group. Renal tubule neoplasms are uncommon in female F344/N rats, and the presence of four neoplasms in the 50 mg/kg bw/d group, combined with the increased incidence of hyperplasia, is suggestive of an association with chemical exposure. However, the absence of an increase in neoplasms in the 100 mg/kg bw/d group in the presence of increased hyperplasia makes the association with chemical exposure uncertain.

Forestomach: The incidences of chronic active inflammation (vehicle control, 1/50; 50 mg/kg bw, 3/50; 100 mg/kg bw, 10/50), epithelial hyperplasia (2/50, 5/50, 13/50), and epithelial ulcer (1/50, 3/50, 11/50) were significantly increased in the forestomach of 100 mg/kg bw females. The severities of these lesions were similar among all groups.

Pancreas: The incidence of pancreatic acinar atrophy in 100 mg/kg male rats was significantly greater than that in the vehicle controls.

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: pancreatic acinar atrophy and nephropathy at the higher dose

Dose descriptor:

LOAEL

Effect level:

50 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: based on non-neoplastic lesions of the skin at all doses

Critical effects observed:

not specified

All dose formulations analysed during the 2 year studies were within 10% of the target concentration.

Conclusions:

Under the test conditions, there was no evidence of carcinogenic activity of the test substance in male rats in any of the tested dose levels. There was an equivocal evidence of carcinogenic activity in female rats based on a marginal increase in the incidences of renal tubule neoplasms. However, the absence of an increase in neoplasms in the 100 mg/kg bw/d group in the presence of increased hyperplasia makes the association with chemical exposure uncertain.

Executive summary:

A two-year dermal study was conducted in F344/N rats to evaluate the carcinogenic potential of 'amides, C8-18 (even-numbered) and C18-unsatd., N,N-bis(hydroxyethyl)' (in the form ofcoconut oil acid diethanolamine condensate).

 

 Doses studied include 0, 50, or 100 mg/kg bw/d test substance (0, 85, or 170 mg/mL in ethanol). 50 male/female test animals were used in each group. 5 exposures per week were given for 104 wk. The animals were observed twice daily, body weights and clinical findings were recorded periodically. Necropsy was performed on all animals and complete histopathology was performed.

 

The survival rates of treated male and female rats were similar to those of the vehicle controls. The mean body weights of dosed males and females were similar to those of the vehicle controls throughout the study. The only chemical-related clinical finding was irritation of the skin at the site of application in 100 mg/kg bw/d females. There were marginal increases in the incidences of renal tubule adenoma or carcinoma (combined) in 50 mg/kg bw/d females. The severity of nephropathy increased with increasing dose in female rats.

 

 Non neoplastic lesions of the skin at the site of application included epidermal hyperplasia, sebaceous gland hyperplasia, parakeratosis, and hyperkeratosis, and the incidences and severities of these lesions increased with increasing dose. The incidences of chronic active inflammation, epithelial hyperplasia, and epithelial ulcer of the forestomach increased with dose in female rats, and the increases were significant in the 100 mg/kg bw/d group.

 

 Under the test conditions, there was no evidence of carcinogenic activity of the test substance in male rats administered 50 or 100 mg/kg bw/d. There was an equivocal evidence of carcinogenic activity in female rats based on a marginal increase in the incidences of renal tubule neoplasms. However, the absence of an increase in neoplasms in the 100 mg/kg bw/d group in the presence of increased hyperplasia makes the association with chemical exposure uncertain.

Data source

Reference

Reference Type:

publication

Title:

NTP TR 479

Author:

NTP

Year:

2001

Bibliographic source:

NIH Publication No. 01-3969

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Reference substance 005

Cas Number:

93-83-4

Test animals

Species:

rat

Strain:

other: F344N

Sex:

male/female

Administration / exposure

Type of coverage:

not specified

Vehicle:

ethanol

Details on exposure:

not reported

Analytical verification of doses or concentrations:

yes

Duration of treatment / exposure:

13 weeks

Frequency of treatment:

5 days/week for 13 weeks

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 25, 50, 100, 200 or 400 mg/kg/day

No. of animals per sex per dose:

10/sex/dose

Control animals:

yes, concurrent vehicle

Details on study design:

no information

Positive control:

not applicable

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Weekly once


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


HAEMATOLOGY: Yes
- Time schedule for collection of blood: On Days 4 and 24 (blood was collected from the retroorbital sinus of clinical pathology study male and
female rats from each dose group)
- Anaesthetic used for blood collection: Yes (carbon dioxide/oxygen mixture)
- How many animals: All animals
- Parameters checked in table [No.?] were examined.: Hematocrit; hemoglobin concentration; erythrocyte, reticulocyte, nucleated erythrocyte, and platelet counts; mean cell volume; mean cell hemoglobin; mean cell hemoglobin concentration; and leukocyte count and differentials


CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: On Days 4 and 24 (blood was collected from the retroorbital sinus of clinical pathology study male and
female rats from each dose group)
- How many animals: All animals
- Parameters checked in table [No.?] were examined: Urea nitrogen, creatinine, total protein, albumin, cholesterol, triglycerides, alanine aminotransferase, alkaline phosphatase, sorbitol dehydrogenase, and total bile acids
OTHER: Organs weighed were heart, right kidney, liver, lung, right testis, and thymus

Sacrifice and pathology:

SACRIFICE: At the end of the 14 wk studies, blood was collected from the retroorbital sinus of all core study rats 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. In addition to gross lesions and tissue masses, the following tissues were examined: 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 (site of application) was examined in all core study animals, and the kidney was examined in core study male and female rats.

Other examinations:

Sperm motility and vaginal cytology: At the end of the studies, samples were collected for sperm motility or vaginal cytology from all rats receiving 100, 200 and 400 mg/kg bw of test material. The following sperm motility parameters were evaluated: spermatid heads per gram of testis, spermatid heads per testis, spermatid count, and epididymal spermatozoal motility and concentration. The left cauda epididymis, epididymis, and testis were weighed. Vaginal samples for cytology evaluations were collected for 12 consecutive days prior to the end of the studies from all female rats. The length of the estrous cycle and the length of time spent in each stage of the cycle were evaluated.

Statistics:

not reported

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Dermal irritation:

effects observed, treatment-related

Mortality:

mortality observed, treatment-related

Body weight and weight changes:

effects observed, treatment-related

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

Clinical biochemistry findings:

effects observed, treatment-related

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Gross pathological findings:

no effects observed

Neuropathological findings:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

not examined

Details on results:

CLINICAL SIGNS AND MORTALITY: Clinical findings included irritation of the skin at the site of application in 100, 200, and 400 mg/kg bw males and females.


BODY WEIGHT AND WEIGHT GAIN: Final mean body weights and body weight gains of 200 and 400 mg/kg bw males and females were significantly less than those of the vehicle controls.



HAEMATOLOGY: At week 14, a minimal microcytic, normochromic, nonresponsive anemia occurred in the 100 and 200 mg/kg bw females and 400 mg/kg bw males and females. The anemia also occurred in the 400 mg/kg bw males and females on Day 24. Increased segmented neutrophil counts occurred in 400 mg/kg bw males and females at week 14 and in 400 mg/kg bw females on Day 24.


CLINICAL CHEMISTRY: Cholesterol concentrations were significantly decreased in 200 and 400 mg/kg bw males and in females administered 100 mg/kg bw or greater; triglyceride concentrations were also decreased in 200 and 400 mg/kg bw males. At week 14, there was a minimal concentration-related increase of serum albumin concentration in all treated groups of females and in 100 mg/kg bw or greater male rats; on Day 24, increased albumin concentration occurred in the 400 mg/kg bw females. There were minimal increases of urea nitrogen concentration that occurred in the 200 and 400 mg/kg bw female rats on Day 24 and at week 14. At week 14, an increase in alanine aminotransferase activity occurred in 50 mg/kg bw or greater male rats. Additionally, alkaline phosphatase activity was increased in 400 mg/kg bw males.



ORGAN WEIGHTS: Kidney weights of females administered 50 mg/kg bw or greater were significantly greater than those of the vehicle control group. Left epididymis weights of 200 and 400 mg/kg bw males were significantly less than those of the vehicle controls, but this was most likely secondary to decreased mean body weights in these groups



HISTOPATHOLOGY: Histopathologic lesions of the skin at the site of application included epidermal hyperplasia, sebaceous gland hyperplasia, chronic active inflammation, parakeratosis, and ulcer. The incidences and severities of these skin lesions generally increased with increasing dose in males and females. The incidences of epidermal hyperplasia in all dosed groups of males and in females administered 50 mg/kg bw/d or greater were significantly greater than those in the vehicle controls. The incidences of renal tubule regeneration in 100, 200, and 400 mg/kg bw females were significantly greater than the vehicle control incidence, and the severities in 200 and 400 mg/kg bw females were increased.

OTHER: Estrous cycle lengths of dosed females were similar to those of the vehicle controls

Effect levels

1

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: renal tubule regeneration, alteration in body weights, haematological and reduced cholesterol and triglyceride levels at the higher doses

2

Dose descriptor:

LOAEL

Remarks:

local effects

Effect level:

25 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

other: minimal severity neoplastic (epidermal and sebaceous gland hyperplasia in males) lesions of the skin at the lowest dose of 25 mg/kg bw/day

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

none

Applicant's summary and conclusion

Conclusions:

Under the test conditions, the No Observed Adverse Effect Level (NOAEL) for systemic effects of the test substance can be considered to be 50 mg/kg bw/d and the LOAEL for local effects at 25 mg/kg bw/d.

Executive summary:

A study was conducted to evaluate the subchronic toxic effects of structurally similar 'amides, C8 -18 (even-numbered) and C18 -unsatd., N,N-bis(hydroxyethyl)' (i.e.,coconut oil acid diethanolamine condensate)when administered by dermal route in F344/N rats. The study was performed in compliance with Food and Drug Administration Good Laboratory Practice Regulations (21 CFR, Part 58).

Groups of 10 male and 10 female rats were administered 0, 25, 50, 100, 200, or 400 mg/kg bw/d of the test substancein ethanol by dermal application for 14 weeks.

 

All rats survived until the end of the study. Final mean body weights and body weight gains of 200 and 400 mg/kg bw/d males and females were significantly less than those of the vehicle controls.Clinical findings included irritation of the skin at the site of application in 100, 200, and 400 mg/kg bw/d malesand females. Hematological changes include minimal microcytic anemia at the end of the treatment period.Decreases in cholesterol and triglyceride concentrations were observed in the higher group rats (i.e.,≥.100 mg/kg bw/d)

Histopathological lesions of the skin at the site of application included epidermalhyperplasia, sebaceous gland hyperplasia, chronic active inflammation, parakeratosis, and ulcer. Theincidences and severities of these skin lesions generally increased with increasing dose in males andfemales. The incidences of epidermal hyperplasia in all dosed groups of males and in females administered 50 mg/kg bw/d or greater were significantly greater than those in the vehicle controls. The incidences of renal tubule regeneration in 100, 200, and 400 mg/kg bw/d females were significantly greater than the vehicle control incidence, and the severities in 200 and 400 mg/kg bw/d females were increased.

 

Under the test conditions, the No Observed Adverse Effect Level (NOAEL) for systemic effects of the test substance can be considered to be 50 mg/kg bw/d and the LOAEL for local effects at 25 mg/kg bw/d.