Amides, C16-18 and C18-unsatd., N,N-bis(hydroxyethyl)

Carcinogenicity

Description of key information

The 2 year chronic dermal studies conducted in rats and mice have shown structurally similar amides, C8-18 and C18-unsatd., N,N-bis(hydroxyethyl) to be carcinogenic in mice based on the induction of liver and kidney tumours but no evidence of carcinogenic effects were observed in rats. Structurally similar, amides, C12, N, N-bis(2 -hydroxyethyl) has also shown evidence of liver carcinogenicity in mice but not in rats. In contrast, amides, C18 (even-numbered) and C18-unsatd., N,N-bis(hydroxyethyl) in 2 year chronic dermal studies in rats and mice showed no evidence of carcinogenic effects. The authors clearly stated that in the studies with amides, C8-18 and C18-unsatd., N,N-bis(hydroxyethyl) and amides, C12, N, N-bis(2 -hydroxyethyl), the presence of DEA as a contaminant was responsible for the observed carcinogenic effects in mice. This suggestion is also further strengthened by more recent research which suggests that DEA is a non-genotoxic carcinogen with a mode of action involving choline depletion, which is a rodent specific mechanism unlikely to be relevant to humans. Therefore in light of all the available information, amides, C16-18(even-numbered) and C18-unsatd., N,N-bis(hydroxyethyl) is not expected to be a carcinogen in humans.

Key value for chemical safety assessment

Carcinogenicity: via dermal route

Link to relevant study records

Reference

Endpoint:

carcinogenicity: dermal

Type of information:

migrated 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 data waiving:

other:

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to other study

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

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

Route of administration:

dermal

Vehicle:

ethanol

Details on exposure:

The test material 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 wk

Frequency of treatment:

Five exposures per week

Post exposure period:

No

Remarks:

Doses / Concentrations:
0, 50 or 100 mg/kg bw (0, 85, or 170 mg/mL in ethanol)
Basis:
nominal conc.

No. of animals per sex per dose:

50/sex/dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Doses of 200 or 400 mg/kg bw 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, the incidences of skin lesions, especially ulceration, were less than at 200 mg/kg bw, and in general, the severities were minimal to mild. Therefore, 100 mg/kg bw was selected as the high dose for this 2-yr study

Positive control:

No

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

Other examinations:

None

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

Mortality:

mortality observed, treatment-related

Body weight and weight changes:

not examined

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:

not examined

Gross pathological findings:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

effects observed, treatment-related

Description (incidence and severity):

equivocal evidence

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 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 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 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 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 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 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 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 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 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.

Relevance of carcinogenic effects / potential:

Yes

Dose descriptor:

NOAEL

Sex:

male/female

Remarks on result:

not determinable

Remarks:

no NOAEL identified. Effect type:carcinogenicity (migrated information)

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 of coconut 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.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Quality of whole database:

Exceeds the information requirements for this tonnage band.

Justification for classification or non-classification

The available data suggests that 'amides, C16-18 (even-numbered) and C18-unsatd., N, N-bis(hydroxyethyl)' is not carcinogenic to humans. Although rodent carcinogenicity studies conducted on structurally similar 'amides, C8-18 and C18-unsatd., N, N-bis(hydroxyethyl)' and 'amides, C12, N,N-bis(2-hydroxyethyl)' provided evidence of carcinogenicity in mice, it was concluded that this was due to the presence of a contaminant - free diethanolamine (DEA) in the test substance. Diethanolamine has been shown to be a non-genotoxic carcinogen in mice. However the proposed mode of action for non-genotoxic mechanism indicates a rodent-specific phenomenon which is not relevant for man. This evaluation is supported by a recent decision of a scientific board by National Toxicology Program (NTP) after a public hearing at which it was decided that DEA should not be listed as a carcinogen under the RoC (Report on Carcinogens) process.

Therefore based on the overall weight of evidence, 'amides C16-18 (even-numbered) and C18-unsatd., N, N-bis(hydroxyethyl)', does not require classification for the carcinogenicity endpoint according to EC criteria (67/548/EEC) and CLP criteria (EC 1272/2008).

Additional information

No carcinogenicity data have been identified on amides, C16-18 (even-numbered) and C18-unsatd., N,N-bis(hydroxyethyl). However, a substantial amount of data is available on other DEA-derived FAA, particularly on amides, C8-18 and C18-unsatd., N,N-bis(hydroxyethyl) , amides, C12, N,N-bis(2-hydroxyethyl) and amides, C18-unsatd., N,N-bis(hydroxyethyl) which have been thoroughly investigated as part of the NTP program. The use of data from the FAA'category for the assessment of the carcinogenicity of amides, C16-18 (even-numbered) and C18-unsatd., N,N-bis(hydroxyethyl) by means of read-across (analogue approach) is justified due to their structural similarities as indicated by a common functional group (i. e., fatty acid alkanol amide) and common breakdown products (e. g., hydroxylation and oxidation of alkyl chains). The structural similarity of C16-18 and C18-unsatd., N,N-bis(2-hydroxyethyl) and C8-18 and C18-unsatd., N,N-bis(2-hydroxyethyl), C12 N,N-bis(2-hydroxyethyl) or C18-unsatd., N,N-bis(2-hydroxyethyl), (“DEA-FAA”) results in similar physico-chemical, human health and environmental properties.

Dermal

A 2 year dermal study was conducted in F344/N rats to evaluate the carcinogenic potential of amides, C8-18 and C18-unsatd., N,N-bis(hydroxyethyl). Doses of 0, 50 or 100 mg/kg bw/day of the test substance (0, 85 or 170 mg/mL in ethanol) were administered to 50 male and female test animals in each group. Five exposures per week were given for 104 weeks. The animals were observed twice daily, body weights and clinical findings were recorded periodically. Necropsy and histopathology was performed on all animals. 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 skin irritation at the site of application in the 100 mg/kg bw/d dose group (females). There were marginal increases in the incidences of renal tubule adenoma or carcinoma (combined) in 50 mg/kg bw/day dose group (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 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/day group. There was no evidence of carcinogenic activity of the test substance in male rats administered 50 or 100 mg/kg bw/day. 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/day group in the presence of increased hyperplasia makes the association with chemical exposure uncertain (NTP report 479, 2001).

In a 2 year dermal study conducted in B6C3F1 mice, the carcinogenic potential of amides, C8-18 and C18-unsatd., N,N-bis(hydroxyethyl) was evaluated. Doses of 0, 100 or 200 mg/kg bw/day of the test substance (0, 50 or 100 mg/mL in ethanol) was administered to 50 male and female mice. Five exposures per week were given for 104 to 105 weeks. 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. Survival of dosed male and female mice was generally similar to that of the vehicle controls. Mean body weights of 100 mg/kg bw/day females from Week 93 and 200 mg/kg bw/day females from Week 77 were less than those of the vehicle controls. The only clinical finding attributed to treatment was skin irritation at the site of application in males administered 200 mg/kg bw/d. The incidences of hepatic neoplasms (hepatocellularadenoma, hepatocellular carcinoma and hepatoblastoma) were significantly increased in male and/or female mice. The incidences of eosinophilic foci in dosed groups of male mice were increased relative to that in the vehicle controls. The incidences of renal tubule adenoma and renal tubule adenoma or carcinoma (combined) were significantly increased in 200 mg/kg bw/day males. Several non-neoplastic lesions of the skin at the site of application were considered treatment-related. Incidences of epidermal hyperplasia, sebaceous glandhyperplasia and hyperkeratosis were greater in all dosed groups of males and females than in the vehicle controls. The incidences of thyroid gland follicular cell hyperplasia in all test groups of males and females were significantly greater than those in the vehicle control groups. Based on the above results, there was clear evidence of carcinogenic activity in male B6C3F1 mice based on increased incidences of hepatic and renal tubule neoplasms and in female B6C3F1 mice based on increased incidences of hepatic neoplasms (NTP report 479, 2001).

It is important to consider that in both the 2 year dermal rat and mouse studies described above, the test substance amides, C8-18 and C18-unsatd., N,N-bis(hydroxyethyl) contained free DEA as a contaminant at a concentration of at least 0.5%. In a recent review by Leunget al.(2005), studies conducted by The NTP Program have indicated that lifetime dermal exposure to DEA increased the incidence and multiplicity of liver tumours in mice but not in rats. DEA was not carcinogenic when tested in the Tg.Ac transgenic mouse model and short term genotoxicity tests have provided negative results. It has been shown that DEA induces mouse liver and kidney tumours by a non-genotoxic mode of action that involves its ability to cause choline deficiency. Choline is an essential nutrient in mammals and therefore this mode of action is qualitatively applicable to human. However there are marked species differences in susceptibility to choline deficiency, with rats and mice being much more susceptible that other mammalian species including humans. These differences are due to the quantitative differences in the enzyme kinetics controlling choline metabolism. The fact that DEA is carcinogenic in mice and not in rats has therefore important implications for human health risk assessment. DEA has also been shown to be less readily absorbed across rat and human skin compared to mouse skin and a NOAEL for DEA-induced choline deficiency in mice has been established to be 10 mg/kg/day, thus indicating that there is a critical level of DEA that must be attained in order to affect choline homeostasis. The lack of carcinogenic response in rats suggest that exposure to DEA did not reach the critical level. Since rodents are more sensitive to choline deficiency that humans, it can be concluded that the carcinogenic effects of DEA in mice is not predictive of similar susceptibility in humans and also suggests that DEA should not be classified as a carcinogen, as the hepatic tumours seen in mice and the proposed mode of non-genotoxic mechanism for liver and renal tumours are not relevant to humans.

In another 2 year dermal study conducted in rats and mice, the carcinogenic potential of structurally similar, amides, C12, N,N-bis(2-hydroxyethyl (LDEA) was investigated. Doses administered were 0, 50 or 100 mg/kg bw/day (0, 85 or 170 mg/mL) to 50 male and 50 female F344/N rats and 0, 100 or 200 mg/kg bw/day (0, 50 or 100 mg/mL) to 50 male and female B6C3F1 mice in each group. Five exposures per week were given for 104-105 weeks (rats) or 105-106 weeks (mice). 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 results revealed no evidence of carcinogenic activity of structurally similar LDEA in male or female rats at any of the dose levels. There was also no evidence of carcinogenic activity in male mice administered 100 or 200 mg/kg bw/day. However there was some evidence of carcinogenic activity in female mice based on increased incidences of hepatocellular neoplasms (NTP report 480, 1999). The authors concluded that the increases seen were associated with the presence of free DEA, present as a contaminant at a concentration of 5% (approximately).

Two year dermal studies were conducted in 50 F344/N male/female rats and 50 B6C3F1 male/female mice to evaluate the carcinogenic potential of amides, C18 (even-numbered) and C18-unsatd., N,N-bis(hydroxyethyl). The test substance contained up to 0.19% free DEA. Rats were administered 0, 50 or 100 mg/kg bw/day of test substance in ethanol (0, 85 or 170 mg/mL) and mice 0, 15 or 30 mg/kg bw/day of the test substance in ethanol (0, 7.5 or 15 mg/mL), 5 exposures per week 5 for 104 (rats) or 105 weeks (mice). 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. 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. Dose dependent increase in irritation (i. e., mild to moderate) and non-neoplastic lesions (i. e., minimal to moderate) of the skin was observed at the site of application in males and females. 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. Based on the results, no evidence of carcinogenic activity was observed at any tof the dose levels tested (NTP report 481, 1999). Survival of the dosed male and female mice was similar to that of the vehicle control groups. The mean body weights of females (Week 76 onwards) were reduced than those of the vehicle control group at 30 mg/kg bw/day. The only significant treatment related clinical finding was irritation of the skin at the site of application in 30 mg/kg bw/day males. The incidences of epidermal hyperplasia, sebaceous gland hyperplasia and chronic active inflammation of the dermis in all dosed groups were significantly increased relative to the vehicle controls at 3 months and 2 years. The increased incidences of hyperkeratosis in dosed males at 3 months and in dosed males and females at 2 years, of parakeratosis in 30 mg/kg bw/day males at 3 months and 2 years, and of ulcer in 30 mg/kg bw/day males and exudate in 30 mg/kg bw/day males and females at 2 years were also attributed to the test substance administration. No significant neoplastic lesions or evidence of carcinogenic activity was observed at any tested dose in skin and lymph nodes. Overall, no evidence of carcinogenic activity was observed at any of the dose levels tested (NTP report 481, 1999).

Justification for selection of carcinogenicity via dermal route endpoint:

2 year dermal rat study conducted by NTP and appropriate to base the assessment on.