6,6',6''-(1,3,5-triazine-2,4,6-triyltriimino)trihexanoic acid, compound with 2,2',2''-nitrilotriethanol (1:3)

Carcinogenicity

Administrative data

Endpoint:

carcinogenicity: dermal

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Well documented study report, acceptable for assessment

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1999

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 451 (Carcinogenicity Studies)

GLP compliance:

yes

Test material

Test material information

Constituent 1

Details on test material:

- Name of test material (as cited in study report): Triethanolamine- Physical state: a clear, colorless, viscous liquid- Analytical purity: approximately 99%- Lot/batch No.: 7G-60 from Texaco Chemical Company (Division of Texaco, Inc., Bellaire, TX)

Test animals

Species:

rat

Strain:

Fischer 344

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Simonsen Laboratories, Inc. (Gilroy, CA)
- Age at study initiation: 6 weeks
- Weight at study initiation: male 21 gram / female 17.5 gram
- Housing: Polycarbonate (Lab Products, Inc., Maywood, NJ), changed weekly
- Diet (e.g. ad libitum): NIH-07 open formula pelleted diet (Zeigler Brothers, Inc., Gardners, PA), available ad libitum, changed weekly
- Water (e.g. ad libitum): Tap water (City of Columbus municipal supply) via automatic watering system (Edstrom Industries, Waterford, WI), available ad libitum
- Acclimation period: 11 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.0 - 23.9
- Humidity (%): 35 - 76
- Air changes (per hr): minimum of 10
- Photoperiod (hrs dark / hrs light): 12 / 12


IN-LIFE DATES: From: 14 November 1988 To: 2 November 1990

Administration / exposure

Route of administration:

dermal

Vehicle:

acetone

Details on exposure:

TEST SITE
- Area of exposure: area extending from the animal’s mid-back to the intrascapular region
- Time intervals for shavings or clipplings: approximately once per week

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Periodic analyses of dose formulations of triethanolamine were conducted by the study laboratory and the analytical chemistry laboratory with gas chromatography. The dose formulations were analyzed at the beginning of the study and every 6 to 10 weeks thereafter; animal-room samples were analyzed every 22 to 26 weeks. Of the doses analyzed, 95% (59/62) of the formulations were within 10% of the target concentrations. All animal-room samples were within 10% of the target concentrations. Results of periodic referee analyses performed by the analytical chemistry laboratory agreed with the results obtained by the study laboratory.

Duration of treatment / exposure:

103 weeks

Frequency of treatment:

5 days per week

Post exposure period:

None

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 32, 63, or 125 mg/kg bw/day (males) and 0, 63, 125, or 250 mg/kg bw/day (females)
Basis:

No. of animals per sex per dose:

60

Control animals:

yes, concurrent vehicle

Details on study design:

Dermal application was chosen as the route of exposure to mimic the principal means of human exposure to triethanolamine and because considerable systemic exposure is achieved with this route.

Positive control:

None

Examinations

Observations and examinations performed and frequency:

Observed twice daily; clinical findings were recorded monthly. Animals were weighed initially, weekly for 13 weeks, monthly thereafter, and at the end of the studies.

Sacrifice and pathology:

Necropsy performed on all animals. Organs weighed were left and right kidneys and liver.
Complete histopathology was performed on all rats. In addition to gross lesions and tissue masses, the tissues examined included: adrenal gland, bone and marrow, brain, clitoral gland, epididymis, esophagus, gallbladder (mice), heart, kidney, large intestine (cecum, colon, and rectum), liver, lung, lymph nodes (mandibular and mesenteric), mammary gland, nose, ovary, pancreas, parathyroid gland, pituitary gland, preputial gland, prostate gland, salivary gland, seminal vesicle, skin (lesions and unaffected skin from site of application; inguinal skin), small intestine (duodenum, jejunum, and ileum), spinal cord and sciatic nerve (if neurologic signs were present), spleen, stomach (forestomach and glandular stomach), testis, thymus, thyroid gland, trachea, urinary bladder, and uterus.

Other examinations:

None

Statistics:

The probability of survival was estimated by the product-limit procedure of Kaplan and Meier.
Statistical analyses for possible dose-related effects on survival used Cox’s method for testing two groups for equality and Tarone’s life table test
to identify dose-related trends.
Neoplasm prevalence was modeled as a logistic function of chemical exposure and time.
Prevalence analysis of Dinse and Lagakos.
Life table test.
Fisher exact test.
Cochran-Armitage trend test.
Parametric multiple comparison procedures of Dunnett and Williams.
Nonparametric multiple comparison methods of Shirley and Dunn.
Jonckheere’s test.
Mann-Whitney U test.
Multivariate analysis of variance.

Results and discussion

Results of examinations

Details on results:

SURVIVAL
The survival rates of males receiving 32 mg/kg and females receiving 250 mg/kg were slightly less than those of the vehicle controls.

BODY WEIGHTS AND CLINICAL FINDINGS
The mean body weight of females administered 250 mg/kg ranged from 9% to 12% less than that of the vehicle controls between weeks 73 and 93; however, at the end of the study, the mean body weight of this group was only 7% less than that of the vehicle controls. Mean body weights of dosed males were similar to those of the vehicle controls throughout the study. Male and female rats receiving triethanolamine had irritated skin at the site of application; in dosed females, the site of application also had a crusty appearance. The number of animals in which these findings were observed increased with increasing dose.

ORGAN WEIGHTS
At the 15-month interim evaluation, the absolute left and right kidney weights and relative right kidney weight of females administered 250 mg/kg were slightly greater than those of the vehicle controls; the organ weights of dosed males were similar to those of the vehicle controls.

PATHOLOGY AND STATISTICAL ANALYSES
Skin
Gross lesions attributed to triethanolamine administration consisted of multiple, small, randomly located, red or brown lesions or crusts at the site of application in females. Increased incidences of nonneoplastic lesions at the site of application in dosed rats were observed at the 15-month interim evaluation and at the end of the 2-year study. Lesions occurred more frequently in females than in males receiving equivalent doses. Lesions consisted of thickened epidermis (acanthosis) and ulceration with associated chronic active inflammation in dosed males and females, as well as erosion in dosed females.
At the 15-month interim evaluation, two males in the 125 mg/kg group had minimal acanthosis at the site of application. Females receiving 125 or 250 mg/kg had acanthosis, inflammation, and ulceration. Acanthosis and inflammation were of mild average severity; the average severity of ulceration was mild in the 125 mg/kg group and moderate in the 250 mg/kg group. At 2 years, the incidence of acanthosis in males administered 125 mg/kg and the incidences of acanthosis, inflammation, and ulceration in dosed cell females were greater than in the vehicle controls; additionally, males in the 125 mg/kg group had greater incidences of inflammation and ulceration than the vehicle controls. The incidences of erosion, which was diagnosed only in areas distinctly removed from ulceration, were significantly greater in females receiving 125 or 250 mg/kg than in the vehicle controls at 2 years.
The epidermis covering the entire site of application was generally mildly thickened (two to four times) relative to that of the vehicle controls, with neutrophils occasionally observed within the epidermis. Ulcers were random and multifocal and were of mild to moderate severity. Ulcers were characterized by complete segmental necrosis of epidermis, with variable erosion of the dermis and associated chronic active inflammation (neutrophils, lymphocytes, and macrophages). Ulcerated areas were covered with cellular debris, keratin, fibrin, and inflammatory cells composing the “crusts” noted grossly. Erosion consisted of necrosis of the superficial layers of epidermis and did not extend into the dermis.
At 2 years, one male in the 125 mg/kg group had a keratoacanthoma at the site of application. However, the incidences of keratoacanthoma and squamous cell papilloma of the skin (all sites) were slightly less in dosed males than in the vehicle controls. No keratoacanthomas or squamous cell papillomas occurred in vehicle control male rats in the only other dermal study with an acetone vehicle in the NTP database; however, the incidences of keratoacanthoma (10%) and squamous cell papilloma (6%) in the vehicle controls in the present study fall within the historical range for these neoplasms in male rats in NTP feed studies (keratoacanthoma, 0%-10%; squamous cell papilloma, 0%-8%). One vehicle control male had a basal cell adenoma at the site of application; this neoplasm did not occur in dosed males. Additionally, one vehicle control female had a squamous cell papilloma and one female in the 63 mg/kg group had a keratoacanthoma away from the site of application; no skin neoplasms occurred in females administered 125 or 250 mg/kg triethanolamine. Squamous cell papillomas were observed in 6 of 1,202 females in NTP feed studies (range 0%-2%) but did not occur in the other dermal study.

Kidney
No kidney lesions were observed at the 15-month interim evaluation; however, the absolute kidney weights of females in the 250 mg/kg group were greater than those of the vehicle controls. At 2 years, the incidence of adenoma of the renal tubule epithelium in males in the 63 mg/kg group was marginally greater than that in the vehicle controls. No renal tubule adenomas occurred in vehicle control male F344/N rats in other dermal study in the NTP database with an acetone vehicle; the incidence of this neoplasm in untreated control males ranged from 0% to 6%. Although the neoplasm incidence observed in the 63 mg/kg group exceeds the incidence in untreated controls in the NTP database, an equal or greater incidence did not occur in males in the 125 mg/kg group, and the neoplasms in all groups were small and were detected only microscopically. Additionally, the incidences of hyperplasia were not increased in dosed males. Because of these uncertain findings, an extended evaluation of the kidneys of vehicle control and dosed males was conducted. In this extended evaluation, additional proliferative lesions (hyperplasia and adenoma) were identified, with similar incidences in all groups; however, the incidence of adenoma was marginally, although not significantly, greater in the 125 mg/kg group than in the vehicle controls. Nephropathy was observed in nearly all male rats in all groups; there was no apparent difference in the severity of this lesion between dosed and vehicle control groups.
The proliferative lesions were phenotypically similar to those spontaneously occurring in F344/N rats. Focal renal tubule hyperplasia consisted of single or multiple adjacent tubule profiles containing three or more layers of epithelial cells that partially or completely filled the tubule lumens and that usually caused slight dilatation of the tubule. The hyperplastic cells were generally slightly larger than normal epithelial cells and were polygonal, with abundant eosinophilic cytoplasm. All adenomas were small (less than 0.9 cm) and expansile and usually consisted of multiple, variably sized tubule profiles, some with necrotic centers. Cells of adenomas were generally the larger than normal and polygonal, with abundant eosinophilic cytoplasm.

Thyroid Gland
The incidence of C-cell adenoma or carcinoma (combined) was marginally greater in female rats in the 250 mg/kg group than in the vehicle controls (0 mg/kg, 1/50; 63 mg/kg, 2/50; 125 mg/kg, 2/50; 250 mg/kg, 6/49). This greater incidence was not considered to be related to the administration of triethanolamine. Thyroid gland C-cell neoplasms are relatively common, spontaneously occurring neoplasms in male and female rats, occurring in 6 of 46 vehicle control females (13%) in the other NTP dermal study with an acetone vehicle and in 175 of 1,196 untreated control females (15%) in NTP feed studies. Further, of the 24 feed studies in the database, no control group had an incidence of less than 6% for C-cell neoplasms. Additionally, proliferative lesions of the thyroid gland C-cells generally represent a morphological and biological continuum, with progression from hyperplasia to adenoma to carcinoma. In this study, there was often difficulty in determining whether the proliferative lesions were adenomas or hyperplasia. The incidences of hyperplasia in dosed females (8/50, 4/50, 10/50, 2/49; Table B4) did not support a treatment effect, and when the incidences of hyperplasia and neoplasms were combined, the results indicated no increased incidences of proliferative thyroid gland C-cell lesions in dosed female rats.

Uterus
The incidence of stromal polyp of the uterus was marginally increased in females in the 125 mg/kg group, but not in the 250 mg/kg group (2/50, 1/50, 8/50, 5/50). Stromal polyps are relatively common, spontaneously occurring, benign neoplasms in female rats, occurring in 3 of 50 vehicle control females (6%) in the other study with an acetone vehicle in the NTP database and in 178/1,202 untreated control females (16%) in NTP feed studies. Also, the vehicle control incidence of 4% is well below the historical incidence for untreated controls, and the incidence in the 125 mg/kg group is the same as the historical incidence for untreated controls. Therefore, the increased incidence of stromal polyp in females in the 125 mg/kg group was not considered to be related to triethanolamine administration,

Pituitary gland
The incidences of hemosiderin pigment in the pituitary gland pars distalis increased with increasing dose in male rats (0 mg/kg, 23/50; 32 mg/kg, 24/50; 63 mg/kg, 32/48; 125 mg/kg, 35/50), and the incidence of angiectasis was also marginally greater in males administered 125 mg/kg than in the vehicle controls (30/50, 36/50, 29/48, 39/50). These are minimal changes in the incidence of common incidental lesions of uncertain biological significance. Conversely, the incidences of these lesions were lower in females administered 250 mg/kg than in the vehicle controls (hemosiderin pigmentation: 33/50, 29/50, 27/50, 22/50; angiectasis: 37/50, 35/50, 36/50, 29/50).

Effect levels

1

Dose descriptor:

NOAEL

Effect level:

250 mg/kg bw/day (nominal)

Sex:

male/female

Basis for effect level:

other: no treatment related carcinogenic effects were observed

Remarks on result:

other: Effect type: carcinogenicity (migrated information)

2

Dose descriptor:

NOAEL

Effect level:

125 mg/kg bw/day

Sex:

male/female

Basis for effect level:

other: increased relative kidney weight in females (not in males, tested up to 125 mg/kg bw/day)

Remarks on result:

other:

Remarks:

Effect type: other: systemic toxicity (migrated information)

3

Dose descriptor:

NOAEL

Effect level:

63 mg/kg bw/day

Sex:

male

Basis for effect level:

other: acanthosis, ulceration, and chronic active inflammation at the site of application

Remarks on result:

other:

Remarks:

Effect type: other: local toxicity (migrated information)

4

Dose descriptor:

NOAEL

Effect level:

< 63 mg/kg bw/day

Sex:

female

Basis for effect level:

other: acanthosis and chronic active inflammation at the site of application

Remarks on result:

other:

Remarks:

Effect type: other: local toxicity (migrated information)

Any other information on results incl. tables

Although the results of the 13-week rat study led to the selection of doses for female rats in the 2-year study that were twice those administered to males, the females were clearly more sensitive to triethanolamine administration, as shown by the much greater incidences and severities of inflammation and irritation at the site of application in females compared to males in the 2-year study. Ulceration occurred in about half the female rats receiving 125 or 250 mg/kg, while only 5 of 50 males receiving 125 mg/kg had similar lesions. The survival rate of female rats receiving 250 mg/kg was less than that of the other groups, although the mean body weight of this group was only slightly decreased during the study, and this decrease was observed only around week 90. There were no skin neoplasms in male or female rats at or away from the site of application that were considered to be related to triethanolamine administration.

Histopathologic findings which were evaluated to determine their relationship with triethanolamine exposure included renal tubule adenomas in male rats and thyroid gland C-cell adenomas and carcinomas and uterine stromal polyps in female rats. The increased incidences of thyroid gland C-cell neoplasms and uterine stromal polyps were not considered to be related to triethanolamine administration. The evidence for a relationship between the renal tubule neoplasms in male rats and triethanolamine administration was considered equivocal. The total number of male rats identified in the combined single and step-section evaluations as having proliferative lesions (hyperplasia and adenoma) of the renal tubule epithelium was 10/50 (vehicle controls), 8/50 (32 mg/kg), 11/49 (63 mg/kg), and 8/50 (125 mg/kg). Although the proliferative lesions were observed only microscopically, those that were identified as adenomas were clearly larger lesions; these appeared in one vehicle control male, two males in the 32 mg/kg group, six males in the 63 mg/kg group, and four males in the 125 mg/kg group. Four of the six adenomas in the 63 mg/kg group were noted during the standard histopathologic evaluation, and thus this incidence may be compared to historical control data. The 8% incidence in the 63 mg/kg group exceeds the historical mean (0.8%) and range (0%-6%) observed in previous untreated control groups from feed studies. However, the lack of both a clear dose response and an increase in incidences of total proliferative lesions in dosed rats leaves doubt that this result could be attributed to triethanolamine administration with certainty. Other examples of the use of an extended evaluation of the kidney and their interpretation have been discussed by Eustiset al.(1994).

In a previous carcinogenicity study in which triethanolamine was administered in drinking water at concentrations of 1% and 2% to groups of 47 to 50 F344/DuCrj rats (Maekawaet al.,1986), two renal tubule adenomas were observed in females in the 2% group; none occurred in the controls or in the 1% group. This finding was discounted by the authors because renal toxicity was observed in females in the 2% group. Doses were halved for females after week 68 because of toxicity. The total triethanolamine intake during the study was reported to be 119 or 232 mg (actually grams) for female rats. For comparison, assuming 100% absorption of material from the skin and average body weights of 350 g for males and 200 g for females, rats in the 125 (male) and 250 mg/kg (female) groups in the current studies would have received total doses of approximately 23 g for males and 26 g for females. Thus, the lack of kidney toxicity in the current study is consistent with the findings of Maekawaet al.(1986). These authors also reported increased incidences of hepatic neoplasms in males and uterine endometrial sarcoma in females. However, these findings were not attributed to triethanolamine administration because in compar­ison to historical incidences, the neoplasm trends reflected low incidences in the control groups rather than increased incidences in the exposed groups.