2,2',2''-nitrilotriethanol citrate

Repeated dose toxicity: dermal

Administrative data

Endpoint:

sub-chronic toxicity: dermal

Type of information:

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

Adequacy of study:

key study

Justification for type of information:

See read-across record in section 13.

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

read-across source

Reference

Endpoint:

sub-chronic toxicity: dermal

Type of information:

experimental study

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

Qualifier:

equivalent or similar to guideline

Guideline:

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

GLP compliance:

not specified

Limit test:

no

Species:

mouse

Strain:

B6C3F1

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Simonsen Laboratories, Gilroy, CA
- Age at study initiation: 39 to 42 days
- Weight at study initiation: 19.2 - 24.6 g (males), 14.7 - 19.6 g (females)
- Fasting period before study: No
- Housing: Individual in polycarbonate cages
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 11 to 14 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.5 - 24
- Humidity (%): 35 - 65
- Air changes (per hr): at least 15
- Photoperiod (hrs dark / hrs light): 12 / 12

Type of coverage:

not specified

Vehicle:

acetone

Details on exposure:

TEST SITE
- Area of exposure: an area extending from the animal's mid-back to dorsal interscapular region
- Time intervals for shavings or clipplings: at least 24 hours prior to initial dose, and once weekly thereafter

REMOVAL OF TEST SUBSTANCE
No data

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 4.0, 2.0, 1.0, 0.5, 0.25 and 0 g/kg bw
- Concentration (if solution): 1120, 560, 280, 140, 70 and 0 mg/ml
- Constant volume or concentration used: yes

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

90 days

Frequency of treatment:

5 days per week

Dose / conc.:

250 mg/kg bw/day

Remarks:

Equivalent to 70 mg/mL

Dose / conc.:

500 mg/kg bw/day

Remarks:

Equivalent to 140 mg/mL

Dose / conc.:

1 000 mg/kg bw/day

Remarks:

Equivalent to 280 mg/mL

Dose / conc.:

2 000 mg/kg bw/day

Remarks:

Equivalent to 560 mg/mL

Dose / conc.:

4 000 mg/kg bw/day

Remarks:

Equivalent to 1120 mg/mL

No. of animals per sex per dose:

20 animals per dose:
- 10 special study animals (designated for periodic urinalysis, haematology, and clinical chemistry determinations)
- 10 base study animals (subject to the collection of clinical observations data, sperm morphology and vaginal cytology evaluations, necropsy with gross examination and tissue collection, and histopathologic examination).

Control animals:

yes, concurrent vehicle

Details on study design:

Post-exposure period: no

Observations and examinations performed and frequency:

DETAILED CLINICAL OBSERVATIONS
- Time schedule: Weekly

DERMAL IRRITATION
- Time schedule for examinations: Daily

BODY WEIGHT
- Time schedule for examinations: Weekly

HAEMATOLOGY
- Time schedule for collection of blood: Week 11
- Anaesthetic used for blood collection: No data
- Parameters examined were erythrocyte count (RBC), haemoglobin (HgB), haematocrit (HCT), leukocyte count (WBC), platelet count, mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), mean corpuscular haemoglobin concentration (MCHC), leukocyte differential count, reticulocyte count, erythrocyte and platelet morphology.

CLINICAL CHEMISTRY
- Time schedule for collection of blood: Week 11
- Parameters examined were sorbitol dehydrogenase (SDH), glutamic-pyruvic transaminase activity (GPT), glutamic-oxaloacetic transaminase activity (GOT), urea nitrogen (BUN), creatinine, total protein, albumin, glucose.

URINALYSIS
- Time schedule for collection of urine: week 1, week 3, week 7 and week 12
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters examined were specific gravity, glucose, protein, microscopic examination of sediment.

Sacrifice and pathology:

- The following tissues were examined: gross lesions and tissue masses (and regional lymph nodes, if possible), blood smear (if required by the pathologist), mandibular and mesentric lymph nodes, salivary gland, sternebrae, femur, or vertebrae, including marrow, thyroids, heart, esophagus, stomach (to include forestomach and glandular stomach), uterus, brain (three sections, including frontal cortex and basal ganglia, parietal cortex and thalamus, and cerebellum and pons), thymus, trachea, parathyroids, small intestine (duodenum, jejunum, ileum), cecum, colon and rectum, liver, prostate, testis, epididymis, seminal vesicle, ovaries, lungs and mainstem bronchi, nasal cavity and nasal turbinates (3), preputial or clitoral glands (paired), pancreas, spleen, kidneys, adrenals, urinary bladder, pituitary, spinal cord and sciatic nerve (if neurology signs were present), eyes (if grossly abnormal), mammary gland (including surface skin), pharynx (if grossly abnormal), skin (lesions in dosed area, unaffected skin in dosed area, and undosed control skin).

- Complete histopathologic evaluation of all tissues listed was performed on all base study mice from the 4.0 and 0 g/kg dose groups. On the basis of those findings, the skin at the site of application was selected as the target tissue, and was examined in mice in all lower dose groups. In addition, any gross lesions detected at necropsy were examined microscopically.

Other examinations:

Sperm morphology and vaginal cytology were evaluated in the control and the three highest dose groups.

Clinical signs:

no effects observed

Description (incidence and severity):

Significant clinical abnormalities were restricted to the site of dermal dose application in both sexes, and occurred only at the highest dose level (4.0 g/kg).

Dermal irritation:

effects observed, treatment-related

Description (incidence and severity):

Significant clinical abnormalities the site of dermal dose application occurred only at the highest dose level (4.0 g/kg).
- Males: Scaliness, irritation, and discoloration at the site of application were observed in 10 males dosed at 4.0 g/kg body weight, and one male in this dose group exhibited erosion of the skin paint area.
- Females: In female mice dosed at 4.0 g/kg, scaliness at the site of application was seen in 8 animals, irritation in 7, and discoloration in 10. Other clinical abnormalities were determined to not be treatment related.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

No deaths occurred during the study that were attributed to dermal application of test substance. No deaths occurred during this sub-chronic study in the base study groups. Mortality did occur in special study groups that included 3 males in the 0 g/kg dose group, 2 in the 0.25 g/kg dose group, 1 in the 1.0 g/kg dose group, and 2 in the 2.0 g/kg dose group. One female in the 2.0 g/kg group also died from complications resulting from blood collection during Week 13; in addition, one female mouse in the 1.0 g/kg dose group was found to be missing during study Week 9.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

- All male dosed groups, on average, gained slightly more weight during the study than did their respective control group. The male control group mean weight gain was 7.8 grams; the mean weight gains for dosed male groups ranged from 8.1 to 8.7 grams.

- All female triethanolamine-dosed groups had mean body weight gains that were slightly depressed relative to control. These depressions were not related to dosage level; the differential weight gains for the dosed groups ranged from -3.3 percent (1.0 and 4.0 g/kg dose groups) to -14.1 percent (0.25 g/kg, or lowest, dose group). Treatment with test substance had no apparent effect on body weight gain.

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):

Haematology was performed on blood collected on study Day 85 from special group mice.
Males:
- Mean data for all dosed male groups were statistically similar to those for the male control group.
- All mean leukocyte differential counts (relative and absolute) for males were statistically similar to control.
Females:
- In females dosed at 4.0 g/kg body weight, there were significant increases in the number of leukocytes and the mean corpuscular haemoglobin concentration. There was also a slight, but statistically significant, decrease in the mean corpuscular volume of the 2.0 g/kg female dose group. All other mean values for dosed female mice were statistically similar to control.
- The only significant change in data for the female mice was an increase in the absolute number of eosinophils.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Males:
- There was a dose-related, statistically significant decrease in serum sorbitol dehydrogenase that occurred in all dosed groups.
- The mean values for all other parameters measured were statistically similar to control.
Females:
- there was also a significant decrease in serum sorbitol dehydrogenase at all treatment levels.
- Additionally, there was an increase in both serum total protein and albumin in the 2.0 g/kg female dose group, and a significant decrease in alanine aminotransferase in the female 1.0 g/kg dose group.

Urinalysis findings:

effects observed, non-treatment-related

Description (incidence and severity):

Urine was collected from special study group mice and analysed on Days 46 and 80.
Males:
- With the exception of decreased number of bacteria for the male 4.0 g/kg dose group at Day 46, the mean values for all parameters evaluated for male mice at both times that urinalysis was performed were statistically similar to control.
Females:
- There was a significant increase in the mean urine volume of the 4.0 g/kg female dose group, and also in the mean urine specific gravity of the 2.0 g/kg female dose group, at study Day 46. The biological significance of these elevations is unclear because they were not present at the Day 80 urinalysis; at study Day 80, all mean values for the female treated groups were statistically similar to control.

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):

- Mean brain absolute weight and weight relative to body weight data for dosed groups were statistically similar to control.
- The mean liver absolute weights, liver to body weight ratios, and liver to brain weight ratios of male and female mice dosed at 4.0 g/kg were significantly increased over control. The mean liver to body weight ratios of the 2.0 and 0.25 g/kg male dose groups were also significantly higher than control.
- Mean right kidney absolute weight, weight relative to body weight, and weight relative to brain weight of the 4.0 g/kg female dose group were significantly higher than control. In addition, the right kidney to body weight ratios of the 4.0 g/kg male dose group, the 2.0 g/kg male and female dose groups, and the 0.25 g/kg female dose group were significantly increased: the mean right kidney to brain weight ratio of the 0.25 g/kg female group was also elevated.
- In females dosed at 4.0 g/kg body weight, there was increased mean spleen absolute weight, spleen to body weight ratio, and spleen to brain weight ratio. The only significant change in thymus weight data was an increase in the mean thymus to body weight ratio for the 2.0 g/kg female dose group.
- There was a significant increase in heart to body weight ratio for both male and female mice dosed at 4.0 g/kg body weight. The mean heart absolute weight and weight relative to brain weight for these groups were also elevated, but not significantly so.
- Group mean lung, left testis, and left epididymis weight values for all triethanolamine-dosed groups were statistically similar to control.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

- Final body weight: The mean weight for each dosed group was statistically similar to that of their respective control group.
- Gross lesions were common on the skin of high dose mice. The compound-related macroscopic lesions at the application site were dermal crusts and/or white/white-scaley skin. Other gross lesions were considered incidental.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Microscopic examination was done on all tissues from mice that received the highest dose (4.0 g/kg) and control (0 g/kg) group mice. The skin was examined to the lowest dose level. Gross lesions were examined in all lower dose groups. Few microscopic lesions were found in organs other than skin.
- The test substance-related skin lesions were chronic-active inflammation and acanthosis. Chronic-active inflammation was seen in 4.0 g/kg male and female mice and in one 2.0 g/kg female mouse.
- The chronic-active inflammation contained epidermal and/or dermal components.
- The epidermal component included acanthotic, hyperkeratotic, focally parakeratotic epidermis; these changes were graded separately as acanthosis. Severely affected epidermis also contained focal haemorrhage, fibrin, serum pockets, pustules, erosions and/or ulcers.
- The dermal component in severely affected mice was characterized by dermal fibrosis, distortion of hair follicles and adnexal organs, and variably severe mixed inflammatory infiltrates consisting of histiocytes, lymphocytes and neutrophils.
- Although only the most severe grades of chronic-active inflammation contained all of the previously listed epidermal and dermal features, the lesion was regarded as a continuum and, therefore, the morphology "chronic-active inflammation" was used in all dose groups.

- Less severe lesions of chronic-active inflammation had reduced acanthosis and lacked one or more of the following components: epidermal haemorrhage, fibrin deposits, serum pockets, pustules, erosions and/or ulcers.
- Additionally, less severe lesions contained fewer of the dermal features of the most severe lesions.
- Where only the term acanthosis was used, the least severe lesion contained 2-3 layers of epidermal cells above the basilar layer.
- Occasionally, there was minimal to mild dermal fibrosis associated with the acanthosis.
- The highest incidence and severity of chronic-active inflammation and acanthosis occurred in high dose males and females.
- Minimal acanthosis was seen in the majority of mice in all lower dose groups.
- Non-compound related microscopic lesions were seen in the lung and uterus.

Histopathological findings: neoplastic:

no effects observed

Details on results:

The compound-induced skin lesions in males and females were chronic-active inflammation and acanthosis. The incidence and severity of inflammation were slightly higher in high dose males than females. In females, however, inflammation extended to one dose lower than in males. Acanthosis was seen in all dose groups; the more severe lesions were seen only in the high dose. The consistently similar incidence of minimal acanthosis in males and females of all lower dose groups suggested that the skin was reactive to, but tolerant of the test material at doses lower than 4.0 g/kg in males and 2.0 g/kg in females.

Dose descriptor:

LOAEL

Remarks:

local effects

Effect level:

250 mg/kg bw/day

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

Dose descriptor:

LOAEL

Remarks:

systemic effects

Effect level:

250 mg/kg bw/day

Sex:

female

Basis for effect level:

organ weights and organ / body weight ratios

Dose descriptor:

NOAEL

Remarks:

systemic effects

Effect level:

1 000 mg/kg bw/day

Sex:

male

Basis for effect level:

organ weights and organ / body weight ratios

Critical effects observed:

no

Cross-reference 2

Reason / purpose for cross-reference:

read-across source

Reference

Endpoint:

sub-chronic toxicity: dermal

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

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

GLP compliance:

not specified

Limit test:

no

Species:

rat

Strain:

Fischer 344

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Simonsen Laboratories, Gilroy, CA
- Age at study initiation: 39 to 42 days
- Weight at study initiation: 59.2 - 85.4 g (males), 57.0 - 79.3 g (females)
- Fasting period before study: No
- Housing: Individual in polycarbonate cages
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 11 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.5 - 24
- Humidity (%): 35 - 65
- Air changes (per hr): at least 15
- Photoperiod (hrs dark / hrs light): 12 / 12

Type of coverage:

not specified

Vehicle:

acetone

Details on exposure:

TEST SITE
- Area of exposure: an area extending from the animal's mid-back to dorsal interscapular region
- Time intervals for shavings or clipplings: at least 24 hours prior to initial dose, and once weekly thereafter

REMOVAL OF TEST SUBSTANCE
No data

TEST MATERIAL
- Amounts applied: 2.0, 1.0, 0.5, 0.25, 0.125 and 0 g/kg bw
- Concentration: 1120, 560, 280, 140, 70 and 0 mg/mL
- Constant volume or concentration used: yes

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

90 days

Frequency of treatment:

5 days per week

Dose / conc.:

125 mg/kg bw/day (nominal)

Remarks:

Equivalent to 70 mg/mL

Dose / conc.:

250 mg/kg bw/day (nominal)

Remarks:

Equivalent to 140 mg/mL

Dose / conc.:

500 mg/kg bw/day (nominal)

Remarks:

Equivalent to 280 mg/mL

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Remarks:

Equivalent to 560 mg/mL

Dose / conc.:

2 000 mg/kg bw/day (nominal)

Remarks:

Equivalent to 1120 mg/mL

No. of animals per sex per dose:

20 animals per dose:
- 10 special study animals (designated for periodic urinalysis, haematology, and clinical chemistry determinations)
- 10 base study animals (subject to the collection of clinical observations data, sperm morphology and vaginal cytology evaluations, necropsy with gross examination and tissue collection, and histopathologic examination).

Control animals:

yes, concurrent vehicle

Details on study design:

Post-exposure period: no

Observations and examinations performed and frequency:

DETAILED CLINICAL OBSERVATIONS
- Time schedule: Weekly

DERMAL IRRITATION
- Time schedule for examinations: Daily

BODY WEIGHT
- Time schedule for examinations: Weekly

HAEMATOLOGY
- Time schedule for collection of blood: Week 11
- Parameters examined were erythrocyte count (RBC), haemoglobin (HgB), haematocrit (HCT), leukocyte count (WBC), platelet count, mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), mean corpuscular haemoglobin concentration (MCHC), leukocyte differential count, reticulocyte count, erythrocyte and platelet morphology.

CLINICAL CHEMISTRY
- Time schedule for collection of blood: Week 11
- Parameters examined were sorbitol dehydrogenase (SDH), glutamic-pyruvic transaminase activity (GPT), glutamic-oxaloacetic transaminase activity (GOT), urea nitrogen (BUN), creatinine, total protein, albumin, glucose.

URINALYSIS
- Time schedule for collection of urine: week 1, week 3, week 7 and week 12
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters examined were specific gravity, glucose, protein, microscopic examination of sediment.

CLINICAL CHEMISTRY
- Time schedule for collection of blood: Week 11
- Parameters examined were sorbitol dehydrogenase (SDH), glutamic-pyruvic transaminase activity (GPT), glutamic-oxaloacetic transaminase activity (GOT), urea nitrogen (BUN), creatinine, total protein, albumin, glucose.

URINALYSIS
- Time schedule for collection of urine: week 1, week 3, week 7 and week 12
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters examined were specific gravity, glucose, protein, microscopic examination of sediment.

Sacrifice and pathology:

- The following tissues were examined: gross lesions and tissue masses (and regional lymph nodes, if possible), blood smear (if required by the pathologist), mandibular and mesentric lymph nodes, salivary gland, sternebrae, femur, or vertebrae, including marrow, thyroids, heart, esophagus, stomach (to include forestomach and glandular stomach), uterus, brain (three sections, including frontal cortex and basal ganglia, parietal cortex and thalamus, and cerebellum and pons), thymus, trachea, parathyroids, small intestine (duodenum, jejunum, ileum), cecum, colon and rectum, liver, prostate, testis, epididymis, seminal vesicle, ovaries, lungs and mainstem bronchi, nasal cavity and nasal turbinates (3), preputial or clitoral glands (paired), pancreas, spleen, kidneys, adrenals, urinary bladder, pituitary, spinal cord and sciatic nerve (if neurology signs were present), eyes (if grossly abnormal), mammary gland (including surface skin), pharynx (if grossly abnormal), skin (lesions in dosed area, unaffected skin in dosed area, and undosed control skin).

- Complete histopathologic evaluation of all tissues listed was performed on all base study rats from the 2.0 and 0 g/kg dose groups. On the basis of those findings, the skin at the site of application (both males and females) and the kidney (females only) were selected as the target tissues. These tissues were examined in rats in successively lower dose groups to a no-effect level; in addition, any gross lesions detected at necropsy were examined microscopically.

Other examinations:

Sperm morphology and vaginal cytology were evaluated in the control and the three highest dose groups.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

The only clinical abnormalities associated with treatment with the test substance occurred at the site of dermal dose application. Other abnormalities were determined not to be compound induced.

Dermal irritation:

effects observed, treatment-related

Description (incidence and severity):

Males:
- Discoloration of the skin was seen in males from all dose groups (including control); it was present in 6 males in the 0 g/kg dose group, and in all 10 base study animals in each of the dosed groups.
- Irritation at the site of application was seen in the three highest dosed groups (0.5, 1.0, and 2.0 g/kg), with incidence increasing and time to onset decreasing with increasing dose level.
- Scaliness was observed only at the 1.0 and 2.0 g/kg levels; this was observed in 1 rat in the 1.0 g/kg group (first seen on Day 13), and in 5 rats in the 2.0 g/kg group (first seen on Day 6).
- Crustiness at the site of application was recorded for 10 rats in the 2.0 g/kg dose group; two males from this group were also observed to have ulceration at the application site.
Females:
- Irritation was observed at the 1.0 (7 rats) and 2.0 (10 rats) g/kg dose levels.
- Scaliness was also seen in female rats from the 1.0 and 2.0 g/kg groups (4 animals dosed at the 1.0 g/kg level, and 5 animals dosed at the 2.0 g/kg level).
- Crustiness was present in 3 female rats dosed at 2.0 g/kg.

Mortality:

no mortality observed

Description (incidence):

All animals in all dose groups survived until scheduled termination.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

The 2.0 g/kg male dose group exhibited a pronounced depression in body weight gain; the differential weight gain, relative to control, for this group was -32.8 percent, whereas those for the other male dosed groups ranged from -4.8 to +8.4 percent. The mean weight gains for all female dosed groups were depressed when compared with that of the female control group; the differential weight gains (relative to control group weight gain) for the 0.125, 0.25, 0.5, 1.0, and 2.0 g/kg female dose groups were -13.1, -10.6, -6.0, -21.4, and -35.9 percent, 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:

effects observed, treatment-related

Description (incidence and severity):

Haematology was performed on blood obtained from special study rats on study Day 80.
- Male rats dosed at 2.0 g/kg exhibited a significantly increased leukocyte count and decreased mean corpuscular volume.
- Significantly decreased mean corpuscular volume was also exhibited by the 2.0 g/kg female dose group; the haematocrit of this group was depressed as well.
- Male rats in the 2.0 g/kg dose group exhibited significant increases in both relative and absolute number of segmented neutrophils and eosinophils, and a significant reduction in the relative number of lymphocytes.
- Female rats treated at the same dosage level (2.0 g/kg) also exhibited a significant increase in relative and absolute numbers of segmented neutrophils, as well as a decrease in relative lymphocyte count.
These haematological changes in high dose rats of both sexes can be attributed to an inflammatory response resulting from dermal irritation. The mean values for all other dosed groups of both sexes were statistically similar to control.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Males:
- The 2.0 and 0.25 g/kg male dose groups exhibited elevated SGOT levels
- The mean SGPT level of the 2.0 g/kg male dose group was also significantly higher than control.
No other statistically significant changes were seen when mean data from the male dosed groups were compared with that of control.

Females:
- The 2.0 g/kg female dose group exhibited elevated serum urea nitrogen, albumin, SGOT, and SGPT.
The only other significant change in the female group mean data was a decrease in serum sorbitol dehydrogenase in rats dosed at 0.5 and 1.0 g/kg.

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

Urine, collected from special study group rats on Days 3, 16, 44, and 86, was analysed.
Males:
- At study Day 3, mean values for all male dosed groups were statistically similar to those of the male control group.
- At study Day 16, the only values statistically different from control were urine protein at the 1.0 g/kg level, and the number of leukocytes at the 0.25 g/kg level.
- By study Day 44, there was a significant reduction in urine protein levels in males dosed at 0.5, 1.0, and 2.0 g/kg; in addition, the specific gravity of urine collected from the 2.0 g/kg male dose group was significantly increased over control.
- At the final urinalysis (Day 86), the specific gravity of urine from rats in the 2.0 g/kg dose group was again significantly elevated, and the urine protein levels for the 0.5, 1.0, and 2.0 g/kg male dose groups were significantly lower than control. The only other parameter statistically different from control was increased urine volume in males dosed at 0.5 g/kg.

Females:
- At study Day 3, there was decreased urine protein concentration at the 0.25 g/kg level and above; the number of crystals found in the urine of rats in the 2.0 g/kg dose group was also significantly greater than control.
- On Day 16, increased number of crystals was also present, in females dosed at 1.0 and 2.0 g/kg.
- Beginning on Day 16, and continuing through Days 44 and 86, the specific gravity of urine from female rats from the two highest dose groups (1.0 and 2.0 g/kg) was significantly greater than that of control females.
- At Day 44, the only other statistically significant changes observed were increased urine protein concentration in females dosed at 0.5 g/kg, and increased number of crystals in females dosed at 2.0 g/kg.
- At Day 86, in addition to the increased urine specific gravity in the two highest dose groups, the 2.0 g/kg females exhibited an increase in urine glucose concentration.

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):

Final body weight:
- The mean final body weights for both male and females dosed at 2.0 g/kg body weight were significantly decreased (p <0.01), compared with those of their respective control groups.

Organ weights:
- The mean brain to body weight values for the 2.0 g/kg male and female dose groups were significantly increased relative to control. In the absence of statistical changes in the absolute brain weight for these groups, this increase in relative weight appears to be the result of depressed final body weights in rats dosed at 2.0 g/kg.
- Statistically significant increases in mean right kidney absolute weights and weights relative to brain weight occurred in both males and females dosed at 1.0 and 2.0 g/kg. The right kidney to body weight ratios of the 0.25, 0.5, 1.0, and 2.0 g/kg male dose groups and the 1.0 and 2.0 g/kg female dose groups were also significantly increased over that of control.
- The mean spleen absolute weight and spleen weight relative to brain weight of the 2.0 g/kg female dose group were significantly decreased, relative to control values. There was a significant increase in the mean spleen to body weight ratio in male rats dosed at 1.0 and 2.0 g/kg.
- Thymus absolute weight and weight relative to brain weight were increased in males dosed at 2.0 g/kg body weight.
- Mean liver to body weight ratios for the 0.5 and 1.0 g/kg male dose groups were significantly elevated over that of control. No statistically significant changes were observed in mean liver weight values for the female dose groups.
- The mean lung absolute weight and lung to brain weight ratio of the 2.0 g/kg male dose group were significantly decreased, relative to control. The mean lung to brain weight of the 0.5 g/kg male dose group was also significantly decreased. The only statistically significant change in mean lung weight values for female rats was increased lung to brain weight ratio at the 0.125 g/kg level.
- No statistically significant changes were seen in mean heart weight data for male and female rats.
- For both testes, there was a significant increase in weight relative to body weight in the 2.0 g/kg male dose group; this was a result of decreased body weight at necropsy.
- No statistical changes were observed for mean left epididymis weight values. The 2.0 g/kg male dose group exhibited decreased right epididymis absolute weight, and increased weight relative to body weight. Right epididymis to body and to brain weight ratios were increased in males dosed at 0.25 g/kg.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Gross lesions were common on the skin of high dose rats. The compound-related lesion seen at the application site was dermal crust. Yellow skin was seen in the lumbar region of treated and control animals. This lesion had no corresponding microscopic lesion and is attributed to application of the vehicle (acetone). Other gross lesions were considered incidental, spontaneous lesions.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

- The compound-related skin lesions were chronic-active inflammation and acanthosis, and were seen in 2.0, 1.0, 0.5, and 0.25 g/kg male rats and in 2.0, 1.0, and 0.5 g/kg female rats.
- The chronic-active inflammation contained epidermal and dermal components.
- The epidermal component was characterized by acanthotic, hyperkeratotic, focally parakeratotic epidermis that occasionally contained rete pegs; these changes were graded separately as acanthosis. Severely affected epidermis contained focal haemorrhage, fibrin and/or mineral deposits, bacterial colonies, serum pockets, pustules, erosions and/or ulcers.
- The dermal component in severely affected rats was characterized bydermal fibrosis, neocapillarization, minimally distorted adnexal organs, and variably severe mixed inflammatory infiltrates consisting of histiocytes, lymphocytes, neutrophils and eosinophils.
- Although only the most severe grades of chronic-active inflammation contained all of the previously listed epidermal and dermal features, the lesion was regarded as a continuum, and, therefore, the morphology "chronic-active inflammation" was used in all dose groups.
- Less severe lesions of chronic-active inflammation had reduced acanthosis, and lacked one or more of the following components: epidermal haemorrhage, fibrin and/or mineral deposits, bacterial colonies, serum pockets, pustules, erosions and/or ulcers.
- Additionally, less severe lesions contained fewer of the dermal features of the most severe lesion.
- In minimal inflammatory lesions the epidermal component was predominant.
- Where only the term acanthosis was used, the least severe lesion contained 2-3 layers of epidermal cells above the basilar layer.
- Occasionally, there was minimal to mild dermal fibrosis associated with the acanthosis.
- In males and females there was a dose dependent reduction in severity and incidence of skin lesions from high to no-effect dose.
- Non-compound related microscopic lesions were seen in the kidney, liver, lung, prostate, ovary, preputial gland, clitoral gland, eye, Zymbal's gland, nose/nasal cavity; and mediastinal lymph node.

Histopathological findings: neoplastic:

no effects observed

Details on results:

The compound-induced skin lesions in males and females were chronic-active inflammation and acanthosis. The incidence, severity, and morphology of the lesions were similar between sexes. In males, however, the compound effect extended to one dose lower than in females. Although there was increased incidence of nephropathy from low to high dose in female rats, the severity of this lesion did not vary between dose groups, and, therefore, it was regarded as incidental. Other microscopic lesions were regarded as incidental and spontaneous or associated with retrobulbar bleeding.

Dose descriptor:

NOAEL

Remarks:

local effects

Effect level:

125 mg/kg bw/day

Sex:

male

Basis for effect level:

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

Dose descriptor:

NOAEL

Remarks:

local effects

Effect level:

250 mg/kg bw/day

Sex:

female

Basis for effect level:

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

Dose descriptor:

NOAEL

Remarks:

systemic effects

Effect level:

125 mg/kg bw/day

Sex:

male

Basis for effect level:

other: increased relative kidney weight

Dose descriptor:

NOAEL

Remarks:

systemic effects

Effect level:

500 mg/kg bw/day

Sex:

female

Basis for effect level:

other: increased absolute and relative kidney weight

Critical effects observed:

no

Materials and methods

Test material

Test material information

Constituent 1

Reference substance name:

2,2',2''-nitrilotriethanol citrate

EC Number:

249-576-7

EC Name:

2,2',2''-nitrilotriethanol citrate

Cas Number:

29340-81-6

Molecular formula:

C6H15NO3.xC6H8O7

IUPAC Name:

tris(2-hydroxyethyl)azanium 3,4-dicarboxy-3-hydroxybutanoate

Test material form:

liquid

Results and discussion

Effect levels

1

Key result

Dose descriptor:

NOAEL

Remarks:

systemic effects

Effect level:

125 mg/kg bw/day

Based on:

test mat.

Sex:

male

Basis for effect level:

other: increased relative kidney weight

Remarks on result:

other: Result read-across source CAS No. 102-71-6

Remarks:

rat

2

Dose descriptor:

NOAEL

Remarks:

systemic effects

Effect level:

500 mg/kg bw/day

Based on:

test mat.

Sex:

female

Basis for effect level:

other: increased absolute and relative kidney weight

Remarks on result:

other: Result read-across source CAS No. 102-71-6

Remarks:

rat

3

Dose descriptor:

NOAEL

Remarks:

local effects

Effect level:

125 mg/kg bw/day

Based on:

test mat.

Sex:

male

Basis for effect level:

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

Remarks on result:

other: Result read-across source CAS No. 102-71-6

Remarks:

rat

4

Dose descriptor:

NOAEL

Remarks:

local effects

Effect level:

250

Based on:

test mat.

Sex:

female

Basis for effect level:

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

Remarks on result:

other: Result read-across source CAS No. 102-71-6

Remarks:

rat

5

Dose descriptor:

NOAEL

Remarks:

systemic effects

Effect level:

250 mg/kg bw/day

Based on:

test mat.

Sex:

female

Basis for effect level:

organ weights and organ / body weight ratios

Remarks on result:

other: Result read-across source CAS No. 102-71-6

Remarks:

mouse

6

Dose descriptor:

NOAEL

Remarks:

systemic effects

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male

Basis for effect level:

organ weights and organ / body weight ratios

Remarks on result:

other: Result read-across source CAS No. 102-71-6

Remarks:

mouse

7

Dose descriptor:

LOAEL

Remarks:

local effects

Effect level:

250 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

Remarks on result:

other: Result read-across source CAS No. 102-71-6

Remarks:

mouse

Target system / organ toxicity

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

250 mg/kg bw/day

System:

integumentary

Organ:

skin

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified