Di(µ-2,2´,2´´-nitrilotris(ethanol)-diperchlorato)dinatrium

Repeated dose toxicity: dermal

Administrative data

Endpoint:

sub-chronic toxicity: dermal

Type of information:

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

Adequacy of study:

weight of evidence

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:

Justification for Read Across is given in section 13 of IUCLID.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1999

Materials and methods

Principles of method if other than guideline:

The substance was evaluated for its cumulative toxic effects of repated exposure via the dermal route to the rats, for 13-weeks.

GLP compliance:

yes

Remarks:

FDA GLP Regulations (21 CFR, Part 58).

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

2,2',2''-nitrilotriethanol

EC Number:

203-049-8

EC Name:

2,2',2''-nitrilotriethanol

Cas Number:

102-71-6

Molecular formula:

C6H15NO3

IUPAC Name:

2,2',2''-Nitrilotriethanol

Test animals

Species:

rat

Strain:

Fischer 344

Remarks:

F344/N

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Simonsen Laboratories (Gilroy, CA).
- Age at study initiation: 6 weeks old.
- Housing: individually in Polycarbonate (Lab Products, Inc., Garfield, NJ) cages, equipped with Beta-Chips hardwood chips (Northeastern Products, Inc., Warrensburg, NY) (changed weekly). Cage filters: DuPont 2024 spun-bonded polyester filter (Snow Filtration Co Cincinnati, OH), changed every 2 weeks. Stainless steel racks were used.
- Diet: NIH-07 open formula pelleted diet (Zeigler Brothers, Inc.,Gardners, PA), available ad libitum, changed weekly
- Water: tap water (City of Columbus municipal supply) via automatic watering system (Edstrom Industries, Waterford, WI), available ad libitum.
- Acclimation period: 11-14 days.
- Other: five male and six female and mice were randomly selected for parasite evaluation and gross observation for evidence of disease. At the beginning of quarantine and at the beginning and the end of the studies, serologic analyses were performed on five male and five female and mice using the protocols of the NTP Sentinel Animal Program.

ENVIRONMENTAL CONDITIONS
- Temperature: 19.4 - 23.9 °C.
- Humidity: 35 - 65 %.
- Air changes: 15 changes per hour.
- Photoperiod: 12 hrs dark / 12 hrs light.
- Other: fluorescent light was used.

Administration / exposure

Type of coverage:

not specified

Vehicle:

acetone

Details on exposure:

TEST SITE
- Area of exposure: the area extending from the animal’s mid-back to the dorsal intrascapular region; the site of application was clipped weekly during the studies.

PREPARATION OF DOSES: all dose formulations were prepared by mixing the substance and acetone to give the required concentration. The dose formulations were prepared once every 2 weeks and were stored at 5 °C in amber glass bottles under a nitrogen head space, protected from light, for up to 3 weeks.

VEHICLE
- Vehicle used: except for the highest dose, which was applied neat, all doses were administered in acetone.
- Justification for choice of vehicle: acetone is miscible with the substance and because acetone rapidly evaporates.
- Amount of vehicle: dose volumes were adjusted weekly, if necessary, according to the average body weights of the dosed groups.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Stability studies of the dermal dose formulations were performed by the analytical chemistry laboratory; stability was confirmed for at least 3 weeks at room temperature in sealed glass vials, under a nitrogen head space, in the dark and for at least 3 hours under animal room conditions (open to air and light). Periodic analyses of dose formulations of the test substance were analysed with gas chromatography. The dose formulations were analyzed at the beginning, midpoint, and end of the studies; animal-room samples of the same dose formulations were also analyzed. All dose formulations and animal-room samples were within 10 % of the target concentrations.

Duration of treatment / exposure:

13 weeks/90 days

Frequency of treatment:

5 days per week for 13 weeks

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

125 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

250 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

500 mg/kg bw/day (nominal)

Doses / concentrations 4

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Doses / concentrations 5

Dose / conc.:

2 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: doses were based on the results of 14- and 16-day comparative studies in which Fischer 344 rats received the test material by the dermal route in acetone, in drinking water, or by inhalation. The results of the dermal studies included skin irritation of a severity that limited the highest dose for the 13-week studies 2000 mg/kg for rats.
- Additional groups of 10 male and 10 female rats designated for clinical pathology evaluations received the same dermal exposures as the core study rats.

Examinations

Observations and examinations performed and frequency:

CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly

DERMAL IRRITATION: Yes. The skin (site of application) of male and female rats in the lower exposure groups were examined until a no-effect level was determined.

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

HAEMATOLOGY: Yes
- Time schedule for collection of blood: during week 12.
- Collection of blood: drawn from the retroorbital sinus of rats anesthetized with a mixture of 70%:30% carbon dioxide:air.
- Parameters checked: hematocrit; hemoglobin concentration; erythrocyte, reticulocyte, and nucleated erythrocyte counts; mean cell volume; mean cell hemoglobin; mean cell hemoglobin concentration; platelet count; and total leukocyte count and differentials.
- Haematology analysis: blood for hematology determinations was placed in tubes containing potassium EDTA as an anticoagulant. Blood for serum analyses was collected in tubes without anticoagulant, allowed to clot at room temperature, and centrifuged, and the serum was separated. Hematology parameters were measured on an Ortho ELT-8/ds hematology analyzer (Ortho Instruments, Westwood, MA). Differential leukocyte counts and morphologic evaluation of blood cells were determined by light microscopic examination of blood films stained with a modified Romanowsky stain. Reticulocyte counts were determined by light microscopy from smears of whole blood stained with new methylene blue.
- Animals tested: rats designated for clinical pathology testing.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: during week 12.
- Collection of blood: drawn from the retroorbital sinus of rats anesthetized with a mixture of 70%:30% carbon dioxide:air.
- Animals tested: rats designated for clinical pathology testing.
- Parameters checked: urea nitrogen, creatinine, glucose, total protein, albumin, alanine aminotransferase, aspartate aminotransferase, and sorbitol dehydrogenase.
- Analysis: clinical chemistry parameters were measured on a Hitachi 704 chemistry analyzer (Boehringer Mannheim, Indianapolis, IN).

URINALYSIS: Yes
- Time schedule for collection of urine: during weeks 1, 3, 7 and 13.
- Metabolism cages used for collection of urine: animals were housed individually in polycarbonate metabolism cages (Maryland Plastics, New York) for a 16-hour collection period.
- Collection of urine: the urine collection containers were immersed in an ice water bath during the sampling period to minimize evaporation and to suppress bacterial growth.
- Animals fasted: Yes.
- Parameters checked: glucose, protein, volume, and specific gravity
- Urine analysis: urine volume was measured and specific gravity was determined with a refractometer (American Optical, Buffalo, NY). Urine chemistry variables were measured on a Hitachi 704 chemistry analyzer.
- Animals tested: rats designated for clinical pathology testing.

OTHER:
SPERM MORPHOLOGY: at the end of the 13-week studies, samples were collected for sperm morphology evaluations from all rats in the 0, 500, 1000, and 2000 mg/kg groups. Male animals were evaluated for sperm morphology, count, and motility. The right epididymis and right testis were isolated and weighed. The tail of the epididymis (cauda epididymis) was then removed from the epididymal body (corpus epididymis) and weighed. Modified Tyrode’s buffer was applied to slides, and a small incision was made at the distal border of the cauda epididymis. The sperm effluxing from the incision were dispersed in the buffer on the slides, and the numbers of motile and nonmotile spermatozoa were counted for five fields per slide by two observers. Following completion of sperm motility estimates, each right cauda epididymis was placed in buffered saline solution. Caudae were finely minced, and the tissue was incubated in the saline solution and then heat fixed at 65E C. Sperm density was then determined microscopically with the aid of a hemacytometer. Four sperm morphology slides were prepared for each animal evaluated. An aliquot of killed sperm suspension was stained in a test tube, spread on a microscope slide under a coverslip, and examined.

VAGINAL CYTOLOGY: at the end of the 13-week studies, samples were collected for vaginal cytology evaluations from all rats in the 0, 500, 1000, and 2000 mg/kg groups. For 7 consecutive days before the scheduled terminal sacrifice, the vaginal vaults of the females were moistened with saline, if necessary, and samples of vaginal fluid and cells were stained. Relative numbers of leukocytes, nucleated epithelial cells, and large squamous epithelial cells were determined and used to ascertain estrous cycle stage (i.e., diestrus, proestrus, estrus, and metestrus).

Sacrifice and pathology:

GROSS PATHOLOGY: Yes.
The brain, left epididymis, heart, right kidney, liver, lungs, spleen, left testis, and thymus were weighed.

HISTOPATHOLOGY: Yes.
- Animals tested: complete histopathology was performed on core study rats in the vehicle control and 2000 mg/kg groups.
- Analysis: tissues for microscopic examination 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.
- Tissues tested: 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, uterus, and vagina (females in vaginal cytology studies only). Additionally, the kidney of female rats, pituitary gland of male and female rats, and skin (site of application) of male and female rats in the lower exposure groups were examined until a no-effect level was reached.

Statistics:

The probability of survival was estimated by the product-limit procedure of Kaplan and Meier (1958). Animals found dead of other than natural causes or missexed were censored from the survival analyses; animals dying from natural causes were not censored. Statistical analyses for possible dose-related effects on survival used Cox’s (1972) method for testing two groups for equality and Tarone’s (1975) life table test to identify dose-related trends. All reported P values for the survival analyses are two sided.

Kaplan, E.L., and Meier, P. (1958). Nonparametric estimation from incomplete observations. J. Am. Stat. Assoc. 53, 457-48
Tarone, R.E. (1975). Tests for trend in life table analysis. Biometrika 62, 679-682.

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Clinical findings related to the substance administration occurred only at the site of application and included irritation, scaliness, and crustiness for males and females.

Dermal irritation:

effects observed, treatment-related

Description (incidence and severity):

Irritation, scaliness and crustiness was observed at the site of application for males and females.
Males also had discoloration, and two males administered 2000 mg/kg had ulceration at the site of application.

Mortality:

no mortality observed

Description (incidence and severity):

Final mean body weights and weight gains of males and females in the 2000 mg/kg groups were significantly (P≤0.01) less than those of the vehicle controls; the mean body weight gain of females in the 1000 mg/kg group was also significantly (P≤0.05) less than that of the vehicle controls.

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Minimal to mild changes occurred only in the 2000 mg/kg groups, and are consistent with the marked chronic active skin inflammation observed microscopically. Mild increases in segmented neutrophil counts occurred in male and female rats in the 2000 mg/kg groups; in males, this change was accompanied by increased leukocyte and eosinophil counts. These findings are consistent with the presence of a mild inflammatory leukogram related to the skin inflammation. Minimal decreases occurred in mean red cell volume in male and female rats administered 2000 mg/kg and in hematocrit in females administered 2000 mg/kg. These changes are consistent with a minimal depression of hematopoiesis related to chronic inflammation.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Serum aspartate aminotransferase activities were mildly increased in male rats receiving 250 mg/kg or greater and in females receiving 2000 mg/kg. The cause of the increased aspartate amino transferase activity is unknown, but this increase could indicate mild cardiac/skeletal muscle or hepatic injury.
Serum alanine aminotransferase activity was minimally increased in males in the 1000 and 2000 mg/kg groups. This enzyme is liver specific in the rat, and increases in serum activity would be consistent with a hepatic effect. However, the activity of sorbitol dehydrogenase, which is also liver specific decreased minimally in females administered 500 or 1000 mg/kg. Additionally, there was no microscopic evidence of hepatic injury or change in absolute liver weight in dosed rats.

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

Minimal increases in albumin and urea nitrogen concentrations, likely representing minimal dehydration, occurred in females that received 2000 mg/kg. Additionally, increased urine specific gravity in females in the 1000 and 2000 mg/kg groups at weeks 7 (day 44) and 13 and in male rats in the 2000 mg/kg group at week 13 would be consistent with dehydration. Dehydration can be a sequela of inflammation. Urine protein excretion was decreased in males in the 2000 mg/kg group on day 16, in males administered 500 mg/kg or greater at week 7, and in males in the 1,000 and 2000 mg/kg groups at week 13. The cause for the decreased protein excretion is unknown.

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Kidney weights were generally greater in males and females administered 500, 1000, or 2000 mg/kg than in the vehicle controls. Other differences in organ weights of males and females administered 2000 mg/kg were considered secondary to the lower body weights of these groups.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Gross lesions attributed to the substance application included crust at the site of application for males and females administered 1000 or 2000 mg/kg
Yellow skin coloration in the lumbar region of vehicle control and treated males was attributed to the application of acetone. Microscopic lesions at the site of application included acanthosis and inflammation, which varied from minimal or mild in the lower dose groups to marked in the 2000 mg/kg groups. In rats with minimal acanthosis, the epidermis was as much as twice the normal thickness. The more severe lesions were focal to multifocal and included a thickened epidermis (three to four times the normal thickness); chronic active inflammation; and erosion, ulceration, or both. The underlying dermis was often thickened by chronic active inflammation, including fibrosis.

Neuropathological findings:

effects observed, treatment-related

Description (incidence and severity):

Dosed females had greater incidences of nephropathy than did the vehicle controls. Nephropathy consisted of minimal to mild, focal or multifocal cortical tubules lined by hyperchromatic, basophilic tubule epithelium (regeneration). Mineralization, which also occurred with greater incidences and severity in dosed females, consisted of tiny lamellated concretions within tubule lumina and/or epithelium. The incidences of nephropathy were not increased in dosed males.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

The incidences of hypertrophy of the pituitary gland pars intermedia were significantly greater in males and females in the 2000 mg/kg group than in the vehicle controls. The pars intermedia is a thin section of tissue composed of compact clusters of polygonal cells that lies between the pars distalis and the pars nervosa. Because of its small size and because of some variation in sectioning of the pituitary gland, the pars intermedia was not always present in the section examined. In the animals with hypertrophy, the pars intermedia was as much as twice the normal size, with a somewhat nodular appearance. Individual cells were moderately enlarged, apparently due to an increase in the amount of cytoplasm.

Histopathological findings: neoplastic:

not examined

Description (incidence and severity):

No statistically significant differences in sperm morphology or vaginal cytology parameters occurred between dosed and vehicle control mice

Effect levels

Dose descriptor:

NOAEL

Remarks on result:

other: not determined/specified

Target system / organ toxicity

Critical effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

No NOAEL was determined in the current study.

Executive summary:

The test material was evaluated for its cumulative toxic effects of repated exposure to rats. For this reason, groups of 10 male and 10 female F344/N rats were topically administered 0, 125, 250, 500, 1000 and 2000 (in acetone) and 4000 (neat) mg/kg bw, 5 days per week, for 13 weeks.

All rats survived to the end of the study. Final mean body weights and weight gains of males and females administered 2000 mg/kg and the mean body weight gain of females administered 1000 mg/kg were significantly less than those of the vehicle controls. Clinical observations included irritation, scaliness, and crustiness of the skin at the site of application for males and females. Males also had discoloration, and two males administered 2000 mg/kg had ulceration at the site of application. Changes in clinical pathology parameters were minor and consistent with inflammation at the site of application. Kidney weights were generally greater in males and females administered 500, 1000, or 2000 mg/kg than in the vehicle controls. Microscopic lesions attributed to the substance administration included acanthosis and inflammation at the site of application nephropathy in females, and hypertrophy of the pituitary gland pars intermedia in males and females. These lesions generally occurred with dose-related increases in incidence and severity in males and females.

No NOAEL was determined in the current study.