Amides, C12-18 and C18-unsatd., N,N-bis(hydroxyethyl)

Carcinogenicity

Key value for chemical safety assessment

Carcinogenicity: via oral route

Link to relevant study records

Reference

Reference 1

Endpoint:

carcinogenicity: dermal

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Not available

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

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 mice to evaluate the carcinogenic potential of the test substance.

GLP compliance:

yes

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: 6 wk
- Housing: Housed individually in Polycarbonate , changed weeklyand rotated every 2 wk
- Diet : NIH-07 open formula pelleted diet, ad libitum
- Water : Tap water via automatic watering system available cleaned every 2 wk, ad libitum
- Acclimation period: 13 d (males); 14 d (females)

ENVIRONMENTAL CONDITIONS
- Temperature : 20.5-26.1°C
- Humidity : 33-64%
- Air changes : 10/hr
- Photoperiod : 12 h dark/12 h light

IN-LIFE DATES: From: Dec. 30, 1992 To: Jan. 06, 1995

Route of administration:

dermal

Vehicle:

ethanol

Details on exposure:

VEHICLE
Purity: ranged from 97 to 103% relative to the reference standard

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Dose preparations were analysed approximately every 2 months using HPLC. All dose formulations analysed during the 2-yr studies were within 10% of the target concentration. In addition to dose formulation analysis prior to dosing, samples collected after dosing (animal room samples) were analysed periodically.

Duration of treatment / exposure:

105-106 wk

Frequency of treatment:

Five exposures per week

Dose / conc.:

0 mg/kg bw/day (nominal)

Dose / conc.:

100 mg/kg bw/day (nominal)

Remarks:

corresponding to 50 mg/mL in ethanol

Dose / conc.:

200 mg/kg bw/day (nominal)

Remarks:

corresponding to 100 mg/mL in ethanol

No. of animals per sex per dose:

50/sex/dose

Control animals:

yes, concurrent vehicle

Details on study design:

Dose selection rationale: Dose selection was based primarily on the increased incidences of a spectrum of skin lesions at the site of application. Doses of 400 or 800 mg/kg bw were associated with high incidences of chronic inflammation and ulceration and were thus considered to be inappropriate for a 2-yr study. A marked reduction in toxic response occurred at 200 mg/kg bw, and 100 mg/kg bw was a no-effect level in male mice. Therefore, 200 mg/kg bw was selected as the high dose for the 2-yr study and 100 mg/kg bw as the low dose.

Positive control:

No

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: At study initiation, at 4-wk intervals during the study, and at necropsy

DERMAL IRRITATION (if dermal study): Yes

BODY WEIGHT: Yes
- Time schedule for examinations: Animals were weighed initially at study initiation, weekly during weeks 1-13, at 4-wk intervals thereafter, and at the end of the study.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: No

CLINICAL CHEMISTRY: No

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:

METHOD OF SACRIFICE: Carbon dioxide asphyxiation

GROSS PATHOLOGY: Yes

HISTOPATHOLOGY: Yes, Complete histopathology was performed on all animals. 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.

Clinical signs:

no effects observed

Description (incidence and severity):

No significant difference in clinical findings was observed between the dosed groups and the vehicle control groups.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Description (incidence):

No significant difference in survival was observed between the dosed groups and the vehicle control groups.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

No significant difference was observed in the mean body weights between the dosed groups at 100 mg/kg bw and the vehicle control groups. The mean body weight of females that received 200 mg/kg bw was 94% that of the vehicle controls by week 33 and remained lower throughout the remainder of the study.

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:

no effects observed

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Liver: The incidences of hepatocellular adenoma or carcinoma (combined) were significantly increased in dosed female groups compared to that in the vehicle control group, as was the incidence of hepatocellular adenoma in 100 mg/kg bw females. These incidences exceeded the historical control ranges for these neoplasms. There were also increases in the incidences of eosinophilic foci in dosed female mice, and the increase was significant in females at 200 mg/kg bw. The incidences of hepatocellular adenoma, carcinoma, and adenoma or carcinoma (combined) were not significantly increased in dosed male mice. These increases were associated with free diethanolamine, which was present as a contaminant of lauric acid diethanolamine condensate.

Thyroid Gland: Incidences of focal hyperplasia of thyroid gland follicular cells were increased in dosed male mice; the incidence in the 200 mg/kg bw group was significantly greater than that in the vehicle control group. There were no corresponding increases in the incidences of follicular cell neoplasms.

Histopathological findings: neoplastic:

no effects observed

Description (incidence and severity):

Skin, site of application: Incidences of nonneoplastic lesions of the skin at the site of application were significantly increased in dosed males and females. The lesions were of minimal to moderate severity and consisted mostly of thickening of the epidermis (epidermal hyperplasia) and sebaceous gland hyperplasia. Compared to the vehicle controls, the incidences of chronic inflammation and hyperkeratosis were significantly greater in all groups of dosed males and females, and the incidences of parakeratosis were significantly greater in males and females administered 200 mg/kg bw. Ulcers occurred in a few mice at 200 mg/kg bw and were indicative of more severe local irritation.

Other effects:

not examined

Relevance of carcinogenic effects / potential:

Yes

Key result

Dose descriptor:

NOAEL

Sex:

male/female

Remarks on result:

not determinable

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

Conclusions:

Under the study conditions, no evidence of carcinogenic activity was reported in male mice. Some evidence of carcinogenic activity in female mice based on increased incidence of hepatocellular neoplasms was associated with free diethanolamine (DEA), which was present as a contaminant of the test substance.

Executive summary:

A study was conducted to evaluate the long-term repeated dose dermal carcinogenicity of the test substance, C12 DEA, in compliance with GLP. Groups of 50 male and 50 female mice were exposed to dermal doses equivalent to 0, 100 or 200 mg/kg/day, 5 d/week, for 105-106 weeks. The animals were observed twice daily. Body weights and clinical findings were recorded periodically. Necropsy and complete histopathology was performed on all animals at test end. Mean body weights of females in 200 mg/kgbw/d dose group were lower than those of the vehicle controls beginning at week 33. The incidences of hepatocellular adenoma or carcinoma (combined) were significantly increased in treated females compared to the vehicle controls, as was the incidence of hepatocellular adenoma in the100 mg/kg bw/day female group. There were dose-related increases in the incidences of non-neoplastic lesions of the skin at the site of application, including epidermal and sebaceous gland hyperplasia, hyperkeratosis, chronic inflammation, and parakeratosis. Treated males had greater incidences of thyroid gland follicular cell focal hyperplasia than did the vehicle controls; the incidence in the 200 mg/kg bw/d group was significantly greater than that in the vehicle control group. Under the study conditions, no evidence of carcinogenic activity was reported in male mice. Some evidence of carcinogenic activity in female mice based on increased incidence of hepatocellular neoplasms was associated with free diethanolamine (DEA), which was present as a contaminant of the test substance (Irwin, 1999). However, recent evidences suggest that DEA should not be classified as a carcinogen, as the hepatic tumours seen in mice and the proposed mode of non-genotoxic mechanism are not relevant to humans/primates.

Reference 2

Endpoint:

carcinogenicity: dermal

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Not available

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

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 rats 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 Farms, Germantown, NY
- Age at study initiation: 6 wk
- Housing: Housed individually in Polycarbonate , changed weekly and rotated every 2 wk
- Diet : NIH-07 open formula pelleted diet, ad libitum
- Water : Tap water via automatic watering system, ad libitum
- Acclimation period: 11 d (males); 12 d (females)

ENVIRONMENTAL CONDITIONS
- Temperature : 21.1-26.7°C
- Humidity : 36-59%
- Air changes : 10/h
- Photoperiod : 12 h dark/12 h light

IN-LIFE DATES: From: Dec. 14, 1992 To: Dec. 14, 1994

Route of administration:

dermal

Vehicle:

ethanol

Details on exposure:

VEHICLE
Purity: ranged from 97% to 103% relative to the reference standard

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Dose preparations were analysed approximately every 2 m using HPLC. All dose formulations analysed during the 2-yr studies were within 10% of the target concentration. In addition to dose formulation analysis prior to dosing, samples collected after dosing (animal room samples) were analysed periodically.

Duration of treatment / exposure:

104-105 wk

Frequency of treatment:

Five exposures per week

Post exposure period:

No

Dose / conc.:

0 mg/kg bw/day (nominal)

Dose / conc.:

50 mg/kg bw/day (nominal)

Remarks:

Corresponding to 85 mg/mL in ethanol

Dose / conc.:

100 mg/kg bw/day (nominal)

Remarks:

Corresponding to 170 mg/mL in ethanol

No. of animals per sex per dose:

50

Control animals:

yes, concurrent vehicle

Details on study design:

Dose selection rationale: Dose selection was based primarily on the increased incidences of a spectrum of skin lesions at the site of application. Doses of 200 and 400 mg/kg bw caused high incidences of chronic inflammation and ulceration in males and females. Final mean body weights and body weight gains of male rats treated with 200 or 400 mg/kg bw were also less than those of the vehicle controls. Therefore, 200 and 400 mg/kg bw were considered inappropriate for the study. There was a very obvious reduction in toxic response of the skin at 100 mg/kg bw, with chronic inflammation and ulceration being absent in females and present in only one male; thus, 100 mg/kg bw was selected as the high dose for the 2-yr rat study. The responses observed at 25 and 50 mg/kg bw were very similar and consisted of only minimal hyperplasia; therefore, 50 mg/kg bw was selected as the low dose for the study.

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: At study initiation, at 4-wk intervals during the study, and at necropsy

DERMAL IRRITATION (if dermal study): Yes

BODY WEIGHT: Yes
- Time schedule for examinations: Animals were weighed initially at the study initiation, weekly during weeks 1-13, at 4-wk intervals thereafter, and at the end of the study.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: No

CLINICAL CHEMISTRY: No

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:

Method of Sacrifice: Carbon dioxide asphyxiation

GROSS PATHOLOGY: Yes

HISTOPATHOLOGY: Yes, Complete histopathology was performed on all animals. 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.

Clinical signs:

no effects observed

Description (incidence and severity):

No significant difference clinical findings was observed between the dosed groups and the vehicle control groups.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Description (incidence):

No significant difference in survival was observed between the dosed groups and the vehicle control groups.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

No significant difference was observed in the mean body weights between the dosed groups and the vehicle control groups.

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:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Changes of minimal to moderate severity occurred in the skin at the site of application in treated male and female rats. The major alterations from normal skin were thickening of the epidermis (epidermal hyperplasia) and sebaceous gland hyperplasia (which usually occurred along with epidermal hyperplasia). Incidences of chronic inflammation, hyperkeratosis, and parakeratosis in all dosed groups were significantly greater than those in the vehicle controls, as were the incidences of ulceration in 100 mg/kg bw males and females. The nonneoplastic skin lesions at the site of application were considered to be indicative of local irritation with no neoplastic or preneoplastic changes.

Histopathological findings: neoplastic:

no effects observed

Description (incidence and severity):

No significant differences was reported between the vehicle control groups and dosed groups in the incidences of neoplasms.

Other effects:

not examined

Details on results:

OTHER FINDINGS: The incidence of forestomach ulcer was significantly lower in males that received 100 mg/kg bw/d than in the vehicle controls. The incidences of inflammation of the nasal mucosa were significantly lower in dosed males than in the vehicle controls. The incidence of chronic inflammation of the liver was significantly lower in females administered 100 mg/kg bw than in the vehicle controls.

Relevance of carcinogenic effects / potential:

Yes

Key result

Dose descriptor:

NOAEL

Effect level:

100 mg/kg bw/day (nominal)

Sex:

male/female

Basis for effect level:

other: Under the test conditions, no evidence of carcinogenic activity for the test substance was reported.

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

Conclusions:

Under the study conditions, no evidence of carcinogenic activity for the test substance was observed at any of the dose levels tested.

Executive summary:

A study was conducted to evaluate the long-term repeated dose dermal carcinogenicity of the test substance, C12 DEA, in compliance with GLP. Groups of 50 male and 50 female rats were exposed to dermal doses equivalent to 0, 50 or 100 mg/kg/day, 5 d/week, during 104-105 weeks. The animals were observed twice daily. Body weights and clinical findings were recorded periodically. Necropsy and complete histopathology was performed on all animals at test end. There were no significant differences between vehicle control and dosed males or females in survival or mean body weights. There were no chemical-related differences in neoplasm incidences. Dose-related increases occurred in the incidences of non-neoplastic lesions of the skin at the site of application, including epidermal and sebaceous gland hyperplasia, hyperkeratosis, chronic inflammation, parakeratosis, and ulcer. Under the study conditions, no evidence of carcinogenic activity for the test substance was observed at any of the dose levels tested (Irwin, 1999).

Reference 3

Endpoint:

carcinogenicity: dermal

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 29 April 1993 to 10 May 1995

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

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 2 year toxicity study in B6C3F1 mice was conducted to evaluate the carcinogenic potential by the repeated dermal exposure to the test substance.

GLP compliance:

yes

Species:

mouse

Strain:

B6C3F1

Sex:

male/female

Details on test animals or test system and environmental conditions:

- Source: Taconic Laboratory Animals and Services (Germantown, NY)
- Age at study initiation: 6 wks
- 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: 11 d and Females: 12 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: 21.1-25.0°C
- Relative humidity: 36-68%
- Air changes: 10/h
- Photoperiod: 12 h dark/12 h light

IN-LIFE DATES: From: 1993-04-29 To: 1995-05-10

Route of administration:

dermal

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.
The dose formulations were stored at room temperature, protected from light, in amber glass bottles for up to 28 days.

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 analysed every 9 weeks during the 2-year study and 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:

2-yr

Frequency of treatment:

5 exposures/wk

Post exposure period:

No

Dose / conc.:

0 mg/kg bw/day (actual dose received)

Dose / conc.:

15 mg/kg bw/day (actual dose received)

Dose / conc.:

30 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

50/sex/dose; 5 M/F (for 3-month interim evaluation in mice)

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose selection is based primarily on the incidences and severities of skin lesions observed at the site of application during the 13-week dermal studies. All groups of mice administered 100 mg/kg bw/day or greater exhibited high incidences of skin lesions at the site of application; thus, 100 mg/kg bw/day or greater were considered inappropriate for the 2-year study.

- The severities of the skin lesions increased with increasing dose in groups administered doses greater than 100 mg/kg bw/day; however, the severities of other lesions generally were increased only slightly between 100 and 800 mg/kg compared to the eightfold increase in dose. Therefore, the skin response appeared to plateau at 100 mg/kg, and higher doses did not produce a proportional increase in response. The incidences of skin lesions in groups administered 50 mg/kg bw/day were slightly less (minimal to mild) than those observed in groups administered 100 mg/kg bw/day. The skin response at the site of application in 50 mg/kg bw/day groups was such that 50 mg/kg bw/day was also considered inappropriate for a 2-year study; however, the slight reduction in incidences and the lower severities observed in the 50 mg/kg bw/day groups compared to those in the 100 mg/kg bw/day groups indicated that 50 mg/kg was below the plateau and at the upper end of a dose range in which skin response at the site of application exhibited a greater dose dependency. Therefore, at doses below 50 mg/kg bw/day, a proportional reduction in incidences and severities of skin lesions at the site of application would be expected. Accordingly, a high dose of 30 mg/kg and a low dose of 15 mg/kg were selected for the 2-year study in mice.

Positive control:

Not included in this study

Observations and examinations performed and frequency:

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Clinical findings were recorded monthly and at the end of the studies.

BODY WEIGHT: Yes
- Time schedule for examinations: Weighed initially, weekly for 13 wks, approximately monthly thereafter and again at the end of the studies

DERMAL IRRITATION (if dermal study): Yes

HAEMATOLOGY: No

CLINICAL CHEMISTRY: No

Sacrifice and pathology:

SACRIFICE: At the end of 3rd month 5 males and females were sacrificed for skin evaluation and at the end of the 2-year study all animals were sacrificed by carbon dioxide asphyxiation.

GROSS PATHOLOGY: Yes

HISTOPATHOLOGY: Yes
- Skin from the site of application was examined from all mice at the 3-month interim evaluation.
- Complete histopathology was performed on all the rats at the end of the study. In addition to gross lesions and tissue masses, the tissues examined were: adrenal gland, bone with marrow, brain, clitoral gland, esophagus, gallbladder (mice), heart with aorta, large intestine (cecum, colon, and rectum), small intestine (duodenum, jejunum and 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 (site of application), spleen, stomach (forestomach and glandular), testis with epididymis and seminal vesicle, thymus, thyroid gland, trachea, urinary bladder, and uterus.
- 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:

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 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).

Clinical signs:

no effects observed

Description (incidence and severity):

The only significant treatment-related clinical finding was irritation of the skin at the site of application in 30 mg/kg bw males (vehicle control, 0/55; 15 mg/kg bw, 1/55; 30 mg/kg bw, 20/55).

Dermal irritation (if dermal study):

effects observed, treatment-related

Description (incidence and severity):

The only significant treatment-related clinical finding was irritation of the skin at the site of application in 30 mg/kg bw males (vehicle control, 0/55; 15 mg/kg bw, 1/55; 30 mg/kg bw, 20/55).

Mortality:

no mortality observed

Description (incidence):

Survival of dosed male and female mice was similar to that of the vehicle control groups.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Mean body weights of dosed males and 15 mg/kg bw females were similar to those of the vehicle controls throughout the study. Mean body weights of 30 mg/kg bw females were less than those of the vehicle controls beginning week 76.

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:

not examined

Gross pathological findings:

no effects observed

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Skin lesions: Minimal to mild non-neoplastic lesions of the skin at the site of application were observed in females more than 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 at 2 years. There was increased incidence of hyperkeratosis in dosed males at 3 months and in dosed males and females at 2-years. The increased incidences of parakeratosis in 30 mg/kg bw/day males at the 3-month interim evaluation and at 2-years and 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.

Histopathological findings: neoplastic:

no effects observed

Description (incidence and severity):

- Skin lesions: No significant treatment related effects were observed. Few neoplastic lesions observed (i.e., one fibrosarcoma in vehicle control female and two fibrosarcomas in females at 15 mg/kg bw/day) were not considered significant as they did not follow a dose dependent pattern.

- Malignant lymphoma: No significant treatment related effects were observed. The increased incidence of the lymphomas observed in females at 30 mg/kg bw/day was found to be similar to the incidences observed in the other dermal studies with ethanol as the vehicle (i.e., comparable to historical control values).

Other effects:

not examined

Relevance of carcinogenic effects / potential:

No, there were no significant neoplasms associated with administration of the test substance.

Key result

Dose descriptor:

NOAEL

Remarks:

carcinogenicity

Effect level:

ca. 30 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no evidence of carcinogenic activity at any tested dose levels

Key result

Dose descriptor:

NOAEL

Remarks:

(systemic effects)

Effect level:

ca. 15 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: body weight changes at 30 mg/kg bw/d

Key result

Dose descriptor:

LOAEL

Remarks:

(local effects)

Effect level:

ca. 15 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: incidences of non-neoplastic lesions of the skin

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

Conclusions:

Under the study conditions, no evidence of carcinogenic activity was observed at any tested dose levels in mice.

Executive summary:

A study was conducted to evaluate the long-term repeated dose dermal carcinogenicity of the test substance, C18-unsatd. DEA, in compliance with GLP. Groups of 50 male and 50 female mice were dermally exposed to 0, 15 or 30 mg/kg bw/day in ethanol at a frequency of 5 d/week for a period of 105 weeks. 5 males and 5 females were considered for the 3 month interim assessment. Survival, clinical findings, body weight and histopathology were evaluated at specific time intervals. Survival of the dosed male and female rats 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 at 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 levels in skin and lymph nodes. Under the study conditions, no evidence of carcinogenic activity was observed at any tested dose levels in mice. (Irwin, 1999).

Reference 4

Endpoint:

carcinogenicity: dermal

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 06 May 1993 to 16 May 1995

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

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 2-year toxicity study in F344/N rats was conducted to evaluate the carcinogenic potential by the repeated dermal exposure to the test substance.

GLP compliance:

yes

Species:

rat

Strain:

Fischer 344

Sex:

male/female

Details on test animals or test system and environmental conditions:

- Source: Taconic Laboratory Animals and Services (Germantown, NY)
- Age at study initiation: 7 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 (PJ 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: 13 d and Females: 14 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: 21.1-23.3°C
- Relative humidity: 31-73%
- Air changes: 10/h
- Photoperiod: 12 h dark/12 h light

IN-LIFE DATES: From: 1993-05-06 To: 1995-05-16

Route of administration:

dermal

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.
The dose formulations were stored at room temperature, protected from light, in amber glass bottles for up to 28 days.

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 analysed every 9 weeks during the 2-year study and 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:

2-yrs

Frequency of treatment:

5 exposures/wk

Post exposure period:

No

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)

No. of animals per sex per dose:

50/sex/dose

Control animals:

yes, concurrent vehicle

Details on study design:

Dose selection rationale: The dose selection was based primarily on the incidences and severities of skin lesions observed at the site of application during the 13-week dermal studies. The doses of 200 and 400 mg/kg bw/day exhibited reduced mean body weight and body weight gain along with high incidences of skin lesions at the site of application; thus were considered inappropriate for a 2-year study. Further, lesions of the skin were also present at the site of application in groups administered 100 mg/kg bw/day; however, the incidence were less than those observed in the 200 and 400 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. Therefore, 100 mg/kg bw/day was selected as the high dose for rats in the 2-year study. In groups treated with 50 mg/kg bw/day, the incidences of skin lesions diminished considerably and lesion severities were minimal. Therefore, 50 mg/kg bw/day was selected as the low dose.

Positive control:

No

Observations and examinations performed and frequency:

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Clinical findings were recorded monthly and at the end of the studies.

BODY WEIGHT: Yes
- Time schedule for examinations: Weighed initially, weekly for 13 weeks, approximately monthly thereafter and again at the end of the studies

HAEMATOLOGY: No

CLINICAL CHEMISTRY: No

Sacrifice and pathology:

SACRIFICE: At the end of the 2-year study animals were sacrificed by carbon dioxide asphyxiation.

GROSS PATHOLOGY: Yes

HISTOPATHOLOGY: Yes
- Complete histopathology was performed on all the rats at the end of the study. In addition to gross lesions and tissue masses, the tissues examined were: adrenal gland, bone with marrow, brain, clitoral gland, esophagus, gallbladder (mice), heart with aorta, large intestine (cecum, colon and rectum), small intestine (duodenum, jejunum and 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 (site of application), spleen, stomach (forestomach and glandular), testis with epididymis and seminal vesicle, thymus, thyroid gland, trachea, urinary bladder, and uterus.
- 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:

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 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).

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

The only significant treatment-related clinical finding was mild to moderate irritation of the skin at the site of application in dosed males and females (males: vehicle control, 0/50; 50 mg/kg bw, 17/50; 100 mg/kg bw, 32/50; females: 3/50, 46/50, 50/50).

Dermal irritation (if dermal study):

effects observed, treatment-related

Description (incidence and severity):

The only significant treatment-related clinical finding was mild to moderate irritation of the skin at the site of application in dosed males and females (males: vehicle control, 0/50; 50 mg/kg bw, 17/50; 100 mg/kg bw, 32/50; females: 3/50, 46/50, 50/50).

Mortality:

no mortality observed

Description (incidence):

Survival of dosed male and female rats was similar to that of the vehicle control groups.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Mean body weights of 100 mg/kg bw males were slightly less than those of the vehicle control group throughout most of the study. Mean body weights of 100 mg/kg bw females were less than those of the vehicle controls from week 24 until the end of the study.

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:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

- Skin lesions: Minimal to moderate non-neoplastic lesions of the skin at the site of application were observed. The major alterations included (thickening of the epidermis, sebaceous gland and epidermal hyperplasia, hyperkeratosis, parakeratosis, chronic active dermal inflammation and ulcer) which were significantly increased in dosed males and females relative to the vehicle control.

- Forestomach lesions: No significant treatment related effects were observed. The increased incidence of hyperkeratosis and ulceration in 50 mg/kg bw/day males were not considered to be treatment related as these effects were not observed in females.

- Testis: No significant treatment related effects were observed.

- Thyroid gland lesions: No significant treatment related effects were observed.

Histopathological findings: neoplastic:

no effects observed

Description (incidence and severity):

- Skin lesions: No significant treatment related effects were observed. Few observed skin neoplasms (one subcutaneous fibroma in one vehicle control male and one subcutaneous fibrosarcoma in each of the 50 and 100 mg/kg bw/day male groups) were not considered to be significant as the incidences did not follow a pattern indicative of an association with the test substance.

- Testis: No significant treatment related effects were observed. Increased interstitial cell adenoma in males at 100 mg/kg bw/day was not considered significant as similar incidences were reported in vehicle controls in historical NTP dermal studies.

- Thyroid gland: No significant treatment related effects were observed. Marginal increase in the incidence of follicular cell adenoma or carcinoma observed in males at 50 mg/kg bw/day was not considered dose related and no follicular cell hyperplasias were observed .

Relevance of carcinogenic effects / potential:

No, there were no significant neoplasms associated with administration of the test substance.

Key result

Dose descriptor:

NOAEL

Remarks:

carcinogenicity

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no evidence of carcinogenic activity at any tested dose levels

Key result

Dose descriptor:

NOAEL

Remarks:

(systemic effects)

Effect level:

ca. 50 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: body weight changes at the LOAEL

Key result

Dose descriptor:

LOAEL

Remarks:

(local effects)

Effect level:

ca. 50 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: irritation and non-neoplastic lesion of the skin at both the tested doses

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

Conclusions:

Under the study conditions, no evidence of carcinogenic activity was observed with the test substance at any tested dose levels in rats.

Executive summary:

A study was conducted to evaluate the long-term repeated dose dermal carcinogenicity of the test substance, C18-unsatd. DEA, in compliance with GLP. Groups of 50 male and 50 female rats were dermally exposed to 0, 50 or 100 mg/kg bw/day in ethanol at a frequency of 5 d/week for a period of 104 weeks. Survival, clinical findings, body weight and histopathology of different organs were assessed at specific time intervals. 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. A dose dependent increase in irritation (mild to moderate) and non-neoplastic lesions (minimal to moderate) of the skin were observed at the site of application in all animals. 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. Under the study conditions, no evidence of carcinogenic activity was observed with the test substance at any tested dose levels in rats (Irwin, 1999).

Reference 5

Endpoint:

carcinogenicity: dermal

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Not available

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

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 material.

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

Duration of treatment / exposure:

104 wk

Frequency of treatment:

Five exposures per week

Post exposure period:

No

Dose / conc.:

0 mg/kg bw/day (nominal)

Dose / conc.:

50 mg/kg bw/day (nominal)

Remarks:

Corresponding to 85 mg/mL in ethanol

Dose / conc.:

100 mg/kg bw/day (nominal)

Remarks:

Corresponding to 170 mg/mL in ethanol

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

Description (incidence and severity):

The only clinical finding attributed to dosing was irritation of the skin at the site of application in 100 mg/kg bw females.

Dermal irritation (if dermal study):

effects observed, treatment-related

Description (incidence and severity):

The only clinical finding attributed to dosing was irritation of the skin at the site of application in 100 mg/kg bw females.

Mortality:

mortality observed, treatment-related

Description (incidence):

Survival rates of dosed male and female rats were similar to those of the vehicle controls.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

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

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

Description (incidence and severity):

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.

Histopathological findings: neoplastic:

effects observed, treatment-related

Description (incidence and severity):

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 bw group but only one neoplasm (an adenoma), in the 100 mg/kg bw group. Renal tubule neoplasms are uncommon in female F344/N rats, and the presence of four neoplasms in the 50 mg/kg bw 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 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.

Other effects:

effects observed, treatment-related

Description (incidence and severity):

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

Relevance of carcinogenic effects / potential:

Yes

Key result

Dose descriptor:

NOAEL

Based on:

other:

Sex:

male/female

Remarks on result:

not determinable

Remarks:

No NOAEL identified. 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 treatment uncertain.

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

Conclusions:

Under the study conditions, there was no evidence of carcinogenic activity of the test substance in male rats at any dose. 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 treatment uncertain.

Executive summary:

A study was conducted to evaluate the long-term repeated dose dermal carcinogenicity of the test substance, C8-18 and C18-unsatd. DEA, in compliance with GLP. Groups of 50 male and 50 female rats were exposed to 0, 50 or 100 mg/kg bw/day of the substance in ethanol by dermal application, 5 times per week, for 104 weeks. Mortality, clinical signs and bodyweight were recorded throughout the study. At necropsy, a gross macroscopic examination and complete histopathology were carried out. The survival rates of treated male and female rats were similar to those of controls. There were no significant differences in bodyweight throughout the groups. The only treatment-related clinical finding was irritation of the skin at the site of application in 100 mg/kg bw/day females. 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. There were marginal increases in the incidences of renal tubule adenoma or carcinoma (combined) in 50 mg/kg bw/day females. The severity of nephropathy increased with increasing dose in female rats. 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. Under the study conditions, there was no evidence of carcinogenic activity of the test substance in male rats at any dose. 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 treatment uncertain (Irwin, 2001).

Reference 6

Endpoint:

carcinogenicity: dermal

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Not available

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

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 mice to evaluate the carcinogenic potential of the test material.

GLP compliance:

yes

Species:

mouse

Strain:

B6C3F1

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 (PJ 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: 13 to 14 d

ENVIRONMENTAL CONDITIONS
- Temperature : 20.6-23.9°C
- Humidity : 30-67%
- Air changes : 10/hr
- Photoperiod : 12 h dark/12 h light

IN-LIFE DATES: From: Jan. 20, 1993 To: Jan. 20, 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 analyzed 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. All dose formulations analysed during the 2-yr studies were within 10% of the target concentration.

Duration of treatment / exposure:

104-105 wk

Frequency of treatment:

Five exposures per week

Post exposure period:

No

Dose / conc.:

0 mg/kg bw/day (nominal)

Dose / conc.:

100 mg/kg bw/day (nominal)

Remarks:

Corresponding to 50 mg/mL in ethanol

Dose / conc.:

200 mg/kg bw/day (nominal)

Remarks:

Corresponding to 100 mg/mL in ethanol

No. of animals per sex per dose:

50/sex/dose

Control animals:

yes, concurrent vehicle

Details on study design:

Dose selection rationale: Exposure to coconut oil acid diethanolamine condensate in the 14 wk study produced only a minimal toxic response in mice except in the skin at the site of application. The incidences of chronic active inflammation as well as several other skin lesions were significantly increased at doses of 200 mg/kg bw and greater in both male and female mice. The incidences of ulceration were increased in males exposed to 400 and 800 mg/kg bw and in females exposed to 800 mg/kg bw. Therefore, 400 and 800 mg/kg bw were considered inappropriate for a 2-year study. However, ulceration was present in only one 200 mg/kg bw male and no females, and the severities of these lesions in all affected groups were minimal to mild. Below 200 mg/kg bw, the incidences of skin lesions decreased markedly with a minor difference in response between 50 and 100 mg/kg bw. Therefore, 200 mg/kg bw was selected as the high dose and 100 mg/kg bw as the low 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 wk 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 mice. In addition to gross lesions and tissue masses, the following tissues were examined: adrenal gland, bone and marrow, brain, clitoral gland, esophagus, gallbladder, 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

Description (incidence and severity):

The only treatment-related clinical finding was irritation of the skin at the site of application in males that received 200 mg/kg bw.

Dermal irritation (if dermal study):

effects observed, treatment-related

Description (incidence and severity):

The only treatment-related clinical finding was irritation of the skin at the site of application in males that received 200 mg/kg bw.

Mortality:

mortality observed, treatment-related

Description (incidence):

The survival of dosed males and 100 mg/kg bw females was similar to that of the vehicle controls. Survival of the 200 mg/kg bw group of female mice was reduced compared to the vehicle control group, but the difference was not significant.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Mean body weights of dosed males were similar to those of the vehicle controls throughout most of the study; those of 100 and 200 mg/kg bw females were less than those of the vehicle controls from wk 93 and 77, respectively.

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

Description (incidence and severity):

Skin: Several nonneoplastic lesions of the skin at the site of application were determined to be chemical related. Incidences of epidermal hyperplasia, sebaceous gland hyperplasia, and hyperkeratosis in all dosed groups of males and females were significantly greater than those in the vehicle control groups. The incidences of ulceration in 200 mg/kg bw males and inflammation and parakeratosis in 200 mg/kg bw females were increased.

Histopathological findings: neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Liver: Dosed male and female mice had significantly greater incidences of hepatic neoplasms (hepatocellular adenoma, hepatocellular carcinoma, and hepatoblastoma (males) than the vehicle controls. There was a morphologic continuum from adenoma to carcinoma, with less differentiation and typical trabecular formations in the carcinomas. Carcinomas were often a centimeter or more in diameter, whereas adenomas were generally smaller and more discrete. Carcinomas metastasized to the lung in a few males and females. Adenomas, carcinomas, and hepatoblastomas displaced normal liver parenchyma, and none contained normal lobular architecture. Hepatoblastomas were characterized by well-demarcated focal areas composed of bundles of deeply basophilic, spindle-shaped cells.

Kidney: The incidences of renal tubule adenoma (1/50, 1/50, 7/50) and of renal tubule adenoma or carcinoma (combined) (1/50, 1/50, 9/50) in 200 mg/kg bw males were significantly greater than those in the vehicle controls. Renal tubule hyperplasia, adenoma, and carcinoma formed a morphological continuum. Adenomas were focal, compressive masses approximately five or more tubules in diameter; carcinomas were morphologically similar to adenomas but were larger and often showed cellular debris and/or mineralization. Renal tubule neoplasms were located in the cortex or outer medulla. Focal proliferative masses less than five tubules in diameter were classified as focal hyperplasia.

Thyroid Gland: The incidences of follicular cell hyperplasia in all dosed groups of males (vehicle control, 11/50; 100 mg/kg bw, 20/50; 200 mg/kg bw, 23/50) and females (27/50, 36/50, 33/50) were significantly greater than those in the vehicle controls. Follicular cell hyperplasia consisted of focal areas of thyroid gland follicles lined with increased numbers of epithelial cells, which formed papillary projections in some instances.

Relevance of carcinogenic effects / potential:

Yes

Key result

Dose descriptor:

NOAEL

Based on:

other:

Sex:

male/female

Remarks on result:

not determinable

Remarks:

No NOAEL identified as hepatic tumours seen in mice and the proposed mode of non-genotoxic mechanism for renal/liver tumours are not relevant to humans/primates.

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

Conclusions:

Under the test conditions, 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. These increases were associated with the the concentration of free diethanolamine present as a contaminant in the diethanolamine condensate. However, recent evidences suggest 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 renal/liver tumours are not relevant to humans/primates.

Executive summary:

A study was conducted to evaluate the long term repeated dose dermal carcinogenicity of the test substance, C8-18 and C18-unsatd. DEA, in compliance with GLP. The doses studied included 0, 100 and 200 mg/kg bw/day of test substance (corresponding to 0, 50, or 100 mg/mL in ethanol). Fifty male/female test animals were used in each group. Five exposures per week were administered for 104 to 105 weeks. The animals were observed twice daily, and body weights and clinical findings were recorded periodically. All animals were necropsied and complete histopathology was performed. Survival of dosed male and female mice was generally similar to that of the vehicle controls. The mean bodyweights of the 100 mg/kg bw/day females from week 93 and of the 200 mg/kg bw/day females from week 77 were lower than those of the vehicle controls. The only clinical finding attributed to treatment was an irritation of the skin at the site of application in males administered 200 mg/kg bw/day. The incidences of hepatic neoplasms (hepatocellular adenoma, hepatocellular carcinoma and hepatoblastoma) were significantly increased in male and/or female mice. The number of eosinophilic foci in dosed groups of male mice was higher than in the vehicle controls. The occurrences 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 gland hyperplasia and hyperkeratosis were greater in all dosed groups than in the vehicle controls and the number of thyroid gland follicular cell hyperplasia in all dosed groups was also significantly greater than those in the vehicle control groups. 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. These increases were associated with the concentration of free diethanolamine present as a contaminant in the test substance. However, recent evidence 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 renal/liver tumours are not relevant to humans/primates (Irwin, 2001).

Reference 7

Endpoint:

carcinogenicity: dermal

Type of information:

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

Adequacy of study:

key study

Study period:

Not available

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Remarks:

KL2 due to RA

Justification for type of information:

Refer to section 13 of IUCLID for details on the category justification.

Reason / purpose for cross-reference:

reference to other study

Principles of method if other than guideline:

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

GLP compliance:

yes

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: 6 wk
- Housing: Housed individually in Polycarbonate , changed weeklyand rotated every 2 wk
- Diet : NIH-07 open formula pelleted diet, ad libitum
- Water : Tap water via automatic watering system available cleaned every 2 wk, ad libitum
- Acclimation period: 13 d (males); 14 d (females)

ENVIRONMENTAL CONDITIONS
- Temperature : 20.5-26.1°C
- Humidity : 33-64%
- Air changes : 10/hr
- Photoperiod : 12 h dark/12 h light

IN-LIFE DATES: From: Dec. 30, 1992 To: Jan. 06, 1995

Route of administration:

dermal

Vehicle:

ethanol

Details on exposure:

VEHICLE
Purity: ranged from 97 to 103% relative to the reference standard

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Dose preparations were analysed approximately every 2 month using HPLC. All dose formulations analysed during the 2-yr studies were within 10% of the target concentration. In addition to dose formulation analysis prior to dosing, samples collected after dosing (animal room samples) were analysed periodically.

Duration of treatment / exposure:

105-106 wk

Frequency of treatment:

Five exposures per week

Dose / conc.:

0 mg/kg bw/day (nominal)

Dose / conc.:

100 mg/kg bw/day (nominal)

Remarks:

Corresponding to 50 mg/mL in ethanol

Dose / conc.:

200 mg/kg bw/day (nominal)

Remarks:

Corresponding to 100 mg/mL in ethanol

No. of animals per sex per dose:

50/sex/dose

Control animals:

yes, concurrent vehicle

Details on study design:

Dose selection rationale: Dose selection was based primarily on the increased incidences of a spectrum of skin lesions at the site of application. Doses of 400 or 800 mg/kg bw were associated with high incidences of chronic inflammation and ulceration and were thus considered to be inappropriate for a 2-yr study. A marked reduction in toxic response occurred at 200 mg/kg bw, and 100 mg/kg bw was a no-effect level in male mice. Therefore, 200 mg/kg bw was selected as the high dose for the 2-yr study and 100 mg/kg bw as the low dose.

Positive control:

No

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: At study initiation, at 4-wk intervals during the study, and at necropsy

DERMAL IRRITATION (if dermal study): Yes

BODY WEIGHT: Yes
- Time schedule for examinations: Animals were weighed initially at study initiation, weekly during weeks 1-13, at 4-wk intervals thereafter, and at the end of the study.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: No

CLINICAL CHEMISTRY: No

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:

METHOD OF SACRIFICE: Carbon dioxide asphyxiation

GROSS PATHOLOGY: Yes

HISTOPATHOLOGY: Yes, Complete histopathology was performed on all animals. 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.

Clinical signs:

no effects observed

Description (incidence and severity):

No significant difference in clinical findings was observed between the dosed groups and the vehicle control groups.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Description (incidence):

No significant difference in survival was observed between the dosed groups and the vehicle control groups.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

No significant difference was observed in the mean body weights between the dosed groups at 100 mg/kg bw and the vehicle control groups. The mean body weight of females that received 200 mg/kg bw was 94% that of the vehicle controls by week 33 and remained lower throughout the remainder of the study.

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:

no effects observed

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Liver: The incidences of hepatocellular adenoma or carcinoma (combined) were significantly increased in dosed female groups compared to that in the vehicle control group, as was the incidence of hepatocellular adenoma in 100 mg/kg bw females. These incidences exceeded the historical control ranges for these neoplasms. There were also increases in the incidences of eosinophilic foci in dosed female mice, and the increase was significant in females at 200 mg/kg bw. The incidences of hepatocellular adenoma, carcinoma, and adenoma or carcinoma (combined) were not significantly increased in dosed male mice. These increases were associated with free diethanolamine, which was present as a contaminant of lauric acid diethanolamine condensate.

Thyroid Gland: Incidences of focal hyperplasia of thyroid gland follicular cells were increased in dosed male mice; the incidence in the 200 mg/kg bw group was significantly greater than that in the vehicle control group. There were no corresponding increases in the incidences of follicular cell neoplasms.

Histopathological findings: neoplastic:

no effects observed

Description (incidence and severity):

Skin, Site of Application: Incidences of nonneoplastic lesions of the skin at the site of application were significantly increased in dosed males and females. The lesions were of minimal to moderate severity and consisted mostly of thickening of the epidermis (epidermal hyperplasia) and sebaceous gland hyperplasia. Compared to the vehicle controls, the incidences of chronic inflammation and hyperkeratosis were significantly greater in all groups of dosed males and females, and the incidences of parakeratosis were significantly greater in males and females administered 200 mg/kg bw. Ulcers occurred in a few mice at 200 mg/kg bw and were indicative of more severe local irritation.

Other effects:

not examined

Relevance of carcinogenic effects / potential:

Yes

Key result

Dose descriptor:

NOAEL

Sex:

male/female

Remarks on result:

not determinable

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

Conclusions:

Under the study conditions, no evidence of carcinogenic activity was reported in male mice. Some evidence of carcinogenic activity in female mice based on increased incidence of hepatocellular neoplasms was associated with free diethanolamine (DEA), which was present as a contaminant of the test substance.

Executive summary:

A study was conducted to evaluate the long-term repeated dose dermal carcinogenicity of the read across substance, C12 DEA, in compliance with GLP. Groups of 50 male and 50 female mice were exposed to dermal doses equivalent to 0, 100 or 200 mg/kg/day, 5 d/week, for 105-106 weeks. The animals were observed twice daily. Body weights and clinical findings were recorded periodically. Necropsy and complete histopathology was performed on all animals at test end. Mean body weights of females in 200 mg/kgbw/d dose group were lower than those of the vehicle controls beginning at week 33. The incidences of hepatocellular adenoma or carcinoma (combined) were significantly increased in treated females compared to the vehicle controls, as was the incidence of hepatocellular adenoma in the100 mg/kg bw/day female group. There were dose-related increases in the incidences of non-neoplastic lesions of the skin at the site of application, including epidermal and sebaceous gland hyperplasia, hyperkeratosis, chronic inflammation, and parakeratosis. Treated males had greater incidences of thyroid gland follicular cell focal hyperplasia than did the vehicle controls; the incidence in the 200 mg/kg bw/d group was significantly greater than that in the vehicle control group. Under the study conditions, no evidence of carcinogenic activity was reported in male mice. Some evidence of carcinogenic activity in female mice based on increased incidence of hepatocellular neoplasms was associated with free diethanolamine (DEA), which was present as a contaminant of the read across substance (Irwin, 1999). However, recent evidence 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 are not relevant to humans/primates.

Reference 8

Endpoint:

carcinogenicity: dermal

Type of information:

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

Adequacy of study:

key study

Study period:

Not available

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Remarks:

KL2 due to RA

Justification for type of information:

Refer to section 13 of IUCLID for details on the category justification.

Reason / purpose for cross-reference:

reference to other study

Principles of method if other than guideline:

A two-year dermal study was conducted in rats 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 Farms, Germantown, NY
- Age at study initiation: 6 wk
- Housing: Housed individually in Polycarbonate , changed weekly and rotated every 2 wk
- Diet : NIH-07 open formula pelleted diet, ad libitum
- Water : Tap water via automatic watering system, ad libitum
- Acclimation period: 11 d (males); 12 d (females)

ENVIRONMENTAL CONDITIONS
- Temperature : 21.1-26.7°C
- Humidity : 36-59%
- Air changes : 10/h
- Photoperiod : 12 h dark/12 h light

IN-LIFE DATES: From: Dec. 14, 1992 To: Dec. 14, 1994

Route of administration:

dermal

Vehicle:

ethanol

Details on exposure:

VEHICLE
Purity: ranged from 97% to 103% relative to the reference standard

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Dose preparations were analysed approximately every 2 m using HPLC. All dose formulations analysed during the 2-yr studies were within 10% of the target concentration. In addition to dose formulation analysis prior to dosing, samples collected after dosing (animal room samples) were analysed periodically.

Duration of treatment / exposure:

104-105 wk

Frequency of treatment:

Five exposures per week

Post exposure period:

No

Dose / conc.:

0 mg/kg bw/day (nominal)

Dose / conc.:

50 mg/kg bw/day (nominal)

Remarks:

Corresponding to 85 mg/mL in ethanol

Dose / conc.:

100 mg/kg bw/day (nominal)

Remarks:

Corresponding to 170 mg/mL in ethanol

No. of animals per sex per dose:

50

Control animals:

yes, concurrent vehicle

Details on study design:

Dose selection rationale: Dose selection was based primarily on the increased incidences of a spectrum of skin lesions at the site of application. Doses of 200 and 400 mg/kg bw caused high incidences of chronic inflammation and ulceration in males and females. Final mean body weights and body weight gains of male rats treated with 200 or 400 mg/kg bw were also less than those of the vehicle controls. Therefore, 200 and 400 mg/kg bw were considered inappropriate for the study. There was a very obvious reduction in toxic response of the skin at 100 mg/kg bw, with chronic inflammation and ulceration being absent in females and present in only one male; thus, 100 mg/kg bw was selected as the high dose for the 2-yr rat study. The responses observed at 25 and 50 mg/kg bw were very similar and consisted of only minimal hyperplasia; therefore, 50 mg/kg bw was selected as the low dose for the study.

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: At study initiation, at 4-wk intervals during the study, and at necropsy

DERMAL IRRITATION (if dermal study): Yes

BODY WEIGHT: Yes
- Time schedule for examinations: Animals were weighed initially at the study initiation, weekly during Weeks 1-13, at 4-wk intervals thereafter, and at the end of the study.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: No

CLINICAL CHEMISTRY: No

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:

Method of Sacrifice: Carbon dioxide asphyxiation

GROSS PATHOLOGY: Yes

HISTOPATHOLOGY: Yes, Complete histopathology was performed on all animals. 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.

Clinical signs:

no effects observed

Description (incidence and severity):

No significant difference clinical findings was observed between the dosed groups and the vehicle control groups.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Description (incidence):

No significant difference in survival was observed between the dosed groups and the vehicle control groups.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

No significant difference was observed in the mean body weights between the dosed groups and the vehicle control groups.

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:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Changes of minimal to moderate severity occurred in the skin at the site of application in treated male and female rats. The major alterations from normal skin were thickening of the epidermis (epidermal hyperplasia) and sebaceous gland hyperplasia (which usually occurred along with epidermal hyperplasia). Incidences of chronic inflammation, hyperkeratosis, and parakeratosis in all dosed groups were significantly greater than those in the vehicle controls, as were the incidences of ulceration in 100 mg/kg bw males and females. The nonneoplastic skin lesions at the site of application were considered to be indicative of local irritation with no neoplastic or preneoplastic changes.

Histopathological findings: neoplastic:

no effects observed

Description (incidence and severity):

No significant differences was reported between the vehicle control groups and dosed groups in the incidences of neoplasms.

Other effects:

not examined

Details on results:

OTHER FINDINGS: The incidence of forestomach ulcer was significantly lower in males that received 100 mg/kg bw/d than in the vehicle controls. The incidences of inflammation of the nasal mucosa were significantly lower in dosed males than in the vehicle controls. The incidence of chronic inflammation of the liver was significantly lower in females administered 100 mg/kg bw than in the vehicle controls.

Relevance of carcinogenic effects / potential:

Yes

Key result

Dose descriptor:

NOAEL

Effect level:

100 mg/kg bw/day (nominal)

Sex:

male/female

Basis for effect level:

other: Under the test conditions, no evidence of carcinogenic activity for the test substance was reported.

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

Conclusions:

Under the study conditions, no evidence of carcinogenic activity for the test substance was observed at any of the dose levels tested.

Executive summary:

A study was conducted to evaluate the long-term repeated dose dermal carcinogenicity of the read across substance, C12 DEA, in compliance with GLP. Groups of 50 male and 50 female rats were exposed to dermal doses equivalent to 0, 50 or 100 mg/kg/day, 5 d/week, during 104-105 weeks. The animals were observed twice daily. Body weights and clinical findings were recorded periodically. Necropsy and complete histopathology was performed on all animals at test end. There were no significant differences between vehicle control and dosed males or females in survival or mean body weights. There were no chemical-related differences in neoplasm incidences. Dose-related increases occurred in the incidences of non-neoplastic lesions of the skin at the site of application, including epidermal and sebaceous gland hyperplasia, hyperkeratosis, chronic inflammation, parakeratosis, and ulcer. Under the study conditions, no evidence of carcinogenic activity was observed at any of the dose levels tested (Irwin, 1999).

Reference 9

Endpoint:

carcinogenicity: dermal

Type of information:

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

Adequacy of study:

key study

Study period:

From 29 April 1993 to 10 May 1995

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Remarks:

KL2 due to RA

Justification for type of information:

Refer to section 13 of IUCLID for details on the category justification.

Reason / purpose for cross-reference:

reference to other study

Principles of method if other than guideline:

A 2 year toxicity study in B6C3F1 mice was conducted to evaluate the carcinogenic potential by the repeated dermal exposure to the test substance.

GLP compliance:

yes

Species:

mouse

Strain:

B6C3F1

Sex:

male/female

Details on test animals or test system and environmental conditions:

- Source: Taconic Laboratory Animals and Services (Germantown, NY)
- Age at study initiation: 6 wks
- 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 (PJ 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: 11 d and Females: 12 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: 21.1-25.0°C
- Relative humidity: 36-68%
- Air changes: 10/h
- Photoperiod: 12 h dark/12 h light

IN-LIFE DATES: From: 1993-04-29 To: 1995-05-10

Route of administration:

dermal

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.
The dose formulations were stored at room temperature, protected from light, in amber glass bottles for up to 28 days.

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 analysed every 9 weeks during the 2-year study and 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:

2-yr

Frequency of treatment:

5 exposures/wk

Post exposure period:

No

Dose / conc.:

0 mg/kg bw/day (actual dose received)

Dose / conc.:

15 mg/kg bw/day (actual dose received)

Dose / conc.:

30 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

50/sex/dose; 5 M/F (for 3-month interim evaluation in mice)

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose selection is based primarily on the incidences and severities of skin lesions observed at the site of application during the 13-week dermal studies. All groups of mice administered 100 mg/kg bw/day or greater exhibited high incidences of skin lesions at the site of application; thus, 100 mg/kg bw/day or greater were considered inappropriate for the 2-year study.
- The severities of the skin lesions increased with increasing dose in groups administered doses greater than 100 mg/kg bw/day; however, the severities of other lesions generally were increased only slightly between 100 and 800 mg/kg compared to the eightfold increase in dose. Therefore, the skin response appeared to plateau at 100 mg/kg, and higher doses did not produce a proportional increase in response. The incidences of skin lesions in groups administered 50 mg/kg bw/day were slightly less (minimal to mild) than those observed in groups administered 100 mg/kg bw/day. The skin response at the site of application in 50 mg/kg bw/day groups was such that 50 mg/kg bw/day was also considered inappropriate for a 2-year study; however, the slight reduction in incidences and the lower severities observed in the 50 mg/kg bw/day groups compared to those in the 100 mg/kg bw/day groups indicated that 50 mg/kg was below the plateau and at the upper end of a dose range in which skin response at the site of application exhibited a greater dose dependency. Therefore, at doses below 50 mg/kg bw/day, a proportional reduction in incidences and severities of skin lesions at the site of application would be expected. Accordingly, a high dose of 30 mg/kg and a low dose of 15 mg/kg were selected for the 2-year study in mice.

Positive control:

Not included in this study

Observations and examinations performed and frequency:

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Clinical findings were recorded monthly and at the end of the studies.

BODY WEIGHT: Yes
- Time schedule for examinations: Weighed initially, weekly for 13 wks, approximately monthly thereafter and again at the end of the studies

DERMAL IRRITATION (if dermal study): Yes

HAEMATOLOGY: No

CLINICAL CHEMISTRY: No

Sacrifice and pathology:

SACRIFICE: At the end of 3rd month 5 males and females were sacrificed for skin evaluation and at the end of the 2-year study all animals were sacrificed by carbon dioxide asphyxiation.

GROSS PATHOLOGY: Yes

HISTOPATHOLOGY: Yes
- Skin from the site of application was examined from all mice at the 3-month interim evaluation.
- Complete histopathology was performed on all the rats at the end of the study. In addition to gross lesions and tissue masses, the tissues examined were: adrenal gland, bone with marrow, brain, clitoral gland, esophagus, gallbladder (mice), heart with aorta, large intestine (cecum, colon, and rectum), small intestine (duodenum, jejunum and 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 (site of application), spleen, stomach (forestomach and glandular), testis with epididymis and seminal vesicle, thymus, thyroid gland, trachea, urinary bladder, and uterus.
- 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:

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 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).

Clinical signs:

no effects observed

Description (incidence and severity):

The only significant treatment-related clinical finding was irritation of the skin at the site of application in 30 mg/kg bw males (vehicle control, 0/55; 15 mg/kg bw, 1/55; 30 mg/kg bw, 20/55).

Dermal irritation (if dermal study):

effects observed, treatment-related

Description (incidence and severity):

The only significant treatment-related clinical finding was irritation of the skin at the site of application in 30 mg/kg bw males (vehicle control, 0/55; 15 mg/kg bw, 1/55; 30 mg/kg bw, 20/55).

Mortality:

no mortality observed

Description (incidence):

Survival of dosed male and female mice was similar to that of the vehicle control groups.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Mean body weights of dosed males and 15 mg/kg bw females were similar to those of the vehicle controls throughout the study. Mean body weights of 30 mg/kg bw females were less than those of the vehicle controls beginning week 76.

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:

not examined

Gross pathological findings:

no effects observed

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Skin lesions: Minimal to mild non-neoplastic lesions of the skin at the site of application were observed in females more than 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 at 2 years. There was increased incidence of hyperkeratosis in dosed males at 3 months and in dosed males and females at 2-years. The increased incidences of parakeratosis in 30 mg/kg bw/day males at the 3-month interim evaluation and at 2-years and 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.

Histopathological findings: neoplastic:

no effects observed

Description (incidence and severity):

- Skin lesions: No significant treatment related effects were observed. Few neoplastic lesions observed (i.e., one fibrosarcoma in vehicle control female and two fibrosarcomas in females at 15 mg/kg bw/day) were not considered significant as they did not follow a dose dependent pattern.

- Malignant lymphoma: No significant treatment related effects were observed. The increased incidence of the lymphomas observed in females at 30 mg/kg bw/day was found to be similar to the incidences observed in the other dermal studies with ethanol as the vehicle (i.e., comparable to historical control values).

Other effects:

not examined

Relevance of carcinogenic effects / potential:

No, there were no significant neoplasms associated with administration of the test substance.

Key result

Dose descriptor:

NOAEL

Remarks:

carcinogenicity

Effect level:

ca. 30 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no evidence of carcinogenic activity at any tested dose levels

Key result

Dose descriptor:

NOAEL

Remarks:

(systemic effects)

Effect level:

ca. 15 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: body weight changes at 30 mg/kg bw/d

Key result

Dose descriptor:

LOAEL

Remarks:

(local effects)

Effect level:

ca. 15 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: incidences of non-neoplastic lesions of the skin

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

Conclusions:

Under the study conditions, no evidence of carcinogenic activity was observed at any tested dose levels in mice.

Executive summary:

A study was conducted to evaluate the long-term repeated dose dermal carcinogenicity of the read across substance, C18-unsatd. DEA, in compliance with GLP. Groups of 50 male and 50 female mice were dermally exposed to 0, 15 or 30 mg/kg bw/day in ethanol at a frequency of 5 d/week for a period of 105 weeks. 5 males and 5 females were considered for the 3 month interim assessment. Survival, clinical findings, body weight and histopathology were evaluated at specific time intervals. Survival of the dosed male and female rats 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 at 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 read across substance administration. No significant neoplastic lesions or evidence of carcinogenic activity was observed at any tested dose levels in skin and lymph nodes. Under the study conditions, no evidence of carcinogenic activity was observed at any tested dose levels in mice. (Irwin, 1999).

Reference 10

Endpoint:

carcinogenicity: dermal

Type of information:

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

Adequacy of study:

key study

Study period:

From 06 May 1993 to 16 May 1995

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Justification for type of information:

Refer to section 13 of IUCLID for details on the category justification.

Reason / purpose for cross-reference:

reference to other study

Principles of method if other than guideline:

A 2-year toxicity study in F344/N rats was conducted to evaluate the carcinogenic potential by the repeated dermal exposure to the test substance.

GLP compliance:

yes

Species:

rat

Strain:

Fischer 344

Sex:

male/female

Details on test animals or test system and environmental conditions:

- Source: Taconic Laboratory Animals and Services (Germantown, NY)
- Age at study initiation: 7 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 (PJ 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: 13 d and Females: 14 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: 21.1-23.3°C
- Relative humidity: 31-73%
- Air changes: 10/h
- Photoperiod: 12 h dark/12 h light

IN-LIFE DATES: From: 1993-05-06 To: 1995-05-16

Route of administration:

dermal

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.
The dose formulations were stored at room temperature, protected from light, in amber glass bottles for up to 28 days.

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 analysed every 9 weeks during the 2-year study and 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:

2-yrs

Frequency of treatment:

5 exposures/wk

Post exposure period:

No

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)

No. of animals per sex per dose:

50/sex/dose

Control animals:

yes, concurrent vehicle

Details on study design:

Dose selection rationale: The dose selection was based primarily on the incidences and severities of skin lesions observed at the site of application during the 13-week dermal studies. The doses of 200 and 400 mg/kg bw/day exhibited reduced mean body weight and body weight gain along with high incidences of skin lesions at the site of application; thus were considered inappropriate for a 2-year study. Further, lesions of the skin were also present at the site of application in groups administered 100 mg/kg bw/day; however, the incidence were less than those observed in the 200 and 400 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. Therefore, 100 mg/kg bw/day was selected as the high dose for rats in the 2-year study. In groups treated with 50 mg/kg bw/day, the incidences of skin lesions diminished considerably and lesion severities were minimal. Therefore, 50 mg/kg bw/day was selected as the low dose.

Positive control:

No

Observations and examinations performed and frequency:

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Clinical findings were recorded monthly and at the end of the studies.

BODY WEIGHT: Yes
- Time schedule for examinations: Weighed initially, weekly for 13 weeks, approximately monthly thereafter and again at the end of the studies

HAEMATOLOGY: No

CLINICAL CHEMISTRY: No

Sacrifice and pathology:

SACRIFICE: At the end of the 2-year study animals were sacrificed by carbon dioxide asphyxiation.

GROSS PATHOLOGY: Yes

HISTOPATHOLOGY: Yes
- Complete histopathology was performed on all the rats at the end of the study. In addition to gross lesions and tissue masses, the tissues examined were: adrenal gland, bone with marrow, brain, clitoral gland, esophagus, gallbladder (mice), heart with aorta, large intestine (cecum, colon and rectum), small intestine (duodenum, jejunum and 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 (site of application), spleen, stomach (forestomach and glandular), testis with epididymis and seminal vesicle, thymus, thyroid gland, trachea, urinary bladder, and uterus.
- 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:

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 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).

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

The only significant treatment-related clinical finding was mild to moderate irritation of the skin at the site of application in dosed males and females (males: vehicle control, 0/50; 50 mg/kg bw, 17/50; 100 mg/kg bw, 32/50; females: 3/50, 46/50, 50/50).

Dermal irritation (if dermal study):

effects observed, treatment-related

Description (incidence and severity):

The only significant treatment-related clinical finding was mild to moderate irritation of the skin at the site of application in dosed males and females (males: vehicle control, 0/50; 50 mg/kg bw, 17/50; 100 mg/kg bw, 32/50; females: 3/50, 46/50, 50/50).

Mortality:

no mortality observed

Description (incidence):

Survival of dosed male and female rats was similar to that of the vehicle control groups.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Mean body weights of 100 mg/kg bw males were slightly less than those of the vehicle control group throughout most of the study. Mean body weights of 100 mg/kg bw females were less than those of the vehicle controls from week 24 until the end of the study.

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:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

- Skin lesions: Minimal to moderate non-neoplastic lesions of the skin at the site of application were observed. The major alterations included (thickening of the epidermis, sebaceous gland and epidermal hyperplasia, hyperkeratosis, parakeratosis, chronic active dermal inflammation and ulcer) which were significantly increased in dosed males and females relative to the vehicle control.

- Forestomach lesions: No significant treatment related effects were observed. The increased incidence of hyperkeratosis and ulceration in 50 mg/kg bw/day males were not considered to be treatment related as these effects were not observed in females.

- Testis: No significant treatment related effects were observed.

- Thyroid gland lesions: No significant treatment related effects were observed.

Histopathological findings: neoplastic:

no effects observed

Description (incidence and severity):

- Skin lesions: No significant treatment related effects were observed. Few observed skin neoplasms (one subcutaneous fibroma in one vehicle control male and one subcutaneous fibrosarcoma in each of the 50 and 100 mg/kg bw/day male groups) were not considered to be significant as the incidences did not follow a pattern indicative of an association with the test substance.

- Testis: No significant treatment related effects were observed. Increased interstitial cell adenoma in males at 100 mg/kg bw/day was not considered significant as similar incidences were reported in vehicle controls in historical NTP dermal studies.

- Thyroid gland: No significant treatment related effects were observed. Marginal increase in the incidence of follicular cell adenoma or carcinoma observed in males at 50 mg/kg bw/day was not considered dose related and no follicular cell hyperplasias were observed .

Relevance of carcinogenic effects / potential:

No, there were no significant neoplasms associated with administration of the test substance.

Key result

Dose descriptor:

NOAEL

Remarks:

carcinogenicity

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no evidence of carcinogenic activity at any tested dose levels

Key result

Dose descriptor:

NOAEL

Remarks:

(systemic effects)

Effect level:

ca. 50 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: body weight changes at the LOAEL

Key result

Dose descriptor:

LOAEL

Remarks:

(local effects)

Effect level:

ca. 50 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: irritation and non-neoplastic lesion of the skin at both the tested doses

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

Conclusions:

Under the study conditions, no evidence of carcinogenic activity was observed with the test substance at any tested dose levels in rats.

Executive summary:

A study was conducted to evaluate the long-term repeated dose dermal carcinogenicity of the read across substance, C18-unsatd. DEA, in compliance with GLP. Groups of 50 male and 50 female rats were dermally exposed to 0, 50 or 100 mg/kg bw/day in ethanol at a frequency of 5 d/week for a period of 104 weeks. Survival, clinical findings, body weight and histopathology of different organs were assessed at specific time intervals. 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. A dose dependent increase in irritation (mild to moderate) and non-neoplastic lesions (minimal to moderate) of the skin were observed at the site of application in all animals. 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. Under the study conditions, no evidence of carcinogenic activity was observed at any tested dose levels in rats (Irwin, 1999).

Reference 11

Endpoint:

carcinogenicity: dermal

Type of information:

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

Adequacy of study:

key study

Study period:

Not available

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Justification for type of information:

Refer to the section 13 for details on the category justification.

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 material.

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

Duration of treatment / exposure:

104 wk

Frequency of treatment:

Five exposures per week

Post exposure period:

No

Dose / conc.:

0 mg/kg bw/day (nominal)

Dose / conc.:

50 mg/kg bw/day (nominal)

Remarks:

Corresponding to 85 mg/mL in ethanol

Dose / conc.:

100 mg/kg bw/day (nominal)

Remarks:

Corresponding to 170 mg/mL in ethanol

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

Description (incidence and severity):

The only clinical finding attributed to dosing was irritation of the skin at the site of application in 100 mg/kg bw females.

Dermal irritation (if dermal study):

effects observed, treatment-related

Description (incidence and severity):

The only clinical finding attributed to dosing was irritation of the skin at the site of application in 100 mg/kg bw females.

Mortality:

mortality observed, treatment-related

Description (incidence):

Survival rates of dosed male and female rats were similar to those of the vehicle controls.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

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

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

Description (incidence and severity):

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.

Histopathological findings: neoplastic:

effects observed, treatment-related

Description (incidence and severity):

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 bw group but only one neoplasm (an adenoma), in the 100 mg/kg bw group. Renal tubule neoplasms are uncommon in female F344/N rats, and the presence of four neoplasms in the 50 mg/kg bw 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 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.

Other effects:

effects observed, treatment-related

Description (incidence and severity):

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

Key result

Dose descriptor:

NOAEL

Based on:

other:

Sex:

male/female

Remarks on result:

not determinable

Remarks:

No NOAEL identified.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 treatment uncertain.

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

Conclusions:

Under the study conditions, there was no evidence of carcinogenic activity in male rats at any dose. 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 treatment uncertain.

Executive summary:

A study was conducted to evaluate the carcinogenicity potential of the read across substance, C8-18 and C18-unsatd. DEA, in compliance with GLP. Groups of 50 male and 50 female rats were exposed to 0, 50 or 100 mg/kg bw/day of the substance in ethanol by dermal application, 5 times per week, for 104 weeks. Mortality, clinical signs and bodyweight were recorded throughout the study. At necropsy, a gross macroscopic examination and complete histopathology were carried out. The survival rates of treated male and female rats were similar to those of controls. There were no significant differences in bodyweight throughout the groups. The only treatment-related clinical finding was irritation of the skin at the site of application in 100 mg/kg bw/day females. 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. There were marginal increases in the incidences of renal tubule adenoma or carcinoma (combined) in 50 mg/kg bw/day females. The severity of nephropathy increased with increasing dose in female rats. 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. Under the study conditions, there was no evidence of carcinogenic activity in male rats at any dose. 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 treatment uncertain (Irwin, 2001).

Reference 12

Endpoint:

carcinogenicity: dermal

Type of information:

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

Adequacy of study:

key study

Study period:

Not available

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Justification for type of information:

Refer to the section 13 for details on the category justification.

Reason / purpose for cross-reference:

reference to other study

Principles of method if other than guideline:

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

GLP compliance:

yes

Species:

mouse

Strain:

B6C3F1

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 (PJ 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: 13 to 14 d

ENVIRONMENTAL CONDITIONS
- Temperature : 20.6-23.9°C
- Humidity : 30-67%
- Air changes : 10/hr
- Photoperiod : 12 h dark/12 h light

IN-LIFE DATES: From: Jan. 20, 1993 To: Jan. 20, 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 analyzed 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. All dose formulations analysed during the 2 yr studies were within 10% of the target concentration.

Duration of treatment / exposure:

104-105 wk

Frequency of treatment:

Five exposures per week

Post exposure period:

No

Dose / conc.:

0 mg/kg bw/day (nominal)

Dose / conc.:

100 mg/kg bw/day (nominal)

Remarks:

Corresponding to 50 mg/mL in ethanol

Dose / conc.:

200 mg/kg bw/day (nominal)

Remarks:

Corresponding to 100 mg/mL in ethanol

No. of animals per sex per dose:

50/sex/dose

Control animals:

yes, concurrent vehicle

Details on study design:

Dose selection rationale: Exposure to coconut oil acid diethanolamine condensate in the 14 wk study produced only a minimal toxic response in mice except in the skin at the site of application. The incidences of chronic active inflammation as well as several other skin lesions were significantly increased at doses of 200 mg/kg bw and greater in both male and female mice. The incidences of ulceration were increased in males exposed to 400 and 800 mg/kg bw and in females exposed to 800 mg/kg bw. Therefore, 400 and 800 mg/kg bw were considered inappropriate for a 2-year study. However, ulceration was present in only one 200 mg/kg bw male and no females, and the severities of these lesions in all affected groups were minimal to mild. Below 200 mg/kg bw, the incidences of skin lesions decreased markedly with a minor difference in response between 50 and 100 mg/kg bw. Therefore, 200 mg/kg bw was selected as the high dose and 100 mg/kg bw as the low 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 wk 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 mice. In addition to gross lesions and tissue masses, the following tissues were examined: adrenal gland, bone and marrow, brain, clitoral gland, esophagus, gallbladder, 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

Description (incidence and severity):

The only treatment-related clinical finding was irritation of the skin at the site of application in males that received 200 mg/kg bw.

Dermal irritation (if dermal study):

effects observed, treatment-related

Description (incidence and severity):

The only treatment-related clinical finding was irritation of the skin at the site of application in males that received 200 mg/kg bw.

Mortality:

mortality observed, treatment-related

Description (incidence):

The survival of dosed males and 100 mg/kg bw females was similar to that of the vehicle controls. Survival of the 200 mg/kg bw group of female mice was reduced compared to the vehicle control group, but the difference was not significant.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Mean body weights of dosed males were similar to those of the vehicle controls throughout most of the study; those of 100 and 200 mg/kg bw females were less than those of the vehicle controls from wk 93 and 77, respectively.

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

Description (incidence and severity):

Skin: Several nonneoplastic lesions of the skin at the site of application were determined to be chemical related. Incidences of epidermal hyperplasia, sebaceous gland hyperplasia, and hyperkeratosis in all dosed groups of males and females were significantly greater than those in the vehicle control groups. The incidences of ulceration in 200 mg/kg bw males and inflammation and parakeratosis in 200 mg/kg bw females were increased.

Histopathological findings: neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Liver: Dosed male and female mice had significantly greater incidences of hepatic neoplasms (hepatocellular adenoma, hepatocellular carcinoma, and hepatoblastoma (males) than the vehicle controls. There was a morphologic continuum from adenoma to carcinoma, with less differentiation and typical trabecular formations in the carcinomas. Carcinomas were often a centimeter or more in diameter, whereas adenomas were generally smaller and more discrete. Carcinomas metastasized to the lung in a few males and females. Adenomas, carcinomas, and hepatoblastomas displaced normal liver parenchyma, and none contained normal lobular architecture. Hepatoblastomas were characterized by well-demarcated focal areas composed of bundles of deeply basophilic, spindle-shaped cells.

Kidney: The incidences of renal tubule adenoma (1/50, 1/50, 7/50) and of renal tubule adenoma or carcinoma (combined) (1/50, 1/50, 9/50) in 200 mg/kg bw males were significantly greater than those in the vehicle controls. Renal tubule hyperplasia, adenoma, and carcinoma formed a morphological continuum. Adenomas were focal, compressive masses approximately five or more tubules in diameter; carcinomas were morphologically similar to adenomas but were larger and often showed cellular debris and/or mineralization. Renal tubule neoplasms were located in the cortex or outer medulla. Focal proliferative masses less than five tubules in diameter were classified as focal hyperplasia.

Thyroid Gland: The incidences of follicular cell hyperplasia in all dosed groups of males (vehicle control, 11/50; 100 mg/kg bw, 20/50; 200 mg/kg bw, 23/50) and females (27/50, 36/50, 33/50) were significantly greater than those in the vehicle controls. Follicular cell hyperplasia consisted of focal areas of thyroid gland follicles lined with increased numbers of epithelial cells, which formed papillary projections in some instances.

Relevance of carcinogenic effects / potential:

Yes

Key result

Dose descriptor:

NOAEL

Based on:

other:

Sex:

male/female

Remarks on result:

not determinable

Remarks:

No NOAEL identified as hepatic tumours seen in mice and the proposed mode of non-genotoxic mechanism for renal/liver tumours are not relevant to humans/primates.

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

Conclusions:

Under the test conditions, 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. These increases were associated with the the concentration of free diethanolamine present as a contaminant in the diethanolamine condensate. However, recent evidences suggest 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 renal/liver tumours are not relevant to humans/primates.

Executive summary:

A study was conducted to evaluate the long-term repeated dose dermal carcinogenicity of the read across substance, C8-18 and C18-unsatd. DEA, in compliance with GLP. The doses studied included 0, 100 and 200 mg/kg bw/day of test substance (corresponding to 0, 50, or 100 mg/mL in ethanol). Fifty male/female test animals were used in each group. Five exposures per week were administered for 104 to 105 weeks. The animals were observed twice daily, and body weights and clinical findings were recorded periodically. All animals were necropsied and complete histopathology was performed. Survival of dosed male and female mice was generally similar to that of the vehicle controls. The mean bodyweights of the 100 mg/kg bw/day females from week 93 and of the 200 mg/kg bw/day females from week 77 were lower than those of the vehicle controls. The only clinical finding attributed to treatment was an irritation of the skin at the site of application in males administered 200 mg/kg bw/day. The incidences of hepatic neoplasms (hepatocellular adenoma, hepatocellular carcinoma and hepatoblastoma) were significantly increased in male and/or female mice. The number of eosinophilic foci in dosed groups of male mice was higher than in the vehicle controls. The occurrences 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 gland hyperplasia and hyperkeratosis were greater in all dosed groups than in the vehicle controls and the number of thyroid gland follicular cell hyperplasia in all dosed groups was also significantly greater than those in the vehicle control groups. 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. These increases were associated with the concentration of free diethanolamine present as a contaminant in the test substance. However, recent evidence 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 renal/liver tumours are not relevant to humans/primates (Irwin, 2001).

Endpoint conclusion

Endpoint conclusion:

no study available

Carcinogenicity: via inhalation route

Endpoint conclusion

Endpoint conclusion:

no study available

Carcinogenicity: via dermal route

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Study duration:

chronic

Justification for classification or non-classification

The available data suggests that the read across substance is not carcinogenic to humans. Although studies provided evidence of carcinogenicity in mice, it was concluded that this was due to the presence of a contaminant - free diethanolamine (DEA, EC no. 203-868-0, CAS no. 111-42-2) - in the test substance. DEA has been shown to be a non-genotoxic carcinogen in mice. However, the proposed mode of action indicates a rodent-specific phenomenon involving choline depletion which is unlikely to be relevant to man. This evaluation is supported by a 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. DEA has also recently been reviewed in the EU under the REACH regulation and no classification for carcinogenicity has been proposed (https://echa.europa.eu/registration-dossier/-/registered-dossier/15770/2/1/?documentUUID=b015c4bf-61f0-4b7b-b858-cfe6f686f088). Therefore, based on the overall weight of evidence of the available information, C12-18 and C18-unsatd. DEA does not require classification for carcinogenicity according to CLP (EC 1272/2008) criteria.

Additional information

A study was conducted to evaluate the long-term repeated dose dermal carcinogenicity of the read across substance, C8-18 and C18-unsatd. DEA, in B6C3F1 mice, in compliance with GLP. The doses studied included 0, 100 and 200 mg/kg bw/day of test substance (corresponding to 0, 50, or 100 mg/mL in ethanol). Fifty male/female test animals were used in each group. Five exposures per week were administered for 104 to 105 weeks. The animals were observed twice daily, and body weights and clinical findings were recorded periodically. All animals were necropsied and complete histopathology was performed. Survival of dosed male and female mice was generally similar to that of the vehicle controls. The mean bodyweights of the 100 mg/kg bw/day females from week 93 and of the 200 mg/kg bw/day females from week 77 were lower than those of the vehicle controls. The only clinical finding attributed to treatment was an irritation of the skin at the site of application in males administered 200 mg/kg bw/day. The incidences of hepatic neoplasms (hepatocellular adenoma, hepatocellular carcinoma and hepatoblastoma) were significantly increased in male and/or female mice. The number of eosinophilic foci in dosed groups of male mice was higher than in the vehicle controls. The occurrences 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 gland hyperplasia and hyperkeratosis were greater in all dosed groups than in the vehicle controls and the number of thyroid gland follicular cell hyperplasia in all dosed groups was also significantly greater than those in the vehicle control groups. 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. These increases were associated with the concentration of free diethanolamine present as a contaminant in the test substance. However, recent evidences suggest 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 renal/liver tumours are not relevant to humans/primates (Irwin, 2001).  

 

A study was conducted to evaluate the long-term repeated dose dermal carcinogenicity of the read across substance, C8-18 and C18-unsatd. DEA, in Fischer 344 rats, in compliance with GLP. Groups of 50 male and 50 female rats were exposed to 0, 50 or 100 mg/kg bw/day of the substance in ethanol by dermal application, 5 times per week, for 104 weeks. Mortality, clinical signs and bodyweight were recorded throughout the study. At necropsy, a gross macroscopic examination and complete histopathology were carried out. The survival rates of treated male and female rats were similar to those of controls. There were no significant differences in bodyweight throughout the groups. The only treatment-related clinical finding was irritation of the skin at the site of application in 100 mg/kg bw/day females. 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. There were marginal increases in the incidences of renal tubule adenoma or carcinoma (combined) in 50 mg/kg bw/day females. The severity of nephropathy increased with increasing dose in female rats. 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. Under the study conditions, there was no evidence of carcinogenic activity of the test substance in male rats at any dose. 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 treatment uncertain (Irwin, 2001).  

  

A study was conducted to evaluate the long-term repeated dose dermal carcinogenicity of the read across substance, C12 DEA, in compliance with GLP. Groups of 50 male and 50 female mice were exposed to dermal doses equivalent to 0, 100 or 200 mg/kg/day, 5 d/week, for 105-106 weeks. The animals were observed twice daily. Body weights and clinical findings were recorded periodically. Necropsy and complete histopathology was performed on all animals at test end. Mean body weights of females in 200 mg/kg bw/d dose group were lower than those of the vehicle controls beginning at week 33. The incidences of hepatocellular adenoma or carcinoma (combined) were significantly increased in treated females compared to the vehicle controls, as was the incidence of hepatocellular adenoma in the100 mg/kg bw/day female group. There were dose-related increases in the incidences of non-neoplastic lesions of the skin at the site of application, including epidermal and sebaceous gland hyperplasia, hyperkeratosis, chronic inflammation, and parakeratosis. Treated males had greater incidences of thyroid gland follicular cell focal hyperplasia than did the vehicle controls; the incidence in the 200 mg/kg bw/d group was significantly greater than that in the vehicle control group. Under the study conditions, no evidence of carcinogenic activity was reported in male mice. Some evidence of carcinogenic activity in female mice based on increased incidence of hepatocellular neoplasms was associated with free diethanolamine (DEA), which was present as a contaminant of the test substance (Irwin, 1999). However, recent evidences suggest that DEA should not be classified as a carcinogen, as the hepatic tumours seen in mice and the proposed mode of non-genotoxic mechanism are not relevant to humans/primates.  

 

A study was conducted to evaluate the long-term repeated dose dermal carcinogenicity of the read across substance, C12 DEA, in compliance with GLP. Groups of 50 male and 50 female rats were exposed to dermal doses equivalent to 0, 50 or 100 mg/kg/day, 5 d/week, during 104-105 weeks. The animals were observed twice daily. Body weights and clinical findings were recorded periodically. Necropsy and complete histopathology was performed on all animals at test end. There were no significant differences between vehicle control and dosed males or females in survival or mean body weights. There were no chemical-related differences in neoplasm incidences. Dose-related increases occurred in the incidences of non-neoplastic lesions of the skin at the site of application, including epidermal and sebaceous gland hyperplasia, hyperkeratosis, chronic inflammation, parakeratosis, and ulcer. Under the study conditions, no evidence of carcinogenic activity for the test substance was observed at any of the dose levels tested (Irwin, 1999).  

  

A study was conducted to evaluate the long-term repeated dose dermal carcinogenicity of the read across substance, C18-unsatd. DEA, in compliance with GLP. Groups of 50 male and 50 female mice were dermally exposed to 0, 15 or 30 mg/kg bw/day in ethanol at a frequency of 5 d/week for a period of 105 weeks. 5 males and 5 females were considered for the 3 month interim assessment. Survival, clinical findings, body weight and histopathology were evaluated at specific time intervals. Survival of the dosed male and female rats 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 at 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 levels in skin and lymph nodes. Under the study conditions, no evidence of carcinogenic activity was observed at any tested dose levels in mice. (Irwin, 1999). 

 

A study was conducted to evaluate the long-term repeated dose dermal carcinogenicity of the read across substance, C18-unsatd. DEA, in compliance with GLP. Groups of 50 male and 50 female rats were dermally exposed to 0, 50 or 100 mg/kg bw/day in ethanol at a frequency of 5 d/week for a period of 104 weeks. Survival, clinical findings, body weight and histopathology of different organs were assessed at specific time intervals. 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. A dose dependent increase in irritation (mild to moderate) and non-neoplastic lesions (minimal to moderate) of the skin were observed at the site of application in all animals. 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. Under the study conditions, no evidence of carcinogenic activity was observed with the test substance at any tested dose levels in rats (Irwin, 1999).